MICROBIAL ANTAGONISMS AND
ANTIBIOTIC SUBSTANCES

LONDON

GEOFFREY CUMBERLEGE
OXFORD UNIVERSITY PRESS

Penicillin crystals

Microbial Antagonisms

AND

Antibiotic Substances

SELMAN A. WAKSMAN

PROFESSOR OP MICROBIOLOGY, RUTGERS

UNIVERSITY; MICROBIOLOGIST, NEW JERSEY

AGRICULTURAL EXPERIMENT STATION

^La vie emfeche la vie" — Pasteur

NEW YORK

THE COMMONWEALTH FUND

1947

COPYRIGHT, 1945, BY

THE COMMONWEALTH FUND

FIRST PRINTING MARCH 1 945

SECOND PRINTING DECEMBER 1 945

SECOND EDITION, REVISED AND ENLARGED

COPYRIGHT, 1947, BY

THE COMMONWEALTH FUND

PUBLISHED BY THE COMMONWEALTH FUND
41 EAST 57TH STREET, NEW YORK 22, N.Y.

PRINTED IN THE UNITED STATES OF AMERICA
BY E. L, HILDRETH & COMPANY, INC.

This hook is ajfectionately dedicated to
BOBILI

who has stimulated me in moments of defressiony

who has been at all times an inspiration in the

search for the unknown, my constant associate

and antagonist

PREFACE TO THE FIRST EDITION

On the basis of their relation to man, the microscopic forms of life may
be classified in two major groups: pathogenic forms that attack living
systems, especially those useful to man and to his domesticated plants
and animals } and saprophytic forms that attack inanimate matter, in-
cluding the universal scavengers and the organisms utilized in industry
and in the preparation of foodstuffs. Between true parasitism — one or-
ganism living in or upon the body of another — and true saprophytism
— one organism merely destroying the waste products and the dead
cells of another — are groups of relationships that may be designated as
antagonistic and associative. In the first of these, one organism is in-
jured or even destroyed by the other, whereas in the second, one or-
ganism assists the other and may in turn be benefited by it.

The antagonistic interrelationships among microorganisms have at-
tracted attention since the early days of bacteriology. Following the
discovery by Pasteur that microbes are responsible for certain human,
animal, and plant diseases, it was established that other organisms, later
designated as antagonists, are able to combat and even destroy the dis-
ease-producing agents. At first the soil was believed to be the natural
habitat of the bacteria that cause epidemics and disease as a whole, but
after careful study the fact was definitely established that very few of
these bacteria survive for long in the soil. On the contrary, the soil was
found to be the natural medium for the development of antagonists
chiefly responsible for the destruction of pathogens. The saprophytic
organisms that influence in various ways the disease-producing bacteria
and fungi were found to inhabit, in addition to the soil, various other
natural substrates, such as manure heaps and water basins.

The activities and potentialities of these antagonistic microbes still
present many problems. Little is known about the nature and mode of
formation of the antibiotic substances they produce, and even less about
the mode of their action. The substances vary greatly in their physical
and chemical properties. Some are soluble in water, others in ether, alco-
hol, or other solvents. Some are thermolabile, others are thermostable.
Some are sensitive to alkalies or to acids, others are not. Some are

viii PREFACE TO THE FIRST EDITION

readily oxidized and destroyed, others are not. Some are subject to de-
struction by specific enzymes. The substances are largely bacteriostatic
in action, to a lesser extent bactericidal. They are selective in their ac-
tion upon bacteria, some affecting largely gram-positive organisms and
others acting alike upon certain gram-positive and certain gram-nega-
tive forms. Some are also fungistatic and fungicidal. Differences are
largely quantitative rather than qualitative.

Some of the substances are highly toxic to animals. Others are either
nontoxic or of limited toxicity and are active in vivo. Some hemolyze
red blood cells, others do not. Those that are hemolytic and moderately
toxic may be useful for application to local infections. Those that are
neither hemolytic nor toxic and are active in vivo may have great im-
portance in combating certain diseases in animals and man.

Some substances are formed by only a few specific organisms, others
may be formed under proper conditions of nutrition by many different
organisms. Some antagonists produce only one type of antibiotic sub-
stance, others form two or even more chemically and biologically dif-
ferent substances.

The ability of an antagonist or its products — antibiotic substances — to
destroy a parasitic microorganism in vivo is influenced by the nature of
the host as well as by the type and degree of the infection. The manner
in which antagonists destroy or modify parasites varies greatly, depend-
ing frequently upon the nature of the antibiotic substances produced.

It is thus clear that the subject is extremely complicated, involving
numerous interrelationships among different biological systems of both
higher and lower forms of life.

In the following pages an attempt is made to present the broad inter-
relationships among microorganisms living in association, either in sim-
ple mixed cultures or in complex natural populations, with special at-
tention to the antagonistic effects. Emphasis is laid upon the significance
of these associations in natural processes and upon their relation to dis-
ease production in man and in his domesticated plants and animals. The
chemical nature of the active — antibiotic — substances produced by vari-
ous antagonists is described and the nature of the antagonistic action as
well as its utilization for practical purposes of disease control is dis-
cussed. However, because concepts of the significance of these phenom-

PREFACE TO THE FIRST EDITION ix

ena are changing so rapidly, no pretense has been made of examining
completely the practical applications of this important subject.

Due to the fact that more detailed studies have been made on the
production, nature, and utilization of penicillin, more information is
presented about this than about any of the other substances. However,
this should not be construed as desire on the author's part to emphasize
this substance.

The subject of antagonistic effects of microorganisms has been re-
viewed in both general treatises (706, 944) and special papers (268,
440, 443, 449, 580, 621, 730, 836, 867, 986) J special attention has been
paid to the occurrence of such organisms in the soil (316, 670, 941).
Advantage was taken of these reviews in the preparation of the com-
prehensive bibliography presented at the end of this monograph. At-
tention is directed also to a recent complete review of the literature on
the nature and formation of penicillin, the historical development of
our knowledge of this agent, method of assaying, and clinical applica-
tion (410).

The author expresses his sincere appreciation to the members of the
staff of the Microbiology Department, New Jersey Agricultural Ex-
periment Station ; to members of the Department of Research and De-
velopment of Merck & Co. and of the Merck Institute for permission
to use reproductions of their work, especially the photograph of strep-
tomycin crystals J to members of the staff of E. R. Squibb & Sons for
supplying the photograph of the penicillin-sodium crystals used as the
frontispiece to this volume j to Mrs. Herminie B. Kitchen for her care-
ful editing of the manuscript j and to the many investigators in the field
whose work has been freely cited both in the form of text or tabular
matter and as illustrative material.

S. A. W.
November i^, ig44

PREFACE TO THE SECOND EDITION

The manuscript of the first edition of this book was completed less than
three years ago. Since then the subject of antibiotics has made phe-
nomenal progress. A number of new substances have been isolated.
Several of those known previously as crude preparations have been
purified, and some have been crystallized. Penicillin has risen from a
metabolic product of certain fungi, promising but difficult to produce,
to one of the most important chemotherapeutic agents now available to
the medical world, and its yield has been increased a hundredfold by
the selection of new strains and by the development of more suitable
media and better conditions of growth. Its chemistry has been com-
pletely elucidated, and the existence of a number of different forms
varying in chemical nature and biological activity has been established.
Streptomycin was a laboratory curiosity late in 19435 now it occupies
an important place as a promising chemotherapeutic agent for the
treatment of certain diseases resistant to penicillin and the sulfa drugs.
This rapid progress of our knowledge of the formation, isolation,
and utilization of antibiotics makes it advisable to bring out a revised
edition of this book. A great deal of new material has been added, but
in order to avoid enlarging the book excessively, it was decided to omit
a number of references, mostly earlier articles of purely historical in-
terest for which the reader is referred to the first edition, and those
dealing with the clinical application of penicillin. Several excellent
volumes on penicillin dealing with its use for disease control have re-
cently been published.

S. A. W.
February 75, 1^4^

CONTENTS

1. Soils and Water Basins as Habitats of Microorganisms i

2. Human and Animal Wastes 19

3. Interrelationships among Microorganisms in Mixed Popula-
tions 36

4. Isolation and Cultivation of Antagonistic Microorganisms j
Methods of Measuring Antibiotic Action 53

5. Bacteria as Antagonists 85

6. Actinomycetes as Antagonists 108

7. Fungi as Antagonists 1 30

8. Microscopic Animal Forms as Antagonists 154

9. Antagonistic Relationships between Microorganisms,

Viruses, and Other Nonspecific Pathogenic Forms 1 63

10. Chemical Nature of Antibiotic Substances 170

1 1 . The Nature of Antibiotic Action 2 1 8

12. Utilization of Antibiotic Substances for Disease Control 261

13. Microbiological Control of Soil-borne Plant Diseases 300

14. The Outlook for the Future 314
Classification of Antibiotic Substances 329
Glossary 331
Bibliography 233
Index of Microorganisms 395
General Index ' 403

62i9i

CHAPTER I

SOILS AND WATER BASINS AS HABITATS \. tt
OF MICROORGANISMS

Although microorganisms inhabit a variety of substrates, from the
dust in the atmosphere, the surface of living plants and plant residues,
and numerous foodstuffs to the living systems of plants and animals,
their natural habitations are soils and water basins.

The soil is by no means an inert mass of organic and inorganic de-
bris. On the contrary, it fairly teems with life. The organisms inhabit-
ing the soil range from those of ultramicroscopic size to those readily
recognizable with the naked eye. Many thousands of species, capable
of a great variety of activities, are represented in the soil. The physical
nature and chemical composition of the soil, the climate, the plant vege-
tation, and the topography influence greatly both the composition of
the microbiological population of the soil and its relative abundance.
One gram of soil contains hundreds, even thousands, of millions of bac-
teria, fungi, actinomycetes, protozoa, and other groups of microorgan-
isms. Under certain conditions, especially when the supply of fresh or-
ganic matter in the form of plant and animal residues is increased, the
number may be much greater. This varied microbiological population
renders the soil capable of bringing about a great variety of chemical
and biological reactions.

Through its diverse activities, the microscopic population inhabiting
soils and water basins forms one of the most important links in the chain
of life on earth. However, its great influence upon numerous phases of
human endeavor has been recognized only within recent years. All
plants and all animals, including man himself, are dependent upon
these organisms to bring about some of the processes essential to the
continuation of life. The growth of annual and perennial plants, the
supply of food for man and animals, and the provision of clothing and
shelter depend largely upon the activities of these microorganisms,
especially the transformations brought about in the state of such ele-
ments as carbon, nitrogen, sulfur, and phosphorus.

2 MICROORGANISMS IN SOILS AND WATER BASINS

Soils and water basins may be regarded as the primary reservoirs for
all living systems inhabiting this planet. Whereas the great majority of
microorganisms are saprophytic in nature, living upon inorganic ele-
ments and compounds and upon the dead residues of plant and animal
life, others have become adapted to a parasitic form of existence and
have learned to thrive upon the living tissues of plants and animals.
Many of these parasites find their way into the soil and into water basins
and may be able to survive there for long periods of time or even in-
definitely.

Although the following discussion is limited primarily to the micro-
biological population of the soil, it also applies, to a greater or lesser
extent, to the microorganisms that inhabit manures made up of animal
excreta, household wastes, and artificially prepared composts and to
those that inhabit water basins, including rivers, lakes, and seas. There
are, however, marked differences in the nature of the microbial popu-
lation of waters and of soils because of the physical and chemical differ-
ences in the composition of these two substrates. Nevertheless, some of
the underlying principles apply to all substrates. There are, for exam-
ple, marked differences in the nature and abundance of the populations
of soil and water and those of milk, sewage, and foodstuffs. Whereas
microorganisms multiply in the latter substrates at a very rapid rate,
those in the soil and in water basins are more nearly static, since the rate
of their multiplication is much slower except under very special condi-
tions, such as the addition of fresh, undecomposed plant and animal
residues or a change in the environment or in the chemical nature of
the substrate.

PHYSICAL PROPERTIES OF THE SOIL

The soil — the surface layer of the earth's crust — comprises three dis-
tinct phases, the gaseous, the liquid, and the solid. The last is largely
inorganic in nature, with varying concentrations of organic constituents
originating from plant and animal residues and found in the soil in dif-
ferent stages of decomposition. The organic substances together with
the living and dead cells of microorganisms that inhabit the soil make
up what is known as soil organic matter or, more often, soil humus. The
soil as a medium for the development of microorganisms is thus mark-

PHYSICAL PROPERTIES OF THE SOIL 3

edly different from the common artificial laboratory media, whether
these be synthetic or consist of products of animal or plant life, upon
which these organisms are grown.

The inorganic soil particles are surrounded by films of colloidal ma-
terials, which are both inorganic and organic in nature. As a rule, the
microorganisms inhabiting the soil adhere to these films, although some
move freely in the water surrounding the particles. Water and air play
essential roles in the soil system and control the nature and extent of the
soil population. The nature and size of the mineral and organic soil
fractions, as well as the phenomena of adsorption, also influence the
abundance, nature, and distribution of microorganisms in the soil. Sandy
soils are better aerated than heavy clay soils j they are, therefore, more
favorable for the growth of aerobic bacteria and fungi. However, since
such soils lack the high water-holding capacity of the heavier soils, they
are more readily subject to the process of drying out, which may result
in a reduction in microbial activities.

Oxygen, another important factor in microbial development in the
soil, becomes available to microorganisms by gaseous diffusion. The
oxygen supply diminishes with increase in depth of the soil. When an
excess of free water is present in the soil, gaseous oxygen cannot pene-
trate very deeply and soil organisms then become dependent upon the
dissolved oxygen which diffuses into the soil solution. Since the rate of
oxygen diffusion is extremely slow, waterlogged soils tend to become
depleted of oxygen. Under these conditions, there are marked changes
in the microbiological population of the soil: the fungi and actinomy-
cetes tend to decrease, and the bacteria, especially anaerobic types, pre-
dominate. Peat bogs are examples of soils in a perpetual anaerobic state j
the microbial population is quite distinct from that of mineral soils.
Semiarid soils, with a much greater diffusion of oxygen into the deeper
soil layers, possess a population which is largely aerobic j in these and
other mineral soils the abundance and nature of the organic matter exert
a decided influence upon the abundance and nature of the microorgan-
isms present.

Th^ microbiological populations of soils, composts, and water basins
are also influenced markedly by seasonal and temperature changes.
Certain microorganisms are capable of active life at temperatures ap-

4 MICROORGANISMS IN SOILS AND WATER BASINS

preaching the freezing point of water j others, known as thermophilic
forms, can withstand very high temperatures, some being active even at
60° to 70° C.

The reaction of the soil is also a factor influencing the nature of the
population. Many microorganisms are active within a very limited
range of /)H values j others, notably many of the fungi, are adapted to
much wider ranges of reaction. In acid soils, larger numbers of fungi
are present, because of the fact that they tolerate more readily the
more acid reactions, which limit bacterial competition. On the other
hand, actinomycetes comprise a large percentage of the microbial popu-
lation of dry and alkaline soils.

CHEMICAL COMPOSITION OF THE SOIL

The solid part of the upper or surface layer (20 to 30 cm.) of the soil
commonly is made up of i to 10 per cent organic matter and 90 to 99
per cent inorganic or mineral matter. The concentration of organic mat-
ter may be even less than i per cent, as in desert and poor sandy soils, or
more than 10 per cent, as in certain virgin prairie soils and, especially,
peat lands which consist of 50 to 99 per cent organic matter, on a dry
basis.

The organic matter of the soil is markedly different in chemical na-
ture from that of plant and animal materials. It contains much less cellu-
lose and hemicelluloses than the majority of plants and is higher in
lignins and proteins. It is characterized by a narrow ratio of the two
important elements carbon and nitrogen, usually about 10: i j it is much
more resistant to microbial decomposition than are plant and animal
residues. It is black, is soluble to a considerable extent in alkalies, and is
partly reprecipitated by acids. These alkali-soluble constituents have
often been designated as "humic acids" or "humic bodies," thus impart-
ing the idea that soil organic matter is made up largely of these "acids"

(94^).

The inorganic constituents of f he soil comprise largely sand, silt, clay,
and, to a more limited extent, a number of soluble and insoluble salts,
notably phosphates, sulfates, and silicates of calcium, magnesium, potas-
sium, iron, aluminum, manganese, zinc, copper, and others. Some of

BIOLOGICAL STATE OF THE SOIL 5

the chemical elements comprise the framework of the soil and are used
to only a limited extent by plant and microbial life. Others form im-
portant nutrients (for example, C, N, S, P, H, and O) or serve as cata-
lysts for the continuation of life (Zn, Fe, Mn, Cu, Mo, B, and even K
are often considered as belonging in this category). The function of
most of these elements in the life of microorganisms is not fully under-
stood. In view of the fact that some of the elements in the latter group
have been found to form important constituents of certain enzyme sys-
tems, the difference between the two functions is not significant.

BIOLOGICAL STATE OF THE SOIL

The abundance of higher plant and animal life in and upon the sur-
face of the soil influences considerably the nature and extent of the
microbiological population. Certain plants harbor in their roots specific
microorganisms that act as symbiontsj this is true of the root nodule
bacteria of leguminous plants and the mycorrhiza-forming fungi found
in orchids, evergreens, and many other plants. Higher plants also
offer a favorable environment for the growth of certain other types
of bacteria and fungi, this specific environment being designated as the
rhizosphere. The bacterial population of the rhizosphere is not very
different qualitatively from that found some distance away from the
plants, except that certain types of bacteria are more prominently repre-
sented.

The growth of plants results in the production of waste materials
and residues left in and upon the soil in the form of roots, leaves,
needles, and other products, all of which offer favorable nutrients for
microbial development. The root systems of plants also bring about bet-
ter aeration of the soil, thus making conditions more favorable for the
development of aerobic organisms. The presence of higher plants often
leads to the development of certain types of bacteria, fungi, and nema-
todes that are pathogenic to the plants, such as the causative agents of
root rots, damping-off diseases, root-galls, and various others. Some of
the pathogens may become well established in the soil and may persist
there long after the specific host plants have been removed. They may
even be able to attack other hosts. Plant life thus exerts a variety of in-

6 MICROORGANISMS IN SOILS AND WATER BASINS

fluences upon the nature and abundance of the soil-inhabiting micro-
organisms.

Higher animals also influence the soil microbiological population.
Cattle and horses on pastures contribute, through their droppings,
energy sources and various other essential nutrients for the develop-
ment of microorganisms. After death, the bodies of animals, from the
smallest insects to man, the lord of creation, also offer available nutri-
ents for the growth of numerous microorganisms. Many animals living
in the soil, such as insects and rodents, become carriers of certain bac-
teria and fungi that are destructive to their hosts j this phenomenon is
often utilized for combating injurious animals. Finally, the numerous
animals living on the surface of the soil leave waste products rich in bac-
teria, fungi, and invertebrate animals, some of which are capable of
causing serious animal diseases (945).

NATURE AND COMPOSITION OF THE SOIL
MICROBIOLOGICAL POPULATION

The microorganisms inhabiting the soil can be divided, on the basis
of their systematic position in the biological kingdom, into the following
eight groups: bacteria, actinomycetes, fungi, algae, protozoa, worms,
insects and other near-microscopic animals, and ultramicroscopic forms.
The last group comprises bodies that range from living systems to
products of living organisms j they possess the property of activating
similar substances and imparting to them their specific activities, as in the
case of phages and viruses.

Five methods are commonly employed for determining the abun-
dance of the various groups of microorganisms inhabiting the soil 3
namely, plate culture, selective culture, direct microscopic methods,
contact slide, and mechanical separation. Each of these has certain ad-
vantages and certain limitations. In many cases, special methods have
been devised to supplement the more common methods.

The plate method is based upon principles similar to those employed
in other branches of bacteriology. Various media are used, both organic
and synthetic. The soil microbiologist has attempted to produce media
that either allow the development of the greatest number and the great-

SOIL MICROBIAL POPULATION 7

est variety of organisms or are particularly favorable for the growth of
certain special types of organisms. None of the media so far employed
allows the growth of the total soil population. The plate method is often
supplemented by the selective culture method, in which a great variety
of media are used in order to obtain a representative picture of the soil
population. Since the number of media required to enable all soil micro-
organisms to develop is virtually limitless, the enrichment methods can
only give a proximate idea of the nature and abundance of the micro-
biological population. Because of the development on the plate of cer-
tain organisms that exert a toxic effect upon others, the plate method
often shows excessive variation in the numbers of bacteria and fungi
(256).

The microscopic methods have been introduced to fill this gap, since
by them the relative abundance of the various groups of organisms
found in soils, composts, or other natural substrates can be established.
Unfortunately, these methods do not allow any differentiation between
living and dead cells, nor do they permit a differentiation between the
various physiological types of microorganisms such as pathogens and
nonpathogens. A further limitation, especially of the contact slide, is
that the fast-growing forms cannot be prevented from overgrowing
the slide and repressing the slow-growing types.

The mechanical separation methods are based upon the use of special
sieves or water emulsions and are utilized for the study of the larger
forms such as insect larvae and nematodes.

The relative abundance of the different groups of microorganisms in
a given soil, as determined by any one of the foregoing methods, varies
with the nature of the soil, amount of organic matter, oxygen sup-
ply, moisture content, temperature, acidity, and buffering capacity
(Table i), as well as with the nature of the higher plants growing in
the given soil (Table 2). Despite all these factors, the microbiological
population of the soil throughout the world has certain definite and
common characteristics and comprises certain well-defined, specific
types. The bacteria usually range in number from a few hundred thou-
sand to several hundred million per gram of soil, though many species
do not develop on the common plate. Fungi are found in the form of
mycelial filaments and as spores and may therefore constitute as large

8 MICROORGANISMS IN SOILS AND WATER BASINS

a mass of living matter as do the bacteria ; their actual number, as deter-
mined by the plate method, may vary from a few thousand to several
hundred thousand per gram of soil. The significance of these results is

TABLE I. INFLUENCE OF SOIL TREATMENT ON NUMBER
OF MICROORGANISMS

REACTION

TREATMENT OF SOIL

OF SOIL

MICROORGANISMS FOUND*

fH

Bacteria

Act

:inomycetes

Fungi

Unfertilized and unlimed

4.6

3,000

1,150

60

Lime only added

6.4

5,410

2,410

23

Potassium salts and phosphates

added

5-5

5^360

1,520

38

Salts and ammonium sulfate

added

4.1

2,690

370

1X2

Salts, ammonium sulfate, and

lime added

5.8

6,990

2,520

39

Salts and sodium nitrate added

5-5

7,600

2,530

47

Stable manure and salts added

5-4

8,800

2,920

73

From Waksman (945).

* In thousands per gram of soil as determined by plate method.

TABLE 2. INFLUENCE OF GROWING PLANTS ON NUMBER
OF MICROORGANISMS IN THE SOIL

SAMPLE OF

PLANT

SOIL TAKEN

MICROORGANISMS FOUND*

Bacteria

Actinomycetes

Fungi

Rye

Near roots

28,600

4,400

216

Away from roots

13,200

3,200

162

Corn

Near roots

41,000

13,400

178

Away from roots

24,300

8,800

134

Sugar beet

Near roots

57,800

15,000

222

Away from roots

32,100

12,200

176

Alfalfa

Near roots

93,800

9,000

268

Away from roots

17,800

3,300

254

From Starkey (877).

• In thousands per gram of soil.

SOIL MICROBIOLOGICAL POPULATION 9

not always clear, since a given colony may have originated from a
hyphal filament, a mass of mycelium, or a single spore. Determination
by the plate method of the number of actinomycetes is subject to the
same limitations j these organisms usually constitute from lo to 50 per
cent of the colonies appearing on common bacterial agar plates.

Algae are numerous in the surface layers of soil only. Protozoa are
present in the soil in an active vegetative or trophic state and in the
form of cysts. The active cells appear when excessive water is present,
even for a few hours j in dry soil, the cysts predominate. Flagellates
are represented by the largest numbers, sometimes approaching a mil-
lion individuals per gram of soil j amebae are next in abundance j cili-
ates are usually found to the extent of a few hundred to several thou-
sand per gram of soil. Nematodes, rotifers, earthworms, and larvae of
numerous insects are also abundant, often forming a large part of the
bulk of the living mass of cell substance.

By means of the selective and enrichment culture methods, several
physiological classifications of bacteria have been recognized. The fol-
lowing descriptive terms are commonly used to designate these groups :
autotrophic vs. heterotrophic, aerobic vs. anaerobic, motile vs. non-
motile, pathogenic vs. saprophytic, psychrophilic and mesophylic vs.
thermophilic, symbiotic vs. nonsymbiotic, and antagonistic vs. non-
antagonistic.

The fungi may be classified into three types: saprophytic and free-
living, mycorrhiza-producing, and plant pathogenic. The most com-
mon groups of soil fungi are found in the genera Rhizo-pus, Mucor,
Penicillium, Aspergillus y Trichoderma, Fusarmm, Cladosforium, and
Cefhalosforium. The soil often harbors an abundant population of
yeasts and fleshy or mushroom fungi. The latter may produce an ex-
tensive mycelium in the soil, binding the particles together and pre-
venting their falling apart.

Various bacteriolytic agents, including specific phages, have also been
demonstrated in the soil. The phage of root-nodule bacteria is of par-
ticular interest. It is readily adsorbed by the soil, but its presence can
easily be established. The repression of spore-forming bacteria and the
abundance of Pseudomonas jiuorescens may be due to the antagonistic
action of the latter.

10

MICROORGANISMS IN SOILS AND WATER BASINS

SOILS AND WATER BASINS AS CULTURE MEDIA

Microorganisms require for their growth and respiration certain
energy sources and certain nutrients, as well as certain conditions favor-
able for their development. Different organisms show considerable
variation in this respect. The mineral elements required for growth and
multiplication are almost invariably present in the soil and to a large
extent also in many water basins. The available energy supply may be
limited, however, and thus usually becomes the most important factor
regulating the abundance and activities of microorganisms in natural
substrates. The autotrophic bacteria depend on the supply of oxidizable
minerals such as ammonium salts, nitrite, sulfur, iron, and manganese,
the oxidation of which makes energy available for their growth. The
heterotrophic organisms are dependent on the carbon compounds
brought into the soil in the form of plant and animal residues as well
as the bodies of many insects, earthworms, and other small animals.
The roots of plants also supply an abundance of easily available sub-
stances for microbial nutrition.

Every organic compound produced in nature finds its way, sooner or
later, into the soil or into lakes and rivers, where it serves as a source
of energy for microorganisms. This energy becomes available to some
of the organisms through anaerobic or fermentative transformation and
to others through aerobic or oxidative processes. The net change in the
energy produced by any one organism or group of organisms is accom-
panied by a loss of free energy by the system to which the culture is

TABLE 3. MULTIPLICATION OF COLIFORM BACTERIA IN STERILE SOIL

BACTERIA

ORGANISM

INOCULATED*

BACTERIA RECOVERED*

After 10 days

After 26 days

Escherichia coli

in soil alone

2,600

149,000,000

138,000,000

Aerobacter aero genes

in soil alone

109,000

48,000,000

42,600,000

in soil and glucose

109,000

1,660,000

240,000,000

From Waksman and Woodruff (978).
* Per gram of soil.

NUTRITION OF MICROORGANISMS 11

confined. The synthesis of new cell material by microorganisms is ac-
companied by a gain of free energy, which must be supplied by other
chemical transformations. Ordinary soils, however, contain microbial
nutrients in concentrations sufficient to support a large number of living
cells. This can be illustrated by the fact that when a soil is sterilized and
then inoculated with a pure culture of bacteria rapid multiplication
takes place (Table 3). When fresh water taken from a lake or the sea is
kept in the laboratory for one or two days, a great Increase in its bac-
terial population occurs.

There is considerable variation in the ease with which a specific or-
ganism can be isolated from a natural substrate and consequently in
the techniques employed. Some microorganisms may be present in
abundance and can be readily isolated. Others are found only in limited
numbers and can be obtained only with considerable difficulty and by
the use of special procedures. Still others can be isolated only after the
natural substrate is treated in such a manner as to favor the multiplica-
tion of the specific organism ; this can be done by enriching the soil with
a nutrient or substance which the particular organism is able to utilize,
or by changing conditions of reaction, by aeration, or by other treat-
ment. Such treatment sometimes results in the development of special
strains or races adapted to the special conditions.

NUTRITION OF MICROORGANISMS IN
NATURAL SUBSTRATES

It was at first assumed that bacteria and other microorganisms possess
a simpler type of metabolism than do higher plants and animals j al-
though some can obtain all the nutrients required for cell synthesis and
energy from simple elements and compounds, others need for their nu-
trition certain highly complicated organic substances. Recently it has
been recognized that various "growth-promoting" substances or vita-
mins play an important role in the nutrition of many microorganisms. It
has also been established that highly complicated enzyme systems are
produced by these lower forms of life, and that many interrelationships
exist among their metabolic processes, the composition of the medium,
and the environmental conditions. One thus begins to realize that the

L 5 3 3 A

12 MICROORGANISMS IN SOILS AND WATER BASINS

metabolism of these microbes is also highly complicated. Most of the in-
formation on their nutrition is based upon their growth on artificial cul-
ture media. In nature, however, these organisms live in associations and
vary considerably in the degree of their interdependence. As yet no
laboratory method has been developed that duplicates these conditions.
Microorganisms vary considerably in their nutrition and energy
utilization, as well as in the breakdown and transformation of the avail-
able nutrients. Certain elements or compounds are required for cell
synthesis. In some cases, certain trace elements as well as varying con-
centrations of growth-promoting substances are also essential. Among
the nutrient elements, nitrogen occupies a prominent place. Consider-
able variation exists in the ability of microorganisms to utilize different
types of nitrogen compounds: some can obtain their nitrogen from a
wide variety of substances j others are restricted to the use of a single
group of compounds such as proteins, amino acids, urea, ammonia, or
nitrate J a few are able to use atmospheric nitrogen. The variety of or-
ganic nitrogenous bodies supplied to microorganisms in soils and in
water basins is limited only by the number of such compounds synthe-
sized by plants and animals. The complex forms of nitrogen are broken
down to simpler compounds j these may be assimilated by organisms
and again built up into complex forms, or they may be utilized only by
other organisms. Microbial activity thus regulates the state of the nitro-
gen in natural substrates and is responsible for the continuous stream
of ammonia and nitrate forming the available sources of nitrogen that
make possible the growth of higher plants.

THE GROWTH OF THE MICROBIAL CELL IN PURE
CULTURE AND IN MIXED POPULATIONS

When nutrients are available in sufficient concentration and when the
environmental conditions are favorable for the development of the
microbial cell in pure culture, growth follows a definite sigmoid-shaped
curve. Slow multiplication is followed by rapid development, until a
certain maximum number of cells within a given volume of medium is
reached} the rate of growth then diminishes. The maximum population
of Aerohacter aero genes grown in a medium containing lactose and

DISEASE-PRODUCING ORGANISMS IN THE SOIL 13

ammonium tartrate increases at first in proportion to the concentrations
of these nutrients but later becomes independent of them. The onset of
the stationary phase may be due to several factors: exhaustion of sub-
stances necessary for growth, change in the reaction of the medium to
one unfavorable for further development, accumulation of toxic prod-
ucts. When the nutrients in the medium are exhausted, addition will
restore growth. When an unfavorable change in reaction has taken
place, the addition of acid or alkali will render the medium again favor-
able. The production of toxic substances in the medium can be counter-
acted usually by the use of heat or by treatment with charcoal, though
some of the injurious bodies may be heat-resistant.

In the presence of other microorganisms, a certain organism may
show reactions markedly different from those obtained in pure culture:
it may produce substances that are either favorable or injurious to the
other cells, it may compete with the other organisms for the available
nutrients or it may render the medium more favorable for their de-
velopment. Some bacteria like Bacillus cereus can attack native proteins
but not amino acids, whereas others like Ps. jiuorescens can attack amino
acids but not proteins j when these two organisms are placed together in
the same medium, their activities supplement one another. Numerous
other instances are found in soil and water of an organism preparing
the substrate for another, ranging from distinct symbioticism, where
one organism depends absolutely for its living processes upon the ac-
tivities of another (symbiosis), to association, where one organism
merely is favored by the growth of another (metabiosis), to the injury
of one organism by another (antagonism), and finally, to the actual
destruction of one by another (parasitism).

INTRODUCTION OF DISEASE- PRODUCI NG
ORGANISMS INTO THE SOIL

Ever since higher forms of life first made their appearance on this
planet they have been subject to attack by microbes. These microscopic
organisms must have gained, at an early stage in the development of
the higher forms, the capacity of attacking them in one manner or an-
other. There is no plant or animal now living that is not subject to in-

14 MICROORGANISMS IN SOILS AND WATER BASINS

fection by different bacteria, fungi, and protozoa. The more advanced
the animal body is in the stage of evolution, the more numerous are its
ills, most of which are caused directly or indirectly by microorganisms.

The microbial agents causing thousands of diseases of plant and ani-
mal life have now been recognized and even isolated and described. In
many cases these disease-producing agents are closely related morpho-
logically to others that lead a harmless existence in soils or water
basins i many of the saprophytes, for instance, are found to be of great
benefit to man and to his domesticated plants and animals. This sug-
gests the probability that pathogenic microorganisms represent certain
strains of soil and water-inhabiting types that have become adjusted to
a parasitic existence. During their life in the host, they multiply at a
rapid rate and produce substances toxic to the body of the host. The re-
sult is that the host is incapacitated for a certain period of time, until it
succeeds in building up resistance against the invading organisms. It
may thus overcome the injurious effect of the pathogen or it may be
killed if such resistance cannot be effected. In the first instance, a tem-
porary or permanent immunity against the specific disease-producing
microbe or its close relatives may result. The host is often able to sur-
vive the attack without being able to destroy the invading microbes j if
it again attains a normal form of life, it is designated as a carrier of the
disease-producing agent.

Pathogenic organisms pass their existence in the living body of the
plant or animal. They spread from one host to another by contact or
through a neutral medium, such as water, milk, or dust where they may
remain alive and active for varying lengths of time, or they reach the
soil or water basins in the excreta of the host. If the host is killed by
the infecting microbes, they may survive for some time upon the rem-
nants of what was once a living animal or plant and thus find their way
into the soil and water basins.

Considering the millions of years that animals and plants have ex-
isted on this planet, one can only surmise the great numbers of microbes
causing the numerous diseases of all forms of life that must have found
their way into the soil or into streams and rivers. What has become of
all these pathogenic bacteria? This question was first raised by medical
bacteriologists in the eighties of the last century. The soil was searched

SAPROPHYTIC ORGANISMS IN THE SOIL 15

for bacterial agents of infectious diseases. It was soon found that, with
very few exceptions, organisms pathogenic to man and animals do not
survive very long. This was at first believed to be due to the filtration
effect of the soil upon the bacteria. It came to be recognized, how-
ever, that certain biological agents are responsible for the destruction
of the pathogenic organisms. These investigations led to the conclusion
that the soil can hardly be considered as a carrier of most of the infec-
tious diseases of man and animals. The fact that many pathogens can
grow readily in sterilized soil but do not survive long in normal fresh
soil tends to add weight to the theory of the destructive effect upon
pathogens of the microbiological population in normal soil.

INTRODUCTION OF SAPROPHYTIC ORGANISMS
INTO THE SOIL

It often becomes necessary to inoculate the soil with organisms not
usually found there. The common practice of inoculating soil with bac-
teria capable of forming root nodules on leguminous plants is a case in
point. It is essential, therefore, to know how long these organisms will
survive. The survival period is influenced greatly by the presence of a
host plant that protects the specific bacteria from attack by antagonistic
organisms. In the absence of the host plant, the bacteria seem to disap-
pear gradually, and reinoculation becomes advisable when the host is
again planted in the given soil. It has been observed also that specific
strains of bacteria tend to deteriorate in the soil, and that it is necessary
to reinoculate the soil with more vigorous strains of the organisms in
question.

Some bacteria, notably members of the Azotobacter group, are able
to fix nitrogen independently of host plants but these organisms are
absent from many soils. The suggestion was made that such soils might
benefit from inoculation. However, it has been found that when soils
and peats are inoculated with A. chroococcum large-scale destruction
of the latter often occurs (814), due, it is believed, to the presence in
th9 soil of antagonistic organisms as well as toxic substances (492, 687,
980).

Certain fungi are unable to grow in fresh nonsterilized soil but are

16 MICROORGANISMS IN SOILS AND WATER BASINS

capable of growing in heated soil. This was found to be due to the fact
that normal soils contain certain substances that render the growth of
the fungus impossible j these substances are destroyed by heating. An
extract of fresh soil acts injuriously upon the growth of the fungus
Pyronema; the injurious effect is partly removed on boiling. Certain
forest soils contain not only antifungal but also antibacterial factors
(630a) which are dialyzable and thermostable j other thermolabile and
nonfilterable substances may be present which neutralize the effect of
the antibiotics.

The survival of microorganisms added to soil or water is thus influ-
enced by the nature of the native soil or water population, the organ-
isms added, the composition of the substrate, and various environ-
mental conditions.

SAPROPHYTIC AND PATHOGENIC NATURE OF
CERTAIN SOIL MICROORGANISMS

Various fungi and actinomycetes causing animal diseases, notably
skin infections, appear to resemble very closely the corresponding soil
saprophytes. It was therefore suggested that many of the dermato-
phytic fungi normally lead a saprophytic existence in the soil but are
also capable of developing on epidermal tissue and bringing about in-
fection of the tissues. This was found to be true especially of species of
S-porotrkhum, various actinomycetes such as those causing lumpy jaw of
cattle, and certain other organisms. Henrici (406) divided fungus in-
fections of animals into two groups : first, superficial mycoses, compris-
ing moniliases and dermatomycoses, that are caused by a variety of
fungi widely distributed in nature 5 and, second, deep-seated infections,
namely, aspergillosis, sporotrichosis, and blastomycosis, with a marked
tendency to restricted distribution. The latter were said to be caused
primarily by saprophytic forms, including varieties capable of chance
survival and of multiplication when accidentally introduced into ani-
mal tissues.

Walker (981) suggested that the partly acid-fast coccoid, diph-
theroid, and actinomycoid organisms that have been cultivated repeat-
edly from leprosy are merely different stages in the life cycle of the

SAPROPHYTIC AND PATHOGENIC MICROORGANISMS 17

same form. The causative agent of leprosy, like certain pathogenic
actinomycetes, is believed to be a facultatively parasitic soil organism,
probably of wide but irregular distribution. Leprosy was thus looked
upon primarily as a soil infection, brought about presumably through
wounds i a secondary means of infection by contagion was not excluded.
A comparison of cultures obtained from rat leprosy, human leprosy,
and bacteria of soil origin led to the conclusion that the strains from all
three sources were identical j human and rat leprosy were said to have
the same etiology and endemiology, finding a normal habitat in the soil.

An interesting relationship has been shown to exist between Texas
fever and the capacity of cattle tick {Boofhilus bovis)y the parasite car-
rier, to persist in the soil (865). The causative agent is an organism
with protozoan characteristics. It persists in southern pastures where
the carriers survive from one season to the next and keep the cattle con-
tinuously infected. The disease is of little importance in northern re-
gions, the ticks being destroyed during the winter. When northern cat-
tle are moved to southern pastures, they become subject to the disease.

Pathogenic microorganisms capable of surviving in the soil have pre-
sented important economic problems to farmers raising hogs, cattle,
poultry, and other domestic animals, but disease incidence through this
source has been greatly diminished by the proper practice of sanitation.
The rotation of crops has been utilized for the purpose of overcoming
these conditions, several years usually being required to render infected
pastures safe for use. The fact that most pathogenic organisms rapidly
disappear when added to the soil makes this problem rather simple j the
prevention of infectious diseases would have presented far more diffi-
cult problems were the infecting agents to remain indefinitely virulent
in the soil. The few disease-producing agents that are capable of per-
sisting, such as anthrax, blackleg, and coccidiosis, have been the cause,
however, of considerable damage to animals.

Of greater economic importance than the survival in the soil of hu-
man and animal pathogenic agents is the fact that the soil harbors a
number of plant pathogens, including not only fungi, bacteria, and
actinomycetes, but also nematodes and insects. Fortunately, the con-
tinued development of these organisms in the soil also leads to the ac-
cumulation of saprophytic organisms destructive to them.

18 MICROORGANISMS IN SOILS AND WATER BASINS

The extent to which virus diseases persist in the soil is still a matter
for speculation. It has been demonstrated that the phage of legume
bacteria may persist and become responsible for a condition designated
as "alfalfa-sick soils" and "clover-sick soils" (178, 49o). In order to
overcome this condition, the breeding of resistant varieties of plants has
been recommended.

CHAPTER 2

HUMAN AND ANIMAL WASTES

And a flace shall thou have without the campy ivhither thou shalt
go forth abroad: and a sfade shalt thou have with thy weapons;
and it shall be, when thou sittest abroad, thou shalt dig therewith,
and shalt afterward cover that which cometh from thee. —
Deuteronomy 2^:1^ and 14.

Human and animal excreta and other waste products, which are or fre-
quently become both offensive and dangerous to public health, sooner
or later find their way into the soil and water basins. The soil also re-
ceives the many residues of growing crops that are annually left on the
land, together with the waste materials of the farm and the home
(942), These wastes contain substances partly digested by man and ani-
mals, and their metabolic waste products, as well as freshly synthesized
material in the form of microbial cells. The microbial population of
such waste materials comprises agents of digestion, some microbes that
are present accidentally, and some that possess the capacity of causing
human, animal, and plant diseases.

These waste materials do not remain long in an unaltered form and
do not accumulate in or on the surface of the soil or in water basins j
otherwise both soil and water long ago would have been rendered un-
sightly, disagreeable bodies, which man would not dare to tread upon
or enter. On the contrary, the soil and the water are capable of di-
gesting all these cast-off materials and of completely destroying their
undesirable characteristics. Through all past ages, the waste products of
plant and animal life have disappeared, whereas the soil and the water
in the rivers, lakes, and seas have remained essentially the same, except
under very special conditions such as those that brought about the pro-
duction of peat in water-saturated basins and, in past geological ages,
the formation of coal. The capacity of soil and water to destroy these of-
fensive wastes is due entirely to the microorganisms that inhabit the
substrates. The important ultimate products of destruction are am-
monia, carbon dioxide, and water j often hydrogen and methane are

20 HUMAN AND ANIMAL WASTES

produced j various mineral compounds, such as phosphates, sulfates, and
potassium salts are also liberated. These mineralized substances are es-
sential for the continuation of plant and animal life on this earth.

Largely because of the activities of the microorganisms inhabiting
soils and water systems, man does not need to worry about the disposal
of plant and animal wastes. These activities need only be regulated, in
order to accomplish the breakdown of complex substances with the
greatest efficiency and the least loss of valuable nutrient elements. The
following principal objectives are usually to be attained: first, the de-
struction of plant and animal pathogens, including pathogenic bacteria
and fungi and disease-producing protozoa, worms, and insects j second,
the liberation of the essential elements required for plant nutrition in
available forms, especially carbon, nitrogen, and phosphorus j and,
third, the formation of certain resistant organic substances, known col-
lectively as humus, which are essential for the improvement of the
physical, chemical, and biological condition of the soil.

STABLE MANURES AND FECAL RESIDUES

IVLicrobial Pofulation

Fresh excreta of animals and man are rich in fecal bacteria, consisting,
on the average, of 5 to 20 per cent bacterial cells. Lissauer (575)
calculated that the bacterial substance of feces ranges from 2,5 to 15.7
per cent of the dry weight, with an average of 9 per cent. Bacteria were
reported to make up 9 to 42 per cent of the bulk of animal stools, the
percentage depending on the composition of the foodstuffs, the nature
of the animal and its condition of health, and other factors {^66).
Since i mg. of dry bacterial substance contains about 4 billion bacterial
cells, the number of these organisms in fecal excreta can be seen to be
very large, although many, if not most, of the cells are no longer in a
living state.

By suitable methods of cultivation, human feces were found (626)
to contain 18 billion bacteria per gram. About 100 billion bacteria may
be produced daily in the human intestine. Human feces are made up,
on an average, of 32.4 per cent bacterial cells amounting to 2,410
million bacteria per milligram of moist material. Feces of healthy

STABLE MANURES AND FECAL RESIDUES 21

persons were shown (301 ) to contain 8.2 to 24.2 per cent bacterial cells j
in those of persons suffering from intestinal disturbances the percent-
age was 20.1 to 40.2. With the development of the microscopic tech-
nique for counting bacteria, much larger numbers of cells were shown
to be present than could be determined by the plate method.

The urine of healthy persons is sterile or very low in bacteria. Be-
cause of the ability of many bacteria to utilize the chemical constituents
of urine, rapid bacterial multiplication takes place in fresh urine, espe-
cially when mixed with animal feces and bedding (811).

The microbiological population of animal excreta is characteristic. In
addition to the common fecal bacteria, it contains fungi, thermophilic
bacteria, and, in herbivorous animals, anaerobic cellulose-decomposing
bacteria (581).

The bacterial population of fresh cow manure was found (833) to
consist of 47.5 per cent cocci {Streptococcus fyogenes, Sarcina sp.,
and Micrococcus candicans) ,21.2 per cent coli-like colonies {Escherichia
coli, A. aero genes y and S. sefticemiae), and many dark colony-forming
types. Other groups represented were Bacteroides, Flavobacteriumy
Pseudomonas, Bacillus^ various anaerobic bacteria, Oidiuniy and many
others. When the manure was allowed to decompose, yellow rods,
fluorescent bacteria, and mesentericus types took the place of the strep-
tococci.

The following heterotrophic bacteria have been demonstrated (811)
in manure : Bacillus subtilis, Bacillus mesentericus. Bacillus cereus, Ba-
cillus tumescenSy Bacillus fetasitesy Pseudomonas fluorescenSy Pseudo-
monas futiduy Salmonella enteritidisy Escherichia coliy Proteus vul-
garis y Micrococcus luteusy Micrococcus candicans y Staphylococcus albusy
Sarcina -flavay Streptococcus pyo genes y and others. Anaerobic bacteria
are also abundant (337).

Pathogenic bacteria may also occur frequently in human feces and
in stable manure 5 Mycobacterium tuberculosis and various hemolytic
streptococci (860), as well as pathogenic anaerobes including Clos-
tridium welchiiy CI. septicumy CI. oedematisy and CI. fallax have been
fpund (484).

The protozoa capable of developing in manure and in urine include
not only saprophytic forms but also certain parasites, such as Tricho-

22 HUMAN AND ANIMAL WASTES

mastric and Trichomonas^ capable of living and even of multiplying
in excreta. The coprophilic protozoa comprise various flagellates, cer-
tain amebae, and ciliates. The liquid part of the manure is considerably
richer than the solid in total number of protozoa as well as in species,
including Polytofna uvellay Cryftochilum nigricans^ and T etramUus
rostratus. These protozoa nearly all feed upon bacteria. The infusoria
may feed upon smaller protozoa, so that forms like Colpdium may not
destroy bacteria at all.

Human and animal excreta also contain a large population of fungi,
chiefly in a spore state. Schmidt (837) divided the manure-inhabiting
fungi into three groups :

Those found only in manure ; their spores are swallowed with the feed,
and they pass unchanged through the digestive tract, though they
are favorably influenced toward germination by the body heat and
digestive fluids of the animal. Their natural multiplication by spores
is impossible without the physiological action of the digestive proc-
esses.

Those that do not have to pass through the digestive tract of an animal in
order to germinate and develop. The representatives of this group
occur in nature only in manure, although some are able to grow also
on other substrates. They can be cultivated both on manure and on
other media, mostly at ordinary temperatures.

Organisms found both in manure and on other substrates. They grow
readily at room temperature on a number of media.

Comfosition and Decomposition

The chemical composition of human and animal excreta, and of
stable manures in general, varies considerably, depending on the nature
of the animal, its age, mode of nutrition, and composition of food-
stuffs (463). As soon as voided, manure begins to undergo rapid de-
composition. This results in the formation of ammonia and various
other nitrogenous degradation products. These give rise to offensive
sm.ells, which are controlled by the conditions of decomposition. From
a sanitary point of view, it is essential that decomposition should be
accompanied by the destruction of the injurious organisms present in
the manure. The fecal organisms gradually disappear and their place

STABLE MANURES AND FECAL RESIDUES

23

is taken by a population concerned in the decomposition of cellulose,
hemicelluloses, and proteins.

The decomposition of complex plant and animal residues leads to a
rapid reduction in carbohydrates and is accompanied by the evolution
of considerable heat, the temperature of the compost reaching as high
as 75° C, as shown in Figure i.

In order to hasten the decomposition of manure, conditions must be
favorable to the activities of microorganisms. It must be properly

2z

1- LU

«/5q 70

_

t^^'

§0 60

1 /

o^-

•'*• J

' /

. z

fej^so

- \\

A :

^.-^

NO BEDDING

.WITH BEDDING

15 20 25 30 35 40 45
COMPOSTING PER.10D IN DAYS

Figure i. Influence of straw bedding upon temperature changes in the
composting of manure. Circles indicate times of turning composts. From

Waksman and Nissen (961).

aerated and well moistened but not saturated with water. By placing the
manure, together with the waste materials of the farm and the home,
in heaps, designated as composts, the decomposition processes can be
controlled so as to lead to heat liberation j this results in the destruction
of the injurious organisms and the conservation of the plant nutrient
elements. When not properly regulated, the decomposition processes
may be wasteful, unsanitary, and unsightly, and may even become a
source of infection to man and his domesticated animals.

24 HUMAN AND ANIMAL WASTES

SEWAGE

Disposal of sewage and other home wastes is one of the important
sanitary problems of men living in industrial and residential centers.
Haphazard methods of disposing of sewage not only lead to conditions
most unpleasant to human habitation but they are dangerous from the
standpoint of infectious diseases.

Sewage abounds in microorganisms that originate not only from hu-
man excreta but also from other household and industrial wastes. The
various saprophytic bacteria present in sewage rapidly attack the or-
ganic constituents and bring about their gradual mineralization. The
destructive action of saprophytic organisms greatly reduces the number
of pathogens (342, 343). Activated sludge, for example, has been
shown (882) to possess a definite and consistent bactericidal action
against the colon bacteria. In addition to antagonistic organisms, active
bacteriophages against nearly all types of intestinal bacteria are present
in sewage. The destruction of pathogens by bacteriolysis thus readily
finds a place in the activated-sludge method of sewage purification.

Dissolved oxygen is generally present when sewage is diluted with
water. As the destruction of the organic matter proceeds rapidly, the
oxygen becomes depleted, so that none is left after a few hours. The
predominant bacterial flora of the water may then become anaerobic,
with the result that the chemical processes of decomposition are com-
pletely changed J hydrogen sulfide, mercaptans, and other foul -smell-
ing substances are then formed. This is accompanied by a typical
anaerobic breakdown of carbohydrates, leading to the formation of vari-
ous organic acids, carbon dioxide, hydrogen, and methane. The nitro-
gen in the protein and urea is transformed to ammonia and various
amines. When sewage is aerated, the anaerobic processes gradually give
way to aerobic processes, as the oxygen diffuses into the liquids or as the
sewage is diluted with water containing dissolved oxygen.

When sewage is freed from solids by sedimentation before discharge,
or when it is aerated sufficiently to maintain the concentration of dis-
solved oxygen, decomposition proceeds rapidly without the production
of the bad odors usually associated with the anaerobic breakdown. The
destruction of the pathogenic bacteria results largely through the ac-

GARBAGE 25

tivities of the saprophytes (809, 1008). For the purpose of promoting
the development of aerobic bacteria, processes employing the use of
intermittent sand filters, broad irrigation, contact beds, trickling filters,
and activated sludge are applied.

The modern methods of sewage purification are based on the long-
known fact that the soil is a destroyer of offensive wastes. In early days,
in fact, the soil handled all sewage problems. Sewage disposal plants in
modern cities are so operated that microorganisms found to be so effi-
cient in the soil are able to act under optimum conditions, resulting in
rapid purification. Sewage freed from most of its organic constituents
can be discharged into a stream and will not deplete the water of its dis-
solved oxygen. Chlorine is frequently employed in the final treatment
to assure the complete destruction of the pathogens.

GARBAGE

The processes involved in the disposal of garbage from the home
are similar to those utilized in the disposal of stable manure rather than
of sewage. At present, garbage usually is destroyed by burning, which
results in great economic waste, or is dumped outside cities, thus creat-
ing centers of infection and unpleasant appearance. More logical and
less wasteful processes are based upon the principle of composting. Sev-
eral of these processes are now utilized in India and China, where eco-
nomic pressure is greatest. By proper handling, a product is formed that
is free from injurious insects, parasitic worms, and bacteria, and that
conserves all the valuable elements essential for plant growth.

DESTRUCTION OF INJURIOUS MICROORGANISMS

Improper methods of disposal of human and animal wastes were
responsible, in the early history of mankind, for many epidemics of
cholera, typhoid, plague, and other diseases. Only in recent years, after
man learned the nature of the spread of these diseases, were proper
methods developed for disposing of human wastes.

Fecal-borne diseases rank with venereal disease and tuberculosis as
the most important infectious diseases of China, because the people

26 HUMAN AND ANIMAL WASTES

do not maintain proper sanitation and because human excreta are used
as fertilizers. Any successful system for the control of these diseases
must be sanitary and at the same time profitable. Of 1,190 persons
examined, 81 per cent were positive for ascaris, with an average egg
count of 14,000 per cubic centimeter. Children had a higher count than
adults, and females a higher count than males. The life habits of the
Chinese people are highly favorable for the spread of ascaris. By a
special process of composting of feces, sufficient heat was produced to
destroy disease-producing organisms and their reproductive bodies.
The compost thus produced is highly effective as a fertilizer (1023).

SURVIVAL OF HUMAN AND ANIMAL PATHOGENS
IN SOIL AND WATER

During the period 1878 to 1890 following the brilliant work of
Pasteur, when bacteriology was still in its infancy, medical bacteriolo-
gists took much interest in soil microbes. This was due largely to the
belief that causative agents of disease that find their way into the soil
may survive there and thus become a constant and important source of
infection. The introduction by Koch, in i88i-, of the gelatin plate
method placed in the hands of the investigator a convenient procedure
for measuring the abundance of the soil population and determining the
survival in the soil of agents causing serious human diseases. In spite of
the fact that this method revealed only a very small part of the soil
population, it enabled the medical bacteriologist to establish beyond
doubt that such organisms tend to disappear in the soil. This resulted in
definite conviction on the part of the public health and medical world
that the soil is seldom a source of infection. It was soon demonstrated
that disease-producing agents die out in the soil at a rather rapid rate,
depending on the nature of the organisms, the soil, climate, and other
conditions.

Organisms that Survive for Long Periods

Only a few disease-producing microorganisms are able to survive in
the soil for any considerable periods of time. These few include the or-
ganisms causing tetanus, gas gangrene, anthrax, certain skin infections,

SURVIVAL OF PATHOGENS IN SOIL AND WATER 27

actinomycosis in cattle, coccidiosis in poultry, hookworm infections,
trichinosis, enteric disorders in man, blackleg in cattle, and Texas fever.
To these may be added the botulinus organism and others producing
toxic substances, as well as bacteria, actinomycetes, and fungi that cause
plant diseases such as potato scab, root rots, take-all disease of cereals,
and damping-off diseases.

Anthrax, a scourge of cattle and sheep, is a persistent survivor in
soil } spores of this organism are known to retain their vitality and viru-
lence for fifteen years. Anthrax survives particularly well in damp re-
gions, especially in soils rich in decomposing organic matter j the hay
and feed from these lands may transmit the disease to animals. The fact
that certain fields carry anthrax infection ("anthrax pastures") was
recognized in Europe long before the nature of the disease was known.
Human infection results from contact with diseased animals or animal
products.

The anaerobic, spore-forming bacteria that cause gas gangrene are
widely distributed in nature. They are found extensively in soils and in
decomposing plant and animal residues. The causation of disease by
these organisms received particular attention during the first world
war, which was fought chiefly in trenches.

Another important pathogenic anaerobe able to survive in soil for
long periods of time is CI. chauvoei, the causative agent of blackleg in
cattle J southern pastures are said to be better carriers of blackleg than
northern pastures. CI. tetani is also widely distributed in the soil and
appears to be associated with the use of stable manures. Wounds in-
fected with soil may lead, therefore, to the development of tetanus or
gas gangrene and must be treated accordingly.

The botulinus organism not only may remain alive in the soil for a
long time (642), but it may also produce there a potent toxin that
causes much loss of water fowl and other wild life. Aeration of the soil
results in the destruction of this toxin by aerobic bacteria (742).

Orgamsms that Survive for Brief Periods
. Other pathogenic bacteria, however, are able to survive in the soil
only for limited periods of time. They are eliminated sooner or later
from the soil, either because of their inability to compete with the soil

28 HUMAN AND ANIMAL WASTES

population or because of their actual destruction by the latter. Although
the pathogens seem to possess considerable resistance toward unfavor-
able soil conditions, they are unable to multiply at rates permitting
their indefinite survival in the soil. The anthrax bacillus and certain
other parasites infesting domesticated and wild animals belong to this
group. Certain insect and animal carriers make possible the survival
and spread of many pathogens in the soil.

The great majority of disease-producing bacteria, however, are able
to survive only for very brief periods outside their respective hosts,
especially in soil and water. It is sufficient to cite the fact that typhoid
and dysentery bacteria, which are known to contaminate watersheds
and water supplies, disappear sooner or later. It has been estimated, for
example, that in sewage sludge free to undergo normal digestion,
typhoid bacteria probably survive for less than 7 days. It was sug-
gested, therefore, that sludge held in a digestion tank for about 10 days
might be applied to the soil for fertilizer purposes without detriment to
public health.

The gram-negative bacteria of the typhoid-dysentery group die out
rapidly in septic material; the typhoid bacteria survive for about 5
days, the Flexner type of dysentery for about 3 days, and the Shiga
bacillus dies out even in a shorter period. If decomposition in the tank
has not advanced far enough, as shown by low alkalinity, the organisms
may survive for a much longer period. The efficiency of ripe tank ef-
fluent to destroy bacteria is believed to be due to both the alkaline re-
action and the presence of antagonistic metabolic products. The destruc-
tion of typhoid and dysentery bacteria in the soil depends on a number
of factors, chief among which are the moisture content and reaction,
and the nature and abundance of the microbiological population. In
moist or dry soils, most of the pathogenic bacteria were found to die
within 10 days (510).

Numerous other pathogenic agents, including those causing some of
the most deadly human and animal scourges — tuberculosis, leprosy,
diphtheria, pneumonia, bubonic plague, cholera, influenza, mastitis and
abortion in cattle, the many poxes — constantly find their way into the
soil in large numbers. They disappear sooner or later, and no one now

SURVIVAL OF PATHOGENS IN SOIL AND WATER

29

ever raises the question concerning the role of the soil as the carrier of
these disease-producing agents or as the cause of epidemics.

This rapid disappearance of disease-producing bacteria in the soil may
be due to a number of factors: (a) unfavorable environment j (b) lack
of sufficient or proper food supply j (c) destruction by predacious agents
such as protozoa and other animals j (d) destruction by various sapro-
phytic bacteria and fungi considered as antagonists j (e) formation by
these antagonists of specific toxic or antibiotic substances destructive to
the pathogens J (f ) in the case of some organisms at least, increase of the
bacteriophage content of the soil resulting in the lysis of some bacteria,
especially certain spore-formers (50).

The course of survival of only a few disease-producing organisms
outside the host has been studied in detail. Sufficient information has
been accumulated, however, to justify certain general conclusions.
When E. colt is added to sterile soil, it multiplies at a rapid rate (Table
3, p. 10), but when added to fresh, nonsterile soil it tends to die out
quickly (Table 4). The rate of its disappearance is independent of re-
action of the soil and of incubation temperature.

In order to illustrate the fate of certain important disease-producing

TABLE 4. SURVIVAL OF BACTERIA ADDED TO SOIL AND THEIR EFFECT
UPON THE SOIL MICROBIOLOGICAL POPULATION

INOCULUM

INCUBATION

ORGANISMS RECOVERED*

Number

Tem-

Coliform

of days

perature

Total

bacteria

Control soil

5

28° C.

21,400

<200

E. colt addedf

5

28° c.

25,600

6,800

E. colt added$

5

28° c.

39>700

3,500

E. coli added

5

37° C.

22,800

4,700

Control soil

33

28° c.

5,900

<I0

E. coli added

33

28° c.

22,100

130

E. coli addedf

33

28° c.

17,600

140

E. coli added

33

37° C.

23,000

<IO

From Waksman and Woodruff (980).

* In thousands per gram of soil.

t Washed suspension of E. coli cells added at start and after 5 days.

% CaCOs added to soil.

30 HUMAN AND ANIMAL WASTES

bacteria which find their way into the soil or into natural water basins, it
is sufficient to draw attention to reports of experiments made on a few
typical pathogens.

The Colon-Typhoid Group of Bacteria

Frankland (293, 294) was the first to establish that Eherthella ty-
fhosa may survive in sterilized polluted water or in pure deep-well
water for 20 to 51 days although it dies out in 9 to 13 days in unsterile
surface water. In other studies (481) it was found that the typhoid or-
ganism was able to survive in sterilized tap water for 15 to 25 days, as
against 4 to 7 days in fresh water j the bacteria died off even more rap-
idly in raw river or canal water, the survival time being reduced to i to
4 days. The degree of survival of the typhoid organism in water was
found to be in inverse ratio to the degree of contamination of the water,
the saprophytic bacteria in the water apparently being responsible for
the destruction of the pathogen. These conclusions were later con-
firmed. Freshly isolated cultures of E. tyfhosa survived a shorter time
than laboratory cultures, high temperatures (37° C.) being more de-
structive than low ones. Sedgwick and Winslow (846) reported that
cells of E. coli rapidly died out in the soil, 99 per cent destruction
occurring in dry soil in 2 weeks, with a longer survival in moist soil.

In general E. tyfhosa is able to survive only a short time in unsteri-
lized soil, much longer in sterile soil. S. Martin (623), for example,
observed that typhoid bacteria survived and grew readily in sterile
soil but when added to well-moistened and cultivated soil they were
rapidly destroyed. The same phenomenon occurred when the patho-
gens were added to a culture of a soil organism in a nutrient medium.
Only in certain soils were conditions favorable for the prolonged
survival of the pathogen. The conclusion was reached that the typhoid
organism is destroyed by the products of decomposition taking place
in the soil. It was further concluded that an antagonistic relation ap-
pears to exist in some soils but not in others and that this is due to the
action of specific antagonistic bacteria present in the particular soils.

Frost (303) also reported that typhoid bacteria were rapidly de-
stroyed when added to the soil. In 6 days, 98 per cent of the cells were

SURVIVAL OF PATHOGENS IN SOIL AND WATER 31

killed, and in the course of a few more days all the cells tended to dis-
appear entirely from the soil. Under conditions less favorable to the
growth of antagonists, the typhoid organism survived not only for
many days, but even for months. The conclusion was reached that when
soil bacteria are given a chance to develop by-products, there results a
marked destruction of typhoid organisms brought into contact with
them.

Among the factors responsible for the disappearance of E. tyfhosa
in water, the presence of certain water bacteria was found to be of spe-
cial importance (924). Rochaix and Vieux (798) demonstrated that
when an achromogenic strain of Pseudomonas aeruginosa was present in
drinking water, it was not accompanied by any other bacteria. Media
inoculated with this organism and E. coli gave, after 1 3 days' incubation,
only cultures of the former. That the two organisms could coexist, how-
ever, was shown by inoculation into sterilized water. Only the actual
development of the antagonist led to the repression of the fecal organ-
ism. The supply of oxygen in the water is important. E. typhosa
added to activated sludge increased within the first 4 to 6 hours j this was
followed by a reduction in 24 hours, and a 99 per cent destruction in sev-
eral days (422). The survival period was shorter in sewage-polluted
than in unpolluted waters, especially when the sewage was aerated.
About 80 per cent reduction of typhoid bacteria was obtained in the
Netherlands East Indies by the passage of sewage through Imhoff
tanks. Digestion of sludge reduced the number further but did not
eliminate the bacteria completely 5 after the sludge was dried no typhoid
bacteria could be found {6S'i)'

A study of microorganisms antagonistic to E. coli resulted in the iso-
lation of organisms from 5 of 44 samples of well water, i of 1 2 sam-
ples of spring water, and 6 of 1 6 samples of surface water. The antag-
onists included 3 strains of Pseudomonas, i each of Sarcina, Micro-
coccus, Flavobacterium, and yeast, 2 actinomycetes, and 3 unidentified
nonspore-forming, gram-negative rods (455).

The survival of E. tyfhosa in manure and in soil is known to be af-
fected decidedly by various saprophytic bacteria. When a carrier was
induced to urinate on a soil, E. fyphosa could be recovered within

32 HUMAN AND ANIMAL WASTES

6 hours from the washings of the soil j however, after 30 hours the or-
ganism could no longer be demonstrated, although the soil was still
moist with the urine (6s5). In the absence of sunlight, the organism
was recovered after 24 hours but not later. When the urine was allowed
to dry on towels, the bacterial cells survived for 10 days because sapro-
phytic microorganisms failed to develop on the dry towels. Other evi-
dence was submitted that E. ty-phosa is destroyed by bacteria grown in
association with it. Moisture was found to be the most important factor
influencing the longevity of typhoid bacteria in the soil j 50 per cent of
the bacteria died during the first 48 hours, the survival of the remainder
extending over a period of months. Even in those investigations where
E. typhosa was detected after 70 or 80 days, the evidence pointed to a
lack of multiplication of these bacteria in the soil 5 when the organism
survived for a shorter time in sterilized than in natural soil, it was
found (616) to be due to the fact that steam heating of soil results in
the formation of bactericidal substances.

E. colt was rapidly crowded out by other organisms in manure
piles. The addition of 9 million cells of E. colt and 13 million cells oi A.
aero genes to a soil resulted, in 106 days, in reductions to 6,000 and
25,000 respectively J in 248 days, both organisms had completely dis-
appeared (856). The occurrence of coliform bacteria in soil depends
entirely on the degree of pollution j soil relatively free from pollution
contains no coliform bacteria or only a small number. No evidence of
multiplication of these bacteria in the soil could be detected (899).

Sea water, as well, appears to have a bactericidal effect upon organ-
isms added to it (959, 1050). This is believed to be due to the presence
of some substance other than salt. Dysentery and typhoid organisms
were found to disappear from sea water in 12 and 16 hours, whereas
paratyphoid organisms survived for 21 and 23 days (915). Harvey
(386) concluded that sea water contains a substance that is inhibitory
to the growth of diatoms j this substance is adsorbed on precipitated
phosphate or animal charcoal, and is destroyed by treatment with
H2O2.

Protozoa were found to be at least partly responsible for the destruc-
tion of the typhoid organism added to water systems (452, 741).

SURVIVAL OF PATHOGENS IN SOIL AND WATER 33

Mycobacterium tuberculosis

The fate outside the hosts of the bacteria causing tuberculosis in man
and in animals has also been studied extensively. Considerable diffi-
culty has often been encountered, however, in demonstrating the pres-
ence of this pathogen, which must be detected usually by guinea pig
inoculation methods. The organism was found to be alive in a dark
room after 157 to 170 days, but not after 172 to 188 daysj in diffused
light, the longevity was only 124 days. In the incubator, the organism
retained its virulence for 2>'i days, but not for 100 days j on ice, virulence
was still evident after 102 days but not after 153 days (657).

Pure cultures of the bovine organism mixed with cow manure and ex-
posed in a 2-inch layer in a pasture remained virulent for 2 months in
sunlight and longer in the shade. Tubercle bacteria were still alive in a
garden soil on the 213th day and dead on the 230th day. They were
alive in buried tuberculous guinea pigs on the 71st day, and dead on
the 99th day. In running water, they survived for more than a year
(86). Mycobacterium tuberculosis survived for 309 days in sputum
kept in darkness, even when completely desiccated j in decomposing
sputum, living organisms could be isolated after 20 days but not after
25 days (871a). Under conditions prevailing in southern England, it
was found ( 1020) that the tubercle organism may remain alive and viru-
lent in cow's feces exposed on pasture land for at least 5 months dur-
ing winter, 2 months during spring, and 4 months during autumn j in
summer, no living organisms were demonstrated even after 2 months.
Under protection from direct sunlight, the survival period was longer.
Feces protected from earthworms yielded viable cells even after 5
months. Virulent bacteria were still present in stored liquid manure at
least 4 months after infection, though during this time a gradual reduc-
tion in virulence of the organism was observed.

The addition of manure to soil was found to favor the survival of the
tubercle bacteria, as indicated by a higher proportion of test animals
becoming tuberculous when the amount of manure added to the soil was
increased (613). Positive tests were obtained for soil and manure after
178 days but not later. The organism survived on grass for at least 49
days. Rhines (780) found that M. tuberculosis multiplied in sterile soil

34 HUMAN AND ANIMAL WASTES

as well as in the presence of certain pure cultures of bacteria j however, a
fungus was found to check the development of the pathogen, especially
in manured soil. In nonsterile soil, the pathogen was slowly destroyed,
the plate count being reduced to about one sixth of the original in
I month. In a study of the survival of avian tubercle bacteria in sewage
and in stream water, there was a reduction, in 73 days, from 48,000 to
1,400 per milliliter in sewage and to 4,200 in water (779).

Other Disease-f reducing Microorganisms

A study of the viability of Brucella rjtelitensis in soil and in water in
Malta brought out the fact that the organism survived in sterile tap
water 42 days and in unsterile tap water only 7 days. It survived 25
days in soil and 69 days in dry sterile soil, but only 20 days in unsterile
manured soil, 28 days in dry natural road dust, 20 days in dry sterile
sand, and 80 days on dry cloth (334, 446).

The rapid destruction of cholera bacteria added to soil was first
pointed out by Houston (451). Similar rapid destruction of the diph-
theria organism was also noted. Serraiia, however, retained its vitality
for 158 days. Vibrio comma also survived for a short time only in feces
(362), different strains showing considerable variability; temperature
was an important factor. During the hot season in Calcutta, the viable
period was somewhat longer than a day, as compared to 7 or 8 days
during the cold season ; the critical cholera months were found to fol-
low directly the cool months. The organism did not survive very long
in fresh water, although the time appeared to be long enough to cause
occasional serious epidemics. It remained alive for 47 days in sea water
(459). The conclusion was reached that although the organism is ordi-
narily destroyed rapidly in water as a result of competition with other
microbes, it may survive in certain instances for some time.

As a result of the evidence presented above and of other information
not reported here, it has gradually become established that the soil
has an enormous purification or sanitation effect upon the pathogenic
bacteria brought into it either by direct excreta, in sewage, or in other-
wise contaminated waters. This effect is of a double kind: (a) physical
adsorption of the bacteria upon the soil, light, porous, sandy soils being

SURVIVAL OF PATHOGENS IN SOIL AND WATER 35

far less efficient in removing the bacteria than heavy loam or clay soils j
(b) biological effect, resulting in the destruction of the bacteria in the
soil. As a result of early studies on the survival of the cholera organism
in the soil, certain soils became recognized as "cholera immune" or as
"cholera destroying" (736).

CHAPTER 3

INTERRELATIONSHIPS AMONG MICROORGAN-
ISMS IN MIXED POPULATIONS

It must not be jor gotten that there are extremes in another di-
rectton, where one of the two associated organisms is injuring
the other, as exemflified by many farasites, but these cases I
leave out of account here. This state of affairs has been termed
antibiosis. — H. M. Ward.

The antagonistic effects of one organism upon another were observed
by many of the early microbiologists. It is sufficient to cite here three
striking examples based upon totally different approaches to the sub-
ject.

In 1876, Tyndall (919), on the basis of the growth of wild cultures
of bacteria and fungi in organic media, spoke of "the struggle for
existence between the Bacteria and the PenkilUum. In some tubes the
former were triumphant j in other tubes of the same infusion the latter
was triumphant. The Bacteria which manufacture a green pigment
appear to be uniformly victorious in their fight with the Penicillium."

In 1877, Pasteur (710) noted that the production of anthrax in sus-
ceptible animals can be repressed by the simultaneous inoculation with
B. anthracis and various other bacteria. This led him to make the
foUov/ing significant suggestion: ". . . on peut introduire a profusion
dans un animal la bacteridie charbonneuse sans que celui-ci contracte le
charbon: il suffit qu'au liquide qui tient en suspension la bacteridie on
ait associe en meme temps des bacteries communes."

In 1879, DeBary (172) emphasized the significance of the antag-
onistic interrelations among microorganisms j when two organisms
are grown on the same substrate, sooner or later one overcomes the
other and even kills it.

These and other observations thus laid the basis for a study of mutu-
alistic effects of microorganisms in natural and in artificial environ-
ments.

SYMBIOSIS AND ANTIBIOSIS 37

SYMBIOSIS AND ANTIBIOSIS

Microbes grow and bring about many metabolic reactions in natural
substrates, such as soils and water basins, in a manner quite different
from those in pure cultures where they are not influenced by the
growth of other organisms. In artificial and natural media, whether
these be synthetic materials, complex organic mashes and infusions used
for the preparation of industrially essential products, or the bodies of
plants and animals, pure cultures of microbes are free from the asso-
ciative and competitive effects of other microbes found in natural sub-
strates. In mixed populations, a number of reactions that do not com-
monly take place in pure cultures are involved. Even in the case of
mixed infections, a pathogen may be preceded or followed by one or
more saprophytes, whereby the processes of destruction brought about
in the living animal or plant body are alleviated or hastened. In the
mixed populations found in natural substrates, the ecological relation-
ships are largely responsible for many of the essential differences in
the behavior and metabolism of the microbes, as compared with the
same organisms growing in pure culture.

Almost all microorganisms inhabiting a natural milieu, such as soil
or water, are subject to numerous antagonistic as well as associative, or
even symbiotic, interrelations. Every organism is influenced, directly
or indirectly, by one or more of the other constituent members of the
complex population. These influences were at first visualized as due
primarily to competition for nutrients. This was well expressed by
Pfeffer, who said that "the entire world and all the friendly and an-
tagonistic relationships of different organisms are primarily regulated
by the necessity of obtaining food." It was soon recognized, however,
that this explanation does not account fully for all the complex inter-
relations among microorganisms in nature.

Symbiotic, or mutualistic, and antagonistic relationships among mi-
croorganisms indicate whether advantages or disadvantages will result
to the organisms from the particular association j the first are beneficial
and the second are injurious and may even be parasitic (41, 982).
When two organisms are capable of utilizing the same nutrients but are
differently affected by environmental conditions such as reaction, air

38 INTERRELATIONSHIPS AMONG MICROORGANISMS

supply, and temperature, the one that finds conditions more suitable
for its development will grow more rapidly and in time be able to
suppress the other. According to Porter (729), the effects produced
by fungi in mixed culture are due either to exhaustion of nutrients or
to the formation of detrimental or beneficial products. When two or
more organisms live in close proximity they may exert antagonistic,
indifferent, or favorable effects upon one another. These potentialities
were later enlarged (1046) to include stimulating, inhibiting, over-
growing, and noninfluencing effects. After considerable experimenta-
tion and speculation, Lasseur (548) came to the conclusion that antago-
nism is a very complex phenomenon and is a result of numerous and
often little-known activities. Antagonism influences the morphology
of the organisms, their capacity for pigment production, and other
physiological processes.

No sharp lines of demarcation can be drawn between associative and
antagonistic effects. Well-defined effects of two symbionts may change
during the various stages of their life cycles or as a result of changes in
the environment. It is often difficult to separate strictly symbiotic phe-
nomena from associations of less intimate nature, frequently desig-
nated as commensalisms. The various stages of transition from obligate
parasitism to true saprophytism can be represented as follows:

Obligate parasitism (cer- Facultative parasitism (spe- Modified parasitism;
tain bacteria, smut fungi) — > cies of Fusarium, Rhizoc- —^ hosts may derive some — >
tonia, and Actinomyces') benefit (certain mycor-

rhiza)

Balanced parasitism (vari- True symbiosis (root- True saprophytism (auto-

ous mycorrhiza) —^ nodule bacteria, lichen — > trophic and heterotrophic

formations) bacteria and fungi).

The phenomena of antagonism do not fit exactly into the above
scheme but are parallel with it: the injurious effects of one organism
upon another range from antagonism of varying degrees of intensity
to the actual living or preying of one organism upon another. The lat-
ter may be classified with the phenomena of parasitism and disease pro-
duction.

Microorganisms inhabiting the soil live in a state of equilibrium

SYMBIOSIS AND ANTIBIOSIS 39

and any disturbance of this equilibrium results in a number of changes
in the microbial population, both qualitative and quantitative. The
ecological nature of this population found under certain specific con-
ditions, as well as the resulting activities, can be understood only
when the particular interrelationships among the microorganisms are
recognized. Because of its complexity, the soil population cannot be
treated as a whole, but some of the processes as well as some of the
interrelations of specific groups of organisms can be examined as sepa-
rate entities. Some have received particular attention, as the relations
between the nonspore-forming bacteria and the spore-formers, the ac-
tinomycetes and the bacteria, the bacteria and the fungi, the protozoa
and the bacteria, and the relations of the bacteria and the fungi to the
insects.

The term "synergism" has been used to designate the living together
of two organisms, resulting in a change that could not be brought about
by either organism alone (440). Microbes living in association fre-
quently develop characteristics which they do not possess when living
in pure culture. For example, Schiller (835) found that when beer
yeasts are placed together with tubercle bacteria in a sugar-containing
but nitrogen-free medium, the yeasts develop antagonistic properties
toward the bacteria and use the latter as a source of nitrogen by secret-
ing a bacteriolytic subtance that is also active outside their cells.
Various bacteria are able to kill yeasts when they are inoculated into
suspensions of the latter in distilled water. The destruction of the
fungus Ofhiobolus, the causative agent of the take-all disease of cereals,
by soil organisms was believed to be a result of the need of a source of
nitrogen by the latter.

Papacostas and Gate (706) suggested applying the term "antibiosis"
to interactions in mixed cultures in vitro and "antagonism" to mixed
infections in vivo. In order to obviate a possible concept that the two
types of interaction, namely in the test tube and in the living body, are
different, it is more appropriate to apply the term "antagonism" to the
unfavorable effects of one living system upon another and "antibiosis"
to the production by one organism of specific chemical substances which
have an injurious effect upon another organism.

40 INTERRELATIONSHIPS AMONG MICROORGANISMS

THE NATURE OF A MIXED MICROBIAL
POPULATION

A mixed microbial population is made up of a great variety of bac-
teria, and often also of fungi, actinomycetes, and protozoa j to these are
added, under certain conditions, various algae, diatoms, nematodes and
other worms, and insects. The specific nature and relative abundance of
the various microorganisms making up a complex population in either a
natural or an artificial environment depend upon a number of factors,
which can be briefly summarized as follows :

The physical nature of the medium in which the population lives: soil,
compost, or manure pile; river, lake, or ocean; sewage system; or
peat bog.

The nature, concentration, and availability of the chemical constituents
of the medium used by the microbes as nutrients, especially the ma-
terials used as sources of energy and for the building of cell sub-
stance. Various organic and inorganic substances, whether complex
or simple in chemical composition, favor the development of specific
groups of microorganisms capable of utiHzing them. For example,
sulfur favors the development of specific sulfur bacteria, and cellu-
lose favors such organisms as are capable of attacking this complex
carbohydrate as a source of energy. In many instances there is con-
siderable competition for the available food material. Organisms that
possess a greater capacity for attacking the particular compound, or
are capable of preventing the development of other organisms by the
formation of substances injurious to the latter, usually become pre-
dominant. Proteins, starches, and sugars can be acted upon by a
great variety of microorganisms. The predominance of one group
may depend not only upon the chance presence of the particular or-
ganism or its capacity for more rapid growth, but also upon its ability
to form alcohols, acids, and other products that influence the growth
of other organisms.

Environmental conditions favorable or unfavorable to the development
of specific organisms. Of particular importance in this connection
are temperature (thermophilic vs. mesophih'c organisms), oxygen
supply (aerobic vs. anaerobic organisms), moisture content (bac-
teria and fungi vs. actinomycetes), reaction (acid-sensitive vs. acid-

ASSOCIATIVE INTERRELATIONSHIPS 41

tolerant forms), as well as the physical conditions of the substrate as
a whole.

The presence and abundance of organisms that produce substances having
a favorable and stimulating or an injurious and toxic effect upon
other organisms, or that may compete for the available nutrients.
The equilibrium in the microbiological population in a natural me-
dium such as soil or water may be upset by the introduction of spe-
cific nutrients, as well as by treatment with chemical and physical
agents whereby certain organisms are destroyed and others stimu-
lated.

The presence of specific microorganisms in a natural medium may be con-
siderably influenced by the presence of certain parasitic or phagocytic
agents. The role of protozoa in controlling bacterial activities by
consuming the cells of the bacteria has been a subject of much specu-
lation. The presence of bacteria, fungi, and nematodes capable of
destroying insects is of great importance in human economy. Many
other relationships, such as the presence of phages against specific
organisms, are often found greatly to influence the nature and com-
position of a specific population.

ASSOCIATIVE INTERRELATIONSHIPS

Numerous instances of associative interrelationships among micro-
organisms are found in nature. These may be grouped as follows:

Preparation or modification of the substrate by one organism whereby it
is rendered more favorable or more readily available for the growth
of another organism. As an illustration one may cite the breakdown
of cellulose by specific bacteria, thereby making the particular en-
ergy source available to noncellulose-decomposing organisms, in-
cluding not only certain bacteria and fungi but also higher forms of
life such as ruminant animals (herbivores) and insects (termites,
cockroaches), which carry an extensive cellulose-decomposing micro-
biological population in their digestive systems. Another illustration
is the breakdown of complex proteins by proteolytic bacteria, result-
ing in the formation of amino acids and polypeptides, which form
f favorable substrates for peptolytic bacteria. The ammonia liberated

from proteins and amino acids supplies a source of energy for nitrify-

42 INTERRELATIONSHIPS AMONG MICROORGANISMS

ing bacteria and a source of nitrogen for many fungi. The ability of
bacteria to concentrate in solution those nutrients that are present
only in mere traces enables animal forms (protozoa) to exist at the
expense of the bacteria ( io6).

Influence upon the oxygen concentration available for respiration. This
involves the phenomenon first observed by Pasteur (709) of con-
sumption of oxygen by aerobic bacteria, thus making conditions fa-
vorable for the development of anaerobes.

Symbiotic interrelationships, where both organisms benefit from the asso-
ciation. The three most important examples found in nature are:
(a) the phenomenon of symbiosis between root-nodule bacteria and
leguminous plants; (b) mycorrhiza formations between certain
fungi and higher plants; (c) lichen formation between algae and
fungi. Certain other interrelations are not strictly symbiotic, but are
found to fall between groups a and c; here belong nitrate reduction
accompanied by cellulose decomposition and nitrogen-fixation with
cellulose decomposition, carried out in each case by two specific
groups of organisms.

Production by one organism of growth-promoting substances that favor
the development of other organisms. The formation of riboflavin by
anaerobic bacteria in the digestive tract of herbivorous animals is an
interesting and highly important phenomenon in the nutrition of such
animals. The production of bacterial growth stimulants by yeasts
and the beneficial action of mixed populations upon nitrogen-fixation
by Avcotohacter are other illustrations of this general phenomenon.
The presence of specific bacteria has been found necessary for the
sporulation of certain yeasts and for the formation of perithecia by
various Aspergilli (825). Various other processes of association have
also been recognized (940).

Destruction by one microorganism of toxic substances produced by an-
other, thereby enabling the continued development of various mem-
bers of the microbiological population.

Modification of the physiology of one organism by another. In the presence
of certain bacteria, Clostridium granulobacter-fectinovorum forms
lactic acid instead of butyl alcohol (873). The presence of Clos-
tridium acetobutylicum in cultures of bacteria producing dextro-lactic
acid and laevo-lactic acid causes such bacteria to form the inactive lac-
tic acid (897) ; intimate contact of the bacteria is essential, the use of
membranes preventing this effect. Pigment formation by Ps.

ASSOCIATIVE INTERRELATIONSHIPS 43

aeruginosa may be weakened when the latter is grown together with
other organisms. E. colt may lose the property of fermenting sugars
when grown in the presence of paratyphoid organisms (462).

Some associations of microorganisms are not so simple. The complex
system of animal infection by more than one organism, with the result-
ing complex reactions in the animal body, is a case in point.

The effect of one organism upon the activities of another can be illus-
trated by the results of the decomposition of complex plant material by
pure and mixed cultures of microbes (Table 5). Trichoderma, a fungus

TABLE 5. DECOMPOSITION OF ALFALFA BY PURE AND MIXED
CULTURES OF MICROORGANISMS

TOTAL

HEMICELLU-

CELLU-

ALFALFA DE-

LOSES DE-

LOSE DE-

NH3-N

ORGANISM

COMPOSED

COMPOSED

COMPOSED

PRODUCED

Per cent

Per cent

Per cent

mgm.

Trichoderma

9-3

4-7

0

61

Rhizopis

6.6

12.8

2.9

53

Trichoderma + Rhizofus

13-7

22.6

10.6

63

Trichoderma + Cunninghamella 15.0

15.4

5-7

47

Trichoderma + ?s. fuorescens

10.5

14.5

6.4

32

Streftomyces 3065

16.6

43-0

23.2

52

Trichoderma + Streftomyces

3065

12.5

14.6

4.8

56

Soil infusion

28.4

40.9

50.8

21

From Waksman and Hutchings (960).

known to be an active cellulose-decomposing organism, did not attack
at all the cellulose of alfalfa and decomposed the hemicelluloses only to
a limited extent} however, the organism utilized the proteins rapidly,
as illustrated by the amount of ammonia liberated. RhizofuSy a non-
cellulose-decomposing fungus, attacked largely the hemicelluloses in
the alfalfa and some of the protein } a small reduction in cellulose was
recorded, probably because of an analytical error. When Trichoderma
was combined with Rhizofus y the former attacked readily both the cel-
lulose and the hemicelluloses. The same effect upon the activity of
Trichoderma was exerted by other noncellulose-decomposing organ-

44 INTERRELATIONSHIPS AMONG MICROORGANISMS

isms, such as the fungus Cunninghamella and the bacterium Ps. fluores-
cens. On the other hand, when Trichoderma was combined with a cellu-
lose-decomposing Stre'ptomyces, there was considerable reduction in the
decomposition of the total plant material as well as of the cellulose and
hemicelluloses. These results further emphasize the fact that two or-
ganisms may either supplement and stimulate each other or exert an-
tagonistic effects. The total soil population is far more active than any
of the simple combinations of microorganisms.

COMPETITIVE INTERRELATIONSHIPS

The following competitive relations among the microscopic forms of
life inhabiting the sea have been recognized ( 15) :

Competition among chlorophyll-bearing diatoms for the available nutri-
ent elements in the water

Competition among the copepods for the available particulate food mate-
rials, notably the diatoms

Competition between individuals belonging to one species and individuals
belonging to another

Competition between young, growing, and reproducing cells and older,
respiring cells

Food competition and space competition

Competition between transitory and permanent populations

Competition between sedentary or sessile organisms and free-moving forms

This list has been enlarged (943) to include other factors that are par-
ticularly prominent in nonaquatic environments:

Degree of tolerance of the immune or resistant varieties and of the less re-
sistant or more sensitive forms to attack by disease-producing or-
ganisms

Fitness for survival of microbes that are able to adapt to a symbiotic form
of life, such as leguminous plants or mycorrhiza-producing plants,
and those that are not so adapted

Survival of parasitic forms that require living hosts for their development,
as contrasted with saprophytes that obtain their nutrients from min-
eral elements or from dead plant, animal, and microbial residues

ANTAGONISTIC INTERRELATIONSHIPS +5

Various special types of competition, for example, competition between
Strains of root-nodule bacteria (Rhizobium), whereby one strain
checks completely the multiplication of other strains, even outside the
plant, the dominant strain then becoming responsible for all the
nodules produced (679).

These phenomena of competition are found not only in natural sub-
strates, such as soil and water, but also in artificial media. When several
microbes are growing in the same culture medium, some will be re-
pressed in course of time whereas others will survive and take their
place. This is due to the fact that these microbes compete for the use of
the same nutrients or that conditions, such as reaction, oxygen supply,
and temperature, are more favorable to some organisms than to others.
Another phenomenon may also be involved, that some organisms may
produce toxic substances that repress the growth of others. In artificial
media, slowly growing tubercle bacteria, diphtheria organisms, and
others will be repressed by the rapidly growing saprophytes. Under
aerobic conditions, aerobic bacteria and fungi will repress yeasts and
anaerobic bacteria, whereas under anaerobic conditions the reverse will
take place. An alkaline reaction will favor the development of bacteria,
an acid reaction will favor the growth of fungi.

ANTAGONISTIC INTERRELATIONSHIPS

When two or more organisms live together, one may become antag-
onistic to the others. The composition of the medium and the conditions
of growth influence the nature and the action of the antagonist j the
metabolism and cell structure of the antagonized organism may be
modified or the cell itself may be destroyed (184). In urine, for
example, staphylococci may become antagonistic to E. coli or vice versa,
depending on the initial numbers of the two groups, on the formation
of metabolic products, or on the exhaustion of nutrients (246). The
toxic substances produced by the antagonists comprise a variety of com-
pounds, ranging from simple organic acids and alcohols to highly com-
plex bodies of protein or polypeptide nature.

Various types of antagonism are recognized. Nakhimovskaia (670)

L 5 B R A

46 INTERRELATIONSHIPS AMONG MICROORGANISMS

concluded that all phenomena of antagonism among microorganisms

can be conveniently classified into four groups :

1. Antagonism in vivo vs. antagonism in vitro. According to some inves-

tigators, only the inhibitive forms of antagonism (in vitro) may be
designated as true antagonisms; the in vivo forms w^ere designated
as phenomena of antibiosis. As pointed out above (p. 38), this
differentiation is no longer recognized.

2. Repressive, bactericidal, and lytic forms of antagonism. One may fur-

ther distinguish between bacteriostatic and bactericidal, fungistatic
and fungicidal forms of antagonism, as well as between antagonism
of function and antagonism of growth.

3. Direct, indirect, and true antagonism.

4. One-sided and two-sided antagonism; antagonism between strains of

the same species and antagonism among strains of different species.

Duclaux (212) was the first to demonstrate that the growth of a
fungus upon a certain medium renders the medium unfavorable for the
further growth of the same organism. Kiister (541) has shown that
culture solutions in which fungi have grown are not suitable for the
germination of freshly inoculated spores but are improved by boiling.
This effect was observed as a result of the growth not only of the same
organism but also of other species. Similar observations were made for
bacteria: Marmorek (620) reported, in 1902, that the growth of
hemolytic streptococci in broth rendered the medium unsuitable for
subsequent growth of the same organism. The production of spores by
bacteria was believed to be caused by the formation of toxic, thermola-
bile organic substances j upon the destruction of these by boiling, the
medium was again made favorable for the growth of bacteria and bac-
terial spores were once more able to germinate. Some of the toxic sub-
stances appeared to be thermostable (668).

Fungi are capable of producing not only growth-inhibiting but also
growth-promoting substances. By means of certain procedures, it was
found possible to separate the two (690). The tendency of fungus
hyphae to turn away from the region in which other hyphae of the
same fungus were growing was explained as a negative reaction to
chemical substances produced by the growing fungus (306). This nega-

ANTAGONISTIC INTERRELATIONSHIPS 47

tive chemotropism was shown to be due to thermolabile staling sub-
stances (358). The phenomenon of staling was often spoken of as vacci-
nation of medium (45), and was ascribed to the action of protein degra-
dation products.

These and other experiments led to the conclusion that many micro-
organisms are capable of producing substances that are injurious to their
own development (iso-antagonistic) or, and sometimes much more so,
to other organisms growing close to them (hetero-antagonistic). The
growth of certain fungi and bacteria in practically pure culture, even in
a nonsterile environment, was believed to be due to this phenomenon.
It is sufficient to mention the lactic and butyric acid bacteria, the citric-
acid-producing species of As-pergillus, the lactic and fumaric-acid-
producing species of RMzofus, and the alcohol-producing yeasts. The
chemical substances produced by these organisms in natural substrates
may be looked upon as protective metabolic products of microorgan-
isms in their struggle for existence. Such products play a highly sig-
nificant part in the metabolism of various organisms, especially those
that grow parasitically upon living plant and animal bodies.

Among the various types of antagonism, the one resulting in the pro-
duction of active substances that can be isolated and purified has re-
ceived the greatest consideration recently. These substances have been
designated as toxins, poisons, antagonistic agents, bacteriostatics, and
antibiotics. The chemical nature of some has been elucidated, but that
of many others is still unknown. Some of these substances are destroyed
by boiling, by exposure to light, or by filtration, whereas others are re-
sistant to heat and to ultraviolet raysj some are readily adsorbed by
certain filters, from which they can be removed by the use of special
solvents such as ether, alcohol, chloroform, and acetone. The concen-
tration of the antagonistic substances produced by many fungi and bac-
teria is greatly influenced by the energy and nitrogen sources in the
medium and by environmental conditions, such as temperature and
aeration.

The three important types of antagonism are (a) the repressive, in-
hibitive, or bacteriostatic, (b) the bactericidal, and (c) the bacteriolytic.
When one bacterium is inoculated into the filtrate of another, the
growth of the first is slower than that of the control. Certain types of

48 INTERRELATIONSHIPS AMONG MICROORGANISMS

antagonism express themselves in the destruction by the antagonist of
the other organisms present in the mixed culture, with or without lysis.
B. mesentericusj for example, is capable not only of depressing but also
of killing the cells of diphtheria and pseudodiphtheria (1052). The
lytic form of antagonism is illustrated by the action of Ps. aeruginosa,
B. brev'ts, and certain other antagonists upon micrococci and various
spore-forming bacteria.

In differentiating between "direct antagonism" and "passive antag-
onism," attention was directed (670) to the fact that the latter depends
not upon the direct action of the antagonist but upon the changed con-
ditions of culture under the influence of the antagonist's growth. This
may comprise a change in ^H and r¥L of medium or an impoverish-
ment of some of the nutrient constituents. "Direct antagonism" was
often distinguished (677) from "indirect antagonism," the first being
limited to those phenomena in which the antagonistic action is con-
nected with the direct action of the living cell, whereas in the second the
metabolic products produced by one organism are injurious to others.
Intestinal bacteria were found (367, 369) to repress the anthrax organ-
ism only when the former were in an active living state. Other investi-
gators (418) designated the action of the living cell itself as "true
antagonism."

Bail (32) suggested that for every bacterium there is a typical
constant number of cells capable of living in a given space. When this
concentration (M) is reached, multiplication comes to a standstill, in-
dependently of exhaustion of the nutrients or formation of toxic sub-
stances. The same phenomenon was believed to hold true when two
bacteria live together (1013): if the limiting cell-in-space concentra-
tions are different for the two organisms, the one with a higher M value
represses the other; however, the weaker species may check the stronger
when planted in sufficient excess (243). It has been suggested (370)
that certain physiological properties of the individual organisms, desig-
nated as "biological activity" and "competitive capacity," must also be
taken into consideration in evaluating this relationship. The fact that
the number of yeast cells reaches a maximum independently of the ini-
tial number of cells added or the concentration of nutrients in a given

ANTAGONISTIC INTERRELATIONSHIPS 4-9

volume of medium has been explained (91) by the amount of oxygen
originally present.

Garre (315) deserves the credit for having first noted that antago-
nism may be either one-sided or two-sided. In the first case, one organ-
ism represses another that is not antagonistic to itj in the second case,
both organisms repress each other. A one-sided antagonism may become
two-sided under certain conditions of culture. E. coli is antagonistic to
E. typhosa; however, if the latter is inoculated into a medium some-
what earlier than the former, E. tyfhosa becomes antagonistic to E. coli

(936).

Although the most common antagonisms are between organisms of
different species, there are numerous instances where one strain of a
species may be antagonistic toward another strain of the same species
(53j 370? 651)- Certain strains may develop antagonistic properties in
the presence of other strains (74). Nonflagellated variants of typhoid
bacteria were repressed by a flagellated form, smooth variants of para-
typhoid bacteria by rough forms, and so on. The fact that all bacterial
cultures stop growing after a certain period of time has been interpreted
to be a result of the antagonistic action of some cells upon others. When
the filtrates of such cultures are added to fresh nutrient media they may
stop the growth of the same species as well as that of other species.

Certain organisms produce pigments in the presence of others j these
pigments are believed to be in some way associated with the phenome-
non of antagonism. In the presence of S. lutea^ V. comma forms a
dark violet pigment that is accompanied by an increase in agglutination
and in virulence (670). The destruction of Dictyostelimn muco-
roides by a red-pigment-forming bacterium was accompanied by an in-
crease in intensity of the pigment (723)5 the blue pigment of Bac-
terium violaceumy however, only delayed the growth of the fungus.
Penicillium ajricanum produces a more intense pigment in contact with
other fungi such as Asfergillus niger; this pigment accumulates in the
mycelium of the latter, which may thereby be killed (186). P. luteum
and Sficaria furfurogenes produce a pigment that is used not only
for purposes of protection, but also for attack upon other organisms,
whereby the latter are killed and stained (669).

50 INTERRELATIONSHIPS AMONG MICROORGANISMS

THEORIES OF THE NATURE OF
ANTAGONISTIC ACTION

The various theories proposed to explain the mechanism of antago-
nistic effects of microorganisms may be summarized under the follow-
ing processes:

Exhaustion of nutrients

Physicochemical changes in medium

Pigment action

Action at a distance

Space antagonism

Enzyme action, either directly by the antagonist or as a result of cell

autolysis, under the influence of the antagonist
Production and liberation of antibiotic substances

Pasteur (710) ascribed the antagonistic effect that aerobic bacteria
have upon the anthrax organism to the consumption of the oxygen by
the former} the unfavorable influence of normal blood upon the growth
of anthrax was believed to be due to competition for the oxygen by the
red blood corpuscles. Freudenreich (298) considered the antagonism
between Ps. aeruginosa and Bacillus anthracis as due to exhaustion of nu-
trients by the former. These studies were soon followed by numerous
other investigations in which the exhaustion of nutrients in the media
was believed to be responsible for the phenomenon of antagonism j the
onset of the stationary phase in bacterial growth was believed (579) to
belong here. Change in ^H of medium, exhaustion of nutrients, and
accumulation of toxic products were also found to be limiting factors.

It thus became apparent, even in the early days of bacteriology, that
certain changes are produced by microbes in the medium in which they
grow which render it unfit for the growth of other organisms. It also
was soon recognized that the problem is more complicated than the
mere exhaustion of nutrients. The changes in relationship produced by
changes in surface tension, in oxidation-reduction potential, in reaction,
and in osmotic pressure were suggested as explanations. Among the
classical examples of the effect of reaction upon the growth of other
organisms is the acidification of milk by lactic acid bacteria. Metchnikov
emphasized the fact that Lactobacillus bulgaricus acts antagonistically

NATURE OF ANTAGONISTIC ACTION 51

not only by means of the lactic acid that it produces but also by the
formation of special substances. The production by bacteria of alkali-
reaction products that have an injurious effect upon the further growth
of the organisms has also been demonstrated (342). These substances
were found to correspond to amino compounds, liberated in the process
of cellular disintegration. Numerous other physical and physicochemi-
cal factors influence the growth of an organism in an artificial medium.
It is to be recalled that the rate of survival of bacterial cells in water or in
salt solution is markedly influenced by the colloids present, the con-
centration of electrolytes, the reaction, and the temperature.

Microbial antagonism was thus looked upon largely as a result of a
series of physical factors, including various radiations such as mytoge-
netic rays, -pH changes, conductivity, electric charge, and surface ten-
sion (525).

Most antagonisms, however, can be explained by the production of
antibiotic substances by the antagonists. Because of the thermolability
of some, sensitivity to chemical reagents, or adsorption on bacterial
filters, considerable difficulty has been experienced in isolating the
active substances. Many of these substances are iso-antagonistic, where-
as others are able to act upon different bacteria. Most of them have been
found to be thermostable.

The first antibiotic recognized as such was pyocyanase, produced by
Ps. aeruginosa (235). Other organisms that produce such substances
are Serratia marcescens (229), Ps. jluorescens {S^^)-, B. mesentericus
(1052), B. mycoides, B. subtilis, and other spore-forming bacteria.
Since the early work at the turn of the century and especially during
the last five or six years, many new antibiotics have been isolated or
demonstrated. These will be discussed in detail later.

The production of these antibiotics by microorganisms is greatly
influenced by reaction, temperature, and aeration of substrate, as well
as by the presence of other organisms. Evidence is still lacking as to
whether these substances may accumulate in the soil and in water,
whether the antagonized organisms are able to overcome their effect,
and whether they are destroyed by other members of the soil or water
microbiological population (365, 976).

Different organisms possess different degrees as well as different

52 INTERRELATIONSHIPS AMONG MICROORGANISMS

mechanisms of antagonism. Often one organism may completely check
the growth of another j later, growth may be resumed, although it will
not be quite normal. Antagonism stimulates spore-production and
brings about deformed growth of the mycelium in fungi or the forma-
tion of gigantic cells in bacteria. The morphological effects produced
by the antagonists comprise changes in form, size, and structure of
hyphae, direction of growth, complete cessation of growth, and ab-
breviation of hyphal segments.

CHAPTER 4

ISOLATION AND CULTIVATION OF

ANTAGONISTIC MICROORGANISMSj METHODS

OF MEASURING ANTIBIOTIC ACTION

In nearly all the earlier work and even in many recent investigations
on the antagonistic properties of microorganisms and the production
of antibiotic substances, two procedures were employed : indiscriminate
testing of pure cultures of bacteria and fungi, commonly taken from
culture collections, for antagonistic effects against one another or against
certain specific or test organisms} and isolation of occasional antagonistic
organisms from old plate cultures, as air contaminants, or from mixed
infections. These studies were carried out either by medical bacteri-
ologists interested in agents capable of suppressing bacterial pathogens
or by plant pathologists interested in organisms capable of inhibiting
the growth of fungi, principally those concerned in the causation of
plant disease. They resulted in the accumulation of considerable infor-
mation concerning antagonistic organisms, the nature of the phenome-
non of antagonism, and, to a more limited extent, the mechanisms in-
volved. Neither of these methods, however, is suitable for a systematic
study of antagonism as a natural process.

The last decade has witnessed a number of systematic attempts to de-
termine the distribution of antagonists in nature, to isolate specific or-
ganisms capable of bringing about the desired reactions, and to estab-
lish the mechanism involved in these reactions. These studies were
undertaken by a group of Russian investigators interested largely in
fungi and actinomycetes as agents antagonistic to other microorganisms
chiefly causing plant diseases, and by American and British investigators
interested in agents active against bacterial pathogens of man.

The early significant, but unrecognized, investigations of Schiller
(835) on forced antagonisms and the studies of Gratia and his asso-
ciates (356, 357) on mycolysates were in direct line of the more re-
cent studies of Dubos (201), who made a systematic attempt to isolate
from specially enriched soils bacteria capable of destroying specific

54 ISOLATION AND CULTIVATION OF ANTAGONISTS

pathogenic organisms. Although it had been previously established that
many spore-forming bacteria are capable of producing substances that
have antibacterial properties, as shown by the work of Pringsheim
(738), Much (664), and others, Dubos was the first to succeed in iso-
lating in crystalline form the active substances involved and in demon-
strating their chemical nature. He utilized for the isolation of the or-
ganisms the soil enrichment culture method. This consisted in adding
repeatedly various pathogenic bacteria to a soil in which, as a result,
antagonistic organisms developed that were capable of destroying the
bacteria i these organisms were then isolated by appropriate procedures.
These investigations, as well as the work of Fleming (261 ) later fol-
lowed by other British investigators (5) on the antibacterial properties
of molds belonging to the Penkillium notatum group, served as the di-
rect stimulus to numerous studies. The entire series of studies led to
the development of simple methods for the systematic isolation of
microorganisms capable of inhibiting the growth of fungi and bacteria,
both pathogenic and saprophytic, and for separating many of the anti-
biotic substances produced by these organisms.

METHODS OF ISOLATING ANTAGONISTIC
MICROORGANISMS

Several methods are now available for the isolation of antagonistic
microorganisms from natural substrates such as soil, stable manure,
composts, sewage, water, and food products. These methods are dif-
ferent in nature, but they are all based on the same principle, that of
bringing a living culture of a bacterium or fungus into close contact with
a mixed natural population, thereby allowing certain members of this
population to develop at the expense of the added culture.

Soil Enrichment Method

By this method a soil is enriched with known living pathogenic bac-
teria. Fresh garden or field soil is placed in glass beakers or pots, and
the moisture of the soil is adjusted to optimum for the growth of aerobic
bacteria, which is about 6s per cent of the water-holding capacity of the
soil (20 to 50 per cent of the moist soil) j the containers are covered

METHODS OF ISOLATION 5 5

with glass plates and placed in an incubator at 28° or 37° C. Washed
suspensions of living bacteria are added to the soil at frequent intervals,
care being taken to avoid puddling it with an excess of the fluid, so con-
ditions will not be made anaerobic. Samples of the enriched soil are
removed at intervals and tested for the presence of organisms antag-
onistic to the bacteria added. Fresh washed suspensions of the living
bacteria are inoculated with the enriched soil as soon as the presence of
antagonistic organisms is demonstrated j this results in the development
of the antagonistic organisms and the destruction of the bacteria in sus-
pension. Transfers are then made to fresh suspensions of the bacteria,
resulting in an enrichment of the antagonist, which can finally be iso-
lated in pure culture (201, 207, 442).

The significance of the soil enrichment method and its application to
the isolation of specific antagonistic organisms has been questioned
(969). It was suggested that whereas there is no question concerning
the multiplication of microorganisms capable of decomposing a given
substance or of secreting enzymes active upon such a substance in re-
sponse to its introduction into the soil, there is still doubt whether
specific antagonistic organisms develop as a result of the introduction
of living cells into the soil. The reason for this was based upon the fact
that antibiotic reactions produced by antagonistic organisms do not
affect bacteria by simple digestive or oxidative mechanisms.

Bacterial A gar Plate Method

This method was first used by Gratia and Dath (357) for the isola-
tion of antagonistic agents, actinomycetes having been found readily
by it.

To isolate antagonistic bacteria, agar (1.5 per cent) is washed in dis-
tilled water, then dissolved in water supplemented by i per cent glucose
and 0.2 per cent K0HPO4. Ten-milliliter portions of the sugar-
phosphate agar are placed in glass tubes and sterilized. The sterile agar
is melted, and the tubes are placed in a water bath kept at 42° C. A
washed, centrifuged suspension of living bacteria, grown on solid or in
liquid media, is then added and thoroughly mixed with the agar. This
"bacterial agar" is poured into a series of Petri plates containing one-
milliliter portions of fresh or enriched soil, diluted i : lOO to i : 10,000

56 ISOLATION AND CULTIVATION OF ANTAGONISTS

times with sterile water. The contents of the plates are thoroughly
mixed in order to distribute the diluted soil suspension in the bacterial
agar. The plates are inverted and incubated at 28° or 37° C.

After I to 10 days' incubation, depending on the nature of the or-
ganism used for the preparation of the plates, the presence of antago-
nists is manifested by the formation of clear zones surrounding their
colonies (Figure 2). The organisms are isolated from these colonies
and are retested for antagonistic properties, either by transfer to fresh
bacterial agar plates or by inoculating solidified agar plates and cross-
streaking with test organisms (956, 978).

In the isolation of antagonistic fungi the same method is followed,
except that it is preferable to make the bacterial agar acid by using
KH2PO4 in place of K2HPO4. The resulting acidity (pH 4.5) inhibits
the growth of bacteria and actinomycetes. Since the soil contains fewer
fungi than bacteria, lower dilutions of soil are employed for this pur-
pose (1:10 to 1:1,000).

This method, like the soil enrichment method, does not always yield
desirable results. As shown in Table 6, some of the most important
antagonists, such as Ps. aeruginosa, S. antibioticus, A. jiavus, and P.
notatum-y do not develop on such a plate since they cause only limited
lysis of bacteria. On the other hand, B. brevis, S. griseus, A. fumigatus,
and A. clavatus cause extensive lysis of gram-positive bacteria and so
can readily be isolated.

Crowded Plate Method

Ordinary field or garden soil is plated out on common nutrient (beef-
peptone) agar, very low dilutions (1:10 to 1:1,000) being used to
enable a large number of bacterial colonies to grow on the plate. The
resultant crowding of these colonies allows the development on the
plate of potential antagonists that are normally present in the soil. The
production of antibacterial substances by these antagonists inhibits the
growth of bacteria in close proximity to them and, in consequence, clear
zones are formed around the colonies (Figure 3). It is possible, by
means of this method, to demonstrate that many strains of spore-form-
ing bacteria possessing antagonistic properties are present in the soil and
can readily be isolated from it.

METHODS OF ISOLATION

57

TABLE 6. GROWTH OF ANTAGONISTIC ORGANISMS ON BACTERIAL WASHED
AGAR MEDIA AND LYSIS OF BACTERIA

ANTAGONISTIC
ORGANISM

Bacteria:
B. brevis
B. simflex
Ps. aeruginosa

Actinomycetes:
5. antibioticus
S. griseus
S, lavendulae
Micromonosfora sp.
N. gardneri

Fungi:

A. clavatus
A. flavus
A. fumigatus
Glioclaiium sp.
P. notatum

MEDIUM CONTAINING WASHED CELLS OF

E. coli S. lutea B. subtilis

Growth Lysis Growth Lysis Growth Lysis

O O

o o

O O

O O

O o

O O

O O

O O

h+ o

H- O

h+ O

h+ o

H- O

From Waksman and Schatz (969).

Note, o indicates no growtii of antagonist or lysis of test bacterium as shown by formation of

clear zone on plate; ± indicates trace; + to I I I I indicates increasing amounts of growth or lysis.

Direct Soil Inoculation Method

Nutrient agar plates are inoculated with the bacteria or fungi for
which antagonists are to be found, and the plates are incubated for 24
to 48 hours at 28° or 37° C. Particles of fresh or enriched soil placed
on the surface of the bacterial or fungus growth on the plate will give
rise to antagonistic organisms that will bring about the killing or even
the lysis of the original culture. By this method, organisms antagonistic
to many bacteria and fungi causing plant and animal diseases have been
isolated (683,685).

For the isolation of bacteria antagonistic to fungi, the latter are
grown on potato agars until they have spread over the plate j particles
of moist soil are then placed on the surface of the mycelium, and the
plates are incubated in a moist chamber. Bacteria lysogenic to the fungi

58 ISOLATION AND CULTIVATION OF ANTAGONISTS

grow out of the soil and gradually dissolve the mycelium until the en-
tire surface of the plate becomes free of the hyphae of the fungus. By
transferring some of the material from the lysed spots, pure cultures of
bacteria have been obtained that are capable of producing destructive
effects upon the fungi, similar to the action of the particles of soil.

To these four methods may be added the "forced antagonism"
method of Schiller (835), previously referred to, which consists in feed-
ing a culture of an organism with another one, thereby forcing the sec-
ond to develop the capacity of destroying the first.

By means of the foregoing methods, as well as various modifications
of them, it was possible to demonstrate that soils, composts, and water
basins contain an extensive population of microorganisms possessing
antibacterial and antifungal properties. When E. coli was used as the
test organism, it was found that although this organism was capable not
only of surviving but actually of multiplying in sterile soil, it died off
very rapidly when added to fresh soil. The rate of its destruction was
greatly increased with every subsequent addition of washed bacterial
cells to the soil. This was accompanied by the development of certain
antagonistic microbes capable of destroying E. coli in pure culture.

A large number of fungi, actinomycetes, and bacteria possessing an-
tagonistic properties have thus been isolated. The nature of the test or-
ganism was found to be of great importance in this connection. When
Stafhylococcus aureus , S. luiea, and B. subtilis were used, a large num-
ber of antagonists could readily be isolated. With E. coUy however, a
much smaller number of microbes thus isolated possessed antagonistic
properties. Certain other gram-negative bacteria, like Brucella abortuSy
were more sensitive than E. coli, whereas certain gram-positive bac-
teria, like B. mycoides and B. cereus, were less sensitive than B. subtilis
(956,958).

Bacteria destructive to fungi, or possessing fungistatic and fungicidal
properties, have also been isolated from soils as well as from the surface
of plants, such as flax, by transferring small sections of soil or plant
stem to plates of fungi growing on potato agarj transfers made from
the lytic spots yielded antagonistic bacteria (686). By the use of this

Figure 2. Development of antagonistic fungi on bacterial-agar plate. From
Waksman and Horning (956).

Figure 3. Bacterial plates made from soil, showing clear zones surround-
ing colonies of antagonistic organisms. From Stokes and Woodward (885).

Antagonistic action of .S. ant'i-
b'toticus upon S. lutea

Antagonistic action of S. ant'i-
btoticus upon B. myco'ides

Bacteriostatic action of actino-
mycin upon 5. lutea

Bacteriostatic action of actino-
mycin upon B. myco'tdes

Figure 4. Antagonistic effects of living organisms and their products. From
Waksman and Woodruff (974).

METHODS OF TESTING ANTAGONISTIC ACTION 59

method, Chudiakov (143) isolated various bacteria antagonistic to
Fusarium. The antagonists were found abundantly in cultivated soils,
but not in flax-sick soils rich in Fusarium. Bamberg (35) demonstrated,
in the soil, bacteria capable of bringing about in 10 days complete de-
struction of Ustiliago zeae and other fungi. Myxobacterium was also
found (473) capable of bringing about the disintegration of fungus
mycelium. Nonspore-forming bacteria, similar to the cultures of
Chudiakov, were isolated and shown to be able to attack a number of
fungi, including species of Fusarium, Sclerotinia, Gleosforium, Acro-
stalagmus, Alternaria, and Zygorhynchus (729).

METHODS OF TESTING THE ANTAGONISTIC
ACTION OF MICROORGANISMS

Once antagonistic organisms have been isolated, it is essential to es-
tablish their bacteriostatic spectrum — that is, their ability to inhibit the
growth of various specific microorganisms. Usually these antagonists
do not affect alike all bacteria and fungi, some acting primarily against
gram-positive bacteria and against only a few gram-negative forms
(mostly cocci), others acting upon certain bacteria within each of these
two groups.

A considerable number of methods have been developed for meas-
uring these antagonistic effects. They measure the selective nature of
the antagonistic action and they can also give quantitative information
concerning the intensity of this activity. Because of the great differences
in the degree of sensitivity of bacteria to the action of the antagonists,
the proper selection of one or more test organisms is highly essential.
S. aureus has been employed most commonly, different strains of this
organism having been found to vary greatly in their sensitivity even to
the same substance. Stre-ptococcus viridans, B. subtilis, Micrococcus ly-
sodeikticus, S. luiea, E. coli, and E. tyfhosa are other organisms that
are frequently employed for testing the activity of antagonists. Al-
though for purposes of concentration and purification of a known sub-
stance a single test organism is sufficient, it has been found advisable
during the isolation of antagonistic organisms and the study of the na-

60 ISOLATION AND CULTIVATION OF ANTAGONISTS

ture of the antibiotic substance or substances that they produce to use
more than one test bacterium, including one or more gram-positive and
one or more gram-negative bacteria.

Most of the methods for testing antagonistic action are based upon
the growth of the test organisms in the presence of the living antago-
nists or of the antibiotic substances produced by them in liquid and on
solid nutrient media. Only a few of these methods are now utilized,
most of them being chiefly of historical interest.

Liquid Media

Several methods using liquid media have been proposed for testing
the antagonistic activities of microorganisms:

Simultaneous inoculation of the medium with the antagonist and the test
organism.

Inoculation of the medium with the antagonist first, followed after 6 to
48 hours by inoculation with the test organism.

Inoculation of the medium with the test organism first, followed, after a
certain interval, by the antagonist.

Effect of the metabolic products of the antagonist upon various micro-
organisms. In 1888, Freudenreich (298) first filtered the culture
through a Chamberland candle and inoculated the filtrate with the
test organisms. The culture filtrate is usually added to the fresh me-
dium, either previously inoculated with the test organism for the
purpose of establishing the lytic effect of the filtrate, or followed by
the test organism, whereby the bacteriostatic action is measured.

Placing a porcelain filter or cellophane membrane between the cultures
of the antagonist and of the test organism. Frankland and Ward
(295) used a filter of the Pasteur-Chamberland type partly filled
with broth and placed in a beaker containing the same kind of broth;
the antagonist and test organism were inoculated into the two lots of
broth, and the effect of each upon the growth of the other was de-
termined. Frost (303) emphasized, however, that, although theo-
retically this is an ideal method, it is open to criticism since motile
bacteria are usually able to grow through the filter after a certain
lapse of time.

Collodion sac method. Collodion sacs, prepared by means of test tubes
from which the bottoms have been cut out, are partly filled with

METHODS OF TESTING ANTAGONISTIC ACTION 61

broth and placed in a flask containing the same kind of broth. The
test organism is inoculated into the medium inside the sac, and the
antagonist into the flask (303).

Solid Media

Solid media have also been used extensively for testing the action of
antagonists. These media offer certain advantages over liquid media.
The following methods are most commonly used:

Simultaneous inoculation of antagonist and test organism. This method,
introduced by Garre (315) in 1887, consists in streaking the an-
tagonist and the test organism on the surface of a solidified agar or
gelatin medium. The streaks are alternate and may be parallel, radi-
ating from a common center, or intersecting at right angles (Fig-
ure 4). If the active substance produced by the antagonist does not
diffuse for any considerable distance into the medium, the method is
not satisfactory. Frost (303) modified this method by inoculating
the whole medium with the test organism and, when the medium
had hardened, streaking the antagonist across the surface. The first
of these came to be known as the anaxogramic method ; the second
is often spoken of as the implantation method. The spotting of the
two organisms on the plate is illustrated in Figure 5.

Successive inoculation of the test organism, after the antagonist has al-
ready made some growth, so as to enable the active substance to dif-
fuse.

Double plate methods (303). A Petri dish is divided into two parts by
means of a small glass tube or rod. After sterilization, one tube of
molten agar is heavily inoculated with the antagonist and poured
into one half of the plate. When the agar has hardened, another tube
of sterile agar is poured into the other half of the plate. Both sides are
then streaked with the test organism, each side being equally inocu-
lated by separate streaking. This can be done by using a loop bent at
nearly right angles; the charged loop is moved from the circumfer-
ence toward the glass rod. The loop is then sterilized, recharged with
the test culture, and the streak continued on the other side of the
plate. The inoculation with the test organism may be made soon

( after the plate is poured, or the antagonist may be given an opportu-
nity to develop before the test organism is streaked thus making the

62 ISOLATION AND CULTIVATION OF ANTAGONISTS

Uelminthosforium (A and B) in- Pestalozzia (A) inhibited by one
hibited by Fusarium (C) species of Penic'tlUum (C) but not

by another (B)

Helminthos for turn (A) inhibited Helminthosfortum (A and B) in-
by a bacterium (C) hibited by a white yeast (C)

Figure 5. Inhibition of fungus development by antagonists. From Porter
(729)-

METHODS OF TESTING ANTAGONISTIC ACTION 63

antagonistic effect more striking. This method has also been used
(258) for testing the antibiotic properties of fungus cultures.

Mixed culture inoculation. The cultures of the antagonist and the test or-
ganism are mixed and inoculated upon the surface of the solidified
agar or before the molten agar has been added to the plate. The colo-
nies of the antagonist will be surrounded by clear sterile zones, free
from any growth of the test organism.

Spot inoculation of the antagonist upon an actively growing culture of a
bacterium or fungus on an agar plate. This method is particularly
convenient for detecting antagonists that possess lytic properties.

A layer of molten sterile agar is used to cover the surface of an antagonist
that has made some growth in a plate, and the surface of the agar
layer is then inoculated with the test organism. The active substance
produced by the antagonist will diffuse through the agar and reduce
the growth of the test bacterium (609).

Semisolid media are used for testing the action of antagonists upon
the motility of bacteria (182).

A number of other methods, usually modifications of those outlined
above, have been used for testing the ability of fungi to produce anti-
biotic substances (724, 1016). Some of these methods, notably the agar
diffusion (cup, paper disc, cylinder) test, are used for the quantitative
estimation of the concentration of the antibiotic in the medium and for
isolation purposes. These methods can indicate the formation not only
of growth-inhibiting but also of growth-promoting substances (99).

Raper et al. (765) removed plugs of agar of constant dimensions
from the fungus cultures being tested and placed them on the surface
of plates seeded with S. aureus. The plates were incubated at 37° C,
and the amount of penicillin present was estimated by the size of the
zones of inhibition. For the purpose of screening many cultures, a
modified Czapek's solution agar, i per cent by volume of corn steep
liquor {^$^ per cent solids) was used} the solution was adjusted to /jH
7.0, and 2 per cent agar was added. Twenty-milliliter portions were
placed in tubes, sterilized, and poured into sterile Petri dishes. The
plates were selected to insure that the agar layers were of uniform
depth. Single colonies were established by suspending spores of the cul-
ture to be tested in melted agar at 45 ° C. The agar was allowed to so-

64 ISOLATION AND CULTIVATION OF ANTAGONISTS

lidify and small amounts were placed with an inoculating needle in the
centers of the agar plates. The plates were incubated at 24° C. for 6
days J then 4 or 5 plugs were removed radially from the agar, the first
being adjacent to the colony margin, and tested as described above

(838).

Various other methods have been proposed for measuring the rate of
production or secretion of antibiotic substances by fungi (726, 963).

METHODS OF GROWING ANTAGONISTIC

ORGANISMS FOR THE PRODUCTION

OF ANTIBIOTIC SUBSTANCES

Once the antagonistic action of any organism has been established,
the next step is to determine the nature of the substance produced and
to measure quantitatively its antibiotic action. Before this can be done,
however, the organism must be grown upon suitable media under
conditions favorable for the maximum production of the antibiotic
substance.

The media used for the production of antibiotics can be classified into
two groups: synthetic and complex organic media. The first contain a
source of carbon, usually glucose, sucrose, or starch (2 to 6 per cent) j a
source of nitrogen, usually nitrate or ammonia sulfate (0.2 to 0.6 per
cent) ; several salts, namely, K0HPO4 orKHoP04 (o.i to 0.2 percent),
MgS04.7H20 (0.05 per cent), KCl (0.05 per cent), and FeS04.7H20
(0.00 1 per cent). Certain supplementary materials such as yeast ex-
tract, meat extract, or corn steep, or trace elements such as ZnS04,
MnS04, or CUSO4 (i to 2 ppm.) may also be added. The organic
media contain a complex form of nitrogen, such as tryptone, peptone,
casein digest j either no other source of carbon is used or a carbohydrate
is added in the form of lactose, glucose, dextrin, starch, brown sugar,
molasses, or similar products as well as several salts similar to those
listed above. Some media are supplemented with CaCOo, others are
not, depending upon the extent of acidity produced by the organism.

The medium may be solid (agar or bran) or liquid, the latter being
the more common. Several types of culture vessels are used, depending
on the condition of aeration. Since so far as is known all the micro-

METHODS OF GROWING ANTAGONISTIC ORGANISMS 65

organisms capable of producing antibiotic substances are aerobic, either
shallow layers of medium ( 1.5 to 2 cm. in depth) are placed in station-
ary vessels (flasks or trays), or shaken cultures are used. In the case of
deep vessels or tanks, the medium is properly stirred and aerated by
forced draft with sterilized and filtered air.

The optimum temperature required for the growth of the antagonis-
tic organisms and the production of antibiotic substances ranges be-
tween 20° and 30° C. The length of incubation varies from 2 to 6 days
for submerged cultures and from 3 to 20 days for stationary cultures.

A knowledge of the preliminary treatment of the inoculum or spore
material is essential. For the growth of spore-forming bacteria, the use
of a pasteurized spore suspension is advisable in order to avoid the vari-
able factor due to vegetative cells. Actinomycetes and fungi are grown
on agar slants in order to obtain abundant spore material for the inocu-
lation of stationary cultures. For submerged cultures, special spore sus-
pensions are produced by growing the organisms in shaken cultures.

The cultures must be tested carefully in order to establish the opti-
mum activity when the culture filtrate is cooled and extraction of active
substance is started.

Tyrothrkin

For the production of tyrothricin, shallow layers of medium are used
most frequently. The media contain complex sources of nitrogen, such
as tryptone, casein hydrolysate, soybean meal digest, and pressed juice
of waste asparagus. Simple substances, such as glutamic acid, aspara-
gine, ammonium salt, plucid citric or malic acid, are also effective in
presence of 0.2 per cent tryptone. Glucose, mannitol, or glycerol (3
to 5 per cent) can be used as the source of carbon, and calcium, magne-
sium, and manganese as required mineral. Maximum yields of more
than 2 gm. per liter are obtained in 10 to 16 days' incubation at 35° C.
(564).

Penicillin

^ For the production of penicillin, the composition of the medium is
highly important. At first a simple glucose-nitrate solution known as
Czapek-Dox medium was used. It was later found that when yeast ex-

66 ISOLATION AND CULTIVATION OF ANTAGONISTS

tract or corn steep liquor was added and brown sugar was used in place
of glucose, the growth of the organism and the production of penicillin
were greatly facilitated (5, 281). The ratio of C and N sources is sig-
nificant. It has been shown, for example, that penicillin is produced
in organic media when the ratio sucrose-peptone is less than i .0, and in
inorganic media when sugar-NaNOg ratio is i.o or lo.o (591).

The following was found (838) to be a suitable medium for maxi-
mum production of penicillin :

Lactose 40.00 gm.

NaNOg 3.00 gm.

MgS04.7H20 0.25 gm.

KH0PO4 0.50 gm.

ZnSOg 0.0 1 gm.

Corn steep liquor 90 ml.
Distilled water to make 1000 ml.

This medium has been variously modified, as by reducing the lactose
to 20 mg. per liter and the corn steep to 40 ml. or by using in its place
25 mg. of dried steep liquor solids (764).

The need for a specific penicillin-promoting substance, such as might
be found in corn steep or in other plant extracts (555)j in order to in-
crease appreciably the yield of the antibiotic agent is of particular in-
terest. Certain amino acids, namely, arginine, histidine, and glutamic
acid, in concentrations of 0.3, 0.3, and 0.4 gm. per liter, respectively,
appear to provide a large part of this stimulating effect (1004). A
proper balance of the concentration of the ions POf , SOf , NO -3 ,
and Mg+-^ is also essential. The proportions of the essential three salts
in optimum solution were found (733) to be KH2PO4 — O.475,
MgS04.7HoO— 0.05, and NaN03— 0.475 j different strains show
marked differences in their response to a change in balance of these
three salts.

This led to the development of different synthetic media, such as the
following (735) J the amounts are given on a liter basis:

Starch 5.0 gm.

Lactose 25.0 gm.

Glucose, crude 5.0 gm.

METHODS OF GROWING ANTAGONISTIC ORGANISMS 67

Glacial acetic acid

6.0 gm.

NaoHPO^

1.6 gm.

K,PO,

2.0 gm.

NH4NO3

4.0 gm.

(NH,),S03

i.O gm.

KNO3

i.O gm.

MgSO^.yHoO

0.25 gm.

FeS04.7H20

0.2 gm.

MnS04.7HoO

0.04 gm.

CUSO4.5H..O

0.005 gin-

Cr (as KoCroO,)

3 Mg

Phenylacetic acid and its derivatives have a marked effect upon peni-
cillin yields 5 frequently amide derivatives are just as effective as the
corresponding acids (887).

With the introduction of the submerged process for the production
of penicillin, it became necessary to find a simple means of obtaining
large numbers of spores. For this purpose, a medium high in calcium
salt appears to be essential (282). Such a medium is as follows:

Sucrose or brown sugar 20.0 gm.

NaNOo 6.0 gm.

KH2PO4 1.6 gm.

MgS04.7HoO 0.5 gm.

CaClo 25.0 gm.

Tap or distilled water to make lOOO ml.

The culture is grown for 4 to 6 days with continuous aeration and agi-
tation.

Various other methods are used for spore production, for the purpose
of inoculating large batches of medium. For surface growth, dry spores
are mixed with a floating and spreading agent, such as whole wheat
flour.

Other media, such as bran (762), have been utilized to a limited
extent for the production of penicillin. However, the submerged proc-
ess, accompanied by agitation and aeration, using one of the above
liquid media has now come into general use for large-scale production
of penicillin.

68 ISOLATION AND CULTIVATION OF ANTAGONISTS

Streftothrkin and Streftomycin

For the production of streptothricin, a tryptone medium with starch
or glucose is used. A typical medium is given here:

Glucose or

starch

10.00 gm.

Tryptone

5.00 gm.

K2HPO4

2.00 gm.

NaCl

2.00 gm.

FeSO^

0.0 1 gm.

Tap water to make

1000 ml.

For stationary cultures, 0.25 per cent agar may be added.

For streptomycin, certain specific organic precursors are required.
The precursors are present in meat extract, in corn steep, and in the
cells of certain microorganisms such as yeasts and actinomycetes. A typi-
cal medium, on a liter basis, consists of:

Glucose

1 0.0 gm.

Peptone

5.0 gm.

Meat extract

5.0 gm.

NaCl

5.0 gm.

Final fn

6.S to 7.0

Tap water to

make

1000 ml.

For spore production, a simple synthetic medium may be used, such
as glucose-asparagine agar, consisting of:

Glucose

lO.O gm.

Asparagine

0.5 gm.

K2HPO4

0.5 gm.

Agar

15.0 gm.

Distilled water to

make

1000 ml.

A synthetic medium has also been suggested (905a) for streptomy-
cin production, consisting of:

Glucose

7.4 gm.

Ammonium lactate

5.4 gm.

KH,P04

2.38 gm.

METHODS OF MEASURING ANTIBIOTIC ACTIVITY 69

K0HPO4 S-65 gm.

MgS04.7HoO 0.98 gm.

ZnS04.7H20 o.oi 1 5 gm.

FeS04.7HoO o.oiii gm.

CUSO4.5H.O 0.0064 gm.

MnClo^HoO 0.0079 gm.

Distilled water to make lOOO ml.

fU 6.95

METHODS OF MEASURING THE ACTIVITY OF
ANTIBIOTIC SUBSTANCES

It has long been recognized that the evaluation of bacteriostatic and
bactericidal substances is controlled to a considerable extent by the
methods employed. These methods are based upon the following fac-
tors: (a) proper selection of the test organism, (b) composition of the
medium used for testing activity, (c) time of action, (d) conditions of
carrying out the test, and (e) nature of the active substance. The results
obtained in a comparison of substances containing the same active prin-
ciple may not be very reliable when different agents are compared, since
these vary greatly in their specific action upon different bacteria. This is
especially true of antibiotics.

In most of the work on chemical disinfectants, which are primarily
bactericidal agents, the death rate of the viable cells has been used as a
basis for evaluation. Different substances have been compared with a
standard, ordinarily phenol. Since antibiotic and chemotherapeutic
substances are primarily bacteriostatic in action, the inhibition of the
growth and multiplication of the test organism is commonly used as a
basis for their evaluation.

In any attempt to select a single standard method for measuring
quantitatively the activity or potency of an antibiotic substance, it is es-
sential to recognize several pertinent facts, which may be briefly sum-
marized as follows:

(Antibiotic (antibacterial, antimicrobial) substances are primarily bac-
teriostatic (or fungistatic) in their action; some substances are also
markedly bactericidal (or fungicidal).

70 ANTIBIOTIC ACTION OF ANTAGONISTS

Antibiotic substances are selective in their action ; they are able to inhibit
the growth of some bacteria in very low concentrations, whereas
much larger amounts are required to affect other bacteria and some
organisms may not be inhibited at all by the particular substance even
in very high concentrations.

Conditions for the bacteriostatic activity of different antibiotic substances
vary greatly. Some substances are not active at all, or their activity
is greatly reduced in some media because of the neutralizing effect of
certain constituents of the media, such as peptone, />-amino-benzoic
acid, or glucose. Other agents require the presence in the medium of
specific constituents for their activity to become effective. The activ-
ity of some is reduced at an acid reaction, whereas that of others
is not affected.

The mechanism of the action of different antibiotic agents is different.
Some agents interfere with bacterial cell division, others with bac-
terial respiration, and still others with utilization by the bacteria of
essential metabolites.

Many antagonistic organisms produce more than one antibiotic substance.
Ps. aeruginosa produces pyocynnase and pyocyanin; B. brevisy grami-
cidin and tyrocidine; P. notatuni, penicillin and notatin; A. fumiga-
tuSy spinulosin, fumigatin, fumigacin, and gliotoxin; A. flavus, asper-
gillic acid and penicillin. The culture filtrate of an antagonistic or-
ganism often differs, therefore, in its activity from that of the
isolated active substance.

The course of production of antibiotic substances by two typical antago-
nistic organisms is illustrated in Figures 6 and 7.

In view of the bacteriostatic nature of antibiotic substances, few of
the methods commonly used for testing the efficiency of antiseptics and
germicides can be employed. This is particularly true of the "phenol
coefficient test," which measures the germicidal action of phenol upon
E. tyfhosa. The limitations of this method, based on the bactericidal ac-
tion of a single substance on a single organism, even as applied to chemi-
cal antiseptics have long been recognized (810).

A number of methods have been developed for determining the ac-
tivity of antibiotic substances. They vary greatly, each having its limita-
tions and advantages. Because of lack of uniformity in the methods, the
results obtained by one are not always comparable with those obtained

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ANTIBIOTIC ACTION OF ANTAGONISTS

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INCUBATION PERIOD IN HOURS

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Figure 7. Production of gliotoxin by Trlchoderma. From Weindling
(989).

by another. The most important methods at present in use are briefly
summarized in the following pages (583).

A gar Streak-Dilution Method

If an unknown antibiotic substance is tested, it is essential to employ
several test organisms in order to throw light upon the selective activity
of the substance on different bacteria, and thus to determine the anti-
biotic spectrum characteristic of each substance. Nutrient agar media
have usually been employed. Sterility is not absolutely essential for
this method, although it is desirable. The unknown substance is diluted
to various concentrations (i, 0.3, o.i, etc.; or i, 0.5, 0.25, etc.) ; these
dilutions are added and thoroughly mixed with definite volumes
(10 ml.) of sterile agar medium, melted and cooled to 42° to 45° C.
The agar is allowed to solidify, and is streaked with three or four test
bacteria, among the most common of which are E. coli, B. mycoides, B.
suhtilisy S. aureus, M. lysodeikticus, S. lutea, M. fhlei, as well as
various other bacteria and fungi. The age of the cultures (16 to

METHODS OF MEASURING ANTIBIOTIC ACTIVITY 73

24 hours) is important. The plates are incubated at 28° or 37° C. for
16 to 24 hours, and readings are made. The highest dilution at which
the test organism fails to grow is taken as the end point. Activity is ex-
pressed in units, using the ratio between the volume of the medium and
the end point of growth or the dilution at which growth is inhibited

(964).

The bacteriostatic and fungistatic activity of several antibiotic sub-
stances is shown in Table 7.

Serial Dilution Method

Once a substance is characterized as regards its selective action upon
specific bacteria, its activity or concentration can be measured more ac-
curately by the liquid dilution or titration method. One test organism is
selected, usually a strain of S. aureus. Different strains may vary in their
action. Definite volumes of the test medium are placed in test tubes and
sterilized (sterility is essential in this method), and various dilutions of
the active substance are added. The dilutions can range in order of 3 :i,
2:1, or even narrower, namely in series of 1.2:1, 1.5:1, etc. The tubes
are inoculated with the test organism and incubated for 16 to 24 hours.
In some cases the medium is inoculated before it is distributed into the
tubes. The highest dilution of the antibiotic giving complete inhibi-
tion of growth, as expressed by a lack of turbidity of medium, is taken
as the end point. Activity is expressed in units as above.

The dilution method has several disadvantages: every assay takes
much time 5 during chemical fractionation, the substance may become
contaminated with bacteria not sensitive to the active substances 5 only
one organism can be used in a single series of tests.

One modification of the method has been adapted for measuring the
activity of penicillin. Several dilutions of the active agent are prepared
and 0.5 ml. portions added to 4.5 cm. quantities of liquid medium in
test tubes. These are inoculated with a standard drop (0.04 ml.) of a
24-hour culture of the test organisms. Complete or partial inhibition is
shown by the absence of turbidity after 24 hours of incubation at 37° C.
Dilutions higher than those required for complete or partial inhibition
gave, after 24 hours of incubation, only a retarding effect (1,5)5 ^ "^i"
croscopic examination (311) indicated defective fission of the bacteria,

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METHODS OF MEASURING ANTIBIOTIC ACTIVITY 75

even though the macroscopic appearance of the culture did not show any
inhibition. Pneumococci and S. viridans show marked strain differences
by this method. In one experiment with Salmonella tyfhiy partial in-
hibition was obtained in a dilution of i : 1 0,000 j however, elongation
of the cells was detected in a dilution of i : 60,000, a concentration
which was considered as a therapeutic possibility (Table 8).

The other modifications of this method either use different test cul-
tures, such as B. sub tilts (285), or have been developed to meet the re-
quirements of the clinician when only small amounts of blood or other
body fluids are available, in which case a very sensitive strain of S.
hemolyticus is used (751). The use of Klebsiella fnemnoniae for as-
saying streptothrycin and streptomycin permits the determination of as
little as 0.05 Mg/ml., giving somewhat more rapid and more accurate
results (191).

Agar Diffusion {Cuf, Pafer Disc, Cylinder) Method (5, 173, 283,
285, 390)

This method, first employed for measuring antiseptics qualitatively
(810), was later developed for quantitative use. A suitable agar me-
dium is inoculated with a test organism {S. aureus or B. subtilis), the
active agent being placed upon the agar within a groove or in a special
small glass cup with an open bottom from which the substance diffuses
into the medium. The rate of diffusion of the antibiotic is parallel to its
concentration. Potency can be calculated by measuring the zone of in-
hibition and comparing it with that of a known standard preparation.
Various modifications of this method have recently been introduced
(286, 937). This method has the advantage of simplicity and con-
venience, since it does not require sterile material and several prepara-
tions or duplicates can be tested on the same plate. The method also
possesses certain disadvantages, however, since it cannot be used for
comparing different substances but is limited to the measurement of
activity of only one type of substance; it cannot be used for the study
of unknowns until a standard has been established for each j it cannot
be used for substances that are not water soluble.

Nutrient agar containing 5 gm. NaCl, 3 gm. meat extract, 5 gm.

76 ANTIBIOTIC ACTION OF ANTAGONISTS

TABLE 8. BACTERIOSTATIC SPECTRUM OF PENICILLIN

DILUTIONS AT WHICH INHIBITORY

ORGANISM AFFECTED EFFECTS WERE OBSERVED

Complete Partial None

N. gofiorrhoeae^ 2,000,ooo > 2,000,000 > 2,000,000

"N . meningitidis 1,000,000 2,000,000 4,000,000

S. aureus 1,000,000 2,000,000 4,000,000

S.fyogenes 1,000,000 2,000,000 4,000,000

B.anthracis 1,000,000 2,000,000 4,000,000

A.bovis 1,000,000 2,000,000 4,000,000

CI. tetani\ . i ,000,000

CI. welchii 1,500,000

CI. sefticum 300,000 1,500,000 7,500,000

CI. oedematiens 300,000 1,500,000

S.viridansX 625,000 3,125,000

Pnetimococcus\ 250,000 500,000 1,000,000

C. difhtheriae (miiis) 125,000 625,000

C. difhtheriae {gravis) 32,000 64,000 128,000

S. gartneri 20,000 40,000 8 0,000

S.tyfhi 10,000 30,000 90,000

Pneu7nococcus\ 9,000 27,000

Anaerobic Streptococcus^ 4,000 8,000 16,000

P. vulgaris 4,000 32,000 6o,000

S. viridans% 4,000 8,000 1 6,000

P.festis 1,000 100,000 500,000

S. iyfhimurium <^ 1,000 8,000 1 6,000

S.faratyfhiB • <I,000 5,000 10,000

Sh. dysenteriae 2,000 4,000 8,000

Br. abortus 2,000 4,000 8,000

Br.melitensis <i,ooo 2,500 10,000

Anaerobic streptococcus <(4,000 <(4,ooo 4,000

V. comma <^ 1,000 1, 000 2,000

E.coli <i,ooo <i,ooo 1,000

K. pieumoniae <^ i ,000 < i ,000 i ,000

Ps. aeruginosa < 1 ,000 < 1 ,000 i ,000

M. tuberculosis <i,000 < 1,000 1,000

L. icterohaemorrhagiae <^ 3,600 <^ 3,600 35600

From Abraham et al. (5). Crude penicillin preparation was used.

•Another strain was inhibited only up to 32,000.

t Grown in Lemco broth; in beef broth complete inhibition reached only 100,000.

X In Pneumococcus, S. viridans, and anaerobic streptococci, different strains appear at different

levels in the table.

METHODS OF MEASURING ANTIBIOTIC ACTIVITY 77

peptone, 15 gm. agar, 1,000 ml. tap water, and adjusted to fH 6.8, is
poured into plates to a depth of 3 to 5 mm. The plates are seeded thor-
oughly with the test organism (S. aureus) by flooding with i: 10 or
1:50 dilution of i6-to-24-hour-old broth culture in sterile water. The
excess fluid may be removed with a pipette. The surface of the agar is
allowed to dry somewhat in the 37° C. incubator for i to 2 hours, the
lids of the plates being raised about i cm. above the bottoms of the
dishes. Sterile short glass cylinders (5 mm. inside diameter) are placed
on the agar, the lower edge of the cylinder sinking into the agar, and are
filled with the test solution. Several cylinders may be placed in one dish.

For measuring the activity of penicillin, the plates are incubated for
12 to 16 hours at 37° C. The diameter of the clear zone around the
cylinder is measured with pointed dividers to the nearest 0.5 mm. The
relation of concentration of penicillin in the solution to the zone of in-
hibition, or the "assay value," is expressed by a curve which is obtained
with standard solutions. This curve tends to flatten out above 2 units of
penicillin per milliliter. The assay value is not influenced by the -pH of
the test material, thickness of the agar, or sterility of the material.

The Oxford unit (O.U.), as determined by this method, is the
amount of penicillin that will just inhibit completely the growth of the
test strain of S. aureus in 50 ml. of medium. Thus, a preparation con-
taining one unit of penicillin per milligram of material just inhibits the
growth of the test organism in a dilution of 1 150,000.

An international standard for penicillin, based upon crystalline ma-
terial, has been adopted.

In one of the modifications of the agar diffusion method, a spore sus-
pension of B. suhtills is used as the test organism. It is grown for sev-
eral days under forced aeration, and the cultures are pasteurized in or-
der to destroy the vegetative cells. The spore suspension is stored in the
cold and used as the stock inoculum j it is titrated in order to determine
the optimum amount for seeding purposes. The lowest level (usually
0.1 to 0.2 ml. per 100 milliliters of agar) that gives a dense, continuous
growth of the organism under the assay conditions is selected as the
optimum.

It has been reported (839) that when B. suhtilis changes from the

78 ANTIBIOTIC ACTION OF ANTAGONISTS

smooth to the rough phase there is a marked increase in resistance to
certain penicillins but not to others. This has a bearing upon a knowl-
edge of the chemical entities present in the penicillin preparations.

This method is also very convenient for measuring the activity of
streptothricin and streptomycin. A standard curve is obtained by filling
the cups in quadruplicate with dilutions of the standard containing lO,
20, 40, 60, 80, and 100 |jg/ml. The dilution of the unknown contains
about 50 Mg/ml. After overnight incubation at 30° C, the inhibition
zones around the cups are measured and plotted to give a standard
curve. The concentrations of the unknowns are read off this curve by
projecting the value of the inhibition zones.

A standard streptomycin agar was developed (582) consisting of
6 gm. peptone, 1.5 gm. beef extract, 3.0 gm. yeast extract, 15.0 gm.
agar, 1,000 ml. distilled water, -pH after sterilization 7.9 ±0.1. The
test strain B. subtilis is grown on agar or in submerged liquid medium.
The cells are suspended in sterile 0.5 M potassium phosphate buffer,
^H 7.0, and pasteurized to kill the vegetative cells. The spore suspen-
sion is counted by plating and is stored at 2° to 4° C. Twenty-milliliter
portions of sterile agar are first poured into the plates 5 the agar is
allowed to harden and is then covered with 4 ml. of the seeded agar
containing about 250,000 spores per ml. of agar. The plates may be
stored at 2° to 4° C. for several days. The test sample is diluted with
equal volume of 0.2 M potassium phosphate buffer, ^H 7.9, and all
subsequent dilutions are made with o. i M buffers. Either paper discs or
cups may be used, 4 to 6 per plate ; in the case of discs 0.08 ml. of sample
is added rapidly to each disc after it has been placed on the agar. A
standard is used on each plate. The plates are incubated at 30° C. for 15
to 30 hours. At 37° C. the zones develop after 4 to 6 hours' incubation.
A typical curve is shown in Figure 8.

Turbidimetric Method

End-point methods have long been recognized as having many limi-
tations. Since it is difficult to determine accurately the end point and
since it takes a relatively much larger amount of an antibiotic substance
to inhibit completely the growth of the test organism as compared with
only 50 or 99 per cent inhibition, the suggestion has been made that

METHODS OF MEASURING ANTIBIOTIC ACTIVITY

79

Figure 8. Daily standard curve of streptomycin on streptomycin assay agar,
incubation 30° C. From Loo et al, (582).

partial Inhibition of growth be measured and, from this, the concentra-
tion of the active substance be calculated in a manner similar to the
measurement of the potency of bactericidal agents. Partial inhibition
can be determined by plating for the number of viable bacteria, as com-

80

ANTIBIOTIC ACTION OF ANTAGONISTS

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PENICILLIN IN B PER. MILLILITER

.35

.40

Figure 9. Relation between penicillin concentration and inhibition of
Stafhylococcus aureus. The penicillin preparation contained 42 Oxford units
per milligram, and the incubation period was sixteen hours at 37° C. From
Foster (280).

pared with the control, or it can be measured by a convenient turbi-
dimeter. The results obtained by this method are more nearly quantita-
tive than those obtained by other methods, as shown in Figure 9. By
proper modifications, the length of time required to obtain a satisfac-
tory reading can be reduced to four hours (482, 610), or even to 90
minutes (280, 554).

The turbidimetric method has also found application in the stand-
ardization of streptomycin. For this purpose, certain noncapsulated
cultures of K. fneumoniae are used. Nutrient broth is inoculated from
a slant and incubated at 37° C. for 22 to 24 hours. A series of dilutions
of streptomycin in nutrient broth is prepared and one-milliliter portions
are added in duplicate to tubes containing 9 cc. of broth inoculated with

2 per cent of the culture. The tubes are incubated at 37° C. for 3 hours,

3 drops of formalin are added to stop growth, and turbidity is deter-
mined in a photoelectric colorimeter. The turbidity measurements are

METHODS OF MEASURING ANTIBIOTIC ACTIVITY 81

plotted against the concentrations of streptomycin and compared with
the standard.

S fecial Methods

Special methods were found to be specific for measuring the action of
certain substances. The ability of tyrothricin to hemolyze red blood
cells served as the basis for measuring the potency of this substance
( 1 8 1 ) : the tyrothricin content is calculated from the amount of hemoly-
sis by the unknown and is read from a standard curve. The inhibition
of growth of a (3-hemolytic streptococcus, group A, as measured by
hemolysin production has been used for assaying the potency of peni-
cillin (747, 1022). Penicillin can also be estimated by its inhibition of
nitrite production by 6". aureus cultures (350). The antiluminescent
test has been utilized not only for measuring the activity of certain sub-
stances but also for determining their possible usefulness. The results
of a comparative study of a number of antibiotic substances by this and
the dilution method are brought out in Table 9.

Other methods suggested for measuring the activity of antibiotic
substances are based upon interference with a given physiological func-
tion of the test organism such as dehydrogenase activity and respiration
(562) upon the prevention of growth of the test organism (pneumo-
coccus) in semisolid tissue culture medium (392), or upon the diffu-
sion of the antibiotic placed upon the surface of inoculated semisolid in
tubes and measurement of the depth of clear zone (27). Although only
a single method is usually employed in the concentration and standard-
ization of a given antibiotic such as penicillin or streptomycin, it is often
advisable to check the results by another method, especially where sev-
eral test organisms are used, in order to obtain an antibacterial spectrum
that will demonstrate that one is still dealing with the same type of
chemical compound.

The results obtained by the various methods for determining the
relative potency of different antibiotics lend themselves readily to
statistical analysis (68, 513, 514).
' Methods have also been developed for detection of chemotherapeu-

82

ANTIBIOTIC ACTION OF ANTAGONISTS

TABLE 9. ANTILUMINESCENT AND ANTIBACTERIAL ACTIVITIES
OF VARIOUS ANTIBIOTIC SUBSTANCES

SMALLEST AMOUNT SHOWING ACTIVITY, IN MICROGRAMS

al/ab ratio

Antiluminescent

test

Antibacterial test*

Tolu-p-quinone

O.I I

Gramicidin

.002

Tolu-p-quinone

.002

Pyocyanase

3

Tyrothricin

.008

Pyocyanase

.07

Clavacin I

II

Penicillin II
Penicillin I
Flavatin

.0156

.06

.256

Clavacin I

.18

Aspergillic acid

15

Gramidinic acid

•23

Sodium clavacinate .18

Gliotoxin

17

AP2it

•31

Clavacin II

.19

Clavacin II

22

Actinomycin

•54

Sulfanilamide
Phenol

<.56
•5

Pyocyanin

47

Aspergillic acid

2.0

Pyocyanin

1-7

Actinomycin

54

Gliotoxin

2.1

Lauryl sulfate

4.6

Streptothricin

56

Streptothricin

2.8

Aspergillic acid

7-5

Sodium clavacinate

94

Fumigacin

13-0

Gliotoxin

8.0

Flavatin

256

Fumigacin

273

Pyocyanin

27.0

Streptothricin

20.0

Lauryl sulfate

273

Pyocyanase

42.0

Fumigacin

21.0

Phenol

1 1 70

Tolu-p-quinone

55.0

Actinomycin
Flavatin

1 00.0
1 000.0

Penicillin I

1650

Lauryl sulfate

59.0

AP2it

>i630

Sulfanilamide

3940

Clavacin I

63.0

Gramidinic acid

>2i75

Gramicidin

>500

Clavacin II

1 13.0

Penicillin I

27,500

Gramidinic acid

>5oo

Sodium

clavacinate

500.0

Tyrothricin

>62,5oo

Tyrothricin

>500

Phenol

2300.0

Gramicidin

> 2 50,000

AP2it

>5oo

Sulfanilamide >7000.0

Penicillin II

>325,ooo

Penicillin II

>50oo

From Rake, Jones, and McKee (748).

* Streptococcus pyogenes used as test organism.

t A tyrothricin-llke preparation.

tic substances in tissues and their secretions, as by the use of jfluorescent
microscopy, penicillin giving a green fluorescence (403). By utilizing
the inactivating effect of penicillinase upon penicillin, it was possible to
work out a method for the evaluation of different forms of penicillin in
blood serum (130).

Several methods are commonly employed for measuring bactericidal

METHODS OF TESTING IN VIVO ACTIVITY 83

action of antibiotic substances. A suspension of washed bacterial cells in
saline or other suitable solution, or a 5-to-i2-hour-old broth culture of
the test organism, is treated with various dilutions or concentrations of
the active substance. After incubation at 37° C. for i to 24 hours, the
number of living cells is determined. If the active substance has lytic
properties or if the test organism undergoes lysis readily, the readings
are simplified. If no lysis occurs, the treated bacterial suspension or cul-
ture is streaked or plated out. The streaking procedure gives only a
relative idea of the extent of bactericidal action. If 50 to 90 per cent
killing of cells Is to be taken as a unit of measurement, the culture Is
plated on a suitable medium and the actual number of surviving cells
are determined.

Some of the foregoing methods can also be utilized for measuring
the fungistatic and fungicidal properties of antibiotic substances. Pro-
tective fungicides may first function as fungistatic agents, others func-
tion better either as fungicidal or as fungistatic agents, and still others
show either a high or a low for both. The growth of Ceratostomella
ulmi was inhibited by actinomycin, clavacin, and hemipyocyanin in con-
centration of 1:100,000 (771, 949).

METHODS OF TESTING THE IN VIVO ACTIVITIES
OF ANTIBIOTIC SUBSTANCES

Ordinary pharmacological, bacteriological, and pathological proce-
dures are used for testing the toxicity and activity of antibiotic sub-
stances In the animal body.

In order to determine the amount of an antibiotic required for the
treatment of a certain infection, It is essential to know not only the re-
sponse of the organism causing the Infection but also the sensitivity
of the particular strain Involved. It is also essential to determine the
concentration of the antibiotic In the body fluids. A number of methods
have been proposed for this purpose, especially for penicillin and strep-
tomycin.

Most of these represent various modifications of the agar diffusion
and serial dilution methods, using a hemolytic streptococcus or some
other suitable test organism, such as S. aureus or B. subtilis. In some

84 ANTIBIOTIC ACTION OF ANTAGONISTS

cases the serial dilution has been combined with the turbidimetric
method. The ability of penicillin to inhibit hemolysin production by
streptococci has also been utilized (8ooa). A comparison of the agar
diffusion, turbidimetric, and serial dilution methods led to the conclu-
sion that the last is the most suitable for routine clinical work (508).
Penicillin-containing material may also be spread over a given area of
a nutrient agar surface, allowing time for absorption of the liquid, and
streaking the surface with standard strains of S. aureus of known sensi-
tivity j on comparing with standard penicillin preparation, it is possible
to determine both the concentration of penicillin and the degree of sen-
sitivity to penicillin of the infecting agent (152).

Fleming (266) proposed a method using a hemolytic streptococcus
as test organism and blood (preferably group O) from which leucocytes
have been removed or inactivated and treated with a clot-inhibiting
substance as a medium. Hemolysis of blood is measured either in a slide
cell or in a capillary tube. The penicillin concentration in the blood is
estimated by the serial dilution method. This method has been vari-
ously modified for clinical assays of penicillin.

A convenient method for measuring the concentration of streptomy-
cin in body fluids is to use the agar diffusion method with an alkaline
medium, low in salt, and a carefully selected test organism (582, 879).

CHAPTER 5

BACTERIA AS ANTAGONISTS

Following the work of Pasteur in 1877 on the antagonistic ef-
fects of bacteria against the anthrax organism, considerable attention
has been centered upon bacteria as agents possessing antibacterial prop-
erties. A systematic study of this phenomenon was first made by Babes
in 1885 (155), who demonstrated that this antibacterial action is due
to the formation of definite chemical substances. Garre (315) first in-
troduced, in 1887, suitable methods, such as the streak test, for demon-
strating the antagonistic effect of one organism upon another. The first
antibiotic substance, pyocyanase, was isolated in 1 899 by Emmerich and
Low (235).

Freudenreich (298) found in 1888 that when certain bacteria were
grown in a liquid medium, the filtrate obtained by passing the culture
through a porcelain candle supported the growth of the typhoid or-
ganism not at all or only very feebly. Garre (315) observed that Ps.
futida inhibited the growth of S. aureus, E. tyfhosa, and Bacillus muco-
sus-cafsulatus but not of B. ant hr acts and other bacteria. It was soon
reported {S^'i)j however, that B. anthrach was also killed by the
Pseudomonas antagonist, whereas the growth of S. aureus and V .
comma was only retarded j no effect at all was exerted upon E, typhosa
or E. coli. In consequence, the antagonist was claimed to be active
against B. anthrach but not against other bacteria. Olitsky (691) con-
cluded that Ps. -fluorescens inhibited the growth not only of E. typhosa
but also of B. anthracis, V. comma, S. marcescens, and S. aureus. These
and other apparently contradictory results were undoubtedly due to
differences in the specific nature of the strains of the organisms used by
the various investigators and to different methods of cultivation.

The presence of Ps. fluorescens in sewage was found (551 ) to reduce
greatly the period of survival of the typhoid organism. The latter did
not develop even in gelatin upon which Ps. fluorescens had previously
grown, and it could not be detected in sterile sewage in which the an-
tagonist was present for seven days. According to Frost (303), E. ty-

86 BACTERIA AS ANTAGONISTS

fhosa can be antagonized by a number of different soil bacteria, of
which Ps. fluorescens exhibits the strongest effect. He observed that al-
though P. vulgaris acted more rapidly, the active substance did not dif-
fuse to so great a distance in the medium, thus pointing to a different in-
hibition mechanism. Mixed cultures showed greater activity than pure
cultures, either because the latter lost their antibiotic property when
grown for a long time on artificial media or because mixed cultures com-
prise two or more species with a greater combined action. The antago-
nistic substances produced by these bacteria were active at 37° C, where-
as at ice-chest temperature the action was delayed so that the pathogen
had an opportunity to develop. This was believed to offer a possible ex-
planation for the fact that when water supplies become contaminated
in cold weather, their power of producing infection is retained for a
longer time than when the contamination takes place in warm weather.

Frost concluded that the phenomenon of antagonism results in
checking the growth of E. ty fhosa as well as in killing the pathogen.
Evidence that antagonistic substances exist in an active state in the soil
or in water appeared to be lacking j rather, the results suggested that
formation of such substances depends on the actual development of
specific antagonistic organisms. Changes in environment, such as tem-
perature, oxygen supply and reaction of the medium, and nature and
concentration of nutrients, were believed to have little or no influence
on the production of the antibiotic substances j these were produced
under conditions favoring growth of the antagonists.

The activity of the influenza organism was found ( 1 02 5 ) to be largely
dependent on the presence of accompanying bacteria. Some of these,
especially micrococci, are favorable to the growth of this organism
whereas others, such as Ps. aeruginosa and B. subtilis, are injurious.

According to Lewis {S^^)-, luxuriant growth of Ps. fluorescens in
manured soil and in protein solution containing B. cereus is due to an-
tagonistic action of the former organism against the latter. Ps. fuo-
rescens also inhibits the growth of B. anthracis, B. megatherium^ V.
comma, Chrom^obacterium, violaceum^, and Rhodococcus. Other species
of the genera Bacillus, Eberthella, Sarcina, Neisseria, and Phytomonas
are somewhat more resistant to the action of Ps. fluorescens. Salmonella
species are less sensitive, whereas E. coli, A. aerogenes, and S. marces-

SPORE-FORMING BACTERIA 87

cens are highly resistant. Ps. fluorescens produces a thermostable sub-
stance which is toxic to all bacteria except the green fluorescent forms
and which is active against actinomycetes but not against fungi. This
substance is water-soluble and dialyzable through collodion and other
membranes.

In addition to the aforementioned bacteria, numerous other groups
were found to contain strains which had strong antagonistic properties
toward bacteria as well as fungi. Some of the antagonists were highly
specific, such as those acting upon the various types of pneumococcij
others were less selective, such as certain soil bacteria that can bring
about the lysis of living staphylococci and inhibit the growth of various
gram-positive and gram-negative bacteria. S. m,arcescens was antagonis-
tic to various spore-forming bacteria. These, in turn, were antagonistic
to sarcinae, bringing about their lysis, to V. comma, and to various
other bacteria. It was further found that the antagonists modified the
physiology of the antagonized organism. When two bacteria were
planted, for example, in the same medium, metabolic products were
formed that were not produced in the culture of either organism alone,
whereas certain decomposition processes were either hastened or re-
tarded (674).

The various antagonistic bacteria can be divided into several groups,
on the basis of their morphological and physiological properties.

SPORE-FORMING BACTERIA

Many aerobic spore-forming bacteria possessing antagonistic proper-
ties have been isolated from a great variety of sources, such as soil, sew-
age, manure, and cheese. Among these, B. subiilis, B. mycoides, B.
mesenterkus, and B. brevis occupy a prominent place, as shown in
Table 10.

Duclaux (212) isolated antagonistic spore- forming bacteria from
cantal cheese, the organisms having been designated as Tyrothrix.
Nicolle (680) obtained from the dust in Constantinople a strain of B.
subtilis that had decided bacteriolytic properties against members of the
pneumococcus group and various other bacteria such as the typhoid, an-
thrax, and Shiga organisms. E. coU and V. comma were most readily

88 BACTERIA AS ANTAGONISTS

TABLE 10. SPORE-FORMING BACTERIA ANTAGONISTIC TO OTHER BACTERIA

ANTAGONIST

ORGANISM AFFECTED

KNOWN PROPERTY

REFERENCES

B. ant kr acts

Anthrax, typhoid, and
lactic acid bacteria

298, 819

B. brevis

Gram-positive bacteria

Produces tyrothricin

201, 202, 208

B. mcsentericus

Many bacteria

Bacteriolytic

419

B. mesentericus

Diphtheria bacteria

Bactericidal

31.984

B. mesentericus

C. difhtheriae

Substance thermola-

738

vulgatus

bile, nonfilterable

B. my c aides

7 to 20 species of
bacteria

Lytic

664

B. mycoides,

Most pathogens and

292

var. cytolyticus

many nonpathogens

B. subtilis

Various bacteria

Bacteriolytic

680

B. subtilis

Various bacteria, espe-
cially certain plant
pathogens

Produces subtilin

453,460

B. subtilis

M. tuberculosis, E.
tyfhosa, etc.

927

B. subtilis

M. tuberculosis

Thermostable sub-

693, 816

and other bacteria

stance produced

B. subtilis-

Mostly living gram-

Lytic

806, 808

mescntericus

positive bacteria and
dead gram-negative
bacteria

B. thermofhilus

5. lutea

Suppresses growth

864

acted upon, staphylococci were less affected, and B. suifestijer least.
The filtrate of the organism grown in peptone broth had strong anti-
biotic properties J it liquefied gelatin and hemolyzed red blood cor-
puscles. When various bacteria cultivated on a solid medium were sus-
pended in physiological salt solution and seeded with the antagonist,
the latter developed abundantly and the bacterial suspensions became

SPORE-FORMING BACTERIA 89

clarified. The lysed solutions of pneumococcus prepared by the use of
the filtrate of B. subtilis could be used for purposes of vaccination. In
this connection, Nicolle spoke of the work of Metchnikoff who had
proved, in 1897, that organisms belonging to the B. subtilis group are
capable of destroying various bacterial toxins.

Rosenthal (806) isolated, from soil and from fecal matter, facultative
thermophilic antagonistic bacteria belonging to the B. mesentericus
group capable of dissolving both living and dead bacteria. The simul-
taneous growth of the antagonist with V. comma and other bacteria
brought about the clarification of the culture of the latter in about 5 or
6 days. These bacteriolytic organisms were designated as "lysobacteria."
It was recognized that the action of antagonists is different from that of
phage in several respects: (a) the filtrate of the antagonist is active
against other bacteria j (b) both living and dead cultures of bacteria are
dissolved J (c) antagonistic action is not so specific as that of phage j (d)
races of E. coli resistant to phage are dissolved by the filtrate of the an-
tagonist. The active substance was believed to be of the nature of an
enzyme. Friedlander's bacillus was not acted upon, possibly because of
the formation of a pellicle by the bacillus. The active substance was
formed in 4 to 5 days but increased in activity after 2 to 3 weeks. It was
essential that a surface pellicle of the organism be maintained. Sub-
merged growth was less favorable. Fresh filtrates had the greatest ac-
tivity, the property being lost after storage for 3 months. The substance
was thermolabile, activity being destroyed at 70° C. The filtrate of an
organism dissolved by the action of the antagonist proved to be as ac-
tive as the filtrate of the culture of the antagonist. It acted injuriously
upon intestinal bacteria not only in vitro but also in vivo.

Much and associates (664) isolated several strains of B. •mycoides
that possessed strong antagonistic properties. The active strains were
said to be found only rarely in nature. They gave a mesentericus-like
growth, producing a pellicle and no turbidity in bouillon. One strain
was able to lyse 20 species of bacteria, another acted upon 18, a third
on 12, and a fourth on only 7. Marked differences were shown to
exist in the degree of antagonistic activity of the different strains.
P. vulgaris, E. tyfhosa, and V. comma were lysed in 24-hour bouillon
cultures as a result of adding pieces of agar containing colonies of the

90 BACTERIA AS ANTAGONISTS

antagonist. A lytic effect was also exerted upon staphylococci (824) and
gram-negative bacteria (504, 505). The substance was precipitated by
10 per cent tungstic acid and lead acetate and was thermostable.

Much and Sartorius (664) came to the conclusion that B. mycoides
Flugge comprises two groups of organisms. One produces branching
colonies on agar and forms no pellicle in meat broth, the flaky growth
dropping to the bottom and the medium remaining more or less clear.
The second group forms flat surface growth similar to that of B. mes-
enterkus on agar and a pellicle on the surface of liquid media. Many
of the pellicle-forming strains have the capacity, in varying degrees, of
dissolving various cultures of bacteria. This is not due to their proteo-
lytic activity, since members of the first group may be more actively
proteolytic. The culture filtrate of the antagonist dissolves the bacteria
but does not destroy their antigenic properties. The lytic substance,
designated as Much-lysin, was said to have a double effect: one, bound
to the living cells of the organism, had nothing to do with phage, and
the other, found in the bacteria-free filtrate, had an apparent similarity
to phage but was distinct from it.

The idea that in the case of bacterial antagonists one is dealing with
specific strains rather than with distinct species was further emphasized
by Franke and Ismet (292). Various strains of B. mycoides, desig-
nated as cytolitkusy were shown to be able to lyse many pathogenic and
nonpathogenic bacteria but not their own cells j the same action was
exerted by the culture filtrate (Table 11). The lytic action of strains of
B. subtilis upon different bacteria, including M. tuberculosis (927),
pneumococci, typhoid, diphtheria, and other organisms, has also been
definitely established.

Pringsheim (738) isolated a strain of B. mesentericus-vulgatus that
had a decided inhibiting effect upon a variety of bacteria, particularly
Corynebacterium difhtheriae. On agar plates the antagonist produced
a circular zone of inhibition, just beyond which was a ring of larger
colonies, indicating a stimulating effect. It was suggested that the an-
tagonist produced a toxin that was stimulating in small doses and in-
jurious in larger concentrations. The active substance was thermolabile
and nonfilterable. The antagonistic properties appeared to be inherent
in the particular strain of an organism and were not increased by serial

SPORE-FORMING BACTERIA

91

passage. The action of filtrates of B. mesenterkus against diphtheria
organisms was considered (984) as highly specific. Other strains of this
organism were reported to be active against Pasteurella festis (244).
Living gram-positive bacteria were found (806) to be more susceptible
than gram-negative organisms to the antagonistic action of spore-form-
ing aerobes J in the case of dead organisms, the reverse was true. Plates
were heavily seeded with the test bacteria and the centers of the plates

TABLE

I. LYSIS OF PATHOGENIC BACTERIA BY VARIOUS STRAINS OF A
SPORE-FORMING ANTAGONIST (cYTOLYTICUs)

ORGANISM LYSED

E. tyfhosa

Paratyphoid A

Paratyphoid B

Shigella

Y bacillus

E. coli

C. diphtherias

Ps. pyocyaneus

S. aureus

S. alius

S. citreus

S. viridis

S. /laemolyticus

S. mucosus

P. vulgaris (Weil-Felix)

Pneumococcus

STRAIN NUMBER OF CYTOLYTICUS

II III VI VII VIII

IV
o

+-H-
O

o

+
o
+
o

From Franke and Ismet (292).

O no clearing.

+ trace but no true clearing.

++ clearing, slight sediment.
+++ clearing without sediment.

inoculated with the antagonist. Inhibition of growth and lysis were used
as measures of antagonistic action.

Hettche and Weber (419) isolated 41 strains of B. mesenterkus
from 25 samples of soil. These were streaked on blood agar, and the
diphtheria organism was used for testing their effect. In 18 strains the
antagonistic action was detected in 24 hours j there was no parallelism

92 BACTERIA AS ANTAGONISTS

between inhibition and hemolysis. Of the 1 8 active strains, 1 1 lost the
property after two transfers and 2 were exceedingly active.

More recently, beginning with the work of Dubos, considerable at-
tention has been devoted to spore-forming bacteria, resulting in the iso-
lation of a number of substances or preparations that have been desig-
nated as tyrothricin, gramicidin S, subtilin, bacitracin, bacillin, sim-
plexin, subtilysin, and endo-subtilysin (815a). These substances are
largely active against gram-positive bacteria j however, some also aifect
gram-negative bacteria and fungi.

Dubos (201) obtained from a soil enriched with various living bac-
teria a gram-negative, spore-bearing bacillus (B. brevis) that had a
marked lytic effect against gram-positive bacteria, including staphy-
lococci and pneumococci. The antagonist was grown for 3 to 4 days in
shallow layers of peptone media at 37° C. The bacterial cells were re-
moved by centrifuging, and the filtrate was acidified, giving a precipi-
tate from which a highly active substance (tyrothricin) was isolated.
On crystallization, two preparations were obtained, namely gramicidin
and tyrocidine, these making up only a fraction of the tyrothricin com-
plex.

Natural substrates, such as soil, sewage, manure, and cheese, were
found (209) to contain various spore-forming bacteria that have
marked antagonistic properties against various gram-positive and gram-
negative bacteria. Hoogerheide (442) obtained from the soil an aero-
bic, spore-forming bacterium that produced a highly active bactericidal
substance J it also prevented the formation of capsules by Friedlander's
bacterium. This substance appeared to be similar to gramicidin. Grami-
cidin S is, however, more like tyrocidine.

Further studies definitely established that strains of spore-forming
bacteria possessing antagonistic properties are widely distributed in the
soil and possess certain physiological characteristics that differentiate
them from the inactive strains. This is brought out in Table 12. The
production of the antibiotic is a function of the growth of the bacterial
cell. The yield of the antibiotic is influenced by the composition of the
medium j the substance is bound to a protein, the bond between the two
being destroyed by trypsin (523).

B. sub tills has been reported by many investigators to exert an an-

SPORE-FORMING BACTERIA

93

tagonistic effect upon many pathogenic bacteria, bringing about their
complete lysis. The time required for such lysis was 48 hours for gram-
positive cocci, 5 days for the typhoid and paratyphoid organisms, and
8 to 12 days for E. coli and M. tuberculosis (693). The action of B.

TABLE 12. BIOCHEMICAL CHARACTERISTICS OF ACTIVE AND INACTIVE
STRAINS OF SPORE-FORMING SOIL BACTERIA

LIQUE-

HY-

pro-

FAC-

DROLY-

STRAIN ACID PRODUCTION FROM

duction

TION OF

SIS OF

GRAM

Dextrose Lactose

Sucrose

OF HoS

GELATIN

STARCH

STAIN

Active Strains

A-2

-

+

+

-

-

A-5

-

+

+

-

-

A-io

-

+

+

-

-

A-21

-

+

+

-

-

A-23

-

+

+

-

-

A.27

+

+

-

-

-

A-34

-

+

+

-

-

Inactive Strains

A-15

+

-

-

+

+

A-31 - +

+

-

+

+

+

A-32 + +

+

-

+

+

+

From Stokes and Woodward (885).

— reaction becoming alkaline.

+ acid produced.

subtilis upon various bacteria is also growth-inhibiting. This property is
due to the formation of one or more antibiotics which have been de-
scribed in the literature under several different names.

Cultures of B. subtilis found (453) to have a high activity against
plant pathogenic bacteria yield an antibiotic that was designated (460)
subtilin. Other preparations designated by the same name (759) have a
strong bacteriostatic, bactericidal, and lytic effect upon a variety of bac-
teria, including B. anthracis, C. di-phtheriae, and Sh. dysenteriae; the
activity of the culture filtrate was about 4 to 1 6 units and there was a
marked parallelism between the antibacterial properties of the filtrate
and its proteolytic action.

A water-soluble, nontoxic, relatively heat-stable compound was iso-

94 BACTERIA AS ANTAGONISTS

lated from other strains of B. subtiUs and named bacitracin (469). Still
another strain of B. sub tills isolated from soil enriched with M. tuber-
culosis yielded an antibiotic designated bacillin (284). This substance
is produced in manganese-containing media and is mostly found in the
cell-free filtrate of the culture. It is adsorbed on norite and eluted with
90 per cent ethyl alcohol, concentrated in vacuo and taken up in water.
It is active against both gram-positive and gram-negative bacteria.
Blood and certain other complex organic materials reduce or destroy
its activity. This is due to the presence of a substance which was desig-
nated antibacillin and which was found to be a peptide (1030).

B. Ucheniformisy related to B. subtilis, was found (107) to produce
an effect against M. tuberculosis. The active substance was present in
the cells of the organism when grown on a synthetic medium. The cul-
ture was acidified to f¥L 2.5 and treated with 3 volumes of 95 per cent
ethanol. The coagulum was autoclaved and extracted on boiling with
0.5 volume of 0.4 per cent acetic acid for 45 minutes. The substance
had an activity against M. fhlei and S. aureus in i : 80,000 dilution, but
not against E. coU. M. tuberculosis hominis was inhibited in i : 20,000
dilution. Preparations of greater purity had an activity of i : 80,000/
gm. The preparation, which was not very toxic to mice, was considered
as a mixture of several substances.

Various other antibiotics have been reported for aerobic spore-form-
ing bacteria. Some of these substances are active against both gram-
positive and gram-negative bacteria. This is true, for example, of colis-
tatin (323a).

Spore-forming bacteria are also able to produce antibiotics antago-
nistic to fungi. B. simplex was found (154) to be antagonistic to
Rhizoctonia solaniy an important plant pathogen. It produced a
thermostable agent that inhibited the growth and even caused the
death of the fungus. When the active substance was added to the soil
it controlled to some extent seed decay and damping-off disease of
cucumbers and peas. It was also active against bacteria (491). It is ad-
sorbed on norite and eluted with methyl alcohol 5 the latter is evapo-
rated in vacuo and the residue is taken up in water. This preparation
was designated as simplexin (287).

B. mesentericus produced on artificial media an antibiotic that sup-

NONSPORE-FORMING BACTERIA 95

pressed the growth of H elminthosforium sativum. It increased sporu-
lation of the fungus, inhibited or retarded spore germination, caused
abnormal hyphal development, and induced mutations in certain
strains of the fungus. The substance was thermostable and diffusible. It
passed through a Berkfeld filter, was absorbed by infusorial earth,
withstood freezing and desiccation, and did not deteriorate readily. It
was destroyed by alkalies but not by acids. It was inactivated or de-
stroyed, however, by certain fungi and bacteria (142).

Various other spore-forming bacteria were found capable of inhibit-
ing the growth of bacteria, fungi, and other lower forms of life. In
many instances, only little is known of the nature of the active agent in-
volved. It is sufficient to illustrate this by an observation that B. h'lru-
denses, growing abundantly in the digestive fluids of leeches and con-
sidered as a symbiont of these animals, exerted a marked inhibitory
effect upon the growth of various bacteria and fungi (845).

Antagonistic relations among entomogenous bacteria have been
demonstrated for the foul brood of the honeybee (441). This inter-
action between B. fopilliae and B. lentimorbus was believed to explain
the mutually exclusive development of the two types of milky disease
in Japanese beetle groups.

NONSPORE-FORMING BACTERIA: PS. AERUGINOSA,

Among the nonspore-forming bacteria, those belonging to the fluo-
rescent, green-pigment and red-pigment producing groups have prob-
ably received the greatest attention as antagonists. Bouchard (78) was
the first to report, in 1888, that the pyocyaneus organism {Ps. aerugi-
nosa) was antagonistic to the anthrax bacillus. It was soon found (131,
298) that when grown on artificial media, this organism affected bac-
teria, including E. tyfhosa, Pfeiferella mallei, V. comma, and Bac-
terium tyrogenes. The growth of staphylococci, micrococci, diplococci,
and spore-forming rods was also reduced. The antagonist inhibited its
own growth as well.

These early observations were amply substantiated (Table 13). Ps.
aeruginosa was shown to be active against E. coli, M. tuberculosis, and

96

BACTERIA AS ANTAGONISTS

a variety of other bacteria. The addition of top minnows (Gambusia)
to water polluted with E. colt caused the disappearance of the bacteria j
this was shown to be due to the inhibiting effect of the pyocyaneus or-
ganism present in the intestinal flora of Gambusia. The presence of this
antagonist in water renders the colon index of the water an unreliable
guide to pollution (388). When a mixture of the antagonist and the
colon organism was incubated, the former tended to outgrow the latter
after 24 hours. Even after sterilization, media in which Ps. aeruginosa
had grown depressed the growth of other microorganisms including

TABLE 13. NONSPORE-FORMING BACTERIA AS ANTAGONISTS TO BACTERIA

ANTAGONIST

ORGANISMS AFFECTED

Ps. aeruginosa

B. anthracis, E. typhosa, V.

comma, etc.

Ps. aeruginosa

Gram-negative bacteria, M.

tuberculosis, and yeasts

Ps. fiuorescens

E. coli, S. marcescens, C.

difhtheriae, B. anthracis,

etc.

Ps. fiuorescens

Actlnomycetes

S. marc esc ens

CI. chauvoei, B. anthracis.

staphylococci, micrococci

S. marcescens

Gram-positive but not gram-

negative bacteria

E. colt

Typhoid, paratyphoid, diph-

theria, staphylococci, and

proteolytic bacteria

E. colt

Other E, coli strains

E. coli

B. anthracis and other spore-

forming bacteria

A . aerogenes

B. anthracis, P. festis

E. tyfhosa

E. tyfhosa, Ps. fiuorescens,

E. coli, B. anthracis

S. far at yf hi

E. coli, B. anthracis, P. festis

KNOWN PROPERTY

Thermostable, filter-
able substance

Depresses growth

Thermostable, filter-
able substance

Lytic action

Colorless, thermo-
stable, lytic sub-
stance

Alcohol-soluble
pigment

Growth-inhibiting

REFERENCES

64, 78, 131,235,

236, 298, 557

451a, 798, 800,
801

244, 303, 315, 334,
417, 418, 420, 421,

446, 563, 566,692

593

51, 229,777

420

53,55, 132,368,
515,685,769,912,

983

3573,681

no, 344,367,369,
457,485, 819,923

244, 367, 369

315,354,923,
936

244, 462, 810, 923

NONSPORE-FORMING BACTERIA

TABLE 13 {continued)

97

ANTAGONIST

Streptococci

Streptococci

Staphylococci

Micrococci

Diplococci and
pneumococci

K. -pneumoniae

P. vulgaris

P. avicida
Myxobacteria

Anaerobic bac-
teria

ORGANISMS AFFECTED

B. anthracis, C. difhtheriae

B. anthracis, Ph. tumefaciens,
S. lactis, P. festis, L. bul-
garicus

Gram-positive bacteria, C.
difhtheriae, P. festis

V. comma, M. tuberculosis,
E. tyfhosa, Br. melitensis

Various bacteria

B. anthracis, C. difhtheriae,
P. festis

B. anthracis, P. festis, CI.
sforogenes

B. anthracis, E. tyfhosa

Plant-disease-producing
bacteria

M. tuberculosis, B. anthracis

KNOWN PROPERTY

Activity not associ-
ated with hemoly-
sis or virulence

Thermostable, non-
filterable substance

Thermolabile sub-
stance

Active filtrate

Thermostable lytic
substance

REFERENCES

53, 110, 187, 233,

303,670,711, 836

70, 244, 802, 1007

53, 155,215,244,

247

213, 214, 580, 625,
670

213, 214, 243, 244,
370, 580,677, 766

677>7"5 853
36, 244, 923, 985

440, 708

S. marcescensy Ps. fluorescenSj and Sacckaromyces cereviseae; spore
formation by the last was favored (800).

The specific antagonistic action of Ps. aeruginosa upon various bac-
teria was found by early investigators to be due to the production of an
active heat-resistant substance. By filtering the culture through a Berk-
feld, evaporating to a small volume, dialyzing through a parchment
membrane, precipitating with alcohol, and drying over sulfuric acid, a
preparation was obtained which was designated as pyocyanase (see
p. 51). It had, even in very low concentrations, a marked destructive
effect upon diphtheria, cholera, typhus, and plague organisms, as well
as on pyogenic streptococci and staphylococci. It rapidly dissolved V.

98 BACTERIA AS ANTAGONISTS

comma cells and in a few seconds rendered inactive such bacterial toxins
as that of diphtheria. Since the bacteriolytic action of pyocyanase was in
direct proportion to the time of its action and concentration, and in in-
verse proportion to the numbers of bacteria acted upon, its enzymatic
nature was believed to be substantiated. The preparation withstood
heating in flowing steam for 2 hours.

It has been established that pyocyanase has a lytic effect against the
diphtheria organism, streptococci, meningococci, the typhoid organism,
pneumococci, P. festis. Vibrio metchnikovi, V. commas and many other
bacteria. There has been considerable disagreement, however, concern-
ing the chemical nature and therapeutic action of pyocyanase, due
largely to the variation in the nature of the preparations obtained.
Kramer, for example, has shown (529) that the activity of the sub-
stance depends on three factors: nature of strain, not all strains being
equally effective} composition of medium, glycerol-containing media
being most favorable} and method of extraction of active substance
from culture media. The enzymatic nature of pyocyanase was not uni-
versally accepted, largely because of the thermostability of the sub-
stance, its solubility in organic solvents, and the fact that temperatures
of o to 37° C. fail to influence its activity (59, 420, 737).

Vs. aeruginosa produces, in addition to pyocyanase, a blue pigment,
pyocyanin. Both substances possess lytic properties, i : 1,000 dilution of
the pigment being able to lyse E. coli in 6 hours. Pyocyanin was said to
be more effective in younger cultures, and pyocyanase in older. Pyo-
cyanin had a bactericidal action also upon S. hemolyticusy S. albus, S.
aureus, C. dl-phtheriae , M. tuberculosis, V. metchnikovi, and the
Y-Ruhr bacillus, but not upon P. vulgaris, E. coli, or the typhoid organ-
ism. In general, gram-positive bacteria were largely affected. Numer-
ous other substances have been isolated from the cells of the organism
or from the culture medium of Ps. aeruginosa. It is sufficient to men-
tion the pyo-compounds and pyolipic acid.

In order to test the action of Ps. aeruginosa upon other bacteria,
Kramer (529) placed a drop of a suspension of this organism upon a
plate inoculated with M. tuberculosis or with V. metchnikovi. In 24
hours, a sterile zone surrounded the colony of the antagonist, the width

COLON-TYPHOID BACTERIA 99

of the zone depending upon the moisture content of the medium, the
degree of diffusion of the active substance, its concentration, and the
resistance of the test bacteria. When either of the two pathogens was
inoculated into liquid media and the antagonist was introduced simul-
taneously or within 24 hours, the latter had a decided bactericidal effect.

No less extensive is the literature on the antagonistic action of the
fluorescent group of bacteria, first established by Garre (315) in 1887
and later by others. Its bacteriostatic spectrum is illustrated in Table
14. The active substance is thermostable, dialyzes through a membrane,
passes through Seitz and Berkfeld filters and is said to be soluble in
chloroform (418, 566). Aerobic culture conditions are favorable to its
accumulation. Members of this chromogenic group of bacteria were also
found to be able to bring about the lysis of infusoria (134).

S. marcescens exerts antagonistic effects against a number of bacteria,
including diphtheria, gonococci, anthrax, and CI. chauvoeiy as well as
fungi causing insect diseases (624). The formation of antibiotic sub-
stances by this organism has been demonstrated by various investi-
gators. These substances are active not only in vitro but also in vivo.
Their formation was believed not to be associated with the production
of the pigment by the organism. Hettche (420), however, asserted that
the bactericidal action of Serratia is closely related to pigment produc-
tion. The pigment was extracted with alcohol and was found capable of
dissolving dead gram-positive bacteria but not gram-negative organ-
isms. Eisler and Jacobsohn (229) ascribed the antagonistic action of
Serratia not to the pigment but to certain water-soluble, thermostable
(70° C. for 30 minutes) lytic substances.

THE COLON-TYPHOID BACTERIA

Members of the colon-typhoid group are not typical soil inhabitants,
although they find their way continuously into the soil and into water
basins. Various organisms belonging to this group have been said to
possess antagonistic properties (440). Bienstock {ss) reported, in
1899, that proteolytic bacteria are repressed by the presence of E. coli
and A. aero genes. Tissier and Martelly (912) emphasized that this
phenomenon occurs only in the presence of sugar, the effect being due

100 BACTERIA AS ANTAGONISTS

TABLE 14. ANTAGONISTIC ACTION OF PS. FLUORESCENS UPON
VARIOUS MICROORGANISMS

ORGANISM PERCENTAGE OF AGED MEDIUM IN THE AGAR

0.5 I.O 2.5 5.0 10 15 20 30 40 50

B. cereus — — +

B. mycoides — — +

B. ant hr ads — +

B. vulgatus — - +

B.subtilis - - +

B, megatherium — +

R. cinnebareus — +

R. roseus - - +

M. fiavus — - — +

N. catarrhalis — — — +

Ps. aeruginosa __________

Ps. fiuorescens __________

S.lutea - - - +

S. marcescens _____ +

S.albus - - +

S. aureus — - — +

S. citreus — — +

K. fneumoniae - - - +

V. comma — +

Ch. violaceum — +

£'. tyfhi - - +

5A. faradysenteriae — — +

5. enteritidis — — — +

5. suisfestifer — — — +

5. pillorum — - — +

£. ^o/i ______ +

^ . aero genes ______ 4.

?>^. bowlesii - — +

5^^:. marianus __________

5<j^. ellifsoideus _______ +

Si7^. fastorianus ________ +

Zy gosac. friorianus — — — — — -- +

Torula sfhaerica __________

i4 . «z^^r __________

From Lewis (566).

+ denotes complete inhibition.

COLON-TYPHOID BACTERIA 101

to the fermentation of the sugar by E. coli, resulting in the production
of acid.

Wathelet (983) observed in 1895 that in mixed culture the colon
bacterium gradually replaces the typhoid organism and this was later
fully confirmed. The occurrence of slowly growing lactose-fermenting
strains of E. colt in stools has been ascribed to the phenomenon of an-
tagonism (462), and the inhibitory action upon E. tyfhosa added to
certain stools was also ascribed to the antagonistic action of E. coli
(681). Different strains of E. coli repress the typhoid organism to a
different extent. The ratio of the two organisms developing on agar was ,
designated as the antagonistic index j an index of 100:20 means that
for every lOO colonies of the colon organism, 20 colonies of typhoid
developed.

Active colon strains may be inhibitive to other strains of the same
organism. The existence of strong and weak antagonistic strains has
been questioned frequently (1034). Many of these strains were ob-
served to have a strong antagonistic action against the pathogenic in-
testinal flora j these results were contested, however (97, 543). The ac-
tion of E. coli of different origin varies, freshly isolated strains being
more active than stock cultures (783, 866). It has also been reported
that fresh, actively growing cultures of E. typhosa inhibit the growth
of E. coli, but that older cultures are not antagonistic (936).

The production by smooth strains of E. coli of a highly specific bac-
teriolytic substance which lyses the cells of a rough strain of this organ-
ism has also been indicated (1045) j this substance was ineffective
against other rough and smooth strains, whereas the filtrates of the
rough strains were inactive upon the smooth strain. The substance is
readily destroyed by heating at 70° to 80° C. E. coli antagonism has
also been correlated (632) with the greater resistance of the strains to
environmental factors, their greater rate of multiplication, and their
greater adaptation to nutrient media.

A bacteriophage was found (574) to develop as a result of the an-
tagonistic action of E. coli against the Shiga bacillus and was said to
ocpur in the intestines where antagonistic conditions are always present.
Gratia (355) found that the filtrates of one race of E. coli inhibited

102 BACTERIA AS ANTAGONISTS

another race and caused an agglutination of the latter in fluid media.
The weakest antagonists were said (387) to belong to the paracolon
group, the strains of medium activity to the colon group, and the
strongest antagonists to the colon-immobilis type. Whenever the feces
were found to contain large numbers of E. colt, no typhoid organisms
were present. The resistance of certain persons to intestinal diseases
was, therefore, ascribed to the high antagonistic colon index. By utiliz-
ing the principle of antagonism of some strains of E. coli against others,
two types of E. coli resistant to the antagonistic substance were isolated
( 176) : one produced giant colonies, the other small punctiform, trans-
lucent colonies.

More recently it was established (357a) that various strains of E. coli
produce a complex mixture of antibiotics, designated as colicines, which
are mostly bacteriostatic against certain other strains of this organism
as well as against other pathogenic enterobacteria. On the basis of their
selective action, concentration, diffusibility, thermostability, and sensi-
tivity to antagonistic organisms, eight groups of substances were listed.
They represent polypeptides readily destroyed by trypsin.

E. coli exerts an antagonistic action also upon S. schottmulleriy C.
di'phtheriae, staphylococci, M. tuberculosis, B. anthracis, various spore-
forming soil bacteria, and putrefactive water bacteria. The action
against anthrax was said to be only temporary (344). It was also sug-
gested (457) that only living cultures of E. coli are active. The simul-
taneous inoculation of S. aureus and E. coli was found (769) to be in-
jurious to the first and not to the second organism j this effect was in-
creased by an increase in the number of E. coli cells in the inoculum.
Gundel and Himstedt (368) have shown that E. coli, but not ^. aerog-
enes, is antagonistic to S. aureus and S. albus.

The term autophage has been used (342) to designate the process of
clearing a water emulsion of dead cells by a culture of an antagonist
such as E. coli. This clearing effect was said to be due to the fact that the
dead cells are used as nutrients by the living organism. The mechanism
of the action was variously explained by a change in the />H value of
the medium or in the oxidation-reduction potential or by a direct enzy-
matic effect. In some cases thermolabile, filterable substances were dem-

COCCI 103

onstrated (369, 618). These substances have been considered to be
either autotoxins (148) or proteolytic enzymes (719). The filtrate of
E. coli was reported (836) to be highly selective in its action, depress-
ing only the dysentery organism of Shiga. Gundel (372) isolated from
a bouillon culture of E. coli thermostable lipoids capable of bringing
about the lysis of the colon organism and other bacteria. The antago-
nistic relations between E. coli and V. comma are well established. The
cholera organism also possesses antagonistic properties (308, 499).

The typhoid organism is also capable of exerting an antagonistic ac-
tion against itself as well as against Ps. jiuorescenSy E. coli, and various
other bacteria, including B. anthracis. The nature of the action is not
clearly understood. Salmonella faratyfhi possesses antagonistic proper-
ties against E. coli, B. anthracis, P. -pestis, and various other bacteria.

COCCI

Numerous cocci have been found to possess antagonistic properties
against other bacteria. Doehle (187) first demonstrated in 1889 that
streptococci are able to antagonize B. anthracis, especially on solid
media. Similar action was exerted against diphtheria bacteria j this ac-
tion was not correlated with the hemolytic properties or the virulence
of the antagonist. Further studies established the effect of various
streptococci against anthrax. This effect was found (no) to be more
pronounced in liquid than in solid media, and to be highly specific as
regards the strain. S. pyogenes was shown to be antagonistic, in vivo, to
B. anthracis and to Phytomonas tumejaciens, even to the extent of sup-
pressing vegetative malformations brought about by the last named
(70). 5. cremoris was active against S. lactis ( 1007), 5. mastidis against
5. lactis and L. acidophilus, and Streptococcus mucosus against P. pestis.
Rogers (802) reported an antagonistic effect of S. lactis against L. bul-
garicus; the active substance was thermostable and would not pass
through a bacterial filter. More recently, certain streptococci were
found (625) to produce a very potent antibiotic which was thermo-
stable and dialyzablej it was active against various gram-positive but
not gram-negative bacteria j it was well tolerated on subcutaneous and

104 BACTERIA AS ANTAGONISTS

intravenous injection, and was believed to offer promise as a chemo-
therapeutic agent,

Freudenreich (298) first emphasized the antagonistic action o£
staphylococci against various bacteria. The list was later enlarged to in-
clude gram-positive acid-resisting forms, corynebacteria, and the plague
organism. Some of these antagonists were found to be able to lyse the
dead cells of their own kind as well as those of various other organisms.
Gundel (372) isolated from staphylococci an active lipoid which had
bactericidal properties. A water-soluble, alcohol-insoluble substance,
said to be an enzyme capable of bringing about the lysis of corynebac-
teria, was also isolated from a strain of staphylococcus (215).

Various micrococci possess strong antagonistic properties. Lode
(580) isolated a micrococcus which affected a variety of microorganisms
three or more centimeters away, the active substances being dialyzable.
An organism related to Micrococcus tetragenus and described as M. an-
tibioticus was found to possess a strong antagonistic action against V.
comma y M. tuberculosis y E. tyfhosaj Ph. tumejaciensy Br. melitensisy
various spore-forming bacteria, numerous cocci, and others.

Diplococci exerted an antagonistic action against various bacteria, in-
cluding pyogenic staphylococci and streptococci in the sputum, spore-
formers, and gram-negative bacteria. They produced, under aerobic
conditions only, a filterable substance that was heat resistant.

The antagonistic action of pneumococci has definitely been estab-
lished. The active substance of these organisms was said to be thermo-
labile, since it was destroyed at 80° to 85° C.j it was produced only
under aerobic conditions. In reviewing the literature on the longevity
of streptococci in symbiosis, Holman (440) observed that many
chances of error are inherent in mixed cultures, particularly with closely
similar organisms j pneumococci, for example, were found to be able to
live for long periods in association with nonhemolytic streptococci.
Peculiar antagonistic relations between pneumococci and staphylococci
were also reported (13). Adaptive alterations could be expected in the
growth of bacteria in mixed cultures (32). Which of the two organisms
antagonizes the other was believed to depend frequently upon the nu-
merical abundance of one or the other (243).

OTHER BACTERIA 105

OTHER AEROBIC AND ANAEROBIC BACTERIA

The antagonistic action o£ K. pneumoniae against B. anthracis has
been reported. Freudenreich (298) found that the filtrate of this an-
tagonist repressed the growth of a number of bacteria, including the
diphtheria and plague organisms.

Other aerobic bacteria were found capable of exerting antagonistic
effects against one or more organisms, these effects varying considerably
in nature and intensity. It is sufficient to mention the action of P. vul-
garis against B. anthracis and P. festis; of Ps. aviseftica against B. an-
thracis and E. tyfhosa; of Bacterium lactis aerogenes against B. an-
thracis and P. festis. B. anthracis is capable of iso-antagonism and of
antagonizing certain other organisms, including E. tyfhosa and Bac-
terium acidi lactici (786). Certain Myxobacteriales have been shown to
be capable of bringing about the lysis of various plant-disease-producing
bacteria 3 a thermostable lytic substance, passing through cellophane but
not through a Seitz filter, was obtained. Although certain bacteria like
Achromobacter lifolyticum were found capable of reducing the patho-
genicity of M. tuberculosis, no active cell-free extract could be ob-
tained (79).

M. tuberculosis produces a water-soluble substance, designated
phthiocol, which in concentrations of 0.05 to o.i per cent inhibited the
growth of various gram-positive and gram-negative bacteria, but not
Ps. aeruginosa (568a).

Bacillus larvae, a gram-negative rod, was found (441) capable of
inhibiting the growth of various gram-positive and gram-negative bac-
teria. The human and bovine strains of M. tuberculosis were also in-
hibited but not the avian strain. The antibiotic was soluble in water but
not in organic solvents. It was adsorbed on activated charcoal but no
eluent could be found. It was moderately heat stable. Its antibiotic ac-
tivity was inhibited by glucose but not by cysteine or sucrose.

The morphology of one bacterium may be considerably modified by
the presence of another. Living cultures of L. bulgaricus influenced the
variation of E. coli from the "S" to the "R" phase, inhibited develop-
ment of the organism, and even brought about its lysis. No active sub-

106 BACTERIA AS ANTAGONISTS

stance could be demonstrated} the lactic acid itself had only a limited
effect (9). Korolev (528) has shown that when a yellow sarcina was
added to solid media a stimulating effect was exerted on the growth of
species of Brucella {Br. melitensisj Br. abortus, Br. suis)-, in liquid
media, however, the activities of these species were repressed, the sar-
cina thus acting as an antagonist. A white staphylococcus exerted an an-
tagonistic action on Brucella species both in liquid and on solid media.

Certain acid-producing aerobes were found capable of inhibiting toxin
production by Clostridium hotulinum in glucose but not in noncarbo-
hydrate media (373). Since acid itself cannot bring about this effect,
Holman (440) suggested that the acid must be active in a nascent state.
A mixture of a Clostridium sforogenes and CI. Botulinum also inter-
fered with the development of the toxin j it was even thought possible
that the first anaerobe might cause the disappearance of toxin already
produced (164, 165). S. aureus, E. coU, P. vulgaris, and other bac-
teria permitted the growth of CI. hotulinum in aerobic cultures, accom-
panied by toxin production (290). However, Streptococcus thermofhi-
lus inhibited the growth of CI. hotulinum, the toxin of the latter being
gradually destroyed (493).

Passini (708) claimed that Bacillus futrificus verrucosus destroyed
M. tuberculosis in nine days. The effect of other anaerobes on the sur-
vival of anthrax spores in dead animals has been extensively studied
(440). Novy (688) reported that the injection into guinea pigs of P.
vulgaris and Clostridium oedem^atiens resulted in rapid death of the
animals and extensive growth of the anaerobe in the animal bodies;
however, the simultaneous inoculation of CI. sforogenes and P. vul-
garis did not result in putrid lesions. According to Barrieu {'^6), the
presence of P. vulgaris and certain nonpathogenic spore-bearing aerobes
in wounds favors, through their proteolytic activity, the virulence of
pathogenic bacteria. Pringsheim (738) grew CI. welchii with Alka-
ligenes fecalis for ten generations on agar slants and could easily detect
in the growth of the latter the opaque colonies of the anaerobe. A lique-
fying sarcina allowed CI. welchii and CI. butyricum to grow in open
tubes. Many war-wound infections were believed (985) to be due to an
association of P. vulgaris with anaerobes, since the former increased the
virulence of CI. ferjringens and others.

OTHER BACTERIA

107

The antagonistic effects of lactic acid bacteria of the L. bulgarkus and
L. acidophilus groups have received considerable attention, especially
in regard to their action against intestinal bacteria. This was believed
to be due to the production of acid by the bacteria rather than to the
formation of specific antagonistic substances. This phenomenon aroused
particular interest because of the function of some of these organisms in
replacing bacterial inhabitants of the human digestive system (526).

Various bacteria also have a marked destructive effect upon plant
pathogenic fungi, as will be shown later. Some produce stable, heat-
resistant, antifungal substances (731 ).

CHAPTER 6

ACTINOMYCETES AS ANTAGONISTS

AcTiNOMYCETES are found in large numbers in many natural sub-
strates. They occur abundantly in soils, composts, river and lake bot-
toms, in dust particles, and upon plant surfaces. Certain species are
capable of causing serious animal and plant diseases.

Actinomycetes, like fungi, produce a mycelium, but they are largely
unicellular organisms of dimensions similar to those of bacteria. Some
of the constituent groups are closely related to the bacteria, others to
the fungi. On the basis of their morphology, the order A ctinomycetales
has been divided into three families, Mycohactenaceae^ Actinomy-
cetaceae^ and Streftomycetaceaey comprising the genera Mycobacte-
riumy Actinomyces y Nocardiay StreftomyceSy and Micromonosfora.
These genera are represented in nature by many thousands of species,
of which several hundreds have been described. A few are shown in
Figure lo.

Comparatively little i^ known of the physiology of actinomycetes.
Some produce certain organic acids from carbohydrates j others prefer
proteins and amino acids as sources of energy, many species being
strongly proteolytic. Some are able to attack starch, with the production
of dextrins and sugar, accompanied by the formation of diastatic en-
zymes. Many reduce nitrates to nitrites. Some attack sucrose and form
the enzyme invertasej others, however, do not. Certain species are able
to utilize such resistant compounds as rubber and lignin. Synthetic
media are favorable for the production of a characteristic growth and
pigmentation. Among the pigments, the melanins have received par-
ticular attention. They range from the characteristic brown to various
shades of black and deep green and are formed in protein-containing
and in some cases also in protein-free media. The other pigments range
from blue, yellow, and orange to various shades of grey.

According to Beijerinck (41), the process of pigment production by
actinomycetes in gelatin media is associated with the formation of a

S. antibiotic lis y important antagonist.
From Waksman and Woodruff (974)

S. hivcjidulary important antagonist

' 4^ -^li

Submerged growth of S. lavendulae.
From Woodruff" and Foster (1031)

S. grlseus, streptomycin-producing str;
Prepared by Waksman and Schat2

*p

V

4

r

>

4

X-

{

^

M. vulgaris.

From Waksman.

Cordon, ar

id Hulpoi (953)

"X^r

Streftomyces 3042, showing close spi
type of branching. Prepared by Starl

Figure 10. Types of actinomycetes.

ACTINOMYCETES AS ANTAGONISTS 109

quinone, which turns brown at an alkaline reaction and in the presence
of oxygen. The action of quinone in the presence of iron was found to
be similar to that of the enzyme tyrosinase. Since an excess of oxygen is
required for the formation of quinone, only limited amounts are found
in deep cultures. The quinone is believed to be formed from the pep-
tone in the medium j although good growth was produced on media
containing asparagine, KNO3, and ammonium sulfate as sources of ni-
trogen, only traces of quinone, if any, were found. The tyrosinase reac-
tion is not involved in the production of all black pigments by actinomy-
cetesj some species produce such pigments in purely synthetic media,
in the complete absence of peptone.

Actinomycetes grow in liquid media in the form of flakes or small
colonies, usually distributed either on the bottom and walls of the con-
tainer or throughout the liquid j often a ring is formed on the surface
of the medium around the wall of the vessel. In many cases, a full sur-
face pellicle is produced, which may be covered with aerial mycelium.
As a rule, the liquid medium does not become turbid, even in the pres-
ence of abundant growth. When grown on solid media, actinomycetes
form small, compact, soft to leathery colonies j a heavy lichen-shaped
mat is produced that may become covered by an aerial mycelium. The
addition of a small amount of agar (0.25 per cent) to a liquid medium is
highly favorable to growth, especially in large stationary containers.

Actinomycetes can also be grown in liquid media in a submerged con-
dition, with suitable agitation and aeration in order to supply oxygen j
the medium may also be kept in shaken state. Growth occurs in the
form of a homogeneous suspension of discrete colonies and mycelial
fragments throughout the liquid. Responses in growth and biochemical
activities as a result of treatments may thus be obtained under more
homogeneous physiological conditions.

Although most actinomycetes are aerobic, some are anaerobic, and
many can grow : t a reduced oxygen tension. The aerobic actinomycetes
commonly found on grasses and in soil are said (511) never to have
been isolated from animal infections. Mixed infections consisting of
anaerobes growing at body temperature together with aerobes have
often been demonstrated. Certain aerobic species also are capable of

no ACTINOMYCETES AS ANTAGONISTS

causing infections in man and other animals, and certain plant diseases
(potato scab, sweet potato pox) are caused by aerobic species of actino-
mycetes.

ANTAGONISTIC PROPERTIES

Many actinomycetes have the ability to antagonize the growth of
other microorganisms, notably bacteria, fungi, and other actinomycetes;
this is brought out in Tables 1 5 and 1 6. The antagonistic species are not
limited to any one genus but are found among three genera, Nocardiay
StreftomyceSy and Mkromonosfora,

Gasperini (322) first demonstrated, in 1890, that certain species of
the genus Strepomyces had a marked lytic effect upon other micro-
organisms. He emphasized that "Streftothrix develops habitually in a
spontaneous manner upon the surface of bacteria and fungi, upon which
it lives to a limited extent in the form of a parasite, due to the faculty
that its mycelium possesses to digest the membrane from these lower
fungi." Greig-Smith (364, 365) found that soil actinomycetes are an-
tagonistic to not only bacteria but also certain fungi j since actinomy-
cetes grow abundantly in normal soils, it was suggested that they may
become an important factor in limiting bacterial development. Lieske
established (571) that specific actinomycetes are able to bring about
the lysis of many dead and living bacterial cells; they are selec-
tive in their action, affecting only certain bacteria such as S. aureus and
S. fyogenesy but not S. lutea, S. marcescens, or Ps. aeruginosa.

Rosenthal (805) isolated from the air an actinomyces species which
he designated as the true biological antagonist of the diphtheria or-
ganism. He inoculated the surface of an agar plate with an emulsion of
the bacteria and inoculated the actinomyces into several spots. At the
end of two days, the plate was covered with the diphtheria organisms,
but the colonies of the actinomyces were surrounded by large trans-
parent zones. In another method utilized, agar was mixed with an emul-
sion of the diphtheria bacteria killed by heat, and the mixture was
poured into plates. After solidification of the medium, the antagonist
was inoculated in several spots on the plates. Its colonies gradually be-
came surrounded by clear zones, thus proving that it produced a lytic

ANTAGONISTIC PROPERTIES 111

TABLE 15. ANTAGONISTIC PROPERTIES OF VARIOUS ACTINOMYCETES

ANTAGONIST

ORGANISMS AFFECTED

KNOWN PROPERTY

REFERENCES

S. alius

Pneumococci, strepto-

Thermolabile sub-

354, 357>

cocci, staphylococci,

stance, causes lysis

looi, 1002

Ps. aeruginosa, etc.

of dead cells

S. alius

Various fungi

Protein, enzyme,
causes lysis of dead
and certain living
bacteria

10-12

S. antibioticus

All bacteria and fungi,

Thermostable sub-

976

especially gram-posi-
tive types

stance, bacterio-
static

S. griseus

Gram-positive and gram-
negative bacteria, not
fungi or anaerobic
bacteria

Produces streptomy-
cin

830

S. lavendulae

Various gram-positive

Produces streptothri-

979

and gram-negative
bacteria

cin

S. fraecox

S. scabies

644

Streftomyces sp.

Bacteria and fungi

Lytic action

322

Streftomyces sp.

Diphtheria

Growth inhibition

805

Streftomyces sp.

B. mycoides, proactino-
mycetes, mycobacteria

Bactericidal action,
with or without
lysis

76,534

Streftomyces sp.

Fusarium

Lytic action

633

A'', gardneri

Gram-positive bacteria

Bacteriostatic action

313,958

Micromonosfora

Gram-positive bacteria

Thermostable active
substance produced

958

Actinomycetes

Dead and living bacteria

Lysis

571

Actinomycetes

Spore-forming bacteria

Repression of growth

364, 1000

Actinomycetes

Gram-positive bacteria

Thermostable sub-
stance, produced on
synthetic media,
resembles lysozyme

536,671

Actinomycetes

Pythium

Thermostable sub-
stance

908

2 ACTINOMYCETES AS ANTAGONISTS

TABLE 1 6. ANTIBACTERIAL SPECTRUM OF CERTAIN ANTAGONISTIC
ACTINOMYCETES

TEST ORGANISM

ZONE OF INHIBITION.

, IN MILLIMETERS

S. violaceus

5. aurantiacus

S. griseus

S. gl obis for us

N. rubra

35

32

0

0

N. corallina

40

45

22

10

N. alba

40

25

0

0

M. rubrum

40

33

10

0

M. citreum

38

37

0

0

M. tuberculosis

8

10

0

0

M. smegmae

10

8

O

0

M. fhlei

20

25

0

0

Corynebacterium sp.

12

10

0

0

E. coli

0

0

0

0

S. aureus

25

19

0

0

M. ruber

35

28

0

0

B. mycoides

30

10

0

0

B. megatherium

33

5

0

0

B. mesentericus

30

2

0

0

B. subtilis

23

I

0

0

B. tumescens

22

0

0

0

Ps. fluorescens

0

0

0

0

Ps. aeruginosa

O

0

0

0

P. vulgaris

0

o

0

0

S. marcescens

0

o

0

0

M. luteus

30

25

0

0

M. candicans

37

22

0

0

M. roseus

42

27

0

0

M. lysodeikticus

38

33

o

0

S. lutea

30

27

0

0

A%, vinelandii

3

0

0

o

Az. chroococcum

5

0

0

0

Rh. leguminosarum

0

0

0

0

Radiobacter

0

0

0

0

From Krassilnikov and Korenlako (534)-

substance that diffused through the agar and dissolved the diphtheria
cells.

Gratia and Dath (357) suspended dead cells of staphylococci and
other bacteria in 2 per cent agar and exposed the plates to the air. A cul-

ANTAGONISTIC PROPERTIES 113

ture of a white actinomyces developed on the plates, each colony being
surrounded by a clear zone of dissolved bacterial cells. By transferring
this culture to a suspension of dead staphylococci in sterile saline, a
characteristic flaky growth was produced, the bacterial suspension be-
coming clarified in 36 hours. When the lysed emulsion was filtered, the
filtrate could again dissolve a fresh suspension of dead staphylococci.
This culture was found able to attack all staphylococci tested as well as
certain gram-negative bacteria, such as Ps. aeruginosa; however, it was
inactive against M. tuberculosis and E. coli. Some antagonistic strains
could also attack E. coli, though this property was readily lost.

This type of antagonism was believed to be widely distributed in na-
ture and to be directed against many bacteria, pathogenic and sapro-
phytic. The culture of the antagonist in bouillon gave a very active
agent, whereas the lysed bacterial suspension was weaker in its action.
The active substance was present extensively in old cultures and was
fairly stable. The material obtained by lysing the suspension of bacteria
by means of an antagonist was designated as "mycolysate." It did not
possess the toxicity of the nonlysed suspension but it preserved its anti-
genic properties {2>S^)' Gratia (354) also reported that actinomycetes
were able to attack living cells of bacteria, except E. coli and E. tyfhosa
which had to be first killed by heat before they could be dissolved.

Welsch (100 1, 1002) made a detailed study of the lytic activity of
an actinomyces culture, presumably identical with that employed by
Gratia and later described as Actinomyces alhus. The culture was grown
in different media, the best results being obtained in very shallow layers
of ordinary bouillon. The active substance present in the filtrate was
designated as "actinomycetin." It was able to dissolve, at least partly,
all dead bacteria, whether killed by heat or by chemicals, gram-positive
or gram-negative, though gram-negative bacteria were, as a rule, more
susceptible. The growing culture of the antagonist brought about better
clarification (lysis) of the bacterial suspension than the filtrate. The
solubilizing properties of the active agent, its susceptibility to heat and
to ultraviolet rays, its size as measured by ultrafiltration, suggested its
protein nature. The kinetics of its action pointed to its being an enzyme.
It was precipitated by acetone, alcohol, and ammonium sulfate. Most
of the gram-negative bacteria were not attacked either by actinomycetin

114 ACTINOMYCETES AS ANTAGONISTS

or by the living culture of the antagonist. Only a few of the gram-
positive bacteria, including certain pneumococci and streptococci, could
be dissolved by sterile actinomycetin. A definite parallelism in the ac-
tivity of the preparation against dead bacteria and of the living culture
against living bacteria suggested that the same substance is concerned
in both cases. The bacteria were therefore divided ( looo), on the basis
of their relation to actinomycetin, into three groups :

Bacteria that were lysed by the culture filtrate; these included pneumo-
cocci and hemolytic streptococci

Bacteria that were not dissolved even by the most active soluble sub-
stance, but which were depressed by the mycelium of the living ac-
tinomyces; these comprised various sarcinae and fluorescens types

Bacteria that were not acted upon by either the living culture or the
actinomycetin preparation ; these included the colon-typhoid and the
pyocyaneus groups, though when the latter were killed by heat or
inactivated by radium emanations, as in the case of E. colt, or were
placed under conditions unfavorable to multiplication, they were dis-
solved by the lytic substance.

The first detailed survey of the distribution of antagonistic organisms
among actinomycetes was made by a group of Russian investigators.
According to Borodulina (76), actinomycetes are able to antagonize
various spore-forming bacteria and to bring about the lysis of their liv-
ing cells. A thermostable substance was produced on agar media. The
activity of this substance was greatly reduced at an alkaline reaction,
whereas an acid reaction favored it. When B. mycoides and an antago-
nist were inoculated simultaneously into peptone media, no inhibitive
effect was obtained, because the bacterium changed the reaction of the
medium to alkaline, thereby making conditions unfavorable for the
production of the antibiotic substance by the antagonist. When the an-
tagonist was first allowed to develop in the medium, before the bac-
terium was inoculated, a strong antagonistic effect resulted, which led
to the elongation of the vegetative cells of B. mycoides; this was due to
a delay in fission and was accompanied by the suppression of spore
formation.

Krassilnikov and Koreniako (534) found that many species of actino-
mycetes belonging to the genus Streftomyces but not Nocardia pro-

ANTAGONISTIC PROPERTIES

115

duced a substance that possessed a strong bactericidal action against a
large number of microorganisms. This substance was particularly active
against nocardias, mycobacteria, and micrococci ; it was less active upon
spore-bearing bacteria and had no action at all on nonspore-forming
bacteria, as illustrated in Table i6. Under the influence of the anti-
biotic factor, the microbial cells were either entirely lysed or killed with-
out subsequent lysis. The action upon spore-bearing bacteria was bac-
teriostatic but not bactericidal. The nonspore-forming bacteria, includ-
ing species of Rhizobiutn and Azotobacter, not only were not inhibited
but were actually able to develop in filtrates of the antagonists.

Of 80 cultures of actinomycetes isolated from different soils, 47 pos-
sessed antagonistic properties, but only 27 of them secreted antibiotic
substances into the medium (Table 17). These agents were capable of
inhibiting the growth of gram-positive but not of gram-negative bac-
teria or fungi. The nature of the action of the various antagonists was

TABLE 17. OCCURRENCE OF ANTAGONISTIC ACTINOMYCETES IN
DIFFERENT SOILS

TOTAL STRAINS

NUMBER OF

STRAINS W^HICH

OF ACTINOMY-

ANTAGONISTIC

LIBERATED TOXIC

NATURE OF SOIL

CETES TESTED

STRAINS

SUBSTANCES

Chernozem

24

10

9

Podzol

11

Solonets

4

High altitude soil

9

Sandy soil

6

Dry desert soil

5

River bank meadow

14

Cultivated soil

7

—

—

—

Total

80

47

27

From Nakhimovskaia (671).

found not to be identical. Some excreted water-soluble substances into
the medium, others did not. All the antibiotic agents were thermo-
stable, since heating for 30 minutes at 1.5 atmospheres only reduced
somewhat their activity. For those antagonists which did not excrete

116 ACTINOMYCETES AS ANTAGONISTS

any substance into the medium, the presence of the growing antagonist
was essential in order to bring about an inhibition of bacterial develop-
ment. On the basis of their sensitivity to the antibiotic substance of
actinomycetes, mycobacteria could be differentiated from nonspore-
forming, especially nodule-forming, bacteria. The production of the
antibiotic substance was highest in synthetic media and was rather weak
or even totally absent in media that contained proteins. The substance
was filterable and was able to resist the effect of radiation.

It was further reported (671) that the antagonistic effects of actino-
mycetes were manifested not only in artificial media but also in soil, the
interrelations here being much more complex. Some of the strains that
produced antagonistic effects in artificial nutrient media were ineffec-
tive under soil conditions. The antagonistic action was more intense
in light podzol soils and was greatly reduced in heavy or chernozem
soils. One of the factors that resulted in a decrease in the antagonistic
properties of actinomycetes in the heavy soils was apparently the high
content of organic matter. By adding peptone to a light soil, the antago-
nistic action of the actinomycetes was greatly weakened. When actino-
mycetes were allowed to multiply in the soil before inoculation with
B. mycoidesy the antagonistic effect was highly pronounced even in the
presence of high concentrations of peptone.

An attempt to isolate an antibiotic substance from some of the soil
actinomycetes was made by Kriss {S'i^)- On the basis of its properties
he was led to conclude that this substance could be classified definitely
with lysozyme. It was insoluble in ether, petroleum ether, benzol, and
chloroform, and was resistant to the effects of light, air, and high tem-
peratures. The optimum reaction for the production of this substance by
Streftomyces violaceus was found to be ^H 7.1 to 7.8, the activity not
being increased by selective cultivation. On the basis of its properties,
this substance could hardly be classified with egg-white lysozyme. It
must be concluded also that the differences in the antibiotic properties
of the various antagonistic actinomycetes isolated by the Russian investi-
gators definitely point to the fact that more than one antibiotic substance
was involved.

In a more recent survey (958) of the distribution of antagonistic ac-
tinomycetes in soils and in composts, it was found that of 244 cultures

ANTAGONISTIC PROPERTIES

117

isolated at random from different soils, 49, or 20 per cent, of the cultures
were actively antagonistic} 57, or 23 per cent, showed some antagonistic
properties; and 138, or 57 per cent, possessed no antagonistic action at
all (Table 18). A somewhat similar distribution of antagonistic prop-
erties was observed among a group of well-identified species taken from
a type culture collection, embracing 161 pure strains. Only one of the
members of the genus Nocardia proved to be antagonistic ; only one of
the Micromonospora forms was active. Most of the antagonists were
found among the members of the genus Stre-ptomyces. These cultures
were also examined for bacteriolytic properties, living S. aureus being

TABLE 15. ISOLATION OF ANTAGONISTIC ACTINOMYCETES
FROM VARIOUS SUBSTRATES

GROUP I

GROUP n

GROUP III

GROUP IV

TTITAT.

Percent-

Percent-

Percent-

Percent-

SOURCE OF CULTURES

Cul-

age of

Cul-

age of

Cul-

age of

Cul-

age of

ORGANISMS ISOLATED

tures

total

tures

total

tures

total

tures

total

Fertile, ma-

nured, and

limed soil

74

20

27.0

5

6.8

I

1-3

48

64.9

Infertile, un-

manured.

limed soil

75

11

14.7

8

10.7

4

5.2

52

69-3

Potted soil

13

I

7-7

I

7-7

0

0

II

84.6

Potted soil, en-

riched with

E. coli

21

I

4.8

4

19.0

4

19.0

12

57.2

Potted soil, en-

riched with

mixtures of

bacteria

15

12

80.0

2

13-3

0

0

I

6.7

Lake mud

9

3

33.3

4

44.4

0

0

2

22.2

Stable-manure

compost

37

'

2.7

20

54.0

4

10.8

12

324

Total

244

49

20.1

44

18.0

13

5-3

138

56.6

From Waksman, Horning, Welsch, and Woodruff (958).

Note. The organisms in group I were the most active antagonists, those in groups II and III had more limited

antigonistic properties, and those in group IV showed no antibacterial effects with the methodg used.

118 ACTINOMYCETES AS ANTAGONISTS

used as the test organism. On this basis, 87 cultures (53.1 per cent)
were found to be inactive, 53 cultures (32.3 per cent) were moderately
active, and 24 cultures (14.6 per cent) were highly active. The conclu-
sion was reached (1000) that bacteriolytic activities against killed bac-
teria and living gram-positive bacteria are widely distributed among
the actinomycetes. Growth-inhibiting properties of actinomycetes were
found to be significantly associated with bacteriolytic action upon living
gram-positive bacteria.

Certain actinomycetes also show antagonistic activities against fungi
(10-12, 908). S. albus was capable of inhibiting the growth of all the
species of fungi tested, an effect shown to be due to the production
of an active substance. By the use of a culture of Colletotrkhum gloe-
osforioideSj the antagonistic activities of 80 type cultures of actino-
mycetes were measured. The antagonist was allowed to grow for 5 days
on maltose agar, at fH 7.4, and the fungus was then inoculated. The
cultures of actinomycetes were divided, on this basis, into three groups:
strong, weak, and noninhibitors. The first group comprised 17.5 per
cent of the cultures j the second, 38.8 per centj and the third, 43.7 per
cent. These results are surprisingly similar to those reported for the
distribution of actinomycetes possessing antibacterial properties, includ-
ing those that were isolated at random from the soil and those taken
from a culture collection.

Meredith (633) made a survey of the distribution of organisms an-
tagonistic to Fusarium oxys forum cubense in Jamaica soils j most of
these antagonists belong to the actinomycetes. The antagonists were not
evenly distributed in the various soil samples, 10 of the d^i samples giv-
ing 44 per cent of the antagonistic organisms. Those actinomycetes that
were antagonistic to Fusarium when grown in their own soil-solution
agar were not always antagonistic when tested in soil-solution agar pre-
pared from other soil. A culture of actinomyces isolated from a compost
produced lysis of the Fusarium. When spores of both organisms were
mixed in an agar medium, the fungus developed normally for two days
but began to undergo lysis on the fifth day, large sections of the my-
celium disappearing. On the seventh day only chlamydospores were ob-
served. In 9 days the fungus completely disappeared, the actinomyces
making a normal growth.

NATURE OF ANTIBIOTIC SUBSTANCES 119

NATURE OF ANTIBIOTIC SUBSTANCES

It has already been established that antagonistic actinomycetes read-
ily produce a variety of different types of antibiotic substances. Some of
these have been isolated and even crystallized and information has been
gained concerning their chemical nature. Others have been obtained in
the form of crude but highly active preparations. Still others are known
but they have not been isolated as yet and have, therefore, been rather
insufficiently studied. So far, eight substances have been definitely
recognized: actinomycetin, actinomycin, streptothricin, streptomycin,
proactinomycin, micromonosporin, litmocidin, and mycetin.

Among the various antagonistic actinomycetes, five species have
been studied in detail and, therefore, deserve particular attention,
namely, S. antibioticus (974), S. lavendulae (979), 5. griseus (830),
A'', gardneri (313), and S. albus (1000).

S. antibioticus produces a highly active antibiotic substance that has
been isolated and described as actinomycin. It was shown to be antago-
nistic to all species of bacteria tested as well as to many fungi (Table
19). Actinomycin is not affected by heat. It is soluble in ether and in
alcohol as well as in other solvents, but in water only in very high dilu-
tions. It is highly toxic to animals.

Several species of actinomycetes are capable of producing actinomy-
cin in both organic and synthetic media, the yield varying with the or-
ganism (955a, 1002a). The addition of 0.25 per cent agar to stationary
cultures increases considerably the growth of the organism and the pro-
duction of actinomycin. The presence of a small amount of starch, phos-
phate, and sodium chloride was also found to be favorable. Actinomy-
cin-producing forms are strictly aerobic, and are able to produce actino-
mycin only when grown either in very shallow layers or under aerated
or agitated submerged conditions.

S. lavendulae is capable of inhibiting the growth of many gram-
negative and gram-positive bacteria. It produces an antibiotic substance
designated as streptothricin.

For the production of streptothricin, the tryptone can be replaced by
a variety of simple nitrogenous compounds, such as glycine (Table
20), alanine, aspartic acid, asparagine, and glutamic acidj the carbo-

120

ACTINOMYCETES AS ANTAGONISTS

TABLE 19. BACTERIOSTATIC SPECTRUM OF ACTINOMYCIN

ACTINOMYCIN ADDED, MILLIGRAMS

ORGANISM

GRAM STAIN

PER LITER OF MEDIUM

O.I

I.O

10

100

S. marcescens

-

3

3

3

3

A . aero genes

-

3

3

3

3*

E. coli (intermediate)

-

3

3

3

3*

E. coli

-

3

3

3

I*

Ps. aeruginosa

-

3

3

3

0

Ps. fluorescens

-

3

3

3

0

Br. abortus

-

3

3

3

0

N. catarrhalis

-

3

3

2

0

E. carotovora

-

3

3

2

0

SA. ga/linarum

-

3

2

2

0

A . stutzeri

-

3

2

I

0

H. fertussis

-

3

3

0

0

Az. vinelandii

-

3

0

0

0

S. cellulosae

+

3

2

I

0

S. calif ornicus

+

3

3

2

0

M. tuberculosis

+

3

3

0

0

CI. welchii

+

3

0

0

0

B. macerans

.+

3

3

0

0

B. megatherium

+

3

0

0

0

B. folymyxa

+

3

0

0

0

B. mycoides

+

I

0

0

0

B. mesentericus

+

I

0

0

0

B. cereus

+

I

0

0

0

B. subtilis I

+

0

0

0

0

B. subtilis II

+

0

0

0

0

• G. tetragena

+

0

0

0

0

S. lutea

+

0

0

0

0

Streptococci and staphylococci +

0

0

0

0

From Waksman and Woodruff (975).

Note, o indicates no growth; i, trace of growth; 2, fair growth; 3, good growth.

* If 200 mg. per liter were added the results were usually as follows: for A. aero genes, fair; for

E. coli (intermediate), trace; for E. coli, no growth.

hydrate may be left out completely, with only limited reduction in ac-
tivity. No growth of the organism is obtained on tryptophane, phenyl
alanine, and certain other forms of nitrogen. Good growth may be ob-
tained with ammonium sulfate or sodium nitrate, but the production of

NATURE OF ANTIBIOTIC SUBSTANCES

121

TABLE 20. GROWTH AND PRODUCTION OF STREPTOTHRICIN BY
S. LAVENDULAE

TREAT-

DAYS

GROWTH

IN MG,

ACTIVITY

IN UNITS

SOURCE OF

MENT OF

OF INCU-

PER 100 ML.

E. ~ '

U. sub-

NITROGEN

CULTURE

BATION

OF MEDIUM

colt

tilis

Tryptone

Shaken

2

346

150

1,000

Tryptone

Shaken

5

253

ICO

1,000

Glycine

Shaken

2

162

30

30

Glycine

Shaken

5

266

100

500

Tryptone

Stationary

8

245

20

200

Glycine

Stationary

8

239

25

150

From Waksman (946).

Note. The organism was grown in i per cent starch medium.

the active substance is limited unless the organism is grown under sub-
merged conditions. Iron appears to play an essential role in the produc-
tion of the active substance. An increase in growth as a result of an in-
crease in the amino-acid concentration, with the same amount of carbo-
hydrate, causes an increase in the production of streptothricin. An in-
crease in growth as a result of an increase in carbohydrate concentration
does not.

When the medium contains one amino acid as the only source of car-
bon and nitrogen, there is gradual increase in the alkalinity of the
medium, resulting in the destruction of the streptothricin. Neither the
growth of the organism nor the production of the streptothricin, how-
ever, is influenced by the reaction of the medium, within certain limits,
even between ^H 4.4 and 8.0 ( 1028). The metabolism of S. lavendulae
and the course of production of streptothricin under stationary and sub-
merged conditions are illustrated in Figure 1 1 . The bacteriostatic spec-
trum of streptothricin is shown in Table 2 1 . It has a certain delayed,
even if limited, toxicity to animals and is active in vivo against both
gram-positive and gram-negative bacteria (792).

Different strains of S. lavendulae differ greatly in their ability to pro-
duce streptothricin. The possibility that other species of Streftomyces
zre also capable of producing streptothricin or closely related com-
pounds, as indicated by somewhat different antibiotic spectra, has also

aamnniiAi ai3d simn

1
j

/ 1

(

>

Q^

<

z

o

1- d

1-

X \

<

o \

1-

u <

w

5 \

i

> \

\

Q^ \

K -

Q

X \

^

\ \

^4. \

li^-A -

1

1 1 \

aaniino jo s?i3±nn"iii^ 99 2i3d sNvajDiiii^M

TABLE 21. INHIBITORY EFFECT OF STREPTOTHRICIN UPON GROWTH
OF VARIOUS BACTERIA

CRUDE STREPTOTHRICIN ADDED,

ORGANISM

B. subtilis

B. mycoides

B. macerans

B. megatherium

B. folymyxa

B. cereus

M. lysodeikticus

S. muscae

S. lutea

A . aerogenes*

A . aero genes

E. coU\

E. colt (4348)

S. marcescens

S. m^cescens

Ps. fluorescensX

Sh. gallinarum

P. fseudotuberculosis

Br. abortus

S. cholerasuis

S. schottmulleri

S. abortivoequtTia

S. tyfhimurium

H. suis

H. influenzae

Br. abortus

Az. agile

Az. vinelandii

Az. chroococcum

Az. indicu?n

M. fhlei

CI. butyricum^

L. casei^

S. a/bus

S. violaceus-ruber

S. lavendulae

;ram

:S per 10 CUBIC CENTIMETERS AGAR

I

0.3

0.1

0.03

O.OI

0

0

0

0

I

2

2

2

2

2

2

2

2

2

2

0

0

0

2

0

2

2

2

2

2

2

2

0

0

I

2

0

0

I

2

0

0

0

2

0

I

2

2

0

0

Tr

2

0

0

0

2

0

Tr

I

2

I

2

2

2

I

2

2

2

2

2

2

2

0

0

0

2

0

0

Tr

2

0

0

0

2

0

0

Tr

2

0

0

I

2

2

0

0

Tr

2

2

0

0

2

2

2

0

0

2

2

2

0

0

0

0

I

0

0

0

2

2

0

0

0

0

2

0

0

0

0

2

0

0

Tr

2

2

0

0

2

2

2

0

0

I

2

2

2

2

2

2

2

0

0

2

2

2

0

0

I

2

2

0

I

2

2

2

2

From Waksman and Woodruff (979).

Note, o indicates no growth; I, limited growth; 2, good growth; Tr, trace of growth.

• Representing 3 distinct strains.

t Representing 5 strains of E. coll obtained from different sources.

t Representing 4 strains.

§ Cultured anaerobically.

124-

ACTINOMYCETES AS ANTAGONISTS

been established (969). Other members of the genus are capable of
forming different antibiotics (498).

S. grlseus produces an antibiotic substance, designated as streptomy-
cin, that is also active against both gram-positive and gram-negative
bacteria. It is similar in its solubility and certain chemical properties to
streptothricinj however, it acts readily against B. mycoides and is more
active than streptothricin against certain gram-negative bacteria, such
as M. tuberculosis, S. marcescens, and Ps. aeruginosa. The bacteriostatic
spectrum of streptomycin is given in Table 22. Streptomycin is also
active in vivo against a variety of other bacteria, as shown later. It is
also active against spirochetes, but it is not active against fungi, ana-
erobic bacteria or viruses (791).

TABLE 22. BACTERIOSTATIC SPECTRUM OF STREPTOMYCIN

ORGANISM

n

ORGANISM

f^S

A. aero genes

0.5-2.5

M. tuberculosis, hominis

0.15

B. anthracis

0.375

N. gonorrhoeae

5.0

B. megatherium

0.25-3.0

P. pestis

0.75-1.5

B. mycoides

0.1-3.8

P. tularensis

0.15-0.3

B. subtilis

0. 1 2-1.0

Ph. fruni

0.25

Br. abortus

0.5-3-75

Pr. vulgaris

0.4-3-0

Br. suis

0.5

Ps. aeruginosa

2.5-25.0

CI. butyricum

8.0

Ps. fiuorescens

12.5

CI. tetani

>i04

S. lutea

0.25

C. difhtheriae

0.375-3.75

5. marcescens

I.O

D. pneumoniae

8.0

5. enteritidis

0.5

E. tyfhi

I -0-37.5

S. schottmillleri

2.0

Er. muriseftica

2-5

Sh. paradysenteriae

0.25-3.75

E. coli

0.3-3-75

S. aureus

o.5->i6.o

H. influenzae

1.56-5.0

S. hemolyticus

2.0->l6.0

H. -pertussis

1.25-3-0

S. viridans

>i6

L. monocytogenes

2.5

V. comma

6.0-37.5

K. -pneumoniae

0.625-8.0

A. bovis

3-75

M. mallei

10->10.0

N. asteroides

12.5

M. avium

lO.O

S. antibioticus

<o.4

M. fhlei

0.12

S. lavendulae

1.25

From Waksman and Schatz (970).

Note. Unit of activity is the number of micrograms of streptomycin per

required to inhibit growth.

iter of suitable medium

NATURE OF ANTIBIOTIC SUBSTANCES 125

One of the important cultural characteristics of the production of
streptomycin is the need for a specific substance in the medium, as found
in meat extract, corn steep, or the mycelium of the actinomyces. On a
medium containing sodium nitrate or an amino acid as a source of nitro-
gen, the organism produces good growth but little antibiotic activity.
However, when the mycelium thus formed in the culture is added to
fresh medium, streptomycin production takes place. This indicates that
the organism is capable of synthesizing the streptomycin "precursor,"
but not in sufficient amounts to influence the production of the antibiotic
in the culture. For rapid and abundant formation of streptomycin, the
presence of a "precursor" in the medium is required. The presence of a
small amount of carbohydrate, such as glucose, is also favorable to
growth and the production of the streptomycin. The reaction of the
medium changes first to acid and later to alkaline, the highest alka-
linity (^H 8.2 to 8.6) corresponding to the maximum production of
the streptomycin. When the culture begins to undergo lysis, there is
another increase in acidity of the culture, accompanied by an increase in
viscosity, due to the formation of slimy material.

Growth and activity reach a maximum in stationary cultures in 8 to
10 days, at 28° C, and in submerged cultures in 2 to 3 days. The latter
depends largely on the nature and amount of inoculum: with a heavy
inoculation of pregerminated spore material the maximum may be
reached in 36 to 48 hours.

Streptomycin is produced only by certain strains of S. griseus. Even
the active culture may gradually deteriorate (832) due to the forma-
tion of inactive strains or spore-free variants, which are unable to form
the antibiotic. For the successful production of streptomycin, it is essen-
tial, therefore, continuously to select active strains from the mother
culture.

The streptomycin is found in the culture filtrate, from which it can
be isolated by suitable methods. However, the mycelium of the organ-
ism appears to contain small amounts of a second antibiotic factor which
is soluble in ether, and which has a bacteriostatic spectrum distinct from
that of streptomycin. This second factor has not been sufficiently
studied.

A'', gardneri produces an active bacteriostatic substance that has been

126 ACTINOMYCETES AS ANTAGONISTS

designated as proactinomycin (313). Its bacteriostatic spectrum is
shown in Table 23. It is produced on both synthetic and organic media.
It is effective chiefly against gram-positive bacteria, although to a more
limited extent than actinomycin.

S. violaceus produces on synthetic media a substance partly soluble
in water, and largely soluble in alcohol, benzene, chloroform, and di-
chlorethane, but not in ether. It gives a deep violet color in alcoholic
solution and is active against gram-positive bacteria, less so against
mycobacteria and corynebacteria, and not at all against gram-negative
bacteria. It was designated as mycetin (245, 533a). Various gram-nega-
tive bacteria, as well as pus, reduce its activity.

A^. cyanea (Pr. cyaneus) produces a pigment, designated litmocidin,
which is related to the anthocyanins and inhibits the growth of various
bacteria in vitro but not in vivo (8oa, 323b).

TABLE 23. BACTERIOSTATIC EFFECT OF PROACTINOMYCIN

APPROXIMATE DILUTION OF
MATERIAL IN MILLILITERS
ORGANISM GIVING HIGHEST EFFECT

D. fneumoniae 1,500,000

S. fyogenes 500,000

S. aureus 500,000

A'', meningitidis 500,000

B. anthracis 500,000

V . comma 6,000

.y. tyfhi, S. faratyfhi B, Shigella, E. coli 2,000

From Gardner and Chain (313).

S. alhus forms a bacteriolytic substance designated as actinomycetin,
which is a protein and is enzymatic in nature. Its lytic action upon bac-
teria was visualized by Welsch (looo) as a two-step reaction: first,
the susceptible cells are killed by the selectively bactericidal lipoid j sec-
ond, those dead cells are dissolved by the bacteriolytic enzyme, which
alone is responsible for the lysis of heat-killed bacteria. The phenome-
non does not take place in complex culture media, since the bactericidal

NATURE OF ANTIBIOTIC SUBSTANCES 127

action of the lipoid is greatly impaired under those conditions j the pres-
ence of the living actinomyces is generally necessary, since free lipoid
should be secreted in the susceptible suspension.

Certain actinomycetes can produce agents that are capable of exerting
a lytic effect not only upon the organisms that produce them but also
upon other organisms ( lOi i ). The formation of an autolytic substance
by a thermophilic actinomyces was also demonstrated (492, 531). The
filtrates of such lysed cultures were said to offer promise in the treat-
ment of actinomycosis caused by Actinomyces bovis (185).

In the case of some actinomycetes, like S. griseus, the lysis of the cells
appears to be a definite stage in the life cycle of the organism. This is
observed particularly in rapidly growing submerged cultures, especially
when they are inoculated not with fresh spores but with cell growth
previously obtained under submerged conditions. The production of
streptomycin is definitely associated with the lysis of the culture j when
lysis progresses, not only does streptomycin formation cease completely
but the substance already formed may be rapidly destroyed.

Despite a seeming similarity in their growth characteristics, and de-
spite the fact that some investigators (356) assumed that all actinomy-
cetes are able to act as antagonists, it is now definitely established (534,
971, 974) that one is dealing here with highly specific types or strains.
Although many actinomycetes yield, either in the culture filtrate or in
the mycelium, an ether-soluble substance that has some bacteriostatic
activity, only certain species, such as S. antibioticusy are capable of pro-
ducing the typical actinomycin.

Both the quantitative production of the substance and the presence of
other substances depend entirely upon the culture of the organism. One
is dealing here both with strain specificity and with species character-
istics.

Strains can be isolated from antagonistic actinomycetes that are com-
pletely inactive. This phenomenon seems to be correlated with the
type of growth of the organism; nonsporulating strains of S. griseus,
for example, do not produce streptomycin. The formation of this anti-
biotic agent is associated definitely with the growth of certain sporulat-
ing strains of this organism (832).

128 ACTINOMYCETES AS ANTAGONISTS

ANTAGONISTIC EFFECTS OF ACTINOMYCETES

AGAINST AGENTS PRODUCING

PLANT DISEASES

Various species of Streftomyces are also strongly antagonistic against
bacteria causing plant diseases, such as Bacterium solanacearum (426).
According to McCormack (593), aerobic conditions are necessary for
the development of the antagonistic properties of actinomycetes ; those
requiring less oxidized conditions are themselves antagonized. B. mega-
theriumy for example, was said to be antagonistic to certain species but
was antagonized by others. Vs. -jtuorescens^ however, was antagonistic
to actinomycetes as a whole, causing their lysis.

Actinomycetes possess antagonistic properties not only against bac-
teria but also against some other actinomycetes. The more aerobic spe-
cies are antagonistic to the less aerobic types. Millard (644) believed
that he succeeded in controlling potato scab caused by Streftomyces
scabies by the use of green manures such as grass cuttings. The develop-
ment of scab on potatoes grown in sterilized soil and inoculated with
S. scabies was reduced by the simultaneous inoculation of the soil with
Streftomyces fraecoXy an obligate saprophyte. By increasing the pro-
portion of the latter organism to the pathogen, the degree of scabbing
on the test potatoes was reduced from 100 per cent to nil. The sterilized
soil provided sufficient nutrients for the development of the antagonist
and only a small increase in the control was obtained when grass cut-
tings were added and sterilized along with the soil.

Sanford (820) was unable, however, to control potato scab by the
inoculation, with S. scabies and S. fraecox, of both steam-sterilized and
natural soil containing different amounts of green plant materials.
These organisms were perfectly compatible on potato dextrose agar, as
well as in a steam-sterilized soil medium. The control of scab, there-
fore, was said to have been due not to the direct action of S. fraecox
but to certain other undetermined microorganisms favored by the pres-
ence of the green manure. S. scabies is very sensitive to various products
of fungi and bacteria. When grown in close proximity to various bac-
teria, the acid production of the latter inhibited S. scabies to a consider-
able degree. Its complete inhibition was not due to the acid reaction

IN VIVO ACTIVITY 129

alone, however, since a certain bacterium was isolated from the soil
which definitely inhibited the growth of this plant pathogen.

Goss (347) observed that the severity of scab is dependent on the
amount of S. scabies present in the soil, which was believed to be con-
trolled by the soil microflora. No evidence was obtained as to whether
the effect of the soil flora on S. scabies was due to specific organisms.
Kieszling (502) isolated two cultures of bacteria which were antagonis-
tic to S. scabies; when added to the soil, these bacteria prevented the
development of scab on potatoes.

IN VIVO ACTIVITY OF SUBSTANCES PRODUCED
BY ACTINOMYCETES

Just as the chemical nature of the antibiotic agents produced by ac-
tinomycetes varies, so does the action of these agents in the animal body.
Some, like actinomycin, are very toxic, whereas others, like streptothri-
cin and streptomycin, have low toxicity and give great promise of prac-
tical application. Because of the activity of streptothricin and strepto-
mycin against gram-negative bacteria and because of the lack of reliable
chemotherapeutic agents active against these bacteria, the utilization of
these substances in the treatment of certain diseases caused by such bac-
teria has become very significant. Some preparations, like actinomyce-
tin, have been utilized in the preparation of a bacterial hydrolysate
(mycolysate) for vaccination purposes. Streptomycin has come to be
recognized as an important chemotherapeutic agent in the treatment of
a number of infectious diseases.

CHAPTER 7

FUNGI AS ANTAGONISTS

The antagonistic interrelationships in which fungi are involved com-
prise the following reactions: (a) the antibacterial activities of fungi j
(b) the antagonistic effects of fungi upon fungi; (c) the effects of bac-
teria and actinomycetes upon fungi; (d) the action of fungi upon in-
sects and other animal forms. From the point of view of practical utiliza-
tion, two aspects deserve special consideration: (a) the utilization of
fungi for combating human and animal diseases; (b) the antagonistic
interrelationships of fungi with o'ther organisms, since fungi comprise
the most important group of microorganisms that cause plant diseases.

ANTIBACTERIAL EFFECTS OF FUNGI

Apparently Gosio is to be credited (268) with having first demon-
strated that a crystalline material produced by a species of Penkillium
has the capacity of inhibiting the growth of a bacterium, namely B. an-
thracis; this substance is now known as mycophenolic acid. Soon after-
ward, in 1897, Duchesne (211) reported that certain green Penicillia
are capable of repressing the growth of various bacteria or of bringing
about their attenuation. Vaudremer (934) demonstrated in 19 13 that
the presence of A. jumigatus results in the attenuation of the cells of
M. tuberculosis.

Vaudremer (934) was the first to attempt the clinical utilization of
a fungus product. He treated "more than 200 patients" suffering from
tuberculosis with extracts of the fungus; although no toxic effects were
observed, the curative properties of the preparation were such as not to
justify any significant conclusions.

Since these early studies a number of fungi have been found to pos-
sess antibacterial properties; this phenomenon has sometimes been
spoken of as mycophagy (935a). Several fungi have been studied in de-
tail, and in some cases one or more antibiotic substances have been iso-
lated (Figure 12). The property of inhibiting the growth of bacteria is

Typical conidial structure of p. woz'rt- Typical penicillus of NRRL

turn NRRL 832. From Raper and 1951- From Raper and Alexander

Alexander (764)

(764)

V

i^, C,, .N^^

i

';i

1 ' i

f-f /

' .' r/

p 1

(:/i'Uk.

1
i

P. citrinum. From Biourge (56) P. chrysogenum. From Biourge (56)

A. clavatus. From Wehmer (9B3a) A. jumigatiis. From Wehmer (983a)
Figure 12. Some typical fungi that produce antibiotic substance^s.

ANTIBACTERIAL EFFECTS 131

not characteristic of any one genus or even species, but of certain strains
within a given species. Different strains may produce distinct variants
of a given antibiotic. This is true especially of penicillin, of which a
number of types have now been isolated, varying both chemically and
in their selective antibacterial properties.

Some organisms produce more than one antibiotic. Two genera,
Penicillium and As-pergillus, have been found to comprise a large num-
ber of antagonistic forms. Several other genera are also known to con-
tain organisms that possess antibacterial properties j very few of these,
however, were ever found among the Phycomycetes j the Basidiomy-
cetes also include a large number of organisms capable of producing
antibiotics.

The active fungi may be divided (956) into the following eleven
groups:

Asfergillus clavatus A. jumigatus

A. flavus-oryzae PenictlliuTn cyclofium-clavijorine

Penicillium luteum-furfurogenum Fusarium-C efhalosforium

Penicillium notatum^-chrysogenum ChaetowJium, and other Ascomycetes

Trichoderm.a-GVtocladium, Basidiom-ycetes

Miscellaneous other fungi

A comparative study of a number of fungi taken from a culture col-
lection brought out ( 1017) the fact that about 40 per cent of the Asper-
gilli {Aspergillus fumaricus, A. fumigatus, As-pergillus schiemannii,
Aspergillus terreus) and 1$ per cent of the Penicillia {Penicillium
chrysogenufHy Penicillium clavijorme, Penicillium funiculosum, Peni-
cillium exfansum) possessed antagonistic properties. In a study of lOO
strains of Aspergilli (1018), 28 were found to be active against S. au-
reus, 16 against E. coli, and 9 against Ps. aeruginosa; strains of ^. niger
were most positive and those of A . versicolor were negative. Among the
Penicillia, in addition to P. notatumy the following were shown ( lOio)
to produce considerable antibiotic action: P. brunneoviolaceumy P.
chloro-leucon, P. citrinum, P. chrysogenum, and P. griseo-roseum, the
last two species producing the penicillin-type of action. The composi-
tion of the medium and the nature of the strain are of great importance.
The active producers of antibiotic substances belong to the group of

132 FUNGI AS ANTAGONISTS

Asymmetrica. Of many Phycomycetes tested, only Phythofhthora
erythroseftka showed some activity. A few Ascomycetes were also
found to be active. Next in importance to the Aspergilli and Penicillia
as producers of bacteriostatic substances are the Basidiomycetes.

A summary of the antibacterial properties of various fungi and of
the antibiotic substances produced by them is given in Table 24. In ad-
dition to the specific strain of the organism, the composition of the me-
dium and the conditions of growth, especially aeration, are most im-
portant in controlling the amount and nature of the antibiotic substance
produced by the organism. Different strains of the same organism
when grown under identical conditions vary greatly in the production
of the antibiotic substance, as shown for A. clavatus (968) and other
fungi (497).

PenicilUum notatum-chrysogenum Grouf

Because of the production by these organisms of penicillin, which has
already found a wide practical application, this group of fungi deserves
special attention. Fleming (261) first observed that a fungus culture
growing on a staphylococcus plate brought about destruction of the bac-
teria, as shown by the fact that the colonies became transparent and
were undergoing lysis. The fungus was isolated in pure culture and was
later identified as P. notatum. It was found to possess marked bacterio-
static and bactericidal properties for some of the common pathogenic
bacteria, largely the gram-positive cocci and the staphylococci, the strep-
tococci, the diphtheria organism, and the gonococci and meningococci ;
bacteria belonging to the colon-typhoid-dysentery group were not af-
fected. The culture filtrate of the fungus was found to contain an active
substance, which was designated as penicillin.

The selected culture of the fungus is grown on one of the media de-
scribed earlier (pp. 65-69). The reaction of the medium changes from
slight initial acidity (;)H 6 to 7) to distinct acidity (^H 3.0), followed
later by alkalinity, finally reaching a pH of 8.0 or even 8.8. A faint to
deep yellow color is produced in the medium. Penicillin production is
usually at its maximum at about /)H 7 and may remain constant for sev-
eral days or may fall again rapidly. In stationary cultures, once a fungus
pellicle has been produced, the medium can be replaced several times,

TABLE 24. ANTAGONISTIC EFFECTS OF SOME REPRESENTATIVE
FUNGI AGAINST BACTERIA

ANTAGONIST

ORGANISMS AFFECTED

ACTIVE SUBSTANCE

REFERENCES

A . clavatus

Gram-negative and gram-

Clavacin, highly

957,968, I0I2

positive bacteria

bactericidal

A . flavus

Streptococci, staphylo-
cocci, and certain gram-
positive bacteria

Aspergillic acid

480, 740, 1006

A.flavus

Mostly gram-positive

Flavicin, similar to.

103,605,950

bacteria

if not identical
with, penicillin

A . fumigatus

Gram-positive bacteria

Fumigacin, glio-
toxin

96,9555957

A . fumigatus

Various bacteria

Fumigatin, spinu-
losin

702

A . fumigatus

M. tuberculosis

Active filtrate

IO5I

and A . albus

A. niger

Gram-positive and gram-
negative bacteria

Aspergillin

532

Chaetomium sp.

Various gram-positive
bacteria

Chaetomin

956

Gliocladium and

Various gram-positive and

Gliotoxin, highly

977

Trichoderma

gram-negative bacteria

bacteriostatic

P. citrinum

Various bacteria

Citrinin

745

P. claviforme

Gram-positive and gram-
negative bacteria

Claviformin

124, 125

P. notatum and

Mostly gram-positive and

Penicillin, active

5. 75>h6,

P. chrysogenum

also certain gram-nega-

in vivo, low tox-

262, 437, 770,

tive {Neisseria, Gone-

icity

956

coccus') bacteria

P. notatum

All bacteria tested, in

Notatin, penatin.

157,517,521.

presence of glucose

penicillin B,
E. coli factor

786, 956

P. piberulum

Various bacteria

Penicillic acid

61,698,703

and P. cyclofium

P. resdculosum

Various bacteria

Crude metabolic
product

62

Penicillium sp.

Gram-negative as well as
gram-positive bacteria

Penicidin

29

134 FUNGI AS ANTAGONISTS

giving fresh lots of penicillin in about half the time required for the
initial growth. Crude penicillin cultures are capable of inhibiting the
growth of staphylococci in dilutions of i : 8oo. Recently, much more
active preparations have been obtained ( i : 1 0,000 ).

Chain et al. (123) were the first to succeed in isolating from the cul-
ture medium of P. notatum a water-soluble, stable, brown powder
which had marked antibacterial activity. This preparation inhibited, in
dilutions of i to several hundred thousand, the growth of many aerobic
and anaerobic bacteria. The active material was relatively nontoxic to
laboratory animals. Intravenous and subcutaneous injections of 10 mg.
or more to mice had little or no effect. The material was active m vivo,
subcutaneous injections saving the lives of mice injected intraperitone-
ally with S. pyogenes or S. aureus. Intramuscular infections of mice
with CI. seftkum were also successfully treated by repeated subcutane-
ous injections of penicillin.

An extensive literature soon began to accumulate on the production
(127, 128, 171, 281), isolation, and identification of penicillin. The
course of its formation in the culture of the organism is illustrated in
Figure 13. Conditions of nutrition were found to be particularly im-
portant. Preparations having an activity of 2,000 Oxford units or 100,-
000,000 dilution units have been obtained. The importance of the dual
nature of P. notatum (the culture being composed of two distinct cell
constituents) must be recognized for maximum penicillin production
(34, 377). The low toxicity of penicillin, its solubility in water, and its
in vivo activity make it an ideal agent for combating disease caused by
gram-positive bacteria.

P. notatum represents an extremely variable group of organisms,
some strains producing considerable penicillin, others producing little
penicillin but large amounts of a second factor, designated as penatin or
notatin. Some strains of a closely related fungus, P. chrysogenumy are
also capable of producing penicillin that is apparently the same as the
penicillin of P. notatum. The P. notatum-chrysogenum group of fungi
is widely distributed in nature, having been isolated from different soils
and from various moldy food products j however, only a few strains
produce enough penicillin to justify their use for the commercial pro-
duction of this substance.

■b3inn"iitN 2)3d siNvaomiiAj ni avons ivnaisaa

C\J O <D «3

a^l^^^l^N aid simn AS^ioid ni

NnilOIN3d

SIA1V2J9 NI 310n"13d JO 1H9I3M ASQ

136 FUNGI AS ANTAGONISTS

Raper et al. (765) summarized the principal characters which dis-
tinguish the P. notatum-chrysogenum group as follows: (a) colonies,
at 10 to 12 days, velvety or loose-textured, plane or radially furrowed,
with sporing areas in blue-green shades j (b) the colonies are generally
characterized by abundant yellow exudate, often collecting in con-
spicuous droplets j (c) reverse of colony yellow, usually becoming
brown with agej the agar medium becomes yellow j (d) the penicillus
usually consists of from 2 to 5 more or less divergent columns of
spores; it represents an asymmetric, biverticillate structure with smooth
walls throughout; (e) the conidia or spores are smooth walled, vary-
ing from globose and sub-globose {P. notatum) to elliptical {P. chry-
sogenum)y and ranging in size from approximately 2.5 to 4.0 p in
diameter.

Among a large number of cultures investigated, two subgroups were
recognized.

Subgroup I comprises those that are characterized by penicillin yields
ranging from about 20 to 35 O.U./ml.; the colonies are loose tex-
tured, comparatively deep, heavily sporing, at first pale blue-green
but becoming darker with age; conidiophores often comparatively
long and rather coarse, bearing large penicilli; conidia typically sub-
globose to definitely elliptical, characteristic of P. chrysogenum.
These strains are common in soil and also occur in considerable abun-
dance in a wide variety of other natural substrates.

Subgroup II comprises the strains that produce 50 to 80 O.U./ml. of
penicillin; the colonies are fairly compact or even close textured,
velvety throughout, dark green with abundant yellow exudate; co-
nidiophores bear penicilli somewhat smaller than those of Subgroup I,
typical of P. notatum. These strains have been isolated from soil,
cheese, fruits, and bread. Different isolations were found to vary
greatly in their capacity to produce penicillin; substrains derived
from these also show marked variation in productivity.

P. notatum lends itself readily to the study of sectorial mutants.
These either can arise spontaneously or can be induced by special treat-
ments, such as irradiation with x-rays or bombardment of the culture
with neutrons (667). This phenomenon can be utilized for genetic
studies (727). Mutants have thus been obtained which produce un-

Figure 14. P. chrysogmum, NRRL 195 1, and derivative strains. From
Raper and Alexander (764).

ANTIBACTERIAL EFFECTS 137

usually high yields of penicillin in submerged culture (475). On ex-
amination in plate culture as well as microscopically, these high-peni-
cillin-yielding mutants resembled the parent strains from which they
were isolated. Some of the high-yielding cultures are relatively stable j
others, however, are quite unstable, giving substrains with substan-
tially greater yields of penicillin. Light-sporing substrains give reduced
yields J white nonsporulating strains are characterized by low yields
(764). Some of the most productive strains were found to vary pri-
marily in biochemical activity, and only little in cultural or structural
details. Some of the variations are brought out in Tables 25 and 16 and
in Figures 14 and 15.

TABLE 25. PRODUCTION OF PENICILLIN IN SUBMERGED CULTURE BY
NRRL 832 AND THREE SELECTED SUBSTRAINS

THIRD DAY

FOURTH

DAY

FIFTH DAY

SIXTH DAY

CULTURE

Penicillin

/>H

Penicillin

/.H

Penicillin

fU

Penicillin />H

O.U./ml.

O.U./ml.

O.U./ml.

O.U./ml.

832 Stock

35

7-9

65

8.1

61

8.3

40 8.5

832.A2

47

7-9

92

8.0

83

8.3

50 8.4

832.A,(6)

37

8.1

62

8.0

75

8.2

52 8.4

832.B3

29

8.2

37

7-7

42

8.1

35 8.4

From Raper and Alexander (764).

TABLE 26. PRODUCTION OF PENICILLIN IN SURFACE CULTURE BY NRRL
I249.B2I, 1950, 1978, AND TWO SUBSTRAINS OF 1 978, A AND B

FOURTH

DAY

FIFTH

DAY

SIXTH :

DAY

SEVENTH DAY

CULTURE

Penicillin

fn

Penicillin

fn

Penicillin

fn

Penicillin fH

O.U./ml.

O.U./ml.

O.U./ml.

O.U./ml.

I249.B2I

76

6.4

185

7.2

177

7-9

135 8.2

1950 Stock

98

7-3

139

7.7

103

8.0

81 8.3

1978 stock

120

6.9

233

7-4

114

7-9

162 8.3

1978A

109

7-3

154

7.6

131

8.0

85 8.3

1978B

124

6.9

262

7-3

246

7.8

190 8.2

From Raper and Alexander (764).

FUNGI AS ANTAGONISTS

□ su

eFACE CULTURE
SUBMERGED CULTURE

D. 20

PENICILLIUM SUBSTRAIN

Figure 15. Comparative production of penicillin by substrains of P. chry-
sogenum 1951.B25 in surface culture and submerged culture. From Raper
and Alexander (764).

As a result of these studies the following conclusions were reached
(763,764):

1. The capacity to produce penicillin as a metabolic product is a group-

specific rather than a strain-specific character.

2. Different members of the P. notatum-chry so genum group vary

greatly in their capacity to produce penicillin.

3. Special strains are particularly suited for certain types of penicillin

production.

For surface production of penicillin, no strain was found to be better
than the original Fleming culture that has been freed from degenerate,

ANTIBACTERIAL EFFECTS 139

mutant strains. This freeing of mutants must be carried out continu-
ously, in order to avoid the degeneration of the culture. By strain se-
lection and improvement of medium (addition of corn steep liquor, use
of lactose), the penicillin yield of such cultures has been increased from
■2 to 6 to more than 200 O.U./ml. (662). For submerged cultures, how-
ever, strains of P. notatum and P. chrysogenum are used which are not
related to the Fleming strain. The best medium for tank production is
about half the concentration of the nutrients used for surface culture.
Pregerminated inocula are used {66'^).

In a study of the metabolism of the penicillin-producing fungi
(308a) it was established that the most important factors for high
yields of penicillin are the nature of the culture, aeration, temperature,
and proper balance among the nutrients of the medium, especially the
relation between the carbon and nitrogen sources (591 )• Specific amino
acids have an influence on the yield of penicillin, especially in sub-
merged culture (375). Yields ranging from 90 to 900 units of peni-
cillin were obtained under submerged conditions of growth.

The rate of utilization of different sugars and oxygen consumption
by penicillin-producing strains of a submerged culture is brought out
in the following summary (519) :

RATE OF UTILIZATION

O2 UPTAKE

SUGAR

gm./l/hour

ml./l/hour

Lactose

0.32

109

Sucrose

0.46

150

Glucose

0.71

300

The lactose is more slowly utilized than the glucose, and less oxygen
is required for penicillin production. The addition of boron to the me-
dium favors lactose utilization and results in a higher rate of respiration
and nitrogen utilization, with a less abundant mycelium, lower am-
monia levels, and higher penicillin yields (520).

At least four different penicillins have been isolated, namely, F, G,
X, and K, two or more being found in the same culture broth. These
penicillins differ in their chemical characteristics, in their antibacterial
spectra, and also in their chemotherapeutic utilization. For example,
penicillin X is more effective in the treatment of gonorrhea than G.

140 FUNGI AS ANTAGONISTS

Various cocci are 6 to 8 times more sensitive to X than to commercial
penicillin, which is largely G (695).

Penicillin or penicillin-like substances are also produced by A. flavus,
A. farasiticus (149), A. giganteus (722), and a variety of other fungi,
largely species of Aspergillus and Penicillium, including A. niger, A.
nidulans, A. oryzae, P. citreo-roseum (281), P. cms to sum (1039), and
others, such as A. jlavifes ( 1005).

Certain species of PenicilUum are also capable of producing other
antibacterial substances, namely, citrinin, penicillic acid, and clavacin,
the first of which is also produced by species of Aspergillus belonging to
the candidus group (906).

Atkinson (29) tested 68 cultures of PenicilUum and found that 18
possessed antibacterial properties. These cultures were divided into
two groups : first, those largely active against gram-positive bacteria and
producing substances like penicillin and citrinin 5 second, those active
also against gram-negative bacteria and producing substances of the
penicillic acid and penicidin types.

Aspergillus -fiavus-oryzae Group

The. A. oryzae members of this group possess only limited antagonis-
tic properties. Many of the A. flavus strains, however, apparently have
the property of producing at least two antibacterial substances when
grown on suitable media and under suitable conditions.

White and Hill (1006) isolated from cultures of a strain oi A. flavus
grown on tryptone media a crystalline substance, aspergillic acid, that
showed antibacterial activity against certain gram-negative as well as
gram-positive bacteria. The substance was produced when the organism
was grown on organic media, but not on synthetic. It was soluble in
ether, alcohol, acetone, or acetic acid, but not in petroleum ether j it was
soluble in dilute acid or alkaline aqueous solutions, and was precipitated
by phosphotungstic acid. Aspergillic acid proved to have relatively high
toxicity, and showed no protective action against hemolytic streptococci
or pneumococci infections in mice.

Glister isolated a culture (338) that also produced an antibacterial
agent with a wide range of activity, both gram-positive and gram-nega-
tive bacteria being inhibited by the culture filtrate. An extract was ob-

ANTIBACTERIAL EFFECTS 141

tained that inhibited the growth of these bacteria in a dilution of ap-
proximately 1 : 200,000.

A. flavus was found (46) to produce frequent variants j two of these
consistently gave far higher yields of aspergillic acid than those re-
ported by White. The substance was found to have wide activity,
especially against gram-positive cocci, but was less active against the
anaerobes of gas gangrene and the gram-negative bacteria.

Bush and Goth (103) isolated from A. flavus a second substance
designated as flavicin. They grew the organism for 6 to 8 days on a
nitrate-glucose medium containing 2 per cent corn steep. The filtrate
was acidified to -pH 2.5 to 3.0 with phosphoric acid and extracted
with purified isopropyl ether. The ether was treated with a slight ex-
cess of o.2A^ NaHCO.j (5 to 10 cc. per liter of culture), giving a yield
of 75 to 100 per cent of active material obtained. Purification was ob-
tained by acidification of the NaHCOg extract with H3PO4 to ^H
2 to 3 and removal of the precipitate, the latter containing most of the
toxicity (due no doubt to aspergillic acid) and the filtrate most of the
activity. The filtrate was treated with ice-cold isopropyl ether, satu-
rated with COo, washed with cold distilled water, and reextracted. The
combined extracts were distilled at 0° C. to dryness under COo. A yel-
low-orange glassy residue was obtained. It had a low toxicity and was
active in vivo.

The similarity to penicillin of the second antibiotic substance pro-
duced by A. flavus has been definitely established (605, 606) by chemi-
cal isolation and composition, solubility and stability, biological be-
havior, low toxicity to animals, and therapeutic activity. A sodium salt
assaying 240 O.U./mg. was obtained chromatographically and gave the
following composition: 45.36 per cent C, 4.16 per cent H, 3.02 per cent
N, and 13.36 per cent Na, [aj^ = + 108° (in water).

Under submerged conditions, A . flavus thus produces two substances,
one of the aspergillic acid type and the other of the penicillin type.
Some strains produce little or no activity in submerged cultures, and
most strains produce very little activity in stationary cultures. No ac-
tivity is produced in synthetic media (950). The culture filtrate of A.
flavus grown on lactose-peptone media was active against Af . tubercu-
losis and other acid-fast bacteria in vitro.

142

FUNGI AS ANTAGONISTS

Aspergillus jufnigatus Group

Four antibacterial substances were isolated from strains of A . fumi-
gatus: the two pigments, spinulosin and fumigatin (702), which are
not selective in their action against bacteria, the colorless fumigacin
that is active largely against gram-positive organisms (957), and glio-
toxin (339, 631). Helvolic acid, isolated from a strain of A. jumigatus
(126, 161), was found (631, 955) to be identical with purified fumi-
gacin.

Fumigacin is active against S. aureus in dilutions of i : 200,000 to
1 : 750,000 and is very stable. The pigment fumigatin, however, was
said to deteriorate on standing, inhibition of S. aureus being reduced
from 1 150,000 to 1 : 25,000 in 7 days. Fumigacin has a certain degree
of resistance to high temperatures. Boiling in aqueous solution for 5 to
10 minutes reduced but did not destroy completely its activity. Heat-
ing at 80° C. for 15 minutes reduced the activity only slightly. When
fumigacin was dissolved in alcohol and precipitated by addition of nine
volumes of water, the alcohol-water solution was found to contain 0.25
mg. per ml. A comparison of the antibacterial activity of fumigacin with
that of the other substances produced by A. jumigatus is given in
Table 27.

A number of fungi, largely Aspergilli and usually members of the
A . jumigatus group, have been found to be able to inhibit the growth

TABLE 27. CHEMICAL PROPERTIES AND BACTERIOSTATIC ACTIVITY OF FOUR
ANTIBIOTIC SUBSTANCES PRODUCED BY ASPERGILLUS FUMIGATUS

MELTING
CRYSTALLI- POINT

SUBSTANCE ZATION ° C. FORMULA

BACTERIOSTATIC ACTIVITY
IN DILUTION UNITS

B. sub-
E. coli S. aureus tills

Spinulosin Purplish-bronze

plates 201 CgHgOg

Fumigatin Maroon-colored

needles 116 CgHgOg

Fumigacin Very fine white

needles 215-220 C32H44O8

Gliotoxin Elongated

plates 195 C13H14O4N2S2 6,000 1,500,000 750,000

1,200 200,000 40,000
1,200 2,000,000 100,000

ANTIBACTERIAL EFFECTS 143

of M. tuberculosis. As pointed out previously, Vaudremer recorded in
1 913 (934) that the fungus produces a thermostable substance which is
responsible for the antituberculosis effect. Zorzoli (1051) reported in
1940 that A. fumigatus produces a thermostable substance (100° C.
for I hour) which interferes with the growth of M. tuberculosis. Ashes-
hov and Strelitz (27) observed a marked action oi A. fumigatus prepa-
rations upon the B.C.G. but not upon the avian strain of M. tuberculo-
sis; the bacteriostatic activity was greater against M. tuberculosis
B.C.G. than against staphylococci, although the bactericidal activity was
lower. Culture filtrates and extracts of various unidentified fungi were
found capable of inhibiting the growth of the organism (647). One
such extract was designated as mycocidinj its effect upon the human
tubercle bacillus was both bacteriostatic and bactericidal (328). Jen-
nings (464) reported that helvolic acid (fumigacin), one of the anti-
biotics produced by A. fumigatus y in concentrations of 1:10,000 in-
hibited completely and in i : 100,000 only partly, the growth of the
tuberculosis organism isolated from sputum.

A . ustus produces in ordinary Czapek-Dox medium with 4 per cent
glucose and o.i per cent yeast extract, after 14 to 19 days' incubation, a
substance that inhibits the growth of M. tuberculosis and M. ranae
(539). This antibiotic can be extracted from the medium with ether and
other organic solvents. The ether residue is dissolved in phosphate
buffer of fH. 1 1 .0. On acidification of the alkaline solvent, a yellow
flocculent precipitate is obtained. This substance inhibited the growth
of M. ranae in a dilution of 1:150,000, and the acid precipitate in
1 : 300,000 dilution. By means of a "countercurrent distribution" the
active agent was separated into two crystalline and one partially crys-
talline preparations (438).

The mycelium of A. ustus was found (188) to contain a group of
antibiotics, one of which was designated as ustin. This substance was
active against gram-positive, including acid-fast, bacteria (1:500,000).
It is inhibited by serum albumins and by lipids.

Aspergillus clavatus Group

This comprises a number of strains that produce highly active anti-
biotic substances. By treating the culture filtrate with charcoal and

144 FUNGI AS ANTAGONISTS

eluting the active substance with ether, Wiesner (1012) obtained a
preparation having a bactericidal potency in dilutions of i : 100,000.
This activity was not inhibited by serum, pus, or urine j strains of bac-
teria that proved to be resistant to sulfonamides or mandelic acid were
inhibited by this material.

The active substance was designated (957) clavacin. It is active
against E. colt and other gram-negative bacteria, as well as against
gram-positive bacteria. It is different in this respect from fumigacin.
Whereas the latter acts much more readily upon B. mycoides than B.
subiilis, clavacin shows the opposite effect — greater activity against B.
subtilis than against B. mycoides. Clavacin possesses a high bactericidal
action, as compared with other antibiotic substances.

A detailed study of its production by a variety of strains of A. clava-
tus was made (968). The marked differences in the physiology of the
different strains of A . clavatus were said to explain the differences in the
production of clavacin by different strains. Those that change the re-
action of the medium to alkaline, for instance, tend to inactivate the
clavacin.

Since clavacin is produced by a number of different fungi, it has re-
ceived a number of designations, including patulin formed by P. fatu-
lum (744), claviformin by P. claviforme (124, 125), and clavatin
(47). It is also produced by strains of P. exfansum-y P. urticaey A. ter-
reuSyA. giganteusy GymnoascuSy and others (24, 486, 501). For species
of Penlcilliufn it was found (578) that glucose as a source of carbon, an
incubation temperature of 20° C, stationary culture, and a source of
iron offer optimum conditions.

Trichoderma and Gliocladmm Grouf

Certain strains of fungi of the genera Trichoderma and Gliocladium
were found to exert a marked antagonistic action against various fungi
and bacteria. An antibiotic substance designated as gliotoxin was iso-
lated and found (82, 989) to be highly bactericidal. In order to produce
this substance, the fungus is grown in a submerged condition in shake-
cultures. An abundant supply of oxygen and a high acidity (/)H 5.0 or
lower) are essential. Ammonium salts as nitrogen sources give better
results than peptone or nitrates. Glucose and sucrose were found to be

ANTIBACTERIAL EFFECTS 145

good carbon sources. It is of particular interest to note that whereas
penicillin and flavicin are produced in media containing complex or-
ganic materials as sources of nitrogen, fumigacin, clavacin, and glio-
toxin are produced in synthetic media, the presence of complex nitrogen
sources often being deleterious.

Gliotoxin was isolated from the culture filtrate by the use of lipoid
solvents, chloroform being most effective. Nonsterilized media ad-
justed to fH. 2.5 to 3.0 could be used for large-scale production, the high
acidity reducing the effect of contaminants (992). Gliotoxin is stable in
neutral and acid solutions at room temperature j at alkaline reactions,
it is very unstable, the rate of decomposition increasing with increasing
alkalinity and temperature. At ^H 2.4, heating to 122° C. for 30 min-
utes did not affect the active substance. With decreasing acidity, espe-
cially at ^H 5.0, it became less thermostable.

Gliotoxin is also produced by a number of other fungi, including P.
obscurum {66$) a.nd A. fumigaius (631).

Certain species of Trichoderma, including T. viridis, produce another
antibiotic substance that is particularly active against fungi, designated
as viridin (84). It is produced when the organism is grown in shallow
layers of nitrate-containing media for 4 to 6 days at 25° C. j the cultures
are characterized by a bright yellow color. It is isolated from the cul-
ture filtrate by extraction with chloroform, evaporation, and recrystalli-
zation from alcohol or benzene. It is stable only in acid solution.

Fusarium Grouf

The ability of species of Fusarium to produce antibiotic substances
was first observed in a survey of the antibacterial properties of fungi, as
pointed out above (p. 131). F. oxys forum was found (112) to pos-
sess antibacterial properties. One of the organisms, namely F. javanl-
cum', was studied in detail. A substance, designated as javanicin, was
isolated (26) from the medium by the use of ether or benzene. It was
removed from the solvent by extraction with aqueous NasCOs. It con-
tained a quinone group but no carboxyl. It was active against gram-
positive, including acid-fast, bacteria in concentrations of i : 50,000 to
1 : 400,000 but had little activity against gram-negative bacteria. It was
relatively nontoxic.

146 FUNGI AS ANTAGONISTS

Basidtomycetes

The larger Basidiomycetes produce bacteriostatic substances that
compare favorably with those formed by Aspergilli and Penicillia. The
testing of the sporophore extract alone may be indicative, but it is not
a fully reliable test for a positive result j the fungus must be cultured
and a strip test made (1014). Of 700 species tested, about 70 gave a
strong positive reaction and lOO a weak reaction against S. aureus
and/or E. coU (1019). In a comparison of 72 genera, one or more
species of 43 genera produced some antibiotic activity j none, however,
was more active than P. notatum and none affected gram-negative
forms (785, 785a).

Polyporin, produced in the culture filtrate and in the sporophores of
Polystktus sanguineus y is a thermostable substance not affected by ^H
changes between 2.0 and 8.0. It passes through a Seitz filter, is not af-
fected by body fluids, is nontoxic, and is active in vitro and in vivo
against various gram-positive {S. aureus j S. viridans) and gram-nega-
tive bacteria {E. ty^hosa, V. comma, etc.). Clitocybe gigantea var.
Candida, a member of the Agaricus group, contains in its cell material a
substance, designated as clitocybin, which is soluble in water, chloro-
form, acetone, and ether. It is destroyed on heating at 70° to 80° C. It
inhibits the growth of various gram-negative bacteria, such as E. coli,
Ps. aeruginosa, E. tyfhosa, and Br. abortus, various gram-positive bac-
teria, and M. tuberculosis. It is fairly toxic to animals: i gm. of the dry
fungus substance is treated for 24 hours with 10 ml. water j i ml. of this
extract will kill a 300 gm. guinea pig in 48 hours. It is effective in ar-
resting the development of tuberculosis in guinea pigs (439). Several
species of Cortinarius and one of Psalliota inhibited various gram-posi-
tive and gram-negative bacteria (30).

Other Groufs

Various other fungi, including A . albus, A . niger, and Monilia albi-
cans, were found (1051) to exert a marked antibacterial action against
human and bovine tubercle bacteria 5 active filtrates were obtained, but
the specific agents were not isolated. Certain dermatophytes, especially
strains of Trichophyton mentagrofhytes, also produce an antibiotic

ACTION AGAINST FUNGI

147

substance when grown in glucose-peptone media. This substance is simi-
lar to penicillin in that it is favored by the addition of corn steep, and
in its antibiotic spectrum, its sensitivity to reaction and temperature,
and its destruction by penicillinase preparations (714).

A number of unidentified molds have been reported to produce pig-
ments which have antibiotic activity against various bacteria (807).
This is true, for example, of P. c'mnah annus. The red pigment ex-
tracted from the mycelium of this fungus inhibited S. aureus and S.
pyogenes in a dilution of i : 5,000. The extract was slightly hemolytic,
although not very toxic (637).

A study of the distribution of antibiotic properties among the fungi
revealed the fact that the Aspergilli and Penicillia are most active and
the Phycomycetes least (Table 28).

TABLE 28. DISTRIBUTION OF ANTAGONISTIC PROPERTIES AMONG
THE FUNGI

TOTAL

PERCENTAGE

ORDER OR

NUMBER

PERCENTAGE

WEAKLY

PERCENTAGE

GENUS

EXAMINED

ACTIVE

ACTIVE

INACTIVE

Phycomycetes

30

-

-

100

Ascomycetes

20

-

-

100

Aspergillus

150

30

20

50

Penicillium

200

20

30

50

Basidiomycetes

730

10

20

70

From Wilkins and Harris (1017, 1018, 1019).

ANTAGONISTIC ACTION OF FUNGI
AGAINST FUNGI

Numerous fungi were found to exert antagonistic effects either
against fungi belonging to the same species or against other fungi
(Table 29). This phenomenon is particularly important in connection
with the study of plant diseases. The effects are selective. The hyphae
of Peziza will kill various Mucorales, whereas different species of As-
fergillus and Penicillium are able to kill Peziza. A single spore of P.
luteum was found capable of germinating in cultures of Citromyces

[48

FUNGI AS ANTAGONISTS

TABLE 29. ANTAGONISTIC INTERRELATIONSHIPS AMONG
DIFFERENT FUNGI

ANTAGONIST

ORGANISMS AFFECTED

REFERENCES

Acrostalagmus s^.

Rhizoctonia

990

Alternaria tenuis

Ofhiobolus

89

A . clavatus

Various fungi

949

A . fiavus

Peziza

773

A . niger

Peziza, Rhizoctonia

773. 933^990

Botrytis allii

Monilia, Botrytis, etc.

933

Botrytis cinerea

Rhizoctonia

990

Cefhalothecium roseum

H elviinthosforium

359

Cunninghamdla elegans

Monilia

933

Fusarium laieritium

Rhizoctonia

990

Fusarium sp.

Deuterofhofna

827

Gliocladium sp.

H elminthosforium, Mucor, etc.

729

H elminthosforium sp.

Colletotrichum, Fusarium,
Botrytis, etc.

729

H. teres

Fusarium, Ustilago, Helmintho-
sforium, etc.

729

H. sativum

Ofhiobolus

89

Mucor sp.

Ofhiobolus, Mucor

89> 837

Penicillium sp.

Peziza, Rhizoctonia, etc.

773

Penicillium sp.

Ofhiobolus, Fusarium, etc.

89

Peziza sclerotiorum

Mucor, Trichothecium, Dematium,

773

Peziza trifoliorum

etc.

Peziza

773

Sclerotium rolfsii

H elminthosforium

729

SterigTnatocystis sp.

A Iternaria

729

Thamnidium elegans

Mucor

837

Torula suganii

Asfergillus, Monascus, etc.

690

Torulosis sp.

Blue-staining fungi

630

Trichoderma lignorum

Rhizoctonia, Armillaria, Phy-
tofhthora, etc.

989, 990

T. lignorum

Rhizoctonia, Pythium, etc.

14^63,933

Verticil Hum sp.

Rhizoctonia

990

From Novogrudsky (683).

and of bringing about their destruction. P. luteum-furfurogenum pro-
duces a thermostable substance, soluble in ether and in chloroform, that
is antagonistic to the growth and acid production of A. niger (705).

ACTION AGAINST FUNGI 149

Coniofhora cerehella was inhibited by a species of PenkilUumy its my-
celium being considerably modified j however, in time the former or-
ganism adapted itself to the latter and overgrew it, its rate of growth
being eventually more rapid than that of a pure culture (380). Certain
fungi are able to parasitize other fungi. The germination of the spores
of one fungus may be reduced by the presence of spores of another

{SS3)-

Different fungi produce different types of fungistatic and fungicidal
substances, some of which are stable, others unstable. These are formed
particularly by the lower fungi or the molds, with the exception of the
Phycomycetes that have so far not been found to produce any antibiotic
substances. Their action consists in modifying or killing the mycelium
of the other fungus, or merely in preventing spore germination. Brom-
melhues (89), studying the effects of H. sativum and Penicillium sp.
against Ofhiobolus graminis, emphasized that the inhibitory action was
due to a toxic substance that was thermostable and diffusible in agar.
In some cases, no relation could be observed between the acidity pro-
duced by one organism and its ability to influence the growth of another
( 1046) i in other cases, as in the mutualistic effects of Sderotium rolfsii
and Fusarium vasinfeaum, the first overgrew completely the second at
f¥L 6.9, whereas in alkaline ranges the reverse took place (804).

Random isolations oi Penicillium cultures and of other soil-inhabiting
fungi were tested for their effects on the virulence of H. sativum on
wheat seedlings grown in steam-sterilized soil (823). Some forms ex-
erted a marked degree of suppression, some had no effect, and others
increased the virulence of the pathogeny marked variations in activity
were observed among the different species of Penicillium. Because
Hyphomycetes were found to be capable of parasitizing the oospores of
Pythium (196), Hyphomycetes were believed to serve as effective
agents in promoting soil sanitation. Various species of Torulosis, in
addition to certain bacteria, are capable of inhibiting the growth of
Dematiaceae, fungi that cause the blue staining of wood pulp (630). A
species of Penicillium (P. gladioli) was found (8ia) to produce an
antibiotic (gladiolic acid) which is actively fungistatic but only weakly
bacteriostatic.

Certain fungi may affect the reproduction of others. Melanosfora

150 FUNGI AS ANTAGONISTS

-pamfeana, for example, normally does not form any perithecia in cul-
ture but is able to do so in the presence of Basis-porium gallarum or
Fusarium monilijorme. This effect was ascribed to a special substance
that resists heating at i io° C. Different fungi have a special influence
on the germination of spores of various ascomycetes and of other fungi
(28, 776), these effects being characteristic of the antagonists.

The edible mushroom Psalliota camfestris exerts a definite antago-
nism against the parasitic fungus Mycogone (135). This phenomenon
has been looked upon as a case of antibody formation. Species of Fusch
rium are able to antagonize the mushroom fungus 5 however, an actively
growing culture of the latter may become antagonistic to the former
(1026). In the destruction of paper pulp by fungi, a marked antago-
nism was shown (341) to take place between different organisms,
especially by Tr'ichoderma lignorum against various species of Fusa-
rium and other fungi, as illustrated in Figures 16 and 17.

Certain species of Trkhoderma and Gliocladium are able to inhibit
the growth of various plant pathogenic fungi, especially R. solani, as
well as of Blastomycoides dermatitidis , a causative agent of human skin
diseases. The active substance, gliotoxin, is liberated during the early
stages of growth. The mycelium of older cultures contains another sub-
stance that is soluble in acetone j this has only an inhibiting effect and is
not fungicidal as is gliotoxin. The fungicidal effect of gliotoxin upon
the germinating spores of Sclera tinia americana and hyphae of R.
solani was found to be greater than that of CUSO4 and less than that of
HgCU.

Various other fungi are able to exert antagonistic effects against plant
pathogens. T. lignorum and A . niger restricted the growth of the fungi
Macrofhomina fhaseoli and R. solaniy which produce cotton root rot,
and reduced the activity of the filtrates of the pathogens causing wilting
of the plants.

Satoh (826) has shown that Ofhiobolus m^iyabeanus produces both
growth-promoting and growth-retarding substances, the first of which
is heat stable and passes through a Chamberland filter j the second is
inactivated at 100° C. and does not pass through a filter. The formation
of two substances by Torula suganii, both of which were thermostable,
however, was also demonstrated (690).

Figure i6. Antagonistic effect of one fungus, Ps. "zonatum (in center),
upon another, T. I'lgnorum. From Goidanich et al. (341).

Figure 17. Attack of an antagonistic fungus. T. llgnorum, upon another
fungus, F. sambiicinum (in center). From Goidanich et al. (341).

ACTION OF BACTERIA AGAINST FUNGI

151

ANTAGONISTIC EFFECTS OF BACTERIA AND
ACTINOMYCETES AGAINST FUNGI

Various bacteria and actinomycetes have marked selective fungistatic
and fungicidal effects (Table 30). Bacteria active against U. zeae were
isolated from corn, these bacteria being capable of destroying the colo-
nies of the smut fungi. The widespread distribution of such bacteria
in the soil was believed to check the multiplication of the pathogenic
fungi. Four types of bacteria antagonistic to smuts and to certain other
fungi have been described (470). Some of these bacteria produce en-
zymes that are able to dissolve the chemical constituents of the cell
walls of the fungus sporidia; they were also found to be active in the
soil against the specific fungi. Brown (93) observed that H. sativum
and a certain bacterium produced thermostable, mutually mhibitmg
substances. The bacterium as well as its metabolic products inhibited the

TABLE 30. ANTAGONISTIC EFFECTS OF BACTERIA AGAINST FUNGI

ANTAGONIST

Achromobacter %^.
Al. faecalis
Bacillus "/)"
B. anthracis
B. mesentericus
B. mycoides
B. simflex
B. subtilis

Bacterium sp.

Bacterium sp.

Myxobacterium

P. vulgaris

Ps. aeruginosa

Ps. juglandis

Ps. fhaseoli

Ps. translucens

Ps. vulgaris

S. marcescens

Spore-forming bacteri

From Novogrudsky (683).

ORGANISMS AFFECTED

REFERENCES

Fusariumy Sclerotinia

143

Helminthosforium

729

Ustilago, Penicillium

35

S. cerevisiae

525

Helminthosforium

142.729

Helminthosforium

729

Rhizoctonia

154

Cefhalothecium roseum

II

Fusarium, Sclerotinia, etc.

729

Ustilago

470

Ustilago

247, 470

Basisforum, Phytofhthora, etc.

506,729

Saccharomyces

525

Dothiorella

247

Fusarium

63,247

Ofhiobolus

87

Ofhiobolus
Beauveria, etc.

87

10, 11, 12,624

a Fungi

35>729

152 FUNGI AS ANTAGONISTS

growth not only of the particular fungus but also of other members of
the same genus, but not of Fusarium- conglutinans . These bacteria pro-
duced a diffusible agent that inhibited the growth of H. sativum
(i 15a). The active substance was not destroyed by autoclaving^ it dif-
fused into fresh agar and water, producing "stale water" that was in-
hibitory to the fungus.

Chudiakov (143) isolated from the soil two bacteria that were capable
of bringing about the lysis of different species of Fusarium as well as
other fungi. These bacteria were found to be widely distributed in most
soils J they were absent, however, in flax-sick soils, in spite of the abun-
dance of Fusarium. When this fungus was added to the soil containing
antagonistic bacteria, it did not develop, and the plants did not become
diseased. The antagonistic action of a variety of other bacteria against
plant pathogenic fungi has been definitely established, as in the case of
B. simflex against Rhizoctoniay P. vulgaris against Phyio-phthora
(488), and B. mesenlericus against H elminthosforiuin (142). B. sim-
plex was grown (491) for 7 days at 28° C. in potato-dextrose medium
containing i per cent peptone, and the active substance was removed by
charcoal and dissolved in alcohol. Different fungi differed in the de-
gree of tolerance to this substance. The majority were repressed when
10 per cent concentration of the stale medium was added to fresh
medium.

The ability to produce a thermostable substance toxic to the plant-
disease-producing fungus Rhizoctonia is widespread among spore-form-
ing bacteria. The toxic substance is insoluble in ether, chloroform, and
benzol, but is soluble in ethyl alcohol. It passes through collodion,
cellophane, and parchment membranes. It is readily destroyed on boil-
ing in alkaline media but is more resistant in acid media.

Nakhimovskaia (672) found that various bacteria are able to inhibit
the germination of rust spores. Nonspore-forming bacteria, such as Ps.
■fluorescens and S. marcescenSj prevented the germination of the spores
of Ustilaga avenaey Ustilaga hordeiy Ustilaga nuda^ and Ustilaga reae.
Spore-forming bacteria, including B. mycoides and B. 'mesenlericus ^ as
well as sarcinae (5. ureae, S. lutea), exerted no antagonistic action on
the rust spores. The presence of these bacteria, however, influenced the
nature of the germination of the spores, which gave rise to mycelium-

ACTION OF BACTERIA AGAINST FUNGI 153

like forms with great numbers of copulating filaments, whereas in the
control cultures yeast-like forms prevailed and copulating cells were
rarely encountered. The presence of a certain concentration of bacterial
cell substance was essential to this antagonistic effect. With a more lim-
ited amount of cell material, the bacteria ceased to inhibit the germina-
tion of the spores but influenced the germination process in the same
manner as do nonantagonistic bacteria, that is, they stimulated the sex-
ual process. An increase in concentration of cell substance, even of non-
antagonistic organisms, would inhibit spore germination.

The common occurrence of the fungus Pyronema conjiuens in freshly
burned-over soils, but not in natural soils, was explained (684) as due
to the destruction of the bacterial antagonists by heating of the soil. Ps.
fuorescens was particularly effective as an antagonizing agent. A com-
parative study of the fungistatic action of substances of bacterial origin
(883) has shown these to be more active than common disinfectants.
Tyrothricin inhibited the growth of animal pathogens in dilutions of
1:5,000 to 1:20,000, pyocyanin in 1:2,000 to 1:5,000, and hemi-
pyocyanin in i : 20,000 to i : 60,000.

Actinomycetes may also exert a marked depressive effect upon the
growth of fungi. The active substances produced by these organisms
show considerable selective action just as in the case of the bacteria.
Actinomycin was found (974) to inhibit the growth of Penicilliuniy
Aspergillus J Ceratosiomella, and yeasts in concentrations of i :50,000 j
larger amounts (1:10,000) were required to inhibit other fungi, in-
cluding Rhizofus and Trichoderma. Streptothricin is less effective
against fungi, although it inhibits the growth of certain yeasts (1031).

In general, antibiotics vary as much in their antifungal as in their
antibacterial effects. Some, like gliotoxin and actinomycin, were found
to be highly active against both parasitic and saprophytic fungi, whereas
others, like chaetomin and streptomycin, had little if any activity. Since
some of the substances, like actinomycin, have a highly toxic effect
upon animal tissues, the selection of a suitable antifungal agent for
chemotherapeutic purposes is limited to a very few promising mate-
rials j among these gliotoxin and streptothricin were mentioned (771).

CHAPTER 8

MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS

The microscopic animal world inhabiting the soil and water basins com-
prises protozoa, insects and insect larvae, nematodes and other worms.
Their relationships to the microbiological flora of soils and waters are
varied. Many, if not most, of these animals feed upon the bacteria and
fungi, as well as upon the smaller animal forms. Some carry a bacterial
population in their digestive tract and appear to depend upon these
bacteria for some of the digestion processes. Many of the animal forms
are parasitized by bacteria and fungi. Some of these forms are subject
to the action of specific substances produced by microbial antagonists.
No detailed discussion will be presented of these varied relationships,
but attention will be directed to a few specific phenomena which have a
bearing on the subject under consideration. The ability of higher ani-
mals to produce antibacterial substances has been amply established.
Some of these substances are well characterized, as in the case of ly-
sozyme found in mammalian tissues and secretions and inhibins found
in fresh human urine (189).

RELATIONS OF PROTOZOA TO BACTERIA

The lower animal forms inhabiting the soil, manure piles, and water
basins often utilize bacteria in the synthesis of their foodstuffs. Al-
though many of the smallest organisms, namely the protozoa, are able
to obtain their nutrients from simple organic compounds and mineral
salts, they frequently depend upon the bacteria to concentrate the nu-
trients present in dilute forms in the natural substrate. It has been
shown (106), for example, that when carbohydrates are present in
water in very low concentration, the protozoa may not be able to use
them in that dilute formj however, the bacteria can assimilate these
carbohydrates and can build up extensive cell substance, and the pro-
tozoa are then able to multiply by consuming the bacteria. Protozoa
are apparently also able to destroy pathogenic bacteria (781).

RELATIONS OF PROTOZOA TO BACTERIA 155

The fact that some of the protozoa feed upon bacteria served as the
basis for a theory designated as the "protozoan theory of soil fertility"
(812). According to this theory, the capacity of protozoa to consume
bacteria is responsible for the limited fertility of certain soils. The bac-
teria were viewed as the sole agents responsible for the liberation of
nutrients in the decomposition of soil organic matter and for the trans-
formation of these nutrients into forms available to higher plants. The
protozoa, because of their capacity to digest bacteria, were looked upon,
therefore, as the agents injurious to soil fertility. The increased fer-
tility which results from the treatment of soil with heat and with cer-
tain chemicals was believed to be due to the destruction of the protozoa,
considered as the "natural enemies of the bacteria."

Subsequent investigations did not support this theory. When proto-
zoa were added to cultures of bacteria responsible for certain specific
processes they did not exert any detrimental effect upon the particular
reactions brought about by the bacteria, despite the fact that they fed
upon and thereby considerably reduced the numbers of these bacteria.
In many cases, the effect of protozoa upon bacterial activities may actu-
ally be considered beneficial (163). This was found to be true for such
processes as the fixation of atmospheric nitrogen, the liberation of
ammonia from proteins, and the formation of carbon dioxide from car-
bohydrates.

Failure to confirm the protozoan theory of soil fertility was due pri-
marily to the fact that several assumptions were made that were not
fully justified, namely, (a) that bacteria are the only important soil or-
ganisms responsible for the decomposition of the soil organic matter j
(b) that protozoa, by consuming some of these bacteria, are capable of
restricting bacterial development and, if so facto, organic matter de-
composition. The fact was overlooked that the soil harbors, in addition
to the bacteria, many fungi and actinomycetes capable of bringing
about the decomposition of plant and animal residues, resulting in the
liberation of ammonia, and that this could take place even if all the bac-
teria were completely eliminated from the soil.

The favorable effect of partial sterilization of soil upon fertility still
remains to be explained. Various other theories have been proposed, the
most logical of which is one based upon a soil condition designated as

156 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS

"microbiological equilibrium" (972). It has also been suggested (527)
that the phenomenon is due to the disappearance of the bacterial antago-
nists in the soil as a result of partial sterilization.

In many cases, however, protozoa are responsible for bringing about
extensive destruction of bacteria. This may find a practical application
in the purification of water and sewage. The action of the protozoa is
due in this case to the actual ingestion of the bacteria (452).

The theory that protozoa may favor soil processes because of the
stimulation of bacterial development and hence the accelerated trans-
formation of soil materials is not always justified. The assumption is
usually made that these processes take place in the soil in a manner simi-
lar to those brought about in artificial culture media, a generalization
that may be justified only in very special cases. No consideration is given
to the fact that the presence of numerous other organisms in the soil
may modify considerably the activities of the protozoa. The use of arti-
ficial media gives only a one-sided conception of the significance of pro-
tozoa in soil processes. Although the more recent claim concerning the
function of protozoa in the soil is based upon more direct experimental
evidence, it is still inadequate, because it gives insufficient consideration
to the numerous elements involved in the complex soil population.

The protozoa make up only a small portion of the soil population,
both in numbers and in the actual amount of cell substance synthesized.
Their ability to reduce bacterial numbers in normal soil is not very sig-
nificant. The indirect method of studying protozoa in solution media,
whereby the types observed and the activities obtained are quite differ-
ent from those occurring in the natural soil, has been largely responsible
for the exaggerated importance attached to these organisms.

One may conclude that the protozoa, by consuming some of the bac-
teria, keep these organisms at a high state of efficiency, thus assisting in
the breakdown of the plant and animal residues in the soil. In other
words, the rate of energy transformation brought about by bacteria and
even the total amount of change produced in the substrate are increased
by the presence of protozoa. Thus, an interrelationship among micro-
organisms which was at first thought to be antagonistic actually has
proved to be associative. The protozoan Oikomonas termo was found
to be capable of living at the expense of a large number of bacteria.

RELATIONS OF PROTOZOA TO BACTERIA 157

namely 83 per cent of those tested. The fact that Oikomonas causes
many species of bacteria to flocculate was suggested as explanation for
the ability of the protozoa to digest these bacteria (381).

The ability of protozoa to destroy bacteria was said (426) to be re-
sponsible for the protection of certain plants against attack by plant
pathogenic bacteria and fungi. This was said to hold true of attack of
potatoes by Bacterium aroideae and of other plants by Pseudomonas
hyacinthi and Pseudomonas ckri, as well as by species of Fusarium and
Penicill'ium,.

Various bacteria may exert a toxic action upon protozoa, thus limiting
the development or bringing about the destruction of the latter (133,
584). Certain plant pathogenic bacteria inedible by amebae were found
to produce a toxin that was harmful to these amebae. In some cases, the
protozoa were capable of developing a certain resistance to specific
bacterial products (721). The toxic action of some bacteria against
Paramecium could be overcome by the presence of a flagellated proto-
zoan Oikomonas (382).

On the basis of the ability of protozoa to utilize bacteria as food,
Singh classified (855) the latter into 3 groups: (a) edible forms, (b)
inedible but harmless to protozoa, (c) forms toxic to protozoa. Pig-
ment-producing bacteria are inedible and some are toxic j these comprise
the Ps. aeruginosa and the S. m^arcescens groups.

Since some amebae, like Hartmanella castellanii, function as phago-
cytes, they are believed (545) to offer excellent material for the study
of the effect of antibiotic substances upon pathogenic bacteria in the
presence of these amebae, the latter not being affected, as by penicillin,
for example.

Certain factors in the medium seem to affect the encystment of pro-
tozoa (9C0) J it remains to be determined to what extent these factors
can be classified with antibiotic substances.

Myxamoebae of the slime mold Dictyostileum discoideum also live
upon bacteria. They are able to utilize the gram-negative somewhat
better than the gram-positive types, with certain few exceptions. Bac-
terial spores are also ingested by these organisms, but they are not di-
gested.

158 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS

RELATIONS OF PROTOZOA TO FUNGI

The presence of Colfoda and other infusoria in an active form was
found to repress the growth of VerticilUum dahUae in culture media
and to prevent infection of tomato plants by this pathogen j Colfoda
was also active in soils and reduced the incidence of wilting (88).

The ability of various fungi to destroy protozoa and nematodes has
been studied in detail by Drechsler (194, 195).

MALARIAL AND TRYPANOSOME PARASITES

In connection with the recent interest in antibiotic substances, con-
siderable work has also been done on the effect of these substances upon
different strains of Plasmodium causing malaria and upon different
trypanosomes causing various tropical diseases.

Weinman found (993) that the general correlation between the
gram-stain of bacteria and their sensitivity to gramicidin also extends
to protozoa (Leishmania, Trypanosoma) and to the Leftosfira tested.
Tyrocidine had a marked effect, in concentration of 5 pg/ml., upon
the flagellates j they remained active for many hours, gradually losing
their motility j a few escaped, giving rise to delayed growth.

Levaditi and Twort (561) demonstrated that trypanosomes are de-
stroyed by B. sub tills and are also partly destroyed by E. coli, but not by
B. frodigiosus, B. m^esentericus, B. fyocyaneus. The active substance,
designated as trypanotoxin, was found to be produced by B. subtilis in
the culture filtrates and in centrifugates. The washed cells of the or-
ganism were inactive. The substance is thermolabile and is destroyed at
70° C. in 20 minutes. It does not pass collodion dialysis membranes. It
is also active in high concentrations against the tic-fever Sfirillum and
Leishmania J but not against Borrelia gallinarum. It is apparently not
very active in vivo, since it did not protect mice against trypanosomes.
Contact between trypanotoxin and trypanosomes in vitro led to the de-
velopment of toxo-resistant strains of the latter. This resistance was
maintained for many generations j however, the new strains do not be-
come more resistant to pyocyanase and other anti-trypanosome re-
agents.

Further studies (560) brought out the following facts: resistant

MICROBIAL CONTROL OF INSECT DISEASES 159

strains did not adsorb the toxin, as did the susceptible strains} the
susceptible trypanosomes were destroyed completely by antiserum,
whereas the resistant forms were also resistant to this antiserum.

A lipid-like substance produced by species of Phycomyces was ac-
tive against Tryfanosoma equiferdum in vitro but not in vivo (830a).

MICROBIAL CONTROL OF INSECT DISEASES

Insects are subject to attack by various groups of microorganisms,
including bacteria, fungi, protozoa, nematodes, and other insects. Many
attempts have been made to control insect pests by the use of pure or
mixed cultures of microorganisms. In this connection the following re-
lationships must be considered: the receptivity of the insect to microbial
attack during its various stages of development} the environmental
conditions favoring the attack on the insect by the disease-producing
organism; the influence of environment upon the virulence of the at-
tacking microbe; the manner in which the parasite attacks the host; the
coordination of the optimum activity of the disease-producing agent
with the abundance of the host and the proper stage of its develop-
ment.

The microbial agents that keep in check the spread of insects, some of
which are highly injurious to plants and animals, are far more impor-
tant than any other methods of control. These microbial agents can be
classified into three groups, depending upon the nature of the host: (a)
microbes that attack economically useful insects and that must be con-
trolled in order to avoid important losses from disease; (b) microbes
that attack injurious insects and that must therefore be favored and en-
couraged; (c) microbial agents infectious to plants, animals, and man
that are spread by insects.

Various bacterial diseases that formerly caused considerable destruc-
tion of silkworms and bees have been controlled, once the nature of the
organisms concerned was established. One of Pasteur's important con-
tributions to microbiology was the control of Flacheria among silk-
worms. However, most of the problems of control of injurious insects
have been difficult to solve. A great number of bacterial, fungus, and
virus diseases of insects are now known, but the many attempts to em-

160 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS

ploy these pathogens in combating the insect hosts have not alwaj's been
successful. The investigations so far carried out in this important field
may be considered as at a very primitive stage.

Metalnikoff (634) compared the bacterial treatment of caterpillars
of Pect'mophora gossypiella with the action of arsenical poisoning. The
dry spores of Bacterhrrn efhest'iae, Bacterium gelechiaey Bacterium 5,
and Bacterium cazaubon, in powder form, were mixed with water at the
rate of i to 4 ounces to 2^-2 gallons of water, with the addition of 4 per
cent of molasses J this preparation was sprayed on the plants t^^'o to four
times, at regular intervals, at the rate of 196 gallons or less per acre.
The best results were obtained for plants treated with B. ephestiaej the
infestation being reduced by about 50 per cent as compared with the
controls. A slightly smaller reduction occurred on plots sprayed with
B. cazauborij while B. gelechiae reduced the infestation by less than 40
per cent. Those plants that were treated with the arsenical spray showed
a reduction of only 18 per cent.

Recently microorganisms have been used for the control of the larvae
of Japanese and other beetles in the soil. A variety of bacteria, fungi,
and nematodes were found capable of destro\-ing these larvae. Once the
attacking microorganisms have become established in the soil, the larvae
and the beetles themselves tend to disappear. Glaser {'^2)5) utilized for
this purpose Neoaflectana glaseri. This parasite possesses great repro-
ductive capacity and is capable of destro}-ing large numbers of grubs.
Glaser demonstrated the presence of this nematode also in localities
where the grub was not present.

Dutk\' (222) described two spore-forming bacteria {Bacillus fo-
filUae and Bacillus lentimorbus) which cause the milky disease of the
larvae of the Japanese beetle. These bacteria are grown in the larvae
and then inoculated into soil. They are capable of infecting the grub,
and are said to be responsible for the reduction in the beetle population.
Bacteria pathogenic to the citrus red scale have also been isolated from
the soil (868).

Fungi have also been utilized for the control of insects. Sweetman
(891 ) emphasized the importance of entomogenous fungi as destructive
enemies of insects. A limitation to their practical importance in the fight
against insects is that the fungi require special conditions for develop-

MICROBIAL CONTROL OF INSECT DISEASES 161

ment, especially high humidity and favorable temperature, which are
not always found under natural conditions.

Glasgow (22^ established that some of the caecal bacteria of Het-
eroptera show a marked antagonism toward other bacteria and proto-
zoan parasites that occur in the intestines of these insects. The caecal
system of the insects was removed and dropped into nutrient bouillon,
where it remained for a month or more without showing any bacterial
growth. This was believed to be proof of the fact that the caecal bac-
teria are antagonistic to ordinary saprophytic and parasitic bacteria and
prevent their development} also they apparently kill these bacteria
when they invade the alimentary canal of the insect.

According to Duncan (216), the bactericidal principle found in dif-
ferent insects and ticks shows differences in regard to the types of bac-
teria affected and the degree of their susceptibility. The gut-contents
of Argas and Stomoxys show the widest range of action j that of bugs,
the least. Spore-forming bacteria are especially affected by material
from Stomoxys, whereas staphylococci appear to be more susceptible to
the action of Argas material. The gut-contents of ticks was found to
have a weak activity upon P. festis, whereas the contents of certain in-
sects favored the growth of the latter. This phenomenon may have a
bearing upon the function of the plague flea. The action of the lethal
principle is greater and more rapid at 37° C. than at room temperature.
The lethal principle has been found to be active for at least six months
when kept in a dry state. It is thermostable, resisting temperatures as
high as 120° C, and is not destroyed by proteolytic enzymes. It appears
to be bound to proteins, since it is precipitated from solution by alcohol
and acetone, but it is not affected by these reagents. It is insoluble in the
common fat solvents. It becomes inactivated when allowed to act upon
bacteria and appears to be adsorbed by killed bacteria, even by species
that are not destroyed by it. This substance does not have the properties
of either bacteriophage or lysozyme.

The presence in certain insects of a variety of other substances, such
as allantoin, which affect bacterial activities has also been established.
These observations give rise to the hope that man may in time succeed
in developing and utilizing microorganisms for the biological control of
injurious insects (881).

162 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS

RELATION OF NEMATODES TO SOIL
MICROORGANISMS

Nematode worms are represented in the soil by a number of sapro-
phytes as well as by many plant and animal parasites. The latter vary
greatly in their relation to the host. The larvae of the cereal parasite
Tylenchus tritici penetrate the wheat seedlings between the leaf
sheaths, near the growing or apical points. When the head is formed,
the larvae enter the flowering parts and form galls. They become sexu-
ally mature, mate, and lay eggs which hatch in the galls, and then be-
come dormant. When the galls fall to the ground and decompose, the
larvae are liberated and proceed to find and attack new plants. Other
nematodes attack plants by feeding upon the roots. The methods of
control require, therefore, a knowledge of their life history. Some
species produce resistant forms or cysts that may survive in the soil for
many years, even in the absence of the host plant. Soil sterilization by
steam or by chemicals is frequently employed as a measure of nematode
extermination.

Antagonistic relationships may be utilized for the control of nema-
todes. Linford et al. (572) found that the root-knot nematode of pine-
apple {Heterodera marioni) may be controlled by heavy applications of
organic material. The decomposition of this material results in a greatly
increased population of saprophytic nematodes in the soil. The decom-
posed organic residues also support large numbers of such other soil
microorganisms destructive to the parasitic nematodes as the nema-
capturing fungi (180, 196), the nontrapping fungal parasites, the
predaceous nematodes, the predaceous mites, and different bacteria ca-
pable of destroying nematodes.

BACTERICIDAL ACTION OF MAGGOTS

Surgical maggots are said to have a bactericidal effect in wounds, in
addition to removing necrotic debris. The presence of an active bacteri-
cidal substance which is thermostable and active against 5. aureus^
hemolytic streptococci, and CI. welchii has been demonstrated (854) in
the maggot LuciUa serkata.

CHAPTER 9

ANTAGONISTIC RELATIONSHIPS BETWEEN

MICROORGANISMS, VIRUSES, AND OTHER

NONSPECIFIC PATHOGENIC FORMS

Antagonistic phenomena in relation to viruses have been but little in-
vestigated. It has been established, however, that certain microorgan-
isms are capable of destroying viruses, and particularly that some vi-
ruses possess the capacity of antagonizing other viruses. The rapid in-
activation of poliomyelitis virus in the process of aeration of sewage
sludge has also been indicated ( 1 1 1 ) .

BACTERIA AND VIRUSES

The ability of certain strains of B. subtilis to inactivate the virus of
rabies has long been recognized. The activity was found to be due to a
substance produced in the culture filtrate j limited experimental evi-
dence pointed to the effectiveness of this substance not only in vitro but
also in vivo (619). When a mixture of the culture filtrate of B. subtilis
and the virus was injected into rabbits, the activity of the virus was sup-
pressed (619). It has been suggested (774a) that this action upon the
virus of rabies and of equine encephalitis is due not to a true antibiotic
but to a proteinase similar to the one which destroys bacterial toxins.

An inactivating effect of B. subtilis upon the virus of vesicular stoma-
titis as well as staphylococcus phage, when in contact with them for 1 5
to 18 hours at 35° C, was also reported (750). This phenomenon has
been explained as due to the process of adsorption. The facts that it is
selective, that the phage cannot be reactivated, and that the virus is
rendered impotent by the action of the bacterium also point to a pos-
sible antagonistic effect. However, different specific antibiotics, includ-
ing penicillin, tyrothricin, and subtilin, when used either alone or in
combination with sulfonamides or acridine, have failed to prevent in-
fection of mice with influenza virus (537).

A "nontoxic" inactivator has been defined (307) as a substance that

164 ANTAGONISMS BETWEEN NONSPECIFIC PATHOGENS

inactivates plant viruses and is not detrimental to most forms of life.
Various microorganisms are capable of producing such inactivators.
Plant viruses differ in their sensitivity to "nontoxic" inactivators. Ac-
cording to Johnson (473) various microorganisms are capable of form-
ing such inactivators against tobacco-mosaic virus. The inactivators pro-
duced by A. aero genes and A. niger are particularly effective against a
variety of plant viruses, but not against all of themj the inactivators
produced by the two organisms appear to be similar. They are com-
paratively heat stable but are slowly destroyed by certain organisms.
They can be concentrated by evaporation of medium. A substance
which was capable of rapidly inactivating the tobacco-mosaic virus was
isolated (895) from yeast. A chemical reaction between the inactivating
principle and the virus was therefore suggested. The inactivator in this
instance was destroyed by heating with i TV NaOH solution, but not by
2 N HCl. It was not a protein and gave on analysis 39.7 per cent C and
5.85 per cent H. The substance was said to be a polysaccharide. A. niger
was also found (307) to form in the medium a substance capable of in-
activating a number of different plant viruses; the effect of the inactiva-
tor was found to be exerted upon the virus itself and not upon the plant.
Of 150 organisms, comprising bacteria, fungi, and actinomycetes,
isolated from different natural substrates as well as from soil enriched
with virus concentrates, only three showed some inactivation of the
fowl pox virus, and, in one case, of the laryngotracheitis virus. The
active principle of one of these organisms was actinomycin, an anti-
bacterial substance known to be highly toxic to animals (477).

ANTIBIOTIC SUBSTANCES, VIRUSES, AND
PHAGES

The first recorded observation on the effect of antibiotics upon vi-
ruses is that of Fukuhara (304) who demonstrated that pyocyanase,
after having been in contact with the viruses of vaccinia, rabies, and
chicken pest, brought about their inactivation, as shown by the fact
that when viruses so treated were inoculated into experimental animals
the respective diseases did not develop.

Most of the viruses, however, appear to be resistant to the action of

ANTIBIOTICS, VIRUSES, AND PHAGES

165

antibiotics J this was found to be true of penicillin and clavacin against
fowl pox inoculated into the chorioallantoic membrane of the chick
embryo (784). Penicillin was also found (707) to be without effect on
the virus of vaccinia, encephalitis, and equine encephalonigelitisj how-
ever, it had an effect, when used in large doses, on the course of infec-
tion of chick embryos with psittacosis and meningopneumonitis. The
possible effect of other antibiotics, such as aspergillin, upon certain
viruses has also been indicated (375).

In a study of phage inactivation, it was found that streptothricin,
streptomycin, and clavacin exerted an effect, whereas penicillin and
actinomycin did not. There was no correlation between the suscepti-
bility of the host cells and that of the phage to an antibiotic agent. In the
case of E. colt host and phage, a concentration of the antibiotic great
enough to inactivate all the viable cells showed progressive decrease
in 24 hours of phage added to such mixtures. With lower concentra-
tions of the antibiotic, the phage multiplied only when the cells were
increasing. Phage in suspensions of streptomycin-treated cells was not

TABLE 31. EFFECT OF PENICILLIN AND STREPTOMYCIN ON S. AUREUS
PHAGE AND ITS HOST. RESULTS X 10°

BACTERIAL CELLs/mL.

plaques/ml.

AFTER TIME SPECIFIED

AFTER TIME SPECI-

TREATMENT UNI Ts/m L.

AT 37° c.^

FIED AT 37°

c.

3

24

48

3

24

48

hours

hours

hours

hours

hours

hours

Culture control

0

320

3300

570

Cells -\- streptomycin

2

.01

4.25

4200

Cells + penicillin

10

.15

.275

55

Cells + phage

o

.01

4700

Phage + broth

0

7.5

3-7

.001

Phage -\- streptomycin

2

120

.04

Phage -\- penicillin

lO

100

.98

Cells + phage -f-

streptomycin

2

.01

.001

,294

.41

.2

Cells + phage +

penicillin

10

.09

.001

,025

From Jones (476).

* Number of cells at start,

166 ANTAGONISMS BETWEEN NONSPECIFIC PATHOGENS

reactivated by dilution after prolonged incubation (Table 31). Peni-
cillin and streptomycin acting on S. aureus phage and its host, at concen-
trations of the substances which had no destructive effect on the phage
alone, showed that no reduction of the phage occurred when placed in
the presence of penicillin-treated cells, whereas a definite decrease took
place in the case of streptomycin-treated cells (476).

A mixture of phage and penicillin caused more rapid killing and lysis
of staphylococci than either alone, thus indicating that the penicillin-
resistant organisms were killed by the phage and vice versa. Penicillin
itself did not affect phage multiplication and did not interfere with its
lytic action (425).

The formation of antiphage agents can be studied by a group of
methods, making use of the phage agar plate, phage streak, and agar-
diffusion or cup tests. Growth of the antagonist upon the phage-seeded
agar, or the diffusion of the antiphage agent into the agar, is followed
by flooding the surface with host-seeded agar. Antiphage action is in-
dicated by a reduced number of plaques or by a zone of bacterial growth
surrounding either the antagonist or the cup containing the antiphage
substance (466).

The use of antibiotics in combating true viruses has so far given only
little encouragement. However, the inhibition of growth of typhus
rickettsiae by penicillin has been established (361).

RELATIONSHIPS AMONG VIRUSES

The cultivation of influenza virus in a simple tissue-culture was
found (20) to render the culture unable to support the growth of a
biologically distinct strain of the virus added 24 hours later. The tissue-
culture, however, was still capable of supporting multiplication of
a related virus such as that of lymphogranuloma venereum. When
two strains of the influenza virus were added to the tissue-culture simul-
taneously, the one added in larger concentration suppressed the growth
of the other.

Numerous reports have been made concerning the interference of one
virus by another, and even of inactivated bacteriophage with the active
agent of the same strain (1047, 1048). Henle and Henle (404) have

RELATIONSHIPS AMONG VIRUSES 167

shown that even an inactivated virus, whether a homologous or a
heterologous strain, is capable of suppressing the development of the
influenza virus.

Jungeblut and Sanders (483) suggested that poliomyelitis in ani-
mals may be aborted by the injection of another virus. A strong antago-
nism was observed between a murine virus mutant (virus passed
through mice for many generations) and the parent strain of the virus.
The murine virus was capable of counteracting large paralytic doses of
poliomyelitis j the two viruses virtually counterbalanced each other.

Other types of antagonism between viruses include that of canine dis-
temper or lymphocytic chorio-meningitis virus against experimental
poliomyelitis (169). An intramuscular injection of a neurotropic strain
of yellow fever virus was found to protect animals against simultaneous
infection with a highly pathogenic viscerotropic strain (447). The an-
tagonistic agent was believed to be a chemical substance produced by the
murine virus, for which the term "poliomyelitis inhibition" was pro-
posed by Jungeblut. The "interference phenomenon" of two viruses
can be used to advantage in bringing about immunity reactions.

The suppression of one strain of yellow fever virus by another, as
well as of equine encephalomyelitis virus and of influenza A by yellow
fever virus, belongs to the same group of phenomena. No neutralizing
antibodies or nonspecific antiviral substances were found in the yellow
fever virus (558).

A similar type of antagonism is frequently observed also among plant
viruses. Yellow mosaic virus will not grow in the tobacco tissue cells al-
ready infected with the agent causing common mosaic disease (608).
Other antagonistic phenomena between plant viruses have been re-
ported (612). The virus of peach-yellow prevented invasion by the
virus of little-peach and the latter prevented invasion by the former
(540). The conclusion was reached (608), therefore, that virus domi-
nation in a plant may be looked upon as a type of antagonism, quantita-
tive in nature, the degree of domination by a given virus being influ-
enced by the host.

Many other instances of virus antagonism have been reported, as
when one virus prevents the multiplication of another and actually re-
places it in plants in which it is established (37). Certain vitamins, such

168 ANTAGONISMS BETWEEN NONSPECIFIC PATHOGENS

as ascorbic acid and thiamin, and certain other organic compounds, such
as cysteine, inhibit the formation of necrosis produced by tobacco-mo-
saic virus. This reaction is reversible, since necroses begin to develop
when the tobacco leaves thus treated are placed in pure water (815).

The ability of bacterial phages to interfere with the development of
other phages has been studied in detail by Delbriick and Luria (175,
586). They have shown that a certain phage, after inactivation by ultra-
violet radiation, retained its ability to interfere with the growth of an-
other phage acting upon the same host. The partly inactivated first
phage is adsorbed by the sensitive bacteria and inhibits their growth
without producing lysis. The partly inactivated phage interferes also
with the growth of the active phage. This interference between bac-
terial phages was explained as due to competition for a "key-enzyme"
present in limited amount in each bacterial cell. This enzyme was also
believed to be essential for bacterial growth.

In order to explain the "mutual exclusion effect" of one virus by
another, a "penetration hypothesis" was proposed ( 1 74) . According to
this hypothesis, the penetration of one virus into the cell renders the
cell membrane impermeable to any other virus j each virus has a char-
acteristic penetration time, and a change of permeability occurs at the
end of this time. The depressor effect consists in competition between
the two viruses for the same substrate.

The function of a co-factor, like tryptophane, was considered to be
either that of a cement substance acting in a specific combination be-
tween virus and host receptive spots or as a coenzyme which enables
the virus particles, during their encounters with the host cells, to be-
come attached to them and attack them (21).

MICROBES AND TUMORS

The ability of certain microbes to bring about hemorrhage in tumors
(455a, 1044) may also be classed among the antagonistic phenomena.
The hemorrhage-producing agent is a polysaccharide and is isolated
only from gram-negative bacteria.

Laszlo and Leuchtenberger (549) described a rapid test for the de-
tection of tumor-growth inhibitors. Inhibition was judged by comparing

ANTITOXIC PROPERTIES OF ANTIBIOTICS 169

tumor sizes and weights in treated and untreated groups of mice bear-
ing sarcoma, after a period of 48 hours of growth. The groups were
matched as to initial size of the tumors. The selective damage said to
be caused by penicillin to sarcoma cells as compared with normal cells
(156) was later shown (567) to be due not to the pure penicillin itself
but to some impurity present in crude penicillin preparations.

The hemorrhagic effect upon the tumors is highly selective, being
characteristic of the sarcoma cells only and does not occur in normal
tissues, with a few minor and slight exceptions. The phenomena of
hemorrhage and necrosis are followed in some cases by a complete and
permanent regression of the tumor. The curative effects of such treat-
ments are still open to question, however (94). The same may be said
of the effect upon tumors of trypanosomes or of the "factors" produced
by them.

The effect of penatin upon sarcoma has been tested and found to be
negative (113).

ANTITOXIC PROPERTIES OF ANTIBIOTICS

The ability of various microorganisms to destroy or neutralize bac-
terial toxins has been definitely established. The substance involved was
designated as an antidotic (759). It is produced by B. subtilis and P.
notatum; however, isolated penicillin had no such effect, although
large doses of this antibiotic protected mice against the action of gono-
coccal endotoxin (685). Clavacin was also found (675) capable of neu-
tralizing tetanus toxin.

CHAPTER 10

CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

Antimicrobial agents are of either chemical or biological origin. The
first comprise inorganic (heavy metals, halogens) and organic (phenols,
arsenicals, dyes, aromatic oils) compounds. The second include a variety
of products of higher plants (quinine, chaulmoogra oil, wheat flour pro-
tein, allicin), higher animals (lactenin, lysozyfne), and microorgan-
isms, to which the term "antibiotic" Is specifically applied.

The property possessed by culture filtrates of many bacteria of inhib-
iting the growth of bacterial cells has long been recognized. The sug-
gestion has even been made that all bacteria, when tested at the right
age and under proper conditions of culture, are able to produce anti-
bacterial substances (71). It is now definitely established, however,
that this property Is characteristic of only certain strains of specific bac-
teria, fungi, and actinomycetes.

The production of antibiotic substances by microorganisms is influ-
enced by the strain of the organism, composition of the medium, incu-
bation temperature, age of the culture, aeration, and certain other
factors.

The more Important antibiotic substances are described briefly in
Table 32. They may be classified on the basis of their origin from spe-
cific microorganisms, their chemical properties, or their biological ac-
tion. Differences between various compounds may often be in degree
rather than In kind. Different organisms may produce the same anti-
biotic j frequently the substance may show minor variations from the
general type, these variations being both chemical and biological. Some
organisms are able to produce more than one antibiotic: B. brevis pro-
duces tyrocldlne and gramicidin j P. notatum, penicillin and penatin;
A. fumigatusy fumlgatin, fumlgacin, splnulosin, and gllotoxinj A.
fiavusj aspergilllc acid and penicillin.

Since the name of an antibiotic often designates only a crude prepara-
tion, considerable confusion has arisen because different names have
been given to the same preparation, or the same name has been applied

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176 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

by different workers to different preparations even when these have
been obtained from the same organism. Witness, for example, the
designation "subtilin" that has been applied in different countries to
preparations obtained from different strains of members of the B. suh-
t'tlis group grown on media of different composition. The fact that other
names, like "bacitracin," "subtilysin," "endo-subtilysin," and "bacillin,"
are given to certain preparations of B. sub tilts does not necessarily
indicate that the substances are different. We must await further in-
formation concerning the chemical and biological properties of these
preparations before their identity can be definitely established. The
name "aspergillin" has been applied to at least four preparations, in spite
of the fact that it was first used to designate the black pigment of A .
niger.

Various names have been used to designate indefinite preparations
produced by unknown organisms. This is true, for example, of "my-
cocidin" produced by certain fungi and active against M. tuberculosis
(328), and of "fungin" and "my coin," terms used to designate anti-
biotics of fungi and actinomycetes, as well as of the term "inhibin" to
designate antibacterial substances present in honey.

On the basis of their solubility, antibiotics may be divided into four
groups :

Group A. Soluble in water at different reactions, and insolutle in ether.
These substances usually represent proteins, organic bases, or adsorp-
tion compounds on protein molecules. Some have been isolated in a
pure state. They comprise the bacterial enzymes acting upon micro-
bial polysaccharides, actinomycetin, microbial lysozyme, streptothri-
cin, streptomycin, penatin, and pyocyanin.

Group B. Soluble in ether and in water at proper reactions. Here belong
many of the important antibiotic substances so far isolated and de-
scribed, namely, penicillin, flavicin, citrinin, clavacin, proactinomy-
cin, penicillic acid, and aspergillic acid.

Group C. Insoluble in ether and in water. These include gramicidin, ty-
rocidine, subtilin, and simplexin.

Group D. Soluble in ether and insoluble in water. These include fumi-
gacin, fumigatin, gliotoxin, actinomycin, pyocyanase, and others.

Some of the antibiotic substances have been crystallized, and infor-

CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 177

mation has been gained concerning the approximate chemical nature of
others j many others are still imperfectly known. On the basis of their
chemical nature, the antibiotic substances may be divided as follows :

Lipoids and various microbial extracts removed by organic solvents, such

as pyocyanase, pyolipic acid, and others
Pigments, namely pyocyanin, hemipyocyanin, prodigiosin, fumigatin,

chlororaphin, toxoflavin, actinomycin, litmocidin, and others
Polypeptides, comprising tyrothricin, gramicidin, tyrocidine, colicines,

subtilin, bacillin, and actinomycetin
Sulfur-bearing compounds, namely the different penicillins, gliotoxin, and

chaetomin
Quinones and ketones, namely, citrinin, spinulosin, clavacin, and peni-

ciHic acid
Organic bases, including streptothricin, streptomycin, and proactinomycin

Oxford (701) classified the known antibiotic substances on the basis
of their chemical structure. Most of the antibiotic substances can thus be
grouped as follows :

I. Compounds containing C, H, and O only

1. Ce group: C6H6O4 — kojic acid

2. C7 group: C7H6O4 — clavacin

3. Cg group: CgHgOe — puberulic acid

C8H8O4 — fumigatin
C8H10O4 — penicillic acid

4. Cio group: C10H00O3 — pyolipic acid

5. Ci3 group: C13H14O5 — citrinin

6. Ci5 group: C15H14O6 — javanicin

7- Ci7 group: C17H20O6 — mycophenolic acid

8. C20 group: CgoHieOe — viridin

9. C32 group: C32H44O8 — fumigacin, helvolic acid

Various other compounds belonging to this group have been isolated,
such as gladiolic acid, CnHjoOg.

II. Compounds containing C, H, O, and N

* I. C12 group: C10H8ON2 — hemipyocyanin

C12H8O4N2 — iodinin
C10H20O2N2 — aspergillic acid

178 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

2. Ci3 group: CjaHioONg — pyocyanin

3. C21 group: C2iH37_390i2N7 — streptomycin

4. C34 group: C34H4(;04N2 — pyo II

5. C41 group: C4iH5gOiiN8 — actinomycin

6. Ci4e group: High molecular weight compounds, such as grami-

cidin and tyrocidine; diplococcin may also be in-
cluded in this group

III. Compounds containing C, H, O, N, and S

1. C9+, namely the penicillin group of compounds which is desig-
nated by the formula C9H11O4SN2.R

2. Ci3 group: C13H14O4N0S0 — gliotoxin

IV. Other compounds, many of which have as yet not been fully identi-

fied. Here belongs ustin, C19H15O5CI3.

On the basis of their toxicity to animals, antibiotic substances may
also be divided into three groups:

Compounds that are nontoxic or but slightly toxic ; here belong penicillin,
streptomycin, flavicin, polyporin, and actinomycetin

Compounds of limited toxicity, including gramicidin, tyrocidine, citrinin,
streptothricin, and fumigacin

Highly toxic compounds, such as actinomycin, gliotoxin, aspergillic acid,
and clavacin

Many of the antibiotic substances are thermostable, others are ther-
molablle ; some pass readily through Seitz and other filters, others are
adsorbed. The various methods of isolation of these substances are based
upon their chemical nature, solubility, and properties of adsorption.

SUBSTANCES PRODUCED BY BACTERIA

Lifoids and Pigments

Ps. aeruginosa, discovered by Gessard in 1882 (329), and formerly
known under the names of Bacterium fyocyaneum and Bacillus fyo-
cyaneusy produces several antibiotic agents, the colorless lipid pyocya-
nase, the pigment pyocyanin, and an alcoholic extract of the bacterial
cells.

Pyocyanase, the first antibiotic substance to be isolated, has had a

SUBSTANCES PRODUCED BY BACTERIA 179

rather interesting history. Emmerich believed that it is an enzyme
(233, 236). Later it was found (766) that all the active substance
could be extracted with lipid solvents j the extraction of the cells of Ps.
aeruginosa with alcohol also gave active antibacterial preparations. A
crystalline product was finally obtained (448) 5 it was soluble in organic
solvents and had a bactericidal effect upon B. anthracisy S. albus, C.
difhtheriae, and a number of other organisms.

In the course of time it was recognized that all the antibacterial ac-
tivity of the lipoid extracted from the medium was due to the presence
of fatty acids, so that the term pyocyanase is now used to designate the
antibiotic lipid, found in the medium and containing unsaturated fatty
acids. Certain well-defined compounds have recently been isolated, such
as pyolipic acid (50a).

Schoenthal (843) obtained three compounds that possessed antibac-
terial properties, namely, pyocyanin, oxyphenazine, and an active oil
that formed insoluble salts with calcium, barium, and heavy metals.
The last appeared to be similar to what had previously been described
as pyocyanic acid, a substance highly active against V. comma. All three
compounds were isolated by extraction with chloroform.

Different strains of Ps. aeruginosa may produce either pyocyanase or
pyocyanin or both, the production of the two not proceeding in a paral-
lel manner. Among the amino acids, alanine and tyrosine were found to
be favorable to pyocyanin production (346), although the effect of
tyrosine is not very significant (346, 461, 573).

The determination of the nature of the antibacterial substances of
Ps. aeruginosa can be carried out in the following manner (418) : the
organism is grown in bouillon for 14 daysj the cultures are heated for a
half hour at 75° C. to kill the living cells j they are then centrifuged,
the liquid is treated with chloroform which extracts the pigment, and
the chloroform solution is concentrated in vacuo at 50° C. j the aqueous
solution remaining after chloroform extraction is acidified with hydro-
chloric acid and again shaken five times with chloroform, thus extract-
ing the fatty acids. It was found that the antibacterial properties are
yery little diminished by removal of the pigment j however, when both
the pigment and the fatty acids are removed, no antibacterial action is
left in the culture. S. aureus is commonly used as the test bacterium.

180 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

The broth culture of the organism may also be first extracted with
ether, giving pyocyanase, and the residue treated with chloroform,
yielding pyocyanin. The solution left after the removal of the blue
chloroform extract may be again treated with ether, giving a yellow
pigment, which also has some activity (529). This pigment is a deriva-
tive of pyocyanin and is often designated (1036) as hemipyocyanin. It
may also be obtained by acidifying pyocyanin with acetic acid and heat-
ing. The fluorescin remaining in the culture after the ether and chloro-
form extraction was found to be inactive. In old cultures, pyocyanin is
changed into a brown pigment, pyoxanthose. A fourth pigment, which
is yellow in transmissible light and fluorescent-green in reflected light,
is produced under certain conditions. It was excreted into the medium
as a leuco base.

Pyocyanase is soluble in ether, benzol, benzene, and petrol ether
(766). It can be separated into several lipoids, the action of which
shows slight variation. This preparation consists of a phosphatide, a
neutral fat, and a free fatty acid. The antibacterial properties have been
attributed to the last constituent (421 ). A definite relation has been ob-
served between the number of double bonds and the activity of the sub-
stance (59, 420). According to Dressel (197), most fatty acids exert
bactericidal and bacteriolytic effects upon gram-positive bacteria,
whereas gram-negative organisms are not lysed. Pyocyanase acts upon
various bacteria, including the colon-typhoid group, though the ability
of the substance to inhibit the growth of this group of bacteria has been
denied by some workers (372).

B. mesenter'icus and other spore-forming bacteria also produce anti-
biotic agents of a lipoid nature. The substance is not affected by heating
for 30 seconds at 100° C. but is weakened at 1 15° C. for 10 minutes. It
is considered similar in its bactericidal properties to pyocyanase.

Alcohol and acetone extracted from B. mesentericus a weakly active
substance (419) that diffused through a cellophane membrane and
could be partly absorbed on a Berkfeld filter. When shaken directly
with ether, the culture lost its antibacterial properties. The ether extract
was concentrated and ammonia added, and the solution was treated with
50 per cent alcohol. The alcohol was then removed, and the residue was
acidified and treated with petrol ether, which brought the active sub-

SUBSTANCES PRODUCED BY BACTERIA 181

stance into solution. The active substance was again dissolved in alcohol
and taken up in ether. The ether solution was washed with water, evapo-
rated, and dried. One liter of a 30-day-old culture of B. mesentericus
gave 1 62 mg. of petrol-ether-soluble fatty acids and an oily substance
of a brownish color. It was neutralized with NaOH solution and tested.
The extract diluted to 1:7,500 killed diphtheria} a 1:1,000 dilution
was required to kill staphylococci. Iso-valerianic acid and oleic acid,
isolated from this material, had a similar bactericidal action. Weaken-
ing of the substance by heating was demonstrated and was believed to
be due to a break in the double bond of the oleic acid.

E. coli exerts an antagonistic effect in vivo when injected subcutane-
ously or when used for feeding. It produces (367, 369) a thermolabile
substance that was considered to be a lipoid in character. However,
some of the antibiotics of E. coli, namely the colicines, appear to be
definitely proteins or polypeptides.

Pyocyanin is a dark blue pigment, red in acid solution, m.p. 133° C,
water soluble and amphotheric. It is extracted with chloroform, then
reextracted by acidulated water. It is characterized by a wide antibiotic
spectrum and high toxicity to animals. This pigment was first studied
by Fordos in 1 860 (277). Since then many contributions have appeared
dealing with formation and nature of this pigment. Several formulae
have been suggested for pyocyanin (461, 935, 1036), one of which is
shown in Figure 18. The structure of pyocyanin has considerable simi-
larity to chlororaphin and iodinin, obtained from Chromobacterium
(596), and two synthetic compounds, phenazine and acridine (939).
Since Ps. aeruginosa is an extremely variable organism, the nature and
abundance of the pigment are variable. Keeping the organism for 5
minutes at 57° C. or cultivating it in liquid egg-albumin has been found
to result in destruction of some of its pigment-producing properties

(330,557).

Hemipyocyanin is found in old cultures of Ps. aeruginosa (843) and
is synthesized (1036) from pyocyanin. It is a yellow pigment, m.p.
158° C, with basic and phenolic properties. It is moderately bacterio-
static and strongly fungistatic (883).

Prodigiosin is produced by S. marcescens. It is insoluble in water and
is active against B. anthracis (1035).

182 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

CH3O.C C=CHp H

HC CH3

I

I I

c

ZC C=CH\ HOC CH

ri2C CH.CO
O

/

COOH
PENICILLIC ACID CLAVACIN

HC C.CH2OH

KOJIC ACID

PHENAZINE

FUMIGATIN

CONH2

CHLORORAPHIN

Figure 18. Structural formulae of some antibiotic substances.

lodinin is a deep purple-bronze pigment, m.p. 236° C. (Figure 18).
It is produced by Ch. iodinum {S9l) and is excreted into the medium.
It is insoluble in water and in acids but soluble in alkali, and is phenolic
in character. It is dissolved in chloroform. It is active against S. hemo-
lyticus, less so against S. aureus and other bacteria.

Violacein, a purple pigment, is produced by Ch. violaceum. This
pigment is active against gram-positive bacteria, except CI. welchii; it

SUBSTANCES PRODUCED BY BACTERIA 183

has little effect upon the gram-negative bacteria, except the meningo-
cocci. Among the fungi, only Blastomyces dermatiditis is susceptible.
The action of the pigment is greatly affected by serum (569).

It may be added here that certain aromatic oils and various fatty acids
possess marked bactericidal properties (518). Unsaturated acids are
more active than saturated acids (1040). Ordinary peptones have also
been found to contain a substance that is active against various bacteria,
especially when small amounts of inoculum are used (202). The active
substance is thermostable and is associated with an acid-precipitated
fraction that is pigmented and changes color upon oxidation and reduc-
tion. The bacteriostatic effect of this material can be corrected by the
addition of reducing agents, such as thioglycollic acid. The bacterio-
static action of dyes is well known and need hardly be discussed here. It
is sufficient to mention, for example, methylene blue and indophenols
in oxidized forms.

Pyo-compounds. Doisy and his collaborators (389) centered their
attention upon the antibiotics present in the Ps. aeruginosa cells. This
group of compounds was designated as Pyo I, Pyo II, Pyo III, and Pyo
IV. The culture of the organism was incubated for 5 weeks, cooled, and
acidified with HCl to ^H 't^.S'-, it was centrifuged, and the precipitate
was extracted with hot 95 per cent ethyl alcohol. The alcohol extract
was diluted with water to 80 per cent alcohol and treated with petro-
leum ether, to remove the fats and fatty acids. The alcoholic solution
was evaporated and the aqueous residue extracted with ether. The ex-
tract was separated into the four fractions listed above, which repre-
sented pure, crystalline, active substances. These fractions were struc-
turally related and were more active against the gram-positive than the
gram-negative bacteria. They were nontoxic to animals.

Polysaccharidases

Among the antibiotic substances of microbial origin may also be in-
cluded the enzyme systems that have the capacity of decomposing the
capsular substance of certain bacteria, thereby rendering them more
readily subject to destruction in the blood stream or in other substrates.
The first enzyme of this type was isolated by Dubos and Avery (204,

184 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

205, 207) from certain soil bacteria. These enzymes are highly specific,
some being able to act only upon one type of pneumococci. As a result
of their action, the pneumococcus cell is rendered susceptible to destruc-
tion by phagocytosis. This enzyme was produced by the soil bacteria
under selective conditions of culture, that is, when the capsular polysac-
charide of the pneumococcus was present in the medium j the only other
substance that could be used for its production was aldobionic acid,
which was derived from the above polysaccharide. Yields of the en-
zyme were increased by increasing the concentrations of the specific
substrate in the medium from 0.0 1 to o.i per cent. Above o.i per cent,
the yields decreased, 0.3 to 0.4 per cent inhibiting the growth of the bac-
terium. The addition of 0.1 per cent yeast extract favored the produc-
tion of the enzyme j proper aeration was essential, the bacterium mak-
ing the best growth in shallow layers of medium. The enzyme was
concentrated by distillation in vacuo and by ultrafiltration. Toxic sub-
stances accompanying the active preparation could be largely removed
by the use of an aluminum gel. The enzyme is associated with a protein
which passes through a collodion membrane with an average pore size
of 10.6 (J, but is held back by pores having a diameter of 8.2 |j. After
filtration, the enzyme can be recovered in solution by immersing the
membrane in distilled water or in physiological salt solution.

Dubos (199) believed that it is possible to develop "adaptive" bac-
terial enzymes against many organic substances. These enzymes exhibit
a great degree of specificity, as in the case of the enzyme that hydrolyzes
the capsular polysaccharide of the pneumococcus. The cell of this or-
ganism contains an enzyme that changes the cell from the gram-positive
to the gram-negative state, but is ineflrective against streptococci or
staphylococci.

Active preparations of the enzyme protected mice against infection
with as many as i ,000,000 lethal doses of the specific pneumococcus. The
enzyme retained its activity for 24 to 48 hours after its injection into
normal micej it also exerted a favorable influence on the outcome of an
infection already established at the time of treatment. A definite rela-
tionship was found to exist between the activity of the enzyme in vitro
and its protective power in the animal body.

SUBSTANCES PRODUCED BY BACTERIA 185

Polyfepides and Proteins

From the tyrothricin complex group of antibiotics produced by B.
brevisy two crystalline compounds have been isolated. They are poly-
peptides resistant to the action of proteolytic enzymes (201, 208, 450,
909). The organism is grown in shallow layers of a suitable medium,
such as one containing i per cent casein digest or tryptone and 0.5 per
cent NaCl in tap water, adjusted to /)H 7.0. After inoculation, the
medium is heated for 20 minutes at 70° C, in order to kill the vegeta-
tive cells of the bacteria, leaving only the spores to develop. The cul-
ture is allowed to grow for 72 hours. The reaction of the culture is then
adjusted to /)H 4.5 by the use of about 3 or 4 cc. concentrated HCl per
liter of culture. A precipitate is formed which is removed by filtration
through paper J it is then suspended in 95 per cent alcohol (20 cc. of
alcohol per liter of culture) and allowed to stand 24 hours. The active
substance is dissolved and is separated from the residue by filtration j
when the alcoholic solution is diluted with 10 volumes of i per cent
NaCl, the substance is precipitated out. It carries all the activity and can
be desiccated in vacuo, over P2O5, giving a yield of about 100 mg. of
final dry substance per liter of culture medium. The protein-free, alco-
hol-soluble active material is tyrothricin. When an attempt was made to
produce tyrothricin in aerated submerged cultures, none was obtained
in complex nitrogenous media j however, simple amino compounds,
like asparagine, gave good growth and yielded the antibiotic substance.
The presence of cystine in the mixture of amino acids appeared to in-
hibit growth (884).

Gramicidin is obtained by treating tyrothricin with a mixture of
equal volumes of acetone and ether, evaporating, and dissolving in
boiling acetone. On cooling, it crystallizes out as spear-shaped colorless
platelets, melting at 228° to 230° C, with a yield of about 10 to 15
grams from 100 grams of the crude material. Gramicidin is soluble in
lower alcohols, acetic acid, and pyridine, and moderately soluble in dry
acetone and dioxanej it is almost insoluble in water, ether, and hydro-
carbons. When a solution containing 20 to 50 mg. per milliliter alcohol
is diluted to i mg. per milliliter, with distilled water or with glucose
solution, an opalescent solution is produced without flocculation. On
dilution with electrolyte solutions, an immediate flocculation occurs.

186 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

The specific rotation of gramicidin in 95 per cent alcohol solution is
approximately [a]^5 _ _|_ ^o^ Qj^ analysis, it gives 62.7 per cent C,
7.5 per cent H, and 13.9 per cent N. The molecular weight, as
determined in camphor, is about 1,400. The empirical formula of
C74H106O14N14 has been suggested. On further study, the molecular
weight of gramicidin was found (911) to present an anomaly in that it
appeared to depend on the nature of the solvent and on the concentra-
tion of the solute, giving values from 600 to 1,200 j isothermal distil-
lation in methanol, however, indicated a molecular weight of 2,700
to 3,100, with an approximate formula of C146H000O2N30. It gave
neither free amino nor carboxyl groups j it contained 10 molecules of
a-amino acids, of which two or three were tryptophane residues. These
and a saturated aliphatic acid, with 14 to 16 carbons, account for about
85 to 90 per cent of the weight of the substance. Amino acids that have
definitely been identified are /-tryptophane, /^-leucine, /-alanine, dl-
valine, and glycine (345, 892). A study of the configuration of the di-
peptide valyvaline separated from gramicidin brought out the fact that
only valines of like configuration have been joined together by the bac-
terium (137). About 45 per cent of the a-amino acids gave the d con-
figuration (449, 450). An unknown hydroxyamino compound has also
been indicated.

The presence of ethanolamine (2-aminoethanol-l) as a component of
gramicidin hydrolysates, which reacts with periodate to yield formal-
dehyde and NHo, has been definitely indicated (893). Actually two
ethanolamine residues may occur in gramicidin, since their destruction
during acid hydrolysis of gramicidin is considerable. The liberation of
some of these amino acids during hydrolysis, such as valine and trypto-
phane, can be measured by their availability to L. arah'mosus (139).

Tyrocidine hydrochloride is moderately soluble in alcohol, acetic
acid, and pyridine ; it is sparingly soluble in water, acetone, and dioxane,
and is insoluble in ether and hydrocarbon solvents. An alcohol solution
can be diluted with water to give a clear solution containing 5 to 10 mg.
per milliliter; electrolytes produce an immediate precipitate. A solu-
tion in distilled water containing i mg. or even less per milliliter has a
low surface tension and behaves like a soap or detergent solution. Un-

SUBSTANCES PRODUCED BY BACTERIA 187

like gramicidin, it precipitates a number of soluble proteins in a manner
similar to some of the cationic detergents.

Tyrocidine is dissolved in four times its weight of boiling absolute
alcohol, to which is added alcoholic HCl (o.i mol. per liter). On cool-
ing, a precipitate is formed. This is recrystallized from absolute metha-
nol plus small amounts of HCl ; clusters of microscopic needles are ob-
tained, melting at 237-239° C, with decomposition; the specific rota-
tion is [a]^5 = — 102° ( I per cent in 95 per cent alcohol). Tyrocidine
analyzes: 59.4 per cent C, 6.8 per cent H, 13.5 per cent N, 2.7 per cent
CI. The molecular weight is about 1,260 or a multiple of this number.
Tyrocidine is a salt of a polypeptide having free basic amino groups.
The ^-amino acids make up 20 per cent of its a-amino groups. The most
probable molecule was shown to contain two amino groups, three amide
groups, and one weakly acidic carboxyl or phenolic group, with a molec-
ular weight of 2,534. Among the amino acids, tryptophane, tyrosine,
and dicarboxylic-amino acids have been detected j concentration of some
of these acids has been established: aspartic acid, 5.1 per centj valine,
7.6 per cent J and leucine, 8.2 per cent (138, 140). Summaries of the
chemical and biological properties of gramicidin and tyrocidine were
made by Hotchkiss (449) and Hoogerheide (443).

The tyrothricin-type of antibiotic substance appears to be widely dis-
tributed among spore-forming aerobic soil bacteria (442, 444, 885).
Preparations obtained from different bacteria appear to be markedly
different in chemical nature and biological activity. This is true, for
example, of the preparation obtained by the following method: A
seven-day-old bacterial culture was treated with 2 to 5 per cent of an
electrolyte and HCl added to give a fH of 4.0. A precipitate was
formed which was centrifuged and extracted with 95 per cent alcohol,
until no more turbidity could be observed after dilution with an equal
volume of water. The alcoholic extracts were evaporated to dryness and
extracted with ether, petroleum ether, and benzol, in which the active
substances are insoluble. The residue was then dissolved in absolute
alcohol, and the concentrated solution dialyzed for 24 hours against
running tap-water and for 24 hours against distilled water. The active
substance was obtained partly in a precipitated form and partly in a

188 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

colloidal solution in the dialysis bag. Upon evaporation of the water, a
highly active, grayish-white powder was obtained. One hundred liters
of medium gave 15 grams of purified active substance. The activity
could be tested by inhibition of encapsulation of Friedlander's bac-
terium j this was brought about by the addition of 4 mg. to i ml. of cul-
ture medium. This preparation was later found to be identical with
gramicidin (443,911).

Gramicidin S (43, 324) is related to the tyrothricin complex, being
tyrocidine in nature. It was found (894) to be a cyclopeptide, with a
stoiochiometric minimum unit formed from one residue each of /-orni-
thine, /-proline, /-valine, /-leucine, and ^-phenylalanine. The unit pos-
sesses one free amino group, no free carboxyl groups, and one residue
of chloride.

Bacitracin is formed by certain strains of B. subtilis grown in shallow
layers of media. A heavy surface pellicle is produced after 3 to 5 days'
incubation at 37° C. The medium is extracted with normal butanol and
concentrated by steam distillation in vacuo, giving a grayish-white
powder. The substance is neutral and water soluble and withstands heat-
ing for 15 minutes at 100° C. without significant loss of activity. It is
stable in acid solution but unstable in alkaline solution above fH 9, and
is not digested by proteolytic enzymes. It is active chiefly against gram-
positive organisms, but the gonococcus and meningococcus are also
susceptible to it. It is active in vivo against experimentally produced
hemolytic streptococcus and gas gangrene infections (469).

Subtilin is produced by certain strains of B. subtilis. It is a polypep-
tide and is readily digested by proteolytic enzymes. It is most active at
/»H 2.2 and gradually becomes inactivated with decreasing acidity. It
is active against various gram-positive bacteria, acid-fast bacteria, and
certain pathogenic fungi (816). Eumycin, produced by certain strains
of B. subtilis and active largely against fungi, actinomycetes, and myco-
bacteria, although showing little effect against staphylococci may also
belong to this group (471). Subtilysin was reported to have a lytic ac-
tion against gram-negative bacteria, none against cocci (925). Some of
the subtilin preparations also have the capacity of inactivating bacterial
toxins, such as diphtheria, tetanus, and others. This property was as-

SUBSTANCES PRODUCED BY ACTINOMYCETES 189

cribed to the presence of a heat-stable substance designated as antidotic

(759).

Diplococcin Is produced by certain lactic acid streptococci. It is a pro-
tein synthesized in the bacterial cells from the amino acids in the me-
dium, and is extracted with cold dilute acetic acid. The active protein is
precipitated by 60 per cent saturation with ammonium sulphate (701 ).
It is active against gram-positive cocci and Lactobacillus species, but not
against gram-negative bacteria.

A thermostable substance was obtained (154) from B. simplex, an
organism capable of bringing about the destruction of various patho-
genic fungi. This antibiotic was later designated as simplexin. It was
produced by the bacterium grown both on synthetic and on organic
media. It can be adsorbed on activated charcoal and recovered from the
latter by the use of hot alcohol.

To what extent substances of bacterial origin that are toxic to brain
tissues, like toxoflavin (C6H6N4O2), are also effective against bacteria
and other microorganisms still remains to be determined. Toxoflavin,
formed by Bacterium cocovenenans , is extracted from the culture satu-
rated with salt by means of chloroform j from this it is recovered by an
aqueous solution and purified (931, 932).

Other bacterial toxins, like botulinus toxin, various amines and
purine bases, and numerous toxins produced by bacteria in living plant
and animal systems, are beyond the scope of this treatise.

SUBSTANCES PRODUCED BY ACTINOMYCETES

Actinom^ycin

Actinomycin is an ether-soluble and alcohol-soluble pigmented sub-
stance produced by certain actinomycetes, notably S. antibioticus. The
culture medium is treated with ether, giving an orange-colored extract.
The residue is evaporated and treated with petrol ether (975).

The purification of actinomycin was effected by chromatographic
adsorption, followed by fractionation of eluate. The orange-brown resi-
due left after treatment with petroleum ether was dissolved in benzene,
filtered, and allowed to pass through a tower packed with aluminum
oxide. On washing the tower with large amounts of benzene, a chro-

190 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

matogram slowly developed. The column was then washed with a solu-
tion of 1 5 parts acetone to 85 parts benzene until the yellow-orange band
approached the bottom of the column. The elution of the pigment from
the column was accomplished finally by further washing with 30 per
cent acetone in benzene until the eluate was faintly yellow in color. The
later eluates were found by assays to contain all the active pigment,
whereas all previous eluates, as well as the fractions remaining on the
adsorbent, showed no bacteriostatic or bactericidal activity.

Pure actinomycin was obtained by concentrating the 30 per cent
acetone-benzene eluates to dryness, then recrystallizing the red solid
residue from acetone-ether mixtures or from ethyl acetate. From these
solvents, the pigment separated as vermilion-red platelets which
melted at 250° C, with slow decomposition. The pigment is readily
soluble in chloroform, benzene, and ethanolj moderately in acetone
and hot ethyl acetate j and slightly in water and ether. The color of the
solid pigment depends on its state of subdivision j when ground very
fine, its color is orange-red (910).

Actinomycin is optically active, a solution of 5 mg. in 2 cc. ethanol
in a I dm. tube having a rotation — i.6o°j [a]'^ = —320° ± 5. Its
molecular weight was found to be around 1,000. Cryoscopic measure-
ments in cyclohexanol and in phenol gave molecular weights of 768 to
780 and 813, respectively. The approximate molecular formula was
found to be C41H56O11N8. Actinomycin exhibits characteristic ab-
sorption in the visible and ultraviolet regions. In ethyl alcohol, it shows
strong absorption at 450 (E| ^ = 200) and between 230 and 250.

Actinomycin is not soluble in dilute aqueous alkali or in dilute min-
eral acids. It is soluble in 10 per cent hydrochloric acid and appears to
be regenerated by diluting such solutions with water. With strong alco-
holic alkali a purple color is formed, which rapidly disappears. Actino-
mycin is readily reduced by sodium hydrosulfite and by stannous
chloride, but is unaffected by sodium bisulfite. With sodium hydro-
sulfite the reduction is characterized by a change in color from red to
pale yellow. The color change is reversed by exposing the reduced pig-
ment to air. The same reversibility of color occurs when the pigment
is subjected to catalytic hydrogenation in the presence of platinum
oxide. The pigment has one or more functional groups capable of re-

SUBSTANCES PRODUCED BY ACTINOMYCETES 191

versible reduction-oxidation (probably quinone in nature) and several
others capable of acetylation (probably hydroxyls). The quinone-like
structure of the pigment is borne out by its sensitivity to alcoholic alkali,
and to hydrogen peroxide in the presence of sodium carbonate. In the
latter instance, the color rapidly disappears and a cleavage seems to
occur.

Actinomycin in alcohol-water solutions is resistant to the action of
heat, being able to withstand boiling for 30 minutes. When such solu-
tions are made acid, however, boiling has a destructive effect upon the
activity of the substance, the extent of destruction being directly pro-
portional to the concentration of acid. The effect of alkali, however, is
much greater. Dilute alkali changes the color of the substance to light
brown, accompanied by a reduction in activity, which can be largely re-
stored when the solution is made neutral again. At a higher alkalinity
(0.25 N), especially at boiling temperature, the activity and reversibil-
ity are destroyed. Exposure of solutions to light for i to 3 months re-
duces the activity of the pigment very little.

Streftothrkin

Streptothricin is produced by Streftomyces lavendulae grown in a
medium containing glucose or starch ( i per cent) as a source of energy,
and tryptone, glycocoU, glutamic acid, or other organic nitrogenous
compound (0.3 to 0.5 per cent) as a source of nitrogen. Sodium nitrate
is a somewhat less favorable source of nitrogen. The organism is grown
in stationary, shallow cultures containing starch as a source of carbon
or glucose and a small amount of agar, or in submerged cultures. The
optimum temperature for the production of streptothricin is 23° to
25° C. (946). The relation between growth of the organism and pro-
duction of the antibiotic substance is brought out in Table 't^'t,.

Streptothricin is soluble in water and in dilute mineral acids, but is
destroyed by concentrated acids. It is insoluble in ether, petrol ether,
and chloroform. In the crude culture-filtrate and in the alcohol-precipi-
tated form, streptothricin is thermolabile, whereas in the purified state
it is thermostable, withstanding 100° C. for 15 minutes. Treatment
with proteolytic enzymes does not reduce its activity. On adjusting the
reaction of the medium, when growth is completed, to ^H 3.5 with

192 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

TABLE 23- GROWTH OF STREPTOMYCES LAVENDULAE AND PRODUCTION
OF STREPTOTHRICIN ON TRYPTONE-STARCH MEDIUM

DRY WEIGHT

NITROGEN

ACTIVITY

INCU-

OF MYCE-

IN MYCE-

IN

UNITS

BATION

STARCH

LIUM IN

LIUM IN

E.

B.sub-

AERATION

IN DAYS

LEFT

MILLIGRAMS

MILLIGRAMS

coli

tilis

Shaken

2

+++

10

5

Shaken

3

+

225

18.2

10

50

Shaken

4

O

293

26.2

75

250

Shaken

6

o

231

17.3

100

300

Shaken

8

0

75

200

Shaken

12

o

142

9.6

30

50

Stationary

7

+++

50

200

Stationary

10

Tr

235

18.8

50

300

Stationary

H

Tr

60

250

From Waksman (946).

acid, a precipitate is produced, the filtrate containing virtually all the
activity.

Streptothricin is completely adsorbed, at neutrality, on charcoal,
from which it can be removed by treatment for 8 to 1 2 hours with dilute
mineral acid or acid alcohol. The acid extract is neutralized and con-
centrated in vacuo y at 50° C, just to dryness j the residue is extracted
with absolute alcohol, filtered, evaporated, and taken up in water. It
can also be precipitated from the neutralized solution with ether or
acetone. Further concentration and reduction in ash content can be ob-
tained by subsequent treatments. On electrodialysis, the active sub-
stance moves to the cathode at fH. 7.0.

Streptothricin has been crystallized as the Reinecke salt (300). The
crystals consist of a cluster of fine platelets which decompose at 192°
to 194° C. after sintering at 184° C. The molecule was found to corre-
spond to the di-reineckate of a base C13H25O7N5 j the a-amino nitrogen
was 20 to 22 per cent of the total nitrogen. The molecule of streptothri-
cin is thus believed to contain at least five nitrogen atoms, two of which
are present as salt-forming basic groups j it is free of O-methyl, N-
methyl, and hydrolyzable acetyl groups. Streptothricin is stable be-
tween fH I and 8.5, but is destroyed by high alkalinity. The activity of

SUBSTANCES PRODUCED BY ACTINOMYCETES

193

the sulfate is 500-530 [ig/mg. One of the more recent modifications
(713a) of the method of isolation of streptothricin comprises the fol-
lowing steps: charcoal adsorption, elution with formic acid in methyl
alcohol-water, partial concentration in vacuo, precipitation with picric
acid, conversion to hydrochloride, chromatography over aluminum
oxide, and precipitation with methyl orange as helianthate. This
preparation had an activity of 830 ng/mgl., with a specific rotation

Streftomycin

Streptomycin is produced, in stationary and shaken cultures, in a
medium containing meat extract, corn steep, soy bean meal, or some
other suitable material. Its maximum production occurs in shaken cul-
tures in 2 to 3 days, and in stationary cultures in 7 to 10 days (830,
971), as shown in Table 34.

TABLE 34. GROWTH OF S. GRISEUS AND PRODUCTION OF STREPTOMYCIN

Incubation

/^g

/>Hof

Growth

medium

in mg.*

Shaken cultures

2 days

10

. 7.8

270

3 days

70

7-7

185

4 days

60

7.8

-

7 days

70

8.2

-

Stationary cultures'

3 days

6

7-7

73

5 days

12

7.8

171

7 days

53

7.9

163

9 days

-

8.3

264

1 2 days

55

-

-

From Schatz, Bugie, and Waksman (830).

* Weight of dry m>'celium produced by S. griseus.

Streptomycin is also a base, like streptothricin, but differs from it in
cl^emical composition, antibacterial spectrum, and lower toxicity for
animals (830, 952). It is highly active against the gram-negative en-
teric group of bacteria and related organisms. A detailed discussion of

194 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

the nature of the antibiotic action of streptomycin and its utilization for
chemotherapeutic purposes is presented elsewhere (pp. 287-296).

It was at first suggested (947) that 3 units of activity be recognized
for measuring streptomycin: an S unit, or the amount of material that
will inhibit the growth of a standard strain of E. coU in i ml. of glu-
cose-free nutrient agar or broth j an L unit, inhibition on a liter basis j a
G unit, inhibition on a dry weight basis of crystalline material. Since
streptomycin base was found to be 1,000 S units per i mg., it was de-
cided to accept the weight of streptomycin as a basis of standardization:
I S unit is thus equivalent to i microgram of the pure base.

Streptomycin can be isolated from the medium by several proce-
dures. In one method (115), culture filtrates of S. griseus assaying 100
to 180 units of streptomycin per ml. served as the starting material.
Several common adsorption agents, such as charcoal, can be used to re-
move the active material from the culture. The substance is then eluted
with hydrochloric acid in 95 per cent ethanol. Anhydrous hydrogen
chloride in methanol is a more convenient reagent, since the crude
streptomycin can be precipitated directly from the methanol solution
with ether. The filtrate is clarified at pH 2 with 0.5 per cent carbon j
this is followed by removal of the streptomycin, at ^H 7, with i per
cent carbon, which is washed successively with water, neutral ethanol,
and neutral methanol, and the streptomycin is eluted by two or three
extractions with o.i N methanolic hydrogen chloride. The alcoholic
extracts are combined and 2 to 3 volumes of ether added, precipitating
the crude streptomycin chloride as a light-brown amorphous powder.
When the methanol solution contains much water, a sticky gum results.
The recovery of the streptomycin by this method varies from 30 to 50
per cent, the product assaying from 150 to 300 micrograms.

For further purification, a faintly acid solution of crude streptomycin
chloride in 70 to 80 per cent methanol is percolated over a sulfuric
acid-washed alumina column (^H 5 to 6) j an inactive fraction giving
a positive Sakaguchi test first appears, followed by a Sakaguchi-negative
fraction. This test parallels the antibiotic action of the fractions. A small
amount of active material remains on the column and can be washed
through by lowering the methanol content of the solvent. This ma-
terial contains sulfate ion but no chloride. The streptomycin sulfate

SUBSTANCES PRODUCED BY ACTINOMYCETES 195

passes through the column less rapidly, since it is less soluble than the
chloride in methanol.

The various streptomycin fractions obtained from the column are
concentrated and lyophilized, giving white amorphous powders. The
most active fractions range from 600 to 900 jjg/mg., and amount to
approximately 80 per cent of the total. Satisfactory results are obtained
only if the crude streptomycin has an activity of about 200 Mg/mg. or
higher. Preparations of lesser purity contain substances which interfere
with the development of the chromatogram. The chloride is soluble in
methanol, less soluble in ethanol, practically insoluble in butyl alcohol,
acetic acid, and pyridine. The sulfate is only slightly soluble in metha-
nol and practically insoluble in the other solvents.

Streptomycin gives a positive Sakaguchi test, the presence of a guani-
dine group being indicated by the fact that alkaline hydrolysis results
in the formation of ammonia and the disappearance of the Sakaguchi
test. Streptomycin also gives a positive test for an hydroxyl group.
Negative tests are obtained in the amino nitrogen, Hopkins-Cole, Mil-
Ion, xanthoproteic, biuret, and Pauly diazo tests. The presence of a
carboxyl group is considered as questionable, since the streptomycin
chloride, obtained by precipitation from methanolic hydrogen chloride
with ether, gives approximately neutral solution. The ultraviolet spec-
trum of streptomycin showed only end-absorption below 230 my,
which makes improbable the presence of an aromatic ring or conjugated
double bonds.

Streptomycin is inactivated rapidly by o.i N sodium hydroxide at
room temperature. It is relatively stable over a -pH range of i to 10 but
is inactivated by i N hydrochloric acid.

Streptomycin was first crystallized as the reineckate salt from water,
in the form of thin plates which decomposed at 162°-! 64° C, the basic
component being (CioHi907_8N3)^. The antibiotic potency of pure
streptomycin lies between 800 and 910 Mg per mg. (299). Streptomycin
can also be isolated (537a) by the method described above for strep-
tothricin. This includes charcoal adsorption, elution with methanolic
formic acid, precipitation with picric acid, conversion to the hydro-
chloride, chromatography with aluminum oxide, and final conversion
to the crystalline helianthate.

196 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

In another procedure (538) the crystalline salt of streptomycin and
/)-(2-hydroxy-l-naphthylazo)-benzenesulfonic acid is prepared from
streptomycin HCl, and orange II. The salt has an activity of 300
Mg/mg. Crystalline streptomycin sulfate was found to have an activity
of 520 ng/mg.

On chromatographic purifications, streptomycin concentrates yielded
a crystalline double salt of streptomycin trihydrochloride and calcium
chloride (Figure 19). This preparation showed that streptomycin has
the composition CsiHg^.ggN^Ois- The double salt is characterized by
constant biological, chemical, and physical properties. It is more satis-
factory than the hydrochloride which is obtained by precipitation. The
double salt can be prepared from streptomycin hydrochloride or from
the crystalline streptomycin helianthate. A cryoscopic molecular weight
determination on streptomycin trihydrochloride in water gave about
800 for the free base, necessary corrections having been made for the
chloride ion and the non-ideal cryoscopic behavior of the trivalent
streptomycin ion (712).

Further studies on the chemistry of streptomycin revealed the fact
that it has the general constitution of a hydroxylated base (streptidine)
attached through a glycosidic linkage to a nitrogen-containing disac-
charide-like molecule. The latter group of the streptomycin molecule
contains a free or potential carbonyl group and a methyl-amino group

(85).

The reaction of the streptomycin with one molecule of water can be
presented as follows:

C.,H3,_3oN,0,, + H,0 ^ CsH.sNeO, + Ci3H,,_,3NO,
Streptomycin Streptidine Streptobiosamlne

Ci3H,3NO, -f H3O -> CeHioO, + C,Hi,N05

Streptobiosamine Streptose N-methyl-a-

/-glucosamine

The basic nitrogen atom in the streptobiosamine is not present as a
primary amino group. The streptomycin molecule was presented
graphically as follows :

Tyrocidine hydrochloride. From
Hotchkiss (449)

Gramicidin, From Hotchkiss
(449)

e--^:^ A^'U !^^^!^^^^*^^^

Fumigacin. From Waksman and
Geiger (955)

Gliotoxin. From Waksman and
Geiger (955)

i/MI^^Hi

T^^v^^^^^^l

r^^'^^^^H

SI

Pi

Citrinin. Prepared hy Tii

Actinomycin. Prepared by Tischler

Figure 19. Crystalline preparations of antibiotic substances.

FiciURE 20. StiLptoiiiycin crystals.

SUBSTANCES PRODUCED BY ACTINOMYCETES

197

CnHasNAs + H.O-

Streptomycin -

NH + C.jH.jNO,

CHNHC - NH2
CHOH

-> Streptidine

Streptobiosamine

C,3H,3NO, + H,0

H
HOC

I
HOCH

I
HOC - CHO

I
HC

H,C

Streptobiosamine >■ Streptose

CHOH

I
CH3NHCH

O HCOH

I
HOCH

I
CH

I
CH,OH

N-methyl/glucosamine

Streptidine was characterized by the following crystalline salts:
dipicrate, sulfate, carbonate, dihydrochloride, dihydroiodide, dihelian-
thate, di-<:^-camphorsulfonate, and chloroplatinate. Streptidine appears
to contain one or more hydroxyl groups, but no primary amino, car-
boxy, methoxy, or carbonyl groups. It formed an octaacetyl derivative

(713).

Streptidine has the molecular formula C8H18N6O4. A further study
of this compound has been made by Carter et al. (114). Streptomycin
hydrochloride was completely inactivated on standing 24 hours in an-
hydrous i.O N methanolic hydrogen chloride without forming a new
basic group. The addition of two volumes of ether completely precipi-
tated the guanidine, which was previously reported by Carter et al.
(115) as one of the functional groups of streptomycin. From the
supernatant solution there is readily obtained an amorphous, optically
active hydrochloride of a nonguanidine base whose properties agree

198 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

with those of the methyl streptobiosaminide dimethyl acetal hydro-
chloride reported by Brink et al. (85). The addition of picric or sulfuric
acid to an aqueous solution of the guanidine hydrochloride gives in-
soluble crystalline salts that are readily recrystallized from hot water.
The analytical data for the salts agree with those of a diguanidine base
of the composition C8H18N6O4. This compound has the same empirical
formula as that suggested by Brink et al. for streptidine, and is pre-
sumed to be identical with it.

Streptidine sulfate was also obtained by allowing a solution of strep-
tomycin chloride in i N sulfuric acid to stand at 37° C. for 45 hours.
The sulfate was precipitated in crystalline form by adding 3 to 5 vol-
umes of acetone to the reaction mixture.

Streptidine was hydrolyzed by refluxing for 48 hours with 6 N alkali
yielding four moles of ammonia and a new base, for which the name
streptamine was proposed. This base was isolated as the slightly soluble
sulfate by neutralizing the hydrolysis mixture with sulfuric acid and
adding an equal volume of methanol. The sulfate was purified by re-
crystallization from aqueous methanol. The hydrolysis of streptidine
proceeded as follows:

CsHjsNeO^ -f 4H2O -> CoHi.NoO^ + 4NH3 -f 2CO2
Streptidine Streptamine

These results appeared to establish the fact that the six nitrogen
atoms of streptidine are present as two monosubstituted guanidine
groups which are replaced by two primary amino groups in strepta-
mine. Further treatment with benzoyl chloride in pyridine yielded a
product melting at 350° to 351 ° C, the analyses of which agreed fairly
well for hexabenzoylstreptamine.

Streptidine reduced two moles of periodatej streptamine, sixj diben-
zoylstreptamine, twoj no formaldehyde was formed from any of these
compounds. The fact that streptamine required six moles of periodate
suggested to Carter et al. (114) that the four hydroxyl and two amino
groups are located on adjacent carbon atoms, pointing to a cyclic struc-
ture, since an open chain molecule should have yielded at least two
moles of formaldehyde and required only five moles of periodate.
Streptidine and streptamine were assigned the following formula :

SUBSTANCES PRODUCED BY ACTINOMYCETES 199

NH-X

A

OH

HO— *v i-NH-X

OH

Streptamine X = H
Streptidine X= — C^ ^tt

When streptomycin chloride is hydrolyzed with i.o N sodium hy-
droxide, for three minutes at ioo° C. or for eighteen hours at 40° C, a
weakly acidic substance, m.p. 161° -162° C. is obtained. It has been
characterized as maltol, namely,

The maltol gives a brilliant violet color with ferric chloride and a
positive iodoform testj it reacts rapidly with nitric acid, and sublimes
readily, even at 100° C. The benzoate melts at 114°-! 15° C. It has
been isolated from hydrolyzates of streptomycin salts ranging in purity
from 280 to 800 Mg/mg. The yields of maltol were about 30 per cent if
one mole was derived from one mole of streptomycin.

It was suggested that the formation of maltol by alkaline hydrolysis
of streptomycin, measuring the ultraviolet absorption in acid solution,
be used as an assay procedure, for the absorption produced is propor-
tional to the initial antibiotic activity in preparations having a potency
of 50 to 800 Mg/mg. The ferric chloride color reaction also appeared
to be useful for this purpose.

Streptomycin can be distinguished from streptothricin by inactiva-
tion with cysteine. This property is not due to the sulfhydryl group
alone. On oxidation of the cysteine, the substance is reactivated (179).
In view of the specific sensitivity of different bacteria to streptothricin

200 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

and streptomycin, not only can they be readily differentiated from one
another by their bacteriostatic spectra, but the admixture of one with
the other can actually be measured quantitatively. B. mycoides can be
used to measure the presence of a small amount of streptomycin with
streptothricin, whereas bacteria resistant to streptomycin can be utilized
for detecting the presence of a small amount of streptothricin or other
antibiotic (858).

Proactinomycin is produced by A^, gardneri grown in soft agar
media, from which it is extracted by organic solvents, such as ether, amyl
acetate, benzene, and carbon tetrachloride. It can be re-extracted in
water by adjusting the fH to 4.0 with HCl or H2SO4. The aqueous
extract is concentrated in vacuo and evaporated to dryness from the
frozen state. A white powder, very easily soluble in water, is obtained.
The yield of the material is 60 mg. from i liter of culture. The sub-
stance is fairly stable, though boiling for 10 minutes at /)H 2.0 or -pYL
7.0 results in a small loss of activity. Boiling at fH. lO.O destroys the
greater part of the antibacterial activity. Proactinomycin has basic prop-
erties and is precipitated from aqueous solution by such base precipitants
as picric acid, picrolonic acid, and flavianic acid.

Proactinomycin is active in a dilution of i : 500,000 or more against
gram-positive cocci, B. ant hr acts y and N. meningitidis ; it is much less
active against gram-negative bacteria and is not very toxic to animal tis-
sues, but definitely more so than penicillin or streptomycin. When
given by mouth it can confer a considerable degree of protection against
intraperitoneal infection with hemolytic streptococci. It is excreted in
the urine and bile, and is absorbed from the alimentary canal. Repeated
injections cause fatty changes in the livers of mice (273).

SUBSTANCES PRODUCED BY FUNGI
Penicillin

Penicillin is produced by various strains of P. notatum and P. chry-
sogenunty as well as by a variety of other fungi. The penicillin-like
nature of an antibiotic substance is usually established by its chemical
and biological properties: extraction in organic solvents at ^H 2 and
re-extraction in water at ^H 7 ; inactivation by acid and alkali j partial
inactivation by heating at 100° C. and ^H 7 for 15 minutes j complete

SUBSTANCES PRODUCED BY FUNGI 201

inactivation by penicillinase and by copper ionsj inactivation by methyl
alcohol J characteristic antibiotic spectrum, such as activity against S.
aureus and not against E. coU (270).

The strain of the organism used, the composition of the medium, and
the conditions of growth greatly influence not only the yield of penicil-
lin but also its chemical nature. Complex organic media containing glu-
cose or brown sugar as a source of carbon are essential. Nitrate is used
as a source of nitrogen j the medium also must contain a phosphate and
certain other minerals. The supplementary addition of a stimulating
substance in the form of yeast extract, corn steep, or certain vegetable
juices is essential for the maximum production of penicillin. Since the
organism produces an acid, probably gluconic, in the medium, some
CaCOg must also be added. The metabolism of P. notatum in relation
to penicillin production is illustrated in Figure 13 (p. 135).

Four methods have been proposed for the growth of the fungus and
the production of penicillin. These are:

Surface growth in shallow liquid media; usually flasks, bottles, and other
containers are employed, the depth of the medium being 1.5 to
2.0 cm.

Submerged growth in liquid media; the vessels must be provided with
proper stirrers and aeration

Surface growth upon semi-solid media, including grain and bran (762)

Circulation of medium through a column, the supporting material being
made up of wood shavings or pebbles; the rate of flow of the me-
dium is very important

Since the various strains of penicillin-producing organisms vary
greatly in their optimum conditions for the production of this antibiotic
substance, different strains must be used for different conditions of cul-
tivation.

Penicillin is formed in the medium when active growth begins and
reaches a maximum soon after the growth maximum, which occurs in
7 to 14 days in stationary cultures and in 3 to 7 days in submerged cul-
tures, at 20° to 25° C.

Penicillin is soluble in ether, acetone, esters, and dioxanej it is mod-

202 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

erately soluble in chloroform, slightly soluble in benzene and in carbon
tetrachloride. It is soluble in water to the extent of 5 mg./ml.

It is inactivated by oxidation and by evaporation at 40° to 45° C. in
acid and in alkaline solutions, although it is fairly stable at fH 5 to 6.
If the solutions are adjusted to ^H 6.8, it retains its potency for
3 months. The crude penicillin does not dialyze through a collodion
membrane and resists heating at 60° to 90° C. for short periods; it
remains active when heated at 100° C. for 5 minutes but not for 10 min-
utes.

The methods of isolation of penicillin from the culture media can be
classified under the extraction and adsorption procedures.

Fleming first reported that penicillin is insoluble in ether. This was
found (146) to be due to the alkaline reaction of the filtrate; for at ^H
2.0 ether removes completely the antibacterial substance. The ether
extract is evaporated with some water in vacuo at 40° to 45° C, the
residual water containing the active substance, which is extremely labile.

For practical purposes, penicillin is extracted from the acidified cul-
ture by means of different organic solvents, such as ether or amyl ace-
tate (6). It is then removed from the solvent by shaking with phos-
phate buffer or with water at ^H 6.7. Since penicillin is rapidly de-
stroyed at a high acidity, the first extraction must be carried out very
quickly and at a low temperature. In the presence of the solvents, peni-
cillin is stable for several days. The aqueous extract may be partly de-
colorized by shaking with charcoal and filtering. The solution is cooled,
acidified, and extracted several times with ether or amyl acetate; the
extracts are passed through an adsorption alumina column, or through
a 2.5 per cent precipitate of an alkaline earth carbonate on silica gel.
Water may often contain a pyrogenic or heat-producing substance that
must be removed from the penicillin.

The following four main zones were recognized in the chromato-
grams, beginning from the top :

1. A dark brownish-orange layer, the depth of which is inversely propor-

tional to the amount of charcoal used for the decolorization ; this zone
contains some penicillin

2. A light yellow layer containing most of the penicillin but none of the

pyrogen

SUBSTANCES PRODUCED BY FUNGI 203

3. An orange layer which contains some penicillin and some or all of the

pyrogen

4. A brownish or reddish-violet layer which contains almost no penicillin;

the pigment disappears on exposure to light

The fourth fraction is discarded, and the others are eluted with
M/15 phosphate buffer (-pH 7.2). The penicillin is again extracted
with ether, then with water, sodium hydroxide being used to adjust the
fH. Since penicillin is destroyed readily in alkaline solution, care must
be taken in adding the alkali. The "nonpyrogenic" or "therapeutic"
fraction, which contains about 80 per cent of the penicillin, is extracted
with pyrogen-free water. It is a deep reddish-orange liquid, yellow in
dilute solution, with a characteristic smell and bitter taste.

Another method for obtaining penicillin has been suggested (638).
In this method, the culture medium was adjusted to fH 3 to 4, satu-
rated with ammonium sulfate and extracted with chloroform. The con-
centrated chloroform extract was treated with phosphate buffer at fH
7.2 to remove the active substance. This process was repeated, the less
active substance being separated from the active fraction by extraction
with chloroform at different ranges. By precipitating the concentrated
extracts from petroleum ether, the free acid form of penicillin was ob-
tained. By saturating the chloroform-benzol solution with dry am-
monia gas, an ammonium salt was obtained which gave a dark yellow
microcrystalline powder. The salt was more stable than the acid form.
By acetylating or benzoylating the ammonium salt a further increase in
stability was obtained. This penicillin was strongly dextrorotatory and
had an adsorption maximum of 2,750 A°. The preparation had an ac-
tivity of 32,000,000 dilution units against hemolytic streptococci,
which corresponds to about 240 Oxford units per milligram.

When ether is used, the medium is adjusted to fH 3, extracted sev-
eral times, the ether extract treated with dilute NaHCOa, the aqueous
solution acidified and again extracted with ether j this is followed by
shaking with excess of BaCOo, separating aqueous phase, filtering, and
evaporating in frozen state (145).

By the adsorption method, activated charcoal or fuller's earth is used
(20 gm./L). The solution is first acidified to /)H 3.6, filtered, neutral-
ized, treated with charcoal, and filtered. Ethanol is used to remove the

204 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

penicillin from the charcoal j the extract is evaporated, acidified, and
treated with ether. Various procedures for large-scale production and
recovery of penicillin have been described (503, 607, 768).

Various modifications of these methods may be employed. In some
cases, «-butyl alcohol is used for extraction. The culture filtrate is ad-
justed to fH 6.4 and ammonium sulfate added, and the penicillin is
extracted. When light petroleum ether and dilute sodium bicarbonate
solution are added to the butyl alcohol extract, the penicillin is brought
back into aqueous solution (48). The problem of drying is very im-
portant (276).

The barium salt was at first considered as the most suitable form for
general use. In this form, penicillin retains its antibacterial activity for
an indefinite period. It is soluble in absolute methyl alcohol but in-
soluble in absolute ethyl alcohol. However, the Na and Ca salts are the
common forms now used. Penicillin forms water-soluble salts with most
heavy metals, except Fe"^+.

Penicillin is unstable and readily inactivated by a number of reagents,
including heavy metal ions, especially Cu, Pb, Zn, and Cd. Penicillin is
stable toward light and atmospheric oxygen, but is oxidized by H^Oo
and KMn04, the antibacterial activity being lost.

In assaying penicillin, both biological and chemical tests are used. Of
the former, the cup assay method is most commonly employed, al-
though the turbidimetric and other tests are also frequently used. The
different forms of penicillin are recognized by the differences in their
effect upon various bacteria, notably S. aureus and B. subtilis.

The world standard for penicillin has been defined as that activity
which is present in 0.6 micrograms of the international penicillin stand-
ard (384). One mg. of crystalline penicillin will thus contain 1,667
Oxford units (O.U.) and will be comparable to 84 million dilution
units against S. aureus (168, 454).

The chemical method for assaying penicillin is based upon the acidity
produced by the action of a standard penicillinase solution upon the
penicillin preparation and titrated to ^H 8.0 {666). The colorimetric
method is based upon its interaction with an intensely colored primary
amine, N-(l-naphthyl-4-azobenzene)-ethylenediamine to give amidic
products containing acidic groups (845a).

SUBSTANCES PRODUCED BY FUNGI 205

Tests are also made for sterility, moisture content, presence of pyro-
genic substances, and toxicity (248).

By means of adsorption, distribution between solvents, and reduc-
tion, a barium salt or penicillin was at first obtained (5, 6) which was
homogeneous by chromatographic analysis and gave 450 to 500 Oxford
units per milligram of dry material. The active substance was found to
be a salt of a strong dibasic acid with fH values approximately 2.3 and
2.5. The molecule contained one carboxyl, one latent carboxylic, two
acetylatable, at least five C-Me groups, and no easily reducible double
bond. The penicillin thus prepared was more sensitive to oxidizing
agents than to reducing agents j it was unstable toward dilute acids and
alkalies, and to heat (loss of COo), primary alcohols, and various heavy
metal ions. Tentative suggestions were made concerning its chemical
nature as follows: (a) a polysubstituted hydroaromatic ring structure j
(b) the acidic groups (carboxyl) not conjugated with the chromophore
responsible for the absorption j (c) the possible presence of a trisubsti-
tuted a-unsaturated ketone grouping.

With the introduction of new cultures for the production of penicil-
lin, with the development of new methods for the growth of the or-
ganism, as submerged vs. stationary, and especially with the employ-
ment of synthetic media, it was found that several forms of penicillin
are produced (735).

P. chrysogenum x 1,612 was found to yield about 100 O.U./ml. The
penicillin molecule is readily synthesized, especially when a phenyl
linkage has been supplied. The addition of 3.3 gm./L of phenylacetic
acid to the medium gave a maximum yield of 244 O.U./ml.

P. notatum 1,984-A yields 40 to 50 O.U./ml. of penicillin on a
purely synthetic medium, in presence of such factors as indole acetic
acid or naphthalene acetic acid. The production of penicillin takes place
in the presence of the following groups:

I. Cysteine (or cystine in presence of a suitable reducing agent such
as sulfite waste liquor)
. 2. The — C — C — N — chain with the proper linkage at each end

II I

O H

206

CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

3. The phenyl ring, or preferably 2 and 3 combined as phenylacetu-
rates, a-phenylacetamide or 3-phenylethylamine.

Several forms of penicillin or "natural penicillins" have been iso-
lated (715). They were all found to have the empirical formula
C9H11O4SN2.R. These forms may be presented as follows:

O H

CH3
I
S C CH3

P = CH3 CH2 CH--CH CH2 C — N— CH — CH
* I \

0=C N CHCO2H

CH3

I
OH S CCH3

Q = ^_^CH2C-N-CH-CH

0=C N CHCO2H

X = HO^

-/

CH3

I

OH S C CH3

^ / /
CH2C-N-CH-CH
I \
0=^C N CHCO2H

CH3
I
O H S CCH3

K^ / /
= CH3 (CH2)6C-N-CH-CH
I \
0=C N CHCO2H

In accordance with the nomenclature employed in the forthcoming
monograph on the chemistry of penicillin, which is being prepared
under the auspices of the National Academy of Sciences, penicillin G is
designated benzylpenicillin j penicillin K, F, and X are designated, re-
spectively, ;?-heptylpenicillin, A"-pentenylpenicillin, and ^-hydroxy-
benzylpenicillin.

SUBSTANCES PRODUCED BY FUNGI 207

The penicillins are strong monobasic acids of fK about 2.8. On treat-
ment with hot dilute mineral acids, penicillins give i molecule of COo,
an amino acid (penicillamine) and other products. The penicillamine
(^-(3,(3-dimethylcysteine) belongs to the d or "unnatural" series of
a-amino acids. Penicillin G yields on hydrolysis phenaceturic acid,
phenylacetamide, and an aldehyde, CiqHuOoN. Phenylacetic acid was
also identified as a hydrolytic product.

The acidic group of penicillin was found to be identical with the
carboxyl group in penicillamine j the addition of water to penicillin
gives a second carboxyl j the new carboxyl breaks down to COo by the
action of hot dilute mineral acid. The dicarboxylic acid thus produced,
designated as penicilloic acid, is presumably the product of the action
of penicillinase on penicillin.

The molecular weight of penicillin ranges from 490 to 510.

Penicillin is very unstable, especially in acid solutions and in an iso-
lated form.

Penatin

P. notatum produces, in addition to penicillin, a second antibacterial
substance designated as E. coli factor, penatin, notatin, and penicillin B.
This substance is a flavo-protein and acts as a glucose-oxidase, oxygen
being required. It is characterized by its action not only upon gram-
positive but also upon many gram-negative bacteria, and by the fact that
the presence of glucose is required for its activity. Its action is inhibited
by the presence of catalase (157, 517, 786).

Flavian

Flavicin, a substance similar in every respect to penicillin, is pro-
duced by A. flavus (950) j another substance, gigantic acid, is produced
by A . giganteus {"]ii). Preparations of flavicin have also been designated
as flavatin (748),aspergillin (103, 876), and flavacidin (606). This sub-
stance was found to be largely penicillin F, with a potency of 1,400
O.U./mg. against S. aureus and a B. subtilis-S. aureus ratio of 0.72.
The variable R group in the general penicillin formula is represented by
— CHo.CHo.CH^CH.CHg. A small amount of penicillin G (R =
— CHo.CgHg) was also detected (299).

208 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

As fer gillie Acid

Aspergillic acid is produced by A. flavus. It is extracted from the
tryptone medium by adsorption on norite and elution with ether. The
pure acid has an m.p. of 93° C. (84° to 96° C.) and an optical activity
of [a]D = +i4°. The formula C12H20O2N2 has been proposed for
this substance. It possesses a hydroxyl group which gives it its acid na-
ture (/)K 5.5). It is stable under acid and alkaline conditions and can
be distilled with steam or in vacuo without loss of activity. When grown
in brown-sugar-containing media, a closely related substance is formed,
having the formula C12H20O3N2 and an m.p. of 149° C, with lower
biological activity. It can also be extracted from the medium, at low
^H, by organic solvents (benzene or heptane). The pure aspergillic
acid (m.p. 96.5° to 97.5° C.) was found to account for only 5 per cent
of the yield, whereas another fraction (m.p. 118° to 121° C.) had
about 75 per cent of the antibiotic activity of the medium (102, 220).

Aspergillic acid is moderately active against various gram-positive
and gram-negative bacteria, including S. aureus y E. coli, and M. tuber-
culosis, as well as fungi. The addition of blood to the medium greatly
reduces this activity. However, the antibiotic potency and the toxic
effect rapidly disappear after systemic or intrathecal administration, the
material being excreted in the urine in very small amounts (102). The
addition of cobaltous ions (i:ioo,ooo) greatly enhances the tubercu-
lostatic properties of aspergillic acid, especially in media low in iron

(349).

Bromo-aspergillic acid is 8 to 10 times as active against S. hemolyticus
as aspergillic acid. Further studies of the chemical nature of the mate-
rial showed it to be a pyrazine derivative (220).

Citrinin

Citrinin is produced by P. citrinum and other fungi ( 745 ) on a syn-
thetic medium, with inorganic salts of nitrogen and with glucose as a
source of carbon. The culture filtrate is acidified with HCl, and the sub-
stance crystallized from boiling alcohol. Citrinin forms a monosodium
salt which, at ^H 7.0 to 7.2, gives a virtually colorless solution in water.
It is a yellow crystalline solid, m.p. 170° to 171° C. (decomp.). It is

SUBSTANCES PRODUCED BY FUNGI 209

/-rotatory (in alcohol) and nearly insoluble in water. It changes in
color from lemon-yellow at /)H 4.6 to orange-pink at ^H 5.6 to 5.8 and
to cherry-red at fH 9.9. The addition of FeCls to the culture solution
gives a heavy buff-colored precipitate, which dissolves in an excess of
reagent to give an intense iodine-brown solution. Citrinin has little if
any activity against gram-negative bacteria and about 50,000 dilution
units against B. sub tills and S. aureus (33, 907).

Penicillic Acid

Penicillic acid was first isolated in 19 13 by Alsberg and Black
(16) as a metabolic product of P. fuberulum. It is also produced by P.
cyclofium. A limited air supply and an acid reaction of the medium
favor the production of this acid. It is isolated (698, 703) by evapora-
tion of the culture solution, the crude acid crystallizing on cooling. It
is purified by recrystallization from hot water. Yields greater than 2
gm. per liter of culture were obtained. It is a monobasic acid, stable,
colorless, appreciably soluble in cold water, giving a series of colorless
and readily soluble salts (61, 487). It is optically inactive, and its m.p.
is 87° C. (anhydrons), 64° to 65° C. (+ HoO). It has limited activity
against gram-positive and gram-negative bacteria. It is also active
against yeasts, and is toxic to animals when injected subcutaneously in
concentrations of 0.2 to 0.3 gm. per kilogram weight.

Fumigatin

Fumigatin is a 3-hydroxy-4-methoxy-2,5-toluquinone or C8H8O4
(Figure 18). It forms maroon-colored crystals, has an m.p. of 116° C,
and is water-soluble (23, 743). It is extracted with chloroform from
acidified medium. It has limited activity against gram-positive bacteria.

All quinones have been divided into three groups on the basis of their
action on Staphylococcus: (a) those that have a markedly weaker anti-
bacterial action than fumigatin, including toluquinone and some of its
derivatives; (b) those that are somewhat more effective than fumi-
gatin, including 3:4 dimethoxytoluquinone ; (c) those with activity
perceptibly greater than that of fumigatin (methoxytoluquinones).
The introduction of -OCH3 into the quinone nucleus results in an in-

210 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

crease in antibacterial activity. The introduction of an OH or the re-
placement of -OCH3 by OH results in a decrease in activity. None of
these quinones, however, has any very striking action on gram-negative
bacteria, such as E. coli (325, 697).

Clavacin

Clavacin is anhydro-3-hydroxy-methylene-tetrahydro-Y-pyrone-2-
carboxylic acid (Figure 18). It is produced by a number of fungi, and
has also been designated claviformin, patulin, clavatin, and expan-
sin. It is colorless, optically inactive, neutral, and readily soluble in
water and most common organic solvents (445, 489) j it has an m.p. of
111° C. It is isolated either by preliminary adsorption on norite fol-
lowed by removal with ether or chloroform, or by the direct treatment
of the culture with ether. The extract is evaporated, leaving a brown
substance J this is treated with a small amount of water, and the aqueous
solution again extracted with ether. Clavacin crystallizes when the
ether solution is concentrated, or after preliminary purification over a
silica gel column.

Clavacin is about equally active against gram-positive and gram-
negative bacteria, its growth inhibition being about 200,000 dilution
units. It is also strongly fungistatic. It is toxic to animal tissues, its
lethal action upon mice being about 25 mg. per kilogram body weight
when given intravenously or subcutaneously.

Clavacin neutralizes the action of tetanus toxin and can thus be dis-
tinguished from isoclavacin and its derivatives. This specific action was
ascribed (739a) to the position of one double bond in clavacin.

Fumigacin

Fumigacin is produced by different strains of A. jumigatus. It is a
colorless, monobasic acid, m.p. 212° C, /-rotatory in chloroform. It is
insoluble in water except as sodium salt, sparingly soluble in methyl
and ethyl alcohols, and readily soluble in acetone, ether, chloroform,
and other organic solvents. It is extracted from the medium either by
preliminary adsorption on charcoal followed by treatment with ether
and alcohol, or by direct extraction of culture in accordance with the
following method (631) : The culture filtrate is acidified to ^H 2 with

SUBSTANCES PRODUCED BY FUNGI 211

phosphoric acid and extracted three times with ether, the combined ex-
tracts equalling the volume of the filtrate. The ether is evaporated to
one-tenth of its volume and the concentrate is shaken repeatedly with
saturated sodium bicarbonate solution, which removes a dark-red pig-
ment. The solution is then exhaustively extracted with 6 per cent so-
dium carbonate solution. The ether phase, on evaporation, yields glio-
toxin. The sodium carbonate solution is acidified and distributed several
times with benzene 5 the partly crystalline residue from the benzene
(7-12 mg. per i L of culture filtrate), on repeated recrystallization
from methanol, yields pure fumigacin in the form of filamentous
needles. Fumigacin melts with some decomposition at 2i5°-220° C,
depending on the rate of heating. [oi]'J — — 132 ±: 2° (0.41 per cent
in chloroform). The ultraviolet absorption curve shows only strong
end absorption below 260 mp with E 'J^^^ = 298 at 234 mp.

Fumigacin is markedly bacteriostatic against gram-positive bacteria,
but not against the gram-negative forms. It is also active against tuber-
cle bacilli, giving complete inhibition in a dilution of i : 10,000, and par-
tial inhibition in i :ioo,000 dilutions (464). It is not very toxic to ani-
mals (126).

Gliotoxin

Gliotoxin is produced by various species of Trichoderma, Glio-
cladium, Asfergillus {A.fumigatus) and PenicilUum {P. obscurum) j2iS
well as various other fungi. It has been analyzed as C13H14O4N2S2
(474). It is rapidly produced in an acidified (/)H 3.0 to 3.5) synthetic
medium when grown in a submerged or shaken condition for 2 to 4
days. It is extracted from the culture medium by the use of chloroform.
The latter is distilled off, and the residue is taken up in a small amount
of hot benzene or 95 per cent alcohol, from which, on cooling, silky
white needles crystallize. It is recrystallized from benzene or alcohol.
It has an optical rotation of WY^ — — 2,39°, and an m.p. of 121° to
122° C. (991).

Gliotoxin is frequently accompanied by one or more other antibiotic
substances, A. jumigatus producing as many as three others. P. ob-
scurum also produces one other. The removal of both from the medium
is brought about by extraction, at ^H 2, with benzene. The addition of

212 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

equal parts of petroleum ether to the concentrated extract results in
the separation of a crude preparation of gliotoxin. Purification is ac-
complished by repeated crystallizations (66$).

Gliotoxin is sparingly soluble in water and readily soluble in alcohol.
It is unstable, particularly in alkaline solutions, and is sensitive to
oxidation and to heating (988) j it is inactivated by heating for 10 min-
utes at 100° C. ( 14). Its potency was found to be destroyed by bubbling
oxygen for 5 minutes.

Gliotoxin is active against various bacteria and fungi. It is toxic to
Rh'fzoctoma hyphae in a dilution of i : 300,000, which is about two-
thirds of the toxicity of HgCls- The crystals, as well as the crude ma-
terial, were found to be toxic also to Trkhoderma, but the minimum
lethal dose was about 40 times greater than that required for Rhizoc-
tonia.

Viridin

Viridin is produced by Trkhoderma viride. It crystallizes in the
form of colorless rod-like prisms, which decompose without melting at
217° to 223° C. It is extracted from the medium with chloroform,
evaporated under reduced pressures, and crystallized from ethyl alco-
hol. It is optically active 5 a i per cent solution in chloroform gives
[a] ^^ = — 222°. The addition of phloroglucinol and HCl to a dilute
alcoholic solution gives a deep reddish-violet color. It is unstable in
aqueous solutions, but stable at ^H 3.5. It is highly fungistatic but not
very bacteriostatic (84).

Other Substances

A number of other antibacterial substances have been isolated from
fungi, but have not been adequately studied either chemically or bio-
logically. It is sufficient to mention the following:

Puberulic acid, a colorless, optically inactive, water-soluble dibasic
acid, with an m.p. of 316° to 318° C, and puberulonic acid, a bright-
yellow acid with an m.p. of 298° C, are produced (62, 704) by various
species of PenkilUum (P. fuberulum) . The first is a quinol and the
second is quinonoid. They have moderate activity against gram-positive
bacteria. P. fuberulum also produces a photosensitive compound.

SUBSTANCES PRODUCED BY FUNGI 213

C17H10N2O2, with an m.p. of 220° C. j it appears in the mycelium after
5 weeks' incubation and has certain antibiotic properties (108).

Penicidin was isolated (29) from a species of PenkilUum. It is
soluble in ether, alcohol, chloroform, and dilute acids, but not in petrol
ether. It is destroyed by bases, and is adsorbed on active charcoal. It is
similar to aspergillic acid in its antiluminescent properties. It is active
against E. tyfhosa.

Chaetomin is produced by a species of Chaetomium {Ch. cochliodes)
grown in complex organic media. It is active largely against gram-posi-
tive bacteria (948). Much larger concentrations of the material are
found in the mycelium of the organism than in the culture filtrate j it
is extracted from the former with acetone and from the latter with
ethyl acetate. It is purified by washing with sodium carbonate, treated
with petroleum ether, followed by chromatographic absorption. Chae-
tomin contains nitrogen and sulfur, but it differs in biological activity
from penicillin and from gliotoxin (326).

Kojic acid (Figure 18) is produced by various species {A. oryzae and
A. efusus). It possesses definite, even if limited, antibacterial proper-
ties and is more active against gram-negative than gram-positive bac-
teria j its antibiotic activity is not inhibited by serum (150, 465). It is
particularly active against species of Leftos-pira (660).

Polyporin is produced by Polystktus sanguineus grown for two to
three weeks in various synthetic media. It is present in both the culture
filtrate and the sporophores of the fungus. It is water soluble, and is ac-
tive (bacteriolytic) against various gram-negative {E. tyfhosa^ V.
comma) and gram-positive (6". aureus^ bacteria. It is nontoxic and
nonhemolytic (77). Its activity is not affected by oral administration,
by passage through a Seitz filter, or by pus and other body fluids and
tissues. It protected animals against V . comma and E. tyfhosa infec-
tions, and neutralized typhoid vaccine (77a).

Mycophenolic acid is produced by P. brevi-com factum. It was so
named by Alsberg and Black (16) in 1913, although it was first iso-
lated by Gosio in 1 896 and is said (268) to be the first antibiotic to have
'been crystallized. It has only limited activity upon certain gram-posi-
tive bacteria, but it has a considerable effect in inhibiting the growth of

214 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

fungi, especially those pathogenic to man and to plants. This effect is
largely fungistatic, giving titers up to i :8o,000 (271).

Glutinosin is produced by Metarrhizimn glutinosum grown on syn-
thetic media (83a). The substance is extracted with ether, n-h\i.ty\ alco-
hol, or petroleum ether. The solvent is evaporated and the material
crystallized from ethyl alcohol in the form of thin, colorless plates, free
from S and N. It does not melt at 300° C. It has specific antifungal ac-
tivity, inhibiting spore germination, but does not possess any antibac-
terial properties.

SUBSTANCES PRODUCED BY YEASTS

According to Fernbach (252), certain yeasts produce volatile sub-
stances which are toxic not only to other yeasts but also to bacteria. Rose
yeasts {Torula suganii), either fresh or heated to 120 to 130° C, were
found (690) to contain a substance which has an antagonistic action
against fungi, especially in the young mycelial stage, but not against
yeasts ; the growth of A . niger was reduced by 60 to 70 per cent and
that of A. oryzae by 25 to 30 per cent. The substance was not found in
the ash of the organism and was not secreted in the filtrate, but re-
mained in the yeast cells. An alkaline reaction was unfavorable to its
formation and action. The active substance was soluble in acetone, alco-
hol, ether, and chloroform, and was adsorbed by kaolin, Seitz filter,
paper, and by the fungus mycelium. It could be removed from the
kaolin by treatment with ether or acetone. Acetone-treated yeast no
longer had an antagonistic effect, but only a stimulating one.

According to Schiller (835), yeasts produce a bacteriolytic substance
only in a state of "forced antagonism," that is, in the presence of staphy-
lococci and certain other bacteria. The substance is thermolabile, since
it is destroyed at 60° C. It is active also outside the cell. More recently
( 151 ), the active substance of yeast was concentrated. In a crude state,
the active material was found to be nonvolatile and readily soluble in
water, in 95 per cent alcohol, and in acetone containing a trace of water.
It was stable at 100° C. at fH 7.3. It contained nitrogen but no sulfur.
Although a positive biuret reaction was obtained, it appeared that the
protein was present as an impurity.

SUBSTANCES PRODUCED BY ANIMALS 215

The ability of Torulosfora utilis var. major to inhibit the growth of
various gram-negative and other bacteria has also been demonstrated

(112).

SUBSTANCES PRODUCED BY ANIMALS

To what extent antibacterial substances produced by animals and
plants should be classified with the true antibiotics is open to question.
In view of the fact, however, that these substances behave in a manner
similar to antibiotics, they can be mentioned here.

Lysozyme

Fleming (260) found that egg white contains an enzyme, designated
as lysozyme, that is active against certain bacteria, notably micrococci,
bringing about their lysis. It is soluble in water and in dilute NaCl solu-
tion. It is precipitated by chloroform, acetone, ether, alcohol, and tol-
uene. It is not acted upon by pepsin or trypsin.

Lysozyme has been demonstrated in most mammalian tissues and
secretions, in certain vegetables, and in bacteria (905). It was found to
be a polypeptide containing 16 per cent nitrogen and 2 to 3 per cent
sulfur and having a molecular weight of 18,000 to 25,000. It is soluble
and stable in acid solution, insoluble and inactivated in alkaline solu-
tions, and inactivated by oxidizing agents (641 ). It diffuses in agar and
through cellophane, and thus is markedly different from bacteriophage
(332). It is fixed on the bacterial cells. It acts primarily upon the
cell membrane of bacteria, the highly viscous component of the bac-
terial cell (the mucoids), especially the sugar linkages of the complex
amino-carbohydrates, being disintegrated by the enzyme. The degrada-
tion of the bacterial polysaccharide to water-soluble products (N-acety-
lated amino-hexose and a keto-hexose) by lysozyme is accompanied by
complete lysis of some of the bacteria. In the case of other lysozyme-
sensitive bacteria, such as B. suhtillsy no lysis occurs j apparently the
morphological structure of these bacteria does not depend exclusively
on the unaltered state of the substrate for lysozyme (242).

The formation of a lysozyme-like material was also demonstrated
(259) for a coccus isolated from dust. A sarcina susceptible to egg-white

216 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES

lysozyme also was found (641 ) to produce an autolytic enzyme similar
to it. It has been suggested that the antibacterial action of saliva may
be due to the presence in it of antagonistic bacteria. The lysozyme of
saliva is known to act primarily upon gram-positive bacteria. A large
number of bacteria were tested (31) for their ability to antagonize
diphtheria and pseudo-diphtheria organisms. Only the spore-forming
B. mesentertcus and B. suhtills groups produced antagonistic substances,
but these bacteria were not found in the saliva. Cultures of bacteria
isolated from the saliva had no antagonistic effect, thus proving that the
action of saliva need not be due to its bacterial content.

Milk

Milk was found (694) to contain several thermolabile bactericidal
substances and two thermostable compounds which acted injuriously
upon lactic acid bacteria. Orla-Jensen emphasized that the growth of
bacteria in milk is influenced by a combination of activators or growth-
promoting substances and of inhibitors, the predominance of one or the
other being determined by various conditions. These substances influ-
ence the development of specific lactic acid bacteria during the spon-
taneous souring of milk.

Other Substances

Various other animal tissues and fluids contain substances which pro-
duce a bacteriostatic effect upon gram-positive bacteria. The method of
isolation of these substances and their selective action are similar to
those of tyrothricin (523a).

SUBSTANCES PRODUCED BY HIGHER PLANTS

It has been known for a long time that certain alkaloids and other
plant extracts possess bactericidal properties. Sherman and Hodge
(851) demonstrated in 1936 that the raw juices of cabbage, turnips,
and horseradish possess antibacterial properties. The active substance
in the juice could be adsorbed on activated carbon and by passage
through fine Berkfeld filters. The substance was thermolabile, being
destroyed at 60° C. in 10 minutes.

SUBSTANCES PRODUCED BY HIGHER PLANTS 217

Osborn (696) examined as many as 2,300 different flowering plants,
of which 134 species gave positive results. The activity against gram-
positive bacteria was far greater than against gram-negative forms 5
cabbage, cauliflower, broccoli, and kohlrabi gave the greatest effect
upon both groups of bacteria. The Ranunculaceae were most active of
all the plants. The stability of the substances as regards wilting and dry-
ing varied greatly. Extracts of honeysuckles {Lonicera tartar jia), espe-
cially of the roots, are very active (583).

Antibacterial substances are thus found to be widely distributed
among higher plants. Some of these substances have been isolated and
even crystallized. Allicin, a diallyl sulfoxide, was isolated from Allium
sativum (iij). Crepin, an a3-unsaturated lactone, has been isolated
from Crefis taraxacifoUa; the empirical formula C14H16O4 has been
suggested for it. It is active against both gram-positive and gram-
negative bacteria (391). Protoanemonin, obtained from buttercups,
Ranunculus J is active against various bacteria and fungi (847). Toma-
tin, an antibiotic occurring in the tomato plant, was found (456a) to be
active against a variety of gram-positive bacteria and fungi, including
both plant and animal pathogens.

Lichens were also found capable of producing antibacterial activity.
Of about 100 forms tested, 52 could inhibit either B. subtilis or S. au-
reus or both ; gram-negative bacteria are generally not susceptible. Al-
though certain lichen constituents were found to possess antibiotic prop-
erties, no specific agent has been isolated (100). Spanish moss also pro-
duces an antibiotic effect (999).

Unbleached wheat flour was shown (889) to contain a protein which
had bacteriostatic and bactericidal activity in vitro; although its activ-
ity was greater against gram-positive organisms, it also had some ac-
tivity against gram-negative types. The antimicrobial action of this
protein can be neutralized by means of a phosphatide (1033), a reac-
tion which may be due to the formation of a lipoprotein that has no
longer any antibiotic activity.

CHAPTER I I

THE NATURE OF ANTIBIOTIC ACTION

Sulfanilamide, -penicillin, and gramicidin can be clearly set afart
from, the classical antiseptics which are general protoplasmic
poisons. All three substances are primarily bacteriostatic rather
than bactericidal in their action. Since they do not destroy the res-
piration of bacteria, one may assume that the inhibition of growth
which they cause depends not upon interruption of the celltdar
metabolism as a whole, but rather upon some subtle interference
with certain individual reactions. To interrupt the pathogenic
career of an infectious agent, therefore, it is not necessary to kill
the invading cell, but only to block one step in its metabolic path
by some specific inhibitor. — Dubos.

ANTIBIOTIC SUBSTANCES AND CHEMICAL
DISINFECTANTS

Since antibiotic substances vary greatly in their origin and in their
chemical nature, they may be expected to vary also in their mode of
action upon the cells of bacteria and other microorganisms, and in the
effect upon the animal tissues when these agents are used for chemo-
therapeutic purposes. Comparatively little is known concerning these
mechanisms. It is known, however, that antibiotic substances act chiefly
by interfering with the growth of the bacterial cell, although in many
cases they are able to bring about the lysis of the cell as well. Because
of the first effect, it has been assumed that antibacterial agents are struc-
turally related to bacterial metabolites that usually function as co-
enzymes (600). In this connection, the following properties of anti-
biotic agents are of particular significance:

Most antibiotics are strongly bacteriostatic and only weakly bactericidal,
though a few are also strongly bactericidal and some are even bac-
teriolytic.

Some substances act primarily in vitro and only to a limited extent in vivo
because of interference of the body tissues with their action; others,
however, act readily upon bacteria in vivo.

ANTIBIOTICS AND CHEMICAL DISINFECTANTS 219

A few antibiotic agents are fairly nontoxic to the animal body ; others are
somewhat more toxic but can still be utilized; and some are so highly
toxic that they offer little promise as chemotherapeutic agents.

Antibiotic agents differ greatly in their solubility : some are water soluble ;
others are alcohol soluble and only slightly soluble in water; and
some are acids and react with alkali solution to form soluble salts.

Some antibiotic agents are stable under a variety of conditions, whereas
others are unstable.

Some antibiotic substances are hemolytic, others have apparently no in-
jurious effect upon blood cells. The latter can be used for general
body treatment, whereas the former are suitable only for local ap-
plications.

Since antibiotic substances are selective in their action upon microorgan-
isms, none can be expected to be utilized as general agents against all
bacteria. This also points to the remarkable physiological differences
in the morphology and physiology of bacterial cells, and to the dif-
ferences in mode of action of the different antibiotics upon various
bacteria.

A comparison of the antibacterial action of the antibiotic substances
produced by two bacteria will serve to illustrate some of the foregoing
points. Pyocyanin, produced by Ps. aeruginosa, inhibits the growth of
many gram-positive and gram-negative bacteria in dilutions as high as
I : lOOjOOOj pyocyanase and hemipyocyanin have less activity upon the
bacteria, but yeasts are more sensitive to them than to pyocyanin. Ty-
rothricin, produced by B. brevis, is far more specific in its action, which
is limited largely to gram-positive bacteria. The sensitivity of patho-
genic fungi to these compounds also differs markedly. Some other
striking differences are found on comparing two types of antibiotic sub-
stances produced by fungi, namely, penicillin and clavacln, and two
substances produced by actlnomycetes, namely, streptothricin and ac-
tlnomycln. The bacteriostatic spectra of these four substances are re-
corded In Table 't^S- The first of each pair has limited toxicity to ani-
mals, and the second is highly toxic. Whereas penicillin acts largely
upon gram-positive bacteria and only upon a few gram-negative organ-
isms, streptothricin acts alike upon certain bacteria within each group.
Clavacln and actlnomycin, both of which are highly toxic, differ simi-
larly In their action upon bacteria, the first being largely active against

220

NATURE OF ANTIBIOTIC ACTION

gram-positive and the second active against members of both groups.
These four compounds show various other differences in the nature of
their antibacterial action. Differences in the bactericidal properties of
other antibiotic substances are brought out in Table 36.

Various attempts have been made to compare the antibacterial action
of antibiotic substances with that of organic antiseptics. According to
Suter (890), the bactericidal action of a compound depends upon cer-
tain physical and chemical characters} a property that determines the
bactericidal action of the compound upon E. ty-phosa may be relatively
unimportant in the case of another organism such as S. aureus. A sub-
stance may have the same activity, as expressed by the phenol coeffi-
cient, against two organisms and still differ markedly in its relative

TABLE 35. BACTERIOSTATIC SPECTRA OF FOUR ANTIBIOTIC SUBSTANCES

GRAM

TEST ORGANISM STAIN

S. aureus +

S. aureus +

S. lutea +

B. subtilis 4-

B. megatherium +

B. mycoides +

CI. welchii +

Actinomyces sp. +

Neisseria sp. -

Br. abortus -

Sh. gallinarum —

Pasteurella sp. —

Hemofhilus sp. -

S. schottmiilleri —

S. aertrycke -

Ps. -fiuorescens —

5. marcescens —

A . aero genes —
E. coli

E. coli -

PENI-
CILLIN

9,500*

I jOOof

38,000*

19,000*

1,900*

5*
i,50ot
i,ooot

2,000t

it

<lt
10*

<5*
<i*
<5*
<it

<5*

ACTINO-
MYCIN

20,000

60,000

60,000

40,000

40,000

1,000

10

20

10

20

<I0

50

<I0

10

<5
<5

STREPTO-
THRICIN

100
750
200

<3

10-50

100
300
100
30
200

<3

5
30

CLAVACIN
100

500

200
100
200

60

6
60
50

100

Note. Activity is indicated in thousands of dilution units per gram.

• Data based on a sample having 470 Oxford units.

t Data reported by Abraham et al. (5), based on a less active preparation.

ANTIBIOTICS AND CHEMICAL DISINFECTANTS 221

TABLE 26. BACTERICIDAL EFFECTS OF PENICILLIN, GRAMICIDIN, AND
TYROCIDINE UPON S. HEMOLYTICUS

NUMBER OF VIABLE ORGANISMsf

INHIBITING

At

At

At

AGENT*

At start

At I hour

3 hours

7 hours

24. hours

Penicillin

1,500

4.300

2,650

420

0

Gramicidin

1,500

2,430

1,140

7

2.4

Tyrocidine

1,500

O.I

0

0

0

From Dawson, Hobby, Meyer, and Chaffee (171).

* 10 |Xg of each preparation was added to i milliliter of culture.

t In thousands per milliliter.

lethal effects. The conclusion was reached that the mechanism of bac-
tericidal action must be considered as a separate problem for each type
of organism, and, one may add, for each type of compound.

Although the major difference in the action of antibiotic substances
and chemical antiseptics is based upon the selective antibacterial nature
of the former, still an attempt may be made to correlate the two types
of compounds. Marshall and Hrenoff (621) constructed a disinfectant
spectrum for antibacterial substances with a flexible blending of differ-
entiated degrees of activity. The first, or ineffective, band covers a
range of dilutions of an agent between zero concentration and the high-
est dilution which still exerts no action on bacteria. The second, or stimu-
lative, band comprises a range of relatively high dilutions in which
there is a slight stimulation of bacterial multiplication; this range is
ordinarily narrow, but it may become broad. The third, or inhibiting, •
and the fourth, or bactericidal, bands merge indistinguishably. The
fifth, or impractical, band covers a range of concentrations of the dis-
infectant that are too great for practical purposes (Figure 21).

By establishing the normal rate of multiplication of bacterial cells in
a given culture without the disinfectant, one can determine the retarda-
tion of that rate by the disinfectant. This rate approaches zero at com-
plete inhibition with no multiplication and no deaths. A further increase
in the concentration of disinfectant results in the death of some organ-
isms per unit of time, and eventually a concentration is reached at which
all organisms die rapidly. Any rate of multiplication greater than zero
but less than normal can be considered as the bacteriostatic zone, and

222

NATURE OF ANTIBIOTIC ACTION

DISINFECTANT "SPECTRUM-

impractical
(insoluble,
too toxic.

OR )

100%

GENTIAN VIOLET

Space about 1/20 to-tol length

Spoce just perceptible (.001%)

Figure 21. Disinfectant spectrum. From Marshall and Hrenoff (621).

the rate less than zero as the bactericidal zone. According to this con-
cept of bacteriostasis, bacterial growth may be delayed under the influ-
ence of a disinfectant for many days or for many hours j or the bacteria
may progressively die over a period of many days.

The following factors influence the selective action of an antibiotic
agent upon bacteria (200): the acidic and basic properties of the bac-
terial cell, the nature and property of its membrane, its permeability,
the relative importance for metabolism and viability of the specific bio-
chemical systems affected by the agent, the activity of autolytic enzymes
in the bacterial cell, as well as others.

Marked differences exist in the degree of sensitivity of various bac-
teria to different antibiotic substances and chemical agents. Gramicidin
is most specific in its action, being limited to the cocci and acting upon
actinomycetes to only a limited extent. Penicillin is next in its selective
action. Actinomycin, tyrocidine, and gliotoxin act primarily upon the
gram-positive organisms and actinomycetes, and much less upon gram-
negative bacteria. The selective action is in contrast to the generalized,
even if more limited, action of phenol and quinone, which act alike on
both gram-positive and gram-negative organisms. Pyocyanase, pyo-

MECHANISM OF ANTIBIOTIC ACTION 223

cyanin, and the culture filtrate of P. notatum (due to the presence of
notatin) are similar in some respects but not in others to the chemical
compound in their action 3 they are found to be generally bacteriostatic
over a wide range of test organisms, no sharp division being obtained
upon the basis of the gram stain. Streptothricin is unique in its action j
the gram-positive spore-former B. suh tills is most sensitive, but the
other spore-former B. mycoides is not affected at all. The gram-negative
E. coll is more sensitive to streptothricin than either M. lysodeiktlcus
or S. lutea. Streptomycin is almost as active against B. mycoides as
against B. subtllls but is less active against fungi. The antibiotics of mi-
crobial origin are generally found to be stronger bacteriostatic agents
than the chemicals tested. A high bacteriostatic effect is not necessarily
accompanied by a correspondingly high bactericidal action. Gliotoxin,
one of the most active bacteriostatic substances among those tested, pos-
sesses lower bactericidal properties than other preparations. Strepto-
thricin and streptomycin, on the other hand, are highly bacteriostatic
and bactericidal against certain gram-negative bacteria.

The specific morphological differences among the bacteria, based
upon the gram stain, as shown by their sensitivity to antibiotic sub-
stances, are thus found to be relative rather than absolute. Most gram-
positive bacteria are more sensitive to the majority of antibiotics than
are gram-negative bacteria. But other antibiotics, such as streptothricin,
streptomycin, and clavacin, act quite differently and show marked
variations within each group.

MECHANISM OF ANTIBIOTIC ACTION

In an attempt to interpret the antibacterial properties of antibiotic
substances, one may benefit from a comparison of the action of these
substances and that of other antibacterial agents. Recent studies of the
mechanism of antibacterial action of chemotherapeutic agents led to
rather definite concepts concerning the nature of this action. The action
was believed to consist in depriving the bacteria of the use of enzymes
or metabolites by various types of interference. When the organisms
ai-e thus inhibited their nutritional requirements are more exacting than
in their normal state. E. coll and S. hemolytlcuSj when inhibited by acri-

L i B R A ft Y

224 NATURE OF ANTIBIOTIC ACTION

flavine components, were found to require for further growth two types
of material not normally added, one of which could best be replaced by
nucleotides, and the other by a concentrate of amino acids, especially
phenylalanine (598, 599).

On the basis of the information now available, the following mecha-
nisms may be tentatively presented here:

The antibiotic substance interferes with bacterial cell division, thus pre-
venting further growth of the organism. The cell, unable to divide,
gradually dies. It has been shown (363), by the use of the mano-
metric method, that certain bactericidal agents in bacteriostatic con-
centrations have no effect on the metabolic rates of bacteria, though
they do inhibit cell multiplication.

The antibiotic interferes with the metabolic processes of the microbial cells
by substituting for one of the essential nutrients. A specific inhibitory
effect may be exerted by those substances that are structurally re-
lated to normal cell metabolites; such substances are taken up by the
cell in competition with normal nutrients but since they are useless to
the cell for further reactions they block the process of growth (253,
596-600). The antibiotic effect of certain polypeptides, such as
gramicidin, may be due to the presence of a (^-amino-acid isomer of
a natural amino acid, /-leucine, required for bacterial growth (289).

The antibiotic, such as aspergillic acid, interferes with the utilization of
iron or with the functioning of the iron-containing enzyme system
(348).

The antibiotic may interfere with the production and utilization of an es-
sential growth-factor required by the cell. The staling effect of a
medium, frequently spoken of in connection with protozoa as "bio-
logical conditioning" of the organism, may serve as an illustration.
Such effects have been overcome by the addition of a mixture of
thiamine, riboflavin, and nicotinamide (374).

The antibiotic agent brings about the oxidation of a metabolic substance
which must be reduced in the process of bacterial nutrition, or other-
wise modifies the intermediary metabolism of the bacterial cell.

The agent combines with the substrate or with one of its constituents,
which is thereby rendered inactive for bacterial utilization.

The agent competes for an enzyme needed by the bacteria to carry out an
essential metabolic process.

MECHANISM OF ANTIBIOTIC ACTION 225

The agent interferes with various enzymatic systems, such as the respira-
tory mechanism of the bacterial cell, especially the hydrogenase sys-
tem (449) and the phosphate uptake by the bacteria accompanying
glucose oxidation, as in the action of gramicidin. Penicillin, for ex-
ample, inhibits the production of coagulase by staphylococci in vitro
(590).

The antibiotic substance may inhibit directly cellular oxidations, particu-
larly those involving nitrogenous compounds, an action similar to
that of propamidine (521).

The antibiotic substance acts as an enzyme system and produces, in the
medium, oxidation products, such as peroxides, injurious to the bac-
terial cell. The glucose oxidase produced by P. notatum catalyzes
the following reaction :

Glucose + HoO + Oo -> Gluconic acid + HoOo.

The antibiotic substance favors certain lytic mechanisms in the cell,
whereby the latter is destroyed; this mechanism may be either sec-
ondary or primary in nature.

The antibiotic substance affects the surface tension of the bacteria, acting
as a detergent; tyrocidine lowers the surface tension of the bacterial
cell, thereby causing its death, possibly by forming a stable complex
with it (200).

The antibiotic substance may interfere with the sulfhydryl group which is
essential for cell multiplication. This was shown by Fildes (254) to
hold true for mercurials and other chemical antiseptics as well as for
true antibiotics such as clavacin and penicillic acid (325 ).

The interaction of sulfhydryl-containing compounds with antibiotics
depends on the nature and concentration of the latter. It has been sug-
gested (118) that the activity and specificity of an antibiotic are func-
tions of several factors, such as its diflusibility into the microbial cell, its
adsorption by various enzyme systems, its reaction with sulfhydryl
groups of the enzymes or with other sulfhydryl-containing substances
adsorbed by the enzyme. Gliotoxin and the active principles of Allium
sativum and Arctium minus showed little specificity in reactivity toward
the thiols, whereas penicillin, streptornycin, and the Asarum canadense
antibiotic reacted more readily with those sulfhydryl compounds which
contained basic amino groups in the vicinity of the — SH. Pyocyanin
had intermediate properties (115b).

226 NATURE OF ANTIBIOTIC ACTION

The theory of inner antagonism has been suggested (509). The bac-
terial cell is said to contain two antagonistic groups, namely coagulants
and lysinsj when the correlation between these two groups is disturbed
the result is either agglutination and precipitation by the first or lysis
by the second. The phage is given as an example of a free inner antago-
nist, the lysinj reproduction of the phage is thus explained by the
lik^ formation of lysins in multiplying cells. The action of antibiotic sub-
stances and resulting cell lysis were also explained by the inner antago-
nism.

It has been postulated (170) that the action of growth-inhibiting
substances may consist in prolongation of the lag phase, reduction of the
growth rate, lowering of stationary population, or hastening the death
of the bacteria. A bactericide has all these effects, whereas a bacterio-
static agent may affect one stage selectively. When organisms are al-
lowed to grow in the presence of an antibacterial agent, a greater con-
centration of the latter is required to bring about a given effect upon the
bacterial culture.

On the other hand, bacteria subjected to the action of an antibiotic
substance may develop mechanisms that render them resistant to the
action of the substance, and some bacteria and fungi even may produce
an enzyme, such as penicillinase, that brings about the destruction of the
antibiotic substance.

The antibacterial action of gramicidin was found (412) to be in-
hibited by a cationic detergent, phemerol, whereas penicillin was not
affected by either gramicidin or two cationic detergents, phemerol and
zephiran. When gramicidin and penicillin were used together, their
effect was only slightly additive (394) j however, penicillin and strepto-
thricin exerted a marked additive effect upon bacteria sensitive to both
of these substances (287).

The inhibition of the antibacterial action of sulfanilamide by
/)-amino-benzoic acid has been explained by the fact that the latter is a
growth factor in bacterial nutrition. Competition for this growth factor
between the bacterial cell and the bacteriostatic agent is responsible for
the inhibition of the agent. In a similar manner pantoyltaurine, which
is related to pantothenic acid as sulfanilamide is to /)-amino-benzoic
acid, will inhibit the growth of hemolytic streptococci, pneumococci,

MECHANISM OF ANTIBIOTIC ACTION 227

and C. difhtheriae, by preventing the utilization of pantothenic acid by
these bacteria, for which it is an essential metabolite. Fildes (253) em-
phasized that "chemotherapeutic research might reasonably be directed
to modification of the structure of known essential metabolites to form
products which can block the enzyme without exhibiting the specific
action of the metabolite." Since /)-amino-benzoic acid has no such action
on penicillin, it is assumed that its mode of action upon bacteria is dif-
ferent from that of sulfanilamide. However, it was suggested that
penicillin as well acts by inhibiting directly one or more enzymes, the
difference being merely one of degree (648).

The antibacterial activity of iodinin is neutralized by quinonesj this
is probably due to the destruction of the iodinin, since the N-oxide is
reduced by the organism (596, 597, 1009). Different anti-inhibitors are
known for other antibiotic substances, as shown later.

Numerous other examples of metabolite-antagonism can be cited.
Since the nature and function of the various metabolites are so diverse,
and there are so many ways of modifying their structure, the principle
of interference with biological processes through the use of analogs of
essential metabolites is considered as established (994). The interfer-
ence is sometimes explained as a direct competition between the metabo-
lite and its analog for some cellular component for which they both
have great affinity. However, in addition to competition, other factors
also operate. The majority of the interferences involve organisms that
are unable to synthesize the essential metabolite the function of which
is disturbed.

Mcllwain recommended the use of an antibacterial index to repre-
sent the minimal value of Ci/Cm, or the ratio of concentration of in-
hibitor (Ci) just sufficient to prevent the growth of the organism, to the
concentration of metabolite (Cm) present. The smaller the antibacterial
index the more effective is the compound, therefore, as an inhibitor.
With S. hemolytkus, the homopantoyltaurine was found to have an
index of 20,000, the pantoyltauramide 2,000, and the pantoyltaurine
500. The indices vary for different organisms. E. coU and P. vulgaris
synthesize their own pantothenate and are not inhibited by these ana-
logs of pantothenic acid. The mechanism of the resistance is at present
unknown.

228 NATURE OF ANTIBIOTIC ACTION

The concentration of the active substance and the composition of the
medium are highly important in modifying the activity of the sub-
stance. Some antibiotic substances, like penicillic acid, lose considerable
bacteriostatic activity when incubated with sterile broth or with sterile
peptone water at fH 7 and 37° C. for i to 3 days (700) j a similar ef-
fect was observed with certain simple amines and amino acids. The con-
centration of the substances reacting with penicillic acid is diminished
on autoclaving the peptone broth in the presence of 2 per cent glucose.
The neutralizing or anti-inhibiting agent interacts with the antibiotic
substance and neutralizes its antibacterial effect either in the absence or
in the presence of the organism.

Since not all antibiotics of microbial origin have been isolated in a
crystalline state, confusion often resulted from the use of crude prepa-
rations. Concentrated and partly purified actinomycetin had no appre-
ciable lytic action upon living cells j however, the presence of a small
amount of a highly bactericidal substance, which was especially active
against gram-positive bacteria, resulted in the lysis of living bacteria by
actinomycetin. This action was thus a result of the activity of at least
two different agents present in one preparation (1002).

ANTIBACTERIAL ACTION

Chain and Florey (122) divided all antibiotic substances into two
groups:

1. Antibiotics which react with protoplasmic constituents and kill
both bacterial and animal cells, comparable to the action of "antisep-
tics." These antibiotics can be further subdivided into (a) those that are
active against both gram-positive and gram-negative bacteria, and (b)
those that exert a selective antibiotic action, usually against gram-posi-
tive organisms, such as gramicidin and actinomycin. The selectivity is
not absolute, since gramicidin acquires strong bactericidal activity
against gram-negative bacteria in the presence of protamines, due to the
fact that protamines remove phospholipids, which inhibit the antibac-
terial action of gramicidin.

2. Antibiotics which react with substances having a specific signifi-
cance in the bacterial cell only. Some of these substances are largely

ANTIBACTERIAL ACTION 229

growth inhibiting and can, therefore, be designated as "bacteriostatics."
The bacteriostatics may be expected to be relatively nontoxic to animal
cells. Antibiotics of this class have possibilities as chemotherapeutic
agents for general administration and for the treatment of systemic in-
fections.

In order to determine whether an antibacterial substance has chemo-
therapeutic potentialities, the effect of the antibiotic on bacterial respira-
tion can be determined by using the Barcro ft- Warburg apparatus.
If respiration is stopped by addition of the antibiotic in dilution of
1 : 1,000, the organisms may be said to have been killed, the substance
being an antiseptic which will be toxic to animal tissues. If, however,
the antibiotic produces little or no effect on respiration of the bacteria,
there is a probability that the substance has chemotherapeutic possi-
bilities.

Chain and Florey further suggested that observations be made on:
(a) the toxicity of the antibacterial substance to leucocytes, a wide gap
between a toxic concentration and a bacteriostatic effect suggesting that
the substance may be useful, at least for local application j (b) the effect
of blood, pus, and tissue extracts on the bacteriostatic activity, inhibition
of activity being due to chemical combination between the active sub-
stance and a tissue constituent or to an inhibitory mechanism similar to
that of /)-amino-benzoic acid for the sulfonamides; (c) the toxicity of
the substance to mice when injected intravenously. Any therapeutically
active substance will be excreted unchanged or little changed in the
urine, since it does not combine with the tissue cells.

Although Dubos (206) believed that none of the in vitro metabolic
screening methods at present available is satisfactory in a search for
new chemotherapeutic agents, Chain and Florey emphasized that those
antibiotics which pass the above biological tests can be expected to be
effective as general chemotherapeutic agents and to be worth further
investigation with mouse protection tests.

Tyrothricin

The phenomenon of antibiotic action by a specific substance can best
be illustrated by the action of tyrothricin upon bacterial cells. Five dis-
tinct stages have been described (201 ) :

230 NATURE OF ANTIBIOTIC ACTION

1. Inhibition of growth. Certain gram-positive bacteria are inhibited by

as little as i microgram or less of the substance per lo milliliters of
nutrient broth or agar, thus giving an activity of i : 10,000,000 or
more.

2. Bactericidal action consists in the killing of the bacterial cells, either in

a washed state and suspended in saline, or in a growing state in broth
culture.

3. Lytic activity comprises the rate of lysis of a suspension of bacterial

cells. Streptococci, for example, are readily lysed by gramicidin,
whereas staphylococci are acted upon more slowly and less com-
pletely.

4. Inhibition of enzyme activity includes dehydrogenases or enzymes of

respiration. Gram-positive cocci, incubated at 37° C, lose their abil-
ity to reduce methylene blue in the presence of glucose, upon addi-
tion of gramicidin. Since inactivation of the dehydrogenase takes
place before any morphological changes are observed in the cells,
lysis was believed to be a secondary process, following cell injury;
hydrolytic enzymes, however, remained unaffected.

5. Protection of animals by the antibiotic substance against infection.

Gramicidin and tyrocidine differ in chemical properties and in bio-
logical activity. Gramicidin acts only against gram-positive bacteria, in-
cluding pneumococci, streptococci, staphylococci, diphtheria bacteria,
and aerobic spore-forming bacilli j meningococci and gonococci are not
readily acted upon. Tyrocidine affects both gram-positive and gram-
negative organisms. Gramicidin causes hemolysis of washed red cells,
this hemolytic action being destroyed on heating. Tyrocidine causes
lysis of many bacterial species. This action, however, is secondary,
autolysis following the death of the cells. Peptones and serum inhibit
the action of tyrocidine, but gramicidin is affected only to a limited ex-
tent by these agents (617).

Tyrocidine behaves as a general protoplasmic poison. The effect of
gramicidin, on the other hand, is reversible. Staphylococci "killed"
with gramicidin and no longer able to grow on organic media can be
made to grow in the presence of certain tissue components. Gramicidin
Is, therefore, not considered as a gross protoplasmic poison, but retains
a good deal of its activity in animal tissues. When applied locally at the
site of infection, gramicidin exhibits definite action against pneumococci

ANTIBACTERIAL ACTION 231

and streptococci. When injected intravenously, however, it is almost
completely inactive against systemic infection. .

It was demonstrated by tissue culture technique (412) that the he-
molytic effect of tyrothricin was due to the presence of gramicidin.
When tyrothricin or gramicidin was heated in an aqueous suspension
there was a loss of hemolytic and bactericidal activity. Tyrocidine,
which is not very hemolytic, showed no marked toxic effect upon the
leucocytic elements of the human blood in amounts up to 100 mg. per
milliliter for 8 hours. Other investigators (757) have reported that the
hemolytic activity of tyrothricin is inherent rather in the tyrocidine
fraction, although gramicidin also exhibits a definite hemolytic action.
The addition of glucose causes only slight inhibition of the hemolytic
effect.

Treatment with formaldehyde results in the lowering of the hemo-
lytic and toxic activity of gramicidin, without reduction of antibacterial
properties J this was interpreted as signifying that these properties do
not necessarily depend upon the same molecular configuration (S^S)-

Gramicidin was found to be effective, in amounts as low as i mg.,
upon a billion gram-positive organisms, whereas tyrocidine acted in 25
to 50 times that concentration in the absence of inhibitors (449, 450).
Tyrocidine appeared to block all the oxidative systems of the bacteria
studied, whereas gramicidin seemed to affect only certain individual
reactions.

Tyrothricin was reported (67) to inhibit enzymatic dehydrogenation
not only of glucose but also of a number of other compounds, such as
lactic acid, fumaric acid, and glutamic acid. Inhibition of dehydrogenase
was parallel to inhibition of growth.

Both substances were found to exert a protective antibacterial action
in mice infected intraperitoneally with susceptible bacteria j gramicidin
protected the animals at a level one-fiftieth as high as that required for
tyrocidine. Both substances are toxic to animals when injected into the
blood stream j they show little toxicity when applied locally by the sub-
cutaneous, the intramuscular, or the intrapleural route j oral adminis-
tr^ition is not accompanied by toxic effects, but such treatment is in-
effective (758).

Gramicidin remains active in the blood stream, but it has only weak

232 NATURE OF ANTIBIOTIC ACTION

bacteriostatic properties and no bactericidal action. Tyrocidine is
strongly bactericidal but it is inactivated by blood serum, hence it is
limited to local applications. No specific effect was exerted by these sub-
stances on respiratory or circulatory systems (793).

According to Dubos (200), the retention of the stain by gram-posi-
tive bacteria indicates a peculiar property of the cell wall of these or-
ganisms. The addition of one microgram of gramicidin to a billion
pneumococci, streptococci, and staphylococci is considered sujEficient to
inhibit the growth of these organisms on subsequent transfers. This
effect v/as said to be due not to an alteration of the protoplasm but to
some specific interference with an essential metabolic function. Bacterial
cells which have been inhibited by the action of gramicidin become
viable again when cephalin is added to the medium. It was suggested
that the ineffectiveness of gramicidin against gram-negative bacteria
may be due to the presence of a phospholipid in these organisms.

Different strains of S. aureus differ in their susceptibility to the ac-
tion of tyrothricin. There is apparent adaptation of the organism to in-
creasing concentrations of the substance. A marked increase in resist-
ance of the infecting organism, after several weeks of therapy, was ob-
served (752). Staphylococci grown in the presence of increasing con-
centrations of gramicidin become resistant to inhibition by this sub-
stance (81, 720).

Both gramicidin and tyrocidine are said (206) to be surface-active
compounds, their antibacterial action being inhibited by phospholipids.
Tyrocidine behaves like a cationic detergent j it is bactericidal in buffer
solutions for all bacterial species so far tested, with the exception of the
tubercle bacillus. Gramicidin influences some energy-using process
which would normally allow carbohydrate and phosphate storage. This
effect is specific, since penicillin and sulfanilamide do not have the same
effect upon the phosphate metabolism of staphylococci. On the other
hand, like many surface detergents, tyrocidine modifies the surface of
the bacterial cell in such a manner that vital soluble metabolites, such
as nitrogen compounds, inorganic phosphate, and phosphate esters are
washed out of the cell. Hotchkiss (449) concluded that although ty-
rothricin and its constituents are more active against gram-positive than
gram-negative organisms, Neisseriae respond more like gram-positive

ANTIBACTERIAL ACTION 233

cocci, and gram-positive, spore-forming bacteria are insensitive to
gramicidin J tyrocidine has more activity against gram-negative organ-
isms and is more bactericidal, whereas gramicidin is primarily bacterio-
static.

Tyrocidine destroys immediately and irreversibly the metabolic ac-
tivity of the bacteria, such as oxygen uptake and acid production. For
most tissue cells, with the exception of spermatozoa, gramicidin is much
less toxic than tyrocidine. It behaves like a specific inhibitor of certain
metabolic reactions. It retains much of its activity in vivo.

Tyrocidine brings about rapid cytolysis of the cells. There is a quan-
titative relation between the concentration of the antibiotic and the
number of cells lysed, namely i mg. for lo^ and o.i mg. for lo^ cells.
The amino acid decarboxylases are not inhibited even by concentrations
of tyrocidine of 0.3 mg./ml. (309).

Other Antibiotics from S-p ore-forming Bacteria

The other antibiotic substances isolated from spore-forming bacteria
are characterized by bacteriostatic spectra quite difFerent from that of
tyrothricin. This is brought out in Table 37. Some of these substances,

TABLE 37. COMPARATIVE ANTIBIOTIC SPECTRA OF SUBSTANCES PRODUCED
BY AEROBIC SPORE-FORMING BACTERIA

TEST ORGANISM

BACILLIN

SUBTILIN

SIMPLEXIN

5". aureus

I.O

1.0

96.0

M. conglomeratus

2.0

1.0

-

D. pteumoniae III

3-0

-

0.4

S. faraiyfhi

0.25

1 0.0

96.0

Pasteur ell a sp.

1.0

-

0.4

E. coli

2.7

1 0.0

2.7

E. tyfhosa

1.4

lO.O

2.7

From Foster and Woodruif (284).

Note. Unit of activity is the amount of antibiotic required to inhibit S. aureus as test bacterium.

like subtilin, are capable of destroying various bacterial toxins, such as
diphtheria, tetanus, and others, as well as hemolysin (759).

Subtilin was found to be similar to gramicidin in its effect upon sur-
face tension, in producing hemolysis, even if more delayed, in killing

234 NATURE OF ANTIBIOTIC ACTION

Entamoeba histolytica in 1 1400,000 dilution, and in cytolyzing T . equi-
ferdum in i :2,000 dilution (20). Extracts of cells of B. subtilis with
ether or chloroform in an acid medium (pH 2.5), redissolved in an
aqueous alkaline solution (^H 8.5), were found to be active against
staphylococci, E. coli, and M. tuberculosis (693).

Penicillin

In his first description of penicillin, Fleming recorded (261) that
"it was noticed that around a large colony of a contaminating mould the
staphylococcus colonies became transparent and were obviously under-
going lysis." Penicillin was referred to by Fleming as a bactericidal
agent and the conclusion was reached that it belonged to the group of
slow-acting antiseptics, since staphylococci were completely killed only
after an interval of 4^ hours, even in a concentration 30 to 40 times
that required for complete inhibition of the culture in broth. Florey
and Florey (275), however, concluded, as a result of in vitro experi-
ments, that penicillin is bacteriostatic and not bactericidal, at least in
concentrations suitable for chemotherapeutic purposes. This led Garrod
to state that "penicillin is in a true sense an antiseptic rather than a
germicide: it does not kill bacteria quickly." The action of penicillin
was found to be affected by changes in temperature, reaction of the sub-
strate, and age of the bacterial culture (320, 321 ).

In addition to its marked bacteriostatic effect, penicillin has also been
found to be decidedly bactericidal j this is accelerated by an increase in
temperature from 4° to 42° C. but is impaired by an increase in acidity
of medium between f¥l 7.0 and 5.0. The rapid drop in the number of
bacteria within the first 15 minutes after application of the penicillin
was interpreted (594) as indicative of its bactericidal action in vivo.
Young cells are particularly susceptible, whereas mature cells are
neither lysed nor readily killed. The bacteriolytic action of penicillin
upon sensitive organisms is greatest at the maximum rate of multiplica-
tion (507, 512). The lysis of bacteria by penicillin depends upon their
ability to produce autolysin. Bacteria are resistant to the lysin when liv-
ing and become sensitive to it after the cells have been killed by penicil-
lin or by other agents. The rate of bacteriolysis is thus controlled by
bacterial multiplication and production of autolysin (913).

ANTIBACTERIAL ACTION 23 5

Penicillin is markedly sporicidal against sensitive organisms j this
action is greater in milk than in water, especially if preceded by sub-
lethal heating of the spores (162). Penicillin is active against spiro-
chetes (399), including Treponema fallidum (225, 296).

Penicillin is thus found to be actively bactericidal in a medium and
an environment in which active multiplication of the bacteria occurs,
since it acts best in good culture media such as broth or serum and poorly
in water or saline solutions. Although penicillin kills large numbers of
sensitive bacteria, it does not always kill all the bacteria present, but
leaves a few cells that are resistant to its action. These soon begin to
multiply, giving rise to a resistant culture.

Penicillin affects a metabolic function of the bacteria during the early
stages of their development. Certain antibacterial substances, like hel-
volic acid, neutralize the effect of penicillin on the bacteria, whereas
others, like sulfanilamide, have a synergistic effect. The latter is espe-
cially well marked with strains of staphylococci that are naturally re-
sistant to penicillin (121).

Although penicillin is active primarily on gram-positive bacteria, it
also has an effect on certain gram-negative bacteria, but not on the colon
organism. Hemophilus, or Brucella. The gram-negative cocci can be
divided into two groups, on the basis of their sensitivity to penicillin :
N. gonorrhoeae, N. intracellular, and A^. catarrhalis, which are sensi-
tive j and A^. -flava and other nonpathogenic Neisseriae, which are not
sensitive.

Some species of Hemophilus, such as H. ducreyi, are as sensitive to
penicillin as is S. aureus, although less so than S. hemolyticus (659).
High potency preparations of penicillin were found (431) to have an
inhibitive effect even on E. coli. The susceptibility of gram-negative
bacteria to penicillin is much greater in synthetic than in complex or-
ganic media; in the case of the latter, various polypeptides and pos-
sibly some amino acids appear to neutralize the effect of penicillin upon
E. coli, the antagonism being partly removed by methionine (852). In
studies on the effect of penicillin on bacteria in urine, it was shown
(402) that 90 times the dose required to eliminate S. aureus will affect
S. faecalis, 240-fold increase will act on P. vulgaris, and 900-fold will
act on £. coli (880). Although Salmonella strains were inhibited by

236

NATURE OF ANTIBIOTIC ACTION

only 2 units of penicillin per i ml. and P. vulgaris, E. ty-phosa, Shigella,
Escherichia, and Aerobacter showed even greater resistance, it was still
believed that concentrations of penicillin in the urine can be attained to
inhibit the growth of these organisms (904).

Penicillin is not active against pathogenic fungi, the growth of which
may actually be stimulated by this antibiotic. However, it has some
activity against A. bovis, the growth of which was inhibited by 0.0 1
O.U./ml. (496), and against certain other actinomycetes (193).

Various forms of penicillin differ in their action upon specific bacteria.
Welch et al. (998) have shown that penicillin X is more effective than
commercial penicillin against certain bacteria but not against others, not
only in the test tube but also in the animal body. These results have
been confirmed, as shown in Table 38. S. aureus and B. subtilis are more
sensitive to penicillin G than to X, on a weight basis j however, peni-
cillin X is more effective than G on certain other bacteria.

TABLE 38. INHIBITION OF GROWTH OF DIFFERENT BACTERIA BY TWO
FORMS OF PENICILLIN (MICROGRAMS OF PENICILLIN PER Ml)

RATIO

G

ORGANISM

PENICILLIN G

PENICILLIN X

X

S. aureus

.040

.060

0.7

B. subtilis

.059

.098

0.6

Pneumococcus Type I

.019

.016

1.2

Pneumococcus Type II

.007

.005

1-4

Streptococcus Group D

2.400

1.700

1.4

Streptococcus Group B

.120

.066

1.8

Streptococcus Group A

.010

.006

1.7

Er. rhusifathiae

.097

.049

2.0

E. coli

81.000

46.900

1-7

From Libby and Holmberg (568).

Note. Unit of activity calculated on the basis of 1,650 units/mg. for pure penicillin G, and 1,000

units/mg. for pure penicillin X.

Different strains of the same organism show marked variations in
their sensitivity to penicillin. For example, a study of 40 strains of
hemolytic S. aureus isolated from patients in an Army hospital (725)
gave a range of sensitivity from complete tolerance of 4 O.U./ml. to

ANTIBACTERIAL ACTION 237

Inhibition by 0.002 O.U./ml. More than 40 per cent of the strains thus
Isolated could be called resistant j this was especially true of the strains
isolated from patients who received penicillin.

The oxygen uptake of suspensions of staphylococci was not inhibited
to any extent by the action of penicillin for 3 hours. In a concentration
of I : ijOOOj after incubation for 24 hours at 37° C, the bacteria gave
larger numbers of colonies on plating (5). Although 0.0 1 to o.i mg. of
penicillin per milliliter was found (432-437) to be sufficient to inhibit
the growth of 2,500,000 hemolytic streptococci (Group A), no con-
clusion could be reached as to whether its action is truly bactericidal or
merely bacteriostatic.

Penicillin inhibits fibrinolysis by sensitive strains of S. -pyogenes; this
phenomenon is believed to be connected with growth inhibition (183).

A comparison was made of the amounts of crude penicillin and
gramicidin required to bring about total inhibition of growth of bac-
teria, on the basis of micrograms per milliliter of culture medium
(rabbit's plasma and a serum extract of chick embryo) . The results were
as follows:

Penicillin

Gramicidin

D. fneumoniae

2.5-5.0

0.5-1.0

S. fyogenes

2.5

5.0

S. sal'tvarius

20-40

2.5-60

S. jae calls

200*

40-60

S. aureus

2.5-10

300*

* Inhibition not complete at

these figures.

The two substances appeared to be as effective against bacteria in cul-
tures containing growth tissue as in cultures in which no tissue was
present (394,395).

Inhibition of growth of 2 to 4 million hemolytic streptococci was ob-
tained by the use of 0.03 \\g penicillin with an activity of 240 to 250
O.U./mg. (432, 437). No inhibition was obtained with peptone,
/)-amino-benzoic acid, blood, or serum. The fact that both penicillin and
sulfonamides act upon some bacteria and are ineffective upon others
suggests a similarity In their mode of action (648). A marked differ-
ence was found, however, in the action of penicillin and sulfonamides,

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ANTIBACTERIAL ACTION

239

the latter merely decreasing the rate of multiplication and the former
actually bringing about a decrease in the number of organisms present.
This is shown in Figure 22. The rates of activity of penicillin, grami-
cidin, and tyrocidine are compared in Table 25 (p- 220). The bac-
tericidal action of penicillin is greatly influenced by the age of the cul-
ture, young cultures being readily killed whereas older cultures are
only little affected. No penicillin is absorbed or destroyed by the bac-
teria.

Penicillin is not very stable (760). It is sensitive to reaction and
temperature changes. The effect of reaction upon the stability of peni-
cillin is shown in Figure 23. The thermostability of pyrogens and their
removal from penicillin preparations are also important (996).

Para-amino-benzoic acid and sulfapyridine were found to have a
synergistic effect on penicillin. A solution of sodium penicillin with
1,200 units per milliliter gave 100 B. subtilis units in a synthetic casein

2 4 6 8 10

REACTION (pHj

2 4 6 8 10

REACTION (pH)

Figure 23. Inactivating effect of reaction upon penicillin. From Foster and
Wilker (283).

240 NATURE OF ANTIBIOTIC ACTION

hydrolyzate medium j the activity was increased to 6,000 by addition
of ^-amino-benzoic acid in dilution of 1:2,500 to i:io,oooj this was
also true in presence of glucose in test medium. A similar, although
somewhat lower, increase took place with S. aureus; no effect was ob-
tained with S. hemolyticus. The addition of a dilute solution of sulfa-
pyridine, which in itself had little inhibiting effect, exerted an even
greater synergistic action upon penicillin. This effect was exerted not
only in vitro but also in vivo (921).

Various gram-negative bacteria have been found to be susceptible to
penicillin in a medium devoid of amino acids j the action of these ap-
parently consists in neutralizing the action of penicillin upon these bac-
teria (431). Various bacteria are capable of producing penicillin-de-
stroying or penicillin-inhibiting substances. Penicillinase is an enzyme j
it is produced by E. coli and other bacteria j it is inactivated by heat and
at fYL 3.0 and 9.0. Anti-penicillin is not an enzyme j it is produced by
B. subtiUs (yiS^) ; it is heat stable.

The ability of penicillin to destroy the bacteroids in cockroaches was
interpreted (95) as indicative of the fact that these bacteroids are not
parasitic but rather symbiotic microorganisms.

Penicillin was also found (409) to be able to inhibit the rate of cell
division of fertilized sea urchin eggs when used in concentrations of
250 to 2,500 O.U./ml. It inhibits the adsorption of methylene blue on
activated charcoal, in concentrations as low as 100 O.U./ml.

Streftothricin and Streptomycin

Streptothricin and streptomycin are active against both gram-positive
and gram-negative bacteria, although they differ in antibiotic spectra
and in toxicity to animals. They are soluble in water but insoluble in
alcohol and other organic solvents. Both have an optimum reaction at
^H 8.0, and both are repressed by glucose and by acid salts. Both are
stable compounds and are highly resistant to the action of microorgan-
isms. However, the two substances can be differentiated in their relation
to cysteine. Streptomycin becomes inactivated by the addition of 3 to 5
mg. of this compound to 100 pg of the antibiotic, whereas strepto-
thricin is not affected by it (179).

The antibacterial activity of streptomycin can be largely or com-

ANTIBACTERIAL ACTION 241

pletely neutralized or antagonized by various chemical agents. These
include glucose and certain other sugars, an anaerobic environment,
certain sulfhydryl compounds, and ketone reagents. In some cases, as
in the action of sugars or the anaerobic environment, the effect on strep-
tomycin may be traced to the acidity produced under these particular
conditions. However, in the effect of cysteine, of cevitamic acid, and of
ketone reagents the inhibition of streptomycin activity may be asso-
ciated with the blocking of the active grouping in the molecule of the
streptomycin. Streptomycin represents too large a molecule to explain
the inactivation of its antibacterial properties by the blocking of a single
group in its molecule. Until the chemistry of streptomycin is more
clearly elucidated, it is difficult to present a suitable theory that would
explain the various effects of streptomycin inactivation (327).

The ability of various bacteria to give rise to strains which are more
resistant to the action of streptothricin and streptomycin has been defi-
nitely established. Certain strains have been obtained that are a hun-
dred or more times as resistant to streptomycin as the original culture.
Such strains are only slightly more resistant to streptothricin, and show
no difference from the mother culture in their sensitivity to penicillin,
clavacin, or antibiotics of spore-forming bacteria. Variations in sensi-
tivity to streptomycin by natural strains of the same organism have also
been obtained (965). This phenomenon has an important bearing upon
the chemotherapeutic utilization of the material.

Streptothricin-resistant strains of L. easel show differences in panto-
thenic acid and biotin sensitivity from the susceptible parent strains
(716).

Actinomycm

Actinomycin is a bacteriostatic agent, active primarily against gram-
positive bacteria. It is extremely toxic to animals, a factor which limits
its practical utilization. One milligram of actinomycin given to mice,
rats, or rabbits intravenously, intraperitoneally, subcutaneously, or
orally proved (796) to be lethal for i kilogram weight of the animals.
Doses as small as 50 Mg per kilogram injected intraperitoneally daily
for 6 days caused death accompanied by severe gross pathological
changes, notably a marked shrinkage of the spleen. Actinomycin is

242 NATURE OF ANTIBIOTIC ACTION

rapidly removed from the blood and excreted. It has no effect upon
bacteriophage or staphylococci, although o. i milligram per cent inhibits
growth as well as blood coagulation by these organisms (675).

A comparison of the effect of actinomycin with that of tyrothricin
and its constituents, tyrocidine and gramicidin, upon the growth of
rhizobia (917) showed that, whereas gramicidin inhibited all strains
alike, the other three substances inhibited the slow-growing rhizobia
much more than the fast-growing ones. Effective and ineffective strains
behaved alike. Of the four antibiotic substances, tyrocidine was usually
bactericidal, actinomycin was bacteriostatic, and the other two pos-
sessed both properties. Some strains of rhizobia were stimulated by lim-
ited concentrations of actinomycin.

Proactinomycin

Proactinomycin, in low concentrations, lengthens the lag phase of
staphylococci, this increase in lag becoming greater in certain media
as the size of the inoculum is reduced. One of the primary effects of
this antibiotic is believed to be the interference with the synthesis or use
of diffusible substances produced by the cells and concerned with the
ending of the lag. When the concentration of proactinomycin is in-
creased above a certain value, the effect is different: after a period of
incipient growth, the cells begin to die. There is thus a qualitative
similarity between the effect of proactinomycin and of penicillin (3).

Clavacin

Clavacin not only is bacteriostatic on gram-negative bacteria but also
possesses marked bactericidal properties, as is brought out in Figure
24. Clavacin has an inhibiting effect upon the following respiratory
enzyme systems: glucose dehydrogenase, succinoxidase, malic acid
dehydrogenase, glycerophosphate dehydrogenase, and tryptophanase
(379). Various animal fluids and organs (serum, liver) contain an
enzyme which converts clavacin into an acid, due to the hydrolysis of
the lactose ringj a marked reduction in its antibiotic action results
{S?>S)' Clavacin is also active against fungi, including species of
Pythium (24) and C. ulmi (949).

\

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Jf 4

a3inn~iikM yad viij3iDvg jo SNonnm

asxnnnii^ aid viagiDvg jo SNomm

244 NATURE OF ANTIBIOTIC ACTION

Fumigacin is active only upon gram-positive bacteria and has a rather
limited bactericidal effect. Its action upon tubercle bacteria has recently
attracted considerable attention (27). Partially purified preparations
inhibited multiplication of the human strain of M. tuberculosis in dilu-
tions as high as i : 1,400,000. Killing effect was exerted by a dilution of
1:500,0005 this was measured by incubation of a heavy suspension of
the bacteria with different dilutions of the substance, incubating 24
hours, and measuring for viability. The avian type of M. tuberculosis
was not affected.

A s-per gillie Acid

Aspergillic acid is active against various bacteria, including the hu-
man strains of M. tuberculosis. It was suggested that its mode of action
is due to interference with the utilization of iron by this organism
(348). The favorable effect of cobaltous ion on the tuberculostatic ac-
tion of this antibiotic has been established (349).

Gliotoxin

Gliotoxin is active against both gram-positive and gram-negative bac-
teria (Table 39). The substance is rather toxic to animals, the minimum
lethal dose being 45 to 6s mg. per kilogram body weight j hematuria
is caused by even lower concentrations (474).

TABLE 39. BACTERIOSTATIC ACTION OF GLIOTOXIN

TEST ORGANISM

ACTIVITY

5. aureus

4,000,000

S. pyogenes

1,000,000

Pneumococci

4,000,000

S. enieritidis

250,000

A . aero genes

200,000

K. fneumoniae

250,000

E. colt

80,000

From Johnson, Bruce, and Dutcher (475).
Note. Units of activity by dilution method.

BACTERIOSTATIC AND BACTERICIDAL AGENTS 245

Other Antibiotic Agents

On comparing the action of citrinin with penicillic acid, the first was
found (698) to act largely upon gram-positive bacteria j the second,
like quinones, had a more widespread action, especially against the
colon-typhoid group.

Considerable speculation has been focused upon the ability of vari-
ous organisms to inhibit the growth of M. tuberculosis. These comprise
various fungi, such as A. fumigatus (27, 870) and P. notatum (863),
bacteria, and a number of actinomycetes including species of Nocardia
and Streftomyces (831, 1029). The antibacterial action of some of
these organisms is believed to be due to the formation of substances
spoken of as tuberculocidins. The specific effects of some of the sub-
stances, which are now recognized, are described in detail elsewhere
(pp. 294-296).

BACTERIOSTATIC AND BACTERICIDAL AGENTS

Fleming (263) divided all selective bacteriostatic agents, exclusive
of the action of oxygen on anaerobic bacteria, into three groups: (a)
physiological agents, including bile, serum, proteolytic enzymes, and
lysozymej (b) microbiological products, comprising the antibiotic sub-
stances j (c) chemicals of known composition, including dyes, salts (po-
tassium, tellurite, mercuric salts), and other agents.

Most of the antibiotics are characterized not only by their bacterio-
static action but also by marked bactericidal properties, the two usually
being parallel (963). Concentrations of an antibiotic substance smaller
than those needed to cause inhibition often stimulate the growth or
metabolic processes of the organism. In this respect these agents are
similar in action to synthetic detergents and to other chemical disin-
fectants, as discussed previously. It may also be of interest to note here
that the antibacterial action of straight-chain mono-amines and di-
amines, amidines, guanidines, and quaternary bases increases with the
length of the chain up to a maximum and then decreases, the gram-
positive bacteria being more sensitive than the gram-negative organ-
isms. Serum increases the activity of the shorter-chain compounds and
decreases that of the longer-chain compounds, depending to a consider-
able extent upon the test bacteria (305).

246 NATURE OF ANTIBIOTIC ACTION

The bactericidal action of antibiotic agents, as influenced by their
concentration, can be illustrated by the action of pyocyanase (Table
40). In a study of the bactericidal action of actinomycin (975) it was
found that the addition of 0.5 mg. of actinomycin to a 10 ml. sus-
pension of E. coli reduced the number of viable cells from 6,400,000
to 493,000, the methylene blue reduction test remaining positive j
I mg. actinomycin reduced the number of cells to 4,800, the reduction
test becoming negative j 2 mg. of the agent brought about complete de-

TABLE 40. BACTERICIDAL ACTION OF PYOCYANASE UPON THREE BACTERIA

B.

ANTHRACIS

E.

TYPHOSA

C. DIPHTHERIAE

Bacteria per

Bacteria per

Bacteria per

Hours

milliliter

Hours

milliliter

Minutes

milliliter

Start

11,060,000

Start

13,125,000

Start

24,150,000

24

6,890,000

3

1,242,000

2

17,850,000

72

1,360,000

9

105,000

180

0

96

654,000

120

329,000

144

0

From Emmerich, Low, and Korschun (237).

struction of all the cells. The bactericidal action of actinomycin seems
to be a result of a chemical interaction similar to that of other anti-
septics. On adding o.i mg. actinomycin to a suspension of E. coli cells
in a 10 ml. buffer solution, the value of the constant K was found to
vary from 0.021 to 0.026 for different periods of incubation. Figure 25
illustrates graphically the effect of different concentrations of actino-
mycin on the death rate of E. coli in buffer solution.

Quinones have a high bactericidal power (153, 977), due not to
their chemical interaction with the cell proteins but to their reactivity
with the simpler cell constituents such as some of the amino acids. Only
a slight difference was found in the apparent activity of quinones
toward yeasts, bacteria, proteins, peptones, peptides, and certain amino
acids. Alcohol increases the germicidal power of the quinones. Acti-
nomycin contains a quinone group ; however, it acts differently toward
gram-positive and gram-negative bacteria j alcohol has no effect upon
its action, thus pointing to marked differences in chemical and biologi-

BACTERIOSTATIC AND BACTERICIDAL AGENTS 247

9

_

-^ - 0 CONTROL

8

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48

INCUBATION PERIOD IN HOURS

Figure 25. Action of actinomycin on E. coli; death rate in buffer solution.
Amounts are given in milligrams of actinomycin per lo milliliters of solu-
tion. From Waksman and Woodruff (977)-

cal nature of this antibiotic agent and of quinones. On the other hand,
many of the antibiotic substances produced by fungi are typical qui-
nones and act as such.

By varying the concentrations of disinfectants, the types of curves of
destruction of bacterial cells were found (728) to range from linear to
an abrupt drop to zero at critical concentrations. This is brought out in a
study of the spirocheticidal action of penicillin (Figure 26).

248

NATURE OF ANTIBIOTIC ACTION

Figure 26. Spirocheticidal action of various amounts of penicillin in vitro.
Inoculum, 4x10* organisms per milliliter. From Eagle and Musselman
(225).

In general, chemical disinfectants act upon bacteria in four different
ways: (a) some affect the lag phase of the growth period, (b) some in-
fluence the mechanism of cell division, (c) some influence the metabolic
processes, and (d) some affect the death rate of the microbes. Similar, if
not greater, variations are found in the nature of the action of antibiotic
substances of microbial origin upon the bacterial cell.

Gramicidin inhibited dehydrogenase activity, since the antagonized
bacteria rapidly lost their capacity to reduce methylene blue in the pres-
ence of glucose. E. coli suspension treated with actinomycin lost its
capacity to reduce methylene blue before the cells were completely
killed. The oxidation of succinic acid by tissue preparations, which re-

BACTERIOSTATIC AND BACTERICIDAL AGENTS 249

quire the cooperation of succinic dehydrogenase and a cytochrome sys-
tem, was strongly inhibited by pyocyanin. This inhibition exhibited cer-
tain interesting peculiarities: in low concentrations, pyocyanin strongly
inhibited the activity of the complete succinic cytochrome system but
had no effect on the oxidation of succinic acid through methylene
blucj in the presence of KCN, pyocyanin acted as an autoxidizable hy-
drogen acceptor similar to methylene bluej glutaminic acid did not af-
fect the inhibitory action of pyocyanin. This inhibitory action was found
to be due not to the formation of oxalacetic acid but to a direct effect on
succinic dehydrogenase. The influence of pyocyanin on bacterial respira-
tion, as well as its ability to function as an accessory respiratory enzyme,
has aroused much interest (228, 302).

A strong lytic action of some of the antibiotic substances, similar in
some cases to the action of enzymes, has also been Indicated. This lytic
mechanism may be a product of the antagonized cell itself. Autolysis
is usually defined as the destruction of some of the essential chemical
constituents of the cell by enzymes originating within the cell.

The lytic effect does not hold true, however, for most of the anti-
biotic substances and for most of the bacterial cells. Since the greatest
bactericidal effect of penicillin occurs during the maximum rate of bac-
terial multiplication and since cells producing autolysin lyse rapidly it
must be concluded that lysis of the cells follows the killing effect of
penicillin (913).

Chain and Duthie (121) called attention to the fact that Fleming re-
corded the slow bactericidal effect of penicillin on Staphylococcus and
its lytic action only under certain conditions. The original statement by
Florey, Chain, and associates that penicillin was mainly bacteriostatic
was based on the fact that it did not influence the oxygen uptake of rest-
ing Staphylococcus y for large numbers of viable colonies were found
after incubation with penicillin for 24 hours. Penicillin was later found
to be definitely bactericidal, but not under unfavorable conditions such
as low temperature or exhausted media. The bactericidal effect can be
increased by substances which favor bacterial growth or decreased by
substances which interfere with bacterial growth, such as sulfadiazine.
During the resting phase of Staphylococcus ^ even large concentrations
of penicillin have no effect on the oxygen uptake. However, during the

250 NATURE OF ANTIBIOTIC ACTION

early lag phase and the logarithmic phase of multiplication, penicillin
has a strong inhibitory effect, completely stopping oxygen uptake, even
in small concentrations. It was concluded that penicillin exerts a bac-
tericidal effect on Staphylococcus before actual division occurs, or after
one division during the logarithmic phase. It appears to interfere with
some metabolic function of the early stages of bacterial development.

Bonet-Maury and Perault (73), using the differential photometer,
suggested that penicillin stops proliferation of S. aureus almost imme-
diately and lysis follows. A second proliferation occurs regularly, fol-
lowed by a second partial lysis and then a second post-lytic growth.
Gardner (312) reported that high penicillin concentrations (50 to lOO
O.U./ml.) slowly kill bacterial spores. B. anthracls spores were more
susceptible than those of B. subtilisy although neither were completely
eliminated by penicillin.

The relation between antibiotics and bacteriophage has attracted con-
siderable attention. Gratia (353) observed a definite relation between
the action of lysozyme and the liberation of bacteriophage. The action
of antibiotic agents, however, is usually quite different from that of bac-
teriophage (352, 677). Filtrates of cultures of homologous bacteria are
able to inactivate the anti-coli phage j at 27° C, the inactivation is pro-
portional to the phage and filtrate concentration j at 0° C, to the square
root of the latter (231). Based upon the formation of iso-antagonistic
substances, a method has been suggested (132) for the differentiation
of bacteria belonging to the typhoid group.

EFFECT OF ANTIBIOTIC SUBSTANCES UPON THE
MORPHOLOGY OF MICROORGANISMS

Emmerich and Saida (238) were the first to report that anthrax bac-
teria undergo morphological changes as a result of the action of pyocya-
nase. Since that early work, the effect of bacterial filtrates upon cell
multiplication and cell growth has been made the subject of many in-
vestigations. It was reported (381), for example, that no complete ces-
sation of the fission process of bacteria results from the action of the
substance, but that growth itself is checked, the action being nonspecific
as far as bacterial species are concerned. The conclusion was reached that

#'.

S. aureus, normal cells. Prepared by
Foster and Woodruff

*^ »

«

S. aureus, penicillin-inhibited cells.
Prepared by Foster and Woodruff

^

y/ /

/

B. subt'iUsy normal cells. Prepared
by Foster and Woodruff

' : !;. '-.■■.' :

% •

■y\ '

1 / ... ^'

iT S •

f ^ /

..^ •-% \ . ^ ni 1

//z. vinelafu/iiy normal cells.
Prepared by Starkey

B. suhtilisy penicillin-inhibited cells.
Prepared by Foster and Woodruff

/Iz. vhu'landlty actinomycin-inhibited
cells. Prepared by Starkey

Figure 27. Influence of antibiotic substances upon the morphology of bactc

EFFECT ON MORPHOLOGY OF MICROORGANISMS 251

this phenomenon is due to the production and accumulation of metabolic
products injurious to growth. Nonspecific antibiotic substances were
demonstrated (692) in filtrates of bacteria. They not only injured
growth of other bacteria but prevented the production of the ectoplas-
mic antigen. These substances could be partly removed by the use of
adsorbents.

The morphology of bacteria is greatly influenced by the presence of
other organisms or their antibiotic substances. In the case of diphtheria
bacteria this is accompanied by a reduction in virulence (417). The spe-
cific effect of the antagonistic B. mesenterkus upon the morphology of
antagonized bacteria has also been established (731). The antibiotic
substances produced by actinomycetes were shown (76) to affect the
growth of B. mycoides as follows: cell division is delayed; the cells be-
come elongated, reaching enormous size and assuming most peculiar
forms 3 spore formation or, with lower concentrations of agent, the ac-
tive substance is repressed j delayed nonspore-f orming variants are pro-
duced with a modified type of growth on nutrient media (Table 41).
The cells of bacteria subject to the action of streptothricin are greatly
enlarged, due to incomplete fission (287, 1031).

Gardner (311) reported that concentrations of penicillin lower than
those required for full inhibition caused a change in the type of growth
of CI. welchn in liquid media. The majority of the cells became greatly
elongated, giving rise to unsegmented filaments ten to twenty times
longer than the average normal cells. The same was found to be true
of a number of other bacteria (Figure 27). Even gram-negative bac-
teria, which are relatively resistant to penicillin, showed the same ef-
fect. Many bacteria produced giant forms as a result of the autolytic
swelling and bursting of the elongated cells. It was recognized that
these changes were due to a failure of fission. Cell growth not accom-
panied by cell division underwent autolysis. Br. abortus and Br. meli-
tensisy which were not inhibited by penicillin even at i : i,000 dilution,
gave no enlargement of the cells but showed vacuolation even in lower
dilutions. CI. welchiiy which was inhibited by i: 60,000 penicillin,
showed filament formation in a dilution of i : 1,500,000. These obser-
vations were fully confirmed. All sensitive bacteria were shown (913)
to undergo lysis to a greater or less extent in cultures containing peni-

252 NATURE OF ANTIBIOTIC ACTION

TABLE 41. INFLUENCE OF CULTURE FILTRATE OF STREPTOMYCES SP. ON
MORPHOLOGY OF BACILLUS MYCOIDES

MORPHOLOGY OF

MACROSCOPIC

DAYS OF

ANTAGONIZED

GROWTH IN

SPORE

ROD

INCUBATION

BACTERIUM

BROTH

FORMATION

FORMATION

Medium

PLUS

10 PER CENT CULTURE FILTRATE

2

Long filaments

X

-

+

4

Filaments have divided

into elongated cells

z

-

+

17

Cells altered

X

-

+

45

Cell fragments of vari-

ous shape and length

z

-

-

Medium

PLUS

5 PER CENT CULTURE FILTRATE

2

Elongated cells

X

-

+

4

Elongated cells

z

-

+

17

Greatly deformed cells

+

-

+

45

Greatly deformed cells

+

-

+

Control

MEDIUM

2

■H-

-

+

4

++

+

+

17

-H-

+

+

45

Deformed cells rare

++

+

-

From Borodulina (76).

X indicates growth of B. mycoides in the shape of fluffy small balls inside liquid.

cillin. Before lysis, the culture becomes more turbid, thus pointing to
the fact that multiplication is essential before death or lysis of the cells.

The phenomena of swelling and lysis were said (86i) to be associ-
ated with the active growth of the bacterial cell. Suspensions of fully
grown bacterial cells showed neither of these effects when added to con-
centrations of penicillin many times higher. It was suggested that peni-
cillin either has some action on the cellular wall of S. aureus or that it
interferes with the assimilation of one or more growth factors necessary
for the fission of the growing cell.

In a study of the effect of penicillin on bacterial spores, Gardner
(312) observed that the spores gradually lose their high refractivity
and become empty ghosts. Weaker concentrations of penicillin allow
the germination of the spores to occur, the spores swelling up and be-

EFFECT ON PHYSIOLOGY OF BACTERIAL CELL 253

coming spherical coccoids which burst j in the case of some bacteria, not
all the spores are killed j however, the great majority of them become
nonviable. In non-nutrient media the spores are little affected, even by
strong concentrations of penicillin. The conclusion was reached, there-
fore, that the action of penicillin on sensitive bacteria has little or no
connection with multiplication or division, although some abnormal
divisions were observed in certain sensitive organisms. The majority of
S. aureus and S. pyogenes growing on agar are checked or killed before
any visible growth or division has occurred.

A growth-depressing substance, which altered the type of growth of
both fungi and bacteria, was also isolated (151) from yeast. Fungi
treated with this substance produced thick gnarled mycelia and formed
no conidia or pigment. Increasing the concentrations of the depressing
agent changed the nature of the colony of E. coli from smooth to rough
and finally to grainy; this was associated with an increase in the length
of the cell and the formation of filaments. When the cultures thus modi-
fied were placed in media free of the agent, normal, highly motile cells
were again produced.

The mechanism of disintegration of the hyphae of a plant pathogenic
fungus Rhizoctonia by an antagonistic fungus Trichoderma as well as
by the antibiotic product of the latter has been described by Weindling
(989). The hyphae are usually killed in less than 10 hours, as shown by
loss of the homogeneous appearance of the protoplasm and of the
vacuolate structure of the hyphae, which become empty or appear to be
filled with granular material. This is brought out in Figure 29, p. 302.

ANTIBIOTIC SUBSTANCES AND THE PHYSIOLOGY
OF THE BACTERIAL CELL

Half a century ago Smith (867) emphasized that bacteria growing
in mixed cultures undergo temporary and even permanent physiologi-
cal modifications. Aside from cell proliferation, the important meta-
bolic processes commonly considered to be affected by antibiotic agents
were oxygen uptake, acid production, and dehydrogenase activity. Some
agents apparently can inhibit cell growth without destroying the viabil-
ity of the cells and their capacity for taking up oxygen.

254 NATURE OF ANTIBIOTIC ACTION

Gramicidin and tyrocidine were believed to affect bacteria by depress-
ing the surface tension of aqueous solutions. This effect was favored by
the addition of organic solvents such as glycerin, which increases the
solubility of gramicidin. The addition of serum resulted in a decrease in
activity of tyrocidine, to a less extent, however, than of gramicidin.
Heat destroyed the bacterial and hemolytic effects of gramicidin, but
the property of altering surface tension was heat-stable. It has further
been shown (405) that gramicidin, after an initial stimulation, inhibited
oxygen consumption of bovine spermatozoa and rendered them im-
mobile ; aerobic as well as anaerobic glycolysis was depressed by about
40 per cent and motility of the spermatozoa impaired. Tyrocidine,
however, caused a small reduction in the oxygen consumption and in
glycolysis. The action of gramicidin upon the metabolic activities of S.
aureus and S. hemolyticus was shown (210) to be influenced by the
composition of the medium, the presence of potassium and phosphate
ions giving a prolonged stimulation of metabolism, whereas ammo-
nium ions favored a depression in oxygen uptake.

The specific effects of basic proteins, such as protamine and histone,
upon the activity of selective inhibitors offered a possible explanation
for the difference in the action of tyrothricin upon gram-positive and
gram-negative bacteria ( 645 ) . These basic proteins also possess antibac-
terial properties. They have the capacity of sensitizing gram-negative
bacteria by means of substances which otherwise act only on gram-
positive forms.

Pneumococci grown in media containing the specific enzymes which
hydrolyze their capsular material are deprived of these capsules and
fail to agglutinate in the specific antiserum. The enzymes do not inter-
fere with the metabolic functions of the cells, but their action is directed
essentially against the capsule. These enzymes were found not only to
exhibit great selectivity but to be highly specific against the particular
polysaccharides.

The respiratory enzymes of bacteria, such as S. aureus, that are sensi-
tive to citrinin were found (643) to be inhibited by this antibiotic, but
not the corresponding enzymes of resistant bacteria, such as E. coU.

The possible interference of penicillin with the metabolism of bac-
teria has attracted considerable attention due to the great practical im-

INHIBITION OF ACTION 255

portance of this antibiotic. The effect was believed to involve the me-
tabolism of nucleotides and nucleic acids (530). Penicillin had no effect
on the release of glucose from polysaccharides or on the oxidation of
glucose or pentose to acetic acid. However, when yeast sodium nuclei-
nate was added to a suspension of cells, a rate curve was obtained which
was similar to the endogenous curve j the acceleration appeared two to
four hours earlier, and the maximum oxidation was higher and was
maintained for a longer period. Penicillin was completely inhibitory to
this reaction.

INHIBITION OF BACTERIOSTATIC AND
BACTERICIDAL ACTION

The formation of specific chemical compounds capable of inhibiting,
inactivating, or even destroying bacteriostatic and bactericidal sub-
stances of microbial origin has been established for a number of anti-
biotic agents.

Yeasts were found to contain a substance which inhibits the action of
sulfanilamide against S. hemolytkus as well as other streptococci and
pneumococci. This substance has been identified with the ^-amino-ben-
zoic acid referred to above. Br. abortus and certain other bacteria also
contain (360) a factor, designated as "p," which specifically inhibits the
bacteriostatic action of sulfanilamide. This factor stimulates markedly
the growth of many bacteria, and is not specific. The sensitivity of sulfa-
nilamide depends on the rate of release of the factor from the bacterial
cell and not on the total amount produced. This factor was believed to
stimulate some enzyme reaction concerned with bacterial reproduction,
whereas sulfanilamide inhibits this reaction. Similar factors have been
isolated from other microorganisms.

To what extent antibiotic substances can be inhibited in their action
against bacteria still remains to be determined. Certain few facts have
so far been established.

Bacteria not inhibited by penicillin were found (4, 570, 1027)
capable of producing a substance which destroys the growth-inhibiting
property of the antibiotic. The substance is enzymatic and was desig-
nated as penicillinase. It is produced by various bacteria, especially
members of the B. subtilis (221, 922) and E. coli groups. It is precipi-

256 NATURE OF ANTIBIOTIC ACTION

tated from the medium with acetone, alcohol, dioxane, sodium tung-
state, and saturated ammonium sulfate solution. Highly purified prepa-
rations have been prepared from B. cereus, i mg. of the product de-
stroying completely 165,000 units of crystalline penicillin in 3 hours at
fH 7.0 and 30° C. (44). In a dry state, penicillinase is thermostable 3 in
solution, it is more readily inactivated by heat. In a purified state, it is
labile 3 at 45° C, 66 per cent of it is destroyed in 20 minutes and 95 per
cent in an hour (611). The inactivation of penicillin by penicillinase is
accompanied by the evolution of COo from the bicarbonate in solution,
thus pointing to acid formation (279). The optimum fVL for the action
of the enzyme is between 8.0 and 9.0.

Penicillinase can be utilized for the purpose of testing the sterility of
penicillin preparations (550). The penicillin, which would otherwise
cause inhibition of growth of the contaminating organism in the test
medium, is destroyed by the enzyme previous to the test. There is no
apparent relation between the resistance of an organism to penicillin
and its ability to produce penicillinase (1027).

Certain bacteria, as E. coli, produce a thermostable fungistatic factor
which must be differentiated from penicillinase j the effect of this factor
consists in reducing the rate of growth of the fungus, rather than in de-
stroying the penicillin (1009).

Bacterial extracts, pus fluids, peptone, and ^-amino-benzoic acid,
which interfere with the action of sulfonamides, do not affect penicillin.

Cephalin and extracts of gram-negative bacteria, of milk, and of
blood serum were found to inhibit the action of gramicidin. Because of
this, cephalin is capable of reviving bacterial cells killed with grami-
cidin. This phenomenon is similar to the inhibition by phospholipins of
the action of synthetic detergents upon bacterial metabolism. Mucin
inhibits the action of tyrothricin, especially on meningococci and pneu-
mococci (192).

Tannic acid is able to neutralize the antibiotic action of actinomycin.
Humus compounds of the soil have a similar capacity. This effect was
believed to be responsible for rendering harmless, to living plant and
animal forms, the actinomycin produced in the soil (976). Ascorbic
acid also has an effect in reducing the activity of this substance. Since
vitamin C is a strong reducing agent and actinomycin is a reversible

ADAPTATION OF BACTERIA TO ANTIBIOTICS 257

oxidation-reduction system, it is conceivable that actinomycin may be
reduced through the action of the vitamin. Such an effect should be
greatly increased under anaerobic conditions, where no reoxidation due
to atmospheric oxygen could occur. Twenty-five to 50 times as great a
neutralizing effect of ascorbic acid upon actinomycin was obtained un-
der anaerobic conditions with CI. butyrkum as a test organism as under
aerobic conditions with B. mycoides. Reduced actinomycin was inactive.
It was concluded, therefore, that the neutralizing action of ascorbic
acid upon actinomycin was due merely to its reducing properties. As far
as the common growth factors are concerned, the action of actinomycin,
like that of penicillin, differs from the mechanism proposed by Woods
(1032) and others for sulfanilamide inhibition.

Among the phenomena of inhibition of antibiotic substances, the ac-
tion of cysteine offers some important practical applications. This sub-
stance inhibits the antibacterial action of penicidin (29), penicillin, cit-
rinin, gliotoxin, clavacin, pyocyanin (116), and streptomycin (327).
It was suggested that this is due to the fact that the mode of action of
certain antibiotics involves their ability to interfere with the normal
functions of the sulfhydryl groups in bacterial metabolism. The rate
of inactivation of penicillin was found (136) to be dependent on the
concentration of penicillin and on the fH of the medium j since thio-
glycolic acid was somewhat less effective and other amino acids, like
cystine and methionine, had no effect, it was suggested that the process
of penicillin inhibition involves both the sulfhydryl and the amino
groups of cysteine. Since i mg. of cysteine was sufficient to inactivate
about 270 to 410 O.U. of penicillin, it was suggested (423) that ad-
vantage be taken of this fact in testing for sterility of penicillin prepa-
rations.

ADAPTATION OF BACTERIA TO ANTIBIOTICS

Different strains of bacteria vary greatly in their susceptibility to the
same antibiotic substance (75). This is true particularly of penicillin
(267) and streptomycin (965). By growing an organism in media con-
taining increasing concentrations of the antibiotic, more resistant strains
can be obtained. The rate and degree of acquired resistance vary for
each antibiotic. For example, certain bacteria showed no resistance to

258 NATURE OF ANTIBIOTIC ACTION

aspergillic acid, slight resistance to gliotoxin, and marked resistance to
penicillin, streptomycin, and pyocyaninj resistance was attained more
rapidly and was lost more slowly for streptomycin. An increase in re-
sistance to one did not affect the sensitivity to other antibiotics (510a).

The resistance of staphylococci to penicillin, or penicillin-fastness,
was found to be reversible j strains were shown to change frequently to
the sensitive state when they were cultured in antibiotic-free media. It
has even been suggested that the reversion may be more rapid in vivo
than in vitro (914). Strains of staphylococci possessing increasing re-
sistance to penicillin were isolated from infections treated with this sub-
stance (756).

In a study of 1 15 strains of staphylococci, 13.9 per cent proved to be
resistant to penicillin j the resistant strains produced penicillinase, but
not the susceptible ones (72). Of 128 strains collected from various
sources before the advent of penicillin treatment, 123 were inhibited
by one-eighth unit or less of penicillin per ml., 2 were inhibited by one-
fourth unit, and 3 by half a unit. The 5 strains showing slightly in-
creased resistance were nonpathogenic. Thirty-one strains from hospital
wards in which penicillin had been used extensively, particularly in
local application to wounds, were found to be largely penicillin resist-
ant. These strains showed no cultural differences from normal strains.
Whether they developed from sensitive parent organisms in the same
wound or were the result of cross infection, either from carriers or air
borne, was not determined (682). When staphylococci were made re-
sistant to penicillin by cultivation in penicillin-containing medium, this
acquired resistance or "fastness" was lost when the cultures were grown
in plain medium. Pneumococci, however, acquired resistance less
readily and maintained it even when subcultured in plain media (914).
Patients treated with penicillin gave a much larger proportion of re-
sistant strains of S. aureus than patients not so treated (725).

Bacterial cells that have become adapted to resist the action of a par-
ticular antibiotic substance are not resistant to the action of others. How-
ever, bacteria "trained" to resist one substance may also acquire resist-
ance to another by a process of adaptation. This was brought out by
Davies et al. (170) in studies on sulfonamides and proflavine. Adapta-
tion of bacteria to an antibacterial agent has been explained as follows:

DIFFERENTIATION OF BACTERIA 259

1. By natural selection from an initially heterogeneous population. This

concept, however, has lost much support, since variations have been
found to occur in strains derived initially from a single cell.

2. By actual modification of the individual cells. This may be due (a) to

the establishment in the cells of a mechanism alternative to that
normally in use or (b) to the quantitative modification of existing
mechanisms.

3. By a change in some center of organization of the cell.

Demerec (177) suggested that the development of strains of S. au-
reus resistant to penicillin is due to the formation of mutants, the non-
resistant cells being eliminated by the penicillin. The degree of resist-
ance can be increased by exposure to larger doses of the antibiotic j this
increase was considered to be a summation of several independent ge-
netic factors for resistance which undergo considerable mutation. Re-
sistant strains retained that property after many transfers.

There are apparently several mechanisms whereby organisms such
as staphylococci develop resistance to penicillin. One consists in the de-
velopment of a penicillin inhibitory in other cases, however, no inhibitor
can be demonstrated (717).

The adaptation of staphylococci to mycophenolic acid is not an easily
reversible phenomenon, thus suggesting that the adaptation consists in
the development of an alternative mode of growth, comprising a modi-
fication of the enzyme systems of the bacteria (2). The ending of the
lag of growth of S. aureus is due to a diffusible growth intermediate
produced by the cells j the primary action of the antibiotic consists
partly in hindering the utilization of this substance. The effect of the
antibiotic consists in a failure of the dividing cells to separate in the
normal manner. These changes do not occur in "trained" cells.

DIFFERENTIATION OF BACTERIA BY MEANS
OF ANTIBIOTIC SUBSTANCES

Because of their selective action upon different bacteria, antibiotic
substances can be utilized for separating bacteria from one another.
Fleming (264) utilized penicillin to isolate Pfeiffer's bacillus and the
pertussis organism of various cocci, diphtheria, and neisseria organisms j

260 NATURE OF ANTIBIOTIC ACTION

the substance was found to behave as the mirror image of tellurite in
inhibiting specific bacteria. Penicillin was also utilized for the separation
of acne bacilli from accompanying staphylococci (158) and for the
separation of streptococci from staphylococci (262) j also for the isola-
tion of Trichomonas vaginalis and of animal viruses (803).

Actinomycin was used to separate gram-negative from gram-positive
bacteria (974).

Tyrothricin has been utilized (886) for the isolation of A^. gonor-
rhoeae from contamination with gram-positive bacteria. Usually a
1 : 15,000 dilution of the substance in "chocolate" agar was found quite
satisfactory for this purpose. By the use of tyrothricin, gram-negative
microorganisms have been isolated from the nasopharynx even in the
presence of overgrowth by gram-positive cocci. Hemophilus influenzae
is resistant to the action of tyrothricin, and its isolation is facilitated by
adding to the media on which it is cultured tyrothricin in a dilution
which inhibits the cocci (842).

Streptothricin was found to help in distinguishing B. tnycoides from
B.subtilis (979).

SUMMARY

Comparatively little is yet known of the mode of action of antibiotic
substances. This field offers great opportunities for research and utiliza-
tion of bacterial activities. The solution to such important problems as
the morphology of the bacterial cell} taxonomic relations of bacteria j
various physiological reactions of microorganisms, especially the phe-
nomenon of adaptation of bacteria to antibiotics and the problem of
bacterial resistance} the mechanism of causation of disease; and the
very control of disease-producing microorganisms — all fundamental
problems in microbiology — will be furthered by knowledge of the ac-
tion of specific antibiotic substances upon bacteria and other micro-
organisms.

CHAPTER 12

UTILIZATION OF ANTIBIOTIC SUBSTANCES FOR
DISEASE CONTROL

Whether gramicidin or any other froduct of Tuicrobic origin will
eventually be found to fulfill certain furfoses better than either
sulfhonamides or any other class of antiseptic remains to be seen.
That several classes of reagent should be comfeting for suprem-
acy in different asfects of a task which not long ago was consid-
ered imfossible of any real fulfillment is a truly remarkable fosi-
tion{25^)'

Microorganisms and products of their metabolism have been utilized
for the control of disease in man, animals, and plants with varying de-
grees of success. On the basis of the observations mentioned above
(p. 26), Pasteur may be considered as the first to advance the subject
of bacteriotherapy. Emmerich (233) reported that anthrax can be con-
trolled by the use of streptococci such as the erysipelas organism 5 these
bacteria were, therefore, looked upon as agents useful in bringing about
immunity against all bacterial infections. Pawlowsky (711) obtained
immunity against anthrax by inoculation with Friedlander's bacillus.
Bouchard (78) was successful in the control of anthrax by means of
Ps. aeruginosa. This organism, however, did not impart any immunity
to the animals, but by the use of a sterilized ten-day-old culture of the
antagonist, healing action was obtained against anthrax infection or at
least its development was delayed (1026a). It was soon demonstrated
(64, 131) that filtrates of Ps. aeruginosa could destroy B. anthracis.
The pressed extract of Ps. aeruginosa was also found to have a protec-
tive effect when injected in the animal simultaneously with the patho-
gen (534a).

Emmerich and Low later employed a cell-free preparation of Ps.
aeruginosa, concentrated to one-tenth its original volume, to treat rabbits
infected with B. anthracis (898). This preparation also destroyed in
vitro a number of other bacteria, including various staphylococci, strep-
tococci, pneumococci, gonococci, C. difhtheriae, V. comma, and Sh.

262 DISEASE CONTROL

faradysenteriae. These studies led to the development of pyocyanase
preparations of varying degrees of activity. Pyocyanase has been used
in the treatment of a variety of diseases including diphtheria and men-
ingitis. In some of these cases, especially of anthrax, treatment was
rather successful (278), in others it was not, due largely to the low
potency of the product (529, 801).

The lack of recognition of the existence of more than one antibiotic
agent in the culture of Ps. aeruginosa led to the disrepute of pyocyanase.
It was soon reported (69), for example, that a Berkefeld filtrate of the
culture had only a weak therapeutic effect, an observation later con-
firmed by Wagner (938). The facts that some strains of Ps. aeruginosa
do not form any pyocyanase and that even active strains may lose the
capacity to produce this antibiotic (529) were other contributing factors
to the gradual disappearance of pyocyanase as a chemotherapeutic
agent.

Various methods of treating severe infections, like anthrax or ma-
lignant tumors, with mild infective agents have been suggested. The
reduction in pathogenicity of one organism by the presence of others
has thus been well recognized. Nonpathogenic organisms apparently
have specific effects upon the pathogens, the development of which was
prevented or even suppressed. The very occurrence of specific types of
pneumococci in healthy individuals and the causation of specific forms
of pneumonia were found to be controlled by the antagonistic effects of
other microorganisms (371).

It was thus definitely established that the growth of B. anthracis
could be inhibited by antagonists (49). Guinea pigs survived large in-
jections of washings from soil previously contaminated by B. anthracis
through the slaughtering of a diseased cow. When cultures of this or-
ganism were isolated from the soil and injected, however, characteristic
disease symptoms resulted. It was suggested that the anthrax spores are
digested by the leucocytes which have been attracted to the site of in-
jection by the accompanying bacteria (32).

Seitz (849), in discussing the problem of mixed infections, cited
many cases not only of decreased but also of increased virulence of the
pathogen as a result of accompanying bacteria. He warned, therefore,
against too sweeping generalizations concerning the healing effect of

MICROBIAL ANTAGONISTS 263

antagonistic bacteria. He believed that in many cases of artificial infec-
tion, the favorable action of the antagonist may have been due entirely
to increased body resistance. Nevertheless, he accepted the possibility
of utilizing the antagonistic effects of microorganisms, for the treatment
of skin surfaces, including those of the intestinal canal and the vagina,
but not for tissue or blood infection.

Until very recent years, attempts to utilize the activities of antagonis-
tic microorganisms for the control of disease did not always meet with
success. This failure may have been due to an insufficient understanding
of the nature of the chemical agent produced by the antagonist, to a
lack of knowledge concerning the mechanism of its action, especially
as regards the production of the active antibacterial substance or anti-
biotic.

MICROBIAL ANTAGONISTS AND DISEASE CONTROL

In 1885, Cantani treated a tubercular patient with a culture of a sapro-
phytic organism, designated as Bacterium tenno; the results were
highly favorable (109). He expressed the hope that other infectious
diseases readily accessible and of a local nature could be effectively
treated with saprophytic bacteria which are antagonistic to the patho-
gens. Following this work of Cantani, Zageri ( 1043) inoculated S. pyo-
genes into animals suffering from anthrax j the rise in temperature
caused by the streptococcus reduced the viability of the anthrax. The
growth of an antagonistic organism was found to change the environ-
mental conditions favorable to the pathogen, thus causing its attenua-
tion.

These results received the immediate attention of other inve;sti-
gators. In most instances saprophytic organisms such as lactic acid bac-
teria or beer yeasts were used, in other cases mild pathogens were em-
ployed. Lorenz and Ravenel (582a), for example, sprayed the throats
of diphtheria carriers with cultures of S. aureus; although the treat-
ment was successful in eliminating the pathogens, the staphylococci
sometimes caused sore throat.

Gate and Papacostas (323) observed that mixed infections were usu-
ally mild, a phenomenon later confirmed. Mixed cultures of the Fried-

26+ DISEASE CONTROL

lander bacillus and of C. difhtheriae gradually gave a predominance of
the former on repeated transfer} the morphology of the diphtheria
organism changed toward a more homogeneous state on staining. The
use of culture filtrates gave no evidence that the diphtheria toxin was
neutralized by the antagonist, either in vivo or in vitro; however, when
the two organisms were grown together no toxin was formed, nor was
toxin produced when the filtrate of the culture of the antagonist was
used to grow C. difhtheriae. The therapeutic use of filtrates was, there-
fore, suggested. Lactic acid bacteria were also employed successfully
(678) in the treatment of diphtheria.

By allowing an antagonist to act upon a disease-producing organism
that has previously been heated to 56° C, a hydrolyzate was obtained
which could be employed as a vaccine. Bezangon (54) treated typhoid
sufferers with a culture of E. tyfhosa lysed by means of Ps. aeruginosa.
Gratia (354) said, however, that this type of hydrolyzate brings about
heat production, but the use of a preparation obtained by means of an
actinomyces, designated as a mycolysate, does not. The use of living
proteolytic bacteria (neocolysin) for treatment of chronic purulent con-
ditions, such as osteomyelitis, gave favorable results} the bacteria were
believed to continue growing as long as there was dead tissue available
(98).

Besredka (52) used culture filtrates of bacteria for the treatment of
various diseases in man. A filtrate of the anthrax organism was em-
ployed for dressings or for intracutaneous injections} the results were
at least as good as those obtained with the bacterial vaccine. Staphy-
lococci and streptococci were also utilized for similar purposes. Besredka
believed that a substance, designated as antivirus, was secreted by the
bacteria into the filtrate. This was said to check further growth of the
bacteria. The mode of action of the antivirus was considered to be dif-
ferent from that of antibodies : the first affects the cells locally by stimu-
lating their resistance} the second acts upon the organism as, a whole
and, through it, against the infecting agents. Antivirus was prepared by
allowing bacteria to grow in ordinary bouillon for a long time, until the
medium became unfavorable for further development of the bacteria.
Staphylococcus antivirus prevented the growth of the staphylococcus or-
ganism in a medium in which it had grown previously. In the presence

MICROBIAL ANTAGONISTS 265

of the homologous antivirus, the organisms underwent active phagocy-
tosis, this action being specific. The antivirus was nontoxic and could
withstand a temperature of ioo° C. It imparted to certain tissues a local
immunity against the specific bacteria.

The favorable therapeutic results obtained from the use of antivirus
have been confirmed, largely in France, Austria, and Germany. The
antivirus apparently acts not upon the bacterium but upon the tissue of
the host in such a way as to produce local immunization, thus prevent-
ing infection. Nonspecific filtrates may cause an occasional increase of
resistance, but the protection produced by specific filtrates is said to
be more intense and more dependable (689, 775). Antivirus therapy
was believed to offer some promise, although it was said not to give con-
sistent results (385) . Further studies of antivirus led to suggestions that
its favorable effects were due entirely to the culture medium ( 8 ) . The
whole question thus appears to be still debatable, with proponents and
opponents of the specific nature of the antivirus effect (119, 627).

The application of bacteriotherapy for the treatment of chronic infec-
tions of the middle ear (739) and actinomycosis in man has also been
suggested. Filtrates of E. typhosa and of E. coU were found (844) to
check the growth of the typhoid organism, whereas E. coli grew readily
in such filtrates} the more sensitive typhoid bacterium was checked
earlier in its growth than the colon organism. In general, E. tyfhosa
was found to be readily inhibited by the growth of antagonistic bacteria.
Because of this, it was believed that pasteurized milk contaminated with
a pathogenic organism presents a particular danger, since no antago-
nists are present to inhibit the rapid multiplication of the pathogen.
Metchnikov (635) suggested utilization of the antagonistic relations
between lactic acid bacteria and proteolytic bacteria for repressing the
growth of the latter. Thus, pure cultures of the former are introduced
into the food system of man, in order to repress in the intestinal canal
the proteolytic organisms that are supposed to bring about intoxication
in the system. In recent years, L. acidophilus, an inhabitant of the hu-
man intestine possessing antagonistic properties against pathogenic in-
testinal bacteria, has come into general use (778). The problem of com-
bating pathogenic intestinal bacteria by means of nonpathogenic forms
(737) has thus been given wide consideration. The utilization of yeasts

266 DISEASE CONTROL

for combating streptococci and staphylococci may also be classified
among the phenomena of antagonism. On the basis of the rapid destruc-
tion of pathogenic bacteria added to natural water, the storage of drink-
ing water in large reservoirs was recommended as an important safe-
guard against the water's becoming a carrier of bacterial diseases (293-

295).

Clinical methods have been proposed for evaluating the results ob-
tained by treating tooth gangrene by means of antagonists (333). Don-
aldson (190) found that CI. sporo genes or a closely related form had a
marked effect in suppressing the growth of pathogenic organisms in
septic wounds. He believed the antagonistic anaerobe is present in the
majority of gunshot wounds, but that its activities are held in abeyance
by the method of wound-dressing. This antagonist acts by virtue of its
proteolytic enzymes which hydrolyze the dead protein, from which the
pathogenic organisms operate, as well as the toxic degradation products
of other organisms.

Dack (165) reported that CL sforo genes formed in the soil was re-
sponsible for destroying the toxin of CI. botulinum.

ANTIBIOTIC SUBSTANCES AS CHEMO-
THERAPEUTIC AGENTS

Numerous attempts were made, before the advent of tyrothricin
which was soon followed by penicillin, to utilize the products of difFer-
ent organisms for the control of bacterial infections in man and in
animals. Attention has already been called to the use of various pyocya-
nase preparations through nearly half a century, beginning with the
work of Emmerich and Low (235, 236), through the work of Nissle
on mutaflor (680a), and finally the recent studies of Bergstrom and
associates ( 50a) . There was no question of the efficacy of many of these
preparations. Attention has also been called to the work of Vaudremer
(934) and others who attempted to utilize fungus preparations for the
control of bacterial infections. It is only within the last seven years,
that is, with the introduction of tyrothricin, that a new chapter has been
opened in chemotherapy.

Of the numerous antibiotics so far isolated, only a very few have

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 267

found practical application in chemotherapy. Among these, penicil-
lin, streptomycin, and tyrothricin occupy a leading place. In a study of
the relative susceptibility of different staphylococci to the bacteriostatic
action of three different antibiotics it was found (676) that no one agent
parallels necessarily the action of another j a strain resistant to one anti-
biotic may be sensitive to another. Also, different staphylococcus strains
may vary in sensitivity to the same antibiotic, some being inhibited by
0.1 unit of penicillin and others being resistant even to 10 units. Hence
a knowledge not only of species sensitivity but also of strain sensitivity
is essential in selecting an antibiotic for the treatment of a given infec-
tion (310).

In any attempt to evaluate an antibiotic substance as a chemothera-
peutic agent, one must consider the interaction between the drug and
the parasite, on the one hand, and the drug and the host, on the other
(601).

Penicillin

Nature of Action. Since the publication of the first edition of this
book, much work has been done on the in vivo activity of penicillin and
the chemotherapeutic use of this important antibiotic agent. The ap-
pearance of the monographs by Herrell (410), Kolmer (522), and
Fleming (262a) make a detailed discussion of this problem unneces-
sary here J discussion is therefore limited to the fundamental principles
involved.

The antibacterial action of penicillin is selective, susceptibility being,
however, one of degree rather than of kind. Some bacteria are in-
hibited by very low concentrations of the drug} others are not in-
hibited at all or only by high concentrations. On the basis of clinical
experience (494), it has been suggested that organisms inhibited by o.i
O.U. or less per ml. are susceptible to penicillin therapy j some organ-
isms do not respond at all or respond only on occasions. Pyogenic cocci,
anaerobic Clostridia, and certain pathogenic gram-negative cocci {Gono-
coccusy Meningococcus y and Micrococcus catarr kalis') are sensitive,
whereas the colon-typhoid, hemophilic chromogenic bacilli and certain
micrococci {Micrococcus favus) are resistant to its action j it has no
effect upon M, tuberculosis (863), Trypanosoma equiferdumy and the

268 DISEASE CONTROL

influenza virus (789). The purest preparation of penicillin so far avail-
able completely inhibited (275) the growth of S. aureus in a dilution
of between i : 24,000,000 and i : 30,000,000. Partial inhibition was ob-
tained up to 1:1 60,000,000. Salmonella organisms were also sensitive.
Certain strains of Brucella are sensitive to penicillin in vilro, this effect
being enhanced by the presence of small amounts of sodium sulfathia-
zole (918). The sensitivity of most strains of S. hemolytkus is similar
to that of the majority of staphylococci and meningococci j pneumo-
coccus and alpha streptococcus strains fall, in sensitivity, between the
beta streptococci and the meningococci (628). Similar variations in
sensitivity to penicillin of different strains of the same organism have
been observed for various other bacteria, such as C. difhtheriae.

A partial list of the organisms susceptible to penicillin is given in
Table 42.

The antibacterial activity of penicillin is not interfered with by sub-
stances that inhibit sulfonamides, namely, bacterial extracts, pus fluids,
tissue autolysates, peptones, and ^-amino-benzoic acid. It is nontoxic in
concentrations far greater than those required for therapeutic purposes.
It is rapidly excreted through the kidneys and frequent administration
is essential in order to maintain a proper blood concentration.

In its biological properties, penicillin has been found, in general, to
resemble sulfonamide drugs, with certain significant differences (5):

The bacteriostatic power of penicillin against streptococci and staphylo-
cocci is greater than that of sulfonamides, even when the tests are
made under conditions optimum for the action of the latter. Satu-
rated solutions of sulfapyridine and sulfathiazole showed no com-
plete inhibition of bacteria on the assay plate, whereas peniciUin, even
in a dilution of 1 1500,000, gave considerable inhibition.

The action of penicillin on streptococci and staphylococci, unlike that of
the sulfonamides, is influenced very little by the number of bacteria
to be inhibited. Bacterial multiplication can be completely prevented
by as low a concentration of penicillin as 1 : 1,000,000, even if the
inoculum contains several million bacterial cells. In the case of
smaller inocula, inhibition occurs in even higher dilutions. This prop-
erty of penicillin is believed to be of great importance in the treat-
ment of heavily infected wounds, on which the sulfonamide drugs
seem to have little beneficial action.

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270 DISEASE CONTROL

The bacteriostatic power of penicillin against streptococci and staphylo-
cocci is not inhibited to any extent by protein breakdown products or
by pus, which neutralize the bacteriostatic action of sulfonamide
drugs. The leucocytes remain active in any concentration of peni-
cillin usually employed in intravenous injection.

Penicillin is active against strains of bacteria that are resistant to the ac-
tion of sulfonamides. It is effective in the treatment of hemolytic
streptococcus, pneumococcus, and gonococcus infections, which are
resistant to sulfonamides. It has not been found effective, however,
in the treatment of subacute bacterial endocarditis (782).

On repeated passage through broth containing penicillin, pneumo-
coccus cultures as well as Sta-phylococcus sp. and S. fyogenes (604) in-
creased in resistance to penicillin. This was accompanied by a propor-
tional loss of virulence. Small colony variants (G forms) of S. albus
showed a specially high resistance to penicillin (840). Two strains of
pneumococcus developed resistance to penicillin as a result of serial
passage through mice treated with it. The degrees of resistance devel-
oped and acquired varied significantly with the strains. In the case of one
strain, resistance was not impaired by 30 serial passages through nor-
mal mice. The development of resistance in vivo was accompanied by
an increase in resistance to penicillin in vitro. The response of the pneu-
mococci to sulfonamides was not altered by the development of resist-
ance to penicillin. The mechanisms whereby staphylococci become re-
sistant to sulfonamides and to penicillin appear to be distinctly differ-
ent. This increase in resistance may be one of the dangers of using
"homemade" penicillin.

Survival of a certain number of cells of staphylococci in a culture
treated with penicillin may be due to the fact that these cells are tem-
porarily in a nondividing state, since the antibiotic kills the bacteria that
are about to divide. Such cells were designated "persisters" (57) } their
descendants are easily killed by the antibiotic. This concept led to the
recommendation of intermittent treatment by penicillin: treatment to
be interrupted to permit the bacteria to multiply and thus become again
vulnerable. This concept has not been universally accepted (321), some
investigators actually warning against too early interruption of peni-

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 271

cillin therapy. Among the gonococci, no naturally resistant strains have
been encountered (544).

Toxicity. As to the toxicity of penicillin, it was found (5) that mice
were little affected by the intravenous injection of lO mg. of penicillin}
they became ill from the use of 20 mg. but recovered shortly. One hun-
dred milligrams of crude penicillin given intravenously to man caused
a shivering attack with a rise of temperature in about an hour. The lat-
ter was due to the presence of a pyrogenic substance in the preparation.
Certain isolated fractions of penicillin had no such pyrogenic effect.
Penicillin was toxic to mice when given intravenously in single doses
of 0.5, i.o, 1.5, and 2.0 gm. per kilogram. More highly purified prepa-
rations were less toxic. Higher concentrations were required for lethal
effect from subcutaneous administration. The toxic dose is 64 times
greater than the effective dose (789).

The relative toxicity of various salts of penicillin was found (997) to
be, in increasing order, Na, Li, NH4, Sr, Ca, Mg, and K. Based on mil-
ligrams of the cation at the LD50 dose of salts of penicillin, the relative
toxicity was Na, Sr, NH4, Ca, K, and Mg. It was concluded that the
toxicity of the salts of penicillin is primarily due to the cations used in
their preparation.

Penicillin is rapidly absorbed and is excreted in the urine, usually
within one hour (755). It does not appear to undergo any change in
passing through the animal body. This fact was taken advantage of, in
the early days when there was a shortage of penicillin, by recovering it
from the urine. An average yield of 30 per cent of the amount adminis-
tered was obtained (872).

The degree of the antibacterial action of penicillin is proportional
to its concentration in the serum, maximum effects against hemolytic
streptococci being produced by concentrations of 0.019-0.1 56 Oxford
units in i ml. of serum. The LD50 for an 18-gram mouse was 32 mg.
of the sodium salt (437). The cardinal symptoms of toxicity were
choking, gasping, and rapid respiration. However, it is relatively non-
toxic in doses used for therapeutic purposes.

Penicillin was thus found to combine the two most desirable quali-
ties of a chemotherapeutic agent, namely, a low toxicity to tissue cells

272 DISEASE CONTROL

and a highly bacteriostatic action against some of the most common and
destructive bacteria with which man may become infected. It was pos-
sible to maintain a bacteriostatic concentration of penicillin in the blood
without causing any toxic symptoms.

Animal Experiments. In animal experiments it was established
that penicillin is an effective chemotherapeutic agent against pneumo-
cocci, including sulfonamide-resistant types. It is superior, in staphylo-
coccus infections, to bacteriophage, sulfa drugs, and specific antitoxins
(468). In experiments with S. aureus, a survival ratio of 2: i was ob-
tained in favor of penicillin as compared with sulfathiazole, correction
being made for the survival of control mice. Penicillin, when adminis-
tered subcutaneously, intravenously, or intraperitoneally, was also
found to be effective against hemolytic streptococci. Generalized staphy-
lococcal infections were cured by penicillin and local lesions healed
during parenteral administration. The best method for administering
penicillin is by the intramuscular route at 3-hour intervals j the blood
should contain enough penicillin (15,000 O.U. dose) to inhibit the
growth of the infecting agent. Intraocular infection caused by D. fneu-
fnontae was checked by local treatment with penicillin in solutions of
0.25 and 0.1 per centj the application was continued for 2 to 4 days

(817).

Since penicillin readily loses its activity in an acid solution, it is used
in the form of the sodium salt. Rabbits excreted in the urine as much as
50 per cent of the penicillin after intravenous injection, but less than
20 per cent after administration into the intestine j some excretion took
place in the bile. The penicillin could not be detected in the blood
within one-half hour after administration. Cats differed in this respect
from rabbits, since they maintained an antibacterial concentration of
penicillin in the blood for at least 1.5 hours after subcutaneous or intra-
venous injection, and for at least 3 hours after intestinal administration.
They differed also in excreting about 50 per cent of the penicillin in the
urine, even when the substance was injected into the intestine. In this
respect man appeared to resemble cats more closely than rabbits.

A comparison of antibiotic agents against the anaerobes causing gas
gangrene placed tyrothricin in first place, followed successively by peni-
cillin, the sulfa drugs, and other antibiotic agents; however, in vivo

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 273

treatment of mice infected intramuscularly with CI. ferjringens placed
penicillin first, with tyrothricin and aspergillic acid at the bottom of the
list. Penicillin also proved superior to sulfonamides and amino acri-
dines in experimental infection with CI. welch'n and CI. oedemat'tens
(603).

The in vivo activity of penicillin against CI. sefticum and other
anaerobes, as well as many other bacterial pathogens, is brought out in
Table 43. A single subcutaneous treatment of mice with 50 units of
penicillin at the time of intramuscular inoculation with CI. welchii
protected 98 per cent of the infected animals, and repeated small doses
gave as good protection as a single large dose. Delay in the institution
of therapy lowered the survival rate, but not appreciably unless the de-
lay was over 3 hours. Local lesions were completely healed within 3
weeks if penicillin was injected repeatedly into the site of infection.

The effectiveness of penicillin has also been tested against various
other infections in experimental animals, with varying degrees of suc-
cess. It was found, for example, that the administration to mice of peni-
cillin in relatively large doses after injection with murine typhus rick-
ettsiae resulted in marked reduction in mortality, particularly when the
initial dosage of the rickettsiae was relatively small (654). Its favorable
effect on infections due to the ornithosis virus was also indicated (400).
It is also effective in the treatment of leptospirosis in experimental ani-
mals (17,547)-

It has been brought out in recent studies that the effects of different
forms of penicillin against the same bacteria are different in the animal
body and in the test tube. Penicillin K gave one-quarter to one-eleventh
in the blood (injected 0.6 mg./kg.) and persisted in demonstrable
levels for only a short time, as compared to F, G, and X. The recovery
of K in the urine was 30 to 35 per cent, as compared to 74 to 91 per
cent in the case of the other forms. In the treatment of experimental in-
fections, K was one-sixth to one-eleventh as active as G, and one-eighth
to one-thirteenth as active as X. These data point to the more rapid in-
activation of penicillin K in the body, resulting in a lower therapeutic
activity (224).

Although the evidence concerning the effectiveness of different
forms of penicillin is still very limited, the conclusions were reached

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ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 275

(147a) that (a) penicillin K is very unstable in the body, so that its
parenteral effectiveness is open to question j (b) penicillins G and X are
stable but they are rapidly excreted, which is a limiting factor in main-
taining a therapeutic level j (c) penicillin X is more active against
certain hemolytic streptococci, where its specific use is indicated.

Chemotherapeutic Action. Penicillin has also found an important
place in the treatment of local and generalized infections in man. Flem-
ing was the first (261, 262) to recommend that it be employed for
dressing septic wounds. It appeared to be superior to dressings contain-
ing purely chemical agents. Isolated penicillin in a dry state was many
times more powerful than the most potent of the sulfonamide com-
pounds. Local applications include those to lesions of the eye produced
by S. aureus, in which early treatment resulted in the elimination of the
organism from the flora of the conjunctival sac (797). Weight for
weight, penicillin was found to be four times as potent as sulfathiazole
and 100 times as potent as sulfanilamide for the treatment of wound
infections (5, 269).

Penicillin proved to be an especially effective agent for the treatment
of staphylococcal and hemolytic streptococcal infections in man (755),
including streptococcal meningitis and others. Many cases of infected
war wounds treated with penicillin gave, in 24 hours, a uniform drop
in the number of gram-positive organisms, including Clostridia, staphy-
lococci, streptococci, and corynebacteria ; the gram-negative bacteria
were not affected. Excellent therapeutic effects were also obtained in
surgical infections. Even crude culture filtrates of P. notatum, applied
locally, gave good results. It can be used in topical application and has
proved to be an ideal agent for the treatment of genito-urinary infec-
tions. It is readily absorbed and excreted following intravenous, sub-
cutaneous, and oral administration j a part of it is inactivated in the
body.

In order to lessen the frequency of effective invasion of the nose by
bacteria and the subsequent infection of the nasopharynx, the local use
of penicillin has been suggested. The material acts as a prophylactic
against bacterial infections of the upper respiratory tract j the course of
a cold can thus be checked by preventing secondary bacterial infection.
The curing of nasal carriers of staphylococci or even the reduction of

276 DISEASE CONTROL

the number of vegetative organisms was considered important in order
to reduce the danger of the carrier as a source of infection to others. It
has found application in oral infections, in acute infections of the ear,
nose, eyes, in bone infections, meningitis, human anthrax, subacute
bacterial endocarditis, and many other infections, such as rat-bite fever.
In the case of some infections, such as those caused by CI. welchii, peni-
cillin must be used in conjunction with good surgical principles. Peni-
cillin can also find application in certain chronic cases, as in the treat-
ment of chronic dermatitis and in preparing infected surfaces of hands
for skin-grafting and infected stumps for amputation.

Penicillin is an effective agent in the treatment of clinical infections
due to sulfonamide-resistant bacteria. Several strains of A^. gonorrhoeae
isolated from patients in whom the infection was resistant to treatment
with sulfonamide preparations were found to be inhibited completely by
penicillin. The number of organisms decreased greatly at the end of i
or 2 hours' contact with the substance, and no viable organisms were
found after 3 to 4 hours' contact. The complete absence of toxicity fol-
lowing the intravenous administration of penicillin, the lack of discom-
fort to the patient, and the rapid disappearance of clinical symptoms
were observed in cases of sulfonamide-resistant gonorrheal infections.
In all the cases reported, in addition to the clinical response noted, nega-
tive bacterial cultures were obtained some time between 1 7 and 48 hours
after the institution of penicillin therapy. Sulfonamide-resistant gonor-
rhea cases responded to injections of 100,000 to 160,000 O.U. Favor-
able responses have also been obtained in the treatment of sulfonamide-
resistant strains causing staphylococcal pneumonia and empyema and
other diseases. The susceptibility of various bacteria to penicillin can be
determined by means of a very simple technique.

A favorable therapeutic response was obtained by administering peni-
cillin intravenously to patients with staphylococcal infections and by
mouth to a baby with a persistent staphylococcal urinary infection. In
patients suffering from meningitis, penicillin was found to be absorbed
more rapidly than in normal persons, and a larger part of the dose was
excreted in the urine.

Penicillin has not been found to be effective in trypanosome infec-

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 277

tions, but has been used successfully in the treatment of relapsnig fever,
although excessive doses were required (223).

Treatment of early syphilis cases with penicillin (614) indicated that
the therapy was responsible for the rapid and complete disappearance of
the infecting agent from the blood stream, as determined by various
tests. Penicillin was found to be actively spirocheticidal (225). A com-
parative study has been made of the action of penicillin and of other
antibiotic agents upon Tre-ponema pallidum (217). The immobilization
of spirochetes in vitro by penicillin preparations was found to be due to
the impurities present (218). The amount of penicillin required to
produce bacteriostatic titers in rabbits infected with Tr. 'pallidum is five
times greater than that needed for adult man by intramuscular injec-
tion. Although highly favorable effects were obtained in the treatment
of various types of syphilis in man, the need for more careful observa-
tions was indicated.

Penicillin was also found to have an effect upon experimental typhus
rickettsiae (361, 654), bovine mastitis (857), and a variety of other
infections.

As a result of treatment with penicillin of 300 patients, it has been
concluded (782) that this material is far superior to any of the sul-
fonamides in the treatment of S>. aureus infections with and without
bacteriemia, including acute and chronic osteomyelitis, cellulitis, car-
buncles of the lip and face, pneumonia and empyema, infected wounds
and burns.

A study of 500 cases of infections treated with penicillin led to the
following conclusions (494, 588) : Penicillin can be administered intra-
venously, intramuscularly, or topically, but is ineffective when given by
mouth. As it is excreted rapidly in the urine, it must be injected continu-
ously or at intervals of 3 to 4 hours. Penicillin was found to be particu-
larly effective in the treatment of staphylococcic, gonococcic, pneumo-
coccic, and hemolytic streptococcus infections, especially sulfonamide-
resistant gonococcic infections, but not bacterial endocarditis. The usual
patient requires a total of 500,000 to i ,000,000 Oxford units, the best
results being obtained when treatment is continued for 10 to 14 days,
10,000 units to be given every 2 to 3 hours at the beginning of treat-

278 DISEASE CONTROL

ment, either by continuous intravenous injection or by interrupted in-
travenous or intramuscular injections. Good results were obtained by
injections of 100,000 to i6o,ooo units over a period of 2 to 3 days. In
the treatment of empyema or meningitis it was found advisable to use
penicillin topically by injecting it directly into the pleural cavity or the
subarachnoid space. Toxic effects were extremely rare. Occasional chills
with fever or headache and flushing of the face were noted.

A summary of the response of different bacteria in septic gunshot
fractures is given in Table 44. Staphylococci and streptococci are rapidly
responsive to penicillin therapy. Anaerobic cellulitis due to the proteo-
lytic bacteria of putrid wound infection responds to penicillin, but the
bacteria may persist in the presence of devitalized tissue or wound
exudates. The pyocyaneus organism is not susceptible to penicillin, but
it is considered to be relatively unimportant as a single pathogen in the
surgical management of the wounds (269).

A summary of the results of extensive use of penicillin in the North
African campaign of World War II led to the conclusion that in the
treatment of recent soft-tissue wounds penicillin brought about the vir-
tual elimination of infection and saved much hospitalization time.
Treatment of fractures also gave good results, though some penicillin-
resistant cocci appeared. Favorable results were also obtained in various

TABLE 44. RESPONSE OF DIFFERENT BACTERIA FOUND IN WOUNDS
TO PENICILLIN TREATMENT

PENICILLIN

RESPONSE

TYPE OF INFECTION

Systemic

Local

Putrid:

Proteolytic Clostridia

+ (large dosage)

+

Proteus vulgaris

0

0

Nonhemolytic streptococci:

Mesophilic

+

+

Thermophilic (5. faecalis)

0

0 (or slight)

Staphylococci

+ (3-5 days)

+ (often necessary)

Hemolytic streptococci

+ (1-3 days)

+ (not essential)

Pseudomonas aeruginosa

0

0

From Lyons (587).

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 279

Other infections. It is suggested that an average of 750,000 units of
sodium penicillin be allowed for systemic treatment and 50,000 units
of the calcium salt for local treatment (275, 319).

Penicillin is thus found to form a valuable addition to the growing
list of chemotherapeutic agents, to help man combat disease-producing
bacteria. It is commonly used not as a pure acid but as either a calcium
or a sodium salt, the former for local applications and the latter for
intramuscular or intravenous treatments (275, 494). Since penicillin
solutions are quite unstable, especially in the form of salts, the dry
preparations are stored and are dissolved either in water or in saline
just before required for use. Certain of the esters (e-butyl) of penicillin
which are inactive in vitro can, when given by the oral route, become
highly active against hemolytic streptococci (639, 640).

In some cases, penicillin failures have been experienced 5 these may
be due to too brief treatment or too small doses, to need for surgical
drainage, or to other complications. Toxic effects may also be produced
due to the penicillin itself or to some of the accompanying impurities.
Sensitivity is often in the nature of allergy ( 159, 592). Further details
on the toxicity and in vivo activity of penicillin are reported by Herrell
(410).

In place of penicillin preparations, crude culture filtrates are occa-
sionally used. Such crude preparations have been designated as vivi-
cillin, hypholin, etc. (500).

This is not the place to discuss in detail the pharmacology and chemo-
therapy of penicillin. An extensive literature has already accumulated
on this subject. Reference is here made to the detailed summaries (274,
495) and books recently published, where indications for penicillin are
listed under infections due to staphylococci, Clostridia, hemolytic strep-
tococci, anaerobic streptococci, pneumococci, gonococci, anthrax, chronic
pulmonary suppuration, meningococci, bacterial endocarditis, murine
erysipelas, Vincent's syphilis, actinomycosis, diphtheria, and certain
others. It is not effective in mixed infections where a gram-negative
flora predominates, in urinary infections due to E. coli and other gram-
negative bacteria, in tuberculosis, in other diseases caused by gram-
negative bacteria, viruses, or pathogenic fungi, in malaria, or in a
variety of other diseases, such as cancer. The manner and extent of ad-

280 DISEASE CONTROL

ministration and amounts required depend largely upon the suscepti-
bility of the infecting strain of the organism.

Clavacin (Claviformin, PatuUn)

The treatment of common colds that were prevalent in an English
naval establishment by the use of clavacin in the form of nasal sprays or
snuffed up by hand gave 57 per cent complete recovery in 48 hours, as
compared with 9.4 per cent for the controls j no ill effects were observed
(744). These results were not confirmed, however, the conclusion hav-
ing been reached that, compared with the natural evolution of the dis-
ease, clavacin has no demonstrable effect on the course of a cold (876).
This substance also proved to be unsatisfactory for the treatment of bo-
vine mastitis by udder infusion (718). In general, it is too toxic to be of
therapeutic usej it acts as a tissue poison and has other undesirable
pharmacological effects (90). To what extent the antifungal action of
clavacin can be utilized for controlling fungus infections remains to be
determined, since it is known to inhibit the growth of Trichophyton
gyfseum in concentrations of 0.02 per cent and in certain cases even
0.0 1 per cent. It had only a slight effect upon other fungi in 0.04 per
cent concentration (416).

Tyrothricin

Dubos (203) reported that 0.002 mg. of gramicidin, one of the two
chemical constituents of tyrothricin, when injected intraperitoneally
into white mice, exerted a therapeutic action against experimental peri-
tonitis caused by pneumococci and streptococci (Table 45). This sub-
stance was found to be effective against five different types of pneumo-
cocci, eleven types of group A streptococci, and three strains of group C
streptococci. It was, however, almost completely ineffective when ad-
ministered into animal tissues by the intravenous, intramuscular, or
subcutaneous route, because of its lack of activity under these conditions,

Tyrothricin exerted a lethal action in vitro on 1 8-hour broth cultures
of S. hemolyticus, S. aureus, and C. diphtherias, in a final dilution of
1 : 1,000,000; freshly isolated strains of meningococcus were affected in
a dilution of i:i00,000 (841). Two monkeys which carried in the
nasopharynx and throat gram-positive hemolytic streptococci and gram-

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 281

TABLE 45. BACTERICIDAL EFFECT OF TYROTHRICIN UPON
DIFFERENT BACTERIA

TYROTHRICIN IN MILLIGRAMS PER MILLILITER OF CULTURE

0.040

0.020

0.0 10

0.004

0.002

O.OOI

0.0

DiPLOCOCCUS PNEUMONIAE,

Type I

Viability*

-

-

-

-

+

++++

Reductasef NR

NR

NR

NR

NR

NR

CR

Lysis§ C

C

C

c

C

P

N

Streptococcus hemolyticus, Group A, Type 6

Viability*

-

-

-

-

+++

-l-K-f

Reductasef NR

NR

NR

NR

NR

PR

CR

Lysis§ N

N

N

N

N

N

N

Staphylococcus aureus

Viability*

-

-

1 1 1 1

1 1 1 1

■f+-H-

1 1 1 !■

Reductasef NR

NR

NR

CR

CR

CR

CR

Lysis§ C

c

N

N

N

N

N

From Dubos and Cattaneo (208).

•—no growth on blood agar, + reduced growth, I I I I abundant growth.

t NR no reduction of methylene blue, PR partial reduction, CR complete reduction.

§ N no lysis, P partial lysis, C complete lysis.

negative hemolytic bacilli showed disappearance of these bacteria within
2 hours following the administration of tyrothricin. Five days after a
single treatment no hemolytic organisms were found in one monkey,
and, in the other, only throat cultures were positive. A second applica-
tion of the material gave completely negative cultures within 3 hours.
No local or general reactions to these treatments were observed. This
material was also administered to 5 human carriers of hemolytic strepto-
cocci, 2 of whom were persistent nasal carriers for two months following
scarlet fever and the other three convalescent in the third week of this
disease. In only one case was an immediate reduction in the number of
streptococci obtained 5 a striking reduction or complete disappearance of
the organisms occurred in the others on the fifth day, after 3 to 4 spray-
ings. These observations were said to be sufficiently encouraging to jus-
tify the use of the material against carriers harboring streptococci, diph-
theria organisms, meningococci, and pneumococci. Injection of 3 to 40
mg. t3^rothricin into the pleural cavity of rabbits with hemolytic strepto-
coccal empyema brought about the sterilization of the pleural cavity and

282 DISEASE CONTROL

enabled the animals to survive. The injection of lo mg. of tyrothricin
into the pleural cavity of normal rabbits produced certain local tissue re-
actions. More than lO mg. produced adhesions, thickening of the
pleura, sterile abscesses, and other disturbances (753).

The susceptibility of fecal streptococci to tyrothricin varies from
strain to strain. Oral administration of the substance may produce in-
hibition of the growth of streptococci in the intestines of mice. This
inhibition was most readily demonstrated when sulfasuxidine was ad-
ministered together with the tyrothricin (799). Application of tyrothri-
cin to ulcers brought about sterilization and healing of local infections.
Application to the mastoid cavity following mastoidectomy also gave
favorable results. In staphylococcic infections, resistant strains may de-
velop during therapy. Certain sulfonamide-resistant strains of S.
pyogenes were eradicated by application of gramicidin (291).

Tyrothricin and tyrocidine exert a bactericidal effect, and gramicidin
is largely bacteriostatic (Figure 28) j the first two are affected by blood
and serum, but not the last. In order to be effective against bacteria,
the organisms must be in contact with the material (790). Gramicidin
is more toxic than tyrocidine, the toxic dose being larger, however, than
the dose necessary to kill most gram-positive cocci.

Both tyrothricin and tyrocidine cause hemolysis of erythrocytes, and
both are leucocytolytic, gramicidin being less so. Both tyrothricin and
gramicidin cause local and general toxic effects when injected into closed
cavities of the body. Small amounts may bring about the sterilization of
local infections without producing general toxic effects, giving only
minimal local reactions. When injected into the skin, tyrothricin and
gramicidin produce local reaction, the latter to a lesser degree. Oral ad-
ministration is ineffective in reducing or destroying organisms which are
susceptible in vitro. Local application of these substances has not been
attended by toxic reactions even when large amounts were applied
(758). Tyrothricin in high concentrations caused cytoplasmic and nu-
clear disintegration of the exudative rabbit polymorphonuclear leuco-
cytes} in lower concentrations, it brought about altered staining reac-
tions. When there was no apparent microscopic injury to the cells,
phagocytosis of pneumococci took place. The presence of serum brought

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284 DISEASE CONTROL

about some protection of the cells from the effects of these substances

(144).

When given intravenously in a 9.5 per cent alcoholic solution, tyro-
thricin was found (896) to be effective in the treatment of chickens in-
fected with Plasmodium gallinaceum'. It has a marked parasiticidal
effect, especially on the extracellular merozoites produced at segmenta-
tion. Prolonged daily treatment of 100 gm. chickens with 0.2 mg. of
material from the beginning of infection lengthens the incubation
period of blood-induced infections, lowers the peak of acute infection,
and prevents fatal relapses. When given in 12 daily doses, it has a
quinine equivalent of about 4, the effective dose being, however, near
the toxic level. Whereas tyrothricin is parasiticidal, quinine is parasiti-
static, the first being also more effective in inhibiting oxygen consump-
tion of the parasites.

Tyrothricin is limited, because of its toxicity, to topical applications j
for that purpose, it has advantages over certain other antibacterial
agents, such as the sulfonamides and penicillin, because of its stability,
its wider range of antibacterial action, its activity in presence of blood
and serum, its low tissue toxicity, and its lack of sensitizing properties
(403a). It is ineffective when incorporated into common ointment
bases (19).

Particularly favorable results were obtained from the use of grami-
cidin in the treatment of chronic mastitis (576). Several cows received
treatment with increasing amounts of gramicidin diluted with distilled
water. Following the morning milking, the residual milk in the cistern
and in the teat was flushed out with 100 to 200 ml. of the gramicidin so-
lution, containing 60 to 240 mg. in 1,000 ml. water j 800 to 900 ml. were
then injected under pressure into the quarter and allowed to remain
until the next milking. Within one hour after the injection, the treated
quarter became distended and rectal temperature began to increase,
reaching 41° C. at the fifth or sixth hour. The temperature returned
to nearly normal in about 3 hours thereafter, the acute swelling having
subsided at the next milking. The streptococci disappeared from most
of the quarters treated, without an appreciable decrease in milk pro-
duction. The fact that streptococci had been eliminated was established

ANTIBIOTICS AS CHEMOTFIERAPEUTIC AGENTS 285

by daily bacteriological examination of the milk over periods ranging
from 1 5 to 8 1 days.

Sterile mineral oil was later found (577) to be a suitable, nonir-
ritating medium for the administration of the gramicidin, though
some of the cows thus treated gave severe reactions. An alcoholic solu-
tion (2 to 3 ml.) of 80 to 120 mg. gramicidin was emulsified in 15 ml.
sterile distilled water, and the emulsion mixed with 25 ml. of heavy
mineral oil. The mixture was injected into the cistern shortly after the
morning milking and allowed to remain until the evening milking. The
treatment was repeated for several days in succession. Of 31 quarters
naturally infected with Streptococcus agalactiae and treated by the
gramicidin-oil mixture, 16 seemed to have responded by a complete dis-
appearance of the streptococci. The infection in some of the cases was of
severe chronic nature.

Less satisfactory results were obtained in the treatment of bovine
mastitis caused by Streftococcus uher'is; of 4 cases treated, only one re-
sponded satisfactorily. The final recommendation was the daily use for
four consecutive days of 20 to 40 mg. gramicidin in oil. These results
were confirmed by various investigators, who reported 60 to 90 per
cent cure after two or three treatments (622).

A comparison of tyrothricin, trypaflavin, and novoxil for eradicating
5. agalactiae from infected udders showed best results for the first. In-
fections with S. uberis and Streftococcus dysgalactiae also responded
well, but not staphylococcic infections (829). The efficacy of the treat-
ment is influenced by several factors, namely, (a) site of chronic infec-
tion, (b) selection of suitable cases, and (c) stage of lactation. In some
experiments, as many as 90 per cent of the cases were cleared up with
tyrothricin (96).

Gramicidin-like preparations were also used successfully in the treat-
ment of local infections in man. The active material must be used lo-
cally on infected cavities which do not communicate with the blood
stream (412), Tyrothricin has also been used (160) successfully in the
treatment of acute otitis media, acute and chronic mastoiditis, and acute
and chronic sinusitis. The substance does not damage the tissue or in-
terfere with wound healing. The growth of most staphylococci, strepto-

286 DISEASE CONTROL

cocci, and pneumococci is inhibited or the organisms are killed. When
applied locally, tyrothricin does not reach the blood stream. It has also
been found that tyrothricin has an excellent therapeutic effect when
used for urethral irrigations in the male ( 198). In a recent summary, it
was reported (528) that tyrothricin can be used successfully as a wet
dressing in surgical infections j it was most effective in hemolytic strep-
tococcal lesions and next in S. aureus and S. alhus lesions. It actually
encouraged the growth of Ps. aeruginosa and C difhtheriae.

Tyrothricin possesses several limitations, from the point of view of
practical utilization: (a) development of bacterial, notably staphylococ-
cus, variants, which become resistant to this agent j (b) inhibition of its
action by phospholipinsj (c) hemolytic action, which prevents its intra-
venous use.

The filtrate of B. mesentericus was found (984) to have a specific bac-
tericidal action on C. difhtheriae in a dilution of 1:1,250. When in-
jected parenterally into guinea pigs, it inhibited the toxic effect of the
diphtheria organism. The filtrate of the antagonist was found useful in
the treatment of diphtheria carriers.

Gramicidin S appears to be quite different from tyrothricin, since it
crystallizes readily when an alcoholic extract of the acid precipitate of
the bacterial culture is allowed to evaporate. It is four times more ef-
ficient in the killing of staphylococci than tyrothricin, although both
have similar toxicity. It is effective against various gram-negative bac-
teria, such as E. coli and P. vulgaris. Aqueous solutions (0.4 to 0.8 mg.
per ml.) were applied daily for 4 to 5 days to gunshot wounds of soft
tissues at the stage of necrosis, to severe burns, abscesses of the abdomi-
nal wall, and anaerobic infections j this was followed by gramicidin
ointments every 2 to 3 days, with highly favorable results. There was
no loss in potency in the presence of pus, no tissue irritation, with a
favorable effect on regeneration and epithelization (324, 589, 850).
The possible relation of the last phenomenon to cell malignancy has
been suggested (546).

Subtilin and Bacitracin

The toxicity index of subtilin has been defined (816) as the ratio of
the highest dilution of this antibiotic that kills embryonic chick heart

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 287

tissue after an exposure period of lO minutes at 37° C. to the highest
dilution that kills S. aureus under the same conditions. A unit of sub-
tilin has been defined as that amount present in i ml. of the highest di-
lution (expressed in mg.) capable of killing S. aureus in 10 minutes at
37° C.

It is relatively nontoxic when tested by the tissue culture method j it
is about 20 times more toxic to S. aureus than to chick heart tissue. It
exerted a suppressive effect upon experimental tuberculosis in guinea
pigs, and protected animals against infection with pneumococcus Type
III and B. anthracis.

Bacitracin appears to have chemotherapeutic potentialities.

Pyocyaneus Prefarations

Although, as pointed out previously, pyocyanase, the first antibiotic
to be isolated, came into disrepute as a potential chemotherapeutic
agent, various preparations of Ps. aeruginosa have continued to receive
attention. One must differentiate among the lipoidal products and
other antibiotics isolated either from the culture medium or from the
bacterial cells of this organism (50a). The lipoid products obtained by
chloroform extract of bacterial cells were found to be active against
various gram-positive bacteria including M. tuberculosis in concentra-
tions of 1:500 (15 minutes) to 1:500,000 (5 hours) j it was effective
not only in vitro but also in vivo, a-oxy-phenazine obtained in a crystal-
line state was active against various gram-positive and gram-negative
bacteria. It was effective against bovine mastitis and Br. abortus in
guinea pigs (1053).

Streftothricin and Streftomycin

Among the antibiotic substances that are active against gram-negative
bacteria both in vitro and in vivo, streptothricin and streptomycin oc-
cupy a prominent place. Their toxicity is low enough to make possible
the administration of doses sufficient to destroy the pathogens in the liv-
ing tissues (792, 794). In the very first experiments, using chicken egg
embryos, mice, and other experimental animals, they gave favorable
results in the treatment of Br. abortus, bacillary dysentery, typhoid
fever, and food poisoning produced by Salmonella organisms and

288 DISEASE CONTROL

other pathogens. Mycobacteria are readily inhibited by both strepto-
thricin (1029) and streptomycin (232, 831). Different strains of M.
tuberculosis vary greatly in their sensitivity to the same substance. The
same is true of the sensitivity to streptomycin of different strains of
Erysifelothrix and various saprophytic and parasitic actinomycetes.

When given parenterally, streptomycin, injected daily in doses of
50,000 units (50 mg. of pure base) per kg, for one month, was well tol-
erated by mice, rats, and monkeys. Injected subcutaneously, 100 \\g
gave protection to mice against 10,000 lethal doses of the pathogen.
Feces of mice fed streptomycin was sterile as far as lactose-fermenting
bacteria were concerned j the total population was considerably reduced.
Seventy per cent of the streptomycin was excreted in the urine in 5 to 6
hours. In order to maintain proper blood concentration it has to be
administered, therefore, by frequent intramuscular injections or by
continuous intravenous drip (746, 791, 795).

Streptomycin was found to be effective in the treatment of a variety
of experimental infections caused by various gram-negative bacteria,
including Br. abortus (478), Sh. galUnarufn (478), S. schottmulleri
i795)y P- tularensis (397), the Friedlander bacillus or Klebsiella
(398), Z). pneumoniae (y<^5), M. tuberculosis (249), and a number of
others, especially the organisms commonly found in urinary infections
(401). In a comparative study of several human strains of M. tuber-
culosis, the bacteriostatic concentration of streptomycin was shown to
vary from 0.095 to 0.78 Mg per ml.j the effect was not influenced
greatly by either the number of organisms or the presence of human
plasma (Table 46). The bactericidal action of streptomycin upon
human tubercle bacilli was slight as compared with its bacteriostatic
action, nearly lOO pg/ml. being required to kill o.i mg. of virulent
bacterial cells (1039).

In the treatment of mice infected with the tularemia organism
(Table 47), the controls died within 96 hours after inoculation, those
receiving 1,000 units or i mg. of pure streptomycin daily, for 10 days,
survived J smaller amounts of streptomycin gave incomplete protection
(397). Infections caused by various other organisms, such as Borrelia
novyi and Leftosfira icterohaemorrhagiae (Table 48), can also be
treated with streptomycin (399). Streptomycin is not effective in the

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 289

treatment of avian malaria (848), and has little activity upon fungi and
anaerobic bacteria.

TABLE 46. EFFECT OF NUMBER OF ORGANISMS AND PRESENCE OF PLASMA

ON THE BACTERIOSTATIC ACTION OF STREPTOMYCIN ON

M, TUBERCULOSIS VAR. H.37

INOCULUM

CONCENTRATION OF STREPTOMYCIN

, MCG.

PER ML.

1.56

0.78

0.39

0.19

0.095

Without Human

Plasma

0.1 mg.

0

0

0

s

M

0

0

S

s

M

0.5 mg.

0

0

S

M

M

0

0

s

M

M

i.omg.

0

0

M

M

M

0

0

M

M

M

Ten Per Cent H

UMAN

Plasma

Added

0.1 mg.

0

0

0

M

M

0

0

0

M

M

0.5 mg.

0

0

G

G

G

0

0

G

G

G

i.omg.

0

0

G

G

G

0

0

G

G

G

From Youmans (1039).

Note. O indicates no growtii ; S, slight growth; M, moderate growth; G, profuse growth.

TABLE 47. EFFECT OF STREPTOMYCIN ON MICE INOCULATED WITH
PASTEURELLA TULARENSIS

NUMBER

MORTALITY

OF MICE

NUMBER OF Ml

[CE THAT

RATE PER

DOSAGE

TREATED

DIED AFTER INOCULATION

100

3

days

4

days

5
days

8-23

days Total

1 000 /Ag per day

30

0

0

0

0 0

0

500 /Ag per day

None

12

30

0
13

0

17

4

0

I 5*
0 30

42
100

From Heilman (397).

* Two other mice died of pneumonia on the twenty-sixth and thirty-second days.

290 DISEASE CONTROL

Some very striking results have been obtained in the treatment o£
experimental tuberculosis in guinea pigs (Table 49). On the basis of an
arbitrarily established index of infection, microscopically determined,
100 represented the maximum possible amount of tuberculosis. The
control animals, sacrificed after 61 days, exhibited an index of 67 as
contrasted to 5.8 for those which had received streptomycin. In another
experiment, the corresponding values were 81.9 for the untreated and
2.8 for the treated animals. The daily administration of streptomycin
per pig varied from 1,387 to 6,000 mg. Two different strains of the
human tubercle bacillus were equally sensitive to streptomycin in vivo
(250).

Streptomycin was pronounced to be "the most effective in vivo
tuberculochemotherapeutic agent which we have studied and which we
have seen reported in the literature." It has been repeatedly shown
that antibacterial agents active against M. tuberculosis in vitro may be
of little value in combating the infection in vivo; since experimental
tuberculosis in guinea pigs may be quite distinct from that produced by
the same organism in man, the application of results obtained in ani-
mals to its possible effect in humans is largely speculative.

The ability of streptomycin to check and retard the normal course of
tuberculosis infection in guinea pigs has been confirmed. By combining
streptomycin and promin, it was possible to obtain results unlike any-
thing heretofore obtained in the treatment of experimental tuberculosis
infections (862). The combination was also found (1040) to have a
marked suppressive effect on experimental pulmonary tuberculosis in
mice.

Both the toxicological and pharmacological properties of strepto-
mycin readily allow its use in the treatment of diseases in man. Pure
streptomycin, as well as many impure concentrated preparations, have
been given at therapeutic levels, in doses as high as 4 gm. daily, with-
out any unfavorable reactions.

Blood levels following intravenous injections can be better main-
tained with streptomycin than with penicillin, detectable amounts of
the first being present for 6 hours compared with 2.5 to 3 hours for the
second. Following parenteral administration, most of the streptomycin
is excreted in the urine. It is distributed throughout the body fluids:

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS

291

blood, urine, ascitic fluid, pleural fluid, aqueous humor, vitreous
humor, amniotic fluid, and bile. Small amounts of the drug appear in
the spinal fluid in healthy individuals, but in a single case of Hemoph-
ilus in-jiuenzae meningitis the spinal fluid contained 25 ng per cc.

TABLE 48. COMPARISON OF THE EFFECT OF STREPTOMYCIN AND

PENICILLIN ON INFECTION WITH LEPTOSPIRA

ICTEROHAEMORRHAGIAE IN HAMSTERS

NUMBER OF

MORTALITY

HAMSTERS

NUMBER OF HAMSTERS THAT

RATE PER

DOSAGE*

TREATED

DIED AFTER INOCULATION

100

250 units per day:
Streptomycin
Penicillin

6

6

7-14

days

5
0

15-22

days

6

Total

6
6

100
100

500 units per day:
Streptomycin
Penicillin

6
6

4
0

0
0

4
0

67
0

800 units per day:

Streptomycin
Penicillin

6
6

0
0

0
0

0
0

0
0

None

12

12

0

12

100

From Heilman (399).

•Streptomycin and penicillin are measured by totally different systems: a streptomycin unit is a

milliliter dilution unit against E. colt; an Oxford unit of penicillin is 1:50,000 against S. aureus.

TABLE 49. EFFECT OF STREPTOMYCIN UPON THE DEVELOPMENT OF
HUMAN TUBERCULOSIS IN GUINEA PIGS

DURATION OF

DURATION OF

INDEX OF INFECTION

infection,

TREATMENT,

DETERMINED

IN DAYS

IN DAYS

MICROSCOPICALLY

Experiment i

8 control animals

43-60

0

81.9

4 treated animals

54

39-54

2.8

Experiment 2

9 control animals

61

0

67

9 treated animals

61

47-61

5.8

From Feldman, Hinshaw, and Mann (250).

292 DISEASE CONTROL

Relatively little transfer of streptomycin occurs between the blood
and the lumen of the gastro-intestinal tract in either direction. Follow-
ing oral administration, levels as high as 9 mg. per gm. were found in
the feces. Because of the poor transfer of the drug across the walls of the
alimentary tract, it was suggested that the drug be used both orally
and parenterally in the treatment of infections such as those in which
the pathogenic organisms are found both in the gastro-intestinal tract
and in the blood stream (230, 393, 1049).

When fed to mice in their diet streptothricin and especially strepto-
mycin produced a very rapid reduction in the numbers of colif orm and
nonlactose-fermenting organisms in the feces. The amount of strepto-
thricin required for effective reduction of the intestinal bacteria was
one-half the toxic dose. Due to the lower toxicity of streptomycin the
dose required was much below the toxic one and maintained the re-
duced number of organisms as long as therapy was continued. A distinct
change in the intestinal flora was thus produced without increasing the
resistance of the organisms (859).

In order to produce toxic manifestations in animals, it is necessary to
administer either extraordinarily large amounts of the pure material or
smaller amounts of certain impure preparations containing a histamine-
like substance. No correlation was obtained between the acute toxicity
of different lots to experimental animals and clinical tolerance by
human beings. The behavior of the material in man cannot, therefore,
be foretold if antibacterial potency alone is assumed to be the criterion
of purity 5 highly concentrated material has on occasion been unsatis-
factory clinically, whereas considerably less active preparations have
often produced no undesirable effects.

Two distinctly different types of toxicity are possible. One, which is
observed only with certain impure preparations, is due to a histamine-
like substance. The general syndrome produced in man consists essen-
tially of nausea, throbbing headache, general malaise, skin rash, pain
in the joints, flushed face, vomiting, and drug fever, the last appearing
two or three days after treatment and therefore not due to bacterial
pyrogen. Local reactions at the sites of injection have also been en-
countered. These toxic manifestations are entirely absent when crystal-
line streptomycin, as well as many impure preparations, are used. By

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 293

employing only pure streptomycin or impure concentrates which do not
possess this factor it is possible to eliminate the undesirable side-re-
actions. The second kind of toxicity, which results in a fatty meta-
morphosis of the liver and kidney of animals, is an inherent property
of streptomycin itself. However, this organ pathology has not been
observed in man.

Streptomycin has been administered to man intramuscularly (every
3 to 4 hours, a daily total of i to 4 gm.), intravenously (intermittent
or continuous, the latter comprising a daily dose of i to 4 gm.), and
subcutaneously (100 mg, per i ml., at 6-hour intervals). It has also
been given orally (0.5 to 4 gm. per day in 4 doses) and by nebulization
into the tracheobronchial tree (25 to 50 mg. per i ml., for a total of 500
mg. daily) as well as by intrathecal administration (414). By the intra-
venous route a high serum concentration is produced very rapidly,
whereas the same level is obtained but more slowly after intramuscular
or subcutaneous injection. However, the blood level falls rapidly fol-
lowing a single dose and must be maintained by repeated administra-
tions. When given orally or by nebulization into the tracheobronchial
tree, no appreciable amount of streptomycin is absorbed into the general
circulation.

The intravenous injection of single doses of 200 mg. of streptomycin
gives an immediate concentration in the serum of 32 ng per ml. This
falls rapidly to 8 Mg at the end of two hours and to i or 2 |jg at the end
of twelve hours. Intramuscular injection of the same amount gives a
peak serum level of 1 6 Mg at the end of one hour, followed by a slower
drop in serum concentration, with detectable amounts after 12 hours.
Streptomycin appears quickly in the urine, 20 to 35 per cent being re-
covered in 2 hours and 50 to 70 per cent in the 12-hour period after
administration of a single dose. The total recovery varies from 1 5 to
85 per cent of the daily dose, with an average of 53 per cent. When
given parenterally, small amounts of streptomycin appear in the spinal
fluid, namely, i to 2 Mg as compared to 25 [ig in the serum. Follow-
ing oral administration, very little streptomycin reaches the serum, and
only 0.2 to 0.5 per cent is found in the urine; however, there is a high
concentration in the feces.

An intravenous injection of 600 mg. gave 32.8 Mg per ml. of blood

294 DISEASE CONTROL

in 15 minutes with a decrease to 4.9 ng in six hours. Following a
similar intramuscular injection, the rise was slower, with 26.5 pg at 3
hours and 8 pg at 6 hours per ml. of blood. The recovery of the strep-
tomycin in the urine varied during the first 24 hours, between 29 and
89 per cent, with an average of 66 per cent.

In the treatment of chronic infections of the urinary tract in man,
streptomycin was found to be effective in destroying such gram-nega-
tive bacteria as P. vulgaris, A. aero genes, E. coli, Ps. aeruginosa, and
Eberthella sp. Four hours after commencement of streptomycin
therapy ( i gm. of streptomycin daily in 8 divided doses given intra-
muscularly), urinary cultures were negative for P. vulgaris and, after 8
hours, for coliform organisms. In one case, the urine became negative
for E. coli two hours after treatment. Though infections of the normal
urinary tract were permanently cleared up, reinfection was likely to
occur in damaged tracts, the catheter serving as the portal of entry for
the new infection.

Streptomycin has also proved effective for the treatment of enteric
and systemic diseases. In five severe to moderately severe cases of
typhoid studied by Reimann (772), streptomycin was not administered
until late in the development of the disease. Nevertheless, the clinical
improvement of three patients coincided with the period of strepto-
mycin therapy. Of the two unsuccessful cases, the treatment of one was
prematurely discontinued because of the limited quantity of strepto-
mycin available.

In a study of 45 patients suffering from a variety of infections, it was
found (414) that good results were obtained in 10 of 13 cases of
moderately severe and severe infections of the urinary tract due to
various pathogenic bacteria. Of 5 cases of infection involving the tra-
cheobronchial tree, satisfactory results were obtained in 4. In some
miscellaneous infections, comprising cases of typhoid fever, undulant
fever, osteomyelitis, cellulitis, peritonitis, cholangitis, meningitis, and
ozena, the results could be considered satisfactory. Of 8 cases of bacteri-
emia, 6 showed recovery. Doubtful results were obtained in 2 cases of
undulant fever associated with bacteriemia. In 4 cases of syphilis it was
doubtful whether treatment with streptomycin was effective, although

ANTIBIOTICS AS CHEMOTHERAPEUTIC AGENTS 295

failure may have been due to the small amount of streptomycin ad-
ministered.

Salmonella as well as E. coli infections lend themselves readily to
treatment with streptomycin. A patient with a colony count of 23 mil-
lion Salmonella in the stool gave a negative stool after 4 days' oral
therapy with i gm. streptomycin daily, the number of E. coli was re-
duced simultaneously to about 1,000} S. jaecalis disappeared and the
Clostridia were reduced from 75,000 to 8,000. P. tularensis is one of the
most sensitive organisms in vitro to the bactericidal action of strepto-
mycin. Parenteral administration of relatively low doses proved suc-
cessful in human tularemia. One patient who began to receive strepto-
mycin on the eighth day of the disease was sent home as cured on the
seventeenth day. In another case with perisplenitis and generalized
infection of the peritoneal cavity, the peritoneal fluid was noninfective
on the sixth day after treatment, whereas usually such fluid is infective
for at least nine months. Seven patients who had received strepto-
mycin all responded promptly.

In a study of 34 tuberculous patients treated with streptomycin for
nine months, streptomycin exerted a limited suppressive effect, es-
pecially on some of the more unusual types of pulmonary and extra-
pulmonary tuberculosis. The reproduction of M. tuberculosis may
have been temporarily inhibited by the treatment} there was no evi-
dence, however, of rapidly effective bactericidal action. It was sug-
gested that studies be made of early pulmonary tuberculosis, tubercu-
losis of the genito-urinary tract, suppurative tuberculous lymphadenitis,
and early miliary and extensive hematogenous forms of tuberculosis
(429).

In a recent summary (494a) of a thousand cases treated with strepto-
mycin, the following results were recorded: In urinary tract infections
(409 cases), the over-all recovery rate was 42 per cent. Of 100 cases of
H. infiien'zae meningitis, recovery was obtained in 79 per cent; the
17 fatal cases received treatment too late. Of 14 cases of meningitis
caused by other gram-negative organisms, 4 died. In 91 bacteremia
cases, 49 recovered, 12 improved, 26 died} again, the fatal cases were
treated too late. Striking results were seen in tularemia (^G'}^ recoveries

296 DISEASE CONTROL

out of 67 cases) and otitis media (7 immediate recoveries out of 8
cases). In pulmonary infections (44 cases), recovery or improvement
was obtained in 61 per cent j streptomycin is extremely effective against
acute Friedlander bacillus infections. No dramatic effects were seen in
acute brucellosis (45 cases). In typhoid (51 cases), streptomycin, if
given early, accelerated return of temperature to normal. Of 26 cases
with Salmonella infection, 12 recovered, 8 died. Encouraging results
were obtained in peritonitis (39 recoveries out of 53 cases).

Preliminary evaluation of streptomycin in tuberculosis was based on
75 cases. The results that have been observed in miliary tuberculosis,
in tuberculosis of the meninges, and in tuberculosis of the larynx, skin,
and renal tract were considered as highly suggestive that streptomycin
exerts a bacteriostatic effect on the growth of the tubercle bacillus in
man. The same can be said for exudative pulmonary tuberculosis. It
was recommended that a minimum period of treatment should be three
to six months with doses of 1.5 to 3.0 gm. a day. The over-all incidence
of untoward side effects was 20.5 per cent. The commonest reactions in
order of frequency were headache, fever, skin eruptions, flushing of the
skin, and vertigo, alone or in various combinations.

For the treatment of tubercular meningitis, intrathecal administra-
tion of 100 mg. daily has given encouraging results, especially when
combined with parenteral administration (152a).

Failures of streptomycin therapy were attributed to the following
factors: treatment of infections not susceptible to streptomycin 5 inade-
quate dosage J development of resistance to streptomycin in vivo;
change in the species of infecting organism during treatment j and
localization of infection in an inaccessible area. In view of the develop-
ment of resistance of bacteria to streptomycin, as can easily be demon-
strated in urinary tract infections, it was recommended (511a) that
high Initial doses be used.

Other Agents

The protective action of the specific enzyme (polysaccharidase) of a
soil bacterium against type III pneumococcus infection has also been
established (31a). The specific protection induced in experimental
animals is determined by the nature of the polysaccharide of the pneu-

ANTIFUNGAL ACTION OF ANTIBIOTICS 297

mococcus type. The polysaccharidase destroys the protective capsular
substance of the pneumococcus, thus rendering it susceptible to phagocy-
tosis.

The toxicity of many other antibiotics to animal tissues prevents their
consideration as potential chemotherapeutic agents. The range of tox-
icity and type of effect vary considerably, from the highly toxic acti-
nomycin (796) to those which are characterized by lesser but still
considerable toxicity, such as citrinin (18).

Virulent strains of M. tuberculosis were found to lose their virulence
in the presence of certain other organisms or their products. According
to Vaudremer, this phenomenon occurs when the tubercle organism is
kept for 24 hours at 39° C. in contact with a filtered extract of A.
jum'igatusy and a similar effect can be exerted by certain bacteria. On the
other hand, extracts of A . fumigatus were used for the treatment of 200
tuberculous patients, with rather inconclusive results (740, 934).

Treatments of intestinal disturbances by the use of antagonistic micro-
organisms, although highly promising, have not been sufficiently in-
vestigated as yet. It may be of interest to note, in this connection, that
the presence in human intestines of E. coli with a high antagonistic in-
dex is considered as important evidence of immunity of certain indi-
viduals to intestinal disturbances (387, 618, 661, 681).

ANTIFUNGAL ACTION OF ANTIBIOTIC SUBSTANCES

The ability of certain antibiotic substances to inhibit the growth of
various fungi has been brought out previously. For test purposes, a
strain of Trichophyton mentagrofhytes (T. gyfseum) is used (240).
The fungus is grown on a peptone-glucose agar medium of f¥L 5.6 to
5.8, and a spore suspension prepared from ten-day-old mycelial growth
by shaking with glass beads. The spore suspension is then streaked over
agar plates containing varying concentrations of the antibiotic, or added
to a series of tubes containing graded concentrations of the agent, in a
manner similar to determination of phenol coefficient,

(Although a large number of substances are thus found to have
definite activity, they vary greatly in their usefulness because of vari-
ation in toxicity. Some, like actinomycin, are extremely toxic j others,

298

DISEASE CONTROL

like clavacin, less so. Only a few appear to offer definite promise (771,
883) for the practical control of fungus infections, notably hemipyo-
cyanin, gliotoxin, and streptothricin. In addition to clavacin and glio-
toxin, certain other fungus products appear to show some promising
action against fungi pathogenic to man. These include mycophenolic
acid and aspergillic acid (818).

The possibility of utilizing antagonistic bacteria for the control of
fungi causing skin infections has also been suggested (129).

TOXICITY OF ANTIBIOTIC SUBSTANCES

The various antibiotic substances obtained from microorganisms vary
greatly in their toxicity to animals. The therapeutic use of many of these
agents, like actinomycin and clavacin, which are highly bacteriostatic,
may be considered as either entirely excluded for the present or limited
to local applications. Some substances, as pyocyanase, penicillin, and
streptomycin, are relatively nontoxic (790); others, like tyrothricin
(793), streptothricin, and fumigacin (788), are slightly toxic j and still

TABLE 50. MAXIMAL TOLERATED DOSE FOR MICE OF VARIOUS
ANTIBIOTICS

SUBCU-

INTRA-

ORAL

TANEOUS

PERITONEAL

INTRAVENOUS

ANTIBIOTIC

ADMIN-

ADMIN-

ADMIN-

ADMIN-

AGENT

ISTRATION

Ibl'KATION

ISTRATION

ISTRATION

Streptothricin

> 2,000

> 1,000

3,000

1,000

Penicillin

> 2,000

1,600

2,000

500

Fumigacin (helvolic

acid)

800

Citrinin

100

100

Pyocyanin

100

80

Tyrocidine

> 1,000

> 1,000

20

1.25

Tyrothricin

>I,000

> 1,000

10

1.2

Gramicidin

> 1,000

> 1,000

10

1.2

Gliotoxin

5

Clavacin (crude)

3-5

Actinomycin

5

0.15

0.15

0.15

From Robinson (788).

Note: Figures represent maximal tolerated dose in mgm./kgm.

TOXICITY OF ANTIBIOTICS 299

others, like actinomycin and clavacin, are highly toxic (796, 966).
Some, like tyrothricin, are hemolytic (396, 617)5 others, like penicil-
lin, streptomycin, and streptothricin, are not (Table 50).

A great deal of additional information has recently accumulated on
the problem of toxicity and chemotherapy of antibiotic substances. Ref-
erence need only be made to the discussions of penicillin, streptomycin,
and some of the more recently isolated antibiotics, such as subtilin and
bacitracin.

CHAPTER 13

MICROBIOLOGICAL CONTROL OF SOIL-BORNE
PLANT DISEASES

The possibility of controlling microorganisms, especially fungi, in the
soil by favoring the development of antagonistic microorganisms is sig-
nificant for several reasons: Fungi are causative agents of some of the
most important diseases of plants and are added constantly and often
quite extensively to the soil, in plant residues and in diseased plant
products J fungi capable of causing certain diseases of animals and of
man also find their way sooner or later into the soil j many soil-inhabit-
ing fungi have a marked antagonistic effect against fungus and bacterial
plant pathogens.

Some fungi that produce plant diseases are able to survive in the soil
for only short periods of timej others become established in the soil
saprophytically and remain capable of attacking living plants when
proper conditions arise. Some of these fungi are specific, their ability to
attack dijEferent plants being limited, whereas others can cause diseases
of a great variety of plants and many survive in infected soil for long
periods. Some plant diseases, as in the case of virus infections, are trans-
mitted by specialized means, as by insect carriers. This complicates fur-
ther the interrelationship among the different organisms, in relation to
plant and animal diseases.

Microorganisms causing diseases of plants may either reduce the
vigor and productivity of the plants or destroy them completely.
Plants appear to develop at times a certain degree of resistance to mi-
crobial infection. Whether this is in the nature of a phenomenon of im-
munization, similar to that of animals, is still a matter of speculation.
Whatever the nature of the reaction, the degree of resistance depends
to a certain extent upon the imperviousness of the outer layers of the
plant tissues to penetration by the parasites, as well as upon the chemi-
cal composition of the plants. It is believed that an acid plant reaction,
combined with the presence of tannins and lignins, retards the growth of
many disease-producing agents. The survival of the pathogens outside

CONTROL OF SOIL-BORNE PLANT DISEASES 301

the host plant is due to the formation of resistant spores which remain
viable in the soil for long periods. Because of this, the growth of many
plants requires a long rotation if this system is to be used as a means of
controlling the specific diseases.

Many fungi and bacteria causing plant diseases were at first thought
capable of surviving in the soil for an indefinite time, even in the ab-
sence of the hosts. It has since been established, however, that, although
the majority of these pathogens are facultative saprophytes, some are
obligate parasites. The first can be grown easily on sterile soil and on
artificial culture media, whereas the second, such as Plasmodiofhora
hrass'icae and Synchytr'mm endohiotkum^ have not been cultivated so far
upon any artificial media and are known to die out in the soil in the ab-
sence of host plants.

Certain soil-borne plant diseases may be caused by more than one or-
ganism. In the pink-root of onions (376), Phoma terrestris is followed
by Fusar'tum malli; in the take-all disease of wheat, O. graminis is fol-
lowed by Fusarium culmorum. This type of sequence occurs with other
diseases, where the primary parasite first attacks the root and is followed
by a succession of other fungi, both parasites and saprophytes. By means
of the direct microscopic technique, the sequence of microorganisms can
be demonstrated in the infected roots of the plants. Certain less special-
ized parasites are able to live saprophytically on the dead tissues,
whereas the saprophytes are found only in the later stages of decom-
position.

It has been suggested (316, 317, 774) that root-infecting fungi be
classified ecologically as soil inhabitants and soil invaders. The first may
be looked upon as primitive or unspecialized parasites with a wide host
range, their parasitism being considered incidental to their saprophytic
existence in the soil. The second group comprises a majority of root-
infecting fungi, the more highly specialized parasites. The presence of
these in the soil is closely associated with the occurrence of the host
plants : in the absence of a host, these fungi die out in the soil, because
of their inability to compete with the soil saprophytes. The close associa-
tion between this group of organisms and their host plants is believed to
be enforced by competition with the microbiological population of the
soil.

302 CONTROL OF SOIL-BORNE PLANT DISEASES

ANTAGONISM 09 SOIL-INHABITING MICRO-
ORGANISMS TO PLANT PATHOGENS

The antagonistic interrelationships among the members of the micro-
biological population of the soil have received particular attention from
the point of view of modifying the virulence of those plant pathogens,
especially the fungi, that find temporary or permanent habitat in the
soil.

In the infection of wheat seedlings by O. graminis, a number of
fungi and bacteria are able to exert a marked antagonistic action against
the pathogen (822). Not only the living cultures of the antagonists,
but, in many cases, the culture filtrates are also effective (542, 1024).
The growth of H, sativum and F. grammearum upon sterilized soil
was completely suppressed (407) by the addition of small amounts of
unsterilized soil or by the simultaneous inoculation with harmless fungi
and bacteria, with the result that no infection occurred when wheat seeds
were inoculated with this soil. Although H. sativum is able to sporulate
readily in sterilized soil, this does not take place in nonsterilized soil,
sporulation being inhibited by the soil microorganisms. Virulence of
H. sativum on wheat seedlings was reduced by 1 1 to 57 per cent by cer-
tain cultures of Penicillium^; Tricho derma reduced virulence by 50 to
58 per cent, Ahsidia glauca by 39 per cent, and A. nidulans by 30 per
cent. Many fungi, however, had no effect on the virulence of the patho-
gen, and some even increased it (823). The fact that root-rot diseases
are less severe on wheat grown on summer-fallowed land than on land
cropped to wheat for several years was believed to be due to the soil
saprophytic microorganisms, which in bare fallow have an advantage
over the pathogenic organisms in competition for food (Figures 29
and 30).

The infection of wheat seedlings by O. graminis in sterile soil was
found to fall off rapidly with the reestablishment of the original soil
microflora (87). It was emphasized, however, that the effect of various
organisms upon the pathogen grown in artificial culture media is no
proof that the same organisms will be able to suppress the virulence of
the pathogen on wheat in soil. An inverse correlation was shown i^^S^^
to exist between the degree of infection and the protective effect of the

'H^^'

Figure 29. An antagonistic fungus, Tr'ichodermay attacking
a plant pathogenic fungus, 5. roljs'n, showing one break of a
septum. From Weindling (990).

Figure 30. Influence of antagonists upon the growth of Hclminthosporium.
Distortion of mycelium by Bacterium sp. (A) and B. rnmosus (B). C is a
normal mycelium. From Porter (729).

1

Figure 31. Antagonism of soil organisms against parasitism of P.
volutum on Agrost'is. From van Luijk (930).

% m^ ^ ^ ^ 0y

i : 1, ^ .'

[

^^ M^ '- 1 ^

1

\ 1

. ■

f^lGURE 32. Inhibiting effect of sterilized liquid medium of P. exfan-
siim versus Pythiuni de Baryanum on lucerne. From van Luijk (930).

SOIL MICROORGANISMS AND PLANT PATHOGENS 303

general soil microflora j this was determined by comparing infection in
an unsterilized soil with that obtained in a sterilized soil. An increase in
soil temperature was found (408) to increase the antagonistic action
of the soil microflora against the parasitic fungi causing cereal root rot.
Various actinomycetes were shown to be antagonistic (908) to species
of Pythium, a root parasite of sugar cane. The phenomenon of antago-
nism was independent of the ^H changes j it has been ascribed to the
formation of a toxic, partly thermostable, principle. A marked influ-
ence of the soil microflora on grass diseases caused by Pythium is illus-
trated in Figures 31 and 32. Clavacin (patulin) was found capable of
inhibiting the growth of various species of Pythium (the cause of
damping-off disease of seedlings) in dilutions of about 1 1400,000, and
of exerting a strong fungicidal action upon Ceratostomella ulmi, the
causative agent of the Dutch elm disease j the last effect could be over-
come partly by certain nutrients in the medium, especially peptone

(949)-

Numerous soil microorganisms are moderately or strongly antago-
nistic to such pathogens as Hyfochnus centrijugus, Hy-pochnus sasakii,
and Sclerotium oryzae sativae (241 ) j culture filtrates from some of the
antagonistic fungi were also able to reduce the damage caused by the
pathogens. Phytofhthora cactorum was found (1003) to be inhibited
in rotted tissues by the antagonistic effects of secondary organisms. In
many cases, the rotting of fruits was suppressed by mixtures of organ-
isms but not by the pathogens alone j the type of rot was also modified,
depending on the temperature and the specific nature of the antagonists
(827). Phytomonas tumefaciens, the causative agent of crown gall in
plants, can be controlled by means of crude penicillin (92).

The stimulating effect of mycorrhizal fungi on the host plant has
been attributed (297) to the capacity of the fungi to inactivate, destroy,
or absorb certain plant-retarding principles found among the organic
constituents of peat and other humus materials, or produced by fungi.
On the other hand, the toxicity of certain soils to mycorrhiza has been
explained (83) as due to the accumulation of antibiotic substances by
the fungus population of the soil. This concept is purely hypothetical
and is not based upon experimental evidence.

Certain fungi are also known (340) to be antagonistic to ants and

304 CONTROL OF SOIL-BORNE PLANT DISEASES

their fungal symbionts. These antagonists are distributed by the insects,
thus spreading agents that are destructive to themselves and to their
fungus gardens.

GENERAL METHODS OF CONTROL OF
SOIL-BORNE DISEASES

Soil sterilization by heat and chemicals has long been practiced as a
method of controlling soil-borne fungus diseases. This phenomenon is
usually designated as partial sterilization of soil, since not all micro-
organisms are killed by these treatments. However, once a soil thus
treated becomes reinfected with a disease-producing organism, the in-
fection may become much more severe. It has, therefore, been sug-
gested (383) that partially sterilized soil be reinoculated with a mix-
ture of saprophytic microorganisms before it is used as a seedbed, so as
to counterbalance the injurious effect of the parasites (Table 51 ).

The importance of the soil microflora in modifying plant diseases
caused by soil-borne pathogens is being realized more and more clearly.
One of the earliest attempts to control a plant disease by microbiological
agents was made in 1 908 by Potter (731). He found that Pseudomonas
destructansy the cause of rot of turnip, produces a potent, heat-resistant
toxin. The bacteria failed to grow in the presence of this toxin, and were
completely killed by the substance. By spraying turnips with this mate-

TABLE 51. EFFECT OF A BACTERIAL ANTAGONIST ON DAMPING-OFF
OF PLANTS IN THE SOIL

PERCENTAGE OF

SEEDS

PLANTED

PRODUCING NORMAL

SEED-

TREATMENT OF SOIL

LINGS IN

2 WEEKS

Cucumbers

Peas

Control soil

35

52

Fresh medium added

65

55

Diluted medium added

61

77

Washed bacterial cells added

58

75

Culture of bacterial antagonist added

55

80

Diluted culture added

87

90

Culture added continuously

81

90

From Cordon and Haenseler (154).

GENERAL METHODS OF CONTROL 305

rial, the disease could be checked j the toxin was more or less specific for
the particular organism. Certain bacteria commonly found in soils were
shown (306) to have a deleterious effect on the growth, in artificial
media, of Ps. citri, which causes citrus canker. This effect was brought
about by inhibiting the growth of and by killing the pathogen. By the
use of an antagonistic bacterium, wheat seedlings were protected from
infection by H elminthosforium sp. (729). In a similar manner, flax
seedlings were protected from Fusarium sp.

A watermelon disease, caused by Phymatotrkhum omnivoruniy was
considerably reduced when certain specific fungi and bacteria were pres-
ent in the soil together with the pathogen (105)} T. lignorum was
observed to attack and kill the hyphae of Phymatotrkhum in culture.
The severity of the seedling blight of flax, caused by Fusarium lini, was
diminished when the pathogen was accompanied in the soil by various
other fungi (901). The pathogenicity of H. sativum on wheat seed-
lings was suppressed by the antagonistic action of Trichothecium
roseum, this effect being due to a toxic substance produced by the latter
(359). T. lignorum prevented infection of wheat (63) by H. sativum^
and Fusarium' culmorum (Table 52). Novogrudsky (685) obtained
protection against infection of wheat with Fusarium by inoculating the
soil with the bacteria isolated by Chudiakov (143), provided the bac-
teria were introduced simultaneously with the fungus or preceded it.

TABLE 52. EFFECT OF TRICHODERMA LIGNORUM ON GERMINATION AND

GROWTH OF BARLEY INFECTED WITH HELMINTHOSPORIUM

SATIVUM IN STERILIZED SOIL

STRAIN OF
H. SATIVUM

Emerged

PERCENTAGE OF PLANTS

Stunted

Contorted leaves

H

H + T

H + SI

H

H + T

H + SI

H

H + T H + SI

21

84

94

94

46

12

6

52

32 15

22

88

94

98

33

8

6

57

27 14

23

86

88

96

25

17

8

78

31 21

24

88

98

94

10

4

3

17

15 10

From Christensen (141).

Notes. Results are based on randomized duplicate pots, each sown with 50 seeds. H, seeds inoculated with
a spore suspension of H. sativum; H+T, seed inoculated with H. sativum plus T. lignorum; H + SI,
seed inoculated with H. sativum and soil with T, lignorum.

306 CONTROL OF SOIL-BORNE PLANT DISEASES

The role of microbiological antagonism in the natural control of
soil-borne fungus diseases of plants has been well emphasized (408,
821 ). Methods for combating plant pathogenic fungi by the use of bac-
teria and other antagonists have been suggested by various investigators
(46,869).

The principles underlying the biological control of soil-borne plant
diseases were outlined by Garrett (316) in terms of the soil population
in a state of dynamic equilibrium. When a given crop is grown continu-
ously in the same soil, the parasitic organisms capable of attacking the
roots of that crop multiply. Organic manures stimulate the develop-
ment of saprophytic organisms in the soil, and are thus able to check
the activity of the pathogens, which are destroyed by the saprophytes.
Either the metabolic processes of the saprophytes check the growth of
the pathogens, or the saprophytes actually attack and destroy the myce-
lium of the pathogens. The microbiological control of plant diseases
was said to be most effective against those organisms which have be-
come highly adapted to a parasitic form of life. The pathogenic O-phiob-
olusy when present in the form of mycelium inside the infected wheat
stubble buried in the soil, is able to tolerate adverse physical soil con-
ditions. Those soil treatments which favor increased activities of the
microbiological population, such as addition of organic matter, partial
sterilization followed by reinoculation with fresh soil, and improvement
in soil aeration, favored loss of viability of the pathogen.

Van Luijk (930) recommended the control of plant parasites by
inoculating the soil with specific microorganisms selected for their an-
tagonistic capacity, or by the addition of the growth products of these
microorganisms. Living soil fungi, including Trkhoderma viridis and
Absidia s-pinosa, exerted an adverse influence upon Rhizoctonia solani
and reduced its pathogenicity to cabbage seedlings (458). Broadfoot
(87) and others (247), however, emphasized that the antagonism of
a saprophyte to a plant pathogen, determined on artificial culture
media, is not a reliable measure of the actual control of the parasite in
the soil. A lack of specific microorganisms in the soil is not a sufficient
factor limiting biological control under natural conditions. Therefore,
no inoculation of soil with an antagonistic organism, such as T. lig-
norum, can have more than a temporary effect in changing the micro-

Figure 33. Sweet-orange seedlings in nonsterilized soil. A, control;
B, Rhi-zoctoma inoculated into soil layer in bottom of jar; C, Rhi-zoc-
tonia as in B, plus Tr'icho derma in top layer of peat. From Weindling
and Fawcett (992).

GENERAL METHODS OF CONTROL 307

biological balance of the soil population. Similar results have been ob-
tained (992) in efforts to control R. solani, or the damping-off of citrus
seedlings (Figure 33), by the use of T. lignorum, and in the action of
B. simplex upon Rhizoctonia in the soil ( 1 54).

A number of antagonistic bacteria were found (502) to be able to pre-
vent scab formation by S. scabies on potatoes. Daines (167) found that
T. Ugnorum produces a diffusible substance which is toxic to S. scabies
in an artificial liquid medium. However, the toxic principle added to
potato soils is rapidly destroyed there by aeration j it can be removed
from solution by charcoal and by soil, where it is destroyed. It was sug-
gested, therefore, that it is highly doubtful whether antagonists will
be found to be of much assistance in combating potato scab in soil. The
physical and biological environments encountered in many cultivated
soils offer an important barrier against the establishment of the antago-
nist. When the latter was added to a 5-day-old culture of S. scabies, it
was greatly inhibited by the scab organism. Soil bacteria are also able to
produce substances toxic to both Trichoderma and Streftomyces alike.
In such a complex physical, chemical, and biological environment as the
natural soil, these antagonistic relationships may thus be modified or
even entirely destroyed.

The application to the soil of organic materials which favor the de-
velopment of antagonists has given much more favorable results than
the use of pure cultures. Fellows (251) obtained field control of the
take-all disease of wheat in Kansas by the application of chicken and
horse manure, alfalfa stems and leaves, boiled oats and barley, as well
as potato flour. Garrett believed (316, 317) that the factor chiefly con-
trolling the spread of pathogenic fungus along the roots of the wheat
plant was the accumulation of carbon dioxide, with a corresponding
lowering of oxygen tension in the microclimate of the root zone. A high
rate of soil respiration was, therefore, said to check the growth of O.
graminis. This can best be maintained, of course, by periodic additions
of organic manures. Materials low in nitrogen were found to be more
effective than those high in nitrogen. Garrett, therefore, postulated the
hypothesis that the soil microflora used the mycelium of the pathogen as
a source of nitrogen, in the process of decomposition of the nitrogen-
poor materials. The addition of nitrogenous substances, in either an or-

308 CONTROL OF SOIL-BORNE PLANT DISEASES

ganic or an inorganic form, was believed to protect the pathogenic or-
ganism against attack by the soil microflora, by offering a more readily
available source of nitrogen. Tyner (920) suggested that the differ-
ences in the microflora associated with the decomposition of different
plant residues are largely responsible for differences in persistence and
virulence of pathogens causing root rot of cereals.

Against some plant pathogens, however, high nitrogenous materials
were found to be very effective. Considerable reduction in the slime-
disease of tomato plants resulted from the addition of green manures
to the soil before planting (928) j organic materials high in nitrogen,
as well as the supplementary addition of nitrogenous materials suffi-
cient for complete decomposition of the organic matter, brought about
greater reduction of the disease. Organic matter was found to be most
effective during the process of decomposition; after it has undergone
extensive decomposition and reached a stage of slow decomposition,
when it is usually designated as humus, it becomes comparatively inert.

It has been recommended (767) that organic composts be used in
forest soils in order to overcome the infertility resulting from the ac-
cumulation of toxic substances. The addition of the compost changes the
microbiological population of the soil, followed by the destruction of
the toxins.

The antagonistic action of soil microorganisms has been utilized for
the control of P. omnivoruniy the root rot of cotton. This pathogen can
be inactivated when organic manures are added to the soil before the
crop-growing season (506). By the use of the contact slide technique,
microbiological antagonism was found (226) to represent the true
mechanism of the control process; the growth of saprophytic organisms
was most profuse in the slides buried in the manured plots, whereas the
mycelium of the pathogen was most abundant on the slides kept in the
unmanured plots. The conclusion was reached that manuring definitely
controls cotton root rot, as a result of the parasitism by bacteria of the
fungal strands of the causative agent of the disease. Continuous growth
of cotton on certain neutral or alkaline soils in southern United States
was believed to bring about an unbalanced soil population in which P.
omnivorum became a dominant organism; this was accompanied by the
absence or only the sporadic presence of Trkhoderma and other molds

GENERAL METHODS OF CONTROL 309

(903). The application of organic matter to such soils results in the de-
struction of most of the sclerotia and mycelium of the pathogen (649).
Microbial antagonists rather than food exhaustion were, therefore, con-
sidered to be responsible for the destruction of the pathogen.

The Sclerotium rot of sugar beets was found (552) to be controlled
by the application of nitrogenous fertilizers. This effect was believed to
be due largely to a change in the metabolism of the fungus or of the
host. It was also suggested, however, that suppression may possibly
be due to a change in the balance of the soil microbiological population.

The possibility of suppressing the growth and eliminating the infec-
tivity of plant pathogens by utilizing the activities of the soil microflora
was demonstrated also for a number of other diseases. It is sufficient to
cite the suppression of Monilia jructigena on apples by various fungi
and bacteria (933), of F. culmorum and H. sativum on wheat (408,
729), and of species of Rhizoctonta on citrus seedlings. These patho-
gens are markedly influenced by T. lignorum, a common soil sapro-
phyte. A species of Trtchoderma was also found to cause a reduction in
the amount of Texas root rot of watermelons caused by P. omn'ivorwm
(105).

The damping-off of citrus seedlings, caused by a number of fungi,
could be suppressed by T . lignorumy which parasitizes the fungi (989,
992). When T. lignorum was inoculated into pots containing Helmln-
thosforium sp. and Fusarium sp., the pathogenic action of these organ-
isms was checked and rendered harmless to plants (63). T. lignorum
was also decidedly antagonistic to Rhizoctonia and Pythium^j organisms
responsible for seed decay and damping-off of cucumbers (14).

The presence of Gihherella on corn inhibited infection due to T.
viridis; seed grains inoculated with the former gave more vigorous
growth than uninoculated seed grains (227). On the other hand, T.
viridis was found able to attack and to destroy the sclerotia as well as the
mycelium of such pathogenic fungi as Corticium rolfsii, Corticium sa-
dakii, and Sderotinia libertiana. The utilization of this organism for
the biological control of plant diseases has, therefore, been suggested

(<427)-

Henry (407) believed that the biological control by the soil micro-
flora could even be directed against internal seed infection, since appre-

310 CONTROL OF SOIL-BORNE PLANT DISEASES

ciable damage to surface-sterilized flax seed was found to occur in steri-
lized but not in unsterilized soil.

A very interesting application of the phenomenon of antagonism is
also found in the use of harmless fungi (Pullularia fullulans) for the
inhibition of harmful ones growing on paper pulp (7).

CONTROL OF PATHOGENIC FUNGI IN SOIL BY

INOCULATION WITH ANTAGONISTIC

MICROORGANISMS

Despite the favorable results obtained from the action, in artificial
culture, of antagonistic bacteria and fungi upon plant pathogens, the
field results have often been rather disappointing. The soil microflora
seems to have no marked effect on certain diseases, such as the seedling
blight of barley J the antagonistic action of the soil population appears
to be insufficient to suppress the injury caused by diseased seed (141).
The addition of T. Ugnorum and certain other fungi and bacteria to in-
fected seed or to sterilized soil inoculated with H. sativum often pre-
vented seedling injury, increased the stand, and decreased the number
of deformed seed. Chudiakov (143) suggested inoculation of seed with
bacteria for the control of flax against infection with Fusarium. It has
been said that wheat seedlings were protected from attack by the simul-
taneous introduction of lytic bacteria with the pathogenic fungus, but
when the bacteria were introduced 24 hours after the fungus they were
unable to protect the wheat sown 3 days later.

On the basis of extensive studies on the control of plant-disease-pro-
ducing fungi by means of antagonistic soil microorganisms, Novogrud-
sky (683, 685) came to the following conclusions: The distribution and
vigor of parasitic fungi are a result, on the one hand, of resistance and
immunity of plants to infection, and, on the other, of the antagonism
between soil microorganisms and pathogenic fungi. Among the numer-
ous forms of antagonism existing between soil microorganisms and
pathogens, those bacteria which produce lysis of fungi deserve particu-
lar attention. The bacteria are widely distributed in nature j they are
able to destroy and to dissolve the mycelium and the spores of different
phytopathogenic fungi, not only in artificial media but also in the soil.

CONTROL OF PATHOGENIC FUNGI 311

The inoculation of sterilized soil with F. graminearum led to the in-
evitable death of wheat plants, but additional inoculation of the soil
with lysogenic bacteria protected the wheat from the disease. The addi-
tion of bacteria to unsterilized soil which has been made sick by con-
tinuous growth of flax markedly lowered the percentage of plants dis-
eased by F. lint.

The term "bacterization" was suggested by the Russian investigators
(46, 673) to designate the process of treatment of seed with lysogenic
bacteria, whereby the plants are protected against pathogenic fungi.
The susceptibility of plant seedlings to infection by fungi could be
decreased not only by the specific antagonistic bacteria, but also by the
presence of other bacteria which are able, in one way or another, to re-
tard the development of the fungi. The conclusion was reached that the
effect of bacteria on germinating seeds is due to the metabolic products
liberated by the bacteria, which are capable of depressing the develop-
ment of parasitic fungi. By treating flax seeds with the culture filtrate of
the antagonistic bacteria, a similar or even greater decrease in the num-
ber of diseased seedlings was obtained. The nature of the active sub-
stance produced by the antagonists was not investigated further. It was
said to accumulate in 5-to-io-day-old cultures. Heating at 80° C. for
10 minutes had no effect upon the substance, but heating at 100° C.
brought about its inactivation.

Jensen (467) concluded that the beneficial result of bacterization is
due not to nitrogen-fixation by A-zotobacter or to production of growth-
promoting substances by microorganisms, but to the protection of the
seedlings against plant pathogens by specific bacterial antagonists.

Although it has now been definitely established that certain organ-
isms can repress or even destroy disease-producing fungi, the utilization
of specific microbial products for the control of plant diseases has made
comparatively little progress so far. Leemann {SS^) tested the action
of various secretions and extracts of microorganisms upon H. sativum.
He concluded that microorganisms, pathogenic or nonpathogenic, can
supply substances useful as preventive measures against plant patho-
gens. No favorable effects upon the development of Rhizoctonia disease
in plants could be observed, however, from the application to soil of cer-
tain bacterial and fungus products.

312 CONTROL OF SOIL-BORNE PLANT DISEASES

The only important procedure which has thus far found a practical
application is the treatment of the soil in such a manner as to modify its
microbiological population, which in its turn can destroy or in other
ways control the activities of plant pathogens. Many such instances can
be cited for illustrative purposes. In the case of root rots of the straw-
berry, it was found that carbohydrate decomposition induces a favor-
able change in the soil microflora from pathogenic or potentially harm-
ful organisms to beneficial or rather innocuous types (424). Studies of
the eradication of cotton root rot led to recommendations of treatments
of soil with organic materials, especially during the period of increased
microbial activity (650). The survival of the fungus is considered
to be limited by microbial interrelationships rather than by food ex-
haustion. During the early stages of incubation, viable sclerotia are de-
stroyed more rapidly than dead sclerotia in soils treated with organic
matter. It was suggested, therefore, that the germination of the scle-
rotia is an important factor for their elimination from the soil. Field
application of organic materials, accompanied by early October plowing,
results in an increase in microbial activity. This brings about a reduc-
tion of the incidence of dead cotton in the succeeding crop and greater
difficulty of finding sclerotia.

It was reported recently (65) that R. solani is able to cause 100 per
cent damping-off of radish seedlings planted at a distance of 4 cm. from
the inoculum 5 when the seeds were planted at a distance of 9 cm. the
damage was 40 per cent. The addition of i per cent ground wheat
straw or dried grass to the soil had a marked depressing eifect upon the
growth of the fungus. This was ascribed to the nitrogen starvation of
the mycelium, accompanied by the rapid utilization of the available soil
nitrogen by the cellulose-decomposing microorganisms multiplying at
the expense of the fresh organic material j carbon dioxide produced by
the cellulose-decomposers was believed to cause a marked fungistatic
action on the Rhizoctonia.

Treatment of the soil with organic materials, which results in the de-
struction of certain plant pathogens by stimulating the development of
saprophytic microorganisms, may be designated as "partial disinfec-
tion." To what extent antibiotic agents produced by plants, such as

SOIL MICROORGANISMS AND LEGUME BACTERIA 313

tomatin of tomato plants, serve to protect the plant against invasion by
fungi and other pathogens still remains to be determined (456a).

SOIL MICROORGANISMS IN RELATION TO
LEGUME BACTERIA

The possible injury caused to leguminous plants by the antagonistic
action of soil bacteria upon the root nodule bacteria has been but little
investigated (524). Certain bacteria belonging to the Pseudomonas and
Achromobacter groups exercise a strong influence upon the nodule bac-
teria in the root zone or rhizosphere of the legumes. Some stimulate,
others depress, and still others have no effect at all upon the growth of
the legume bacteria. The stimulating effect of non-nodule bacteria upon
the nodule bacteria often is very striking {_SZli)'

CHAPTER 14

THE OUTLOOK FOR THE FUTURE

The production of antibiotic substances by microorganisms under con-
trolled laboratory or factory conditions and the utilization of these sub-
stances for disease control are very recent developments. It has been
known for more than half a century that certain microbes are capable
of combating others, and it has even been suggested that saprophytic
microorganisms may in time be utilized for controlling the growth of
pathogenic forms. Until recently, however, these suggestions were
largely speculative. Such positive facts as were available were merely
isolated items, the full significance of which was not sufficiently well
recognized. This is true, for example, of the claims and counterclaims
concerning pyocyanase and pyocyanin, two antibiotics of bacterial ori-
gin, the antibacterial properties of which have long been known. It is
true, also, of certain antibiotics produced by fungi, such as gliotoxin and
the A. jumigatus factory by actinomycetes, such as actinomycetin and
lysozymej as well as by various other bacteria, such as B. mesenterkus
factor, B. simplex factor. The existing confusion is due largely to the
fact that the utilization of these antibiotic substances for disease control
gave varying results that were frequently rather disappointing.

In 1929, Fleming observed that the growth of a mold, which was
later identified as P. notatum, on a plate seeded with staphylococci pre-
vented the growth of these bacteria. In liquid media, this mold pro-
duced a soluble substance, designated as penicillin, that inhibited pyo-
genic cocci and members of the diphtheria group but not gram-negative
rods. Fleming, however, did not go beyond the suggestion that this sub-
stance be utilized for disease control or beyond the statement that
it might prove useful because of these antibacterial properties. This
contribution received no further attention for more than a decade, with
the exception of very few experiments that served to prove Fleming's
original observations. The full realization of the fact that we are dealing
here with an entirely new field of biology and chemotherapy that is

OUTLOOK FOR THE FUTURE 315

bound to enrich the subject of control of disease came only a little more
than half a decade ago.

In 1939, Dubos announced that new antibacterial agents can be iso-
lated from soil microorganisms that are active not only in vitro but also
in vivo. He proceeded with the isolation of such substances in a novel
and unique manner, which consisted in enriching the soil with patho-
genic bacteria, isolating from such soil specific antagonistic organisms
capable of destroying the pathogens, and finally producing the specific
antibacterial substance by cultivating the organism on suitable media.
This work pointed to a new method of approach to the isolation of anti-
biotic substances and attracted the immediate attention of bacteriolo-
gists, chemists, and medical investigators. One no longer had to depend
for the isolation of antagonistic organisms upon mere air contamina-
tions of exposed bacterial plates, or upon accidental observations of the
destructive action of one microbe upon another. One could now proceed
systematically with the isolation of microorganisms capable of destroy-
ing sfecijic disease-producing bacteria. The mechanisms whereby such
destruction was brought about were found to be due to the production
of characteristic substances, known as antibiotics. The isolation of each
antibiotic required special methods, because of its specific chemical na-
ture. Since most of the antibiotics are selective in their action upon dif-
ferent bacteria, affecting some and not others, the method proved to be
of great importance in pointing a way to the isolation of a variety of
chemical agents active upon gram-positive bacteria as well as gram-
negative forms.

It was not sufficient, however, merely to isolate these protoplasmic
poisons from the cultures of the antagonistic microorganisms. It was
essential to determine the effect of animal tissues upon the action of
these agents and to establish their selective activity upon the bacteria in
the animal body as compared to the test tube. However, once it was
recognized that some of these antibiotics act in vivo against a variety of
diseases, the branch of medical science known as chemotherapy acquired
a new group of tools. This work led to a reexamination of the potentiali-
ties of penicillin, which in turn led to the second important contribution,
namely, the work of Chain and Florey and their co-workers. These in-

316 OUTLOOK FOR THE FUTURE

vestigators succeeded in obtaining crude active preparations of peni-
cillin which, they demonstrated, had a marked effect upon various
gram-positive and certain other bacteria in the animal body. The im-
portance of this discovery was accentuated by the great need of the
moment and the urgency for new methods of combating infections re-
sulting from World War II. These investigations immediately at-
tracted universal attention, and were soon followed by remarkable de-
velopment in the manufacture, isolation, and study of the chemical na-
ture of this highly important antibiotic agent. Penicillin fully deserves
the designation "Wonder Drug" given to it by the popular press.

These two epoch-making contributions were rapidly followed by a
series of investigations that resulted in the isolation of a number of
other antibiotics. Among these, streptomycin deserves special considera-
tion because of its activity against various gram-negative bacteria and
M. tuberculosisy which are not affected by tyrothricin or penicillin, and
because of its limited toxicity to animals and its activity in vivo.

It was soon recognized that one is not dealing here with only three or
more types of chemical compounds capable of destroying various
pathogenic bacteria and fungi, but that a new field of science bordering
on microbiology, chemistry, pharmacology, pathology, and chemo-
therapy was being opened that was bound to result in many practical
applications. The fact that many of these agents, including penicillin,
are produced by several different organisms and, further, the fact that
many of these compounds are formed in several chemical modifications
open to the chemist new fields for the synthesis of types of compounds
heretofore unknown, and point out to the medical world new ways of
combating infections and epidemics.

The rapid progress made in the utilization of antibiotics in so brief a
period of time can best be illustrated by the following two citations:

On May 4, 1940, Garrod (3 1 8), in discussing the use of antiseptics in
wounds, wrote: "Only a few years ago it was thought impossible to kill
bacteria within the body with chemicals and likely always to remain so.
This belief was shaken by the discovery of a urinary antiseptic which
really worked, and it was shattered by the introduction of Prontosil,
with all its manifold consequences. Are we still to deny the possibility of
killing bacteria which are merely lying on a body surface?"

OUTLOOK FOR THE FUTURE 317

Less than four years later, Florey (274) summarized the value of
penicillin: "i. As a preventive of infection in wounds, enabling a po-
tentially septic wound to be treated in much the same way as an aseptic
one, 2. in the promotion of healing in burns and for ensuring the suc-
cess of skin grafts, 3. in infections (due to sensitive organisms) either
(a) chronic, or (b) of such severity as to render the prospect of death
likely, which have not responded to other forms of treatment, 4. in
acute infections due to sensitive organisms, 5. in the rapid curing of
gonorrhoea including sulphonamide-resistant cases, 6. in pneumonia,
7. probably in gas gangrene, but here numbers have been few and meth-
ods not fully tried out."

In commenting upon the remarkable developments in the use of
penicillin, Keefer (493a) stated: "It can be said without reservation
that penicillin is the most remarkable chemotherapeutic agent that has
been discovered up to the present time. In the first place, it is a sub-
stance of extremely great antibacterial power. One mg. of crystalline
penicillin is capable of inhibiting the growth of staphylococci in 82.5
liters of broth. It is non-toxic in maximum therapeutic doses in man. It
is highly efFective against a wide variety of microorganisms of the
gram-positive group. It is effective in both gonorrhea and syphilis. It is
responsible for the reduction of the fatality rate in more diseases than
any other chemotherapeutic agent j it shortens the clinical course of
many infectious disorders, and it saves many days of illness and dis-
ability. When one realizes that this can be accomplished by an agent
that destroys bacteria and other microorganisms such as the Treponema
pallidum without injury to the tissues of the host, it deserves to be called
remarkable. No other known agent can do as much."

Only one year later, the remarkable effectiveness of streptomycin
was summarized by Waksman and Schatz (970) as follows:

"7;^ VIVO streptomycin has sucessfully controlled experimental infec-
tions due to Salmonella schottmUllerij Pseudomonas aeruginosa, Pro-
teus vulgaris, Shigella gallinarum, Brucella abortus, Klebsiella fneu-
moniae, Pasteurella tularensis and 'Mycobacterium tuberculosis.
Against the gram-positive Di-plococcus pneumoniae and Stafhylococ-
cus aureus protection was afforded by somewhat larger doses. Strepto-
mycin has also protected against spirochetal infections due to Borrelia

318 OUTLOOK FOR THE FUTURE

novyi and Leftosfira icterohaemorrhagka. Following the oral or
parenteral administration of streptomycin, no unfavorable effects have
been encountered which could not be attributed largely to the presence
of impurities in the preparations. To date, streptomycin has been ad-
ministered for infections of the urinary tract, typhoid, brucellosis,
Klebsiella infections, tularemia, tuberculosis, and wounds."

It appears, therefore, that certain generalizations concerning possible
future developments in the field of antibiotic substances are justified.

A SEARCH FOR NEW ANTIBIOTIC AGENTS:
A PROBLEM FOR THE MICROBIOLOGIST

Although more than sixty compounds or preparations possessing bac-
teriostatic and fungistatic properties have already been isolated from
microorganisms, there is sufficient evidence that a great many others
can be obtained without too great difficulty, if enough organisms are
studied in greater detail. In this connection, three methods of approach
have been followed: (a) testing organisms found in culture collections
for antibacterial activity in general, followed by a detailed study of one
or more substances produced by one or more organisms j (b) isolating
specific organisms, such as members of the P. notatun-ir-P . chrysogenum
groups, from different soils and from moldy food materials and testing
them for the production of penicillin, in the hope of finding more active
organisms than those now known to exist j (c) enriching the soil with
specific bacteria, followed by the isolation of organisms capable of in-
hibiting the growth of or of destroying such bacteria.

A summary of the various steps essential to the isolation of a suitable
chemotherapeutic agent are given in Figure 34. A number of surveys
have now been made concerning the distribution of organisms capable
of producing antibiotics among bacteria, actinomycetes, and fungi.
Only very few such organisms were selected for more detailed investi-
gation, chiefly because of the great amount of time and experimentation
required for the isolation of any new type of substance. The selection of
a particular antibiotic for isolation and further study is largely gov-
erned by its specific antimicrobial spectrum, its action upon particular

OUTLOOK FOR THE FUTURE 319

Isolation of antagonistic organisms from soils, coTufosts, manures, and
sewage

a. Common agar media; colonies Isolated at random from plates

b. Washed agar-bacterial media; zone-forming or other colonies se-

lected

c. Low dilutions; colonies producing inhibition of other organisms

selected

d. Special selective media

Testing of isolated colonies

a. Streak method; tested against gram-positive and gram-negative bac-

teria

b. Agar plugs removed from plates in which antagonist is growing

c. Liquid media containing bacterial suspension inoculated with an-

tagonist

d. Agar media inoculated with test culture, followed by inoculation

with antagonist

Production of antibiotic substances

a. Synthetic and organic media used

b. Filtrate tested by convenient method

c. Study of surface vs. submerged culture

D etermination of antibacterial sfectruTn and general -prefer ties of
tyfe substance -produced

a. Bacteriostatic spectrum by suitable methods

b. Chemical and physical properties of crude preparation

c. Pharmacological properties and in vivo activity

Recognition of type substance on basis of antibacterial properties

a. Bacteria: pyocyanase, pyocyanin, tyrothrlcin, and subtilin types

b. Fungi: penicillin, gliotoxln, clavacin, and peniclllic acid types

c. Actinomycetes: actlnomycetin, actinomycin, streptothricin, and

streptomycin types

Isolation of antibiotic substance

a. By use of organic solvents

b. By adsorption on charcoal, and removal by acid solutions or sol-

vents

c. Other methods

Isolated antibiotics

a. Antibacterial, physical, chemical, and pharmacological properties

Chemother apeutic uses

Figure 34. Isolation and preparation of antibiotic substances.

320 OUTLOOK FOR THE FUTURE

bacteria, its toxicity to animals, and its activity in vivo. The following
illustrations will suffice:

Of all the aerobic spore-forming bacteria known to produce anti-
biotic substances, only B. brevis and B. subtilis have been utilized for
the isolation of tyrothricin, bacitracin, and subtilin. It is known, for
example, that various strains of B. niesentericuSy B. mycoideSy B. sim-
fleXy and other spore-forming bacteria are capable of producing anti-
biotics, some of which are markedly different chemically, biologically,
or in selective activity. It is sufficient to call attention to such prepara-
tions as bacillin, simplexin, and others. A more detailed study of these
organisms and the substances produced by them is bound to enlarge
greatly our knowledge of this group of chemical compounds and their
therapeutic potentialities.

Of all the nonspore-forming bacteria possessing antagonistic prop-
erties found in soils and water basins, only two have been studied in de-
tail : Ps. aeruginosa has been utilized for the production of pyocyanase,
pyocyanin, and the various pyo-compounds, and Ch. iodinum for the
production of iodinin. It is known, however, that a large number of
other nonspore-forming bacteria are capable of producing a variety of
antibiotic substances, the chemical nature and biological activities of
which are still but little understood.

Only very few of the antibiotic substances produced by actinomycetes
have so far been investigated, isolated, or concentrated; namely, acti-
nomycin, proactinomycin, streptothricin, and streptomycin. Even these
few substances, however, differ markedly in chemical nature and in
biological activity. In view of the fact that as many as 20 to 40 per cent
of all the actinomycetes are known to be capable of producing antibiotic
substances, many of which undoubtedly differ from those already iso-
lated, the wealth of material that is awaiting investigation can only be
surmised. Some of these possibilities have been definitely indicated.

The production of antibiotic agents by fungi likewise has been but
insufficiently studied. The following pertinent facts may direct attention
to the many problems still awaiting investigation :

(a) Some antibiotics, like penicillin, gliotoxin, clavacin, aspergillic acid,
penicillic acid, and citrinin, are produced by a number of different

OUTLOOK FOR THE FUTURE 321

organisms; the nature of the organism often influences not only the
yield of the substance but also its chemical nature and its biological
activity. A number of other substances have been added to this list,
namely, fumigacin, viridin, penicidin, and others.

(b) The mode of nutrition and the manner of growth of a single organ-
ism have often been found to influence the concentration and the na-
ture of the antibiotic substance, v^^hich may be formed in one me-
dium and not at all or in much lower amounts in another medium.
Some organisms are greatly favored in the production of antibiotic
substances by the presence in the medium of certain vitamin-like
complexes. Moreover, the formation of a substance is usually asso-
ciated with a certain stage of growth of the organism, since the sub-
stance is produced at one time and then rapidly destroyed, the range
of its accumulation often being very narrow. These facts point to
certain fundamental aspects in the physiology of the organism pro-
ducing the antibiotic substance that are still little understood.

(c) Certain organisms, such as members of the Fusarium group, produce
bacteriostatic substances, the action of which, however, is rapidly
overcome by the bacteria. This points to problems on the stability of
the antibiotic substance and on the adaptation of bacteria to the sub-
stance.

(d) Although it is known that certain yeasts produce antibiotic substances,
either of an adaptive or of a nonadaptive kind, very little is known
concerning the nature and mode of action of such substances.

(e) Different strains of the same group of organisms, especially under
different conditions of culture, may produce the same type of sub-
stance which varies both chemically and biologically. This is brought
out by the formation of different penicillins. The same principle no
doubt holds for other antibiotics.

These and many other problems are awaiting solution. The micro-
biologist is faced with a new field of research second only to that of the
very discovery of the causation of disease by microorganisms.

THE OPPORTUNITY FOR THE CHEMIST

'The chemist has been searching far and wide for new chemotherapeu-
tic agents. He has synthesized many thousands of compounds, only

322 OUTLOOK FOR THE FUTURE

a few of which have proved to be of practical value. The chemist has
started from a certain lead, such as the arsenical group in the salvarsan
type of compounds and the sulfa-radical in the sulfanilamides. The dis-
covery of new chemical agents possessing antibacterial or antifungal
properties offers the chemist many new models to draw upon for varied
types of syntheses.

Although only very few antibiotics have so far been isolated and
their chemical nature determined, it is already well established that we
are dealing here with a great variety of chemical compounds. It is suffi-
cient to mention, first of all, the penicillins and other sulfur-bearing
compounds, such as gliotoxinj the organic bases, notably streptomycin
and streptothricinj the polypeptides, especially tyrothricinj the oxida-
tion-reduction systems, namely pyocyanin and actinomycin j the ketones
and quinones, such as clavacin and many others. Compounds like acti-
nomycin, which are highly active against bacteria but also highly toxic
to animals, may possibly be modified in such a manner as to reduce their
toxicity without impairing their activity. This is also true of simpler
compounds, such as the less toxic but also less active clavacin and glio-
toxin. The solution of the chemical nature of penicillin offers many
tempting opportunities for the synthetic chemist.

Doubtless most of the compounds that prove to be useful as chemo-
therapeutic agents will sooner or later be synthesized. The contribution
of the bacteriologist may be all but forgotten in the light of the forth-
coming chemical developments, but even the bacteriologist will be
grateful for new tools to help combat disease-producing agents.

THE FIELD OF CHEMOTHERAPY

The utilization of the activities of antagonistic microorganisms for
the control of human and animal diseases has only begun. Many prac-
tices in surgery and many old-time remedies are based on the creation
of conditions favorable to the development of antagonistic microbes.
Plaster treatment of wounds, without the use of antiseptics, has often
given marvelous results. Such wounds have been found to contain
aerobic bacteria with no one group predominating, except that Ps. aeru-
ginosa tends to become more numerous when the healing process has

OUTLOOK FOR THE FUTURE 323

been established. It still remains to be determined whether this organ-
ism exerts a favorable effect due to its antagonistic properties or is only
another wound-infecting agent.

Of particular importance is the development of the manufacture of
antibiotic substances. Largely because of the stimulus given by World
War II when the need for new antibacterial agents became very acute,
an intensive study was made of the practicability of utilizing some of
the agents already known, and search was made for new ones. Among
these, penicillin occupies a leading place. A large number of great con-
cerns in this country, in Great Britain, and elsewhere are engaged in the
manufacture of this drug by utilizing several strains of P. notatum and
P. chrysogenum. An intensive search for new strains resulted in the
isolation of cultures that have increased a hundred-fold the yield of
penicillin from the same lot of medium. The development of deep cul-
ture technique greatly reduced the labor involved. New agents capable
of inhibiting the growth of and destroying other pathogens resistant
to the action of penicillin have been isolated. Most significant of these
is streptomycin.

The progress made in the isolation of antibiotic substances from many
microorganisms has not kept pace with their evaluation as chemothera-
peutic agents. In discussing antimicrobial agents of biological origin,
Dubos (200) emphasized that students of infectious diseases are pri-
marily concerned with the action of these substances upon certain strains
and stages of the parasites, with the mechanism of their action upon the
susceptible cells, and with physiologic and pathologic effects on the host.
Mcllwain (600), on the other hand, suggested that animal testing in
chemotherapy is not necessarily much nearer to the conditions under
which the drug will be finally used than are properly chosen in vitro
conditions i although in vitro testing does not reproduce all the condi-
tions of the normal environment of the parasite, it is less likely, under
present conditions of testing, to introduce new and unknown factors
than is testing in another host. The in vitro and in vivo studies of an
agent are considered as complementary.

The utilization of fungi and bacteria against plant diseases has also
been variously attempted. The main difficulty involved is to estab-
lish the antagonist in the soil. This can be done by modifying soil

324 OUTLOOK FOR THE FUTURE

conditions, as by the addition of stable manure or other plant and animal
residues, in order to favor the development of the antagonist.

Thfc activities of antagonistic microorganisms are also utilized for
combating injurious insects and other lower animal forms destructive to
plants and to animals. Among the insects, the Japanese and other
Asiatic beetles have been treated rather successfully by the use of nema-
todes and certain specific bacteria. Extensive use has already been made
of these bacteria, by inoculating the soil with grubs heavily infected
with them.

Comparatively little is yet known of the ability to control, by means
of antibiotic agents, diseases caused by protozoa, such as malaria and
trypanosomes, as well as virus infections. The hope of finding anti-
biotics active against such resistant diseases as tuberculosis has recently
gained much ground with the discovery that streptomycin has a re-
markable tuberculostatic effect and that it may find a place in the treat-
ment of certain forms of tuberculosis.

These instances suffice to arouse hope that even greater progress can
be expected in the control of disease by utilization of the activities of
antagonistic microorganisms. So far, most energies have been directed
to the treatment of acute infections caused by bacteria. Less is known
of chronic infections. Whether or not man will ever be able to control
all diseases caused by the numerous microscopic and ultramicroscopic
forms of life through the utilization of the activities of antagonistic
microorganisms and antibiotics, he will have gained sufficient knowl-
edge from the mode of action of these organisms, and of the substances
produced by them, to justify further hope in the possibilities thus
opened.

MODE OF ACTION OF ANTIBIOTIC SUBSTANCES:
A FIELD FOR THE PHYSIOLOGIST

Finally, there remains the fourth important group of problems in-
volved in the study of antibiotics, namely, the mode of action of these
substances upon bacteria and other microorganisms. The fact that dif-
ferent agents vary greatly in their bacteriostatic and bactericidal action
upon different organisms is well established. A number of mechanisms

OUTLOOK FOR THE FUTURE 325

have been propounded, some of which hold true for one substance and
some for more than one. Each of these mechanisms involves some ex-
tremely puzzling physiological problems. To take only two illustra-
tions :

(a) If a given substance interferes with the utilization by the bacteria
of a certain metabolite in the medium, as in the relation of sulfa-drugs to
^-amino-benzoic acid, one must assume that the sensitive bacteria re-
quire the metabolite in question and the resistant forms do not, or that
the resistant bacteria synthesize larger concentrations of the particular
metabolite than the sensitive forms. Since the sensitivity of the bacteria
to an antibiotic is often more of degree than of kind, the assumption
would be that the metabolite is either required in different concentra-
tions by the various organisms or is synthesized to a different extent.

(b) The adsorption of the antibiotic substance by the bacterial cell,
rendering the cell incapable of multiplying or dividing, points to an-
other type of mechanism that may be rather common. This may often
express itself in the abnormal enlargement of the cell. A clear under-
standing of this phenomenon will have to await a better knowledge of
the mechanism of cell fission. Should one assume that the resistant cells
and the sensitive cells divide by different mechanisms?

All these and many other problems point directly to the fact that a
better understanding of the physiology of the microbial cell will be
gained from a clearer appreciation of the mode of action of antibiotics
upon the cell.

It is thus to the smallest of living systems, the microbes, that we must
look for the solution of some of the most important problems that have
faced man as well as his domesticated and friendly animals and plants.

CLASSIFICATION OF ANTIBIOTIC SUBSTANCES

GLOSSARY

BIBLIOGRAPHY

INDEX OF MICROORGANISMS

GENERAL INDEX

CLASSIFICATION OF ANTIBIOTIC SUBSTANCES

PRODUCED BY BACTERIA

Bacillin {B. subtilis)
Bacitracin {B. subtilis)
Colicine {E. coli)
Colistatin (aerobic spore-formers)
Diplococcin {Streftococci)
Endo-subtilysin {B. subtilis)
Eumycin (5. subtilis)
Gramicidin {B, brevis)
lodinin {Ch. iodinum)
Phthiocol {M. tuberculosis)
Prodigiosin (5. marcescens)
Pyo compounds {?s. aeruginosa)

Pyocyanase {Ps. aeruginosa)
Pyocyanin {Ps. aeruginosa)
Pyolipic acid {Ps. aeruginosa)
Simplexin {B. simflex)
Subtilin {B. subtilis)
Subtilysin {B. subtilis)
Toxoflavin {B. cocovenenans)
Trypanotoxin {B. subtilis)
Tyrocidine {B. brevis)
Tyrothricin {B. brevis)
Violacein {Ch. violaceum)

PRODUCED BY ACTINOMYCETES

Actinomycetin (5. albus)
Actinomycin (S. antibioticus)
Litmocidin (A^. cyanea)
Micromonosporin {Micromonosfora
sp.)

Mycetin (5, violaceus)
Proactinomycin {N. gardneri)
Streptomycin {S. griseus)
Streptothricin {S. lavendulae)

PRODUCED BY LOWER FUNGI

Aspergillic acid {A. flavus)
Aspergillin {A. niger)
Chaetomin {CA. cochliodes)
Citrinin {P. citrinin, A. candidus)

* Clavacin (^4. clavatus)

* Clavatin (^4. clavatus)

* Claviformin {P. claviforme)
§ Corylophillin {P. corylofhylum)
§ E. coli factor {P. notatum)

* Expansin {P. exfansum)
t Flavacidin {A. fiavus)
f Flavatin {A. fiaz'us)
f Flavicin (^4. flavus)
$ Fumigacin {A. fumigatus)
Fumigatin {A. fumigatus)
t Gigantic acid (^4. giganteus)
Gladiolic acid {P. gladioli)
Gliotoxin {Trichoderma, Gliocla-

dium, A. fumigatus)
Glutinosin {M. glutinosum)

Note. Terms marked with the same symbol are synonyms.

X Helvolic acid (^4. fumigatus)

Javanicin {F. javanicum)

Kojic acid {A. oryzae)

Notalysin {P. notatum)

§ Notatin {P. notatin)

f Parasiticin {A. farasiticus)

* Patulin {P. fatulum)

§ Penatin {P. notatum)

Penicidin {Penicillium sp.)

Penicillic acid {P. fuberulum, P. cy-
clofium)

Penicillin {P. notatum, P. chryso-
genum)

§ Penicillin B {P. notatum)

Puberulic acid {P. piberulum)

Puberulonic acid {P. fuberulum)

Spinulosin {A. sfinulosum, A. fumi-
gatus)

Ustin (a. ustus)

Viridin {T. viridis)

330 CLASSIFICATION OF ANTIBIOTIC SUBSTANCES

PRODUCED BY HIGHER FUNGI

Clitocybin {CI. gigantea) Polyporin {P. sanguineus)

PRODUCED BY ALGAE
Chlorellin {Chlorella sp.)

Although the antibacterial products o£ higher forms of life are not
in a strict sense antibiotic substances, they can be mentioned here be-
cause of their similar properties.

PRODUCED BY HIGHER PLANTS (PHYTONCIDEs)

Allicin {Allium sativum) Pinosylvin {Pinus silvestris)

Crepin {Crefis tar akaci folia) Protoanemonin {Anemone fulsatilla)

Flour protein Tomatin (tomato plant)
Inhibin (honey)

PRODUCED BY ANIMALS

Erythrin (red blood cells) Lysozyme (eggs, tears, and various

Lactenin (milk) organs)

GLOSSARY

Antagonism. The phenomenon of a living organism inhibiting the
growth or interfering with the activities of another living organism
as a result of the creation of unfavorable conditions in the medium
or the production of a specific antimicrobial substance.

Antagonist. An organism having the capacity to inhibit the growth or
interfere with the activity of another organism.

Antagonistic substance. A term frequently used to designate a substance
that neutralizes the bacteriostatic action of an antibiotic substance.

Antibacterial index. The ratio of concentration of an inhibitor sufficient
to inhibit the growth of an organism to the concentration of a metabo-
lite.

Antibiosis. The inhibition of growth of one organism by another.

Antibiotic or antibiotic substance. A chemical substance produced by
microorganisms which has the capacity to inhibit the growth of bac-
teria and other microorganisms and even to destroy them,

Antidotic or antidotic substance. A chemical substance produced by
microorganisms which has the capacity to inactivate the injurious
effect of bacterial toxins.

Anti-inhibitor y inhibitor ^ suf pressor are terms of similar significance to
antagonistic substance.

Bactericidal. Causing the death of bacteria.

Bacterioantagonistic. Inhibiting the growth of bacteria.

Bacteriolytic. Causing not only the death of bacteria but also their lysis
or disintegration.

Bacteriostatic. Inhibiting the growth of bacteria.

Bacteriostatic or antibiotic spectrum. A range of inhibition of growth of
different bacteria by different concentrations of an antibiotic sub-
stance. It may be expressed graphically, the bands of the spectrum
representing the concentrations of the substance.

Bacteriotherafy. The use of living bacteria for the treatment of infec-
tious diseases.

Biostatic complex. The sum total of factors that limit microbial develop-
ment in a medium. The absence of such factors may result in the for-
mation of toxic products.

332 GLOSSARY

Chemotherafy. The use for the treatment of infectious diseases of
chemical agents that are effective against the parasites and are toler-
ated by the host.

Fungicidal. Causing the death of fungi.

Fungistatic. Inhibiting the growth of fungi.

Inactivatovy nontoxic. A substance that inactivates plant viruses and is
not detrimental to most forms of life.

Inhibitor or inhibitive substance. A term variously applied, but usually
used to designate a substance that inhibits the growth of bacteria and
other microorganisms.

Lysogenesis. The production by an organism of substances that cause
the lysis of bacterial cells.

Lysozyfne. A substance produced by living tissues (white of egg, tears,
and also certain microorganisms) that is capable of dissolving living
bacterial cells, especially certain micrococci.

Mycocidal. A substance having tuberculocidal properties.

Mycoin. A term suggested to designate antibiotics of fungal and acti-
nomycete origin.

Oxford unit {O.U.). A standard for penicillin adopted by international
agreement and often designated as international unit (I.N.). One
mg. of crystalline penicillin is equivalent to 1.650 O.U. One unit in-
hibits the growth of a standard strain of S. aureus in 50 ml. of meat
broth.

Phytoncide. A chemical substance produced by higher green plants
which has the capacity to inhibit the growth of bacteria and other
microorganisms.

Streptomycin unit (S.U.). A dilution unit against E. coli as test organ-
ism, or the amount of material that will inhibit the growth of E. coli
in I ml. of medium J equivalent to i pg of streptomycin base.

Tryfanotoxin. A substance that has a destructive effect upon trypano-
somes.

Tuberculocidin. An antibiotic substance having tuberculocidal prop-
erties.

Tuberculostatic. A substance having a bacteriostatic effect upon mem-
bers of the M. tuberculosis group.

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INDEX OF MICROORGANISMS

Absidia glauca, 302

Ab. sfinosa, 306
A chromobacter, 151, 313

A. lifolyticum, 105

A. stutzeri, 120
Acrostalagmus, 59, 148
Actinomyces, 108, 220

See also Streftomyces

A. albus, 113, 269

A. bovis, 76, 124, 127, 236, 269
A ctinomycetaceae, I o 8
Actinomycetales, 108
Actinomycetes. See General Index
Aerobacter, 236

A. aerogenes, 10, 12, 21, 32, 86,
96, 99, 100, 102, 120, 123, 124,
164, 220, 244, 294
Agrostis, 303 (Fig. 31)
Algae, 6, 9

Alkali genes fecalis, 106, 151
AlliuTTt sativum, 217, 225, 330
Alternaria, 59, 148

A. tenuis, 148
Amebae, 9, 23
Anemone Pulsatilla, 330
Anthrax organism. See General Index
Arctium minus, 225
Argas, 161
Armillaria, 148
Asarum canadense, 225
Ascaris, 26

Ascomycetes, 131, 1 3 2, 147
Aspergilli, 42, 131, 132, 142, 146, 147
Asfergillus, C), ^-j, 131, 140, 1 46, 147,
148, 153, 211

A . albus, 1 3 3

i4. candidus, 140, 171, 329

^. clavatus, 57, 74, 130 (Fig. 12),

131, i32> i33> H3> H4> H8,

171, 329
yl. ejfusus, 173, 21 3
^. flavifes, 140

Asfergillus (cont.)

^. ^^«j, 56, 57, 70, 133, 140, 141,

148, 170, 172, 207, 208, 329
A. flaz'us-oryzae, 131, 140, 329
A. fumigatus, 56, 57, 70, 130 (Fig.

12), 131, 133, 142, 143, 145,

170, 171, 172, 173, 175, 210,

211, 244, 297, 314
A. nidulans, 140, 302
A. niger, 49, 100, 131, 133, 140,

146, 148, 150, 164, 171, 176,

214, 329
A. oryzae, 140, 173, 213, 214, 329
A. farasiticus, 140, 1 72, 329
A . schiemannii, 131
A. s-pinulosum, 329
A. terreus, 131, 144
A. ustis, 143, 175, 329
A symmetrica, 132
Azotobacter, 15, 42, 115, 311
Az. agile, 123

Az. chroococcum, 15, 112, 123
Az. indicum, 123
Az. vinelandii, 1 1 2, 120, I 23, 25 I

(Fig. 27)

Bacillus "D," 151
Bacillus, 21, 86

B. anthracis, 36, 50, 76, 85, 86,

93j 96, 97^ ioo> 102, 103, 1

124, 126, 130, 151, 179, 1

200, 246, 250, 261, 262, 2

287
B. brevis, 48, 56, 57, 70, 71, 87,

92, 170, 173, 175, 185, 2

320, 329
B. hirudenses, 95
B. leniimorbus, 95, 160
B. lichenijormis, 94
B. macerans, 120, 123
5, megatherium, 86, 1 00, 112, I

123, 124, 128, 220
B. mesentericus, 21, 48, 51, 87,

o5>
81,
69,

19.

20,

396

INDEX OF MICROORGANISMS

Bacillus (cont.)

89, 90, 91, 94, 112, 120, 151,
152, 158, 180, 181, 216, 251,
286, 314, 320

B. TTiesentericus-vul gatus, 88, 90

B. mucosus-cafsulatus, 85

B. mycoides, 51, 58, 59 (Fig. 4), 72,
87, 88, 89, 90, 100, III, 112,
114, 116, 120, 123, 124, 144,
151, 152, 175, 200, 220, 223,
251, 252, 257, 260, 320

B. mycoides-cytoliticus, 88, 90

B. fetasites, 21

B. folymyxa, 120, 123

B. fopillae, 95, 160

B. frodigiosus, 158

B. pitrificus verrucosus^ 106

B. fyocyaneus. See Ps. aeruginosa

B. ramosus, 302 (Fig. 30)

B. simflex, 57, 94, 151, 152, 175,
188, 307, 314, 320, 329

B. subtilis, 21, 51, 57, 58, 59, 72,
74, 75> 77, 78, 83, 86, 87, 88,
89, 90, 92, 93, 94, 100, 112,
120, 121, 123, 124, 142, 144,
151, 158, 163, 169, 171, 172,
175, 176, 188, 192, 204, 207,
209, 215, 216, 217, 220, 223,
234, 236, 239, 243, 250, 251
(Fig. 27), 255, 260, 269, 320,

329

B.suifestifer, 88

B. thermo-philus, 88

B. tumescens, 1 1 2

B. vulgatus, 100
Bacteria, 36
Bacterium, 5, 151, 160, 302 (Fig. 30)

B. acidi lactici, 105

B. aroideae, 1 5 i

B. cazaubon, 160

B. cocovenenans, 188, 329

B. efhestiae, 160

B. gelechiae, 160

B. lactis aero genes, 105

B. fyocyaneum. See Ps. aeruginosa

B. solanacearum, 128

Bacterium (cont.)

B. termo, 263

B. tyro genes, 95

B. violaceum, 49
Bacteroides, 21

Basidiomycetes, 131, 132, 146, 1 47
Basisforium, 151

B. gallarum, 150
Beauveria, 151

BlastOTuycoides dermatitidis, 150, 183
Boofhilus bovis, 17
Borrelia gallinarum, 158

Bor. novyi, 269, 288, 317

Bor. recurrentis, 269
Botrytis, 148

B. aim, 1 48

B. cinerea, 148
Botulinus organism, 27
Brucella, 106, 235, 268

Br. abortus, 58, 76, 106, 120, 123,
124, 146, 220, 251, 255, 287,
288, 317

Br. melitensis, 34, 76, 97, 104, 106,
251

Br. suis, 106, 124

C efhalosforium, 9,131
Cefhalothecium roseum, 148, 151
Ceraiostomella, i 5 3

C. ulmi, 83, 242, 303
Chaetomium, 131, 133, 213

Ch. cochliodes, 171, 213, 329

Chlorella, 171, 330

Chromobacterium, 181

Ch. iodinum, 173, 182, 320, 329
Ch. violaceum, 86, 1 00, 175, 1 82,

329

Citromyces, 147

Cladosforium, 9

Clitocybe Candida, 146

C. gigantea, 146, 1 72, 330

Clostridium, acetobutylicum,, 42
CI. bifermentans, 269
CI. botulinum, 1 06, 266, 269
CI. butyricum, 106, 123, 124, 257
CI. chauvoer, 27, 96, 99, 269

INDEX OF MICROORGANISMS

397

Clostridium ( con t . )

CI. fallojc, 2 1

CI. granulobacter-fectinovorum^ 42

CI. histolyiicum, 269

CI. novyi, 269

CI. oedematiens, 76, 1 06, 273

CI. oedematis, 21, 106, 269

CI. ferfringens, 106, 273

CI. sefticum, 21, 76, 134, 269, 273,
274

CI. sordelli, 269

CI. sforogenes, 97, 106, 266, 269

CI. tetani, 27, 76, 1 24, 269

CI. tetanomorfhum, 269

CI. welchti, 21, 76, 106, 120, 162,
182, 220, 251, 269, 273, 276
Colletotrichum, 148

C. gloeosforioides, 1 1 8
Colfidium, 22
Colfoda, 158
Ccniiofhora cerebellar 149
Corticium rolfsii, 309

C. sadakii, 309
Cortinarius, 146
Corynebacteria, 104
CorynebacteriuTTiy 112

C. difhtheriae, 76, 88, 90, 91, 93,
96, 97, 98, 102, 124, 179, 227,
246, 261, 264, 268, 269, 280,
286
Crefis taraxacifolia, 217, 330
Cryftochilum nigricans, 22
Cryftococcus neofornums, 74
Cunninghamella, 43, 44

C. elegans, 148

Dematiaceae, 149
Dematium, 148
Deuterofhoma, 148
Dictyostileum discoideum, 157

Z). mucoroides, 49
Diplococci, 95, 97, 104
Diflococcus fneumoniae, 124, 1 26,

237, 269, 272, 288, 317
Diflococcus pneumoniae I, 281

Z), pneumoniae III, 233

Dothiorella, i 5 i
Dysentery bacteria, 28

Eberthella, 86, 294
£■. rhusiopathiae, 269
£. /)i/>^/, 100, 123
i". /y^Aox^, 30, 31, 32, 49, 59, 70,
85, 86, 88, 89, 95, 96, 97, loi,
104, 105, 113, 146, 174, 213,
220, 233, 236, 246, 264, 265

Entamoeba histolytica, 234

Erzvinia carotovora, 1 20

Erysipelothrix, 288
Er. murisepica, i 24
Er. rhusifathiae, 236

Escherichia coli, 10, 21, 29, 30, 31,
32, 43, 45, 49, 57, 58, 59, 72,
74, 76, 85, 86, 87, 89, 91, 93,
94, 95, 96, 98, 99, 100, loi,
102, 103, 105, 106, 112, 113,
114, 117, 120, 121, 123, 124,
126, 131, 133, 142, 144, 146,
158, 165, 172, 181, 192, 194,
201, 207, 208, 210, 220, 223,
227, 233, 234, 235, 236, 240,
244, 246, 247, 248, 253, 254,
255, 256, 265, 279, 286, 291,
294, 295, 297, 329, 332

Flavobacterium, 21,31
Fluorescent bacteria, 95
Friedlander's bacillus, 92
Fusarium, 9, 38, 59, 62, 74, 1 1 1, 118,
131, 145, 148, 150, 151, 152,
157, 305> 309, 310, 321

F. conglutinans , 152

F. culmorum, 301, 305, 309

F. graminearum, 302, 311

F. javanicum, 141, 173, 329

F. lateritium, 148

F. lini, 305, 311

F. main, 301

F. monilijorme, 150

F. oxys forum cubense, 118, 145

F. sambucinum, 150 (Fig. 17)

F. vasinfectum, 1 49

398

INDEX OF MICROORGANISMS

Gafkya tetragena, 1 20

Gambusia, 96

Gas-gangrene organisms, 27

Gibber ella^ 309

Gleosforium, 59

Gliodadium, 57, 131, 133, 144, 148,

150, 173, 211, 329

Gonococcus, 133, 267
Gymnoascus, 144

Hartnianella castellanii, i 5 7
H elminthosforiuTn, 62, 148, 151, I 52,
302 (Fig. 30), 305, 309

H. sativum, 95, 148, 149, 151, I 5 2,
302, 305, 309, 310, 311

H. teres, 148
Hemofhilus, 220, 235

H. ducreyi, 235, 269

H. influenzae, 123, 124, 200, 291,
295

H. fertussis, 120, 124

H. suis, 123
Heterodera marioni, 162
Hyphomycetes, 149
Hyfochfius centrifugus, 303

//, sasakii, 303

Klebsiella, 288, 318

AT. fneumoniae, 75, 76, 80, 97, 100,
105, 124, 244, 317

Lactobacillus, 189

L. acidofhilus, 103, 107, 265

Z,. arabinosus, 186

L. bulgaricus, 50, 97, 103, 1 05, 107

Z. c^j^i, 123, 241
Leishmania, i 5 8
Left OS fir a, 158, 213

L. icterohaemorrhagiae, 76, 269,
288, 291, 318
Listerella mo?iocytogenes, 124
Lonlcera tartar jia, 217
Lucilia sericata, 162

Macrofhomina fhaseoli, 150

Malleomyces mallei, 1 24
Melanosfora famfeana, 1 49
Meningococci, 98, 230
Meningococcus, 267
Metarrhizium glutinosum, 173, 214,

329
Micrococci, 95, 97, 115
Micrococcus, 31

il/. antibioticus, 104

M. candicans, 21, 112

Af. catarrhal is, 267

Af. conglomeratus, 233

M. _^^c«j, 100, 267

M. luteus, 21, 112

M. lysodeikticus, 59, 72, 112, 123,

223, 269
Af . roseus, 1 1 2
M. tetragenus, 104
Micromonosfora, 57, 108, lio. III,
117, 173, 329
M. vulgaris, 108 (Fig. 10)
Monascus, 148
Monilia albicans, 1 46
Af. fructigena, 309
Mucor, 9, 148
Mucorales, 147
Mycobacteriaceae, 108
Mycobacterium, 108
Af. avium, 124
A/, citreum, 1 1 2
Af. />A/^i, 72, 94, 112, 123, 124
TVf. ranae, 143
Af. rubrum, 112
A/, smegmae, 1 1 2

Af. tuberculosis, 21, 33, 76, 88, 90,
93> 94, 95>96, 97, 98, 102, 104,
105, 106, 112, 113, 120, 124,
130, 133, 141, 143, 146, 174,
175, 176, 208, 234, 244, 245,
267, 287, 288, 289, 290, 295,

297, 317, 329> 332
Mycococcus ruber, 112
My CO gone, i 50
Myxobacteria, 97
Myxobacteriales, 105
Myxobacterium, 59, 151

INDEX OF MICROORGANISMS

399

Neisseria, 86, 133, 220, 235

N. catarrhal is, lOO, 120, 232, 235,

269
A^. jlaz'a, 235
iV. gonorrhoeae, 76, 124, 235, 260,

269, 276
iV. intraceUularis, 235, 269
"N . meningitidis, 76, 126, 200
Neoaflectana glaseri, 160
Nocardia, 108, IIO, 114, 115, 117,

245
N. alba, 1 1 2
N. asteroides, 124
N. corallina, 1 1 2
A^. cyanea, lib, 173, 329
A'', gardneri, 57, ill, 119, 125, 1 74,

200, 239
N. rubra, 1 1 2

Oidium, 21
Oikomonas, i 5 7

O. termo, 156
Ofhiobolus, 39, 148, 151

O. graminis, 149, 302, 307

O. miyabeanus, 150

Paramecium, i 57
Paratyphoid bacteria, 91, 96
Pasteurella, 220, 233
P. avicida, 97
P. ^^/^w, 76, 91, 96, 97, 98, 103,

105, 124, 161
P. tulai-ensis, 124, 288, 289, 295
Pectinofhora gossyfiella, 160
Penicillia, 131, 132, 146, 147
Penicillium, 9, 36, 62, 130, 131, 133,
140, 144, 147, 148, 149, 151,
153, 157, 174, 213, 302
P. africanum, 49
P. brevi-com factum, 173, 213
P. brunneoviol aceum, 131
P. chloro-leucon, 131
P. chrysogenum, 130 (Fig. 12), 131,
136, 137 (Fig. 14), 138, 139,
i73> 174, 200, 205, 323, 329
P. cinnabarinus, 147

Penicilliu7n ( con t . )
P. citreo-roseum, 1 40
P. citrinum, 130 (Fig. 12), 131,

133, 171, 208, 329
P. claviforme, 131, 1 33, 144, 171,

329
P. corylofhylu7Ji, 329
P. crustosum, 1 40
P. cyclofium, 131, 133, 174, 209,

329
P. cyclofiuin-clavijomie, \ 3 i
P. exfansum, 131, 144, 171, 303

(Fig. 33), 329
P. funiculosu?n, 1 3 1
P. gladioli, 149, 172, 329
P. griseo-roseum, i 3 i
P. liUeum, 49, 147
P. luteum-furfurogenu??!, 131, 148
P. notatum, 54, 56, 57, 70, 130
(Fig. 12), 131, 132, 133, 134,
135, 136, 139, 146, 169, 170,
173, 174, 200, 201, 205, 207,
223, 225, 275, 314, 323, 329
P. notatum-chrysogenum, 131, 132,

133, 134, 136, 138, 318
P. obscurum, 145, 211
P. fatulum, 144, 171, 329
P. fuberulum, 133, 174, 209, 212,

329

P. resticulosufn, 133

P. sanguineus, 174

P. sfinulosum., 175

P. urticae, 144
Pestalozzia, 62
Peziza, 147, 148

P. sclerotiorum, 148

P. trifoliorum, 148
Pfeijferella jnallei, 95
Phoma terrestris, 301
Phy corny ces, 159
Phycomycetes, 131, 132, 147
Phymatotrichum, 305

Ph. omnivorum, 305, 308
Phytomonas, 86

Ph. bowlesii, 1 00

Ph. fruni, 124

400

INDEX OF MICROORGANISMS

Phytomonas (cont.)

Ph. tumefaciens, c)j, 103, 104, 303
Phytofhthora, 151, 152

Ph. cactorum, 303

Ph. erythroseftica, i 3 2
Pinus silvestrisy 330
Plasmodinfhora brassicae, 30 1
Plasmodium, 158

PI. gallinaceum, 284
Pneumococci, 92, 97, 104, ill, 114,

230, 244, 254,258
PneuTnococcus, 76, 91, 238
Pneumococcus, Type I, 236
Pneumococcus, Type II, 236
Pneumococcus, Type III, 296
Polystictus saftguineus, 146, 213, 330
Polytoma uvella, 22
Proteus vulgaris, 21, 76, 86, 89, 91,
97) 98, 105, 106, 112, 124, 151,
152, 227, 235, 236, 278, 286,

294, 317
Psalliota, 146

Ps. camfestris, 150
Pseudoeurotium zonaium, 1 50 (Fig.

17)

Pseudomonas, 21, 31, 85, 313

Ps. aeruginosa, 31, 42—43, 48, 50,
51, 56, 57, 70, 76, 86, 91, 95,

96, 97, 98, 100, 105, no, III,

112, 113, 120, 124, 131, 146,

151, 157, 158, 174, 175, 178,

179, 181, 183, 219, 261, 262,

264, 278, 286, 287, 294, 317,
320, 322, 329

Ps. aviseftica, 105

Ps. citri, 157, 305

Ps. destructans, 304

Ps. fiuoresce?is, 9, 13, 21, 43, 44,
51, 85, 86, 96,97, 100, 103, 112,
120, 123, 124, 128, 152, 153,
220

Ps. hyacinthi, 157

Ps. juglandis, i 5 1

Ps. fhaseoli, i 5 i

Ps. pttida, 21, 85

Ps. fyocyaneus. See Ps. aerugifiosa

Pseudo7nonas (cont.)

Ps. translucens, i 5 i
Pullularia pillulans, 3 i o
Pyronema, 16

P. confiuens, 153
Pythium, in, 148, 149, 242, 303,

309

P. de Baryanum, 303 (Fig. 32)
P. voluUwi, 303 (Fig. 31)

Radiobacter, 112
Ranunculus, 217
Rhizobium, 45, 115

Rh. leguminosarum, 112
Rhlzoctonia, 38, 148, 151, 152, 212,
253, 307, 309, 31I) 312

R. solani, 94, 150, 306, 307, 312
Rhizofus, 9, 43, 47, 153
Rhodococcus, 86

R. cinnebareus, lOO

R. roseus, 100
Rickettsia frowazekii, 269

Saccharomyces, 151

Sac. cereviseae, 97, 151

5^^. ellipsoid eus, 100

5ac. marianus, 1 00

Sac. fastorianus, lOO
Salmonella, 86, 235, 268, 287, 295

5. ab or tivo equina, 123

S. aertrycke, 220

5. cholerasuis, 123

S. enteritidis, 21, 1 00, 124

5. gartneri, 76

S. faratyfhi, 76, 126, 233

S. fullorum, 100

5. schott??iulleri, 123, 124, 220, 288,

317
5. suifestifer, lOO
5. tyfhi, 75, 76, 126
S. tyfhimuriuvi, i 26
Sarcina, 21, 31, 86
S. jlava, 21
S. lutea, 49, 57, 58, 59 (Fig. 4), 72,

88, 100, iio, 112, 120, 123,

124, 152, 220, 223

INDEX OF MICROORGANISMS

401

Sarcitm (cont.)

S. ureae, 152
Sclerotinia, 59, 151

S. americana, i 50

S. libertiana, 309
Sclerotium, 309

S". oryzae sativae, 303

S. rolfsii, 148, 302 (Fig. 29)
Serratia, 34, 99

S. marcescens, 51, 85, 86, 87, 96,
97) 99j 100, no, 112, 120, 123,
124, 151, 152, 157, 174, 181,
220, 329
Shiga bacillus, 87, loi
Shigella, 91, 126, 236

Sh. dysenteriae, 76, 93, 100

Sh. gallinarum, 120, 1 23, 220, 288,

317
Sh. faradysenteriae, 124, 261—262

Sficaria fuvpurogenes, 49

Sfirillum, 1 58
Sf. minus, 269

Sforotrichum, 16

Staphylococci, 45, 92, 95, 96, 97, 102,
104, III, 112, 120, 172, 225,
230, 232, 258, 260, 268, 270,
278

Stafhy loco ecus, 209, 249, 250, 270
S. albus, 21, 91, 179, 238, 269, 270,

286
5. aureus, 58, 59, 63, 72, 73, 75, 76,
77, 80, 81, 83, 84, 85, 91, 94,
98, 100, 102, 106, no, 112,
117, 124, 126, 131, 134, 142,
146, 147, 162, 165, 166, 172,
179, 182, 201, 204, 207, 208,
209, 213, 217, 220, 232, 233,
235, 236, 237, 240, 243, 244,
250, 252, 253, 254, 258, 259,
263, 268, 269, 272, 274, 275,
277, 280, 281, 286, 287, 291,

317, 332
S. citreus, 91, lOO
S. muscae, 123
5. viridis, 91
Sterigmatocystis, 148

Stomoxys, 161

Streptococci, 76, 97, 103, 104, in,
230, 231, 260, 268, 269, 270,
278, 329
Streptococcus Group A, 236
Streptococcus Group B, 236
Streptococcus Group C, 236
S. agalactiae, 285
S. bovis, 269
5. cremoris, 103, 172
S. dysgalactiae, 285
S. enteritidis, 244

5. faecalis, 235, 237, 269, 278, 295
S. hemolyticus, 75, 91, 98, 124, 182,
208, 221, 223, 227, 235, 238,
240, 254, 255, 268, 269, 280,
281
5. lactis, 97, 103, 269
5. mastidis, 103
S. mucosus, 91, 103
S. nonheTnolyticus , 269
S. fyogenes, 21, 76, 82, 103, IIO,

126, 134, 147, 237, 244, 253,
263, 269, 270, 274, 282

S. salivarius, 237, 269
.?, sefticemiae, 2 1
5. thermofhilus, 106
5. uberis, 285

S. z'iridans, 59, 75, 76, 124, 146,
269
Sfrepomyces, 43, 44, 108 (Fig. lo),
no, ni, 114, n7, 121, 128,

245, 307
S. albus, 98, 100, 102, ni, n8,

n9, 123, 126, 171, 329
S. antibioticus, 56, 57, 59 (Fig. 4),

108 (Fig. 10), ni, n9, 124,

127, 171, 189, 329
S. aurantiacus, n 2

S. calif ornicus, 120

5". cellulosae, 120

5. equinus, 269

S. globisforus, 1 1 2

5. griseus, 57, 108 (Fig. 10), ni,

n2, n9, 124, 125, 127, 175,

193, 194. 329

402

INDEX OF MICROORGANISMS

Streftomyces (cont.)

S. lavendulae, 57, 108 (Fig. 10),

III, 119, 121, 122, 123, 124,

175, 191, 192, 329
S. fraecox, ill, 128
5. scabies, iii, 128, 129, 307
S. violaceus, 112, 116, 125, 173,

329

S. violaceus-ruber, 123
Streftomycetaceae, 108
Streftothrix, 1 1 o
Synchitrium endobioticum, 301

Tetramitus rostratus, 22
Thamnidium elegans, 148
Torula sfhaerica, 1 00

T. suganii, 148, I 50, 214
Torulofsis, 148, 149
Torulosfora utilis, var. major, 2 1 5
Trefonema fallidum, 235, 269, 277,

317
Trichoderma, 9, 43, 44, 72, 131, 133,

144, 145, 150, 153, 173, 211,

212, 253, 302, 302 (Fig. 29),

307, 308, 309, 329
T. lignorum, 148, 150, 150 (Fig.

17)5 3055 306, 307, 309, 310
T. viridis, 145, 175, 212, 306, 309,

329

Trichomastric, 21—22

TrichomojiaSy 22

T. vaginalis, 260
Trichofhyton gypeum, 280, 297

T. mentagrofhytes, 74, 146, 297
Trichothecium, 148

T . roseum, 305
Tryfanosoma, 158

T. equiferdum, 159, 234, 267
Tylenchus tritici, 162
Typhoid bacteria. See General Index
Tyrothrix, 87

Ustilago, 148, 151
U. avenae, 152
JJ . hordei, i 5 2

t/. ««<2?d, I 5 2

C7. z^d^, 59, 151, 152

Yerticillium, 148
Y. dahliae, i 5 8
Vibrio comma, 34, 49, 76, 85, 86, 87,
89, 95, 96, 97, 98, 100, 103,
104, 124, 126, 146, 179, 213,
261
V . metchnikovi, 98

Y-bacillus, 91

Yeasts. See General Index

Zygorhynchus, 59
Zygorsaccharomyces friorianus, 100

GENERAL INDEX

See also Index of Microorganisms, page 395

Abortion of cattle, survival of organism

in soil, 28
Acridine, I 8 1, 182
Actinomyces lysozyme, 116
Actinomycetes, 1 08-1 10

antagonistic properties of, I lO-l 1 8
as antagonists. Fig. 4 (p. 59), 108-
129
against actinomycetes, 128
against agents producing plant dis-
eases, 128-129
against bacteria, no— 118
against fungi, 118, 1 51-153, 303
in soil, 1,6, 8, 9, no, 1 1 5-1 18
physiology, 108

substances produced by, 53-54, 1 19—
127, 198-200, 251, 329
in vivo activity, 129
types. Fig. 10 (p. 108)
Actinomycetin, 113-114, 119, 126,
129,171,314,319,329
chemical and biological properties,

171
effect on bacteria, 126, 228
therapeutic value, 129
toxicity, 178
Actinomycin, 83, 1 19-120, 165, 171,
219, 228, 241, 242, 298, 299,
319, 320, 329
antiluminescent activity, 81, 82
bactericidal action, 246—248
bacteriostatic action, Fig. 4 (p. 59),

220
chemical nature, I 89-191
compared with other antibiotics, 74,

219—223
compared with proactinomycin, 126
compared with tyrothricin, 242
crystals, Fig. 20 (p. 209)
differentiation of bacteria, 260
effect on bacteria, Fig. 5 (p. 62),

Fig. 27 (p. 251)
effect on fungi, 74, 153

Actinomycin {com.)

in vivo activity, 129

neutralizing agent, 256-257

toxicity, 178, 241, 298-299
Actinomycosis, 27, 127
Adaptation of bacteria to antibiotics,

257-259
Adaptive enzymes, 184
Aerobic bacteria as antagonists, 105-

106
Agar diffusion method, 63, 75-78, 84
Agar method for testing antagonistic ac-
tion, 61-63
Agar streak-dilution method, 72-73
Alfalfa, decomposition of, 43—44
Alfalfa-sick soils, 1 8
Algae, 6, 9, 330
Allicin, 170, 217, 330
Amebae, 9, 157
Anaerobic bacteria, 21, 105—107, 266,

272, 273
Anaxogramic method, 61
Animal excreta, See Human and animal

wastes
Animal pathogens, survival of, 26-35
Animals, microscopic

as antagonists, 154-162

In soil, 6

substances produced by, 215-216,
330
Antagonism, 13, 53-59

defined, 331

effect on organisms, 38—39

metabolite, 227

types,_45, 47-48
Antagonist, defined, 331
Antagonistic action, 50—52, Fig. 5 (p.

62)
Antagonistic index, loi, 102
Antagonistic interrelationships among

microorganisms, 36-39, 44-49
Antagonistic microorganisms

isolation and cultivation, 53—69

404

GENERAL INDEX

Antagonistic microorganisms (coni.)
production of antibiotic substances,
49, 64-69
measurement of activity of sub-
stances, 69-83
measurement of bactericidal action,

83-84
measurement of in vivo activity,

83-84
methods of growing organisms,
64-69
utilization for disease control, 263-

266
See also Actinomycetes; Animals,
microscropic; Bacteria; Fungi
Antagonistic substance, defined, 331
Anthrax, 85, 87, 88, 95

survival of organism in soil, 17, 27
treatment, 261

use of culture filtrate in treating in-
fections, 261
Antibacillin, 94
Antibacterial action, inhibition of,

225-228
Antibacterial index, 227, 33 1
Antibacterial spectrum, 319
Antibiosis, 37-39, 331
Antibiotic, defined, 1 70, 33 1
Antibiotic action, mechanism of, 223-

228
Antibiotic spectrum, defined, 331
Antibiotic substances

as means of difi'erentiation of bac-
teria, 259-260
antifungal action, 297-298
bactericidal action, 221, 245-250
bacteriostatic spectra, 220
chemical nature. See Chemical nature

of antibiotic substances
classification, 170, 228-229, 329-

330
compared with chemical antiseptics,

218-223
crystalline preparation, 197
defined, 69-70, 331
disease control. See Disease control
inhibition, 255-257
in vivo activity, 3 1 5
isolation, 3 1 9
manufacture, 319

Antibiotic substances (cont.)

mode of action, 70, 218, 228, 324-

325
produced by actinomycetes, 53, 119—

127, 189-200
produced by bacteria, 178—189
produced by fungi, 200-214
produced by yeasts, 214-215
production, 51, 70—72, 319
structural formulae, 182
See also sfecific substances

Antidotic, 189, 33 1

Antifungal substances, 107, 297-298

Anti-inhibitors, 227-228, 331

Antiluminescent test, 81, 82

Anti-penicillin, 240

Antiphage agents, 166

Antiseptics, 218-223, 228, 3 1 6-3 1 7

Antivirus, 263—264

Ants, fungi antagonistic to, 303

Aromatic oils as bacteriostatic agents,
183

Ascaris, 26

Ascomycetes, 147, 150

Ascorbic acid, eifect on actinomycin,
256

Aspergillic acid, 70, 133, 1 40, 141,
170, 171, 208, 213, 244, 258,
273, 320, 329
antiluminescent activity, 82
chemical nature, 171, 208
eff"ect on bacteria, 82, 224, 244
in experimental infection with CI.

ferfringens, 273
toxicity, 178

Aspergillin, 133, 165, 329

Assay value, 77

Associative interrelationships among
microorganisms, 41—44

Autolysin, 234

Autolysis, 251

Autolytic substance, 127

Autophage, 1 02

Autotoxins, 103

Bacillin, 92, 94, 171, 233, 320,

329
Bacillus mesentericus filtrate, bacteri-
cidal action of, 286

GENERAL INDEX

405

Bacitracin, 92, 94, 171, 188, 286-287,

299, 320, 329
Bacteremia, treatment, 295
Bacteria

aerobic and anaerobic, 9, 1 05-1 07
agents destructive to, 245-250
actinomycetes, I lO-i 1 8
insects, 159— 161
fungi, 107
maggots, 162

protozoa, 21-22, 154-158
substance found in milk, 216
substance produced by yeast, 214—

215
ticks, 161
as antagonists, Fig. 2 (p. 58), 85-
107
against agents producing plant dis-
eases, 151— 153
against bacteria, 85— 1 07
against fungi, 57, 58, 94-95.
1 51-153, Fig. 30 (p. 302),

307 _

against insects, 159— 161

against protozoa, I 5 7

against viruses, 163-164
cocci, 103-104
colon-typhoid group, 30—32, 99-

103 _

differentiation by means of antibiotic

substances, 259—260
effect on tumors, 168-169
enzymes, 224-225
fastness, 258

fluorescent bacteria, 95—99
in fecal matter, 19-22
in soil, 7-10, 26-35
lysis, 83, 87, 88, 91
metabolism, 224

nonspore-forming bacteria, 95—104
spore-forming bacteria, 87-95
substances produced by, 178-179,

329

survival in soil, 26-35

virulence, 297
Bacterial agar plate method, 55-56, 57
Bacterial cell division, interference by

antibiotic substances, 224
Bactericidal, defined, 331

Bactericidal action

methods of measuring, 79, 82, 83

of insects, 161
Bacterioantagonistic, defined, 331
Bacteriolytic agents in soil, 9
Bacteriolytic, defined, 331
Bacteriophage, 101, 161, 166, 168, 250
Bacteriostatic action, 69—72

inhibition, 255-257
Bacteriostatic and bactericidal agents.

See Bacteria, agents destructive to
Bacteriostatic, defined, 331
Bacteriostatic spectrum, defined, 59,

319, 331
Bacteriostatics, 47, 229
Bacteriotherapy, 261, 265, 331
Bacterization, 31 1
Basidiomycetes, antibiotics produced by,

131, 132, 146, 147
Biological conditioning, 224
Biological control of insects, 161
Biological control of plant diseases,

306-313
Biostatic complex, defined, 331
Blackleg organism in soil, 17, 27
Blood cells, hemolysis of, 84

as test of antibiotic activity of tyro-
thricin, 81

by gramicidin, 231, 254

by tyrocidine, 231

by tyrothricin, 231, 299
Blue staining of pulp, 149
Botulinus, 27, 189
Bovine mastitis

survival of organism, 28

treatment with clavacin, 280

treatment with gramicidin, 284-285
Bovine spermatozoa, 254
Bovine tuberculosis in manure, 33
Bran as a medium, 67
Bromo-aspergillic acid, 208
Brucella organism, survival of, 34
Bubonic plague organism, survival of,
28

Catlonic detergent, 232
Cattle tick, relation to Texas fever, 1 7
Cellulose decomposing organisms, 43
Cephalin, 256

406

GENERAL INDEX

Cevitamic acid, effect on streptomycin,

241
Chaetomin, 74, 133, 153, 171, 213,

329

Cheese as a source of antagonistic organ-
isms, 92
Chemical composition of soils, 4—5
Chemical nature of antibiotic substances,
I 70-2 1 7
classification of substances, 170—178
substances produced by actinomy-

cetes, 189-200
substances produced by animals, 215-

216
substances produced by bacteria,

178-189
substances produced by fungi, 200—

214
substances produced by higher plants,

2 1 6-2 1 7
substances produced by yeasts, 214-
215
Chemist, problems for, 321-322
Chemotherapy, 266-297, 322-324,

332
Chlorellin, 171, 330
Chlororaphin, i 81-182
Cholera bacteria
as antagonists, 103
survival, 28, 34
Cholera immune soils, 35
Chromogenic bacteria as antagonists, 99
Citrinin, 133, 140, 171, 182, 208-

209, 245, 254, 298, 320, 329
chemical nature, 171, 177
crystals, Fig. 20 (p. 209)
structural formula, 182
toxicity, 178, 298

Citrus canker, 305

Clavacin, 83, 133, 140, 169, 171, 182,

210, 219, 242-243, 298, 299,
303, 319, 320, 329

antiluminescent activity, 82
bactericidal action, 243
bacteriostatic spectrum, 220
chemical nature, 171, 1 77
compared with other antibiotics, 74,

219-221
effect on bacteria, 74, 210, 223, 242,

243

Clavacin (coni.)

effect on fungi, 242

effect on virus, 165

production, 144, 210

structural formula, 182

therapeutic value, 280

toxicity, 178, 280, 298
Clavatin, See Clavacin
Claviformin, See Clavacin
Clitocybin, 146, 172, 330
Clover-sick soils, 1 8
Coagulase, 225
Cocci, 259

as antagonists, 103— 1 04
Coccidiosis organism, survival in soil, 17
Colds

treatment with clavacin, 280

treatment with penicillin, 275
Colicines, 102, 172, 329
Coliform bacteria in soil, 10, 29
Colistatin, 94, 172, 329
Collodion sac method of testing antago-
nistic action, 60— 61
Colon index, 96
Colon-typhoid bacteria, 245

as antagonists, 99— 103

in manure, 21

in soil, 30-32
Competition among microorganisms,

44-45,
Corylophillin. See Penatin
Cotton root rot, control of, 312
Crepin, 330

Crowded plate method, 56-57
Cultivation of antagonistic microorgan-
isms, methods, 64-69
Cup method, 63, 75-78
Cylinder method, 63, 75-78
Cysteine, effect on streptomycin, 241
Cytolytic bacteria, 91

Damping-off disease, 27, 94, 309, 312
Definitions of terms, 331—332
Dehydrogenases, 81, 248-249, 253
Dermatophytes, 146
Differentiation of bacteria by means of

antibiotic substances, 259—260
Dilution method, 73-74, 84
Diphtheria, 48, 88, 90, 91, 259
survival of organism, 28

GENERAL INDEX

407

Diphtheria {cont.)

treatment of carriers with filtrate of
B. niesentericuSy 286
Diplococcin, 172, 189, 329
Direct antagonism, 46, 48
Direct microscopic method, 7
Direct soil inoculation method, 57—

59
Disease control

of fecal-borne diseases in China, 25-

26
of plant disease. See under Plants
of soil-borne diseases, 14-15, 304-

313

toxicity of antibiotic substances, 298

use of antibiotic substances, 261-299

use of microbial antagonists, 263—

266

Disinfectant spectrum, 222

Disinfectants, chemical, compared with

antibiotic substances, 218—223
Double plate method, 61-63
Dyes as bacteriostatic agents, 245
Dysentery bacteria, survival of, 28

E. colt

antagonists of, 31—32

influence of enrichment of soil with

organism, 29—30
survival of organism, 29, 31—32
E. colt factor. See Penatin
Endo-subtilysin, 92, 173, 329
Enzyme action, inhibition by antibi-
otics, 50, 254
Enzymes acting on polysaccharides,

183-184
Equilibrium among soil microorganisms,

38-39
Erythrin, 330
Eumycin, 172, 188, 329

Fastness of bacteria to antibiotics, 258

Fecal-borne diseases, 25-26

Fecal residues, 20-23

Fertilizer

effect on microbial population, 8, 26
use for disease control, 309

Fibrinolysis, 237

Flavacidin. See Flavicin

Flavatin. See Flavicin

Flavicin, 70, 133, 141, 172, 207

chemical nature, 172, 207

toxicity, 178
Flax blight, 305
Flax-sick soils, i 52
Flour protein, 170, 217, 330
Fluorescent bacteria as antagonists, 95—

99.
Fluorescin, I 80
Food competition, 44
Forced antagonism, 53, 58, 214
Formulae, structural, of antibiotic sub-
stances, 182
Freudenreich's method of testing an-
tagonistic action, 60
Fumigacin, 70, 133, 1 70, 172, 2lO-
21 1, 244, 298, 321, 329
antiluminescent activity, 81, 82
chemical nature, 172, 177, 210-211
crystals. Fig. 20 (p. 209)
effect on bacteria, 74, 244
production, 142—143, 144
toxicity, 178, 298
Fumigatin, 70, 1 33, 142, 170? I72>
182, 209-210, 329
chemical nature, 172, 177
structural formula, 182
Fungi

agents destructive to, 83, 99, 147-

153

actinomycetes, 118,151-153,303
bacteria, 63, 83, 94, I30-147j

151-153

substance produced by yeast, 62,

253
as antagonists, 56, Fig. 2 (p. 58),

Fig. 17 (p. 150), 130-153
against agents producing plant dis-
eases, 151—153
against bacteria, 130-147
against fungi, 62, 147-150, Fig.

29 (p. 302)
against insects and other animal
forms, 160—161
in fecal matter, 22
in soil, 1, 6, 8, 9, 15-16, 301
pathogenic to plants, control of,

306-313
relation to protozoa, 158

408

GENERAL INDEX

Fungi (coni.)

substances produced by, i, 130, 200-
214, 329 ^

survival in soil, 3 00
Fungicidal, defined, 332
Fungistatic, defined, 332
Fungistatic factor, 256
Fungistatic properties, 58
Fungus infections of animals, 16

Gangrene, 27, 266

Garbage disposal, 2 1 5

Garre's method of testing antagonistic

action, 61
Gigantic acid. See Flavicin
Gladiolic acid, 149, 172, 329
Gliotoxin, 70, 133, 142, 144-145,
170, 173, 211-212, 213, 244,
258, 298, 314, 319, 320, 329
antiluminescent activity, 81, 82
chemical nature, 173, 178, 21 1-2 1 2
compared with other antibiotics, 74,
222

crystals, Fig 20 (p. 209)

effect on bacteria, 73, 212, 223, 244

effect on fungi, 73, 150, 153

medium, 144

production, 72, 144-145

toxicity, 178, 244, 298
Glucose-oxidase, 225
Glutinosin, 173, 214, 329
Gonorrhea, treatment with penicillin,

276
Gramicidin, 70, 92, 170, 173, 228,
242, 261, 298

antiluminescent activity, 81, 82

chemical nature, 173, 178, 185-186

compared with other antibiotics, 74,
221

compared with penicillin, 221, 222,

237> 239
compared with tyrocidine, 221, 230
crystals, Fig. 20 (p. 209)
effect on bacteria, 82, 232
hemolytic action, 231, 254
inhibition, 256

mode of action, 226, 230, 248, 254
therapeutic value, 231, 280

Gramicidin (coni.)

toxicity, 178, 230, 280, 282, 283,

298
Gramicidin S, 92, 188, 286
Gramidinic acid, 82
Gram-negative bacteria in soil, 28-32
Gram stain and sensitivity to antibiotic

substances, 223
Growth-promoting substances, 46, 63,

150, 224

Helvolic acid. See Fumigacin
Hemipyocyanin, 82, 153, 179, 181,

219
Hemolysin production method of meas-
uring antibiotic activity, 83
Hemolytic action. See Blood cells, he-
molysis of
Hetero-antagonism, 47
Heterotrophic bacteria in manure, 21
Histone, 254

Human and animal wastes, 1 9-3 5
composting of, 308
destruction of microorganisms, 25-

26
garbage, 25

manure and fecal residues, 20—22
composition and decomposition,

22-23
microbial population, 20-22, 92
sewage, 24—25
survival of pathogens, 26-35
Humic acids, 4
Humus compounds, effect on actinomy-

cin, 256
Hydrogenase, 225
Hypholin, 229

Hyphomycetes, antibiotics produced by,
149

Inactivator, nontoxic, defined, 163—

164, 332
Inactivators of viruses, 163-166
Indirect antagonism, 46, 48
Infections, wound, 316-317

treatment, 275, 278
Influenza organisms, 28, 86
Infusoria, lysis of, 99
Inhibins, 330

GENERAL INDEX

409

Inhibition

of antibacterial action, 255-257

of antibiotic action, 255-257, 259
Inhibitive substance, defined, 332
Inhibitor, defined, 331-332
Inner antagonism, 226
Inorganic constituents of soil, 4-5
Insects

activity of fungi against, 1 59-161

bactericidal action of, 161

control of, 15 9-1 61

in soil, 6, 9, 17
Interference phenomenon, 167
Intestinal disturbances, treatment of,

265, 298
In vivo activities of antibiotic substances,

methods of testing, 83-84
lodinin, 173, 181-182, 320, 329

chemical nature, 173, 177

structural formula, 182
Iron utilization, 224
Iso-antagonism, 47, 250
Isolation of antagonistic microorgan-
isms, methods. See Methods of iso-
lating antagonistic microorganisms

Japanese beetle, control of, 160, 324
Javanicin, 145, 173, 329

Ketones, 241

Kojic acid, 173, 182, 213, 329

Lactenin, 1 70, 330

Lactic acid bacteria, 107, 265

Lactose fermenting bacteria, 1 01

Lauryl sulfate, 82

Leeches, 94

Leguminous plants, 3 I 3

Leprosy, 17, 28

Lichens, 217

Lipoids, 103, 104, 287

Liquid media for testing antagonistic

action, 60— 61
Litmocidin, 1 19, 126, 173, 329
Lysin of bacteria, 90, 226

of actinomycetes, 127

of bacteria, 56, 57, 83, 87,

105, 127, 251-252
of fungi, 152

Lysobacteria, 89
Lysogcnesis, defined, 332
Lysogcnic bacteria, 57-58
Lysozymc, 161, 170, 215-216, 245,
250, 3I4> 330

defined, 332

effect on bacteria, 2 1 5-2 1 6

of actinomycetes, 116
Lytic action

of actinomycetes, 110-114, 126-

127
of antibiotic substances, 93, 98, 105,
225, 249

Maggots, bactericidal action of, 162

Malarial parasites, 158-159

Maltol, 199

Manure. See Human and animal wastes

Manuring of soils, 308

Mastitis. See Bovine mastitis

Measurement of antibiotic activity,
methods. See Methods of measur-
ing antibiotic activity

Mechanical separation method of de-
termining abundance of organisms
in soil, 7

Mechanism of antibiotic action. See
mider Antibiotic substances

Medium, 60-63, 64-69

effect of aeration on antibacterial ac-
tivity of fungi, 137, 138-139
staling, 47

Meningitis, treatment of, 275, 278,
295, 296

Metablosis, 13

Metabolic processes of cells, interfer-
ence by antibiotic substances, 252—

255
Metabolic products, effect of, 60
Metabolite antagonism, 227
Methods of determining abundance of

microorganisms In soil, 6—9
Methods of growing organisms for pro-
duction of antibiotic substances,
64-69
Methods of isolating antagonistic micro-
organisms, 53-59' 319
bacterial agar plate, 55-56
crowded plate, 56
direct soil inoculation, 57—58

410

GENERAL INDEX

Methods of isolating antagonistic mi-
croorganisms (cont.)
"forced antagonism," 53, 58, 214
soil enrichment, 54-55
Methods of measuring antibiotic ac-
tivity, 69-83
agar diffusion or "agar cup," 78
agar streak-dilution, 72—73
antiluminescent test, 82
lysis of red blood cells, 84
serial dilution, 73—75
turbidimetric, 78-81
Methods of measuring bactericidal ac-
tion, 82-83
Methods of testing antagonistic action

of microorganisms, 59-69
Methods of testing in vivo activity of

antibiotic substances, 83—84
Microbial cell, physiology of, 325
Microbiological equilibrium, 156
Microbiological population, 3-4
Microbiologist, problems for, 318—321
Micrococci as antagonists, 104
Micromonosporin, 119, 329
Microorganisms

disease-producing, i 3—1 5
in animal excreta, 20—23, 85—87
in soil, 1-2, 6-9, 3 1 > 3 2, 3 3-3 5
nutrition of, 1 1— 12
See also Actinomycetes ; Animals,
microscropic; Antagonistic micro-
organisms; Bacteria; Fungi;
Mixed cultures
Microscopic methods of determining
abundance of microorganisms in
soil, 7
Milk, bactericidal action of, 216
Milky disease of larvae, 94, 160
Mixed culture inoculation for testing

antagonistic action, 63
Mixed cultures, 86, 104

antagonistic interrelationships, 45-

49 _ ^

associative interrelationships, 41—44
competitive interrelationships, 44-45
growth of microbial cells, 12—13, 40
mutualistic relationships, 36-39, 41-

42
nature, 40-41
Mixed infections, 263-264

Mixed population. See Mixed cultures
Mode of action of antibiotic sub-
stances, 70, 218, 228, 324-

325
Morphology, 103, 223, 250-253
Much-lysin, 90
Mushroom fungi, 9
Mutaflor, 266

Mutants of P. notatum, 136, I 39
Mutations of bacteria, 259
Mutualistic relationships among micro-
organisms, 36-41
Mycetin, 119, 126, 173, 329
Mycobacteria and streptomycin, 288,
289-291, 296
in soil, 17

inhibition by streptomycin and strep-
tothricin, 288
Mycocidal, defined, 332
Mycoin, defined, 332
Mycolysate, 53, 113, 129, 265
Mycophagy, I 30

Mycophenolic acid, 130, 173, 213, 259
Mycorrhlzal fungi, 303
Mytogenetic rays, 51

Natural selection of bacteria, 259

Nematodes, 158, 159, 160, 162

Neocolysin, 265

Nitrite production method for measur-
ing activity of penicillin, 81

Nitrogenous materials, effect on fungi,
307-308

Nonspore-forming bacteria, 95-104

Notalysin, 173

Notatin. See Penatin

Nutrition of microorganisms, 11-12,
223

Organic matter of soil, 4
Organic media, 64
Otitis media, 295, 296
Oxford unit, 77, 204, 332
Oxygen supply of soil, 3
Oxyphenazine, 179, 287

Pantoyltaurine, 226
Paper-disc method, 63, 75-78
Paper-pulp fungi, 150, 310

GENERAL INDEX

41:

Para-amino-benzoic acid, effect on
penicillin, 226-227, 229, 239-
240, 256, 325

Paracolon bacteria, 102

Parasiticin, 329

Parasitism, 13, 37-38

Paratyphoid bacteria, 93

Partial disinfection of soil, 312

Partial sterilization of soil, 155,

304

Passive antagonism, 48

Pathogenic organisms
in fecal wastes, 21—22
in soil, 14-15, 16-18, 26-35

Patulin. See Clavacin

Penatin, 70, 133, 169, 170, 173, 207,

329

Penicidln, 133, 140, 174, 213, 320,

Penicillamine, 207

Penicillic acid, 133, 140, 174, 182,
209, 228, 245, 319, 320, 329
chemical nature, 174, 177
structural formula, 182
Penicillin, 81, 131, 163, 169, 170,
174, 213, 298, 299, 319, 320,

329
administration, 272
animal experiments, 272—275
antiluminescent activity, 81, 82
as prophylactic, 275
assay, 204-205
bactericidal action, 221, 234—235,

249-250
bacteriostatic spectrum, 76, 220, 236
chemical nature, 174, 178, 200-

207
chemotherapy, 274, 275—280
compared with

chemical substances, 70, 221—222

other antibiotics, 219, 222, 232,
237, 242

sulfanilamide, 237

sulfathiazole, 235

sulfonamide, 237-240, 268, 269,
277
crystals, frontisfiece
differentiation of bacteria by, 259-

260
discovery, 132, 314—316

Penicillin (cont.)

effect of other agents, 226

effect of reaction, 234

effect on bacteria, 80, 225, 234,

236-237, 247-248, Fig. 27 (p.

251), 252
effect on cell morphology, 251—253
effect on fungi, 236
effect on metabolism, 255
effect on phage, 165
effect on sea urchin eggs, 240
effect on virus, 165, 166
failures, 279

inactivation, 239, 240, 255-256
inhibitor, 259
in wounds, 3 1 7
isolation, 202-204
lysis of bacteria by, 234
measurement, 73, 75—78, 80-85
medium, 65—67
mode of action, 234—240
pharmacology, 279
production, 132-140, 200-201,

205
resistance of bacteria to, 235, 257—

259, 270
selective action, 267—269
sporocidal action, 235, 252
submerged culture for production,

137-139
surface culture for production, 137-

138
therapeutic value, 266, 267—280,

317
toxicity, 178, 267-269, 271, 272,

276
Penicillin B. See Penatin
Penicillin F, 139, 206, 272, 275
Penicillin G, 139, 206, 273, 275
Penicillin K, 139, 206, 273, 275
Penicillin X, 139, 140, 2o6, 236, 273,

275
Penicillinase, 82, 147, 226, 230, 255—

256, 258
Penicillin-like substances, 140
Peptones as bacteriostatic agents, 183
Persisters, 270
Pfeiffer's bacillus, 259
Phage in soil, 6, 9

412

GENERAL INDEX

Phage of bacteria, 163, 165, 166, 226,

250
Phenazine, 181, 182
Phenol, 69, 70, 82, 222

coefficient, 70
Phthiocol, 105, 174, 329
Phycomycetes, 131, 132
Physiologist, field for, 324-325
Physiology of bacteria, effect of anti-
biotic agents, 87, 253-255, 325
Pigment formation and antagonism,

42-43, 95, 99
Pigments, 49, 108
Pinosylvin, 330
Plant viruses, 167
Plants

agents pathogenic to, 18, 300— 301
antibacterial agents produced by,

216-217, 225, 330
bacteria pathogenic to, 93
bactericidal action of juice, 216
diseases of, control, 300-313
by fungi, 253, 305-310
by use of antagonistic microorgan-
isms, 93, 304-313
fungi pathogenic to, 302-313
influence on microbial population of
soil, 5, 8
Plate culture method, 6-7
Pneumococci as antagonists, 104, 105
Pneumonia organism, survival, 28
Poliomyelitis, 167
Polypeptides, 185-189
Polyporin, 146, 174, 178, 213, 330
Polysaccharidases, 183-184, 296-297
Potato scab, 128-129, 307
Precursor for streptomycin, 68, 125
Proactinomycin, 119, 126, 174, 242,
320, 329
chemical nature, 200
compared with actinomycin, 126
Prodigiosin, 174, 181, 329
Production of antibiotic substances,
methods of growing organisms for,
64-69
Protamine, 228, 254
Protoanemonin, 217, 330
Protozoa

in manure and urine, 21—22
in soil, 6, 9, 42

Protozoa {cont.)

relation to bacteria, 22, 32, 154-157

relation to fungi, 158
Protozoan theory of soil fertility, 155
Pseudodiphtheria, 48
Puberulic acid, 174, 212—213, 329
Puberulonic acid, 174, 329
Pure cultures, 12-13, 86
Pyo-compounds, 98, 1 74, 183, 320,

329
Pyocyanase, 51, 70, 85, 97, 174, 258,
314, 319, 320, 329

antiluminescent activity, 82

bactericidal action, 246

chemical nature, 98, 174, 180

effect on bacteria, 82, 219, 222, 250

isolation, 1 78-1 80

practical value, 98, 262, 266, 287

toxicity, 298

virus Inactivation, 164
Pyocyaneus organism as antagonist, 95,

Pyocyanic acid, i 79
Pyocyanin, 70, 98, 153, 174, 298,
314, 319, 320, 329

antiluminescent activity, 82

chemical nature, 1 74, 178, 1 8 1, 1 82

effect on bacteria, 82, 98, 219, 222-
223, 249

inhibitory action, 225

isolation, 178-180

structural formula, 182

toxicity, 298
Pyolipic acid, 98, 175, 179, 329
Pyoxanthose, I 80
Pyrogenic substances, 239

Quinine, 170, 209-210, 284
Quinones, 245

effect on bacteria, 22, 227, 246-247

Radiations, 51

Repressive antagonism, 48

Resistance of bacteria to antibiotics,

257-259, 270
Respiration of bacteria, 229
Respiratory infections, 275
Rhizosphere, 5, 313
Rickettsiae, affected by penicillin, 166
Root-inhabiting fungi, 301
Rust spores, inhibition of, 152

GENERAL INDEX

413

Saliva, antibacterial action of, 216
Saprophytic organisms

for disease control, 263

in soil, 15-18
Saprophytism, 38
Sarcoma cells, 169
Sea water, bactericidal action of, 32
Seed inoculation, 3 1 1
Selective culture method, 7
Semisolid media, 63, 81
Serial dilution method, 73-74, 84
Sewage, 24-25, 85, 92
Silkworms, destruction of, 159
Simplexin, 92, 94, 175, 189, 233, 320,

329
Simultaneous inoculation method, 60,

61, 102, 106

Skin diseases, 150

Slime disease, 308

Smuts, bacteria antagonistic to, 151

Soil

actinomycetes in, 6, 8, g, 1 1 5-1 1 8

as culture medium, 10— 1 1

as habitat of microorganisms, 1-2

bacteria in, 6, 8, 9, 1 1, 92

biological state, 5—6

chemical composition, 4—5

effect of heating, 304

effect on plant growth. Fig. 34 (p.

254)
fungi in, i, 6, 8, 9-10, 149
invaders, 301

microbial population. See Soil micro-
organisms
pathogens in, 16-18, 27-36
physical properties, 2-4
saprophytes in, 15, 18
treatment, effect on microorganisms,
8
Soil-borne diseases, 300—313
methods of control, 14—15
Soil enrichment method, 53—55
Soil inoculation method, 57-59
Soil microorganisms, 6—9, 34—35
effect of bacteria, 29
equilibrium, 38
nature of, 16— i 8
Solid media for testing antagonistic ac-
tion, 61-63, 64

Space antagonism among microorgan-
isms, 44, 50
Spectrum, bacteriostatic, 59, 76, 319,

331
Spinulosin, 70, 133, 142, 170, 175,

329

chemical nature, 175
Spirocheticidal action of penicillin,

235, 247-248
Spore-forming bacteria

antibiotics produced by, 233—234

as antagonists, 56, 87-95
Spore germination, 149
Spores, preparation of, 65, 68
Sporicidal action of penicillin, 235
Spot inoculation method, 63
Stable manures, 20-23
Staling of medium, 47, 152
Staphylococcus aureus infections, treat-
ment with penicillin, 275, 278
Staphylococci as antagonists, 104
Sterilization of soil, 304

effect on coliform bacteria, 10

partial, 155, 304
Streptamine, 198, 199
Streptidine, 197-199
Streptococcal infections, treatment of,

275
Streptococci as antagonists, 103
Streptomycin, 75, 127, 128, 153, 165,
175> 299, 319, 326, 329

activity, 193-194

administration, 290, 292, 293-294

agar, 78

agar diffusion method for measuring
activity, 84

bacteriostatic spectrum, 1 24

chemical nature, 175, 178, 193-200

chemotherapy, 266, 287-296

compared with other antibiotics, 74

crystals, Fig. 19 (p. 196)

curve, 79

discovery, 316

effect on bacteria, 74, 124, 223,
317-318

effect on phage, 165

inactivation, 195, 199-200, 225,
240-241

in vivo activity, 83

isolation, 194

414

GENERAL INDEX

Streptomycin {cont.)
lysis of organism, 127
media, 68-69
precursor for, 125
production, 119, 124-125, 193
resistance of bacteria to, 241, 257—

259
toxicity, 178, 287, 290, 292-293
tuberculosis treatment, 288, 289—

296
tularemia treatment, 288, 289
urinary infections, treatment, 288,
294, 296
Streptomycin unit, defined, 332
Streptothricin, 75, 82, 11 9-1 24, 129,
153, 165, 175, 240-241, 298,
299. 3195 320, 329
bacteriostatic spectrum, 220
chemical nature, 175, 191— 193,

199-200
compared with other antibiotics, 74,

219-223, 226
diiferentiation of bacteria by, 260
effect on bacteria, 74, 123, 223, 251
effect on fungi, 74, 124
media, 68-69
production, 11 9-1 24
resistance of bacteria to, 241
therapeutic value, 287, 288, 292
toxicity, 178, 287
Structural formulae of antibiotic sub-
stances, 182
Submerged growth, 65, 67, 109
Subtilin, 88, 92, 163, 175, 188, 233,

286-287, 299, 319, 320, 329
Subtilysin, 92, 188, 329
Successive inoculation method, 61
Sulfanilamide

antiluminescent activity, 82
compared with penicillin, 226-227,

235, 237-238
effect on bacteria, 82
inhibition of antibacterial action,
255
Sulfhydryl groups, 255
Sulfonamide, 232, 258
bacteria resistant to, 276
compared with penicillin, 268, 269,
277
Suppressor, defined, 3 3 i

Surface tension, 225, 254

Surgical infections, treatment of, 275,

278
Symbiosis, 13, 37—39, 42
Synergism, 39

Synthetic media, 64, 66-67, 68
Syphilis, treatment with penicillin, 277

Take-all disease, 39

Tannic acid, effect on actinomycin, 56

Temperature for growth of antagonists,

.65
Testing antagonistic action, methods,

59-69
Testing in vivo activity, methods, 83—

84
Tetanus organisms, survival of, 27
Tetanus toxin, 169
Texas fever, 17

Thermolabile substances, 46, 89
Thermophilic actinomycetes, 127
Thermophilic bacteria, 89
Ticks, bactericidal action of, 161
Tissue culture, 81
Tolu-/)-quinone, 82
Tomatin, 330
Toxic substances, 46
Toxicity of antibiotic substances, 178,

229, 241, 286-287, 297, 298-

299
Toxin destruction, 169, 233
Toxin of bacteria, 163
Toxin production, 42, 47, 106, 304
Toxoflavin, 189, 329
True antagonism, 46, 48
Trypanosome parasites, 158—159, 276-

277
Trypanotoxin, 158, 175, 329, 332
Tubercle bacillus, 28, 33—34

inhibition by microorganisms, 146,

245
in human and animal wastes, 21
Tuberculocidin, 245, 332
Tuberculosis, chemotherapy of, 263,

289-296
Tuberculostatic, defined, 332
Tularemia, treatment of, 288, 289
Tumors, 168—169
Turbidimetric method, 78-8 1

GENERAL INDEX

415

90,

295,

Typhoid organism, 85-86, 87, 8i
93, lOi, 242
as antagonist, 103
in soil, 28, 30-32, 85-86
treatment of, 264, 265, 294,
296
Typhus rickettsiae, 166, 273
Tyrocidine, 70, 92, 170, 175, 298,

329 .

bactericidal action, 282

chemical nature, 175, 178, 186-187

compared with gramicidin, 221-

222, 230
compared with penicillin, 239
crystals, Fig. 20 (p. 209)
effect on bacteria, 70, 225, 232
effect on protozoa, 158
hemolytic effect, 231
mode of action, 221, 230—233,
therapeutic value, 231
toxicity, 178, 298
Tyrothricin, 65, 81, 88, 92, 153,

175, 242, 266, 298, 316,

320,329
adaptation of bacteria to, 232
administration, 282
antiluminescent activity, 82
bactericidal action, 281, 282
chemical nature, 175
differentiation of bacteria by, 260
effect on bacteria, 82, 219, 230
hemolytic effect, 231, 299
inhibition, 230, 256
limitation, 286

mode of action, 229-233, 254
production, 71, 92
therapeutic value, 272-273, 280-

286

254

163,
3i9>

Tyrothricin (coni.)

toxicity, 178, 230, 282, 284

Udder infections. See Bovine mastitis
Ultramicroscopic forms in soil, 6
Urinary infections, 275, 294, 295, 296
Urine, bacterial composition of, 21
Ustin, 142, 175, 329
Utilization of antibiotic substances. See
Disease control

Vaccination of medium, 47
Violacein, 175, 182, 329
Viridin, 145, 175, 212, 321, 329
Viruses

antagonisms among, 163-169

inactivators of, 164—166
Vitamins, 1 1, 42
Vivicillin, 279

Wastes, human and animal. See Human
and animal wastes

Water

as culture medium, 10— II

as habitat of microorganisms, 1—2

Wonder drug, 316

Worms, 9

Wound infections, 316—317
treatment, 275, 278

Yeasts, 45

in soil, 9

substances produced by, 2 1 4-2 1 5,
253> 255, 321

utilization, 68, 265-266
Yellow fever virus, 167