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MICROBIAL ANTAGONISMS AND 
ANTIBIOTIC SUBSTANCES 



LONDON 

HUMPHREY MILFORD 

OXFORD UNIVERSITY PRESS 



Microbial Antagonisms 

AND 

Antibiotic Substances 

SELMAN A. WAKSMAN 

PROFESSOR OF MICROBIOLOGY, RUTGERS 

university; microbiologist, new JERSEY 

AGRICULTURAL EXPERIMENT STATION 



"L^ Vie em^eche la vie*'' — Pasteur 



NEW YORK 
THE COMMONWEALTH FUND 

1945 



COPYRIGHT, 1945, 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 book is affectionately dedicated to 

BOBILI 

who has stimulated me in moments of defression^ 

who has been at all times an inspiration in the 

search for the unknown^ my constant associate 

and antagonist 



PREFACE 

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 J 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, others are not. Some are readily oxidized 



viii PREFACE 

and destroyed, others are not. Some are subject to destruction by spe- 
cific enzymes. The substances are largely bacteriostatic in action, to a 
lesser extent bactericidal j some are also fungistatic and fungicidal. 

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- 
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 



PREFACE ix 

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 (83, 229) and special papers (134, 166, 
251, 256, 354, 355, 539, 540, 584, 616, 796, 799, 800, 838) ; special at- 
tention has been paid to the occurrence of such organisms in the soil 
(620, 794). Advantage was taken of these reviews in the preparation of 
the comprehensive bibliography presented at the end of this mono- 
graph. Attention is directed also to a recent complete review of the lit- 
erature on the nature and formation of penicillin, the historical develop- 
ment of our knowledge of this agent, method of assaying, and clinical 
application (678). 

The author expresses his sincere appreciation to the members of the 
staff of the Microbiology Department, New Jersey Agricultural Ex- 
periment Station j 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 j to Dr. G. A. Harrop 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 careful 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 75, 1944 



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 38 

4. Isolation and Cultivation of Antagonistic Microorganisms} 
Methods of Measuring Antibiotic Action 55 

5. Bacteria as Antagonists 80 

6. Actinomycetes as Antagonists 102 

7. Fungi as Antagonists 124 

8. Microscopic Animal Forms as Antagonists 143 

9. Antagonistic Relationships between Microorganisms, Vi- 
ruses, and Other Nonspecific Pathogenic Forms 152 

10. Chemical Nature of Antibiotic Substances 156 

11. The Nature of Antibiotic Action 189 

12. Utilization of Antagonistic Microorganisms and Antibiotic 
Substances for Disease Control 221 

13. Microbiological Control of Soil-borne Plant Diseases 246 

14. The Outlook for the Future 259 
Classification of Antibiotic Substances 270 
Glossary 271 
Bibliography 273 
Index of Microorganisms 331 
General Index 339 



58352 



CHAPTER I 

SOILS AND WATER BASINS AS HABITATS 
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 SOILS 

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 



PHYSICAL PROPERTIES OF SOILS 3 

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- 
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 is marked change in 
the microbiological population of the soil : the fungi and actinomycetes 
tend to decrease, and the bacteria, especially the 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. X \3\>» ^'^/^S^ 



4 MICROORGANISMS IN SOILS AND WATER BASINS 

The 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- 
proaching 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 fH 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 SOILS 

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: 1 5 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" 
(922). 



BIOLOGICAL STATE OF THE SOIL 5 

The inorganic constituents of the 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 
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 true 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 (919). 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- 



6 MICROORGANISMS IN SOILS AND WATER BASINS 

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- 
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. 

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 soilj 
namely, plate culture, selective culture, direct microscopic methods, 
Contact slide, and mechanical separation. Each of these has certain ad- 



SOIL MICROBIOLOGICAL POPULATION 7 

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- 
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 

(--57). 

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 



8 MICROORGANISMS IN SOILS AND WATER BASINS 

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 

TABLE I. INFLUENCE OF SOIL TREATMENT ON NUMBER 
OF MICROORGANISMS 





REACTION 


TREATMENT OF SOIL 


OF SOIL 




f¥L 


Unfertilized and unlimed 


4.6 


Lime only added 


6.4 


Potassium salts and phosphates 




added 


5-5 


Salts and ammonium sulfate 




added 


4.1 


Salts, ammonium sulfate, and 




lime added 


5.8 


Salts and sodium nitrate added 


5-5 


Stable manure and salts added 


5.4- 



MICROORGANISMS FOUND* 

Bacteria Actinomycetes Fung 
3,000 1,150 60 

5,410 2,410 23 

5,360 1,520 38 

2,690 370 112 



6,990 


2,520 


39 


7,600 


2,530 


4-7 


8,800 


2,920 


73 



From Waksman (925). 

* 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 (848). 

* In thousands per gram of soil. 









SOIL MICROBIOLOGICAL POPULATION 9 

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 
a mass of living matter as do the bacteria j 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 
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 Rhizofus, Mucor^ 
Penkilliumy Aspergillus, Trkkoderma, Fusarium, Cladosforium, and 
Cefhalosforium. The soil often harbors an abundant population of 
yeasts and fleshy or mushroom fungi. The latter may produce an ex- 



10 MICROORGANISMS IN SOILS AND WATER BASINS 

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 fluorescens (139, 140) may be due to the 
antagonistic action of the latter. 



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 



SOILS AND WATER BASINS AS CULTURE MEDIA 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 bacterium 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- 

TABLE 3, MULTIPLICATION OF COLIFORM BACTERIA IN STERILE SOIL 





BACTERIA 


ORGANISM 


INOCULATED* 


Escherichia coli 




in soil alone 


2,600 


Aerobacter aero genes 




in soil alone 


109,000 


in soil and glucose 


109,000 



BACTERIA RECOVERED* 

After 10 days After 26 days 

149,000,000 138,000,000 

48,000,000 42,600,000 

1,660,000 240,000,000 



From Waksman and WoodruflF (949). 
* Per gram of soil. 

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 j 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 that would favor the rapid development of the organism in ques- 
tion. Special strains or races of microorganisms may often be developed 
as a result of such treatment, which tends to favor the adaptation of the 
organisms present in the soil to a particular process. ■ ^iKl C.37S. 



-.,.jii«^c: 



12 MICROORGANISMS IN SOILS AND WATER BASINS 

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 
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; others are restricted to the use of a single 
group of compounds such as proteins, amino acids, urea, ammonia, or 
nitrate; 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; 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- 



GROWTH OF THE MICROBIAL CELL 13 

gen in natural substrates and is responsible for the continuous stream 
of ammonia and nitrate forming the available sources of nitrogen that 
makes 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 J the rate of growth then diminishes. The maximum population 
of Aerobacter aero genes grown in a medium containing lactose and 
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. It has been shown (936), for example, that certain bacteria 
like Bacillus cereus can attack native proteins but not amino acids, 
whereas others like Pseudomonas fluorescens can attack amino acids but 
not proteins J when these two organisms were placed together in the 
same medium, their activities supplemented one another. Numerous 



14 MICROORGANISMS IN SOILS AND WATER BASINS 

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 DI SE ASE- PRODUCI N G 
MICROORGANISMS 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- 
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 those which lead a harmless existence in soils or water 
basins J 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 



SAPROPHYTIC ORGANISMS IN THE SOIL 15 

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 
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 (32). 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 



16 MICROORGANISMS IN SOILS AND WATER BASINS 

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. 

The ability of other bacteria, notably members of the Azotobacter 
group, to fix nitrogen independently of host plants and the fact that 
these organisms are absent in many soils led to the suggestion that such 
soils might benefit from inoculation. It has been found, however, that 
when soils and peats are inoculated with Azotobacter chroococcum 
large-scale destruction of the latter often occurs isil^)- This is believed 
to be due to the presence in the soil of antagonistic organisms as well as 
toxic substances (477, 648, 951). 

Certain fungi are unable to grow in fresh nonsterilized soil but are 
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 was found to act injuriously upon the growth of the 
fungus Pyronema; the injurious effect was partly removed on boiling 
(500). 

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 



SAPROPHYTIC AND PATHOGENIC MICROORGANISMS 17 

SforotrichufUy various actinomycetes such as those causing lumpy jaw of 
cattle, and certain other organisms. Henrici (396) 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 j 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 (952) 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 
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 J 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 ; 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)^ the parasite car- 
rier, to persist in the soil (836). 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 



18 MICROORGANISMS IN SOILS AND WATER BASINS 

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. 

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" (169, 474). In order to 
overcome this condition, the breeding of resistant varieties of plants has 
been recommended. 



CHAPTER 2 

HUMAN AND ANIMAL WASTES 

A7td a place shalt thou have without the camf, whither thou shalt 
go forth abroad: and a spade shalt thou have with thy weapons ; 
and it shall he, when thou sittest abroad, thou shalt dig therewith, 
and sh-alt 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 (439, 
922). 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 

Microbial Population 

Fresh excreta of animals and man are rich in fecal bacteria, consisting, 
on the average, of 5 to 20 per cent bacterial cells (802). Lissauer (533) 
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 as much as 9 to 42 per cent of the bulk of animal 
stools, the number depending on the composition of the foodstuffs, the 
nature of the animal and its condition of health, and other factors (364). 
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. Osborne and Mendel (659) removed from the feces of 
white rats the residual food material and found that the bacterial cells 
made up 23 to 41 per cent of the total material j the nitrogen content of 
these cells varied from 10.7 to 12.2 per cent. Since the removal of the 
residual foodstuffs consisted in treatment with ether, alcohol, and acid, 
some of the bacterial cell constituents were also removed j the actual 



STABLE MANURES AND FECAL RESIDUES 21 

concentration of bacterial substance may, therefore, have been even 
greater. 

It has been reported (441 ) that i ml. of the intestinal contents of cat- 
tle contains 10 billion cells capable of development. By suitable methods 
of cultivation, human feces were found (588) to contain 18 billion 
bacteria per gram. Determination (542) of the number of bacteria in 
stable manure gave 1 1.6 billion cells per gram of material by the plate 
method, and, by the dilution method, 5 billion peptone-decomposers, 
100 million urea-decomposers, and 2.5 million cellulose-decomposers. 
About 100 billion bacteria may be produced daily in the human in- 
testine. Human feces are made up, on an average, of 32,4 per cent bac- 
terial cells amounting to 2,410 millions of bacteria per milligram of 
moist material. Feces of healthy persons were shown (300) to contain 
8.2 to 24.2 per cent bacterial cells j in those of persons suffering from 
intestinal disturbances the percentages were 20.1 to 40.2. With the de- 
velopment of the microscopic technique for counting bacteria, much 
larger numbers of cells were shown to be present than could be deter- 
mined 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 (775). 

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 (543). 

Various methods have been developed for permitting the prefer- 
ential development of certain types of bacteria. Gram-negative bacteria 
in the feces can be repressed by certain reagents (839) j gram-positive 
bacteria can be repressed by the addition of antibiotic substances such as 
actinomycin (Table 4). 

The bacterial population of fresh cow manure was found (796) to 
consist of 47.5 per cent streptococci {Streftococcus -pyogenesy Sarcina sp., 
and Micrococcus candkans) ,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 BacteroideSy Flavobacteriuniy 



22 



HUMAN AND ANIMAL WASTES 



TABLE 4. EFFECT OF ACTINOMYCIN ON THE MICROBIOLOGICAL 
POPULATION OF CERTAIN NATURAL SUBSTRATES 







MILLIGRAMS 








DILU- 


OF ACTINO- 








TION 


MYCIN PER 








FOR 


10 MILLILITERS 


COLONIES 


TYPES OF BACTERIA 


SUBSTRATE 


PLATING 


OF AGAR 


ON PLATE 


ON PLATE 


Air-dry soil 


1,000 





Numerous 


Largely gram-positive, 
many spore formers 




1,000 


O.OI 


Fewer 


Gram-negative 




1,000 


O.IO 


96 


Gram-negative 




1,000 


1. 00 





None 


Fresh soil 


1,000 





Numerous 


Largely gram-positive 




1,000 


O.OI 


Fewer 


Gram-negative 




1,000 


O.IO 


Few 


Gram-negative 




1,000 


1. 00 





None 


Fresh milk 


100 





790 


Gram-positive and 
gram-negative 




100 


O.OI 


346 


Gram-negative 




100 


O.IO 


251 


Gram-negative 




100 


1. 00 


I 


Gram-negative 


Fresh sewage 


1,000 





1,248 


Mostly gram-negative 




1,000 


O.OI 


1,172 


Gram-negative 




1,000 


O.IO 


1,131 


Gram-negative 




1,000 


1. 00 


121 


Gram-negative 



From Waksman and Woodruflf (945). 

PseudomonaSy Bacillus^ various anaerobic bacteria, Oidium, 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 (775) 
in manure : Bacillus subtilisj Bacillus mesentericus y Bacillus cereus. Ba- 
cillus tumescenSy Bacillus fetasiteSy Pseudomonas fiuorescenSy Pseudo- 
monas futiday Salmonella enteritidisy Escherichia coliy Proteus vul- 
garis y Micrococcus luteusy Micrococcus candicansy Staphylococcus alhusy 
Sarcina jiavay Streptococcus -pyogeneSy and others. Anaerobic bacteria 
are also abundant (329). 



STABLE MANURES AND FECAL RESIDUES 23 

Pathogenic bacteria may also occur frequently in human feces and 
in stable manure j Alycobacter'min tuberculosis and various hemolytic 
streptococci (830), as well as pathogenic anaerobes including Clos- 
tridium voelchiiy Clostridium se-pticum^ Clostridium^ oedematis y and 
Clostridium jallax have been found (468). 

The protozoa capable of developing in manure and in urine include 
not only saprophytic forms but also certain parasites, such as Tricho- 
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 Polytoma uvella^ Cryftochilum nigricans y and Tetramitus 
rostratus. These protozoa nearly all feed upon bacteria. The infusoria 
may feed upon smaller protozoa, so that forms like Colpidium may not 
destroy bacteria at all. 

Human and animal excreta also contain a large population of fungi, 
chiefly in a spore state. Schmidt (801) 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. 

Composition 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- 



24 



HUMAN AND ANIMAL WASTES 



stuffs (454). As soon as voided, manure begins to undergo rapid de- 
composition. This results in the formation of ammonia (140) and vari- 
ous other nitrogenous degradation products (290). These give rise to 
offensive smells, which are controlled by the conditions of decomposi- 
tion. 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 is taken by a population concerned in the decomposition of 
cellulose, hemicelluloses, and proteins (922). 

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 80° 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 




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



50 55 60 65 



Figure I. Influence of straw bedding upon temperature changes in the 
composting of manure. Circles indicate times of turning composts. From 
Waksman and Nissen (940) 



SEWAGE 25 

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. 



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 (334). Activated sludge, for example, has been shown 
(853) to possess a definite and consistent bactericidal action against the 
colon bacteria. In addition to antagonistic organisms, active bacterio- 
phages against nearly all types of intestinal bacteria are present in sew- 
age. 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- 



26 HUMAN AND ANIMAL WASTES 

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- 
tivities of the saprophytes (772, 980). 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 has 
conserved all the valuable elements essential for plant growth. 



SURVIVAL OF PATHOGENS IN SOIL AND WATER 27 

DESTRUCTION OF INJURIOUS MICRO- 
ORGANISMS 

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. According to Win- 
field (990), fecal-borne diseases rank with venereal disease and tuber- 
culosis as the most important infectious diseases of China, because the 
people do not maintain proper sanitation and because human excreta are 
used as fertilizers. Any successful system for the control of these dis- 
eases must be sanitary and at the same time profitable. The composting 
method can meet these requirements. 

To illustrate this point, it is sufficient to consider an analysis (990) 
of the occurrence of Ascaris lumbricoides, its transmission, and its rela- 
tion to Entamoeba histolytica. 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 com- 
posting of feces, sufficient heat was produced to destroy disease-produc- 
ing organisms and their reproductive bodies. The compost thus pro- 
duced is highly effective as a fertilizer. 

Many other natural substrates, like saliva (704), possess antibacterial 
properties due to the antagonistic action of their own bacterial popula- 
tions. Though antagonistic microorganisms may persist in soil or in 
other natural substrates, substances toxic to bacteria soon tend to be 
destroyed (444). 

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 



28 HUMAN AND ANIMAL WASTES 

may survive there and thus become a constant and important source of 
infection. The introduction by Koch, in 1881, 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, 
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 j 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 5 the hay 
and feed from these lands may transmit the disease to animals. The fact 
that certain fields carry anthrax infection 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 



SURVIVAL OF PATHOGENS IN SOIL AND WATER 29 

these organisms received particular attention during the first world 
war, which was fought chiefly in trenches (957). 

Another important pathogenic anaerobe able to survive in soil for 
long periods of time is Clostridium chauvoeiy the causative agent of 
blackleg in cattle j southern pastures are said to be better carriers of 
blackleg than northern pastures. 

Clostridium tetani is also widely distributed in the soil and appears to 
be associated with the use of stable manures, Nicolaier (638, 639) 
found, in 1884, that tetanus could be produced in experimental animals 
by the injection of soil samples, 69 positive results being obtained from 
140 inoculations. This organism is believed to occur in the soil in the 
form of spores ; its mode of survival, however, is not sufficiently known 
because of a lack of careful study. 

The botulinus organism not only may remain alive in the soil for a 
long time (602), 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 (710), 

Thus we see that pathogenic spore-forming bacteria are always found 
in the soil. Other pathogens are able to survive in the soil only for lim- 
ited periods of time. They are eliminated sooner or later from the soil, 
either because of their inability to compete with the soil population or 
because of their actual destruction by the latter. Although the patho- 
gens seem to possess considerable resistance toward unfavorable soil con- 
ditions, they are unable to multiply at rates permitting their indefinite 
survival in the soiL The anthrax bacillus and certain other parasites in- 
festing domesticated and wild animals belong to this group. Certain in- 
sect and animal carriers make possible the survival and spread of many 
pathogens in the soil. 

Organisms that Survive for Brief Periods 

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 



30 HUMAN AND ANIMAL WASTES 

example (997), that in a sewage sludge free to undergo normal diges- 
tion, 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 j 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 (488). 

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 
ever raises the question concerning the role of the soil as the carrier of 
these disease-producing agents or as the cause of severe or even minor 
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} (e) formation by 
these antagonists of specific toxic or antibiotic substances destructive to 
the pathogens} (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 (49). 

The course of survival of only a few disease-producing organisms 



SURVIVAL OF PATHOGENS IN SOIL AND WATER 



31 



outside the host has been studied in detail. Sufficient information has 
been accumulated, however, to justify certain general conclusions. 
When E. coli is added to sterile soil, it multiplies at a rapid rate, but 
when added to fresh, nonsterile soil it tends to die out quickly (Table 5 ) . 
The rate of its disappearance is independent of the reaction of the soil 
and of the temperature of incubation, but a marked increase in the num- 
ber of soil organisms antagonistic to E. coli accompanies the disappear- 
ance (Table 6). 

TABLE 5. 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. coli addedf 


5 


28° c. 


25,600 


6,800 


E. coli addedt 


5 


28° c. 


39,700 


3>5oo 


E. coli added 


5 


37° C. 


22,800 


4,700 


Control soil 


33 


28° C. 


5,900 


<io 


E. coli added 


33 


28° c. 


22,100 


130 


E. coli added! 


33 


28'^ c. 


17,600 


140 


E. coli added 


33 


37° C. 


23,000 


<I0 



From Waksman and Woodruff (951). 

* In thousands per gram of soil. 

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

i CaCOs added to soil. 



TABLE 6. INFLUENCE OF ENRICHMENT OF SOIL WITH ESCHERICHIA COLI 
ON NUMBER OF ANTAGONISTIC MICROORGANISMS 



Control soil 

Enriched soil 

Enriched soil + CaCOgf 



TOTAL MICRO- 
ORGANISMS PER 
GRAM OF SOIL 



ANTAGONISTS* PER GRAM OF SOIL 

After 65 days After 1 1 7 days After 1 1 7 days 



500,000 
4,000,000 
6,000,000 



1,150,000 
5,700,000 
4,700,000 



9,100,000 
40,000,000 
36,300,000 



From Waksman and Woodruff (949). 

* An antagonistic colony is one surrounded by a halo on the E. coli plate. 

t This container received fewer enrichments with E. coli than the one without CaCOj 



32 HUMAN AND ANIMAL WASTES 

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

Frankland (295, 296) was the first to establish that Eberthella ty- 
-phosa may survive in sterilized polluted water or in pure deep-well 
water for 20 to 5 1 days although it died out in 9 to 1 3 days in unsterile 
surface water. In other studies (464) it was found that the typhoid or- 
ganism is 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 being apparently responsible for 
the destruction of the pathogen. These conclusions were later confirmed 
(777). Freshly isolated cultures of E. tyfhosa survived a shorter time 
than laboratory cultures, higher temperatures (37° C.) being more de- 
structive than lower ones (438). Although some investigators (1007) 
claimed that E. coli may survive in soil for 4 years, others (789) found 
that it disappeared rapidly. Viable typhoid bacteria were recovered 
(774) from polluted soil after lOO days in unsterilized soil, and after 
16 months in sterilized soil. Sedgwick and Winslow (811) reported 
that cells of E. coli rapidly die 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, but much longer in sterile soil (831). S. Martin (586), for 
example, observed that typhoid bacteria survived and grew readily in 
sterile soilj however, when added to a well-moistened and cultivated 
soil they were rapidly destroyed. The same phenomenon occurred 
when the pathogens 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 decomposi- 
tion taking place in the soil. It was further concluded that an antagonis- 



SURVIVAL OF PATHOGENS IN SOIL AND WATER 33 

tic relation appeared to exist in some soils but not in others and that this 
was due to the action of specific antagonistic bacteria present in the par- 
ticular soils. 

Frost (302) also reported that typhoid bacteria are rapidly destroyed 
when added to the soil. In 6 days, 98 per cent of the cells were killed, 
and in the course of a few more days all the cells tended to disappear 
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 destruc- 
tion of typhoid organisms brought into contact with them. 

The survival of typhoid and dysentery bacteria in soil has been the 
subject of many other investigations (256, 577, 685). 

Among the factors responsible for the disappearance of E. typhosa 
in water, the presence of certain water bacteria was found to be of spe- 
cial importance (899). Rochaix and Vieux (760) 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. coU gave, after 13 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 oxygen supply of the water is important (975). E. tyfhosa 
added to activated sludge increased within the first 4 to 6 hours ; this was 
followed by a reduction in 24 hours, and a 99 per cent destruction in sev- 
eral days (411). 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 j after the sludge was dried no typhoid 
bacteria could be found (613). 

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- 



34 HUMAN AND ANIMAL WASTES 

coccus, Flavobacterium, and yeast, 2 actinomycetes, and 3 unidentified 
nonspore-forming, gram-negative rods (445). 

The survival of E. tyfhosa in manure and in soil is known to be re- 
pressed decidedly by various saprophytic bacteria. When a carrier was 
induced to urinate on a soil, E. tyfhosa could be recovered within 
6 hours from the washings of the soilj however, after 30 hours the or- 
ganism could no longer be demonstrated, although the soil was still 
moist with the urine (616). 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. tyfhosa is destroyed by bacteria grown in 
association with it (382). Moisture was found (785) 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 sur- 
vival of the remainder extending over a period of months. 

E. coli was rapidly crowded out by other organisms in manure 
piles (623). The addition of 9 million cells of E. coli and 13 mil- 
lion cells oi A. aerogenes to a soil resulted, in 106 days, in reductions to 
6,000 and 25,000 respectively} in 248 days, both organisms had com- 
pletely disappeared (828). The occurrence of coliform bacteria in soil 
depends entirely on the degree of pollution ; soil relatively free from 
pollution contains no coliform bacteria or only a small number. No evi- 
dence of multiplication of these bacteria in the soil could be detected 

(873). 

Sea water, as well, appears to have a bactericidal effect upon organ- 
isms added to it (937, 1014). 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 1 2 and 1 6 hours, whereas 
paratyphoid organisms survived for 21 and 23 days (887). Protozoa 
were found to be at least partly responsible for the destruction of the 
typhoid organism added to water systems (250, 440, 709, 815). 

Mycobacterium tuberculosis 

The fate outside the hosts of the bacteria causing tuberculosis in man 
and in animals has also been studied extensively. Considerable diffi- 



SURVIVAL OF PATHOGENS IN SOIL AND WATER 3 5 

culty has often been encountered, however, in demonstrating the pres- 
ence of this pathogen, which must be detected usually by guinea pig 
inoculation methods (894). The organism was found to be alive in a 
dark room after 157 to 170 days, but not after 172 to 188 daysj in dif- 
fused light, the longevity was only 124 days 5 in the incubator, the or- 
ganism retained its virulence for 33 days, but not for 100 daysj on ice, 
virulence was still evident after 102 days but not after 153 days (618). 

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 
(85). 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 but not after 25 
days (792, 842). Under conditions prevailing in southern England, it 
was found (987) 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 (574). Positive tests were obtained for soil and manure after 
178 days, but not later. The organism survived on grass for at least 49 
days. Samples of milk of tuberculous cows, kept frozen and examined 
periodically, gave positive tests even after 2 years and 8 months (551 ). 
Rhines (746) found that M. tuberculosis multiplied in sterile soil as 
well as in the presence of certain pure cultures of bacteria 5 however, a 
fungus was found to check the development of the pathogen, especially 



36 HUMAN AND ANIMAL WASTES 

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 (745). 

Other Disease-froducing Microor ganisms 

A study of the viability of Brucella melltensls 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 (326, 430, 431, 432). 

The rapid destruction of cholera bacteria added to soil was first 
pointed out by Houston (437). Similar rapid destruction of the diph- 
theria organism was also noted. Serratia, however, retained its vitality 
for 158 days. Vibrio comma also survived for a short time only in feces 
(358), 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 
(450). 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. 

THE SOIL POPULATION 

The nature of the soil population can thus be considered as more or 
less dynamic, its modification being controlled by the addition of or- 
ganic matter and by soil treatments. The introduction of foreign organ- 
isms tends not to change the nature of the population, but merely to 
stimulate the development of such members as are capable of destroying 
the foreign organisms. The production of antibiotic substances by mem- 



THE SOIL POPULATION 37 

bers of the soil population may also be directly responsible for the rapid 
destruction of the added organisms. It has been shown (635), for ex- 
ample, that subsurface soils contain microbiological populations that 
are smaller, less versatile, and less adaptable than surface soil popula- 
tions. Some factor in the subsurface soils was believed to cause the pre- 
vention of the rapid development of the introduced organisms. Anti- 
biotic or inhibitory substances were said to be responsible for this effect, 
these substances being of microbial origin. Aqueous extracts of soil did 
not adversely affect the growth of soil bacteria in vitro, but alcohol ex- 
tracts reduced their activity in the soil and in artificial culture media. 



CHAPTER 3 

INTERRELATIONSHIPS AMONG MICROORGAN- 
ISMS IN MIXED POPULATIONS 

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

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 {S"^^)- This was well expressed 



SYMBIOSIS AND ANTIBIOSIS 39 

by Pfeffer (684), who said that "the entire world and all the friendly 
and antagonistic 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 com- 
plex interrelations among microorganisms in nature. 

De Bary (165) was the first to emphasize, in 1879, the significance 
of the antagonistic interrelations among microorganisms j when two or- 
ganisms are grown on the same substrate, sooner or later one overcomes 
the other and even kills it. This phenomenon was designated "anti- 
biosis" (42, 953). Symbiotic, or mutualistic, and antagonistic relation- 
ships among microorganisms indicate whether advantages or disadvan- 
tages will result to the organisms from the particular association; the 
first are beneficial and the second are injurious and may even be para- 
sitic. Kruse (508) asserted that, when two organisms are capable of 
utilizing the same nutrients but are diflFerently affected by environ- 
mental conditions such as reaction, air 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 
(695), the effects produced by fungi in mixed culture are due either to 
exhaustion of nutrients or to the formation of detrimental or beneficial 
products. E. F. Smith (829) emphasized that when two or more or- 
ganisms live in close proximity they may exert antagonistic, indifferent, 
or favorable effects upon one another. These potentialities were later 
enlarged (loii) to include stimulating, inhibiting, overgrowing, and 
noninfluencing effects. After considerable experimentation and specula- 
tion, Lasseur (513, 514) came to the conclusion that antagonism is a 
very complex phenomenon and is a result of numerous and often little- 
known activities. Antagonism influences the morphology of the organ- 
isms, their capacity of 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- 



40 INTERRELATIONSHIPS AMONG MICROORGANISMS 

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 
(943). 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 
conditions, 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 (425). Microbes living in association fre- 
quently develop characteristics which they do not possess when living 
in pure culture. Schiller (797, 798), for example, found that when beer 
yeasts are placed together with tubercle bacteria in a sugar-containing 
but nitrogen-free medium, the yeasts develop antagonistic properties 



THE NATURE OF A MIXED MICROBIAL POPULATION 41 

toward the bacteria and use the latter as a source of nitrogen j the yeasts 
secrete a bacteriolytic substance 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 (312) to be a result of the need of a 
source of nitrogen by the latter. 

The term "autoantibiosis" has been used (670) to designate the 
phenomenon of self-inhibition or "staling" of medium as a result of the 
previous growth of the organism in this medium. 

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 utilizing 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 of 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- 



42 INTERRELATIONSHIPS AMONG MICROORGANISMS 

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. mesophilic organisms), oxygen 
supply (aerobic vs. anaerobic organisms), moisture content (bac- 
teria and fungi vs. actinomycetes), reaction (acid-sensitive vs. acid- 
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 



Large root u ith bacterial cells and 
filaments of actin()m\cetes 




V 



^■«*^ 



Root hair with rod-shaped bacteria in 
colonies and short chains 







■.-'i 



Terminal portion of root hair show- 
intj bacteria in form of mantle 






Root hair undergoing attack by 
bacteria and actinomycetes 



i ^ 



"4:^?: 



^' ■^^t^ '^'^ 



'■■•'£ii « 'w?< - ■•■■ % 



Masses of coccoid bacteria growing 
< about a funijus filament 



■ vt , • ■ > 

Dense colonies of bacteria sur 
rounding root hairs 



Figure 2. Relationships between microorganisms and root systems of higher plants. 
From Starkey (848). 



ASSOCIATIVE INTERRELATIONSHIPS 43 

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 
favorable substrates for peptolytic bacteria. The ammonia liberated 
from proteins and amino acids supplies a source of energy for nitrify- 
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 (102). 

Influence upon the oxygen concentration available for respiration. This 
involves the phenomenon first observed by Pasteur (673) of con- 
sumption of oxygen by aerobic bacteria, thus making conditions fa- 
vorable for the development of anaerobes (650—652, 843). 

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 Azotobacter 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 (612, 787 ). Various other processes of association 
have also been recognized (920). 



44 



INTERRELATIONSHIPS AMONG MICROORGANISMS 



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-fect'movorum forms 
lactic acid instead of butyl alcohol (845). The presence of Clos- 
trid'tum acetobutyVicum in cultures of bacteria producing dextro-lactic 
acid and laevo-lactic acid causes such bacteria to form the inactive lac- 
tic acid (870) ; intimate contact of the bacteria is essential, the use of 
membranes preventing this effect. The presence of A. aero genes 
modifies the optimum temperature for nitrogen-fixation by Axoto- 
bacter (749). Pigment formation by P^. aeruginosa may be weak- 
ened when the latter is grown together with other organisms. E. coli 
may lose the property of fermenting sugars when grown in the 
presence of paratyphoid organisms (453). 

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 7). Trichoderma, a fungus 



TABLE 7. 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 





61 


Rhizofus 


6.6 


12.8 


2.9 


53 


Trichoderma + Rhizofus 


13-7 


22.6 


10.6 


63 


Trichoderma + Cunningharnella i 5 .0 


15.4 


5-7 


47 


Trichoderma + Ps. jluorescens 


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 (938). 



COMPETITIVE INTERRELATIONSHIPS 45 

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 j however, the organism utilized the proteins rapidly, 
as illustrated by the amount of ammonia liberated. Rhizofus^ a non- 
cellulose-decomposing fungus, attacked largely the hemicelluloses in 
the alfalfa and some of the protein j a small reduction in cellulose was 
recorded, probably because of an analytical error. When Trichoderma 
was combined with Rhizofus, 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- 
isms, such as the fungus Cunnmghamella and the bacterium Ps. fuores- 
cens. On the other hand, when Trichoderma was combined with a cellu- 
lose-decomposing Streftomyces, 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 (i8) : 

Competition among chlorophyol-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 (924) to include other factors that are par- 
ticularly prominent in nonaquatic environments : 



46 INTERRELATIONSHIPS AMONG MICROORGANISMS 

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 

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, as shown by Nicol and Thornton (637). 

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 5 its 
metabolism and cell structure may become modified or the cell itself 
may be destroyed (174). In urine, for example, staphylococci may be- 



ANTAGONISTIC INTERRELATIONSHIPS 47 

come antagonistic to E. coli or vice versa, depending on the initial num- 
bers of the two groups, on the formation of metabolic products, or on 
the exhaustion of nutrients (247). The toxic substances produced by 
the antagonists comprise a variety of compounds, ranging from simple 
organic acids and alcohols to highly complex bodies of protein or poly- 
peptide nature. 

Various types of antagonism are recognized. Nakhimovskaia (627) 
concluded that all phenomena of antagonism among microorganisms 
can be conveniently classified into four groups of reactions: 

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

tigators (513, 514), only the inhibitive forms of antagonism {in 
vitro) may be designated as true antagonisms; the in vivo forms 
were designated as phenomena of antibiosis. Usually, however, this 
differentiation is not 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 
(228). 

Duclaux (208) 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 (509) 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 (583) 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; upon the destruction of these by boiling, the 
rnedium was made again favorable for the growth of bacteria and bac- 
terial spores were able to germinate again. Some of the toxic substances 



48 INTERRELATIONSHIPS AMONG MICROORGANISMS 

appeared to be thermostable j Nadson and Adamovic (625) showed 
that certain metabolic products of microorganisms, even when heated to 
120° C, may have a strong influence upon the subsequent growth of 
the organisms. 

Fungi are capable of producing not only growth-inhibiting but also 
growth-promoting substances (509, 547). By means of certain proce- 
dures, it was found possible to separate the two (654). 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 (304). This nega- 
tive chemotropism was shown to be due to thermolabile staling sub- 
stances (352). 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 Asfergillus, the lactic and fumaric acid- 
producing species of Rhizofus, 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 



ANTAGONISTIC INTERRELATIONSHIPS 49 

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 substance produced by many fungi and bac- 
teria was found (240, 641 ) to be 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 (299). Certain 
types of antagonism express themselves in the destruction by the an- 
tagonist of the other organisms present in the mixed culture, with 
or without producing a lytic effect, B. mesenterkus^ for example, is 
capable not only of depressing but also of killing the cells of diphtheria 
and pseudodiphtheria (1016), The lytic form of antagonism is illus- 
trated by the action of Ps. aeruginosa^ Bacillus hrevis, and certain other 
antagonists upon micrococci and various spore-forming bacteria. 

In differentiating between "direct antagonism" and "passive antag- 
onism," attention was directed (627) 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 rH of medium or an impoverish- 
ment of some of the nutrient constituents, "Direct antagonism" was 
often distinguished (634) 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 (365, 367) to repress the anthrax organ- 
ism only when the former were in an active living state. Other Investi- 
gators (407) designated the action of the living cell itself as "true 
antagonism." 

Bail (31) 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- 
ciependent of exhaustion of the nutrients or formation of toxic sub- 
stances. The same phenomenon was believed to hold true when two 



50 INTERRELATIONSHIPS AMONG MICROORGANISMS 

bacteria live together (983, 984): if the limiting cell-in-space concentra- 
tions are different for the two organisms, the one with a higher M value 
represses the other j however, the weaker species may check the stronger 
when planted in sufficient excess (244). It has been suggested (368) 
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 (634, 983, 984). 
Brown (90) explained the fact that the number of yeast cells reaches a 
maximum independent of the initial number of cells added or the con- 
centration of nutrients in a given volume of medium by the amount of 
oxygen originally present. 

Garre (311) 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. coU is antagonistic to 
E. tyfhosa; however, if the latter is inoculated into a medium some- 
what earlier than the former, E. tyfhosa becomes antagonistic to E. colt 

(324,915). 

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 
(52, 368, 611). Certain strains may develop antagonistic properties in 
the presence of other strains (77). 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 (627). The destruction of Dktyostelium muco- 
roides by a red-pigment-forming bacterium was accompanied by an in- 



NATURE OF ANTAGONISTIC ACTION 51 

crease in intensity of the pigment (690); the blue pigment of Bac- 
terium violaceum, however, only delayed the growth of the fungus. Ac- 
cording to Doebelt (177), Pemc'illlum ajricanum produces a more in- 
tense pigment in contact with other fungi such as Aspergillus niger; this 
pigment accumulates in the mycelium of the latter, which may thereby 
be killed. Nadson (626) demonstrated that some fungi {Penicillium 
luteum and Spcaria furfuro genes) produce a pigment that is used not 
only for purposes of protection, but also for attack upon other organ- 
isms, whereby the latter are killed and stained. 

DISTRIBUTION OF ANTAGONISTIC PROPERTIES 

Numerous microbes found among the bacteria, fungi, actinomycetes, 
and protozoa possess the capacity of bringing about injurious or de- 
structive effects upon other microorganisms belonging to their own 
groups or to others. In some instances, the antagonistic effects are ob- 
tained only in the presence of the antagonizing organism; in many 
other cases, excretion products consisting of definite chemical com- 
pounds are produced by the antagonist. A few of these products have 
been isolated and have been found to be effective against certain few 
specific organisms or able to act upon a great variety of organisms 
(920). The wide distribution of antagonistic properties among micro- 
organisms is brought out in subsequent tables. 

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 

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

autolysis, under the influence of the antagonist 
Production and liberation of toxic substances 
Pigment action 
' Action at a distance 
Space antagonism 



52 INTERRELATIONSHIPS AMONG MICROORGANISMS 

Pasteur (672, 674, 675) ascribed the antagonistic effect of aerobic bac- 
teria upon the anthrax organism to the consumption of the oxygen by 
the former j 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 (299) 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 (539) to 
belong here. The change in -pW of medium and the accumulation of 
toxic products were also found to become limiting factors. Palevici 
(667) added fruit juice to a stale medium and brought about improve- 
ment in bacterial growth, thus suggesting the exhaustion of growth- 
promoting substances as the cause of staling. Broom (89) emphasized, 
however, that the effect was due to addition of nutrients, including 
glucose. 

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 relationships produced by changes in 
surface tension, in oxidation-reduction potential, in reaction, and in os- 
motic pressure were suggested as explanations (24, 627, 827). 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 
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 (334). 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 



NATURE OF ANTAGONISTIC ACTION 53 

salt solution is markedly influenced by the colloids present (991), the 
concentration of electrolytes (816), the reaction (897), and the tem- 
perature (36). 

Microbial antagonism was thus looked upon (496) largely as a re- 
sult of a series of physical factors, including various radiations such as 
mytogenetic rays (9, 679, 814), fH. changes, conductivity, electric 
charge, and surface tension. 

Most antagonisms, however, can be explained by the production of 
toxic substances by the antagonists. Because of their thermolability, 
sensitivity to chemical reagents, or adsorption on bacterial filters, con- 
siderable difficulty has been experienced in isolating the active sub- 
stances. Many of these substances have been found to be iso-antagonistic 
(autotoxins [141]), whereas others are able to act upon different bac- 
teria. Most of them have been found to be thermostable. 

The first antibiotic substance recognized as such was pyocyanase 
(235), soluble in alcohol, ether, and chloroform. Somewhat similar 
substances appear to be produced by Serratia marcescens ( 230) , Ps. fluo- 
rescens {s2S)jB. mesentencus ( 10 1 6) , and Bacillus mycoides. Whereas 
Emmerich and Low (236) considered pyocyanase to be a proteolytic 
enzyme, others (370, 410, 668, 679) found it to be a lipoid. Since that 
early work and especially during the last five years, many new agents 
have been isolated or demonstrated. These will be discussed in detail 
later. It is sufficient to mention gramicidin and tyrocidine, produced by 
B. brevis, which are polypeptides 5 citrinin and fumigatin, which are 
quinone-like in nature j actinomycin, aspergillic acid, and iodinin, which 
are nitrogenous ring compounds j gliotoxin, which is a sulfur-bearing 
compound J streptothricin, streptomycin, and proactinomycin, which are 
nitrogenous bases. Some of the most important compounds (penicillin) 
have not as yet been sufficiently elucidated. Certain microbial pigments 
(pyocyanin, hemipyocyanin, prodigiosin) have also received considera- 
tion as bacteriostatic and fungistatic agents. 

The production of antibiotic substances by various 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 



54 INTERRELATIONSHIPS AMONG MICROORGANISMS 

(361), whether the organisms thereby affected are able to overcome 
their effect, and whether they are destroyed by other members of the 
soil or water microbiological population (947, 951). 

Different organisms possess different degrees as well as different 
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. Distortions were found to be produced 
in Alternar'ia (231) and in HelTninthosforium (695) by a bacterial 
antagonist. The morphological effects produced by the antagonists com- 
prise changes in form, size, and structure of hyphae, direction of growth, 
and complete cessation of growth and abbreviation of hyphal segments. 

In surveying the phenomena of antagonism among microorganisms, 
Porter (695) reached the conclusion that, among bacteria, the spore- 
formers are strong inhibitors. Actinomycetes also exhibit strong inhibi- 
tory action against most filamentous fungi. Phycomycetes usually 
neither cause inhibition nor are inhibited j the Basidiomycetes contain 
very few organisms possessing antagonistic properties. Ascomycetes 
and Fungi Imperfecti vary greatly in their ability to produce antibac- 
terial substances; some yeasts are strong inhibitors. Certain algae, no- 
tably species of Chlorella, produce a substance (chlorellin) that inhibits 
the growth of various gram-positive and gram-negative bacteria (701). 



CHAPTER 4 

ISOLATION AND CULTIVATION OF 

ANTAGONISTIC MICROORGANISMS^ 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 j 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 
(797? 798) on forced antagonisms and the studies of Gratia and his as- 
sociates (349, 350) on mycolysates were in direct line of the more re- 
cent studies of Dubos (190), who made a systematic attempt to isolate 
from specially enriched soils bacteria capable of destroying specific 



56 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 
(705), Much (621), 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 enrichment culture method. This consisted in adding 
repeatedly various pathogenic bacteria to a soil which, as a result, be- 
came enriched with antagonistic organisms capable of destroying the 
bacteria added j these organisms were then isolated by appropriate pro- 
cedures. The isolation of the specific substances will be described later 
(page 156). 

These investigations, as well as the work of Fleming (265) and 
other British investigators (3, 7, 8, 113) on the antibacterial properties 
of molds belonging to the PenicilUum notatum group, served as the di- 
rect stimulus to numerous studies that followed. The entire series of 
studies led to the development of simple methods for the systematic iso- 
lation of microorganisms capable of inhibiting the growth of fungi and 
bacteria, both pathogenic and saprophytic (857, 934), and for separat- 
ing many of the antibiotic substances produced by these organisms. 



METHODS OF ISOLATING ANTAGONISTIC 
MICROORGANISMS 

Four, and possibly five, 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 different in nature, but they are all based on the same prin- 
ciple, 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 



METHODS OF ISOLATION 57 

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 
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 (427). 

Bacterial Agar Plate Method 

This method was first used by Gratia and Dath (350) 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 K^HPO^. 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 
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 3). The organisms are isolated from these colonies 



. V 
" L I IS R A R V 



lA-S^i- 



58 ISOLATION AND CULTIVATION OF ANTAGONISTS 

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 (934, 949). 

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 K0HPO4. 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 (i: loto i: 1,000). 

By the use of the soil enrichment and bacterial agar plate methods, 
it is possible to demonstrate that ordinary soils contain a large popula- 
tion of microorganisms that are antagonistic to bacteria, including both 
gram-negative and gram-positive forms. The number of antagonists can 
be greatly increased when the soil is enriched with living cells of 
bacteria. 

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 4). 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 (857). 

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. These organisms will bring about the 
killing or even the lysis of the original culture. By this method, or- 




Figure 3. Development of antagonistic fungi on hacterial-agar plate. From 
Waksman and Horning (934). 




Figure 4. Bacterial plates made from soil, showing clear zones surround- 
ing colonies of antagonistic organisms. From Stokes and Woodward (857). 




Antagonistic action of i\ (Uiti- 
hioticiis upon S. lutea 



Antagonistic action of 5. ayit'i- 
hiot'icus upon B. rn^co'idcs 




Bacteriostatic action of actin( 
m)'cin upon iS\ luttui 



I^actcnostatic action of acti 
m\'cin upon R. rnxcoidn 



Figure 5. Antagonistic effects of living organisms and their products. P'rom 
Waksman and Woodruff (945). 



METHODS OF ISOLATION 59 

ganisms antagonistic to many bacteria and fungi causing plant and ani- 
mal diseases have been isolated (644, 646). 

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 
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 (798), 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. 

Isolation of Antagonistic Microorganisms from Soil 

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 y S. lutea, and B. subtilis were used, a large num- 
ber of antagonists could readily be isolated. With E. coli, however, a 
much smaller number of microbes thus isolated possessed antagonistic 
properties. Certain other gram-negative bacteria, like Brucella abortus, 
were more sensitive than E. coli, whereas certain gram-positive bac- 



60 ISOLATION AND CULTIVATION OF ANTAGONISTS 

teria, like B. mycoides and B. cereus, were less sensitive than B. subtilis 

(934, 936). 

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 (647). By the use of this 
method, Chudiakov (130) isolated various bacteria antagonistic to 
Fusanum. The antagonists were found abundantly in cultivated soils, 
but not in flax-sick soils rich in Fusanum. Bamberg (37) 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 (457) 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 Fusanum , Sclerotinia^ Gleos-porium, Acro- 
stalagmus, Alternarla^ and Zygorhynchus (695). 

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 



METHODS OF TESTING ANTAGONISTIC ACTION 61 

the same substance. Streftococcus viridans, B. subtilis, Micrococcus ly- 
sodeikticus, S. lutea, E. coU, 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- 
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 (302, 627). 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 (299) 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 
(297) 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- 



62 ISOLATION AND CULTIVATION OF ANTAGONISTS 

termined. Frost (302) 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 
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 (302). 

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 (311) 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 5). If the active substance produced by the antagonist does not 
diffuse for any considerable distance into the medium, the method is 
not satisfactory. Frost (302) 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 (705). The spotting 
of the two organisms on the plate is illustrated in Figure 6. 

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. This method, suggested by Garre (311), consists in allowing 
the antagonist to produce a good growth on the surface of the me- 
dium; the mass of growth is then removed, and the test organism 
inoculated into the same medium. 

Double plate methods (302). 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 



METHODS OF TESTING ANTAGONISTIC ACTION 



63 




Hel7ninthosforln7n (A and B) in- Pestaloz-zm (A) inhibited by one 
hibited by Fusarium (C) species of Pcnicillium (C) but not 

by another (B) 




Helminthosforiuni (A) inhibited Helminthosforium (A and B) in- 
by a bacterium (C) hibited by a white yeast (C) 



Figure 6. Inhibition of fungus development by antagonists. From Porter 
(695). 



64 ISOLATION AND CULTIVATION OF ANTAGONISTS 

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 
antagonistic effect more striking. This method has also been used 
(261, 267, 270) 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 (844). This method is particu- 
larly convenient for detecting antagonists that possess lytic prop- 
erties. 

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 difiFuse through the agar and reduce 
the growth of the test bacterium (571). 

Semisolid media are used for testing the action of antagonists against 
motility of bacteria (173). 

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 antibiotic substance pro- 
duced by the antagonist and to measure quantitatively this antibiotic 
action. Before this can be done, however, the organism must be grown 
upon suitable media and suitable conditions must be established for the 
favorable production of the antibiotic substance. 



METHODS OF GROWING ANTAGONISTIC ORGANISMS 65 

The media used for the production of antibiotic substances can be 
classified into two groups: synthetic media and complex organic media. 
The first contain a source of carbon, usually glucose (2 to 6 per cent) j 
a source of nitrogen, usually nitrate (0.2 to 0.6 per cent), as well as sev- 
eral salts, namely, K0HPO4 or KH2PO4 (o.i to 0.2 per cent), 
MgSO^.yH.O (0.05 per cent), KCl (0.05 per cent), and FeS04.7H.O 
(0.00 1 per cent) 5 certain supplementary materials, such as yeast ex- 
tract, meat extract, or corn steep, and other salts, such as NaCl (0.05 
to 0.5 per cent), 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 glucose, starch, brown sugar, 
molasses, or similar products as well as several salts similar to those 
listed above. Some media are supplemented with CaCO.j, and others 
are not, depending upon the extent of acidity produced by the organism. 

The medium may be solid or liquid, but the latter type is more com- 
mon. Agar and bran are used as solid media. Several types of culture 
vessels are used, depending on the condition of aeration. Since so far as 
is known all the microorganisms capable of producing antibiotic sub- 
stances are aerobic, either shallow layers of medium (1.5 to 2 cm. in 
depth) are placed in stationary vessels (flasks or trays), or deep vessels 
(tanks) are filled with the medium and properly aerated by forced draft 
with sterilized and filtered air. 

For the production of penicillin, a constant-flow apparatus similar to 
the quick-vinegar process has been suggested (134), the medium trick- 
ling over a column of wood shavings. The establishment and operation 
of large-scale production of penicillin under submerged conditions have 
been described in detail by Callaham (103). 

The optimum temperature required for the growth of the antagonis- 
tic organisms and production of the 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 



66 ANTIBIOTIC ACTION OF ANTAGONISTS 

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. 



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 antibiotic agents. 

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; they are bactericidal (or 
fungicidal) only to a limited extent, although some substances may 
possess marked bactericidal properties. 

Antibiotic substances are selective in their action; they are able to inhibit 



METHODS OF MEASURING ANTIBIOTIC ACTIVITY 67 

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 or /"-amino-benzoic 
acid. Other agents require the presence in the medium of specific 
constituents for their activity to become effective. The activity of 
some is reduced at an acid reaction (287, 1002), 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 pyocyanase and pyocyanin ; B. brevis, grami- 
cidin and tyrocidine; Streftomyces antibioticus, actinomycin A and 
B ; P. notatuniy penicillin and notatin ; Asfergillus fumigatus, spinu- 
losin, fumigatin, fumigacin, and gliotoxin; Asfergillus flavus, asper- 
gillic acid and flavicin. 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 7 and 8. 

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 (735, 773). 

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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The Agar Plate-Dilution Method 

If an unknown antibiotic substance is tested, it is essential to employ 
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1^2 



^ ^ • 2 « , 



. - " ID CU 



o o j= (J Uh D 
n, ^ .S * -)-++ 



METHODS OF MEASURING ANTIBIOTIC ACTIVITY 71 

1 6 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 

(948). 

The bacteriostatic spectra of a group of antibiotic substances com- 
pared with certain chemical agents are shown in Table 8. Different bac- 
teria show different degrees of sensitivity to the different substances. A 
comparison with the action of phenol can result in what may become 
known as the "bacteriostatic phenol coefficient" for each active sub- 
stance. 

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. In some cases, Streftococcus hemolyticusy B. subtilis, and others 
have been used for measuring the activity of a substance against gram- 
positive bacteria, and E. coli for gram-negative bacteria. Definite vol- 
umes of the test medium are placed in test tubes and sterilized (sterility 
is essential in this method), and various dilutions of the active sub- 
stance are added. The dilutions can range in order of 3 : i , 2 : i, or even 
narrower, namely in series of i .2 : i , i .5 : i , 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 substance giving complete inhibition of 
growth, as expressed by a lack of turbidity of medium, is taken as the 
end point. Activity is expressed in units as above (804). 

The dilution method has two disadvantages (276) : first, every assay 
takes much time j second, during chemical fractionation, the substance 
may become contaminated with bacteria not sensitive to the active sub- 
stances. 

One modification of the method has been adapted for measuring the 
actjivity 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 



72 ANTIBIOTIC ACTION OF ANTAGONISTS 

TABLE 9. BACTERIOSTATIC SPECTRUM OF PENICILLIN 

DILUTIONS AT WHICH INHIBITORY 

ORGANISM AFFECTED EFFECTS WERE OBSERVED 

Complete Partial None 

l^l . gonorrhoeae* 2,000,000 >2,000,000 )>2,ooo,ooo 

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

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

S. -pyogenes 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. tetaniif i ,000,000 

CI. zoelchii 1,500,000 

CL sefticum 300,000 1,500,000 7,500,000 

Cl.oedematiens 300,000 1,500,000 

5. viridans% 625,000 3,125,000 

Pneumococcus\ 250,000 500,000 1,000,000 

C. difhtheriae {mitis) 125,000 625,000 

C. difhtheriae {graz'is) 32,000 64,000 128,000 

5". gartneri 20,000 40,000 8o,000 

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

PneumococcusX 9,000 27,000 

Anaerobic streptococcuslj! 4,000 8,000 16,000 

P. vulgaris 4,000 32,000 60,000 

S.viridansX 4,000 8,000 16,000 

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

S. tyfhimurium < 1,000 8,000 16,000 

5. faratyfhi B < i ,000 5 ,000 I o,000 

5^. dysenteriae 2,000 4,000 8,000 

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

Br. melitensis <i,000 2,500 I0,000 

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

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

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

K. fneuTnoniae <( i ,000 <^ i ,000 i ,000 

Ps. aeruginosa <^ i ,000 < i ,000 i ,000 

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

L. icterohoemorrhagioe < 3,600 <( 3,600 3j6oo 

From Abraham et al. (7). 

* 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 73 

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 (2, 7) j a nii- 
croscopic examination (308) indicated defective fission of the bacteria, 
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 tyfhi, partial in- 
hibition was obtained in a dilution of i : 10,000 j however, elongation 
of the cells was detected in a dilution of i : 6o,000, a concentration 
which was considered as a therapeutic possibility (Table 9). 

The Agar Diffusion or "Agar Cuf" Method (7, 284, 285, 385) 

This method, first employed by Reddish (735) and by Ruehle (773) 
largely for qualitative purposes, was later developed (7, 385) for 
quantitative use. A suitable agar medium is inoculated with the test or- 
ganisms {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 active substance is parallel to its concentration. By meas- 
uring the zone of inhibition and comparing it with that of a known 
standard preparation, the potency of the active substances can be calcu- 
lated. This method has the advantage of simplicity and convenience, 
since it does not require sterile material and several preparations 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 unknown. 

Nutrient agar containing 5 gm. NaCl, 3 gm. meat extract, 5 gm. 
peptone, 15 gm. agar, 1,000 ml. tap water, and adjusted to ^H 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 
I : so 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 



74 ANTIBIOTIC ACTION OF ANTAGONISTS 

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 zone around the cylinder 
is measured to the nearest 0.5 mm. by means of pointed dividers. 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 fH of 
the test material, the thickness of the agar, or the sterility of the ma- 
terial. 

The "Oxford unit," as determined by this method, is the amount of 
penicillin that will just inhibit completely the growth of the test strain 
of 5. aureus in 50 ml. of medium. Thus, a preparation containing one 
unit of penicillin per milligram of material just inhibits the growth of 
the test organism in a dilution of 1:50,000 (7, 273, 385). 

One of the modifications of this method (285) consists in using a 
spore suspension of B. subtilis 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. 

This method is also very convenient for measuring the activity of 
streptothricin. A standard curve is obtained by filling the cups in quad- 
ruplicate with dilutions of the standard containing 10, 20, 40, 60, 80, 
and 100 streptothricin units per milliliter. The dilution of the unknown 
contains about 50 units per milliliter. After overnight incubation at 
30° C, the inhibition zones around the cups are measured and plotted 
to give a standard curve. The units of the unknowns are read off this 
curve by projecting the value of the inhibition zones. 



METHODS OF MEASURING ANTIBIOTIC ACTIVITY 



75 



The agar cup method has also been utilized (869) for comparing 
the disinfectant action of chemical antiseptics. S. aureus and B. suhtilu 
were found to be most sensitive to the action of aliphatic alcohols. Vari- 
ous modifications of this method have been introduced, including the 
use of paper discs treated with known dilutions of the active preparation 
(818,917,985). 

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 
partial inhibition of growth be measured and, from this, the concentra- 
tion of the active substance be calculated in a manner similar to the 



24 










2.8 


^^ 


» 













• \ 










"ao 


- V, 






_ 


2.4^ 


<0 


* 











lU 




V 








u 




•, 









5i6 


- 


\ 


^^ 


( - 


2.0 3 


a 




\* 








UJ 

Oi 
UJ 

1- 

w 8 


- 


»^ 




- 


1 
cj 

1.6 ^ 

>- 
1- 

1.25 


2 














2 


- 


^^ 


- 


0.8 


< 








"■"-l-..^ 




Q 




1 


1 1 1 t 1 "'^ 




n /I 





.05 


.10 .15 .20 .25 .30 .35 .40" 1 






PENICILLIN IN 5 PER. MILLILITER 





Figure 9. Relation between penicillin concentration and inhibition of 
Staphylococcus aureus. The penicillin preparation contained 42 Oxford units 
per milligram, and the incubation period was sixteen hours at 37° C. From 
Foster (281). 



76 ANTIBIOTIC ACTION OF ANTAGONISTS 

measurement of the potency of bactericidal agents. Partial inhibition 
can be determined by plating for the number of viable bacteria, as com- 
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 in nature than those obtained by other methods (281, 520), as 
shown in Figure 9. By proper modifications, the length of time re- 
quired to obtain a satisfactory reading can be reduced to four hours 
(4^5) 572)5 or even to 90 minutes (281, 520). 

S fecial Methods 

Certain 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 72) : 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 (715, 989). Penicillin can also be estimated by its inhibition of 
nitrite production by S. aureus cultures (343). The antiluminescent 
test has been utilized (716, 717) not only for measuring the activity of 
certain substances but also for determining their possible usefulness. 
The results of a comparative study of a number of antibiotic substances 
by this and the dilution methods are brought out in Table 10. 

Various other methods have been suggested for measuring the activ- 
ity of antibiotic substances. Some are based upon interference with a 
given physiological function of the test organism such as dehydrogenase 
activity and respiration, others upon the prevention of growth of the 
test organism (pneumococcus) in semi-solid tissue culture medium 
(387). Although only a single method is usually employed in the con- 
centration and standardization of a given antibiotic substance such as 
penicillin, it is often advisable to check the results by another method, 
especially where several test organisms are used, in order to ascertain 
that one is still dealing with the same type of chemical compound. 

Some of the above methods can also be adapted to the determination 
of the concentration of antibiotic agents in the body fluids and exudates 
(719). 



METHODS OF MEASURING BACTERICIDAL ACTIVITY 77 

TABLE 10. 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.II 


Gramicidin 


.002 


Tolu-p-quinone 


. .002 


Pyocyanase 


3 


Tyrothricin 


.008 


Pyocyanase 


.07 


Clavacin I 


" 


Penicillin II 
Penicillin I 
Flavatin 


.0156 

.06 

.256 


Clavacin I 


.18 


Aspergillic acid 


15 


Gramidinic acid 


.23 


Sodium clavacinate .18 


Gliotoxin 


17 


AP2lt 


•31 


Clavacin II 


.19 


Clavacin II 


22 


Actinomycin A 


•54 


Sulfanilamide 
Phenol 


<-56 
•5 


Pyocyanin 


47 


Aspergillic acid 


2.0 


Pyocyanin 


1-7 


Actinomycin A 


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 


II 70 


Tolu-p-quinone 


55.0 


Actinomycin A 
Flavatin 


1 00.0 
1 000.0 


Penicillin I 


1650 


Lauryl sulfate 


59.0 


AP2lt 


>i630 


Sulfanilamide 


3940 


Clavacin I 


63.0 


Gramidinic acid 


>2i75 


Gramicidin 


>5oo 


Clavacin II 


II 3.0 


Penicillin I 


27,500 


Gramidinic acid 


>500 


Sodium 

clavacinate 


500.0 


Tyrothricin 


>62,5oo 


Tyrothricin 


>500 


Phenol 


2300.0 


Gramicidin 


>250,000 


AP2it 


>500 


Sulfanilamide >7000.0 


Penicillin II 


>325,ooo 


Penicillin II 


>50oo 











From Rake, Jones, and McKee (716). 

* Streptococcus pyogenes used as test organism. 

t A tyrothricin-Iike preparation. 



METHODS OF MEASURING BACTERICIDAL 
ACTION OF ANTIBIOTIC SUBSTANCES 

Several methods are commonly employed for measuring bactericidal 
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 



78 ANTIBIOTIC ACTION OF ANTAGONISTS 

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 
determined. 

Various modifications of this method have been developed. In one 
such modification (607), the bacterial cells are suspended for 15 to 30 
minutes in various dilutions of the active substance j the cells are then 
centrifuged oflF, washed, and cultured. This procedure can be utilized 
for substances that have a rapid bactericidal action. Its value is limited, 
however, by the fact that most antibiotic substances do not kill bacteria 
so rapidly as do chemical antiseptics (p. 189). 

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 (550). The growth of Ceratosto- 
mella ulmi was inhibited by several substances, comprising actinomycin, 
clavacin, and hemipyocyanin in concentration of i: 100,000 (803). 

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. Some of the results obtained are presented 
in subsequent chapters. 

ISOLATION AND UTILIZATION OF 
ANTIBIOTIC SUBSTANCES 

The isolation of antagonistic microorganisms from natural sub- 
strates, the determination of the nature of the antibiotic substances pro- 



UTILIZATION OF ANTIBIOTIC SUBSTANCES 79 

duced by them, and the utilization of such substances for chemothcra- 
peutic purposes involve ten distinct steps which may be briefly sum- 
marized as follows: 

1. Enrichment of soil or water with specific organisms against which an- 

tagonists are to be obtained. 

2. Plating of the enriched or unenriched soil or water upon special bac- 

terial agar. 

3. Isolation of the antagonistic organisms from the plates or other culture 

media. 

4. Testing of the isolated culture for bacteriostatic and fungistatic proper- 

ties against a variety of bacteria or fungi. 

5. Growing the antagonist in suitable media and testing the filtrate or 

extract of the culture for the presence of the antibiotic substance. 

6. Separation and concentration of the antibiotic substance from the cul- 

ture medium, and determinations of its bacteriostatic spectrum. 

7. Chemical isolation of the antibiotic substance. 

8. Determination of the chemical nature of the active substance. 

9. Testing of the bacteriostatic and bactericidal properties of the sub- 

stance in intra. 
10. Animal experimentation and practical application; study of toxicity, in 
vivo activity, and therapeutic action. 



CHAPTER 5 

BACTERIA AS ANTAGONISTS 

Following the early work of Pasteur (674) 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 (150), followed by Garre in 1887 and Freudenreich in 1888. 
Freudenreich (299) demonstrated 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 (311) observed that Ps. 
-putida inhibited the growth of S. aureus, E. tyfhosa, and Bacillus muco- 
sus-cafsulatus but not of B. anthrac'ts and other bacteria. It was soon 
reported (524), however, that B. anthracis was also killed by the 
Pseudomonas antagonist, whereas the growth of 5. aureus and V . 
comma was only retarded j no effect at all was exerted upon E. tyfhosa 
and E. coli. In consequence, the antagonist was claimed to be active 
against B. anthracis but not against other bacteria. Olitsky {6ss) con- 
cluded that Ps. fuorescens inhibited the growth not only of E. tyfhosa 
but also of B. anthracis, V . comma, S. marcescens, and S. aureus. These 
and other apparently contradictory results were undoubtedly due to 
diflFerences 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. fiuorescens in sewage was found (517) 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 (326, 430, 431, 432), and it could not be detected in sterile sew- 
age in which the antagonist was present for seven days. According to 
Frost (302), E. tyfhosa can be antagonized by a number of different 
soil bacteria, of which Ps. fluorescens exhibits the strongest effect. He 
observed that although P. vulgaris acted more rapidly, the active sub- 
stance did not diffuse to so great a distance in the medium, thus point- 



BACTERIA AS ANTAGONISTS 81 

ing to a different inhibition mechanism. Mixed cultures showed greater 
activity than pure cultures, either because the latter lose their antibiotic 
property when grown for a long time on artificial media or because 
mixed cultures comprise two or more species with a greater combined 
action. The antagonistic substances produced by these bacteria were ac- 
tive at 37° C, whereas at ice-chest temperature the action was delayed 
so that the pathogen had an opportunity to develop. This was believed 
to offer a possible explanation for the fact that when water supplies 
become contaminated in cold weather, their power of producing infec- 
tion 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. tyfhosa 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 (993) 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 (525), 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. The former 
also inhibits the growth of B. anthracis, Bacillus megatherium^ V . 
commay Chromobacterium 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- 
cens are highly resistant. Ps. fuorescens produces a thermostable sub- 
stance which is toxic to all bacteria except the green fluorescent forms 



82 BACTERIA AS ANTAGONISTS 

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, as in the case of those acting upon the various types of pneumo- 
cocci (819, 821, 822) J others were less selective, as in the case of certain 
soil bacteria that can bring about the lysis of living staphylococci and 
inhibit the growth of various gram-positive and gram-negative bacteria 
(820). S. marcescens was shown (61) to be antagonistic to B. subtilis, 
B. mycoidesy and B. megatherium. These spore-formers, 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 modi- 
fied 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 
retarded (632). 

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

SPORE-FORMING BACTERIA 

Many strains of aerobic spore-forming bacteria possessing antagonis- 
tic properties and differing in morphological, cultural, and physiologi- 
cal characteristics have been isolated from a great variety of sources, 
such as soil, sewage, manure, and cheese. Among these bacteria, B. sub- 
tilisy B. mycoides, B. mesentericus, and B. brevis occupy a prominent 
place, as shown in Table 11. It was established (205) that some of 
these bacteria produce in peptone media an alcohol-soluble, water- 
insoluble substance endowed with bactericidal properties. 

Duclaux (208) was among the first to isolate and describe antagonis- 
tic spore-forming bacteria. Cantal cheese was their origin, and the or- 
ganisms were designated as Tyrothrix. NicoUe (640) isolated from the 
dust in Constantinople a strain of B. subtilis that had decided bacterio- 



SPORE-FORMING BACTERIA 



83 



lytic properties, especially against members of the pneumococcus group 
as well as against various other bacteria such as the typhoid, anthrax, 
and Shiga organisms. E. coli, V. comma, and staphylococci were less 
affected, and Bacillus suifestifer was least acted upon. The filtrate of 
the organism grown in peptone broth had strong antibiotic properties} 

TABLE II. SPORE-FORMING BACTERIA ANTAGONISTIC TO OTHER BACTERIA 



ANTAGONIST 

B. ant hr acts 

B. brevis 

B. mesentericus 

B. mesentericus 

B. mesentericus 
vulgatus 

B. mycoides 
B. mycoides 



B. mycoides, 
var. cytolyticus 

B. subtilis 

B. subtilis 



B. subtilis 



B. subtilis- 
mesentericus 



B. therm-ofhilus 



ORGANISM AFFECTED 

Anthrax, typhoid, and 
lactic acid bacteria 

Gram-positive bacteria 

Many bacteria 

Diphtheria bacteria 

C difhtheriae 

7 to 20 species of 
bacteria 

M. tuberculosis 



Most pathogens and 
many nonpathogens 

Various bacteria 

Various bacteria, espe- 
cially certain plant 
pathogens 

M. tuberculosis, E. 
tyfhosa, etc. 

Mostly living gram- 
positive bacteria and 
dead gram-negative 
bacteria 

5". lutea 



KNOWN PROPERTY REFERENCES 
299, 781, 827 

Produces tyrothricin 190,191,203 

Bacteriolytic 408 

Bactericidal 28, 956 

Substance thermola- 705 
bile, nonfilterable 

Lytic 61,620,621 

Thermostable sub- 482, 483 

stance produced, 
precipitated by 
tungstic acid 

294 

Bacteriolytic 61,640 

442 



903 

Lytic 768, 770 



Suppression of 835 

growth 



84 BACTERIA AS ANTAGONISTS 

it liquefied gelatin and hemolyzed red blood corpuscles. When various 
bacteria cultivated on a solid medium were suspended in physiological 
salt solution and seeded with the antagonist, the latter developed abun- 
dantly and the bacterial suspensions became clarified. The lysed solu- 
tions 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 or- 
ganisms belonging to the B. subtilis group are capable of destroying 
various bacterial toxins. Humfeld and Feustel (442) recently demon- 
strated that an acid extract of B. subtilis cultures has a very high activity 
against certain plant pathogens. This substance was designated as sub- 
tilin. 

Rosenthal (768) 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 5 (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. colt 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 (620) isolated several strains of B. mycoides 
that possessed strong antagonistic properties. The active strains were 



SPORE-FORMING BACTERIA 85 

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 1 8, a third on 
12, and a fourth on only 7. Marked differences were shown (621) to 
exist in the degree of antagonistic activity of the different strains. 
P. vulgaris, E. ty-phosa, and V. comma were lysed in 24-hour bouillon 
cultures as a result of adding pieces of agar containing colonies of the 
antagonist. A lytic effect was also exerted upon staphylococci (786) and 
M. tuberculosis (482). The active substance (483) was precipitated by 
10 per cent tungstic acid and lead acetate and was thermostable. 

Much and Sartorius (621) 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- 
entericus 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 (294). Various strains of B. mycoides, desig- 
nated as cytoliticusy 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 12). The lytic action of strains of 
B. subtilis upon different bacteria, including M. tuberculosis (903), 
pneumococci, typhoid, diphtheria (62), and other organisms has also 
been definitely established. 

Pringsheim (705) isolated a strain of B. mesentericus-vulgatus that 
had a decided inhibiting effect upon a variety of bacteria, particularly 



86 



BACTERIA AS ANTAGONISTS 



C orynehacterium difhtherlae. 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 
passage (1016). The action of filtrates of B. mesenterkus against diph- 
theria organisms was considered (956) as highly specific. Other strains 
of this organism were reported to be active against Pasteurella pestis 
(246). Living gram-positive bacteria were found (768) to be more 
susceptible than gram-negative organisms to the antagonistic action of 
spore-forming aerobes j in the case of dead organisms, the reverse was 
true. Plates were heavily seeded with the test bacteria and the centers 



TABLE 12. LYSIS OF PATHOGENIC BACTERIA BY VARIOUS STRAINS OF A 
SPORE-FORMING ANTAGONIST (CYTOLYTICUS) 



ORGANISM LYSED 




STRAIN NUMBER OF CYTOLYTICUS 






I 


II 


III 


VI 


VII 


VIII 


IV 


E. ty-phosa 


+++ 


-H-f 




-H- 


+++ 








Paratyfhoid A 


+ 


-hH- 




+ 


-H- 


-H-f 


-K-l- 


Paratyfhoid B 





+ 




-F 


++ 


-1^ 





Shigella 


-H- 


4-f 


-H- 


+ 


++ 


+f 





Y bacillus 


-1- 


^H- 




4- 


++ 





-h 


E. coli 


-H-+ 


4-H- 




-H- 


+-H- 








C. difhtheriae 


-t-H- 


++ 




+ 


-H- 




+ 


Ps. fyocyaneus 





-H- 




-1- 


-H-+ 







S. aureus 


+ 










H^-l- 




-f-F 


S. alius 


-h 


-1- 







-H-+ 




-f- 


S. citreus 


4-f+ 









HH-+ 




-f++ 


S. viridis 


+ 






+ 


-f-H- 




-f-H- 


S. Ivaemolyticus 


-H- 






-f- 


-F+ 




4^+ 


S. mucosus 


^ 






-t- 


-H- 




+f+ 


P. vulgaris (Weil-Felix) 


++ 






-1- 


+ 


-1- 


^H- 


Pneumococcus 


-H-f 






+++ 


+^ 








From Franke and Ismet (294). 

no clearing. 

+ trace but no true clearing. 




++ clearing, 
-t-H- clearing 


slight sediment, 
without sediment. 







SPORE-FORMING BACTERIA 87 

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

Hettche and Weber (408) isolated 41 strains of B. mesentericus 
from 25 samples of soil. These were streaked on blood agar, and the 
diphtheria organism was used for testing their effect. In 1 8 strains the 
antagonistic action was detected in 24 hours j there was no parallelism 
between inhibition and hemolysis. Of the 18 active strains, 1 1 lost the 
property after two transfers and 2 were exceedingly active. 

Dubos (190) isolated 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 staphylo- 
cocci 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. 
Dubos and Hotchkiss (205) soon demonstrated the presence in natural 
substrates, such as soil, sewage, manure, and cheese, of various spore- 
forming bacteria that have marked antagonistic properties against vari- 
ous gram-positive and gram-negative bacteria. 

Hoogerheide (427) isolated from the soil an aerobic, 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 (885). 

It has thus been definitely established (857) that strains of spore- 
forming bacteria possessing antagonistic properties are widely distrib- 
uted in the soil and possess certain physiological characteristics that 
differentiate them from the inactive strains. This is brought out in 
Table 13. 

Spore-forming bacteria are also able to produce substances antagonis- 
tic to fungi (231, 695, 734, 738). Cordon and Haenseler (149) iso- 
lated an organism {B. simplex) that was antagonistic to Rhizoctonia 
solani, an important plant pathogen. The antagonist produced a thermo- 
stable substance that inhibited the growth and even caused the death of 
the fungus. When the substance was added to the soil it controlled to 
some extent seed decay and damping-off disease of cucumbers and peas. 
Christensen and Davies (128) found that a strain of B. mesentericus 



88 BACTERIA AS ANTAGONISTS 

produced on artificial media an active substance that suppressed the 
growth of Helminthosforium sativum. It increased sporulation 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 de- 
stroyed by alkalies but not by acids. It was inactivated or destroyed, 
however, by certain fungi and bacteria. 



TABLE 13. 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-2 I 


- 


- 


+ 


+ 


- 


- 


A-23 


- 


- 


+ 


+ 


- 


- 


A.27 


- 


+ 


+ 


- 


- 


- 


A-34 


- 


- 


+ 


+ 


- 


- 


Inactive Strains 














A-15 


- 


+ 


- 


- 


+ 


+ 


A-31 


+ 


+ 


- 


+ 


+ 


+ 


A-32 + 


+ 


+ 


- 


+ 


+ 


+ 


From Stokes and Woodward (857). 












— reaction becoming alkal! 


ine. 




+ acid produced. 







NONSPORE-FORMING BACTERIA: PS. AERUGINOSA, 
PS. FLUORESCENS, AND S. MARCESCENS 

Among the bacteria, those belonging to the fluorescent, green- 
pigment and red-pigment producing groups have probably received 
the greatest attention as antagonists. Bouchard ( 8 1 ) first reported, in 
1888, that Ps. aeruginosa was antagonistic to B. anthracis; the presence 



NONSPORE-FORMING BACTERIA 



89 



of the antagonist was shown (62, 1 20) to reduce considerably the action 
of the pathogen. When grown on artificial media the pyocyaneus or- 
ganism was found (299) to be strongly antagonistic to a number of 
bacteria, including E. tyfhosa, Pjeijferella 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 14). Ps. 
aeruginosa was shown to be active against E. coU, M. tuberculosis (62, 
81, 120, 760, 999), and a variety of other bacteria (522). The addition 
of top minnows {Gambusia) to water polluted with E. coli caused the 
disappearance of the bacteria j this was shown to be due to the inhibit- 
ing effect of the pyocyaneus organism 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 (384). When a mix- 
ture of the antagonist and the colon organism was incubated, the former 
tended to outgrow the latter after 24 hours {2)S(>)- Even after steriliza- 



TABLE 14. NONSPORE-FORMING BACTERIA AS ANTAGONISTS TO BACTERIA 



ANTAGONIST 

Ps. aei-uginosa 
Ps. aeruginosa 
Ps. fluorescens 



ORGANISMS AFFECTED 

B. anthraciSy E. typhosa, V . 
comma, etc. 

Gram-negative bacteria, M. 
tuberculosis, and yeasts 

E. coli, S. marcescens, C. 
difhtheriae, B. ant hr acts, 
etc. 



Ps. fluorescens Actinomycetes 

5. marcescens CI. chauvoei, B. anthracts, 

staphylococci, micrococci 



5. marcescens 



Gram-positive but not gram- 
negative bacteria 



KNOWN PROPERTY 

Thermostable, filter- 
able substance 

Depresses growth 

Thermostable, filter- 
able substance 



Lytic action 

Colorless, thermo- 
stable, lytic sub- 
stance 

Alcohol-soluble 
pigment 



REFERENCES 
62, 81, 120, 235, 
236, 299 

62, 81, 760, 762, 

763,999 

246, 302, 311, 326, 

406, 407, 409, 410, 

430-432, 524, 525, 

656, 827 

50, 209, 230, 743, 
764 

409 



90 



BACTERIA AS ANTAGONISTS 
TABLE 14 {continued) 



ANTAGONIST 

E. coli 



E. coli 
E. coli 



ORGANISMS AFFECTED 

Typhoid, paratyphoid, diph- 
theria, staphylococci, and 
proteolytic bacteria 

Other E. coli strains 

M. tuberculosis and spore- 
forming bacteria 



KNOWN PROPERTY 

Growth-inhibiting 



REFERENCES 
52, 54,61,93, 121, 
366,491,643, 736, 

759, 876, 886, 954 

643 

105,336,365,367, 
448, 469, 781, 823, 



A . aerogenes 
E. tyfhosa 

S. faratyfhi 
Streptococci 

Streptococci 

Staphylococci 
Staphylococci 

Micrococci 

Diplococci and 
pneumococci 

K. fneumoniae 

P. vulgaris 

P. avicida 
Myxobacteria 

Anaerobic bac- 
teria 



B. anthracis, P. festis 

E. tyfhosa, Ps. fiuorescens, 
E. coli, B. anthracis 

E. coli, B. anthracis, P. festis 

B. afithracis, C. difhtheriae 



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

Dead cells of various bacteria 

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

V . com?na, 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-discase-producing 
bacteria 

M. tuberculosis. B. anthracis 



Activity not associ- 
ated with hemoly- 
sis or virulence 

Thermostable, non- 
filterable substance 



Thermolabile sub- 
stance 

Active filtrate 



Thermostable lytic 
substance 



246,365, 367 
224, 311, 347,898, 

246, 453, 823, 898 

52, 105, 131, 178, 
233, 302,627,676, 
799, 800 

69, 246, 765, 979 



351,994,996 
52, 150, 213, 214, 
246, 248, 861 

21 1, 212, 541, 627 



21 1, 212, 244, 246, 

368, 540, 541,634, 

669,733 

52, 54, 246, 302, 

634,676, 823 

40, 246, 426, 649, 
898,958 

299, 672 

841 

425,671 



NONSPORE-FORMING BACTERIA 91 

tion, media in which Ps. aeruginosa had grown depressed the growth 
of other microorganisms including S. marcescens, Ps. fluorescens, and 
Saccharomyces cereviseae; spore formation by the last was favored 
(762). 

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 (120, 299). By filtering the culture 
through a Berkfeld, evaporating to a small volume, dialyzing through 
a parchment membrane, precipitating with alcohol, and drying over 
sulfuric acid, Emmerich and Low (236) obtained a preparation which 
was designated as pyocyanase. It was soluble in water and highly bac- 
teriolytic. 

Pyocyanase was at first looked upon as an enzyme belonging to the 
class of nucleases. It was found to have, 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 . comma cells and in a few seconds rendered in- 
active 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 inverse 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. Other 
proteolytic systems of bacteria are known to be resistant to high tem- 
peratures and to remain active even when kept in a moist state for 1 5 to 
30 minutes at 100° C. ( i ). Pyocyanase was believed to be transformed 
in the bodies of animals into high molecular proteins which still re- 
tained the bacteriolytic action of the free enzyme. 

Since the early work of Emmerich and Low, an extensive literature 
has accumulated on the nature of pyocyanase. Its lytic effect has been 
established against diphtheria (52), streptococci (237), meningococci, 
the typhoid organism, pneumococci (yb(i)^P. festis ( 246) , Vibrio metch- 
nikovi (501), V. comma (692), and many others (794). There has been 
considerable disagreement, however, concerning the chemical nature 
and therapeutic action of pyocyanase (234, 322), due largely to the 
variation in the nature of the preparations obtained (540). Kramer 
(501), for example, has shown that the activity of the substance de- 
pends on three factors: nature of strain, not all strains being equally 



92 BACTERIA AS ANTAGONISTS 

effective j composition of medium, glycerol-containing media being 
most favorable J and method of extraction of active substance from cul- 
ture media. 

The enzymatic nature of pyocyanase was not universally accepted, 
largely because of the thermostability of the substance (489). Dietrich 
(171) ascribed the action of pyocyanase to a change in osmotic pressure, 
Raubitchek and Russ (733) emphasized that the solubility of the sub- 
stance in ether, chloroform, or benzol is not indicative of an enzyme, 
nor is the fact that temperatures of o to 37° C. fail to influence its ac- 
tivity {55, 185,409,669,919). 

Ps, aeruginosa was found to produce (409), in addition to pyocya- 
nase, a blue pigment, pyocyanin. Both substances possess lytic proper- 
ties, 1:1,000 dilution of the pigment being able to lyse E. coli in 
6 hours (366). The pigment was believed (501) to act only on gram- 
positive bacteria. Pyocyanin was said (407) to be more effective in 
younger cultures, and pyocyanase in older. 

In order to test the action of Ps. aeruginosa upon other bacteria, 
Kramer (501) 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 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 the two pathogens were inoculated into liquid 
media and the antagonist was introduced simultaneously or within 24 
hours, the latter had a decided bactericidal effect. No bactericidal fil- 
trate could be obtained. These results were confirmed (634, 919), the 
conclusion being reached that the active molecules do not pass through 
the ultrafilter (949). Pyocyanin had a bactericidal action also upon 
S. hemolyticus, S. albus, S. aureus, C. difhtheriae, M. tuberculosis, V. 
metchnikovi, and the Y-Ruhr bacillus, but not upon P. vulgaris, E. coli, 
or the typhoid organism. In general, gram-positive bacteria were 
largely affected. 

More recently, Schoental (809) succeeded in isolating three anti- 
bacterial substances from the chloroform extracts of cultures of Ps. 
aeruginosa: (a) a blue pigment, pyocyanin j (b) a yellow pigment, 
tf-oxyphenazine, a derivative of pyocyanin j and (c) an almost colorless 



NONSPORE-FORMING BACTERIA 93 

bacteriolytic substance most readily found in old culture media. None 
of these substances was enzymatic in nature. Schoental found that pyo- 
cyanin had a strong bactericidal activity, but its high toxicity and insta- 
bility made it unpromising for therapeutic purposes. The action of 
^-oxyphenazine against many bacteria made it comparable to the fla- 
vinesj however, it was less toxic and non-irritant. The third antibacterial 
substance had a marked lytic action on vibrios, being bactericidal in a 
concentration of i : 1 0,000 and bacteriostatic in i : 100,000. 

No less extensive is the literature on the antagonistic action of the 
fluorescent group of bacteria, first established by Garre (311) in 1887 
and later by others (50, 246, 302, 326, 430-43^, 462, 524, 525, 6s 5, 
827). Hettche and Vogel (407) described the inhibiting effect of 
strains of this organism on the growth of various gram-negative and 
gram-positive bacteria (Table 15). The active substance is thermo- 
stable, dialyzes through a membrane, and passes through Seitz and 
Berkfeld filters (525). It is said (407) to be soluble in chloroform. 
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 (123). Rahn (711) observed the 
phenomenon of iso-antagonism, which is associated with the formation 
of a thermolabile substance that does not pass through a filter. Certain 
bacteria were found (228, 711) to be favored by their own metabolic 
products, whereas others had an adverse effect j the products of the 
first group were usually thermostable and nonfilterable, and the second 
were thermolabile (at 60° to 100° C.) and were destroyed by light. 

5. marcescens is known to exert antagonistic effects against a number 
of bacteria, including diphtheria, gonococci, anthrax (743, 764), and CI. 
chauvoei (209), as well as fungi causing insect diseases (587). The for- 
mation of antibiotic substances by this organism has been demonstrated 
by various investigators. These substances are active not only in vitro 
but also in vivo ( 743 ) . Their activity increases with the age of the cul- 
ture. Their formation was believed not to be associated with the pro- 
duction of the pigment by the organism. They were also of a nonlipoid 
nature. Hettche (409), however, asserted that the bactericidal action 
of Serratia is closely related to pigment production. The pigment was 
extracted with alcohol and was found capable of dissolving dead gram- 



94 BACTERIA AS ANTAGONISTS 

TABLE 15. 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. anthracis 


- 


+ 






B. vulgatus 


- 


- 


+ 




B. subtilis 


- 


- 


+ 




B. megat/ierium 


- 


+ 






R. cinnebareus 


- 


+ 






R. roseus 


- 


- 


+ 




M. -flavus 


- 


- 


- 


+ 


N . catarrhal is 


- 


- 


- 


+ 


Ps. aeruginosa 


- 


- 


- 




Ps. fiuorescens 


- 


- 


- 




S. lutea 


- 


- 


- 


+ 


S. marcescens 


- 


- 


- 


- - + 


5. albus 


- 


- 


+ 




S. aureus 


- 


- 


- 


+ 


S. citreus 


- 


- 


+ 




K. pneumoniae 


- 


- 


- 


+ 


V. comma 


- 


+ 






Ch. violaceum 


- 


+ 






E. tyfhi 


- 


- 


+ 




Sh. faradysenteriae 


- 


- 


4- 




S. enteritidis 


- 


- 


- 


4- 


S. suisfestifer 


- 


- 


- 


4- 


S. fullorum 


- 


- 


- 


4 


.E. coli 


- 


- 


- 


_ _ _ + 


A . aero genes 


- 


- 


- 


_ _ _ + 


Ph. b owl e sit 


- 


- 


+ 




Sac. marianus 


- 


- 


- 




Sac. ellifsoideus 


- 


- 


- 


- - - - + 


Sac. fastorianus 


- 


- 


- 


----- + 


Xygosac. friorianus 


- 


- 


- 


- - - - + 


Torula sfhaerica 


- 


- 


- 




A . niger 


- 


- 


- 





From Lewis (525). 

+ denotes complete inhibition. 



COLON-TYPHOID BACTERIA 95 

positive bacteria but not gram-negative organisms. Eisler and Jacobsohn 
(230) 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 (425). Bienstock (54) reported, in 1899, 
that proteolytic bacteria are repressed by the presence of E. coU and 
A. aerogenes. Tissier and Martelly (886) emphasized that this phe- 
nomenon occurs only in the presence of sugar, the effect being due to 
the fermentation of the sugar by E. colt, resulting in the production 
of acid. 

Wathelet (954) observed in 1895 that in mixed culture the colon 
bacterium gradually replaces the typhoid organism and this was later 
fully confirmed (141, 383, 491, 619, 643, 799, 800, 876). The occur- 
rence of slowly growing lactose-fermenting strains of E. coli in stools 
has been ascribed to the phenomenon of antagonism (453), and the 
inhibitory action upon E. tyfhosa added to certain stools was also as- 
cribed to the antagonistic action of E. coli (643), 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 antagonis- 
tic index 5 an index of 100:20 means that for every 100 colonies of the 
colon organism, 20 colonies of typhoid developed. Manteufel (581), 
however, ascribed this antagonistic action of E. coli to the exhaustion of 
nutrients in the medium. 

Active colon strains were found to be inhibitive to other strains of 
the same organism. The existence of strong and weak antagonistic 
strains has been questioned frequently (1005). Nissle (642) ascribed 
to many of these strains a strong antagonistic action against the patho- 
genic intestinal flora. These results were contested, however (98, 512). 
The action of E. coli of different origin varies (750), freshly isolated 
strains being more active than stock cultures (837). It has also been 



96 BACTERIA AS ANTAGONISTS 

reported that fresh, actively growing cultures of E. tyfhosa inhibited 
the growth of E. coli, older cultures not being antagonistic (915). The 
antagonistic properties of E. coli were often believed to be associated 
with the formation of unstable, thermolabile lytic substances, that 
would not pass through a filter (365, 367, 580). 

A bacteriophage was found (532) to develop as a result of the an- 
tagonistic action of E. coli against the Shiga bacillus and was said to 
occur in the intestines where antagonistic conditions are always present. 
A similar effect was observed by Fabry (245), due to the antagonistic 
stimulus of E. coli by a strain of S. albus. Gratia (348) found that 
the filtrates of one race of E. coli inhibited another race and caused an 
agglutination of the latter in fluid media. According to Hashimoto 
(383), the weakest antagonists belong to the paracolon group, the 
strains of medium activity to the colon group, and the strongest an- 
tagonists to the colon-immobilis type. Whenever the feces were found 
to contain large numbers of E. coli, no typhoid organisms were present. 
The resistance of certain persons to intestinal diseases was, therefore, 
ascribed to the high antagonistic colon index. By utilizing the principle 
of antagonism of some strains of E. coli against others, two types of 
E. coli resistant to the antagonistic substance were isolated ( 168) : one 
produced giant colonies, the other small punctiform, translucent 
colonies. 

E. coli exerts an antagonistic action also upon Salmonella schottmUl- 
leri (305), C. difhtheriae (52, 905), staphylococci (366, 491), M. tu- 
berculosis (6So,6Si)yB.anthracis (105,365-367,781, 823, 898), vari- 
ous spore-forming soil bacteria (469), and putrefactive water bacteria 
(759). The action against anthrax was said to be only temporary (336). 
It was also suggested (448) that only living cultures of E. coli are ac- 
tive. The simultaneous inoculation of S. aureus and E. coli was found 
(736) to be injurious to the first and not to the second organism j this 
effect was increased by an increase in the number of E. coli cells in the 
inoculum. Gundel and Himstedt (366) have shown that E. coli, but 
not A. aero genes y is antagonistic to S. aureus and S. albus. 

The term autophage has been used (334) 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 



COCCI 97 

dead cells are used as nutrients by the living organism. The mechanism 
of the action was variously explained by a change in the fH 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- 
onstrated (141, 26Sy 36^, 580). These substances have been considered 
either as autotoxins (141) or as proteolytic enzymes (683). According 
to Schilling and Califano (799, 800), the filtrate of E. colt depressed 
only the dysentery organism of Shiga. From a bouillon culture of E. 
coliy Gundel (370) isolated thermostable lipoids capable of bringing 
about the lysis of the colon bacteria and other bacteria. The antagonistic 
relations between E. coli and V. comma have been well established, the 
cholera organism also possessing antagonistic properties (307, 480). 

The typhoid organism is also capable of exerting an antagonistic ac- 
tion against itself, as well as against Ps. fuorescens (311) and E. coli 
(323). Similar antagonistic effects (224) have been obtained against 
various other bacteria, including B. anthracis (823, 898). The nature 
of the action is not clearly understood. Salmonella -paratyphi possesses 
antagonistic properties against E. coli (453), B. anthracis (898), P. 
pestis (246), and various other bacteria. 

It may be of interest to record here that E. coli isolated from persons 
affected by rectal cancer are able to convert substances related to bile 
acids to carcenogenic bodies. The possibility of synthesis of such sub- 
stances has also been suggested (186). 



COCCI 

Numerous cocci have been found to possess antagonistic properties 
against other bacteria. Doehle (178) first demonstrated in 1889 that 
streptococci are able to antagonize B. anthracis y especially on solid 
media. Similar action was exerted against diphtheria bacteria (52, 71, 
131? 799j 800), a phenomenon apparently not correlated with the 
hemolytic properties or the virulence of the antagonist. Further studies 
established the effect of various streptococci against anthrax (233, ^dG^ 
676). According to Cantani ( 105) this effect is more pronounced in liq- 
uid than in solid media, and is highly specific as regards the strain. S. 
pyogenes was shown to be antagonistic, in vivo, to B. anthracis and to 



98 BACTERIA AS ANTAGONISTS 

Phytomonas tumefacienSy even to the extent of suppressing vegetative 
malformations brought about by the latter (69). Streftococcus cremoris 
was active against Streftococcus lactis (979), Streftococcus mastidis 
against S. lactis and Lactobacillus acidophilus^ and Streftococcus muco- 
sus against P. festis (246). Rogers (765) reported an antagonistic effect 
of S. lactis against L. bulgaricus; the active substance was thermostable 
and was unable to pass through a bacterial filter (150, 178, 669, 676). 

Freudenreich (299) first emphasized the antagonistic action of 
staphylococci against various bacteria. The list was later enlarged to in- 
clude gram-positive acid-resisting forms (211), corynebacteria (52, 
213, 214), and the plague organism (246). Some of these antagonists 
were found to be able to lyse the dead cells of their own kind (351, 994- 
996) as well as those of various other organisms. Gundel (370) 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 corynebacteria, was isolated from a strain 
of staphylococcus (213, 214). 

Various micrococci possess strong antagonistic properties. Lode 
(541 ) 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 Micro- 
coccus antibioticus (211) was found to possess a strong antagonistic ac- 
tion against V . comma^ M. tuberculosisy E. tyfhosa. Ph. tumejacienSy 
Br. melitensisy various spore-forming bacteria, numerous cocci, and 
others (627). Diplococci exerted an antagonistic action against various 
bacteria (541 ), including pyogenic staphylococci and streptococci in the 
sputum (363), spore-formers, and gram-negative bacteria (447). They 
produced, under aerobic conditions only, a filterable substance that was 
heat resistant. 

The antagonistic action of pneumococci has definitely been estab- 
lished (212, 244, 246, 368, 571, 634, 669, 733). The active substance 
of these organisms was said (541) to be thermolabile, since it was de- 
stroyed at 80° to 85° C. } it was produced only under aerobic conditions. 
In reviewing the literature on the longevity of streptococci in symbiosis, 
Holman (425) observed that many chances of error are inherent in 
mixed cultures, particularly with closely similar organisms j pneumo- 



OTHER BACTERIA 99 

cocci, for example, were found to be able to live for long periods to- 
gether with nonhemolytic streptococci. Peculiar antagonistic relations 
between pneumococci and staphylococci were also reported (15). Adap- 
tive alterations could be expected in the growth of bacteria in mixed 
cultures (31). Which of the two organisms antagonizes the other was 
believed to depend frequently upon the numerical abundance of one or 
the other (244). 



OTHER AEROBIC AND ANAEROBIC BACTERIA 

The antagonistic action of Klebsiella pneumoniae against B. anthracis 
has been reported (216, 676, 823). Freudenreich (299) found that the 
filtrate of this antagonist repressed the growth of a number of bacteria 
including the diphtheria (52, 634) and plague (246) 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 (246, 898)5 of Ps. aviseftica 
against B. anthracis (672) and E. tyfhosa (299) j of Bacterium lactis 
aerogenes against B. anthracis (365, 367) and P. festis (246). B. an- 
thracis is capable of iso-antagonism (781, 827) and of antagonizing cer- 
tain other organisms, including E. tyfhosa and Bacterium acidi lactici 
(299, 827). Certain Myxobacteriales have been shown (841) to be ca- 
pable of bringing about the lysis of various plant-disease-producing bac- 
teria j a thermostable lytic substance, passing through cellophane but 
not through a Seitz filter, was obtained. Although certain bacteria like 
Achromohacter lifolyticum were found capable of reducing the patho- 
genicity of M. tuberculosis^ no active cell-free extract could be ob- 
tained (82). 

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- 
stance could be demonstrated j the lactic acid itself had only a limited 
effect (11). Korolev (499) has shown that when a yellow sarcina was 
added to solid media a stimulating effect was exerted on the growth of 



100 BACTERIA AS ANTAGONISTS 

species of Brucella {Br. melitensis, 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 (372). Since acid itself cannot bring about this effect, 
Holman (426) suggested that the acid must be active in a nascent state. 
A mixture of a Clostridium sf or genes and CI. hotulinum also inter- 
fered with the development of the toxin ; it was even thought possible 
that the first anaerobe might cause the disappearance of toxin already 
produced (158, 1 59, 463 ) . 5. aureus, E. coli, P. vulgaris, and other bac- 
teria permitted the growth of CI. hotulinum in aerobic cultures, accom- 
panied by toxin production (291 ). However, Streftococcus thermofhi- 
lus inhibited the growth of CI. hotulinum, the toxin of the latter being 
gradually destroyed (478). 

Passini (671) 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 
(425). Novy (649) reported that the injection into guinea pigs of P. 
vulgaris and Clostridium oedematiens resulted in rapid death of the 
animals and extensive growth of the anaerobe in the animal bodies j 
however, the simultaneous inoculation of CI. sforogenes and P. vul- 
garis did not result in putrid lesions (426). According to Barrieu (40), 
the presence of P. vulgaris and certain nonpathogenic spore-bearing 
aerobes in wounds favors, through their proteolytic activity, the viru- 
lence of pathogenic bacteria. Pringsheim (705) grew CI. welchii with 
Alkali genes fecalis for ten generations on agar slants and could easily 
detect in the growth of the latter the opaque colonies of the anaerobe. 
A liquefying sarcina allowed CI. welchii and Clostridium butyricum to 
grow in open tubes. Weinberg and Otelesco (958) believed that many 
war-wound infections are due to an association of P. vulgaris with 
anaerobes, since the former increased the virulence of Clostridium fer- 
fringens and others. 

The antagonistic effects of lactic acid bacteria of the L. hulgaricus and 
L. acidofhilus groups have received considerable attention (76), espe- 



OTHER BACTERIA 101 

dally in regard to their action against intestinal bacteria. This was be- 
lieved to be due to the production of acid by the bacteria rather than to 
the formation of specific antagonistic substances (590). This phenome- 
non aroused particular interest because of the function of some of these 
organisms in replacing bacterial inhabitants of the human digestive 
system (497). 



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 Actinomycetales 
has been divided into three families, Mycobacteriaceaey Actinomy- 
cetaceaey and Streftomycetaceaey comprising the genera Mycobacte- 
riuniy Actinomyces y NocardiUy StreftomyceSy and IVLicromonosfora. 
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 is known of the physiology of actinomycetes. 
Some produce certain organic acids from carbohydrates (287, 1002)^ 
others prefer proteins and amino acids as sources of energy, many spe- 
cies being strongly proteolytic. Some are able to attack starch, with the 
production of dextrins and sugar, accompanied by the formation of 
diastatic enzymes. Many reduce nitrates to nitrites. Some attack sucrose 
and form the enzyme invertasej others, however, do not. Certain spe- 
cies 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 re- 
ceived particular 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 pig- 
ments range from blue, yellow, and orange to various shades of grey. 

According to Beijerinck (43), the process of pigment production by 
actinomycetes in gelatin media is associated with the formation of a 
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 





S. antthioticus, important antagonist. 
From Waksman and Woodruff (945) 



S. lavendulaCy important antagonist 




Submerged growth of S. iavendulae. 
From Woodruff and Foster (1002) 



\. 



Thermophilic Streftomyces. From 

Waksman, Umbreit, and 

Cordon (944a) 



'X 



\>r 






M. vulgaris. From Waksman, 
Cordon, and Hulpoi (931) 



Streftomyces 3042, showing close spiral 
type of branching. Prepared by Starkey 



Figure 10. Types of actinomycetes. 



ACTINOMYCETES AS ANTAGONISTS 103 

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, KNO;., 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- 
cetes (945) i 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 (287, 926, lOOi, 1002). 
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 at a reduced oxygen tension. The aerobic actinomycetes 
commonly found on grasses and in soil are said (490) 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 
causing infections in man and other animals, and certain plant diseases 
(potato scab, sweet potato pox) are caused by aerobic species of actino- 
mycetes. 



104 ACTINOMYCETES AS ANTAGONISTS 

ANTAGONISTIC PROPERTIES 

Many actinomycetes have the ability to antagonize the growth of 
other microorganisms, notably bacteria, fungi, and other actinomycetes j 
this is brought out in Tables 1 6 and 1 7. The antagonistic species are not 
limited to any one genus but are found among three genera, NocardiUy 
StreftomyceSy and Micromonosfora. 

Gasperini (317) first demonstrated, in 1890, that certain species of 
the genus Streftomyces 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 (360, 361) found that soil actinomycetes are able 
to antagonize 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 limiting bacterial development. Lieske 
(527) demonstrated that specific actinomycetes are able to bring about 
the lysis of many dead and living bacterial cells j they are selective in 
their action, affecting only certain bacteria such as S. aureus and S. -pyo- 
genes , but not S. lutea, S. marcescens, or Ps. aeruginosa. 

Rosenthal (767) 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 
substance that diffused through the agar and dissolved the diphtheria 
cells. 

Gratia and Dath (350) suspended dead cells of staphylococci and 
other bacteria in 2 per cent agar and exposed the plates to the air. A cul- 



ANTAGONISTIC PROPERTIES 



105 



TABLE I 6. ANTAGONISTIC PROPERTIES OF VARIOUS ACTINOMYCETES 



ANTAGONIST 


ORGANISMS AFFECTED 


KNOWN PROPERTY 


REFERENCES 


S. alius 


Pneumococci, strepto- 


Thermolabile sub- 


347,350, 




cocci, staphylococci, 


stance, causes lysis 


971-973 




Ps. aeruginosa, etc. 


of dead cells 




S. albus 


Various fungi 


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


12-14 


S. antibiottcus 


All bacteria and fungi, 


Thermostable sub- 


947 




especially gram-posi- 


stance, bacterio- 






tive types 


static 




S. lavendulae 


Various gram-positive 


Organic base, water- 


950 




and gram-negative 


soluble 






bacteria 






S. fraecox 


S. scabies 




604, 605 


Streftomyces sp. 


Bacteria and fungi 


Lytic action 


317 


Strefiomyces sp. 


Diphtheria 


Growth inhibition 


767 


Streftomyces sp. 


B. mycoides, proactino- 


Bactericidal action. 


80, 504 




mycetes, mycobacteria 


with or without 
lysis 




Streftomyces sp. 


Fusarium 


Lytic action 


595 


N. gardneri 


Gram-positive bacteria 


Bacteriostatic action 


309, 936 


Micromonosfora 


Gram-positive bacteria 


Thermostable active 
substance produced 


936 


Actinomycetes 


Dead and living bacteria 


Lysis 


527 


Actinomycetes 


Spore-forming bacteria 


Repression of growth 


360, 970 


Actinomycetes 


Gram-positive bacteria 


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


507, 628 


Actinomycetes 


Pythium 


Thermostable sub- 
stance 


884 



106 



ACTINOMYCETES AS ANTAGONISTS 



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- 



TABLE 17. ANTIBACTERIAL SPECTRUM OF CERTAIN ANTAGONISTIC 
ACTINOMYCETES 



TEST ORGANISM 


ZONE OF INHIBITION, 


, IN MILLIMETERS 






S. violaceus 


5. aurantiacus 


5. griseus 


5. globisporus 


A'', rubra 


35 


32 










N. corallina 


4-0 


45 


22 




10 


N. alba 


4-0 


25 










M. rubrum 


40 


33 


10 







M. citreum 


38 


37 










M. tuberculosis 


8 


10 










M. smegmae 


10 


8 










M.fhlei 


20 


25 










Corynebacterium sp. 


12 


10 










E. coli 
















S. aureus 


25 


19 










M. ruber 


35 


28 










B. mycoides 


30 


10 










B. megatherium 


33 


5 










B. mesentericus 


30 


2 










B. subtilis 


23 


I 










B. tumescens 


22 













Ps. fiuorescens 
















Ps. aeruginosa 
















P. vulgaris 
















S. marcescens 
















M. luieus 


30 


25 










M. candicans 


37 


22 










M. roseus 


42 


27 










M. lysodeikticus 


38 


33 










S. lutea 


30 


27 










Az. vinelandii 


3 













Az. chroococcum 


5 













Rh. leguvmiosarum 
















Radiobacter 

















From Krassilnikov and Koreniako (504). 



ANTAGONISTIC PROPERTIES 107 

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 other gram-negative bacteria, such as Ps. aeruginosa; however, 
it was inactive toward 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 (349). Gratia (347) 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 (972, 973) made a detailed study of the lytic activity of an 
actinomyces culture, presumably identical with the one 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 
(971). It was precipitated by acetone, alcohol, and ammonium sulfate. 
Most of the gram-negative bacteria were not attacked either by actino- 
mycetin 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 activity of the preparation against dead bacteria and of the living 



108 ACTINOMYCETES AS ANTAGONISTS 

culture against living bacteria suggested that the same substance is con- 
cerned in both cases. The bacteria were therefore divided (970), 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 (80), 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 (504) found that many species of actino- 
mycetes belonging to the genus Streftomyces but not Nocardia pro- 
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 j it was less active upon 



ANTAGONISTIC PROPERTIES 109 

spore-bearing bacteria and had no action at all on nonspore-forming 
bacteria, as illustrated in Table 17. 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 Rhizobium and Azotobacter, not only were not inhibited 
but were actually able to develop in filtrates of the antagonists. 

Nakhimovskaia (628) found that antagonistic actinomycetes are 
widely distributed in nature. Of 80 cultures isolated from different 
soils, 47 possessed antagonistic properties, but only 27 secreted anti- 
biotic substances into the medium (Table 18). These agents were ca- 
pable of inhibiting the growth of gram-positive but not of gram-nega- 
tive bacteria or fungi. The nature of the action of the various antagonists 

TABLE 18. OCCURRENCE OF ANTAGONISTIC ACTINOMYCETES IN 
DIFFERENT SOILS 





TOTAL STRAINS 


NUMBER OF 


STRAINS WHICH 




OF ACTINOMY- 


ANTAGONISTIC 


LIBERATED TOXIC 


NATURE OF SOIL 


CETES TESTED 


STRAINS 


SUBSTANCES 


Chernozem 


24 


10 


9 


Podzol 


II 


7 




Solonets 


4 


4 




High altitude soil 


9 


6 




Sandy soil 


6 


5 




Dry desert soil 


5 


4 




River bank meadow 


14 


7 




Cultivated soil 


7 


4 


2 


Total 


80 


47 


27 



From Nakhimovskaia (628). 

was found not to be identical. Some secreted 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 
any substance into the medium, the presence of the growing antagonist 
was essential in order to bring about an inhibition of bacterial develop- 



110 ACTINOMYCETES AS ANTAGONISTS 

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 (628) 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 those strains 
that produced antagonistic effects in artificial nutrient media were inef- 
fective 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 (507). 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 f¥L 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 (936) of the distribution of antagonistic ac- 
tinomycetes in soils and in composts, it was found that of 244 cultures 
isolated at random from different soils, 49, or 20 per cent, of the cultures 
were actively antagonistic j 57, or 23 per cent, showed some antagonistic 



ANTAGONISTIC PROPERTIES 



111 



properties} and 138, or 57 per cent, possessed no antagonistic action at 
all (Table 19). 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 

TABLE 19. ISOLATION OF ANTAGONISTIC ACTINOMYCETES 
FROM VARIOUS SUBSTRATES 







GROUP I 


GROUP II 


GROUP III 


GROUP IV 




TOTAL 




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 


I I 


14.7 


8 


10.7 


4 


5.2 


52 


69-3 


Potted soil 


13 


I 


7-7 


I 


7-7 








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 








I 


6.7 


Lake mud 


9 


3 


33-3 


4 


44.4 








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 (936). 

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

antagonistic properties, and those in group IV showed no antibacterial effects with the methods used. 



members of the genus Nocardia proved to be antagonistic j only one of 
the Micromonospora forms was active. Most of the antagonists were 
found among the members of the genus Streftomyces. These cultures 
were also examined for bacteriolytic properties, living S. aureus being 
used as the test organism. On this basis, 87 cultures (53.1 per cent) 



112 ACTINOMYCETES AS ANTAGONISTS 

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 (970) 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 (Table 20). 

TABLE 20. BACTERIOLYTIC AND BACTERIOSTATIC PROPERTIES OF 
VARIOUS SPECIES OF ACTINOMYCETES 











BACTERIOSTASIS 






BACTERIOLYSIS 


bacteriolysis 


OF B. SUBTILIS 






BY LIVING 


BY BROTH 


BY AQUEOUS 


ORGANISMS 




ORGANISMS* 


FILTRATEf 


EXTRACTS 


Group I. Actinom 


VCETES Hi 


[GHLY Bacteriostatic to B. subtilis 


S. antibiodcus 







- 


-H- 


S. californicus 




+ 


C 





S. candidus 




-H- 


c,s 


++ 


S. cellulosae 




+ 


c 





S. griseus (3326b) 




+ 


c 





S. lavendulae 




+ 


c 


++ 


S. reticuli 




+ 


c 





S. roseus 




+ 


C 


+ 


S. ruber 




+ 


- 





S. sap-ophyticus 




-H- 


c,s 





S. scabies (3031) 




-1- 


c 





Strefiomyces s^. (3069) 


-H- 


c 





5. albus (G) 




-H- 


c,s 





Streftomyces sp. (33 


187) 


-K- 


c,s 





N. gardneri 







c 


-H- 


Micromonosfora sp. 







- 


-H- 



Group II. Actinomycetes Moderately Bacteriostatic to B. subtilis 

S. albus {^T,()\) -H- C, S o 

S. cretaceus + c O 

5. albus, var, ochraleuceus -H- C, S O 

5. annulatus + — O 

S. aureus -h CO 

5. bovis -J- CO 

S.f radii -H- C, S o 



ANTAGONISTIC PROPERTIES 

TABLE 20 {continued) 



113 











BACTERIOSTASIS 






BACTERIOLYSIS 


BACTERIOLYSIS 


OF B. SUBTILIS 






BY LIVING 


BY BROTH 


BY AQUEOUS 


ORGANISMS 




ORGANISMS* 


FILTRATE t 


EXTRACTS 


Group II {contmued) 










S. griseus 




++ 


C,S 





S. halstedii 




+ 


C 





S. hominis 




++ 


C,S 





S. lifmanii 




+ 


c 





S. mtcroflavus 




+ 


c 





S. odortfer 




++ 


- 





S. fraecox 




+ 


c 





S. rutgersensis 




-H- 


C,S 





S. samfsonii 




-H- 


C,S 





S. scabies (3352) 




+ 


- 





5". scabies (302 1) 




-hH 


c 





5. setonii 




-H- 


c,s 





S. tetanusemus 




++ 


c,s 





S. coelicolor (3033) 




+ 


Not tested 


Not tested 


Streftomyces^'p. (Lleske, 


No. 


23) ++ 


c,s 





Streftomyces sp. (Lieske, 


No. 


25a) 4-f 


c 






From Waksman, Horning, Welsch, and Woodruff (936). 

* No activity against 5. aureus is indicated by o, moderate activity by +, high activity by ++. 

t Lysis of heat-killed E. coli is indicated by C (high activity) and c (moderate activity); lysis of 

living 5. aureus is indicated by S ; — indicates no activity. 



Actinomycetes also show antagonistic activities against fungi (12- 
14, 844). S. albusy for example, was capable of inhibiting the growth 
of all species of fungi tested, an effect shown to be due to the production 
of an active substance. By the use of a culture of Colletotrichum gloe- 
osforioidesy 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 f¥L 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} the second, 38.8 per cent; and the third, 43.7 per 
cent. These results are surprisingly similar to those reported for the 
distribution of actinomycetes possessing antibacterial properties, includ- 



114 ACTINOMYCETES AS ANTAGONISTS 

ing those that were isolated at random from the soil and those taken 
from a culture collection. 

Meredith (595) 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 (iG 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 

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, six substances have been definitely recog- 
nized j namely, actinomycetin, actinomycin, streptothricin, streptomy- 
cin, proactinomycin, and micromonosporin. 

Among the various antagonistic actinomycetes, five species have 
been studied in detail and, therefore, deserve particular attention, 
namely, S. antibiotkus (945), Streftomyces lavendulae (973), Stref- 
tomyces griseus (795), Nocardia gardneri (309), and 5. albus (970). 

S. antibiotkus produces two highly active antibiotic substances that 
have been isolated and described as actinomycin A and B. The first of 
these has been studied in greater detail. It was shown to be antagonistic 
to all species of bacteria tested as well as to many fungi, as brought out 



NATURE OF ANTIBIOTIC SUBSTANCES 115 

in Table 2 1 . The organism produces dark-brown to black pigments on 
media containing protein and peptone. It 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 dilutions. It is highly toxic to animals. Actinomycin 

TABLE 21. BACTERIOSTATIC SPECTRUM OF ACTINOMYCIN 







ACTINOMYCIN 


ADDED, MILLIGRAMS 


ORGANISM 


GRAM STAIN 




PER LITER OF MEDIUM 








O.I 


I.O 


10 


I GO 


5. marcescens 


- 




3 


3 


3 


A . aero genes 


- 




3 


3 


3* 


E. coli (intermediate) 


- 




3 


3 


3* 


E. coli 


- 




3 


3 


I* 


Ps. aeruginosa 


- 




3 


3 





Ps. -fluorescens 


- 




3 


3 





Br. abortus 


- 




3 


3 





N. catarrhalis 


- 




3 


2 





E. carotovora 


- 




3 


2 





Sh. gallinarum 


- 




2 


2 


o 


A . stutzeri 


- 




2 


I 





H. fertussis 


- 




3 








Az. vinelandii 


- 













S. cellulosae 


+ 




2 


I 





S. calif ornicus 


+ 




3 


2 





M. tuberculosis 


+ 




3 








CI. welchii 


+ 













B. macerans 


+ 




3 


o 





B. megatherium 


+ 













B. folymyxa 


+ 










o 


B. mycoides 


+ 




o 








B. mesentericus 


+ 




o 





o 


B. cereus 


+ 




o 








B. subtilis I 


+ 


o 


o 








B. subtilis II 


+ 


o 











G. tetragena 


+ 











o 


S. lutea 


+ 


o 





o 





Streptococci and staphylococci 


+ 








o 






From Waksman and Woodruff (946). 

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

* rf 200 mg. per liter were added the results were usually as follows: for A. aerogenes, fair; for 

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



16 



ACTINOMYCETES AS ANTAGONISTS 



is produced in both organic and synthetic media, the addition of a small 
amount of agar increasing considerably the growth of the organism and 
the production of the active substance. The addition to the medium of a 
small amount of starch, as well as of phosphate and sodium chloride, was 
also found to be favorable. S. antibioticus is strictly aerobic, and is able to 
produce the active substance only under aerobic conditions that can be 
brought about by growing it either in very shallow layers or in aerated 
or agitated submerged growth. Actinomycin is extracted directly from 
the medium by means of ether j the ether is then evaporated and the 
substance taken up in alcohol. Further purification is accomplished by 
means of petrol ether and passage through a chromatographic column, 
as shown later (p. 171). 

Streftomyces lavendulae is capable of inhibiting the growth of many 
gram-negative bacteria as well as of various gram-positive forms. The 
antibiotic substance produced by this organism was designated as strep- 
tothricin. The organism is grown in a tap-water medium containing i .0 
per cent glucose, 0.5 per cent tryptone, 0.2 per cent K0HPO4, 0.2 per 
cent NaCl, 0.00 1 per cent FeS04, and 0.25 per cent agar. The glucose 



TABLE 22. COMPARATIVE ACTIVITY OF TWO STRAINS OF S. LAVENDULAE 







TREAT- 


DAYS 


GROWTH 
IN MG. 


ACTIVITY 
IN UNITS 


STRAIN 


SOURCE OF 


MENT OF 


OF INCU- 


PER 100 ML. 


E. 


B. sub- 


NUMBER 


NITROGEN 


CULTURE 


BATION 


OF MEDIUM 


colt 


tilts 


8 


Tryptone 


Shaken 


2 


346 


150 


1,000 


H 


Tryptone 


Shaken 


2 


361 


150 


750 


8 


Tryptone 


Shaken 


5 


253 


100 


1,000 


H 


Tryptone 


Shaken 


5 


296 


100 


500 


8 


Glycine 


Shaken 


2 


162 


30 


30 


14 


Glycine 


Shaken 


2 


146 


30 


30 


8 


Glycine 


Shaken 


5 


266 


100 


500 


14 


Glycine 


Shaken 


5 


271 


30 


150 


8 


Tryptone 


Stationary 


8 


245 


20 


200 


14 


Tryptone 


Stationary 


8 


- 


75 


300 


8 


Glycine 


Stationary 


8 


239 


25 


150 


14 


Glycine 


Stationary 


8 


- 


75 


200 



From Waksman (926). 

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



NATURE OF ANTIBIOTIC SUBSTANCES 117 

can be replaced by starch, in which case the presence of agar is unneces- 
sary. When grown in submerged or agitated cultures, the agar is left 
out. The tryptone can be replaced by a variety of simple nitrogenous 
compounds, such as glycine, alanine, aspartic acid, asparagine, and glu- 
tamic acid (Table 22)} the carbohydrate may be left out completely, 
with only limited reduction in activity. No growth of the organism is 
obtained on tryptophane, phenyl alanine, and certain other forms of 
nitrogen. With ammonium sulfate or sodium nitrate good growth may 
be obtained but the production of the active substance is limited unless 
the organism is grown under submerged conditions. Iron appears to 
play an essential role in the production of the active substance. An in- 
crease in growth as a result of an increase in carbohydrate concentration 
does not result in an increase in streptothricin content, but an increase 
in growth as a result of an increase in the amino-acid concentration, with 
the same amount of carbohydrate, causes an increase in the production 
of streptothricin. When the medium contains one amino acid as the only 
source of carbon and nitrogen, there is a gradual increase in the alka- 
linity of the medium, resulting in the destruction of the streptothricin. 
Neither the growth of the organism nor the production of the strepto- 
thricin, however, is influenced by the reaction of the medium, within 
certain limits, even between /)H 4.4 and 8.0 (lOOi). The metabolism 
of S. lavendulae and the course of production of streptothricin under 
stationary and submerged conditions are illustrated in Figure 1 1 . Meth- 
ods of isolation of streptothricin and its chemical properties are de- 
scribed later (p. 173). Its bacteriostatic spectrum or action against vari- 
ous bacteria is shown in Table 23. It has only limited toxicity to animals 
and is active in vivo against both gram-positive and gram-negative bac- 
teria (755). 

Certain strains of Streftomyces griseus produce an antibiotic sub- 
stance, designated as streptomycin, that is also active against both gram- 
positive and gram-negative bacteria. It is similar in its solubility and 
various chemical properties to streptothricin j however, it acts readily 
against B. mycoides and is more active than the latter against certain 
gram-negative bacteria, such as Ps. aeruginosa. The organism grows 
well in stationary liquid media containing meat extract or corn steep. 
Streptomycin is active in vivo against a variety of bacteria, some of 






1 


7 ~l 




zl 






UJ, 


1 




:i: 


1 


■2. 


< 


1 


O 


I 


J 












/cf 


o 




/< 


D 


/ " 


/^ 


Q 


O 


q; 


/ 


/P 


Q. 


/ 


//? " 


z 


f 


ho 


o 


W^ 


4 


Q/ 


''»>^ 


\ 


I 


>-^ 


\ 


1- 






o 




\\ 


1- 




» \ 


a 

UJ 




v\ 


q; 




\ \ 


(- 






10 


1 1 1 


1 1 



D 

O 

CM Z 



asmnniiAj 2i3d s±iNn 




3siniinD JO s^BinniiPN 99 2i3d si^vyonim 



TABLE 23. INHIBITORY EFFECT OF STREPTOTHRICIN UPON GROWTH 
OF VARIOUS BACTERIA 







CRUDE STREPTOTHRICIN ADDED, 




ORGANISM 




MILLIGRAMS PER 10 CUBIC CENTIMETERS AGAR 




3 


I 


0.3 


O.I 


0.03 


O.OI 


B. subtilis 

















I 


B. mycoides 


2 


2 


2 


2 


2 


2 


B. macerans 


2 


2 


2 


2 


2 


2 


B. megatherium 














I 


2 


B. folymyxa 








2 


2 


2 


2 


B. cereus 


2 


2 


2 


2 


2 


2 


M. lysodeikticus 











I 


2 


2 


S. muscae 











I 


2 


2 


S. lutea 














I 


2 


A . aerogenes* 








I 


2 


2 


2 


A . aero genes 











Tr 


2 


2 


E. coli-\ 














2 


2 


E. colt (4348) 








Tr 


I 


2 


2 


5. marcescens 





I 


2 


2 


2 


2 


S. marcescens 


I 


I 


2 


2 


2 


2 


Ps. fluorescensX 


2 


2 


2 


2 


2 


2 


Sh. gallinarum 














I 


2 


P. fseudotuberculosis 











Tr 


2 


2 


Br. abortus 














2 


2 


S. cholerasuis 











Tr 


2 


2 


S. schottmillleri 











I 


2 


2 


S. abortivoequina 











Tr 


2 


2 


S. tyfhimurium 











2 


2 


2 


H. suis 











2 


2 


2 


H. influenzae 

















I 


Br. abortus 














2 


2 


Az. agile 

















2 


Az. vinelandii 

















2 


Az. chroococcum 











Tr 


2 


2 


Az. indicum 











2 


2 


2 


M. fhlei 











I 


2 


2 


CI. butyricum\ 


2 


2 


2 


2 


2 


2 


L. casei^ 











2 


2 


2 


S. albus 











I 


2 


2 


S. violaceus-ruber 
















5. lavendulae 





I 


2 


2 


2 


2 



From Waksman and Woodruff (950). 

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

* Representing 3 distinct strains. 

t Representing 5 strains of £. co// obtained from different sources. 

J Representing 4 strains. 

§ Cultured anaerobically. 



120 ACTINOMYCETES AS ANTAGONISTS 

which, like Ps. aeruginosa, are rather resistant to streptothricin (460a, 

795)- 

A^. gardneri produces an active bacteriostatic substance which has been 
designated as proactinomycin (309). Its bacteriostatic spectrum is 
shown in Table 24. It is produced both on synthetic and organic media. 
Its action is largely directed against gram-positive bacteria, although to 
a more limited extent than that of actinomycin. Its isolation and chemi- 
cal nature are brought out later (p. 175). 

TABLE 24. 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 

F. cholerae 6,000 

5. tyfhiy S. paratyphi B, Shigella, E. coli 2,000 

From Gardner and Chain (309). 

S. alhus produces a bacteriolytic substance designated as actinomyce- 
tinj it has been described on page 107. This substance is a protein and 
is enzymatic in nature. It has not yet been isolated in a pure state. Its 
lytic action was visualized by Welsch (971 ) 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 
action of the lipoid is greatly impaired under those conditions j the pres- 
ence of living actinomyces is generally necessary, since free lipoid 
should be secreted in the susceptible suspension. 

Wieringa and Wiebols (981) observed that certain actinomycetes 
can produce lytic agents that are capable of exerting a lytic effect not 
only upon the actinomycetes themselves but also upon other organisms. 



ACTION AGAINST PLANT DISEASES 121 

The formation of an autolytic substance by a thermophilic actinomyces 
was also demonstrated (477, 502). The filtrates of such lysed cultures 
were said to offer promise in the treatment of actinomycosis caused by 
Actinomyces bovis (175). 

Despite a seeming similarity in their growth characteristics, and de- 
spite the fact that some investigators (349, 973) assumed that all ac- 
tinomycetes are able to act as antagonists, it is now definitely established 
(504, 945) that one is dealing here with highly specific types or even 
strains. For example, an examination of many species for an active sub- 
stance similar to actinomycin brought out the fact that only S. antibioti- 
cus was capable of producing this substance. Although many other 
forms yielded an ether-soluble substance that had some bacteriostatic 
activity, it could not be compared in chemical nature and in biological 
action with actinomycin (944, 946). 

ANTAGONISTIC EFFECTS OF ACTI N O M YCETES 

AGAINST AGENTS PRODUCING 

PLANT DISEASES 

Various species of Streftomyces are also strongly antagonistic against 
bacteria causing plant diseases, such as Bacterium solanacearum (414). 
According to McCormack (552), aerobic conditions are necessary for 
the development of the antagonistic properties of actinomycetes j 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. Ps. fluorescenSy however, was antagonistic 
to actinomycetes as a whole, causing their lysis. 

Actinomycetes possess antagonistic properties not only against bac- 
teria but also against other actinomycetes (552), The more aerobic spe- 
cies are antagonistic to the less aerobic types. Millard (604) 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 ( 605 ) . By increasing the 
proportion of the latter organism to the pathogen, the degree of scab- 



122 ACTINOMYCETES AS ANTAGONISTS 

bing on the test potatoes was reduced from lOO 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 cuttings were added and sterilized along with the soil. 

Sanford (782) was unable, however, to control potato scab by the 
inoculation, with S. scabies and S. -praecoXy 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 (605), 
therefore, 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 was found (782) to be very sensi- 
tive to various products of fungi and bacteria. When grown in close 
proximity to various bacteria, the acid production of the latter inhibited 
S. scabies to a considerable degree. Its complete inhibition was not due 
to the acid reaction alone, however, since a certain bacterium was iso- 
lated from the soil which definitely inhibited the growth of this plant 
pathogen. 

Goss (342) 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 (481 ) 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. 

The ability of antibiotic substances produced by actinomycetes to 
exert a marked inhibiting effect upon plant pathogenic bacteria has been 
established (930). 

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- 



IN VIVO ACTIVITY 123 

tical application. Because of the activity of streptothricin and strepto- 
mycin against gram-negative bacteria (460a, 597, 752) and because of 
the lack of reliable chemotherapeutic agents active against these bac- 
teria, the utilization of these substances in the treatment of certain dis- 
eases caused by such bacteria becomes very significant (p. 243). Some 
preparations, like actinomycetin, have been utilized in the preparation 
of a bacterial hydrolysate (mycolysate) for vaccination purposes. 



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 j (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 other organisms, since fungi comprise 
the most important group of microorganisms that cause plant diseases. 

ANTIBACTERIAL EFFECTS OF FUNGI 

Duchesne (207) was the first to report, in 1897, that certain green 
Penicillia are capable of repressing the growth of various bacteria or of 
bringing about their attenuation. Vaudremer (912) demonstrated in 
19 13 that the presence oi A.fumigatus results in the attenuation of the 
cells of M. tuberculosis. 

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 (914). 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 
not characteristic of any one genus or even species, but of certain strains 
within a given species. Some organisms produce more than one active 
substance. Two genera, Penicillium and Aspergillus, have been found 
to comprise a large number of antagonistic forms. Several other genera 
are also known to contain organisms that possess antibacterial proper- 
ties; very few of these, however, were ever found among the Phycomy- 
cetes and Basidiomycetes. The known fungi capable of producing anti- 
biotic substances may be divided (934) into the following ten groups: 

Aspergillus clavatus A . fumigatus 

A. jiavus-ory'z.ae Penicilliwrn cyclofium-clavijorme 




p. 7iofatu7fi, sporulating bodies 
(X530). Prepared by Foster 



P. notdtumy submerged growth 
(X530). Prepared by Foster 



! \ / t ~-~'!i' 



W 



P. c'ltrinum. From Bioiirge 
(54a) 



fft 



^ 



% /■ 



/u. » 



/ ;/// f 



41 



, A. clavatus. From Wehmer 
(955) 




P. chrysogenum. From Bfourgf 
(54a) 




A. fumigatus. From 
Wehmer (955) 



Figure 12. Some typical fungi producing antibiotic substances. 



TABLE 25, ANTAGONISTIC EFFECTS OF SOME REPRESENTATIVE 
FUNGI AGAINST BACTERIA 



ANTAGONIST 


ORGANISMS AFFECTED 


ACTIVE SUBSTANCE 


REFERENCES 


A . clavatus 


Gram-negative and gram- 


Clavacin, highly 


935>942, 982 




positive bacteria 


bactericidal 




A . flavus 


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


Aspergillic acid 


461, 708,978 


A . flaz'us 


Mostly gram-positive 


Flavicin, similar to, 


100, 565, 929 




bacteria 


if not identical 
with, penicillin 




A . fumigatus 


Gram-positive bacteria 


Fumigacin, glio- 
toxin 


95,933>935 


A . fumigatus 


Various bacteria 


Fumigatin, spinu- 
losin 


663 


A . fumigatus 


M. tuberculosis 


Active filtrate 


1015 


and i4. albus 








Chaetomium sp. 


Various gram-positive 
bacteria 


Chaetomin 


934 


Gliocladium and 


Various gram-positive and 


Gliotoxin, highly 


948 


Trichoierma 


gram-negative bacteria 


bacteriostatic 




P. citrinum 


Various bacteria 


Citrinin 


714 


P. claviforme 


Gram-positive and gram- 
negative bacteria 


Claviformin 


114, 115 


P. notatum and 


Mostly gram-positive and 


Penicillin, active 


3> 7, 79> i35> 


P. chrysogenu7n 


also certain gram-nega- 


in vivo, low tox- 


266,424,737, 




tive (Neisseria, Gono- 


icity 


934 




coccus) bacteria 






P. notatum 


All bacteria tested, in 


Notatin, penatin, 


59, i5i,493> 




presence of glucose 


penicillin B, 
E. coli factor 


494> 751.934 


P. fuberulum 


Various bacteria 


Penicillic acid 


56, 57,661, 


and P. cyclofium 






664 


P. resticulosum 


Various bacteria 


Crude metabolic 
product 


58 


Pe7iicilliu7Ti sp. 


Gram-negative as well as 
gram-positive bacteria 


Penicidin 


26 



126 



FUNGI AS ANTAGONISTS 



Penlcillium luteum-furfurogenum 
Pemc'ilUumnotatum-chrysogenimi 
Tr-ichoderma-Gliocladium 



Fusarium-Cefhalosforiufn 
Chaetormum and other Ascomycetes 
Miscellaneous other fun2:i 



A comparative study of a number of fungi taken from a culture col- 
lection brought out (986) the fact that about 40 per cent of the Asper- 
gilli {Aspergillus fumarkuSy A. jum4gatusj Aspergillus schiemannii, 
Aspergillus terreus) and 25 per cent of the Penicillia (Penicillium 
chrysogenum, Penicillium daviforme, Penicillium funiculosum, Peni- 
cillium exfansum) possessed antagonistic properties. Out of many 
Phycomycetes tested, only Phythophthora erythroseftica showed some 
activity. A few Ascomycetes were also found to be active. A summary 
of the antibacterial properties of various fungi and of the antibiotic 
substances produced by them is given in Table 25. In addition to the 
specific strain of the organism, the composition of the medium and the 
conditions of growth, especially aeration, are most important in con- 
trolling the amount and nature of the antibiotic substance produced by 
the organism, as shown in Table 26. The fact that different strains of 



TABLE 26. EFFECT OF AERATION, AS ILLUSTRATED BY DEPTH OF MEDIUM, 
ON ANTIBACTERIAL ACTIVITY OF SEVERAL FUNGI 





VOLUME OF 
MEDIUM PER 




. ACTIVITY IN UNITS 








ONE-LITER 


FLASK, 


E. 


B. 


B. 






CULTURE 


m MILLILITERS 


coli 


mycoides 


subtilis 


S. lutea 


C haetomium sp. 


100 







20 


20 




600 


A. fumigatus 20 


100 







300 


150 




800 


A. fumigatus 20 


300 







300 


60 




800 


A. fumigatus 84 


100 







600 


300 


>i 


,000 


A. fumigatus 84 


300 







300 


100 


>i 


,000 


P. luteum 1 08a 


100 


















P. luteum 1 08a 


300 










20 




10 


P. notatum F 


100 







3 


15 






P. notatum F 


700 




10 


10 


>IOO 






P. notatum W 


100 







45 


70 




70 


P. notatum W 


700 




100 


80 


450 




150 



From Waksman and Horning (934). 

Note. Cultures were incubated 5 to 6 days at 28° C. 



ANTIBACTERIAL EFFECTS 



127 



the same organism when grown under identical conditions vary greatly 
in the production of the antibiotic substance is brought out in Table 27. 

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 
first consideration. Fleming (265) 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 j 



TABLE 27, PRODUCTION OF CLAVACIN BY FIFTEEN STRAINS OF 
ASPERGILLUS CLAVATUS 









ACTIVITY OF 












5 -DAY-OLD 






STRAIN 






CULTURE IN 






NUMBER 


fYl OF MEDIUM 


E. COLI UNITS 


CLAVACIN 


ISOLATED 




5 


14 




Yield in 


Activity, E. colt 




days 


days 




grams per liter 


units per gram 


120 


6.5 


8.4 





0.016 


25,000 


121 


4.2 


6.7 


75 


1.442 


1,000,000 


122 


4-5 


8.0 





0.035 


4,000 


123 


4.6 


4-5 


20 


0.467 


120,000 


124 


6.2 


8.4 





0.016 


8,000 


125 


3.2 


3-9 





0.248 


600 


126 


6.3 


8.2 





0.039 


20,000 


127 


7.4 


8.1 





0.007 


8,000 


128 


6.7 


8.0 





0.017 


7,000 


129 


3-6 


6.8 


100 


0.950 


400,000 


I29T 


6.6 


5-9 


20 


0.512 


80,000 


130 


4.8 


4-7 


10 


0.323 


500,000 


I30T 


6.9 


7-9 


10 


0.050 


4,000 


131 


6.9 


7.8 


10 


0.035 


5,000 


164 


4.3 


4.6 


30 


0.430 


1,000,000 



From Waksman and Schatz (942). 

Note. Eight-day-old culture was used for extraction of the clavacin. 



128 FUNGI AS ANTAGONISTS 

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. 

A glucose-nitrate solution was used as the basic medium for the pro- 
duction of penicillin. It was supplemented with yeast-extract or corn 
steep liquor, or brown sugar was employed in place of glucose j the 
growth of the organism and the production of the antibacterial sub- 
stances were thus greatly facilitated (7, 282, 804). The reaction of the 
medium was found to change from slight acidity initially (^H 6 to 7) 
to distinct acidity (^H 3.0), followed later by alkalinity, finally reach- 
ing a /)H 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 
-pH. 7 and may remain constant for several days or may fall again rap- 
idly. Aerobic conditions are essential for the formation of penicillin. 
Once a fungus pellicle has been produced, the medium can be replaced 
several times, giving fresh lots of penicillin in about half the time re- 
quired during the initial growth period. Crude penicillin cultures are 
capable of inhibiting the growth of staphylococci in dilutions of i : 800 j 
recently, even more active preparations were obtained. 

There is considerable variation in sensitivity to penicillin among bac- 
teria belonging to the same group : 27 strains of enterococci and 6 strains 
of S. lactis were shown to be resistant to the action of this agent, whereas 
13 strains of S. viridans were susceptible (79). The ability of a strain 
to resist the action of penicillin can be greatly increased by successive 
transfers of the culture to media containing this substance (564, S^^)-' 

Chain et al. (113) 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. sefticum were also successfully treated by repeated subcutane- 
ous injections of penicillin. 





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(\j rvj - - CO ^ o 


1 1 1 1 1 1 
Hd 


1 


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1 1 1 1 1 1 r 


fM 




f X o o o 1 






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/ / 






/ \ <-/ 






/ < ,?/ 






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/ \ */ <'■ 






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V-' A ^ 






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1 1 1 1 1 




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SlAlvaO NI 3-10ni3d JO XH0I3M K&Q 



130 FUNGI AS ANTAGONISTS 

An extensive literature soon began to accumulate on the production 
(5, 117, 118, 164, 282, 332, 422), isolation (113, 469), and identifica- 
tion (6, 8) 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 important. 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. notatu?n (the culture being 
composed of two distinct cell constituents) must be recognized for maxi- 
mum penicillin production (34, 376). 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. 232). 

In addition to true penicillin, P. notatum was found to produce an- 
other substance, which in glucose-containing media is active against not 
only gram-positive but also gram-negative bacteria. It was designated 
as the E. colt factor, penatin, notatin, and penicillin B (p. 179). 

P. notatum represents an extremely variable group of organisms, 
some of the strains producing considerable penicillin, others producing 
little penicillin but large amounts of notatin. Some strains of a closely 
related fungus, P. chrysogenum, are also capable of producing peni- 
cillin that is apparently the same as the penicillin of P. notatum. The 
P. notatum-chryso genum group of fungi is widely distributed in nature, 
having been isolated from different soils (919) and from various moldy 
food products J however, only a few strains produce enough penicillin 
to justify their use for the commercial production of this substance 
(732). Members of the A. flavus group of fungi, as well as strains of 
A. niger, Aspergillus nldulans, A. oryzae, Penicillium citreo-roseum 
(282), i4. gtganteus (688), yl. parasiticus (142), and others, are also 
capable of producing penicillin or closely related compounds. 

Among the other fungi that produce antibiotic substances largely ac- 
tive against gram-positive bacteria may be listed Aspergillus flavifes 
(976), Chaetomium cochliodeSy and others. 

Certain species of Penicillium are also capable of producing other 
antibacterial substances, namely, citrinin, penicillic acid, and claviformin 
(p. 181), the first of which is also produced by certain species of Asper- 
gillus belonging to the candidus group ( 883) . 



ANTIBACTERIAL EFFECTS 131 

Atkinson (26) tested 68 cultures of Penicillium 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 citrininj second, those active 
also against gram-negative bacteria and producing substances of the 
penicillic acid and penicidin types. 

Asfergillus jlavus-oryzae Group 

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

White and Hill (978) isolated from cultures of a strain of this or- 
ganism 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, in England, isolated a strain of A. flavus (330) that also 
produced an antibacterial agent with a wide range of activity, both 
gram-positive and gram-negative bacteria being inhibited by the culture 
filtrate. An extract was obtained that inhibited the growth of these bac- 
teria in a dilution of approximately i : 200,000. 

Jones, Rake, and Hamre (461 ) demonstrated that A . flavus of White 
produces frequent variants 5 two strains were isolated and were found 
to give consistently far higher yields of the antibiotic substance, asper- 
gillic acid, than those reported by White. The substance was found to 
have wide activity, being very active against gram-positive cocci and less 
active against the anaerobes of gas gangrene and the gram-negative ba- 
cilli. No significant differences were found in the spectrum of activity as 
shown by filtrates or by solutions of purified aspergillic acid. 



132 FUNGI AS ANTAGONISTS 

Bush and Goth (lOO) 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 fH 2.5 to 3.0 with 5 |j phosphoric acid and extracted 
with purified isopropyl ether. The ether was treated with a slight ex- 
cess of o.2A^ NaHC03 (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 5 [i H3PO4 to fH 
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 CO2, washed with cold distilled water, and reextracted. The 
combined extracts were distilled at 0° C. to dryness under CO2. A yel- 
low-orange glassy residue was obtained. It had a low toxicity and was 
active in vivo. In its properties it resembled penicillin. 

McKee and MacPhillamy (56s, 56^) further established, by chemi- 
cal isolation and composition, solubility and stability, biological behav- 
ior, low toxicity to animals, and therapeutic activity, that the second anti- 
biotic substance produced by A . flavus is similar to penicillin. A sodium 
salt, assaying 240 O.U./mg. was obtained chromatographically and 
gave the following composition : 45.36 per cent C, 4. 1 6 per cent H, 3.02 
per cent N, and 13.36 per cent Na, [ajo = + 108° C. (in water). 

Waksman and Bugie (929) have shown by means of the bacterio- 
static spectrum that, under submerged conditions, diflFerent strains of 
A. flavus produced two substances, one comparable to aspergillic acid 
and the other to penicillin. Some strains produced little or no activity 
in submerged cultures, and most strains produced very little activity in 
stationary cultures. No activity was produced in synthetic media. The 
production of gigantic acid by a species of A . giganteus and parasiticin 
hy A. -parasiticus (142) appears to be comparable to that of flavicin. 

Aspergillus jumigatus Group 

Four antibacterial substances were isolated from strains of A . jumi- 
gatus: the two pigments, spinulosin and fumigatin {66"^), which are 
not selective in their action against bacteria, the colorless fumigacin 



ANTIBACTERIAL EFFECTS 



133 



that Is active largely against gram-positive organisms (935) and glio- 
toxin (331, 593). Helvolic acid, isolated from a strain of A. fumigatus 
(116, 155) was found (593, 933) 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 : 50,000 to 1 125,000 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. fumigatus is given in 
Table 28. 



TABLE 28. 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 CgHgO^ 

Fumigacin Very fine white 

needles z 15-220 CgsH^^Og 

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 



Vaudremer (912) reported that a group of patients suffering from 
tuberculosis were treated with extracts of A. jumigatuSy with varying 
degrees of success. The disease-producing organism (M. tuberculosis) 
was rendered nonpathogenic by such treatment. 



134 FUNGI AS ANTAGONISTS 

Asfergillus davatus Growp 

This comprises a number of strains that produce highly active anti- 
biotic substances. By treating the culture filtrate with charcoal and 
eluting the active substance with ether, Wiesner ( 982 ) obtained a prepa- 
ration having a bactericidal potency in dilutions of i : 100,000. This ac- 
tivity was not inhibited by serum, pus, or urine j strains of bacteria that 
proved to be resistant to sulfonamides or mandelic acid were inhibited 
by this material. 

The active substance was designated (935) as clavacin. A detailed 
study was made of its production by a variety of strains of A. dava- 
tus. The substance was found to be active against E. coli and other 
gram-negative bacteria, as well as against gram-positive bacteria. 
It is distinct, in this respect, from fumigacin. Whereas the latter acts 
much more readily upon B. mycoldes than B. subtilisy clavacin shows 
the opposite effect — greater activity against B. subtilis than against B. 
mycoldes. Clavacin possesses a high bactericidal action, as compared 
with other antibiotic substances. It has been suggested (942) that the 
marked differences in the physiology of the different strains of A. da- 
vatus explain the differences in the production of clavacin by different 
strains. Those that change the reaction of the medium to alkaline, for 
instance, tend to inactivate the clavacin (Table 27). 

Since clavacin is produced by several fungi, this substance has re- 
ceived a number of designations, including patulin produced by Pen'i- 
cillium fatulum (713), claviformin by P. davijorme (114, 115), and 
clavatin (47). It is also produced by strains of P. exfansuniy A. gigan- 
teus, GymnoascuSy and other fungi (470). 

Trichoderma and GUodadium Grouf 

Certain strains of fungi of the genera Trichoderma and GUodadium 
were found to exert a marked antagonistic action against various fungi 
and bacteria. An antibiotic substance designated as gliotoxin was iso- 
lated and found (962) 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 {f¥L 5.0 or 
lower) are essential. Ammonium salts as nitrogen sources give better 



ACTION AGAINST FUNGI 135 

results than peptone or nitrates. Glucose and sucrose were found to be 
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 /)H 2.5 to 3.0 could be used for large-scale production, the high 
acidity reducing the effect of contaminants (966). 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 -pH 2.4, heating to 122° C. for 30 min- 
utes did not affect the active substance. With decreasing acidity, espe- 
cially at -pH 5.0, it became less thermostable. As pointed out above, 
gliotoxin is also produced by A. jumigatus (593). 

Other Groups 

Various other fungi, including A. albus, A. niger, and Monilia albi- 
cans, were found (1015) to exert a marked antibacterial action against 
human and bovine tubercle bacteria j active filtrates were obtained, but 
the specific agents were not isolated. The nature of the antibiotic sub- 
stances produced by the other groups listed above has not been suffi- 
ciently studied. 



ANTAGONISTIC ACTION OF FUNGI 
AGAINST FUNGI 

Numerous fungi were found to exert antagonistic effects either 
against fungi belonging to the same species (64, 87, 152, 304, 547, 578, 
801, 851) or against other fungi (Table 29). This phenomenon is par- 
ticularly important in connection with the study of plant diseases ( 1 60, 
225,644,695,738, 815, 851,852,959, loii). The effects are selective 
in nature. The hyphae of Peziza will kill various Mucorales, whereas 
different species of Aspergillus and Penicillium are able to kill Peziza 



136 



FUNGI AS ANTAGONISTS 



(738). A single spore of P. luteum was found (955) capable of germi- 
nating in cultures of CitromyceSy and of bringing about their destruc- 

TABLE 29. ANTAGONISTIC INTERRELATIONSHIPS AMONG 
DIFFERENT FUNGI 



ANTAGONIST 

Acrostalagmus sp. 

A Itemaria tenuis 

A. clavatus 

A . flavus 

A . niger 

Botrytis allii 

Botrytis cinerea 

C efhalothecium roseum 

Cunninghamella elegans 

Fusarium lateritium 

Fusarium sp. 

Gliocladium sp. 

H elmifUhosforium sp. 

H. teres 

H. sativum 
Mucor sp. 
Penicillium sp. 
Penicillium sp. 
Peziza sclerotiorum 

Peziza trifoliorum 
Sclerotium rolfsii 
SterigVMtocystis sp. 
Thamnidium elegans 
Torula suganii 
Torulofsis sp. 
Trichoderma lignorum 

T. lignorum 

Verticillium sp. 



ORGANISMS AFFECTED 

Rhizoctonia 

Ofhiobolus 

Various fungi 

Peziza 

Peziza, Rhizoctonia 

Monilia, Botrytis, etc. 

Rhizoctonia 

H elminthosforium 

Monilia 

Rhizoctonia 

Deuterofhoma 

H elmiiithosforiuTn, Mucor, etc. 

CoUetotrichum, Fusariu?n, 

Botrytis, etc. 
Fusarium, Ustilago, Helmintho- 

sforium, etc. 
Ofhiobolus 
Ofhiobolus, Mucor 
Peziza, Rhizoctonia, etc. 
Ofhiobolus, Fusarium., etc. 
Mucor, Trichothecium, Dematiu?n, 

etc. 
Peziza 

H elminthosforium 
A Itemaria 
Mucor 

Asfergillus, Monascus, etc. 
Blue-staining fungi 
Rhizoctonia, ArTuillaria, Phy- 

tofhthora, etc. 
Rhizoctojiia, PythiuTn, etc. 

Rhizoctonia 



REFERENCES 
964 
88 
928 

738,911,964 

911 

964 

353 
911 
964 

790 
695 
695 

695 



88, 801 
738 

88,955 
738 

738 
695 
695 
801 
654 
592 
962-964 

17, 60, 91, 

911 

964 



From Novogrudskjr (644). 



ACTION AGAINST FUNGI 137 

tion. P. luteum-furpurogenum produces a thermostable substance, solu- 
ble in ether and in chloroform, that is antagonistic to the growth and 
acid production of A. niger (668). Coniofhora cerehella was inhibited 
by a species of Penicillium, its mycelium being considerably modified j 
however, in time the former organism adapted itself to the latter and 
overgrew it, its rate of growth being eventually more rapid than that 
of a pure culture (377). Certain fungi are able to parasitize other fungi: 
Pi ftocef halts, for instance, attacks various species of Penicillium and As- 
fergillus (176). The germination of the spores of one fungus may be 
reduced by the presence of spores of another (519). 

Different fungi produce different types of fungistatic and fungicidal 
substances, which may be stable or unstable in nature. 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 (88), studying the effects of H. sativum and Penicillium sp. 
against Ofhiobolus graminis, emphasized that the inhibitory action was 
due to a toxic substance, thermostable in nature 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 
( lOi I ) j in other cases, as in the mutualistic effects of Sclerotium rolfsii 
and Fusarium vasinjectum, the first overgrew completely the second at 
^H 6.9, whereas in alkaline ranges the reverse took place (766). 

Random isolations oi Penicillium cultur&s 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 (785). 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 (184), Hyphomycetes were believed to serve as effective 
agents in promoting soil sanitation. Various species of Torulopsis, in 
addition to certain bacteria, are capable of inhibiting the growth of 
Dematiaceae, fungi that cause the blue staining of wood pulp (592). 

Certain fungi may affect the reproduction of others. Melanos-pora 



13S FL*NGI AS ANTAGONISTS 

pampeana^ for example, normally does not form any peritheda in cul- 
ture but is able to do so in the presence of Basis porium gallarum or 
Fiisjriuf77 momliforme. 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 
(25, 742), these effects being characteristic of the antagonists. 

The edible mushroom, PsalUota campestris^ exerts a deffnite antago- 
nism against the parasitic fungus My co gone (124). This phenomenon 
has been looked upon as a case of antibody formation. %>edes of Fusor- 
r'lum are able to antagonize the mushroom fungus \ however, an actively 
growin-g culture of the latter may become antagonistic to the former 
(998). In the destruction of paper pulp by fungi, a marked antagonism 
was shown {^I'l) ^^ take place between different organisms, especially 
h\ Trichoderma Ugnorum against various species of Fusarium and other 
fungi, as illustrated in Figures 14 and 15. 

Certain spedes of Trichoderma and Gliodadium are able to inhibit 
the growth of various plant pathogenic fungi, especially R. solani, as 
well as of Blastomycoides derm-atitisy a causative agent of human skin 
diseases (962—964). The active substance, gliotoxin, is liberated during 
the early stages of growth. The mycelium of older cultures contains an- 
other substance that is soluble in acetone j this has only an inhibiting ef- 
fect and is not fungicidal as is gliotoxin. The fungicidal effect of glio- 
toxin upon the germinating sp>ores of Sclerotwla americana and hv'phae 
of R. solani was found to be greater than that of CUSO4 and less than 
that of HgCU. 

\'arious other fungi are able to exert antagonistic effects against plant 
pathc^ns. T. lignorum and A . mger restricted the growth of the fungi 
Macrophomina phaseoU and R. solani^ which produce cotton root rot, 
and reduced the activity of the filtrates of the pathogens causing wilting 
of the plants C911). 

Satoh (788) has shown that Ophiobolus miyaheanus 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 ICX)^ C. and does not pass through a filter. The formation 
of two substances by Torula suganiiy both of which were thermostable, 
however, was also demonstrated (654). 







%.. 



FiGLTRE 14. Antagonistic etirect ot one tungus, P$. ■zmsatum (in center), 
upon another, T. Ugnarttm. From Gofdaniich et al. C333). 




FiGLTiE 15. Attack of an antagonstic i 
fundus, F. sambiicinttm (in center). Frc 



•w, upon anodier 
(333)- 



ACTION OF BACTERIA AGAINST FUNGI 



139 



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 (37) from corn, these bacteria being capable of destroying the 
colonies 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 (456). Some of these bacteria produce en- 
zymes that are able to dissolve the chemical constituents of the cell 
walls of the fungus sporidiaj they were also found to be active in the 



TABLE 30. ANTAGONISTIC EFFECTS OF BACTERIA AGAINST FUNGI 



ANTAGONIST 


ORGANISMS AFFECTED 


REFERENCES 


Achromobacter sp. 


Fusarium, Sclerotinia 


130 


Al. faecalis 


H elminthosforium 


695 


Bacillus «Z)» 


Ustilago, Penicillium 


37 


B. anthracis 


S. cerevisiae 


496 


B. mesentericus 


H elminthosforium 


128,695 


B. mycoides 


H elminthosforium 


695 


B. simplex 


Rhizoctonia 


149 


B. subtilis 


Cefhalothecium, roseum 


13 


Bacterium sp. 


Fusarium^ Sclerotinia, etc. 


695 


Bacterium sp. 


Ustilago 


456 


Bacterium sp. 


A Iternaria 


231 


Myxobacterium 


Ustilago 


248, 456 


P. vulgaris 


Basisforum, Phytofhthora, etc. 


485,695 


Ps. aeruginosa 


Saccharomyces 


496 


Ps. juglandis 


Dothiorella 


248 


Ps. fhaseoli 


Fusarium 


60, 248 


Ps. translucens 


Ofhiobolus 


86 


Ps. vulgaris 


Ophiobolus 


86 


S. marcescens 


Beauveria, etc. 


12, 13, 14, 587 


M. tuberculosis 


Pythium 


910 


Spore-forming bacteria 


Fungi 


37,231,695, 
734,738 



From Novogrudsky (644). 



140 FUNGI AS ANTAGONISTS 

soil against the specific fungi. Brown (92) observed that H. sativum 
and a certain bacterium produced thermostable mutually inhibiting 
substances. The bacterium as well as its metabolic products inhibited the 
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 (108). 
The active substance was not destroyed by autoclavingj it diffused into 
fresh agar and water, producing "stale water" that was inhibitory to 
the fungus. 

Chudiakov (130) 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 Rhizoctonia, P. vulgaris against Phytofkthora 
(472), and B. mesentericus against H elminthosforium (128). B. sim- 
flex was grown (475) 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 by 10 
per cent concentration of the stale medium 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 (629) found that various bacteria are able to inhibit 
the germination of rust spores. Nonspore-forming bacteria, such as Ps. 
fuorescens and S. marcescenSy prevented the germination of the spores 
of Ustilaga avenaey Ustilaga hordeiy Ustilaga nuda, and Ustilaga reae. 
Spore-forming bacteria, including B. m-ycoides and B. mesentericus, as 
well as sarcinae {S. ureae, S. lutea), exerted no antagonistic action on 



ACTION OF BACTERIA AGAINST FUNGI 



141 



the rust spores. The presence of these bacteria, however, Influenced the 
nature of the germination of the spores, which gave rise to mycelium- 
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 confluens in freshly 
burned-over soils, but not in natural soils, was explained ( 645 ) 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 
(855) 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 



TABLE 31. FUNGISTATIC AND FUNGICIDAL ACTION OF ANTIBIOTIC 
SUBSTANCES UPON CERATOSTOMELLA ULMI 





MILLIGRAMS 


1 OF SUBSTANCE 


PER 


6 CC. OF 


SUBSTANCE 




NUTRIENT BROTH 








Complete fungi- 


Partial fungi- 




Fi 


jngicidal action 




static action 


static action 






in 48 hours 


Penicillin 















Actinomycin 


O.I 


0.03 






O.I 


Streptothricin 















Clavacin 


0.15 


0.045 






<o.i5 


Fumigacin 





5.0 









Hemipyocyanin 


0.5 


O.I 






0.1 


Gliotoxin 


0.5 


O.I 






^(^ 



From Waksman and Bugie (928). 




142 FUNGI AS ANTAGONISTS 

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 (945) to inhibit the growth of Penicillium, 
As-pergilliSy Ceratostom-ellay 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 (1002). 
A comparison of the fungistatic activity of several antibiotic substances 
upon the causative agent of Dutch elm disease is brought out in 
Table 31. 

ACTIVITY OF FUNGI AGAINST INSECTS AND 
OTHER ANIMAL FORMS 

A number of fungi are capable of parasitizing insects and other ani- 
mal forms. Comparatively little is known concerning the production 
of antibiotic substances by these animal parasites. 



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 (262, 264) and in- 
hibins found in fresh human urine (180). 



INTERRELATIONSHIPS BETWEEN PROTOZOA 
AND 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 (102), 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- 



144 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS 

tozoa are then able to multiply by consuming the bacteria. Protozoa 
are apparently also able to destroy pathogenic bacteria (747). 

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" 
(776). 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 (156, 591, 630). This was found 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. It has been suggested that the presence of protozoa in the 
soil may keep the bacteria at a level of maximum efficiency (157). 

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. 



RELATIONS OF PROTOZOA AND BACTERIA 145 

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 
"microbiological equilibrium" (943). It has also been suggested (498) 
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 (440, 595, 743). 

The idea (157) 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 (157) is based upon more direct experi- 
mental evidence, it is still inadequate, because it gives insufficient con- 
sideration 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 



146 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS 

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 (943). The protozoan Oikomonas termo was 
found capable of living at the expense of a large number of bacteria, 
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 (378). 

The ability of protozoa to destroy bacteria was said (414) 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 citri, as well as by species of Fusarium and 
Penicillium. 

Various bacteria may exert a toxic action upon protozoa, thus limiting 
the development or bringing about the destruction of the latter (122, 
545, 687). Certain plant pathogenic bacteria inedible by amebae were 
found to produce a toxin that was harmful to these amebae. The toxin, 
however, appeared to be without effect on the flagellate Cercomonas, 
which could eat all these bacteria partly or completely (826), In some 
cases, the protozoa were capable of developing a certain resistance to 
specific bacterial products (687). 

Certain factors in the medium seem to affect the encystment of pro- 
tozoa (874) 5 it remains to be determined to what extent these factors 
can be classified with antibiotic substances. 



RELATIONS OF PROTOZOA TO FUNGI 

The presence of Colfoda and other infusoria in an active form was 
found to repress the growth of Verticillium dahliae in culture media 
and to prevent infection of tomato plants by this pathogeny Colfoda 
was also active in soils and reduced the incidence of wilting (87). 

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. The ability of various fungi to destroy protozoa and nema- 
todes has been studied in detail by Drechsler (183). 



INSECT DISEASES AND MICROBIAL CONTROL 147 

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. Because of the war, 
however, the results thus obtained have not yet been published. They 
are highly interesting and offer promise of added application of these 
substances. 

Weinman found (967) that the general correlation between the 
gram-stain of bacteria and their sensitivity to gramicidin also extends 
to protozoa {Leishmaniay Tryfanosoma) and to the Leftosfira tested. 
Tyrocidine had a marked effect, in concentration of 5 y per ml., upon 
the flagellates 5 they remained active for many hours, gradually losing 
their motility} a few escaped giving rise to delayed growth. 

INSECT DISEASES AND MICROBIAL CONTROL 

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 (867). 

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- 



148 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS 

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

Metalnikoff (596) compared the bacterial treatment of caterpillars 
of Pectinofhora gossyfiella with the action of arsenical poisoning. The 
dry spores of Bacterium efhestiae. Bacterium gelechiae^ Bacterium^ 5, 
and Bacterium cazaubon, in powder form, were mixed with water at the 
rate of i to 4 ounces to 2>4 gallons of water, with the addition of 4 per 
cent of molasses; this preparation was sprayed on the plants two 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. efhestiae^ the 
infestation being reduced by about 50 per cent as compared with the 
controls. A slightly smaller reduction occurred on plots sprayed with 
B. cazaubofiy 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 1 8 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 destroying these larvae. Once the 
attacking microorganisms have become established in the soil, the larvae 
and the beetles themselves tend to disappear. Glaser (327) utilized for 
this purpose Neoaflectana glaseri. This parasite possesses great repro- 
ductive capacity and is capable of destroying large numbers of grubs. 
Glaser demonstrated the presence of this nematode also in localities 
where the grub was not present. 

Fungi have also been utilized for the control of insects. Sweetman 
(867) emphasized the importance of entomogenous fungi as destructive 



INSECT DISEASES AND MICROBIAL CONTROL 149 

enemies of insects. A limitation to their practical importance in the fight 
against insects is that the fungi require special conditions for develop- 
ment, especially high humidity and favorable temperature, which are 
not always found under natural conditions. 

Dutky (219) described two spore-forming bacteria {Bacillus fo- 
filliae 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 (840). 

Glasgow (328) 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 j also they apparently kill these bacteria 
when they invade the alimentary canal of the insect. 

According to Duncan (215), 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 J whereas staphylococci appear to be more susceptible to 
the action of Argas material. The gut-contents of ticks was found to 
have a v/eak 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 



150 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS 

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 (849). 

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 tritki 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 wheat plants 
and cereal plants. 

Some nematodes attack plants by feeding upon the roots. The meth- 
ods 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. (528) 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- 



BACTERICIDAL ACTION OF MAGGOTS 151 

capturing fungi (170, 184), the non-trapping fungal parasites, the 
predacious nematodes, the predacious mites, and different bacteria ca- 
pable of destroying nematodes. 

BACTERICIDAL ACTION OF MAGGOTS 

Surgical m.aggots are said to have a bactericidal effect in wounds, in 
addition to removing necrotic debris. Simmons (825) demonstrated in 
the maggot Lucilia sericata the presence of an active bactericidal sub- 
stance which is thermostable and active against S. aureus, hemolytic 
streptococci, and CI. welchii. 



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 ( io6). 

BACTERIA AND VIRUSES 

B. subtilis was found (718) capable of inactivating the virus of vesicu- 
lar stomatitis as well as staphylococcus phage, when in contact with 
them for 15 to 18 hours at 35° C. This phenomenon has been ex- 
plained as due to the process of adsorption. The facts that it is selective 
in nature, that the phage cannot be reactivated, and that the virus is ren- 
dered impotent by the action of the bacterium, all point to an antagonis- 
tic eflFect rather than mere physical adsorption. The virus of rabies is 
said to be influenced in certain ways by B. subtilis , the culture filtrate of 
the organism suppressing the activity of the virus when a mixture of the 
two is injected into rabbits (173). 

However, different antibiotic substances, including penicillin, ty- 
rothricin, and subtilin, when used either alone or in combination with 
sulfonamides or acridine, have failed to prevent infection of mice with 
influenza virus (508). 

A "nontoxic" inactivator has been defined (306) as a substance that 
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 (457, 458) various microorganisms are capable of 
forming such inactivators against tobacco-mosaic virus j A. aero genes 



INTERRELATIONSHIPS AMONG VIRUSES 153 

was found to produce inactivators against a number of viruses. Taka- 
hashi (868) isolated from yeast a substance which was capable of rap- 
idly inactivating the tobacco-mosaic virus. A chemical reaction between 
the inactivating principle and the virus was therefore suggested. The 
inactivator in this instance was destroyed by heating with i N NaOH 
solution, but not by 2 A^ 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. Fulton (306) demonstrated that A. niger forms in the 
medium a substance capable of inactivating a number of different plant 
viruses J the effect of the inactivator was found to be exerted upon the 
virus itself and not upon the plant. 

INTERRELATIONSHIPS AMONG VIRUSES 

Andrews (20) reported that the cultivation of influenza virus in a 
simple tissue-culture rendered 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 (1012, 1013). Henle and Henle (394) have 
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 (467) 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. 

Various other types of antagonism between viruses have been demon- 
strated, as in the case of canine distemper or of lymphocytic chorio- 



154 ANTAGONISMS BETWEEN NONSPECIFIC PATHOGENS 

meningitis virus against experimental poliomyelitis (162). An intra- 
muscular injection of a neurotropic strain of yellow fever virus was 
found to protect animals against simultaneous infection with a highly 
pathogenic viscerotropic strain (433). The antagonistic agent was be- 
lieved to be a chemical substance produced by the murine virus, for 
which the term "poliomyelitis inhibition" was proposed by Jungeblut. 
The "interference phenomenon" of two viruses can be used to advan- 
tage in bringing about immunity reactions. 

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 (569). 
Other antagonistic phenomena between plant viruses have been ob- 
served by McWhorter (573). Kunkel (510) demonstrated that the 
peach-yellow virus prevented the invasion of the virus of little-peach 
and that the latter prevented invasion of the former. McKinney (569) 
concluded that virus domination in a plant may be looked upon as a 
type of antagonism, quantitative in nature, the degree of domination 
by a given virus being influenced by the host. 

The ability of bacterial phages to interfere with the development of 
other phages has been studied in detail by Delbriick and Luria (167, 
546). 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. 



BACTERIA AND TUMORS 

The ability of certain bacteria to bring about hemorrhage in tumors 
(446, 10 10) may also be classed among the antagonistic phenomena. 
Laszlo and Leuchtenberger (515) described a rapid test for the detec- 
tion of tumor-growth inhibitors. Inhibition was judged by comparing 



BACTERIA AND TUMORS 155 

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 of sar- 
coma cells, as compared with normal cells, said to be caused by penicillin 
(150a) was later shown (525a) to be due not to the pure penicillin it- 
self but to some impurity present in crude penicillin preparations. 



CHAPTER 10 

CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

CLASSIFICATION 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), higher animals (lactenin, lysozyme), and microorganisms, 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 (506). The 
suggestion 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 (70). It is now definitely established, however, 
that this property is characteristic of only certain strains of specific bac- 
teria, fungi, and actinomycetes. 

Antibiotic substances of microbial origin are primarily bacteriostatic 
in nature. They are selective in their action. Some substances affect 
largely gram-positive bacteria j their action upon gram-negative bacteria 
is more limited as regards both the kinds affected and the concentration 
required to bring about growth inhibition. Other substances may inhibit 
alike the growth of certain members of both groups of bacteria. One is 
fully justified, therefore, in speaking of a characteristic bacteriostatic 
spectrum for each antibiotic substance. The production of antibiotic sub- 
stances by specific microorganisms is influenced by the strain of the or- 
ganism, the composition of the medium, the temperature of incubation, 
the age of the culture, aeration, and certain other factors. Antibiotic sub- 
stances also vary greatly in their mode of action upon the bacterial cells, 
in their toxicity to animals, and in their practical utilization for the treat- 
ment of human and animal diseases. 

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- 



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160 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

tion. Differences between various compounds may often be in degree 
rather than in kind. Different organisms may produce the same kind of 
compound. Many organisms are able to produce more than one anti- 
biotic substance: B. hrev'ts produces tyrocidine and gramicidin j P. no- 
tatum forms penicillin and notatinj 5. antibiot'icus produces actino- 
mycin A and Bj ^4. fumigatus forms fumigatin, fumigacin, spinulosin, 
and gliotoxinj A. flavus produces aspergillic acid and flavicin. 

On the basis of their solubility, the antibiotic substances may be di- 
vided into three groups : 

Group A. Soluble in water at different reactions, and insoluble in ether. 
These substances usually represent polypeptides, proteins, organic 
bases, or adsorption compounds on protein molecules. Most of them 
have not been isolated in a pure state. They comprise the bacterial 
enzymes acting upon microbial polysaccharides, actinomycetin, mi- 
crobial lysozyme, streptothricin, streptomycin, notatin, and pyo- 
cyanin. 

Group B. Soluble in ether and in water at proper reactions. Here belong 
some of the most important antibiotic substances so far isolated and 
described, namely, penicillin, flavicin, citrinin, clavacin, proactino- 
mycin, penicillic acid, and aspergillic acid. 

Group C. Insoluble in ether and in water. These include gramicidin, ty- 
rocidine, subtilin, and the B. simflex factor. 

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

Some of the antibiotic substances have been crystallized, and infor- 
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 lipoid-like bodies, including pyocyanase and certain little 
known microbial extracts 

Pigments, namely, pyocyanin, hemipyocyanin, prodigiosin, fumigatin, 
chlororaphin, toxoflavin, and actinomycin 

Polypeptides, comprising gramicidin, tyrocidine, subtilin, and actino- 
mycetin 

Sulfur-bearing compounds, such as gliotoxin and chaetomin 



SUBSTANCES PRODUCED BY BACTERIA 161 

Quinones and ketones, namely, fumi'gatin, citrinin, spinulosin, clavacin, 

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

On the basis of their biological activity, the antibiotic substances also 
vary considerably. They may be divided into three groups: 

Primarily bacteriostatic agents, such as penicillin, actinomycin, and pro- 
actinomycin 

Substances which are bactericidal but not bacteriolytic, including pyocya- 
nase, gliotoxin, fumigacin, clavacin, and pyocyanin 

Bacteriolytic substances, namely, gramicidin, actinomycetin, and lysozyme 

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, pyocyanase, 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- 
molabile ; 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 

Lipoids and Pigments. Ps. aeruginosa, discovered by Gessard in 
1882 (320) and formerly known under the names of Bacterium fyo- 
cyaneuni and Bacillus fyocyaneus, was the first organism found to pro- 
duce two antibiotic agents, the colorless pyocyanase and the pigment 
pyocyanin. Pyocyanase, believed to be of the nature of an enzyme, is 
now recognized as a lipoid containing unsaturated fatty acids {SS^ 4^9? 
410). Recently this organism was shown to form (809) three com- 
pounds that possess antibacterial properties, namely, pyocyanin, 



162 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

a-hydroxyphenazine, and an oil that forms insoluble salts with calcium, 
barium, and heavy metals. The last appears to be similar to what has 
previously been described as pyocyanic acid, a substance strongly lytic 
to 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 (27, 320), although the effect 
of tyrosine is not very significant (340, 341, 452, 529). It was suggested 
(407) that the antagonistic action of young cultures of Ps. aeruginosa 
is due primarily to the presence of pyocyanin, whereas in older cultures 
pyocyanase is largely concerned. 

The determination of the nature of the antibacterial substances of 
Ps. aeruginosa can be carried out in the following manner (407) : 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, on removing the pigment, the 
antibacterial properties are very little diminished 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. 

In most cases the broth culture of the organism is 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 (501 ). This pigment is a deriva- 
tive of pyocyanin and is often designated ( 1006) 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. 



SUBSTANCES PRODUCED BY BACTERIA 163 

may be produced under certain conditions. It was excreted into the me- 
dium as a leuco base. 

Pyocyanase is soluble in ether, benzol, benzene, and petrol ether. It 
can be separated (370) 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 attrib- 
uted to the last constituent (410). A definite relation has been observed 
between the number of double bonds and the activity of the substance 
(SSy 409). According to Dressel (185), most fatty acids exert bacteri- 
cidal 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 (370). 

Many commercial pyocyanase preparations have been found to be of 
little practical value. This is believed to be due largely to a lack of recog- 
nition of the importance of strain specificity, conditions of cultivation 
of organism, and methods of extraction of the active substance (501, 

763). 

Since Ps. aeruginosa is an extremely variable organism, the nature 
and abundance of the pigment are also influenced by these conditions. 
Keeping the organism for five 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 (129, 321, 522). 

Pyocyanin was first studied by Fordos in i860 (279). Since then 
many contributions have appeared dealing with formation and nature 
of this pigment. Several formulae have been suggested for pyocyanin 
(452, 603, 913, 1006), one of which is shown in Figure 16. The struc- 
ture of pyocyanin has considerable similarity to chlororaphin and io- 
dinin, obtained from Chromobacterium {SSS^ SS^) ^^^ two synthetic 
compounds, phenazine and acridine (919). 

Besides Ps. aeruginosa, spore-forming bacteria, including B. mesen- 
tericus, were also found to produce antibiotic agents of a lipoid nature. 
The cell-free filtrate of this organism killed diphtheria bacteria in 
4 minutes (1016), but when diluted to i per cent it required 24 hours 
to effect a kill. The substance was not affected by heating for 30 seconds 



164 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

at 100° C. but was weakened at 1 15° C. for 10 minutes. It is considered 
similar in its bactericidal properties to pyocyanase. 

Alcohol and acetone extracted from B. mesenterkus a weakly active 
substance (408) 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 



1 ! ! 

c c c 

CH,O.C C=CH2 H2C C=CH\ HOC CH H3CI 

II I I I > II II , 

HC CH3 H2C CH.CO/ HC C.CH2OH li yOCHj 

I V V „ 

O 



COOH 
PENICILLIC ACID 



CLAVACIN 



KOJIC ACID 




H3C 





PYOCYANIN 




PHENAZINE 



FUMIGATIN 



CONH2 




CHLORORAPHIN 




Figure i6. Structural formulae of some antibiotic substances. 



SUBSTANCES PRODUCED BY BACTERIA 165 

acidified and treated with petrol ether, which brought the active sub- 
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 j 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. colt exerts an antagonistic effect in vivo when injected subcutane- 
ously or when used for feeding. It produces (365, 367) a thermolabile 
substance that was considered to be a lipoid in character. According to 
Hettche (408, 409), one is dealing, in the case of bactericidal constitu- 
ents of the bacterial cell, with lipoids that contain unsaturated fatty 
acids. 

Chromobacterium iodinum produces {SSSi SS^) ^ purple-bronze pig- 
ment designated as iodinin and found to be a di-N-oxide of dihy- 
droxyphenzine. This substance inhibits the growth of streptococci (S. 
hemolyticus) in concentrations of 1.2 to 2.0 x io"° M. 

It may be added here that certain aromatic oils possess marked bac- 
tericidal properties. Ordinary peptones have also been found to contain 
a bacteriostatic substance that is active against various bacteria, especially 
when small amounts of inoculum are used (191). The active substance 
is thermostable and is associated with an acid-precipitated fraction that 
is pigmented and changes color upon oxidation and reduction. The bac- 
teriostatic effect of this material can be corrected by the addition of re- 
ducing agents, such as thioglycollic acid. The bacteriostatic 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 (197). 

PoLYSACCHARiDASES. Among the antibiotic substances of microbial 
origin may also be included the enzyme systems that have the capacity 
of decomposing the capsular substance of certain bacteria, thereby ren- 
dering them more readily subject to destruction in the blood stream or 



166 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

in other substrates. The first enzyme of this type was isolated by Dubos 
and Avery (195, 199, 202) from certain soil bacteria. These enzymes 
are highly specific, some being able to act only upon one type of pneu- 
mococci. As a result of their action, the pneumococcus cell is rendered 
susceptible to destruction by phagocytosis (819,821,822). This enzyme 
was produced by the soil bacteria under selective conditions of culture, 
that is, when the capsular polysaccharide of the pneumococcus was pres- 
ent in the medium j the only other substance that could be used for its 
production was aldobionic acid, derived from the above polysaccharide. 
Yields of the enzyme were increased by increasing the concentrations of 
the specific substrate in the medium from o.Oi to o.i per cent. Above 
0.1 per cent, the yields decreased, 0.3 to 0.4 per cent inhibiting the 
growth of the bacterium. The addition of o. i per cent yeast extract fa- 
vored the production of the enzyme j proper aeration was essential, the 
bacterium making the best growth in shallow layers of medium. The 
enzyme was concentrated by distillation in vacuo and by ultrafiltration. 
Toxic substances 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 Mj but is held back by pores having a diameter of 8.2 m- 
After filtration, the enzyme can be recovered in solution by immersing 
the membrane in distilled water or in physiological salt solution (30, 
195,293,337). 

Dubos (188) 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 ineffective 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 mice ; it also exerted a favorable influence on the outcome of an 
infection already established at the time of treatment. A definite rela- 



SUBSTANCES PRODUCED BY BACTERIA 167 

tionship was found to exist between the activity of the enzyme in vitro 
and its protective power in the animal body. 

Polypeptides. The credit for first isolating, in crystalline form from 
spore-forming aerobic soil bacteria, specific chemical compounds of the 
polypeptide type is due Dubos (190, 193, 203, 436, 530). The antago- 
nistic organism {B. brevis) is grown in shallow layers of a medium 
containing i per cent casein digest or tryptone and 0.5 per cent NaCl 
in tap water, adjusted to /jH 7.0. After inoculation, the medium is 
heated for 20 minutes at 70° C, in order to kill the vegetative cells of 
the bacteria, leaving only the spores to develop. The culture 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 dis- 
solved and is separated from the residue by filtration j when the alco- 
holic solution is diluted with 10 volumes of i per cent NaCl, the sub- 
stance is precipitated out. It carries all the activity and can be desiccated 
in vacuo, over PoO-,, giving a yield of about 100 mg. of final dry sub- 
stance per liter of culture medium. The protein-free, alcohol-soluble ac- 
tive material was designated as tyrothricin. When an attempt was made 
to produce tyrothricin in aerated submerged cultures, none was ob- 
tained in complex nitrogenous media 5 however, simple amino com- 
pounds, like asparagine, gave good growth and yielded the antibiotic 
substance. The presence of cystine in the mixture of amino acids ap- 
peared to inhibit growth ( 856) . 

Tyrothricin can be separated into two crystalline preparations, grami- 
cidin and tyrocidine. Gramicidin is obtained by treating tyrothricin with 
a mixture of equal volumes of acetone and ether, evaporating, and dis- 
solving 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 hydrocarbons. When a solution containing 20 to 50 mg. per 



168 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

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 floc- 
culation occurs. 

The specific rotation of gramicidin in 95 per cent alcohol solution is 
approximately [a]^^ = -|- 5°. On 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 
C74H106N14O14 has been suggested. On further study, the molecular 
weight of gramicidin was found (885) 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 i,200j isothermal distil- 
lation in methanol, however, indicated a molecular weight of 3,100. 
Sulfur and carbon analyses of gramicidin flavianate gave a molecular 
weight of 3,000. It gave neither free amino nor carboxyl groups (126, 
434). Gramicidin is a polypeptide with 10 molecules of a-amino acids, 
of which two or three are 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 substance. Amino acids that have definitely been iden- 
tified are /-tryptophane, <i-leucine, /-alanine, ^/-valine, and glycine 
(339). A study of the configuration of the dipeptide valyvaline sepa- 
rated from gramicidin brought out the fact that only valines of like 
configuration have been joined together by the bacterium (125). About 
45 per cent of the a-amino acids gave the d configuration (435, 436, 
530). An unknown hydroxyamino compound has also been indicated 

(339)- 

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 j 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- 
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 



SUBSTANCES PRODUCED BY BACTERIA 



169 



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 HClj clusters of microscopic needles are ob- 
tained, melting at 237-239° C, with decomposition j the specific rota- 
tion is [a]'s = _ 102° ( I per cent in 95 per cent alcohol). Tyrocidine 



BACILLUS BREVIS BG 
PEPTONE CULTURES 



ACID pH 4.8 



PRECIPITATE 




ALCOHOL EXTRACT 



precipitate 
(tyrothricin) 



ACETONE -ETHER 



SOLUBLE PART 



CRYSTALLIZATION 

FROM ACETONE 

(GRAMICIDIN) 



INSOLUBLE PART 



CRYSTALLIZATION 

FROM ALCOHOL + HCL 

(TYROCIDINE HYDROCHLORIDE) 



Figure 17. Preparation of tyrothricin, gramicidin, and tyrocidine. From 
Dubos (192). 



170 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

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, including aspartic acid, have been de- 
tected (126, 434). 

The tyrothricin type of antibiotic substance appears to be widely dis- 
tributed among spore-forming aerobic soil bacteria (427, 428, 857). 
The following method for its extraction has also been employed: A 
seven-day-old bacterial culture was treated with 2 to 5 per cent of an 
electrolyte and HCl added to give a ^H 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 
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 (427a, 885). 

A thermostable substance was obtained (149) from B. simplex, an 
organism capable of bringing about the destruction of various patho- 
genic fungi. It was produced by the bacterium grown both on synthetic 
and organic media. It can be adsorbed on activated charcoal and recov- 
ered 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 



. 


v_^- ¥ 


/ 


^^^-V |\ 


<^ 


^^%^^ 


^ 


^w\ 


m 


¥l // y 


^jI 


'm / 


"7 





Tyrocidine hydrochloride. From 
Hotchkiss (435) 




.-•^\ 







.*^\ #.->, v^^ 



Fumigacin. From Waksman and 
Geiger (933) 













Gramicidin. From Hotchkiss 
(435) 



^ 



\ 




Gliotoxin. From Waksman and 
Geiger (933) 




Citrinin. Prepared bv Timonin 



Actinomycin. Prepared by Tischler 



Figure 18. Crystalh'ne preparations of antibiotic substances. 



SUBSTANCES PRODUCED BY ACTINOMYCETES 171 

and other microorganisms still remains to be determined. Toxoflavin, 
formed by Bacterium cocovenenanSy 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 (908, 909). 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 

The antibacterial substances produced by actinomycetes can be di- 
vided into three groups : 

Water-soluble and alcohol-insoluble compounds of the protein type, in- 
cluding actinomycetin (346, 347, 971-973), micromonosporin, and 
the compounds of the lysozyme type (507) 

Ether-soluble and alcohol-soluble pigmented compounds, including ac- 
tinomycin 

Basic substances, soluble or insoluble in ether and soluble in aqueous or al- 
cohol acid solution, including streptothricin, streptomycin, and pro- 
actinomycin 

AcTiNOMYCiN is an ether-soluble and alcohol-soluble pigmented sub- 
stance produced by only a few organisms, notably S. antibiotkus. The 
culture medium is treated with ether, giving an orange-colored extract. 
The residue is evaporated and separated (946) into two fractions: 
A, soluble in ether and in alcohol but not in petrol ether, giving a clear 
yellow-colored solution when diluted with water j B, soluble in ether 
and petrol ether, soluble with difficulty in alcohol, and giving a turbid 
suspension with water. Actinomycin A is bright redj it possesses ex- 
tremely high bacteriostatic properties but is rather slowly bactericidal. 
Actinomycin B is colorless j it has comparatively little bacteriostatic 
action but possesses strong bactericidal properties. Despite the fact that 
the organism produces a dark-brown pigment on organic media, actino- 
mycin does not possess the enzymatic properties of a tyrosinase. 

The purification of actinomycin A 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, 



172 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

filtered, and allowed to pass through a tower packed with aluminum 
oxide. On washing the tower with large amounts of benzene, a chro- 
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 A 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; when ground very 
fine, its color is orange-red. 

Actinomycin A is optically active, a solution of 5 mg. in 2 cc. ethanol 
in a I dm. tube having a rotation — 1.60°; [afj = —32.0° ± 5. Its 
molecular weight was found to be around i ,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 C41H56N8O11. Actinomycin A 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 A 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 A 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 



SUBSTANCES PRODUCED BY ACTINOMYCETES 173 

is subjected to catalytic hydrogenation in the presence of platinum 
oxide. The pigment has one or more functional groups capable of re- 
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. 

Streptothricin is produced by Stre-ptomyces lavendulae grown in a 
medium containing glucose or starch ( i per cent) as a source of energy, 
and tryptone, glycocoll, 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. (926). The relation between growth of the organism and pro- 
duction of the antibiotic substance is brought out in Table 33. 

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 ^.s with 



174 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

acid, a precipitate is produced, the filtrate containing virtually all the 
activity. 

Streptothricin is completely adsorbed, at neutrality, on charcoal 
(norit A), from which it can be removed by treatment for 8 to 12 hours 
with dilute mineral acid or acid alcohol. The acid extract is neutralized 
and concentrated in vacuo, at 50° C, just to dryness j the residue is ex- 
tracted with absolute alcohol, filtered, evaporated, and taken up in 

TABLE 33. GROWTH OF STREPTOMYCES LAVENDULAE AND PRODUCTION 
OF STREPTOTHRICIN ON TRYPTONE-STARCH MEDIUM 





INCU- 




DRY WEIGHT 
OF MYCE- 


NITROGEN 
IN MYCE- 


ACTIVITY 
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 





293 


26.2 


75 


250 


Shaken 


6 





231 


17-3 


100 


300 


Shaken 


8 









75 


200 


Shaken 


12 





142 


9.6 


30 


50 


Stationary 


7 


+++ 






50 


200 


Stationary 


10 


Tr 


235 


18.8 


50 


300 


Stationary 


14 


Tr 






60 


250 


FromWaksman (926). 













water. It can also be precipitated from the neutralized solution with 
ether. These preparations usually contain 40 to 50 per cent inorganic 
matter and 2 to 3 per cent nitrogen, on an ash-free basis. Further con- 
centration and reduction in ash content can be obtained by subsequent 
treatments. On electrodialysis, the active substance moves to the cath- 
ode at fH. 7.0. A highly active fraction has been isolated by chromato- 
graphic adsorption on aluminum oxide. 

Streptothricin acts as a base, with an optimum at /)H 8.0. It is re- 
pressed by dextrose and by acid salts. Bacteria subject to the action of 
streptothricin show greatly enlarged cells, due to incomplete fission 
(287, 1002). 

Another substance, designated as streptomycin (795), is similar in 



SUBSTANCES PRODUCED BY FUNGI 175 

many respects to streptothricin, although it differs somewhat in its anti- 
bacterial spectrum and its lower toxicity for animals. 

Proactinomycin is produced by N. 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 ^H 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 fVL 2.0 or fH 
7.0 results in a small loss of activity. Boiling at />H 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. 

SUBSTANCES PRODUCED BY FUNGI 

The early studies of the phenomenon of staling accompanied by the 
production of antibacterial and antifungal substances (83), some of 
which could be removed from the acidified medium by ether or by col- 
loidal clay (700), have recently been superseded by more exact and 
detailed chemical studies. Only a few of the many antibiotic substances 
produced by fungi have so far been identified, however. Some are pro- 
duced in complex organic media, others in simple synthetic media. Only 
the more important substances will be discussed here. Among these, 
penicillin occupies a leading place because of its low toxicity and its ac- 
tivity in vivo. 

Penicillin is produced by various strains of P. notatum and P. 
chrysogenum, and probably by a variety of other fungi (272a, 940a). 
The penicillin-like nature of an antibiotic substance is usually estab- 
lished by its biological and chemical properties: activity against S. au- 
reus and not against E. colt; extraction in organic solvents at fH 2 
and re-extraction in water at fH 7 ; inactivation by acid and alkali j par- 
tial inactivation by heating at 100° C. and /)H 7 for 15 minutes j com- 
plete inactivation by penicillinase and by copper ionsj inactivation by 
methyl alcohol (272a). 



176 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

The strain of the organism used, the composition of the medium, and 
the conditions of growth greatly influence the yield of penicillin. Com- 
plex organic media containing glucose 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 supple- 
mentary addition of a stimulating substance in the form of yeast extract, 
corn steep, or certain vegetable juices is essential for the maximum pro- 
duction of penicillin. Since the organism produces an acid, probably glu- 
conic, in the medium, some CaCOg must also be added. The metabolism 
of P. notatum in relation to penicillin production is illustrated in Fig- 
ure 13 (page 129). 

Four methods have been proposed for the growth of the fungus and 
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 (730) 

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 produced in the medium when active growth begins but 
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- 
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 /»H 5 to 6. 



SUBSTANCES PRODUCED BY FUNGI 177 

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 6o° to 90° C. for short periods j it 
remains active when heated at 100° C. for 5 minutes but not for 10 min- 
utes (737). 

Fleming first reported that penicillin is insoluble in ether. This was 
found (135) to be due to the alkaline reaction of the filtrate 5 for at fH 
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 (7, 8). 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 j 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 
(109). Water may often contain a pyrogenic or heat-producing sub- 
stance 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 

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 

fThe fourth fraction is discarded, and the others are eluted with 
M/15 phosphate buffer (-pH 7.2). The penicillin is again extracted 



178 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

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 8o 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. 

By means of adsorption, distribution between solvents, and reduc- 
tion, a barium salt or penicillin was finally obtained (3, 7, 8) 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 ^H values approximately 2.3 and 
2.5y having a formula of C24H;..OioNoBa. 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 pre- 
pared 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 polysubsti- 
tuted 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 trisubstituted a-unsaturated ketone 
grouping. 

Another method for obtaining penicillin has been suggested (598). 
In this method, the culture medium was adjusted to -pH 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. The analysis of the penicillin prepared by this 
method was found to fit best the formula Ci4Hic)NO,( or C]4Hi-N05 -f- 



SUBSTANCES PRODUCED BY FUNGI 179 

HoO. This penicillin was strongly dextrorotatory and had an adsorp- 
tion maximum of 2,750 A°. The preparation had an activity of 32,000,- 
000 dilution units against hemolytic streptococci, which corresponds to 
about 240 Oxford units per milligram. 

The most suitable form for general use is the barium salt. In this 
form, it retains its antibacterial activity for an indefinite period. The Ba 
salt of penicillin is soluble in absolute methyl alcohol, but is insoluble in 
absolute ethyl alcohol. Penicillin forms water-soluble salts with most 
heavy metals, except Fe^"^"^. It is inactivated by a number of heavy metal 
ions, especially Cu, Pb, Zn, and Cd. Penicillin is stable toward atmos- 
pheric oxygen, but is oxidized by H0O2 and KMn04, the antibacterial 
activity being lost (2, 5, 1 1 1 ). 

In assaying penicillin, tests are made for potency by one of several 
procedures: sterility, moisture content, presence of pyrogenic sub- 
stances, and toxicity (249). 

Several derivatives of penicillin have been obtained: 

1 . Penicillamine, a degradation product ( 6) , is produced by hydroly- 
sis of the barium penicillin for one hour at 100° C. with N/io sulfuric 
acid J the formula C«HiiN04.HCl has been suggested for itj the pres- 
ence of a glyoxal nucleus has been indicated. 

2. Penillic acid (210), a dextrorotatory substance having a pale blu- 
ish fluorescence, is insoluble in ether and soluble in butyl alcohol; it is 
produced by keeping penicillin in aqueous solution at ^H 2.0 ; it has 
some of the properties of an amino acid. 

3. Methyl, ethyl, «-butyl, and benzohydryl esters of penicillin have 
been prepared (599, 600) ; these are insoluble in neutral or slightly al- 
kaline buffers, but soluble in benzene j these esters are much less active 
than true penicillin m vitro but are active in vivo. 

The chemical nature and mode of action of the second antibacterial 
substance produced by P. notatum- are given in Table 34. This sub- 
stance is a 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 (151, 416, 751). 

Flavicin, a substance found (100, 461, s^s^ 5^7, 929) to be similar 



180 



CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 



TABLE 34. NATURE OF SECOND ANTIBIOTIC FACTOR PRODUCED BY 
PENICILLIUM NOTATUM 



PHYSICAL AND 




DESIGNATION 




CHEMICAL 


Penatin 


Notatin 


Penicillin B 


PROPERTIES 


(493) 


(57>i5i) 


(751,902) 


Solubility 


Soluble in water, 


Soluble in water. 


Soluble in water 




insoluble in or- 


insoluble in or- 






ganic solvents 


ganic solvents 




Adsorption or pre- 


Adsorbed on kaolin, 


Precipitated by 


Adsorbed on ben- 


cipitation 


at ;>H 4.0, eluted 


acetone or 


zoic acid, pre- 




with sodium phos- 


tannic acid 


cipitated by 




phate, precipitated 




acetone 




by dioxane and 








phosphotungstic 








acid 






Chemical nature 


Protein 


Flavoproteln 


Protein 


Glucose required 


Required 


Required 


Glucose and cer- 


for activity 






tain other car- 
bohydrates re- 
quired 



Ox\-gen required 
for activity 

Activity against 
gram-negative 
bacteria in addi- 
tion to gram- 
positive bacteria 

Sensitivity to alka- 
lies 

Biological nature 



Apparently required Required 



Required 



Decidedlv active 



Sensitive 



Hydrogen peroxide 
produced; not 
verv toxic 



Highly active Definitely active 



Activity de- 
stroyed at />H 
above 8.0 



Glucose-oxidase 



Fairly toxic to 
animals 



Note. In view of the fact that the three preparations have been obtained by somewhat diflFerent 
chemical procedures and in view of the variation of the strains of P. notatum producing this sub- 
stance, there is a possibility that the different preparations may vary in chemical nature and pos- 
sibly also in biological behavior. 



SUBSTANCES PRODUCED BY FUNGI 181 

in every respect to penicillin, is produced by A. flavus (929) j the same 
is true of gigantic acid produced by A. giganteus (688). Preparations 
of flavicin have also been designated as flavatin (716, 717), aspergillin 
(100), flavacidin (461), and parasiticin. 

AsPERGiLLic Acid is produced by A. -flavus (461, 978) grown on 
tryptone-glucose media, as shown previously (page 131). The pure 
acid has an m.p. of 93° C. (84° to 96'^) and has optical activity of 
[ajo = +14°- The formula CioHoqNoOo has been proposed for this 
substance. It possesses a hydroxyl group which gives it its acid nature 
(^K S-S)- It is stable under acid and alkaline conditions and can be dis- 
tilled 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 CioHooO.-jNo and an m.p, of 149°, with lower bio- 
logical activity. Aspergillic acid is active against both gram-positive and 
gram-negative bacteria. 

CiTRiNiN was isolated from Penicillium ckrinum (714). It is pro- 
duced by growing the organism on a synthetic medium, with inorganic 
salts of nitrogen and with glucose as a source of carbon. The culture fil- 
trate is acidified with HCl, and the substance crystallized from boiling 
alcohol. Citrinin forms a monosodium salt which, at ^H 7.0 to 7.2, gives 
virtually colorless solutions in water. Its bacteriostatic activity is much 
lower than that of penicillin (33). It is a yellow crystalline solid, m.p. 
170-171° (with decomposition). Its formula is C10H14O5. 

Citrinin is a strong acid, changing in color from lemon-yellow at /)H 
4.6 to orange-pink at fYl S-^ to 5.8 and to cherry-red at /)H 9.9 (352). 
Addition of FeCla to the culture solution gives a heavy buff-colored 
precipitate, which dissolves in excess of reagent to give an intense iodine- 
brown solution (871 ). It has little if any activity against gram-negative 
bacteria and about 50,000 dilution units against B. subtilis and S. aureus. 

Penicillic Acid was first isolated in 1 9 1 3 by Alsberg and Black (19) 
as a metabolic product of Penicillium fuberulum. A limited air supply 
and an acid reaction of the medium favor the production of this acid, to 
which the chemical formula C8H10O4 was given. This acid is a rather 
weak antibiotic substance active largely against gram-positive bacteria j 
however, it is more active against gram-negative bacteria than penicillin, 
giving complete inhibition of E. coli m concentrations of 1:50,000, 



182 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

whereas penicillin does not inhibit this organism even in concentrations 
of 1 : 1,000. It was found to possess antibiotic properties also against 
yeasts, and to be toxic to animals when injected subcutaneously in con- 
centrations of 0.2 to 0.3 gm. per kilogram weight. 

More recently, penicillic acid was isolated (661, 664) by evapora- 
tion of the culture solution, the crude acid crystallizing on cooling. It 
was purified by recrystallization from hot water. Yields greater than 
2 gm. per liter of culture were obtained. The acid is a stable, colorless 
compound which is appreciably soluble in cold water and gives a series 
of colorless and readily soluble salts (s^j 470a). 

Penicillic acid was shown to have the constitution y-keto-p-methoxy- 
S-methylene-A-a-hexenoic acid, which exists in both the keto and lactone 
forms : 



CHo=CH(CH3)COC(OCH3)=CHCOOH - 



CHo=CH-(CH3)-C(OH)-C(OCH,)=CH-CO 

i o ! 

FuMiGATiN. Among the other quinones isolated from fungi, fumi- 
gatin deserves consideration (21, 712). It is a 3-hydroxy, 4-methoxy, 
2 : 5-toluquinone or C8H8O4 (Figure 16, page 164). 

All quinones have been divided into three groups on the basis of their 
action on Stafhylococcus: (a) those that have a markedly weaker anti- 
bacterial action than fumigatin, including toluquinone and some of its 
derivatives i (b) those that are somewhat more effective than fumi- 
gatin, including 3:4 dimethoxytoluquinone 5 (c) those with activity 
greater than that of fumigatin (methoxytoluquinones). The introduc- 
tion of -OCH3 into the quinone nucleus results in an increase in anti- 
bacterial activity. The introduction of an OH or the replacement 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. coU (660). Electrode potentials of quinones have been found to be 
unrelated to their bacteriostatic action against E. coli, but for S. aureus, 
the reduction potentials fall within certain limits (417, 6G6). 

Clavacin is produced by A. clavatus (935), P. fatulum (patulin 
[713]), P. clavijorme (114, 115),/*. exfansum (22), and probably a 
number of other fungi (470). It can be isolated from the culture filtrate 



SUBSTANCES PRODUCED BY FUNGI 183 

either by preliminary adsorption on norit followed 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 thus 
isolated (429, 473) from A. davatus cultures showed the following 
chemical properties: melting point, 109-110° C.j empirical formula, 
C7H6O4J molecular weight (cryoscopic in benzophenone) 154^ semi- 
carbazone, darkens at 200°, decomposes at 290° C. j 2,4-dinitrophenyl- 
hydrazone, darkens above 190°, decomposes at about 300° C. j lactone 
group indicated by slow reaction with alkali; saponification number 70 
(evidently molecule cleaved); Zerewitinoff determination (in ;?-butyl 
ether) shows slightly less than one active hydrogen per mol; esterifica- 
tion by the acetic anhydride-pyridine method shows one hydroxyl per 
mol. Clavacin, a neutral optically inactive compound, darkens and 
loses activity in the presence of alkali, reduces Fehling's solution 
strongly on heating, and readily decolorizes alkaline permanganate; 
it does not react with aqueous ferric chloride or Schiff's reagent, and re- 
duces ammoniacal silver nitrate. 

Clavacin (patulin) is anhydro-3-hydroxymethylene-tetrahydro-Y- 
pyrone-2-carboxylic acid, for which a formula has been suggested (713), 
as shown in Figure 1 6. 

Clavacin is soluble in water and in most of the more common organic 
solvents except light petroleum. It is about equally active against gram- 
positive and gram-negative bacteria, its growth inhibition being about 
200,000 dilution units. Its lethal action upon mice is about 25 mg. per 
kilogram body weight, when given intravenously or subcutaneously. 

Claviformin isolated (114, 115) from P. clavijorme has recently 
been shown to be identical with clavacin and patulin (47, 927). 

FuMiGACiN is produced by different strains of A. jumigatus. It can 
be extracted from the culture medium either by preliminary adsorption 
on active charcoal followed by treatment with ether and alcohol, or by 
direct extraction of culture in accordance with the following method 
(593): The culture filtrate is acidified to /)H 2 with phosphoric acid 
and extracted three times with ether, the combined extracts equalling 



184 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

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 pigment. The solution 
is then exhaustively extracted with 6 per cent sodium carbonate solu- 
tion. The ether phase, on evaporation, yields gliotoxin. The sodium 
carbonate solution is acidified and distributed several times with ben- 
zene j 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 215-220° C, depending on the rate 
of heating, [a]^^ =:= — 132 ± 2° (0.41 per cent in chloroform). The 
ultraviolet absorption curve shows only strong end absorption below 
260 m|_i with Ej^ =298 at 234 mp. Fumigacin is practically in- 
soluble in water, sparingly soluble in cold methanol and ethanol, and 
more readily soluble in acetone, ethyl acetate, benzene, and ether. It is 
easily dissolved by chloroform, acetic acid, and dioxane. 

The following reactions are negative: ferric chloride. Legal, fuchsin 
sulfurous acid, Tollens, Molisch, Rosenheim, Hammersten (for cholic 
acid), Jaffe-Tortelli, digitonin. The Zimmerman reaction with m-dini- 
trobenzene for ketones is strongly positive. In the Chabrol-Charonnet 
test for bile acid (phosphoric acid and vanillin) a strong red color is 
obtained. Likewise, the Liebermann-Buchard test gives an intense 
blood-red color. Fehling's solution is slowly but perceptibly reduced 
at 100° C. The formula that has been suggested is Cof)H..8-4o07. Puri- 
fied fumigacin has recently been shown to be identical with helvolic acid, 
isolated from a strain of A . jumigatus. 

Gliotoxin was isolated from cultures of Trichoderma, Gliocladium, 
and A. jumigatus (459, 960, 961), as well as from other fungi (593, 
933), the greatest activity being produced in 2 days. It was extracted 
from the culture medium by the use of chloroform. The latter was dis- 
tilled off, and the residue taken up in a small amount of hot benzene or 
95 per cent alcohol, from which, on cooling, silky white needles crys- 
tallized. It was recrystallized from benzene or alcohol. It was found 
(965) to have a molecular weight of 347, an optical rotation of [a]^^ = 
— 239°, and an m.p. of 121° to 122° C. It analyzed C14H16N0O4S0, 
later shown (459) to be C13H14N2O4S0. 



SUBSTANCES PRODUCED BY YEASTS 185 

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 (961 ) j it is inactivated by heating for 10 min- 
utes at 100° C. ( 17). Its potency was found to be destroyed by bubbling 
oxygen for 5 minutes. 

Gliotoxin is toxic to Rhizoctonia hyphae in a dilution of i : 300,000, 
which is about two-thirds of the toxicity of HgCL. The crystals, as well 
as the crude material, were found to be toxic also to Trichoderma, but 
the minimum lethal dose was about 40 times greater than that required 
for Rhi-zoctonia. Its antibacterial properties are brought out later. 

Other Substances. Several other antibacterial substances have been 
isolated from fungi, but have not been adequately studied either chemi- 
cally or biologically. It is sufficient to mention the following: 

Puberulic acid, CgHsOe, a colorless crystalline dibasic acid, with an 
m.p. of 316-318°, and puberulonic acid, C8H4O6, a bright-yellow acid 
with an m.p. of 298° C, produced (38, 58, 66$) by P. fuberulum. The 
first is a quinol and the second is quinonoid. They have little activity 
against gram-negative bacteria and some activity against gram-positive 
types. 

Penicidin was isolated (26) from a species of Penkillium. 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 was found 
to be active against E. tyfhosa. 

Chaetomin is produced by a species of Chaetommm {Ch. cochliodes) 
grown in complex organic media. It is active largely against gram-posi- 
tive bacteria (930). 

Kojic acid (Figure 16), produced by A. oryzae, apparently also pos- 
sesses some antibacterial properties, more against gram-negative than 
gram-positive bacteria (282). 



SUBSTANCES PRODUCED BY YEASTS 

According to Fernbach (253), 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 (654) to contain a substance which has an antagonistic action 



186 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

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 (798), 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 
(144), 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 /)H 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. 

MICROBIAL LYSOZYME 

The enzyme lysozyme or an antibiotic substance similar to it has 
often been reported to be produced by microorganisms. Fleming (264) 
found that the lysozyme of &gg white was soluble in water and in dilute 
NaCl solution. It was precipitated by chloroform, acetone, ether, alco- 
hol, and toluene. It was not acted upon by pepsin or trypsin. It was par- 
ticularly active against micrococci, bringing about their lysis. 

Lysozyme has been found in nearly all mammalian tissues and secre- 
tions, in certain vegetables, and in bacteria (880). It was found to be a 
polypeptide containing 1 6 per cent nitrogen and 2 to 3 per cent sulfur 
and having a molecular weight of 1 8,000 to 25,000. It is soluble and 
stable in acid solution, insoluble and inactivated in alkaline solutions, 
and inactivated by oxidizing agents (601). It diffuses in agar and 
through cellophane, and thus is markedly different from bacteriophage 



MISCELLANEOUS ANTIBIOTIC SUBSTANCES 187 

(323). It is fixed on the bacterial cells (264), 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 (601), being disintegrated by the enzyme. The 
degradation of the bacterial polysaccharide to water-soluble products 
( N-acetylated amino-hexose and a keto-hexose) by lysozyme is accom- 
panied by complete lysis of some of the bacteria. In the case of other 
lysozyme-sensitive bacteria, such as B. subtilis, no lysis occurs 5 appar- 
ently the morphological structure of these bacteria does not depend ex- 
clusively on the unaltered state of the substrate for lysozyme (243), 

The formation of a lysozyme-like material was demonstrated by 
Fleming (262) for a coccus isolated from dust. A sarcina susceptible 
to egg-white lysozyme also was found (601) 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 (704). 
The lysozyme of saliva is known to act primarily upon gram-positive 
bacteria (880). Auerswald (28) tested a large number of bacteria for 
their ability to antagonize diphtheria and pseudo-diphtheria organisms. 
Only the spore-forming B. mesentericus and B. subtilis groups pro- 
duced 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 bac- 
terial content. 



MISCELLANEOUS ANTIBIOTIC SUBSTANCES 

In connection with the antibiotic substances of microbial origin, at- 
tention may be directed to certain substances, of apparently similar na- 
ture, of plant and animal origin. No attempt will be made to discuss 
here the antibacterial blood reactions, including antibody formation, 
precipitin reactions, agglutination, phagocytosis j lysin formation} ac- 
tion of various body fluids j and other animal and plant reactions against 
bacteria and in response to bacterial infection. 

Unbleached wheat flour was shown (864) to contain a protein which 
had bacteriostatic and bactericidal activity in vitro; although this activ- 
ity was greatest against gram-positive organisms, it also had some ac- 



188 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 

tivity against gram-negative types. The antimicrobial action of this 
protein can be neutralized by means of a phosphatide (1004), a reac- 
tion which may be due to the formation of a lipoprotein that has no 
longer any antibiotic activity. Sherman and Hodge (817) demon- 
strated that the fresh juice of several plants has a marked bactericidal 
effect. 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. It has been re- 
cently demonstrated (658) that antibacterial substances are widely dis- 
tributed among plants. 

Milk was found (657) 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. 



CHAPTER I I 

THE NATURE OF ANTIBIOTIC ACTION 

Sulfanilamide^ fenicillin-j and gramicidin can be clearly set afart 
from the classical antiseptics which are general frotoflasmric 
foisons. All three substances are frimarily bacteriostatic rather 
than bactericidal in their action. Since they do not destroy the res- 
piration of bacteria y one may assume that the inhibition of growth 
which they cause defends not upon interruption of the cellular 
metabolism as a whole ^ but rather upon some subtle interference 
with certain individual reactions. To interrupt the pathogenic 
career of an infectious agenty therefore y it is not necessary to kill 
the invading celly but only to block one step in its metabolic path 
by some specific inhibitor. — Dubos (192). 

ANTIBIOTIC SUBSTANCES AND CHEMICAL 
DISINFECTANTS 

Since antibiotic substances vary greatly in their origin and in their 
chemical nature, they would be expected also to vary 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 (560). In this connection, the following properties of anti- 
biotic agents are of particular significance : 

Most antibiotic substances are strongly bacteriostatic in nature and only 

weakly bactericidal, though a few are also strongly bactericidal, and 

some are even bacteriolytic. 
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. 



190 NATURE OF ANTIBIOTIC ACTION 

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 antibacterial substances are hemolytic; others have apparently no 
injurious effect upon the blood cells. The latter can, therefore, be 
used for general body treatment, whereas the former are suitable 
only for local applications. 

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 the bacterial cells, and to the 
differences in the mode of action of the different antibiotic substances 
upon the various bacteria. 

A comparison of the antibacterial action of the antibiotic substances 
produced by two bacteria v^^ili 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 
1 : 1 00,000 J pyocyanase and hemipyocyanin have less activity upon the 
bacteria, but yeasts are more sensitive to them than to pyocyanin. Ty- 
rothricin, produced by B. hrev'ts, 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 (855). Other 
striking differences are found on comparing two types of antibiotic sub- 
stances produced by fungi, namely, penicillin and clavacin, and two 
substances produced by actinomycetes, namely, streptothricin and ac- 
tinomycin. The bacteriostatic spectra of these four substances are re- 
corded in Table 35. The first of each pair has a 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. 
Clavacin and actinomycin, both of which are highly toxic, differ simi- 
larly in their action upon bacteria, the first being largely active against 



ANTIBIOTIC SUBSTANCES AND DISINFECTANTS 



191 



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 Tables ^6 and 37. 

Various attempts have been made to compare the antibacterial action 
of antibiotic substances with that of organic antiseptics. According to 
Suter (866), the bactericidal action of a compound depends upon cer- 
tain physical and chemical characters j a property that determines the 
bactericidal action of the compound upon E. typhosa 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 


PENI- 


ACTINO- 


STREPTO- 




TEST ORGANISM 


STAIN 


CILLIN 


MYCIN 


THRICIN 


CLAVACIN 


5. aureus 


+ 


9,500* 


20,000 


200 


100 


S. aureus 


+ 


IjOOof 


- 


- 


- 


S. lutea 


+ 


38,000* 


60,000 


100 


500 


B. subtilis 


+ 


1 9,000* 


60,000 


750 


200 


B. megatherium 


+ 


1,900* 


40,000 


200 


100 


B. mycoides 


+ 


5* 


40,000 


<3 


200 


CI. welchii 


+ 


i,50ot 


1,000 


- 


- 


Actinomyces sp. 


+ 


i,ooot 


10 


10-50 


- 


"Neisseria sp. 


- 


2,OOOt 


20 


- 


- 


Br. abortus 


- 


2t 


10 


100 


- 


Sh. gallinarum 


- 


2t 


20 


300 


- 


Pasteur ell a sp. 


- 


it 


<I0 


100 


- 


Hemofhilus sp. 


- 


- 


50 


30 


- 


S. schottmulleri 


- 


<lt 


<I0 


200 


60 


S. aertrycke 


- 


10* 




- 


- 


Ps. jiuorescens 


- 


<5* 


10 


<3 


6 


S. marcescens 


- 


<i* 


<5 


5 


60 


A . aerogenes 


- 


<5* 


<5 


30 


50 


E. coli 


- 


<it 


- 


- 


- 


E. coli 


- 


<5* 


5 


100 


100 



Note. Activity is indicated in thousands of dilution units per gram. 

* Our own data, based on a sample having 470 Oxford units. 

t Data reported by Abraham et al. (i), based on a less active preparation. 



192 



NATURE OF ANTIBIOTIC ACTION 



TABLE 26. BACTERICIDAL EFFECTS OF PENICILLIN, GRAMICIDIN, AND 
TYROCIDINE UPON S. HEMOLYTICUS 



INHIBITING 




AGENT* 


At start 


Penicillin 


1,500 


Gramicidin 


1,500 


Tyrocidine 


1,500 



NUMBER OF VIABLE ORGANISMsf 

At At At 

At I hour 3 hours 7 hours 24 hours 

4,300 2,650 420 o 

2,430 1,140 7 2.4 

0.1 o 00 



From Dawson, Hobby, Meyer, and Chaffee (164). 

* 10 Y of each preparation was added to i milliliter ot culture. 

t In thousands per milliliter. 



TABLE 37. BACTERIOSTATIC AND BACTERICIDAL ACTION OF FUMIGACIN 
AND CLAVACIN 











CLAVACIN, 






FUMIGACIN 




CRUDE MATERIAL 




Bacteriostatic actionf 


Bacteri- 


Bacterio- 


Bacteri- 






Crude 


cidal 


static 


cidal 


TEST ORGANISM* 


Crystals^ 


material^ 


action^ 


actionf 


action:]: 


A . aerogenes 


<40,ooo 


<40,ooo 


>200 


50,000 


20 


E. coli 


<40,ooo 


40,000 


>200 


100,000 


20 


■S. schottmuUeri 


<40,ooo 


<40,ooo 


>200 


60,000 


20 


Salmo?tella sp. 












(Breslau type) 


<40,ooo 


<40,ooo 


>200 


75,000 


20 


S. choleraesuis 


<40,ooo 


<40,ooo 


>200 


150,000 


5 


B. megatherium 


1,250,000 


1,000,000 


20 


100,000 


5 


B. cereus 


500,000 


500,000 


200 


125,000 


5 


B. mycoides 


1,250,000 


500,000 


200 


200,000 


2 


B. subtilis 


750,000 


500,000 


200 


200,000 


2 


S. aureus 3 


750,000 


750,000 


200 


100,000 


2 


S. aureus 2 


500,000 


250,000 


>200 


60,000 


20 


S. aureus H 


750,000 


500,000 


>200 


75,000 


20 


5. aureus W i 


750,000 


500,000 


200II 


200,000 


5 


5, aureus W2 


500,000 


500,000 


200II 


200,000 


2 


5. lutea 


4,000,000 


3,750,000 


20 


500,000 


2 



From Waksman, Horning, and Spencer (935). 

* Staphylococcus cultures and gram-negative pathogens incubated at 37° C, others at 28° C. 

t Units of activity ^ dilution in plate or tube inhibiting growth completely. 

% Micrograms required to kill bacteria in i milliliter portions of 6-hour-old cultures. 

§ Water saturated solution used. 

If Crude mother liquor, from which 2 lots of crystals were removed on basis of dried material in 

solution. 

II Incomplete sterilization of culture even in 24 hours 



ANTIBIOTIC SUBSTANCES AND DISINFECTANTS 



193 



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 (584) 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 j this range is 
ordinarily narrow, but it may become broad. The third, or inhibiting, 
and the fourth, or germicidal, 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 19). 

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- 



DISINFECTANT "SPECTRUM' 




BACTERICIDAL 

11111111111 



HI 



Space obout I/20 totol length 




impractical 
(insoluble, 
too toxic, 

OR ) 



100% 



GENTIAN VIOLET 




Space just perceptible (-OOlX) 



Figure 19. Disinfectant spectrum. From Marshall and Hrenoff (584). 



194 



NATURE OF ANTIBIOTIC ACTION 



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 (Figure 20). Any rate of multiplication 



LOGARITHM 

OF 

BACTERIA 

PER 
MILLILITER 


^^^^^-^-"'''^ RATE = 


I^nT--. 




TIME 



Figure 20. Disinfectant spectrum and rates of bacterial growth. From Mar- 
shall and Hrenoff (584). 



greater than zero but less than normal can be considered as the bacterio- 
static zone, and the rate less than zero as the bactericidal zone. Accord- 
ing to this concept of bacteriostasis, bacterial growth may be delayed 
under the influence 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 (189): the acidic and basic properties of the bac- 
terial cell, the nature and property of its membrane, its permeability, 



ANTIBIOTIC SUBSTANCES AND DISINFECTANTS 195 

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- 
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 j 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. suht'd'is is most sensitive, but the 
other spore-former B. mycoides is not affected at all. The gram-negative 
E. colt is more sensitive to streptothricin than either M. lysodeiktkus 
or S. lutea. Sulfanilamide has a definite, even if limited, retarding effect 
upon the growth of various organisms. The antibiotic substances of 
microbial 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, on the other hand, is highly bacteriostatic and bactericidal 
against certain gram-negative bacteria. 

The specific morphological differences among the bacteria, based 
upon the gram stain (205), as shown by their sensitivity toward anti- 
biotic substances, are thus found to be relative rather than absolute. 
Most of the gram-positive bacteria are more sensitive to the majority 
of antibiotic substances than are the gram-negative bacteria. But other 
an,tibiotic agents, such as streptothricin, streptomycin, and clavacin, act 
quite differently and show marked variations within each group. 



196 NATURE OF ANTIBIOTIC ACTION 

MECHANISM OF ANTIBIOTIC ACTION 

In an attempt to interpret the antibacterial activities 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 (254). The nutritional 
requirements of the organisms thus inhibited are more exacting than in 
their normal state, E. coll and S. hemolyticus, when inhibited by acrifla- 
vine 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 (557, 559). 

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 (359), 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 substance interferes with the metabolic processes of the mi- 
crobial cells, by substituting for one of the essential nutrients. It has 
been suggested (290a) that the antibiotic effect of certain polypep- 
tides, such as gramicidin, may be due to the presence of a ^-amino 
acid isomer of a natural amino acid, /-leucine, required for bacterial 
growth. 

The antibiotic substance may interfere with the vitamin utilization of the 
organism. The staling effect of a medium, frequently spoken of in 
connection with protozoa as "biological conditioning" of the organ- 
ism, may serve as an illustration. Such effects have been overcome 
by the addition of a mixture of thiamine, riboflavin, and nicotin- 
amide (373). 

The antibiotic agent brings about the oxidation of a metabolic substance 



MECHANISM OF ANTIBIOTIC ACTION 197 

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. 

The agent interferes with various enzymatic systems, such as the respira- 
tory mechanism of the bacterial cell, especially the hydrogenase sys- 
tem (435) and the phosphate uptake by the bacteria accompanying 
glucose oxidation, as in the action of gramicidin. Penicillin, for ex- 
ample, was shown (892) to be capable of inhibiting the activity of 
urease. It was later proved (8ioa), however, that this was due not 
to the penicillin itself but to certain impurities in crude penicillin 
preparations. 

The antibiotic substance may inhibit directly cellular oxidations, particu- 
larly those involving nitrogenous compounds, an action similar to 
that of propamidine (494). 

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 (153, 492, 
751, 902) catalyzes the following reaction: 

Glucose -j- Oo — ^ Gluconic acid -f- H2O2. 
Xanthine oxidase acts in a similar manner (531, 79l)- 

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 (189). 

The antibiotic substance may interfere with the sulfhydryl group which is 
essential for cell multiplication. This was shown (254a) to hold true 
for mercurials and other chemical antiseptics. The possible inter- 
relationship between the sulfhydryl group and true antibiotics has re- 
cently been indicated (109a). 

On the other hand, bacteria subjected to the action of an antibiotic 
substance may develop mechanisms that render them resistant to the 



198 NATURE OF ANTIBIOTIC ACTION 

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 (391, 401) to be 
inhibited 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 (388) 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 (538, 813, 1003). 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 pan- 
toyltaurine, which is related to pantothenic acid as sulfanilamide is to 
^-amino-benzoic acid, will inhibit the growth of hemolytic streptococci, 
pneumococci, and C. difhtheriae, by preventing the utilization of panto- 
thenic acid by these bacteria, for which it is an essential metabolite 
(558). Fildes (254) emphasized 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." The antibac- 
terial activity of iodinin is neutralized by quinones 5 this is probably due 
to the destruction of the iodinin, since the N-oxide is reduced by the or- 
ganism {SSS-) SS^)- Different anti-inhibitors are known for other anti- 
biotic substances, as shown later. 

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 /)H 7 and 37° C. for i to 3 days (662) 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 



ANTIBACTERIAL ACTION 199 

substance and neutralizes its antibacterial effect either in the absence or 
in the presence of the organism. 

Since only few antibiotic substances of microbial origin have been 
isolated in a crystalline state, confusion often resulted from the use of 
crude preparations. Welsch (971, 972) found that concentrated and 
partly purified actinomycetin had no appreciable lytic action upon liv- 
ing cells. However, the presence of a small amount of a highly bac- 
tericidal 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. 

ANTIBACTERIAL ACTION 

Two antibiotic agents have recently received special consideration, 
tyrothricin and penicillin. They will be considered here in further 
detail. 

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 (190) : 

1. Inhibition of growth. Certain gram-positive bacteria are inhibited by 

as little as I microgram or less of the substance per 10 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 or 

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 



200 NATURE OF ANTIBIOTIC ACTION 

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. 

Tyrothricin is made up of two compounds, gramicidin and tyroci- 
dine, that differ in their chemical properties and in their biological ac- 
tivity (205). 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 (391, 579). 

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 the infected area, gramicidin exhibits definite action against in- 
fection with pneumococci and streptococci. When injected intrave- 
nously, however, it is almost completely inactive against systemic in- 
fection. 

It was demonstrated by tissue culture technique (401) 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 (728) 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 



ANTIBACTERIAL ACTION 201 

glucose caused only slight inhibition of the hemolytic effect. 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 (435, 436). Tyrocidine ap- 
peared to block all the oxidative systems of the bacteria studied, whereas 
gramicidin seemed to affect only certain individual reactions. Both sub- 
stances were found to exert a protective antibacterial action in mice in- 
fected intraperitoneally with susceptible bacteria 5 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 both are leucocytolyticj they show little toxicity when applied 
locally by the subcutaneous, the intramuscular, or the intrapleural 
route J oral administration is not accompanied by toxic effects, but such 
treatment is ineffective (729). 

Gramicidin remains active in the blood stream, but it has only weak 
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 (756). 

According to Dubos (189, 201), the retention of the stain by gram- 
positive bacteria indicates a peculiar property of the cell wall of these 
organisms. The addition of 0.00 1 mg. of gramicidin to a billion pneu- 
mococci, streptococci, staphylococci, and others is considered sufficient 
to inhibit the growth of these organisms on subsequent transfers. This 
effect was 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 become inhibited under the action of gramicidin be- 
come viable again when cephalin is added to the medium. It was sug- 
gested that the ineffectiveness of gramicidin on gram-negative bacteria 
may be due to the presence of a phospholipid in these organisms. 

Tyrothricin did not exert any effect upon staphylococcus bacterio- 
phage (633). It did, however, inhibit the fibrinolytic activity of heta- 
hemolytic streptococci as well as of the supernatant liquids of these bac- 
teria but not of partially purified fibrinolysin. Although it prevented 
the neutralization of hemotoxin by antitoxin, it did not inhibit the pro- 
tective action of antitoxin against the toxin in mice (64). This substance, 



202 NATURE OF ANTIBIOTIC ACTION 

as well as actinomycin and clavacin, inhibited the coagulation of rabbit 
plasma by staphylococci but did not prevent coagulation by sterile cul- 
ture filtrates of these organisms j none of these three substances de- 
stroyed the toxin, nor did they enhance its hemolytic or lethal action 

(64). 

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 in one patient (720). Various other observations have been made 
(686) that staphylococci grown in the presence of increasing concentra- 
tions of gramicidin become resistant to inhibition by this substance 
(84,689). 

Both gramicidin and tyrocidine are said (196) 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 j it destroys immediately and irreversibly their meta- 
bolic activity, 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. 

It remains to be determined to what extent the action of tyrocidine, 
as well as of other antibiotic substances, can be reversed by detoxication 
with high molecular anions in a manner similar to their action upon sur- 
face active cations. The bacteria were said (901) to function as cationic 
exchanges, both the surface and the adsorbability depending on the 
structure of the cation. The bacterial action of surface-active cations and 
of toxic metallic ions and dye cations was considered as a phenomenon 
of ionic exchange by bacteria. Harmless cations could thus exert a pro- 
tective action on bacteria against the toxic cations. 

Penicillin 

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, Hemofhilus, or Brucella. The gram-negative cocci can be 



ANTIBACTERIAL ACTION 203 

divided into two groups, on the basis of the action of penicillin: first, 
Neisseria gonorrhoea. Neisseria intracellular y and Neisseria catarrhalisy 
which are sensitive j and second, A^. flavus and other nonpathogenic 
Neisseriae, which are not sensitive (261, 266, 267). 

Different strains of S. aureus vary little in their susceptibility to the 
action of penicillin j however, by growing the organism in increasing 
concentrations of this substance, it is possible to obtain more resistant 
cultures. Strains of staphylococci possessing increasing resistance to peni- 
cillin were also isolated from infections treated with the substance 

(727)- 

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 1 : 1,000, after incubation for 24 hours at 37° C, the bacteria gave 
larger numbers of colonies on plating (7). Although 0.0 1 to o.i mg. of 
penicillin per milliliter was found (418-424) to be sufficient to inhibit 
the growth of 2,500,000 hemolytic streptococci (Group A), no conclu- 
sion could be reached as to whether its action is truly bactericidal or 
bacteriostatic. 

A comparison was made of the amounts of penicillin and gramicidin 
required to bring about total inhibition of growth of bacteria, 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. pneumoniae 


2.5-5.0 


0.5-1.0 


S. fyogenes 


2.5 


5.0 


S. salivarius 


20-40 


2.5-60 


S. fae 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 (388, 389). 

Inhibition of growth of 2 to 4 million hemolytic streptococci was ob- 
tained by the use of 0.03 y penicillin with an activity of 240 to 250 Ox- 
ford units per milligram (418-424). Peptone, ^-amino-benzoic acid, 







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ANTIBACTERIAL ACTION 



205 



blood, or serum exerted no inhibiting effect, A marked difference was 
found in the action of penicillin and sulfonamides, the latter merely de- 
creasing the rate of multiplication and the former actually bringing 
about a decrease in the number of organisms present. This is brought 
out in Figure 21. The rates of activity of penicillin, gramicidin, and 
tyrocidine are compared in Table 36 (page 192). The bactericidal ac- 
tion of penicillin is not accompanied by lysis. No penicillin is absorbed 
or destroyed by the bacteria. 

Penicillin is not very stable j it is sensitive to reaction and temperature 
changes. The effect of reaction upon the stability of penicillin is shown 
in Figure 22. 

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. suhtilu units in a synthetic casein 





57 HOURS 





2 4 6^ 

REACTION (pHJ 



2 4 6 8 10 

REACTION (pH) 



Figure 22. Inactivating effect of reaction upon penicillin. From Foster and 
Wilker (283). 



206 NATURE OF ANTIBIOTIC ACTION 

hydrolyzate medium j the activity was increased to 6,000 by addition 
of /)-amino-benzoic acid in dilution of 1 12,500 to i : 10,000 j this was 
also true in presence of glucose in test medium. A similar, although 
somewhat lower, increase took place in case of S. aureus; no effect was 
obtained on S. hemolytkus. 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 (896). 

Attention was directed previously to the production by P. notatum 
of an oxidative enzyme. It is interesting to find that P. chrysogenum 
also forms an oxytropic glucose-dehydrogenase that is not susceptible to 
CO and cyanide (523) ; the glucose is oxidized to gluconic acid. The 
active substances produced by both groups are thus similar (832). 

Other Antibiotic Agents 

Of the other agents, actinomycin, streptothricin, clavacin, gliotoxin, 
and several other mold products deserve particular attention. 

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 (757) to be lethal for i kilogram weight of the animals. 
Doses as small as 50 y 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 rapidly re- 
moved 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 {6'^';})). 

A comparison of the effect of actinomycin with that of tyrothricin 
and its constituents, tyrocidine and gramicidin, upon the growth of 
rhizobia (890) 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- 



BACTERIOSTATIC AND BACTERICIDAL AGENTS 207 

sessed both properties. Some strains of rhizobia were stimulated by lim- 
ited concentrations of actinomycin. 

Streptothricin is far less toxic than these four substances. It acts 
largely upon gram-negative bacteria, and thus is quite distinct from the 
other four antibiotic agents. In this respect, it is similar to clavacin, 
which, however, is far more toxic. 

Clavacin not only is bacteriostatic on gram-negative bacteria but pos- 
sesses marked bactericidal properties, as is brought out in Table 37 
(page 192) and in Figure 23. Fumigacin, on the other hand, is active 
only upon gram-positive bacteria and has far more limited bactericidal 
action. 

Gliotoxin is active against both gram-positive and gram-negative bac- 
teria (Table 38). The substance is rather toxic to animals, the minimum 
lethal dose being 45 to 6$ mg. per kilogram body weight j hematusia 
is caused by even lower concentrations (459). 

TABLE 38. BACTERIOSTATIC ACTION OF GLIOTOXIN 
TEST ORGANISM ACTIVITY 

S. aureus 4,000,000 

5. -pyogenes 1,000,000 

Pneumococcl 4,000,000 

5. enteritidis 250,000 

A. aerogenes 200,000 

K. fneumoniae 250,000 

E. coli 80,000 

From Johnson, Bruce, and Dutcher (459)- 
Note. Units of activity by dilution method. 

On comparing the action of citrinin with penicillic acid, the first was 
found (661) to act largely upon gram-positive bacteria j the second, 
like quinones, had a more widespread action, especially against the 
colon-typhoid group, 

BACTERIOSTATIC AND BACTERICIDAL AGENTS 

Fleming (268) divided all selective bacteriostatic agents, exclusive 
of the action of oxygen on anaerobic bacteria, into three groups: (a) 







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BACTERIOSTATIC AND BACTERICIDAL AGENTS 



209 



physiological agents, including bile, serum, proteolytic enzymes, and 
lysozymej (b) microbiological products, comprising the antibiotic sub- 
stances} (c) chemicals of known composition, including dyes, salts (po- 
tassium, tellurite, mercuric salts), and other agents. 

Concentrations of antibiotic substance smaller than those needed to 
cause inhibition often stimulate growth of the organisms or of their 
metabolic processes (705). In this respect these agents are similar in 
their action to synthetic detergents (35) 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 (303). 

The bactericidal action of antibiotic agents, as influenced by their 
concentration, can be illustrated by the action of pyocyanase (Table 39). 
In a study of the bactericidal action of actinomycin (946) 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 39. BACTERICIDAL ACTION OF PYOCYANASE UPON THREE BACTERIA 



B. 


ANTHRACIS 


E. 


TYPHOSA 


M. TUBERCULOSIS 




Bacteria per 




Bacteria per 




Bacteria per 


Hours 


milliliter 


Hours 


milliliter 


Hours 


milliliter 


Start 


11,060,000 


Start 


13,125,000 


Start 


2,105,000 


24 


6,890,000 


3 


1,242,000 


3 


980,000 


72 


1,360,000 


9 


105,000 


8 


71,500 


96 


654,000 






24 





120 


329,000 










H4 














From Emmerich, Low, and Korschun (237). 



210 



NATURE OF ANTIBIOTIC ACTION 



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 0.02 1 to 0.026 for different periods of incubation. Figure 24 
illustrates graphically the effect of different concentrations of actino- 
mycin on the death rate of E. coli in buffer solution. 



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12 18 24 30 36 

INCUBATION PERIOD IN HOURS 



Figure 24. Action of actinomycin on E. colt; death rate in buffer solution. 
Amounts are given in milligrams of actinomycin per 10 milliliters of solu- 
tion. From Waksman and Woodruff (948). 



BACTERIOSTATIC AND BACTERICIDAL AGENTS 211 

Quinones have a high bactericidaJ power (145, 147, 615, 948), due 
not to their chemical interaction with the cell proteins but to their re- 
activity with the simpler cell constituents such as some of the amino 
acids (146, 148). Only a slight difference was found in the apparent 
activity of quinones toward yeasts, bacteria, proteins, peptones, pep- 
tides, and certain amino acids. Alcohol increases the germicidal power 
of the quinones. Actinomycin 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 biological nature of this antibiotic agent and of quinones. 
On the other hand, many of the antibiotic substances produced by fungi 
are typical quinones and act as such. 

By varying the concentrations of disinfectants, the types of curves of 
destruction of bacterial cells were found (694) 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 25). 

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. colt 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- 
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 
blue J 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 



212 



NATURE OF ANTIBIOTIC ACTION 




Figure 25. Spirocheticidal action of various amounts of penicillin In vitro. 
Inoculum, 4 x 10* organisms per milliliter. From Eagle and Musselman 
(unpublished). 



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 (227, 301, 854). 

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. It is to be re- 
called that autolysis has usually been defined (865) as "the breaking 
down and solution of some of the essential chemical constituents of the 
cell by agencies (enzymes) originating within the cell," This does not 
hold true, however, for most of the antibiotic substances. 



EFFECT ON MORPHOLOGY OF MICROORGANISMS 213 

The relation between antibiotics and bacteriophage has attracted con- 
siderable attention. Gratia (345) observed a definite relation between 
the action of lysozyme and the liberation of bacteriophage. The action 
of antibiotic agents, however, usually exhibits a marked distinction from 
that of bacteriophage (218, 344, 634). Filtrates of cultures of homolo- 
gous bacteria are able to inactivate the anti-coli phage j at 27° C, the 
inactivation is proportional to the phage and filtrate concentration j at 
0° C, to the square root of the latter (232). Based upon the formation 
of iso-antagonistic substances, a method has been suggested (121) 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 (378), 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 
this phenomenon is due to the production and accumulation of metabolic 
products injurious to growth. Nonspecific antibiotic substances were 
demonstrated (6s6) 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 (406). The spe- 
cific effect of the antagonistic B. mesentericus upon the morphology of 
antagonized bacteria has been established by Pringsheim (705). The 
antibiotic substances produced by actinomycetes were shown (80) to 
affect the growth of B. mycoides as follows: cell division is delayed} the 
cells become elongated, reaching enormous size and assuming most pe- 



214 NATURE OF ANTIBIOTIC ACTION 

culiar forms j spore formation or, with lower concentrations of agent, 
the active substance is repressed j delayed nonspore-forming variants 
are produced with a modified type of growth on nutrient media (Table 
40). 

TABLE 40. 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 


X 


- 


+ 


17 


Cells altered 


X 


- 


+ 


45 


Cell fragments of vari- 









ous shape and length x — - 

Medium plus 5 per cent culture filtrate 

2 Elongated cells x - + 

4 Elongated cells x - + 

17 Greatly deformed cells + - + 

45 Greatly deformed cells + — + 

Control medium 

2 ++ - + 

4 ++ + + 

17 ++ + + 

45 Deformed cells rare ++ + — 

From Borodulina (80). 

X indicates growth of B. mycoides in the shape of fluffy small balls inside liquid. 

Gardner (308) reported that concentrations of penicillin lower than 
those required for full inhibition caused a change in the type of growth 
of CI. welchii 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 hold true 
for a number of other bacteria (Figure 26). 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 



S. atirriis, normal cells. Preparctl by 
Foster and Woodrutl" 




S. tiiirrns, pcmcillin-inhihitccl cclU. 
Prepared by Foster and Woodruff 



^ 



'I / 



/ 



/ 



B. subtilisy normal cells. Prepared 
by Foster and Woodruff 






"A%. mnlandu^ normal cells. 
Prepared by Starkey 



B. subtilis, penicillin-inhibited cells. 
Prepared by Foster and Woodruff 




A-z. v'lnlamiii^ actinomycin-inhibited 
cells. Prepared by Starkey 



Figure 26. Iniluence of antibiotic substances upon the morphology of bacteria. 




Figure 27. Mechanism of antibacterial action as illustrated by the gradual 
diffusion of an antibiotic substance in a bacterial agar plate. EflFect of strepto- 
mycin on B. subtilis. 



EFFECT ON PHYSIOLOGY OF THE BACTERIAL CELL 215 

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- 
tensis, 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. "xelchiiy which was inhibited by i : 6o,000 penicillin, 
showed filament formation in a dilution of i : 1,500,000. The phenom- 
ena of swelling and lysis were said ( 833 ) to be associated with the active 
growth of the bacterial cell. Suspensions of fully grown bacterial cells 
showed neither of these effects when added to concentrations of peni- 
cillin many times higher. It was suggested that penicillin 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. 

A growth-depressing substance, which altered the type of growth of 
both fungi and bacteria, was also isolated (144) 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. colt from smooth to rough 
and finally to grainy j 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 Rhvzoctoma by an antagonistic fungus Trichoderma as well as 
by the antibiotic product of the latter has been described by Weindling 
(962). 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 either become empty or as if 
filled with granular material. 

ANTIBIOTIC SUBSTANCES AND THE PHYSIOLOGY 
OF THE BACTERIAL CELL 

Half a century ago Smith (838) emphasized that bacteria growing 
in mixed cultures undergo temporary and even permanent physiologi- 



216 NATURE OF ANTIBIOTIC ACTION 

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. 

Gramicidin and tyrocidine were believed to affect bacteria (390, 391, 
579) by depressing 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 (395) that gramicidin, after an initial stimu- 
lation, inhibited oxygen consumption of bovine spermatozoa and ren- 
dered them immobile J aerobic as well as anaerobic glycolysis was de- 
pressed by about 40 per cent and motility of the spermatozoa impaired. 
Tyrocidine, however, caused a small reduction in the oxygen consump- 
tion and in glycolysis. The action of gramicidin upon the metabolic ac- 
tivities of S. aureus and S. hemolyticus was shown (206) to be influ- 
enced by the composition of the medium, the presence of potassium and 
phosphate ions giving a prolonged stimulation of metabolism, whereas 
ammonium 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 (606). 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. This is brought out in Figure 28. 

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 (193). These enzymes were found not 
only to exhibit great selectivity but to be highly specific against the 
particular polysaccharides. 



EFFECT ON PHYSIOLOGY OF THE BACTERIAL CELL 217 



150 


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Figure 28. Effect of tyrothricin and protamine on the respiration of E. coli 
at 38° C. and />H 5.3. Number of cells per vessel: 5x10^. Concentrations: 
tyrothricin 1:15,000; protamine 1:3,000; mixture, tyrothricin 1:15,000, 
protamine 1:3,000. From Miller, Abrams, Dorfman, and Klein (606). 



218 NATURE OF ANTIBIOTIC ACTION 

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 5. hemolytkus as well as other streptococci and 
pneumococci. This substance has been identified (771) with the 
^-amino-benzoic acid referred to above. No relationship could be estab- 
lished, however, between the growth-promoting properties and anti- 
sulfanilamide activity of the yeast extract (544). 

Br. abortus and certain other bacteria also contain (354, 355) a fac- 
tor, designated as "p," which specifically inhibits the bacteriostatic ac- 
tion of sulfanilamide. This factor stimulates markedly the growth of 
many bacteria, and is not specific. The sensitivity of sulfanilamide de- 
pends 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 yeast (870) and from hemolytic streptococci (538). It is also 
known that certain substances, like methionine, inhibit the growth- 
stimulating effect of biotin (259). 

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, lOOO) to be ca- 
pable of producing a substance which destroys the growth-inhibiting 
property of the antibiotic agent. The penicillin-destroying substance is 
believed to be an enzyme, since it is destroyed by heating at 90° C. for 
5 minutes. It was designated as "penicillinase." The optimum fYi of its 
action was found to be 8 to 9. The presence or absence of this enzyme 
in bacteria is independent of the sensitivity of the organism to peni- 
cillin. 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 



DIFFERENTIATION OF BACTERIA 219 

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 (35). 
Mucin inhibits the action of tyrothricin, especially on meningococci and 
pneumococci (182). 

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 (947). Ascorbic acid 
also has an effect in reducing the activity of this substance. Jungeblut 
(466) demonstrated that vitamin C, interacting with atmospheric oxy- 
gen, leads to the production of destructive peroxides in a medium. Since 
vitamin C is a strong reducing agent and actinomycin is a reversible 
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. butyricum as a test organism as under 
aerobic conditions with B. niycoides. 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 
(1003) and others for sulfanilamide inhibition. 



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 (269) utilized penicillin to isolate Pfeiffer's bacillus and the 
pertussis organism of various cocci, diphtheria, and neisseria organisms j 
the substance was found to behave as the mirror image of tellurite in 
inhibiting specific bacteria. Penicillin was also utilized for the separation 



220 NATURE OF ANTIBIOTIC ACTION 

of acne bacilli from accompanying staphylococci (153) and for the sepa- 
ration of streptococci from staphylococci (266). 

Actinomycin was used to separate gram-negative from gram-positive 
bacteria (945). 

Tyrothricin has been utilized (858) 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 injiuenzae 
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 (808). 

Streptothricin was found to help in distinguishing B. mycoides from 
B. subtilis (950). 

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, 
various physiological reactions of microorganisms, the mechanism of 
causation of disease, and the very control of disease-producing micro- 
organisms — all fundamental problems in microbiology — will be fur- 
thered by knowledge of the action of specific antibiotic substances upon 
bacteria and other microorganisms. 



a 



CHAPTER 12 v'^^^^il*^ 

UTILIZATION OF ANTAGONISTIC MICROORGAN- 

ISMS AND ANTIBIOTIC SUBSTANCES 
FOR DISEASE CONTROL 

Whether gra?mcidm or any other product of mtcrob'ic origin will 
eventually be found to fulfill certain furfoses better than either 
sulfhonamides or any other class of antiseftic remains to be seen. 
That several classes of reagent should be competing for suprem- 
acy in different aspects of a task which not long ago was consid- 
ered impossible of any real fulfilment is a truly remarkable posi- 
tion (343a)- 

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. As early as 1 877, Pasteur (675) noted that the produc- 
tion of anthrax in susceptible animals can be repressed by the simultane- 
ous inoculation with B. anthrads and various other bacteria. This led 
him to make the following significant suggestion: ". . . on peut intro- 
duire 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." 

Pasteur may thus be looked upon 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 j these 
bacteria were, therefore, looked upon as agents useful in bringing about 
immunity against all bacterial infections. Pawlowsky (676) obtained 
immunity against anthrax by inoculation with Friedlander's bacillus. 
Bouchard (81) 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 a delay in the course of its development. Rabbits infected with an- 
thrax were also cured by means of a pyocyanase preparation (872)} 
many other cases of successful treatment of anthrax with pyocyanase 



222 DISEASE CONTROL 

have been reported (280). The pressed extract of Ps. aeruginosa had a 
similar effect when injected in the animal simultaneously with the 
pathogen (505). 

Various methods of treating severe infections, like anthrax (97) or 
malignant tumors (138), with mild infective agents have been sug- 
gested. The reduction in pathogenicity of one organism by the presence 
of others has thus been well recognized (62, 120, 239). Nonpatho- 
genic organisms apparently have specific effects upon the pathogens, the 
development of which was prevented or even suppressed. The very oc- 
currence 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 (369). 

It was thus definitely established that the growth of B. anthrads 
could be inhibited by antagonists (48). Guinea pigs survived large in- 
jections of washings from soil previously contaminated by B. anthrads 
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 leukocytes which have been attracted to the site of in- 
jection by the accompanying bacteria (31). 

Seitz (812), 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 
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, provided it did 
not concern tissue or blood infection, but only skin surfaces, including 
those of the intestinal canal and the vagina. 

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 antagonizing or- 
ganism, to a lack of knowledge concerning the mechanism of its action. 



MICROBIAL ANTAGONISTS 223 

or to the variability of the antagonizing agent as regards strain specific- 
ity and the production of the active antibacterial substance. 



MICROBIAL ANTAGONISTS AND DISEASE CONTROL 

In 1 885, Cantani treated a tubercular patient with a culture of a sapro- 
phytic organism, designated as Bacterium termo; the results were 
highly favorable (104). 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 ( 1009) 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 investi- 
gators (226, 844). 

Gate and Papacostas (318) observed that mixed infections were usu- 
ally mild, a phenomenon later confirmed. Mixed cultures of the Fried- 
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 
i^'T^G) 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 (347). Bezangon (53) treated 
typhoid sufferers with a culture of E. typhosa lysed by means of Ps. 
aeruginosa. Gratia (347) said, however, that this type of hydrolyzate 
will bring about heat production, but the use of a preparation obtained 



224 DISEASE CONTROL 

by means of an Actinomyces, designated as a mycolysate, will not. The 
use of living proteolytic bacteria (neocolysin) for treatment of chronic 
purulent conditions, such as osteomyelitis, gave favorable results j the 
bacteria were believed to continue growing as long as there was dead 
tissue available ( 99 ) . 

Besredka (51) 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 j 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 
of the homologous antivirus, the organisms underwent active phagocy- 
tosis, this action being specific. The antivirus was nontoxic and could 
withstand a temperature of 100° 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. Although unspecific filtrates may cause an occasional in- 
crease of resistance, the protection produced by specific filtrates is said to 
be more intense and more dependable {Gs'},-, 741). Antivirus therapy 
was believed to offer some promise, although it was said not to give con- 
sistent results (381). Further studies of antivirus led to suggestions that 
its favorable effects were due entirely to the culture medium (6). The 
whole question thus appears to be still debatable, with proponents and 
opponents of the specific nature of the antivirus effect ( 1 10, 589). 



MICROBIAL ANTAGONISTS 225 

The application of bacteriotherapy for the treatment of chronic infec- 
tions of the middle ear (706) and actinomycosis in man has also been 
suggested. Filtrates of E. tyfhosa and of E. coli were found (810) 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 (596a) 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. acidofhilusy an inhabitant of the hu- 
man intestine possessing antagonistic properties against pathogenic in- 
testinal bacteria, has come into general use (744). The problem of com- 
bating pathogenic intestinal bacteria by means of nonpathogenic forms 
(703) has thus been given wide consideration. The utilization of yeasts 
for combating streptococci and staphylococci may also be classified 
among the phenomena of antagonism (893). On the basis of the rapid 
destruction of pathogenic bacteria added to natural water, the storage 
of drinking water in large reservoirs was recommended as an important 
safeguard against the water's becoming a carrier of bacterial diseases 
(295-297). 

Clinical methods have been proposed for evaluating the results ob- 
tained by treating tooth gangrene by means of antagonists (325). Don- 
aldson (181) found that CI. sforogenes 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. 



226 DISEASE CONTROL 

Dack (159) reported that CI. sforo genes formed in the soil was re- 
sponsible for destroying the toxin of CI. botuUnum. 



ANTIBIOTIC SUBSTANCES AND DISEASE CONTROL 

In discussing the subject of antiseptics in war-time surgery, Fleming 
emphasized that the treatment of war wounds has become far more 
satisfactory during the second world war than it was during the first. 
It is now known, for instance, that carbolic acid lacks value inside the 
human body, as demonstrated by a diminution in efficiency with in- 
creasing concentrations, due to its destructive effect upon the blood 
leukocytes and body tissues. Dyes have been found also to be of little 
value, since they are absorbed by the cotton used in dressing the 
wounds. Fleming (260) warned against placing too much faith in anti- 
septics belonging to the sulfonamide group, since they are not general 
antiseptics but have specific effects upon certain bacteria, and their ac- 
tion is neutralized by chemicals, by pus, and by dead bacterial cells. 
They are, therefore, of little value in the treatment of seriously septic 
wounds, in which pus and bacteria are inevitably present. Their major 
importance is due chiefly to their great solubility, since they dissolve to 
form high concentrations in the wound. 

In view of these limitations in the use of chemical antiseptics, bacterio- 
static and bactericidal (antibiotic) agents produced by microorganisms 
may find particular application. Among the substances formerly utilized 
for this purpose pyocyanase has received special consideration (239). 
Unfortunately, the variation in the nature of the preparation of this 
material and the difficulty of keeping it in an active condition for very 
long periods of time have prevented its wider usefulness. Among the 
more recent preparations, penicillin occupies a leading place. 

Penicillin 

Nature of Action. The action of penicillin upon bacteria is chiefly 
bacteriostatic and not bactericidal (260). Penicillin shows in vitro a high 
degree of specificity j pyogenic cocci, anaerobic Clostridia, and certain 
pathogenic gram-negative cocci {GonococcuSy Meningococcus y and Mi- 
crococcus catarrhalis) are sensitive, whereas the colon-typhoid, hemo- 



ANTIBIOTIC SUBSTANCES 227 

philic chromogenic bacilli and certain micrococci {Micrococcus flavus) 
are resistant to its action (263) ; however, it has no effect upon M. tu- 
berculosis (834), Trypanosoma equiferdum^ and the influenza virus 
(753). The purest preparation of penicillin so far available completely 
inhibited (276) the growth of S. aureus in a dilution of between 
1 : 24,000,000 and 1 130,000,000. Partial inhibition was obtained up to 
1 : 1 60,000,000. Salmonella organisms were also sensitive. The antibac- 
terial activity of penicillin is not interfered with by substances that in- 
hibit sulfonamides, namely, bacterial extracts, pus fluids (7), tissue 
autolysates, peptones, and ^-amino-benzoic acid. It is nontoxic in con- 
centrations far greater than those required for therapeutic purposes 
(163, 164). However, 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 (7) 
which may be summarized as follows : 

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 could be completely pre- 
vented by as low a concentration of penicillin as 1 : 1,000,000, even 
if the inoculum contained several million bacterial cells. In the case 
of smaller inocula, inhibition occurred in even higher dilutions. This 
property of penicillin is believed to be of great importance in the 
treatment of heavily infected wounds, on which the sulfonamide 
drugs seem to have little beneficial action. 

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 leukocytes remain active in any concentration of peni- 
cillin usually employed in intravenous injection. 



228 DISEASE CONTROL 

Penicillin is active against strains of bacteria that are resistant to the ac- 
tion of sulfonamides (273, 566, 881). It is effective in the treat- 
ment of hemolytic streptococcus, pneumococcus, and gonococcus in- 
fections, which are resistant to sulfonamides. It has not been found 
effective, however, in the treatment of subacute bacterial endocar- 
ditis (748). 

On repeated passage through broth containing penicillin, pneumo- 
coccus cultures as well as Stafhylo coccus sp. and 5. -pyogenes (564) 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 (806). 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 (846, 847). 

Toxicity. As to the toxicity of penicillin, it was found (7) that mice 
were little affected by the intravenous injection of 10 mg. of penicillin j 
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 (753). 

The relative toxicity of various salts of penicillin was found (967) to 
be, in increasing order, Na, NH4, Sr, Ca, Mg, and K. Based on milli- 
grams of the cation at the LD-,o dose of salts of penicillin, the relative 



ANTIBIOTIC SUBSTANCES 229 

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 not inactivated by saliva, bile, or succus entericus, but is 
destroyed rapidly by gastric juice, due not to the pepsin but to the HCl 
in the juice (724, 725). 

Penicillin is slowly absorbed and excreted, usually within one hour, 
in the urine (725). The degree of its antibacterial action is proportional 
to its concentration in the serum, maximum effects against hemolytic 
streptococci being produced by concentrations of 0.019-0. 156 Oxford 
units in i ml. of serum. The LD50 for an 18-gram mouse was 32 mg. 
of the sodium salt (422, 424). The cardinal symptoms of toxicity were 
choking, gasping, and rapid respiration. However, it is relatively non- 
toxic in doses used for therapeutic purposes (163, 164, S^^, 753). 

Penicillin was thus found to combine the two most desirable quali- 
ties of a chemotherapeutic agent, namely, a low toxicity to tissue cells 
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, and to recover a large proportion 
of the substance from the urine j this recovered penicillin could then 
be used again. 

Animal Experiments. In animal experiments (699) it was estab- 
lished that penicillin is an effective chemotherapeutic agent against 
pneumococci, including sulfonamide-resistant types. In experiments 
with S. aureus, a survival ratio of 2 : i was obtained in favor of penicillin 
as compared with sulfathiazole, correction being made for the survival 
of control mice. Penicillin, when administered subcutaneously, intra- 
venously, or intraperitoneally, was also found to be effective against 
hemolytic streptococci (418). Generalized staphylococcal infections 
were cured by penicillin and local lesions healed during parenteral ad- 
ministration. The best method for administering penicillin was by the 
intramuscular route at 3-hour intervals j the blood should contain 
enough penicillin (15,000 Oxford unit dose) to inhibit the growth of 
the infecting agent (276, 399, 404). Intraocular infection caused by 
D. fneumoniae was checked by local treatment with penicillin in solu- 



230 DISEASE CONTROL 

tions of 0.25 and o.i per cent j the application was continued for 2 to 4 
days (780). 

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. The 
excretion of penicillin could be blocked by simultaneous administration 
of diodrast (723-725). 

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 j however, in vivo 
treatment of mice infected intramuscularly with CI. ferfringens placed 
penicillin first, with tyrothricin and aspergillic acid at the bottom of the 
list (562)- Penicillin also proved superior to sulfonamides and amino 
acridines in experimental infection with CI. welchii and CI. aedematiens 

The in vivo activity of penicillin against CI. se-pticum and other 
anaerobes, as well as many other bacterial pathogens ( 1 1 3 ) , is brought 
out in Table 41. A single subcutaneous treatment of mice with 50 
Florey 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 un- 
less the delay was over 3 hours. Local lesions were completely healed 
within 3 weeks if penicillin was injected repeatedly into the site of in- 
fection (371). 

An intravenous injection of 20 mg. of the sodium salt of penicillin 
was without apparent effect on a mouse, and human leukocytes survived 



o >^ 



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ffi Oh 



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232 DISEASE CONTROL 

for an hour in a i per cent solution (273). The efficacy of penicillin 
in protecting mice against streptococcal infections is brought out in 
Table 42. 

TABLE 42. IN VIVO EFFICACY OF PENICILLIN COMPARED WITH SULFANILAMIDE 
IN STREPTOCOCCUS HEMOLYTICUS INFECTIONS IN MICE 



DAILY 


daily dosk 


NUMBER OF MICE OF ORIG 


INAL 20 SURVIVING AFTER 


DOSE IN 


1 


[N OXFORD 


I 


2 


3 


4 


5 


6 


7 


8 


9 


10 


MILLIGRAMS 




UNITS 


day 


days 


days 


days 


days 


days 


days 


days 


days 


days 


Penicillin 


























0.0625 




3-75 























0.125 




7-5 


16 





















0.250 




15.0 


18 


4 


I 


I 


I 


I 


1 


I 







0.500 




30.0 


20 


4 



















i.o 




60.0 


20 


20 


18 


15 


15 


15 


15 


13 


9 


9 


2.0 




120.0 


20 


20 


20 


20 


20 


20 


20 


20 


20 


20 


4.0 




240.0 


20 


20 


20 


20 


20 


20 


20 


20 


20 


20 


Sulfanilamide 
























0.5 






20 





















1.0 






20 


20 


20 


10 


8 


8 


5 


4 


I 


I 


2.0 






20 


20 


20 


20 


20 


20 


20 


20 


14 


3 


4.0 






20 


20 


20 


20 


20 


20 


20 


19 


19 


16 


8.0 






20 


20 


20 


20 


20 


20 


20 


20 


20 


18 


16.0 






20 


20 


20 


20 


20 


20 


20 


20 


20 


20 


Controls 


























Culture dilut: 


Ion 


10-* 























Culture diluti 


ion 


io-« 
























From Robinson (752). 

Notes. Infection: 0.5 cc. of lO"* 6-hoiir-old culture dilution in broth. Treatment: Penicillin given sub- 
cutaneously and sulfanilamide given orally immediately after the inoculation of bacteria, then every 
3 hours day and night for 5 days. 

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 of penicillin in 
relatively large doses to mice after injection with murine typhus rick- 
ettsiae resulted in marked reduction in mortality, particularly when the 
initial dosage of the rickettsiae was relatively small (614). 

Chemotherapeutic Action. Penicillin has also found an important 



ANTIBIOTIC SUBSTANCES 233 

place in the treatment of local and generalized infections in man. Flem- 
ing was the first (265, 266) 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 (267, 271 ). Local applications include those to lesions of the eye 
produced by S. aureus, in which early treatment resulted in the elimina- 
tion of the organism from the flora of the conjunctival sac (758). 
Weight for weight, penicillin was found to be four times as potent as 
sulfathiazole and 100 times as potent as sulfanilamide for the treat- 
ment of wound infections (7, 272). 

Penicillin proved to be an especially effective agent for the treatment 
of staphylococcal and hemolytic streptococcal infections in man (725), 
including streptococcal meningitis (270). 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, staphylococci, 
streptococci, and corynebacteriaj the gram-negative bacteria were not 
affected. Excellent therapeutic effects were obtained. Even crude cul- 
ture filtrates of P. nolatum, applied locally, gave good results (242, 
707). 

In order to lessen the frequency of effective invasion of the nose by 
bacteria and the subsequent infection of the nasopharynx, the use of 
penicillin as an antiseptic snuff was suggested (166). The material acts 
as a prophylactic against bacterial infections of the upper respiratory 
tract } the course of a cold could thus be checked by preventing second- 
ary bacterial infection. The curing of nasal carriers of staphylococci or 
even the reduction of the number of vegetative organisms was consid- 
ered important in order to reduce the danger of the carrier as a source of 
infection to others. Penicillin can also find application in certain chronic 
cases, as in the treatment of chronic dermatitis and in preparing infected 
surfaces of hands for skin-grafting and infected stumps for amputation 

(277)- 

Penicillin is an effective agent in the treatment of clinical infections 
due to sulfonamide-resistant bacteria (39, 136, 137, 143,252,400,576). 
Se\'eral strains of A^. gonorrheae, isolated from patients in whom the 
infection was resistant to treatment with sulfonamide preparations. 



234 DISEASE CONTROL 

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' con- 
tact. The complete absence of toxicity following the intravenous admin- 
istration of penicillin, the lack of discomfort to the patient, and the rapid 
disappearance of clinical symptoms were observed in cases of sulfonam- 
ide-resistant gonorrheal infections. In all the cases reported, in addi- 
tion to the clinical response noted, negative bacterial cultures were ob- 
tained some time between 1 7 and 48 hours after the institution of peni- 
cillin therapy. Sulfonamide-resistant gonorrhea cases responded to in- 
jections of 100,000 to 160,000 Oxford units (136, 479, 549, 863). Fa- 
vorable responses have also been obtained in the treatment of sulfon- 
amide-resistant strains causing staphylococcal pneumonia and empyema 
(44, 6s) and other diseases. The susceptibility of various bacteria to 
penicillin can be determined by means of a very simple technique (879). 

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 (723, 725). 

Penicillin has not been found to be effective in trypanosome infec- 
tions, but has been used successfully in the treatment of relapsing fever 
(29, 393), although excessive doses were required (220, 221). 

Treatment of early syphilis cases with penicillin (575) 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 (112, 271, 275, 523a, 805). Penicillin was found to be actively 
spirocheticidal (222). A comparative study has been made of the action 
of penicillin and of other antibiotic agents upon Treponema -pallida 
(2iy). The administration, at 3-hour intervals for a period of 15 hours, 
of 20,000 units of penicillin intramuscularly was found satisfactory in 
the control of gonorrhea in men (879a, 907). Penicillin was also found 
to have an effect upon experimental typhus rickettsiae (357, 614). 

As a result of treatment of 300 patients with penicillin, it has been 
concluded (748) that this material is far superior to any of the sulfonam- 



ANTIBIOTIC SUBSTANCES 23 5 

ides in the treatment of S. aureus infections with and without bacteri- 
emia, including acute and chronic osteomyelitis, cellulitis, carbuncles 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 (479, 549) : 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- 
ment, either by continuous intravenous injection or by interrupted in- 
travenous or intramuscular injections. Good results were obtained by 
injections of 100,000 to 160,000 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 43. 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. Pyocyaneus is not susceptible to penicillin and is considered to 
be relatively unimportant as a single pathogen in the surgical manage- 
ment of the wound (272, 548). 

In view of the inefficacy of the sulfa drugs for the treatment of 
burns, a detailed study has been made (133) of the utilization of peni- 
cillin applied to the wound in the form of a cream. In 54 wounds thus 
treated, penicillin had a lOO per cent effect upon the hemolytic strepto- 
coccal flora, in 7 cases the strains being insensitive to sulfonamide. 
The staphylococci also disappeared, although somewhat more slowly. 
Healing was usually rapid and no toxic effects were observed. Gram- 



236 DISEASE CONTROL 

negative bacteria {E. coli, P. vulgaris, Ps. aeruginosa) y when present, 
were not affected, as further shown by Bodenham (67). 

A summary of the results of extensive use of penicillin in the North 
African campaign of the present war 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 
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 (276, 316). 

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 (276, 479). 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. Although penicillin has so far proved in- 
effective when administered orally, certain of its esters (e-butyl) that 



TABLE 43. RESPONSE OF DIFFERENT BACTERIA FOUND IN WOUNDS 
TO PENICILLIN TREATMENT 





PENICILLIN 


RESPONSE 


TYPE OF INFECTION 


Systemic 


Local 


Putrid: 






Proteolytic Clostridia 


+ (large dosage) 


+ 


Proteus vulgaris 








Nonhemolytic streptococci: 






Mesophilic 


+ 


+ 


Thermophilic (S. faecalis) 





(or slight) 


Staphylococci 


+ (3-5 days) 


+ (often necessary) 


Hemolytic streptococci 


+ (1-3 days) 


+ (not essential) 


Pseudomonas aeruginosa 









From Lyons (548). 



ANTIBIOTIC SUBSTANCES 237 

are inactive in vitro can, when given by the oral route, become highly 
active against hemolytic streptococci (599, 600). 

Production. Because of the limited amounts of penicillin available 
at the present time, many attempts have been made by physicians and 
hospitals to grow P. notatum on a suitable medium and use the crude 
culture filtrate for the treatment of wounds and infections. Since such 
cultures cannot be standardized and their activity cannot always be de- 
termined and since unforeseen toxic substances may be produced by cer- 
tain contaminants in the culture, this practice should not be encouraged, 
unless carefully supervised by properly qualified bacteriologists. 

The production of penicillin-destroying enzymes by bacteria and 
fungi (4, 379, 516) can be utilized for the purpose of testing the steril- 
ity of penicillin preparations. 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. No apparent 
relation was said ( lOOO) to exist between the resistance of an organism 
to penicillin and its ability to produce penicillinase, a fact not generally 
accepted (73-75). The mode of action of the enzyme is still not clearly 
understood, although there is apparently an increase in the number of 
carboxyl groups, as measured by ^H change ( lOOo). 

Clavacin {Patulin) 

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} no ill effects were observed 
(713). These results were not confirmed, however, the conclusion hav- 
ing been reached that, compared with the natural evolution of the dis- 
ease, patulin has no demonstrable effect on the course of a cold (847a). 
This substance also proved to be unsatisfactory for the treatmnt of bo- 
vine mastitis by udder infusion (681 a). 

Tyrothricin 

Dubos (193) 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- 



238 DISEASE CONTROL 

tonitis caused by pneumococci and streptococci (Table 44). 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. 

TABLE 44. 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* 


- 


- 


- 


- 


+ 


1 1 1 1 


Reductasef NR 


NR 


NR 


NR 


NR 


NR 


CR 


Lysis§ C 


C 


C 


C 


C 


P 


N 


Streptococcus hemolyticus, Group A, Tyi 


pe6 








Viability* 


- 


- 


- 


- 


+++ 


1 1 1 1 


Reductasef NR 


NR 


NR 


NR 


NR 


PR 


CR 


Lysis§ N 


N 


N 


N 


N 


N 


N 


Staphylococcus aureus 














Viability* 


- 


- 


^K+ 


+H-f 


++-++ 


-H-+4- 


Reductasef NR 


NR 


NR 


CR 


CR 


CR 


CR 


Lysis§ C 


C 


N 


N 


N 


N 


N 



From Dubos and Cattaneo (203). 

*— no growth on blood agar, + reduced growth, MM abundant growth. 

t NR no reduction of methylene blue, PR partial reduction, CR complete reduction. 

§ N no lysis, P partial lysis, C complete lysis. 

Tyrothricin exerted a lethal action m vitro on 1 8-hour broth cultures 
of S. hemolyticus^ S. aureus, and C. difhtheriae, in a final dilution of 
1 : 1,000,000; freshly isolated strains of meningococcus were affected in 
a dilution of i: 100,000 (807). Two monkeys which carried in the 
nasopharynx and throat gram-positive hemolytic streptococci and gram- 
negative hemolytic bacilli showed disappearance of these bacteria within 
1 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. 



ANTIBIOTIC SUBSTANCES 239 

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 ; 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. tyrothricin into the pleural cavity of rabbits with hemolytic strepto- 
coccal empyema brought about the sterilization of the pleural cavity and 
enabled the animal to survive. The injection of 10 mg. of tyrothricin 
into the pleural cavity of normal rabbits produced certain local tissue re- 
actions. In excess of 10 mg., adhesions, thickening of the pleura, sterile 
abscesses, and other disturbances were produced (721). 

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 (761 ). 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 (722). Certain sulfonamide-resistant strains of 
iS". fyogenes were eradicated by application of gramicidin (292). 

Tyrothricin and tyrocidine exert a bactericidal effect, and gramicidin 
is largely bacteriostatic (Figure 29) 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 (754). Gramicidin 
is more toxic than tyrocidine, the toxic dose being larger, however, than 
the dose necessary to kill most gram-positive cocci. Penicillin is one- 
tenth as toxic as gramicidin (402, 403). 

Both tyrothricin and tyrocidine cause hemolysis of erythrocytes, and 
both are leukocytolytic, gramicidin being less so. Both tyrothricin and 
gramicidin cause local and general toxic effects when injected into closed 







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ANTIBIOTIC SUBSTANCES 241 

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 m vitro. Local application of these substances has not been 
attended by toxic reactions even when large amounts were applied 
(729). Tyrothricin in high concentrations caused cytoplasmic and nu- 
clear disintegration of the exudative rabbit polymorphonuclear leuko- 
cytes j 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 
about some protection of the cells from the effects of these substances 
(132). Tyrothricin does not inhibit mitosis or migration of fibroblasts 
or activities of leukocytes, following direct applications to tissue culture 

(693). 

The filtrate of B. mesentericus was found (956) to have a specific bac- 
tericidal action on C. difhtheriae in a dilution of i: 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. 

Particularly favorable results were obtained from the use of grami- 
cidin in the treatment of chronic mastitis (535). 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 i ,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 



242 DISEASE CONTROL 

by daily bacteriological examination of the milk over periods ranging 
from 1 5 to 8 1 days. 

Sterile mineral oil was later found (536, 537) to be a suitable, non- 
irritating 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 3 1 quarters 
naturally infected with Streptococcus agalactiae and treated by the 
gramicidin-oil mixture, 26 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 uberis; of 4 cases treated, only one re- 
sponded satisfactorily. The final recommendations consisted in using 
20 to 40 mg. gramicidin in oil, daily, for four consecutive days. These 
results were confirmed by various investigators (16, 888), who re- 
ported 60 to 90 per cent cure for two to three treatments (585). 

A comparison of tyrothricin, trypaflavin, and novoxil for eradicating 
S. agalactiae from infected udders showed best results for the first. In- 
fections with S. uberis and Streptococcus dysgalactiae also responded 
well, but not staphylococcic infections (793). 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. Too ex- 
tensive administration may damage the secretory tissues (622). In 
some experiments, as many as 90 per cent of the cases were cleared up 
with tyrothricin (95). 

Gramicidin-like preparations were also used successfully in the treat- 
ment of local infections in man, such as osteomyelitis (553), and for 
various local administrations, such as conjunctivitis, as well as for in- 
fected diabetic and ulcerating lesions of cancer ( 1007). The active ma- 
terial must be used locally on infected cavities which do not communi- 
cate with the blood stream (3 1 8a, 401 ). Tyrothricin has also been used 
(78, 154) successfully in the treatment of acute otitis media, acute and 



ANTIBIOTIC SUBSTANCES 243 

chronic mastoiditis, and acute and chronic sinusitis. The substance does 
not damage the tissue or interfere with wound healing. The growth of 
most staphylococci, streptococci, and pneumococci is inhibited or the or- 
ganisms 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 (187). 

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 5 (b) inhibition of its 
action by phospholipinsj (c) hemolytic action, which prevents its intra- 
venous use. 

Tyrothricin was found to produce no lesions in the gastro-intestinal 
tract (728, 968), but it is not very active when administered by mouth, 
since it is destroyed by the proteolytic enzymes of the digestive system. 

Streftothrkin 

The fourth agent that was found to offer practical possibilities is 
streptothricin. This agent gave favorable results for the treatment of 
Br. abortus grown on chicken egg embryos. Its toxicity is low enough 
to make possible the administration of doses sufficient to destroy this 
pathogen in the living tissues (Table 45). Both the in vitro and the in 
vivo activities of streptothricin against gram-negative bacteria sug- 
gested the probability that it will prove useful in the local treatment of 
infected wounds and burns, bacillary dysentery, typhoid fever, and food 
poisoning produced by Salmonella organisms (755, 756a). 

Other Agents 

The protective action of the specific enzyme (polysaccharidase) of a 
soil bacterium against type III pneumococcus infection has also been 
established (30, 293). The specific protection induced in experimental 
animals is determined by the nature of the polysaccharide of the pneu- 
mococcus type. The polysaccharidase destroys the protective capsular 
substance of the pneumococcus, thus rendering it susceptible to phagocy- 
tosis. 

The possibility of utilizing antagonistic bacteria for the control of 
fungi causing skin infections has also been suggested (119). 



244 



DISEASE CONTROL 



Virulent strains of M. tuberculosis were found to lose their virulence 
in the presence of certain other organisms or their products. According 
to Vaudremer (912), this phenomenon occurs when the tubercle or- 
ganism is kept for 24 hours at 39° C. in contact with a filtered extract 
oi A. jumigatusy and a similar effect can be exerted by certain bacteria 
(708). On the other hand, extracts of A. jumigatus were used for the 
treatment of 200 tubercular patients with rather inconclusive results 
(912). 



TABLE 45. IN VIVO EFFICACY OF STREPTOTHRICIN COMPARED WITH 
SULFADIAZINE IN SALMONELLA AERTRYCKE INFECTION IN MICE 













NUMBER OF 






daily treat- 


original 


m: 


ICE SURVIVING AFTER 




ment 


IN 


number 


I 


2 


3 


4 


5 


INFECTION* 


milligrams! 


OF mice 


day 


days 


days 


days 


days 


Streptothricin 


















IO-* 


I 




10 


10 


5 


5 


4 


2 


I0-* 


2 




10 


10 


10 


10 


10 


10 


I0-* 


4 




10 


10 


10 


10 


10 


10 


10* 


8 




10 


10 


10 


10 


10 


10 


Sulfadiazine 


















10-* 


16 




10 


10 


10 


10 


4 


4 


Control 


















10-* 






10 













io-« 






10 













io-« 






10 













10-^ 






10 














From Robinson (752). 

* 0.5 cc. of a 6-hour-olcl culture dilution in broth. 

t Streptothricin given subcutaneously immediately after inoculation of bacteria. 



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. coU with a high antagonistic in- 
dex is considered as important evidence of immunity of certain indi- 
viduals to intestinal disturbances (383, 580, 619, 643). 



TOXICITY OF ANTIBIOTIC SUBSTANCES 



245 



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 or clavacin, which are highly bacteriostatic, 
may be considered as either entirely excluded for the present or limited 
only to local applications. Some substances, as pyocyanase, penicillin, 
and streptothricin, are relatively nontoxic (754) j others, like tyrothri- 
cin (756) and fumigacin (752), are slightly toxicj and still others, like 
actinomycin and clavacin, are highly toxic (757, 941). Some, like ty- 
rothricin, are hemolytic (390, 579) j others, like penicillin, actinomycin, 
and streptothricin, are not (Table 46). 



TABLE 46. ACUTE TOXICITY TO MICE OF VARIOUS ANTIBIOTIC AGENTS 









SUBCU- 


INTRA- 








ORAL 


TANEOUS 


PERITONEAL 


INTRAVENOUS 


ANTIBIOTIC 




ADMIN- 


ADMIN- 


ADMIN- 


ADMIN- 


AGENT 




ISTRATION 


ISTRATION 


ISTRATION 


ISTRATION 


Streptothricin 




>2,000 


> 1 ,000 


3,000 


1,000 


Penicillin 




> 2,000 


1,600 


2,000 


500 


Fumigacin (he 


Ivolic 










acid) 








800 




Citrlnin 




100 




100 




Pyocyanin 






100 


80 




Tyrocidine 




> 1,000 


> 1 ,000 


20 


1.25 


Tyrothricin 




> 1,000 


> 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 (752). 

Note: Figures represent maximal tolerated dose in mgm./kgm. 



CHAPTER I 3 

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 ; 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 timcj 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 different 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 247 

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 Plasm-odiofhora 
hrasskae and Synchytrmm endoh'iotkumy 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 (375), Phoma terrestrls is followed 
by Fusarium malli; in the take-all of wheat (314), 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 (313, 739) that the 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 (312a). 



248 CONTROL OF SOIL-BORNE PLANT DISEASES 

ANTAGONISM OF SOI L- I N H AB ITI NG 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 (23,310,554,696). 

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 (784). Not only the living cultures of the antagonists, 
but, in many cases, the culture filtrates are also effective (511, 992). 
The growth of H. sativum and F. graminearum upon sterilized soil 
was completely suppressed (397) 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 11 to 57 per cent by cer- 
tain cultures of Penicillium; Trichoderma reduced virulence by 50 to 
58 per cent, Absidia 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 (7B5). 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 30 
and 31). 

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 (86). 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 (617) 
to exist between the degree of infection and the protective effect of the 



SOIL MICROORGANISMS AND PLANT PATHOGENS 249 

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 (312, 398) to increase the antagonistic ac- 
tion of the soil microflora against the parasitic fungi causing cereal root 
rots. 

Various actinomycetes were shown to be antagonistic (884) to species 
of Pythiuniy a root parasite of sugar cane. The phenomenon of antago- 
nism was independent of the f¥l 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 (691 ) is 
illustrated in Figures 32 and 33 (906). Clavacin (patulin) was found 
capable of inhibiting the growth of various species of Pythium (cause of 
damping-off disease of seedlings) in dilutions of about 1 1400,000 (22), 
and of exerting a strong fungicidal action upon Ceratostomella ulmiy 
the causative agent of the Dutch elm disease j the last effect could 
partly be overcome by certain nutrients in the medium, especially 
peptone (928). 

Numerous soil microorganisms are moderately or strongly antago- 
nistic to such pathogens as Hyfochnus centrifugus, Hyfochnus sasakii, 
and Sderotium 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 (974) to be inhibited in the rotted 
tissues by the antagonistic effects of secondary organisms. In many 
cases, the rotting of fruits was suppressed by mixtures of organisms as 
compared with the pathogens j the type of rot was also modified, de- 
pending on the temperature and the specific nature of the antagonists 
(790). 

The stimulating effect of mycorrhizal fungi on the host plant has 
been explained (298) by 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. 

Certain fungi are also known (332) to be antagonistic to ants and 
their fungal symbionts. These antagonists are distributed by the insects, 
thus spreading agents that are destructive to themselves and to their 
fungus gardens. 



250 CONTROL OF SOIL-BORNE PLANT DISEASES 

GENEP.AL METHODS OF CONTROL OF 
SOIL-BORNE DISEASES 

Soil sterilization by heat and chemicals has long been practiced as a 
method of control of 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 (380, 682) that partially sterilized soil be reinoculated with a 
mixture of saprophytic microorganisms before it is used as a seedbed, 
so as to counterbalance the injurious effect of the parasites (Table 47). 

TABLE 47. 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 (149). 

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 1908 by Potter (697). 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- 
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 (304) to have a deleterious effect on the growth, in artificial 







-^T 



Figure 30. An antagonistic fungus, Trichodermay attacking 
a plant pathogenic fungus, S. rolfsiiy showing one break of a 
septum. From Weindling (963). 




Figure 31. Influence of antagonists upon the growth of Helminthosforiuni. 
Distortion of mycelium by Bacterium sp. (A) and B. rnmosus (B). C is a 
normal mycelium. From Porter (695). 



m^ m~-tr- 



\\ 



^ M^ ^ uM M M 



Figure 32. Antagonism of soil organisms against parasitism of P. 
volutum on Jgrostis. From van Luijk (906). 




r ^i» - ^,, ^ 1 i 







Figure 33. Inhibiting eflFect of sterilized liquid medium of P. exfan- 
sum versus Pyth'mm de Baryanum on lucerne. From van Luijk (906 ) . 



GENERAL METHODS OF CONTROL 251 

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 elminthosforiuni sp. (695). In a similar manner, flax 
seedlings were protected from Fusarium sp. 

A watermelon disease, caused by P hymatotrichwm omnlvoruniy was 
considerably reduced when certain specific fungi and bacteria were pres- 
ent in the soil together with the pathogen (91, lOi) j T. Ugnorum was 
observed to attack and kill the hyphae of Phymatotrichum in culture. 
The severity of the seedling blight of flax, caused by Fusarium Uni, was 
diminished when the pathogen was accompanied in the soil by various 
other fungi (875). 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 
(353). T. Ugnorum prevented infection of wheat (60) by H. sativum 
and Fusarium culmorum (Table 48). Novogrudsky (646) obtained 
protection against infection of wheat with Fusarium by inoculating the 

TABLE 48. EFFECT OF TRICHODERMA LIGNORUM ON GERMINATION AND 

GROWTH OF BARLEY INFECTED WITH HELMINTHOSPORIUM 

SATIVUM IN STERILIZED SOIL 



STRAIN OF 
H, SATIVUM 




Emerg 


ed 


PERCENTAGE OF PLANTS 

Stunted 


Contorted leaves 




H 


H + T 


H + SI 


H 


H + T 


H + Sl 


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 (127). 

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. Ugnorum; H + SI, 
seed inoculated with H. sativum and soil with T. Ugnorum. 



soil with the bacteria isolated by Chudiakov (130), provided the bac- 
teria were introduced simultaneously with the fungus qr preceded it. 
The role of microbiological antagonism in the natural control of soil- 
borne fungus diseases of plants has thus been well emphasized (85, 



252 CONTROL OF SOIL-BORNE PLANT DISEASES 

398, 783, 784). Methods for combating plant pathogenic fungi by the 
use of bacteria and other antagonists have been suggested by various in- 
vestigators (46, 503, 841). 

The principles underlying the biological control of soil-borne plant 
diseases were outlined by Garrett (312) in terms of the soil population 
in a state of dynamic equilibrium. When a given crop is grown continu- 
ously in the sam.e soil, the parasitic organisms capable of attacking the 
roots of that crop multiply (72). Organic manures stimulate the de- 
velopment of saprophytic organisms in the soil, and are thus able to 
check the activity of the pathogens, which are destroyed by the sapro- 
phytes. Either the metabolic processes of the saprophytes check the 
growth of the pathogens, or the saprophytes actually attack and destroy 
the mycelium of the pathogens. The microbiological control of plant 
diseases was said to be most effective against those organisms which have 
become highly adapted to a parasitic form of life. The pathogenic 
Ofhiobolus, when present in the form of mycelium inside the infected 
wheat stubble buried in the soil, is able to tolerate adverse physical soil 
conditions. 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 (906) 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 Trichoderma viridis and 
Absidia sfinosa, exerted an adverse influence upon Rhizoctonia {Cor- 
ticium) solanl and reduced its pathogenicity to cabbage seedlings (449). 
Broadfoot (86) and others (248), however, emphasized that the an- 
tagonism of a saprophyte to a plant pathogen, determined on artificial 
culture media, is not a reliable measure of the actual control of the para- 
site in the soil. A lack of specific microorganisms in the soil is not a suffi- 
cient factor limiting biological control under natural conditions. There- 
fore, no inoculation of soil with an antagonistic organism, such as T . 
llgnoruniy can have more than a temporary effect in changing the micro- 
biological balance of the soil population. Similar results have been ob- 



GENERAL METHODS OF CONTROL 25 3 

tained (966) in efforts to control R. solani, or the damping-off of citrus 
seedlings (Figure 34), by the use of T. lignorum, and in the action of 
B. sifnflex upon Rhizoctonia in the soil (149), 

A number of antagonistic bacteria were found (48 1 ) to be able to pre- 
vent scab formation by S. scabies on potatoes. Daines (161) found that 
T. lignorum 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 (313, 314) 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- 



254 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 (895) 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 (904) 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 5 after it has undergone 
extensive decomposition and reached a stage of slow decomposition, 
when it is usually designated as humus, it becomes comparatively inert 
(878). 

The antagonistic action of soil microorganisms has been utilized in 
several areas of the United States for the control of P. omnivorumy the 
root rot of cotton. It was shown (484-486) that this pathogen can be 
inactivated when organic manures are added to the soil before the crop- 
growing season. Eaton and King (223) demonstrated, by the use of 
the contact slide technique, that microbiological antagonism represents, 
in this case, the true mechanism of the control process; the develop- 
ment 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 (345) 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 J this was accompanied by the absence or only the sporadic 
presence of Trichoder-ma and other molds (878). The application of 
organic matter to such soils results in the destruction of most of the 
sclerotia and mycelium of the pathogen (609). Microbial antagonists 




Figure 34. Sweet-orange seedlings in nonsterilized soil. A, control; 
B, Rhi%octonia inoculated into soil layer in bottom of jar; C, Rh'i-zoc- 
tonta as in B, plus Tr'ichodcrma in top layer of peat. From Weindling 
and Fawcett (966). 



GENERAL METHODS OF CONTROL 25 5 

rather than food exhaustion were, therefore, considered to be respon- 
sible for the destruction of the pathogen. 

The Sclerotium rot of sugar beets was found (518) 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 the possibility exists that the 
suppression is 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 (911), of F. culmorum and H. sativum on wheat (398, 
695), and of species of Rhizoctonia on citrus seedlings. These patho- 
gens are markedly influenced by T. lignorum, a common soil sapro- 
phyte. A species of Trichoderma was also found to cause a reduction in 
the amount of Texas root rot of watermelons caused by P. omnivorum 
(lOi). 

The damping-off of citrus seedlings, caused by a number of fungi, 
could be suppressed by T. lignorum, which parasitizes the fungi (962, 
966). The addition of Trichoderma spores to acid sterilized soils pre- 
vented the damping-off of the seedlings. When T. lignorum was inocu- 
lated into pots containing Helminthosforium sp. and Fusarium sp., the 
pathogenic action of these organisms was checked and rendered harm- 
less to plants (60). T. lignorum was also found (17) to be decidedly 
antagonistic to Rhizoctonia and Pythium, organisms responsible for 
seed decay and damping-off of cucumbers. 

The presence of Gibherella on corn inhibited infection due to T . 
viridis; seed grains inoculated with the former gave more vigorous 
growth than uninoculated seed grains (226). 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 Sclerotinia lihertiana (S. sclerotiorum) . The utilization of 
this organism for the biological control of plant diseases has, therefore, 
been suggested (415). 

Henry (398) believed that the biological control by the soil micro- 



256 CONTROL OF SOIL-BORNE PLANT DISEASES 

flora could even be directed against internal seed infection, since appre- 
ciable damage to surface-sterilized flax seed was found to occur in steri- 
lized but not in unsterilized soil. 



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 
(127) to be insufficient to suppress the injury caused by diseased seed. 
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 ( 130) 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 (646) 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 
fungi, including species of Fusarium, Colletotrichum, and other phyto- 
pathogenic fungi. The lytic effect takes place not only in artificial me- 
dia, but also in the soil. The inoculation of sterilized soil with F. grami- 



CONTROL OF PATHOGENIC FUNGI 257 

nearum led to the inevitable death of wheat plants, but additional inocu- 
lation of the soil with lysogenic bacteria protected the wheat from the 
disease. The addition of bacteria to unsterilized soil which has been 
made sick by continuous growth of flax markedly lowered the per- 
centage of plants diseased by F . lint. 

The term "bacterization" was suggested by the Russian investigators 
(46, 130, 631, 647) 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 (455) concluded that the beneficial result of bacterization is 
due not to nitrogen-fixation by Azotobacter 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 (521) 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. 

The only important procedure which has thus far found a practical 



258 CONTROL OF SOIL-BORNE PLANT DISEASES 

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 (413). 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 (610). 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 (63) that R. solani is able to cause lOO per 
cent damping-off of radish seedlings planted at a distance of 4 cm. from 
the inoculum j 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 effect 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 called "partial disinfection." 

The possible injury caused to leguminous plants through the antago- 
nistic action of soil bacteria upon the root nodule bacteria has been but 
little investigated (495). 



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 of very recent origin. It has been known 
for more than half a century that certain microbes are capable of com- 
bating others, and it has even been suggested that saprophytic micro- 
organisms may in time be utilized for controlling the growth of patho- 
genic 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 antibiotic substances of bacterial origin, 
the antibacterial properties of which have long been known. It is true 
also of certain antibiotic substances produced by fungi, such as gliotoxin. 
The existing confusion is due largely to the fact that the utilization of 
these antibiotic substances for disease control gave results that v/ere 
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 
bound to enrich the subject of control of disease came only about half a 
decade ago. 

In 1939, Dubos announced that new antibacterial agents can be iso- 



260 THE OUTLOOK FOR THE FUTURE 

lated from soil microorganisms that are active not only m 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 enriched soil specific antagonistic 
organisms capable of destroying the pathogens, and finally isolating the 
antibiotic substances from the bacteria. This work pointed to a new 
method of approach to the isolation of antibiotic substances and attracted 
the immediate attention of bacteriologists, chemists, and medical inves- 
tigators. One no longer had to depend for the isolation of antagonistic 
organisms upon mere air contaminations 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 destroying sfecific disease-producing bac- 
teria. The mechanisms whereby such destruction was brought about 
were found to be due to the production of characteristic substances, 
known as antibiotic agents. The isolation of each required special meth- 
ods, because of its specific chemical nature. Since most of the antibiotic 
substances are selective in their action upon different 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 agents active upon gram- 
positive bacteria as well as gram-negative forms. 

It was not sufficient, however, merely to isolate these protoplasmic 
poisons. 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 antibiotic substances act in 
vivo against a variety of diseases, that branch of medical science known 
as chemotherapy acquired a new group of tools. This work led to a re- 
examination of the potentialities of penicillin, which in turn led to the 
second important contribution, namely, the work of Chain and Florey 
and their co-workers. These investigators succeeded in obtaining crude 
active preparations of penicillin, which, they demonstrated, had a 
marked effect upon various gram-positive bacteria In the animal body. 
The importance of this discovery was accentuated by the great need of 
the moment and the urgency for new methods of combating infections 
resulting from World War II. These investigations immediately at- 



THE OUTLOOK FOR THE FUTURE 261 

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 antibiotic substances. It was soon recognized that one is not deal- 
ing here with only two types of chemical compounds capable of destroy- 
ing various pathogenic bacteria and fungi, but that a new field of science 
bordering on microbiology, chemistry, and pathology was being opened 
that was bound to result in many chemotherapeutic applications. The 
fact that many of these agents, including penicillin, are produced by sev- 
eral different organisms and, further, the fact that many of these com- 
pounds are produced in different 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 antibiotic substances in 
so brief a period of time can best be illustrated by the following two 
citations : 

On May 4, 1 940, Garrod ( 3 1 5 ) , 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? " 

Less than four years later, Florey (275) 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 
acutfe infections due to sensitive organisms, 5. in the rapid curing of 
gonorrhoea including sulphonamide-resistant cases, 6. in pneumonia. 



262 . THE OUTLOOK FOR THE FUTURE 

7. probably in gas gangrene, but here numbers have been few and meth- 
ods not fully tried out."* 

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 some fifty compounds or preparations possessing bacterio- 
static and fungistatic properties have already been isolated from micro- 
organisms, there is sufficient evidence that many more can be obtained 
without too great difficulty, if enough organisms are studied in greater 
detail. In this connection, three methods of approach have been fol- 
lowed: (a) testing organisms found in culture collections for antibac- 
terial 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. notatumr-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 organ- 
isms than those now known to exist j (c) enriching the soil with specific 
bacteria, followed by the isolation of organisms capable of inhibiting the 
growth of or of destroying such bacteria. 

Several surveys (26, 282, 504, 628, 644, 934, 936, 986) have already 
been made concerning the distribution of organisms capable of produc- 
ing antibiotic substances among certain groups of bacteria and fungi. 
Only very few such organisms were selected for more detailed investi- 
gation. The reasons for this are quite obvious and are based largely 
upon the great amount of time and experimentation required for the 
isolation of any one substance. The selection of a particular substance 
was largely governed by its specific antibiotic spectrum, or its activity 
upon particular 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 has been utilized for the isolation of 

* Further information on this subject is found in the various reports listed in Chapter 12 of 
this book and in a group of papers presented before a symposium on antibiotic agents (65, 163, 
189, 399a). 



THE OUTLOOK FOR THE FUTURE 263 

tyrothricin. It is known, for example, that various strains of B. mesen- 
tericuSy B. mycoideSy B. subtiUsy and B. simplex are capable of produc- 
ing antibiotic substances, some of which are markedly different chemi- 
cally, biologically, or in selective activity. 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. aerugwosa has been utilized for the production of pyocyanase 
and pyocyanin, and C. iodinum for the production of iodinin. It is 
known, however, that a large number of other nonspore-forming bac- 
teria 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 j namely, ac- 
tinomycetin, actinomycin, streptothricin, and proactinomycin. 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 that 
have already been isolated, the wealth of material that is awaiting in- 
vestigation can only be surmised. Some of these possibilities have been 
definitely indicated. Here belong the lysozyme-like agents discussed by 
Russian investigators (507), micromonosporin which is active largely 
against gram-positive bacteria, and streptomycin (795). The latter was 
found, on the one hand, to resemble streptothricin in its chemical prop- 
erties and activity in vivo, and, on the other hand, to differ from it in its 
antibacterial spectrum. 

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 antibiotic substances, like penicillin, clavacin, and gliotoxin, are 
produced by a number of different organisms; the nature of the or- 



264 THE OUTLOOK FOR THE FUTURE 

ganism often influences not only the yield of the substance but its 
chemical nature and its biological activity. 

(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, which 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. 

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 
very few of which have proved to be of practical chemotherapeutic 
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 discovery of new chemical agents possessing anti- 
bacterial or antifungal properties offers the chemist many new models 
to draw upon for varied types of syntheses. 

Although only very few antibiotic agents have so far been isolated, 



THE OUTLOOK FOR THE FUTURE 265 

and even fewer crystallized, it is already well established that we are 
dealing here with a great variety of chemical compounds. It is sufficient 
to mention the polypeptides (tyrothricin), oxidation-reduction systems 
(pyocyanin, actinomycin), sulfur compounds (gliotoxin), quinones 
(citrinin), various other non-nitrogenous simple (clavacin) and more 
complex (fumigacin) compounds, a variety of nitrogenous compounds 
comprising both bases (streptothricin, proactinomycin) and acids (peni- 
cillin). Compounds, like actinomycin, that are highly active against bac- 
teria 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 ac- 
tive clavacin and gliotoxin. Many a chemist is awaiting the solution of 
the problem of the chemical nature of penicillin before beginning new 
syntheses. 

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. The same 
may be said of the control of plant diseases. Many practices in surgery 
and many old-time remedies are based on the creation of conditions fa- 
vorable to the development of antagonistic microbes. Consider, for ex- 
ample, the method of cast surgery developed during the Spanish Civil 
War. To what extent the application of pure cultures of antagonists 
may improve these and similar practices still remains to be determined. 
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 
been established (889). It still remains to be determined whether this 
organism exerts a favorable effect due to its antagonistic properties or 
is only another wound-infecting agent. 



266 THE OUTLOOK FOR THE FUTURE 

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. As these lines are written, a 
large number of great concerns 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 
is being made for new agents capable of inhibiting the growth of and 
destroying other pathogens resistant to the action of penicillin. 

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 (189) emphasizes 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 (560), on the other hand, believes 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 j 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 (472). The main difficulty involved is to es- 
tablish the antagonist in the soil. This can be done by modifying soil 
conditions, as by the addition of stable manure or other plant and animal 
residues, in order to favor the development of the antagonist. 

The 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 



THE OUTLOOK FOR THE FUTURE 267 

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, virus infections, and certain bacterial diseases such as 
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, he will have gained sufficient knowledge 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 antibiotic substances, namely, the mode of action 
of these substances upon bacteria. The fact that different agents vary 
greatly in their bacteriostatic and bactericidal action upon different, bac- 
teria is well established. A number of mechanisms have been pro- 
pounded, some of which hold true for one substance and some for more 
than one. Each of these mechanisms involves some extremely puzzling 
physiological problems. To take only two illustrations: 

(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 substance is often more of degree than of kind, as in the 
case of actinomycin, the assumption would be that the metabolite is 



268 THE OUTLOOK FOR THE FUTURE 

either required in different concentrations 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 antibiotic 
substances upon the bacterial cell. 

It is thus to the smallest of living systems, the microbe, 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 MICROORGANIS?vlS 

GENERAL INDEX 



CLASSIFICATION OF ANTIBIOTIC SUBSTANCES 



PRODUCED BY ACTINOMYCETES 

Actinomyces lysozyme {Streftomyces 

sp.) 
Actinomycetin (5. albus) 
Actinomycin (5. antibioticus) 



Micromonosporin {Micromonospora 

sp.) 
Proactinomycin {N. gard?ieri) 
Streptomycin (5. griseus) 
Streptothricin (5. laz'endulae) 



PRODUCED BY ALGAE 

Chlorellin {Chlorella i'p.) 



PRODUCED BY BACTERIA 

B. sim-plex factor {B. simflex) 
Diplococcin (Streptococci) 
Gramicidin {B. brevis) 
lodinin {Ch. iodinum) 
Pyocyanase {Ps. aeruginosa) 
Pyocyanin {Ps. aeruginosa) 



Subtilin (5. subtilis) 
Toxoflavin {B. cocoveftenans) 
Tyrocidine {B. brevis) 
Tyrothricin {B. brevis) 
Violacein {B. z'iolaceuni) 



PRODUCED BY FUNGI 

Aspergillic acid {A. fa-jus) 
■ Chaetomin {Ch. cochliodes) 

Citrinin {P. citrinum, A. candidus) 
*Clavacin {A. clavatus, etc.) 
*C]avatin {A. clavatus) 
*Claviformin {P. clavifornie) 
'\E. coli factor {P. notatum) 
JFIavicin {A. flavus) 
§Fumigacin {A, fumigatus) 

Fumigatin {A. fumigatus) 
iGigantic acid {A. giganteus) 

Gliotoxin {Trichoderma^ Gliocla- 
dium, A . fumigatus) 
§Helvolic acid {A. fumigatus) 



Kojic acid {A. oryzae) 
fNotatin {P. notatum) 
^Parasiticin (^4. parasiticus) 
*Patulin {P. fatulum) 
fPenatin {P. notatum) 
Penicidin {Penicillium sp.) 
Penicillic acid {P. pdberulum, 

P. cyclofium) 
Penicillin {P. notatu7n, P. chrysoge- 
ntim) 
f Penicillin B (P. 7iotatum) 
Puberulic acid {P. fuberulum) 
Spinulosin {A. sfinulosum, A. fumi- 
gatus) 



Note. Terms marked with the same symbol are synonyms. 



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. 

Antibiosis. The inhibition of growth of one organism by another. 

Antibiotic. Inhibiting the growth or the metabolic activities of bacteria 
and other microorganisms by a chemical substance of microbial origin. 

Antibiotic substance, antibiotic. A chemical substance, of microbial ori- 
gin, that possesses antibiotic properties. 

Anti-inhibitor, inhibitor, suppressor 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. 

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. 

Fungicidal. Causing the death of fungi. 

Fungistatic. Inhibiting the growth of fungi. 

Inactivator, nontoxic. A substance that inactivates plant viruses and is 
not detrimental to most forms of life. 



272 GLOSSARY 

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. 
Lyso-zyme. 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. 



BIBLIOGRAPHY 



1. Abbott, A. C, and Gildersleeve, N. A study of the proteolytic enzymes 
and of the so-called hemolysins of some of the common saprophytic bacteria. 
J. Med. Research 10:42-62 (1903). 

2. Abraham, E. P. Mode of action of chemotherapeutic agents. Lancet 2: 
761-762 (1941). 

3. Abraham, E. P., Baker, W., Chain, E., Florey, H.W., Holiday, E.R., 
and Robinson, R. Nitrogenous character of penicillin. Nature 149:356 
(1942). 

4. Abraham, E. P., and Chain, E. An enzyme from bacteria able to destroy 
penicillin. Nature 146:837 (1940). 

5. Abraham, E. P., and Chain, E. Purification of penicillin. Nature 149: 
328 (1942). 

6. Abraham, E. P., Chain, E., Baker, W., and Robinson, R. Penicilla- 
mine, a characteristic degradation product of penicillin. Nature 151:107 
(1943). 

7. Abraham, E. P., Chain, E., Fletcher, C. M., Gardner, A. D., Heat- 
ley, N. G., Jennings, M. A., and Florey, H. W. Further observations 
on penicillin. Lancet 2: 177-188 (1941). 

8. Abraham, E. P., Chain, E., and Holiday, E. R. Purification and some 
physical and chemical properties of penicillin; with a note on the spectro- 
graphic examination of penicillin preparations. Brit. J. Exper. Path. 23: 
103-120 (1942). 

9. Acs, L. Ueber echte mitogenetische Depressionen, Bakterienantagonismus 
und mitogenetische Strahlung. Zentralbl. f. Bakteriol., I, Or. 127:342— 
350(1933). 

10. Aldershoff, H. Untersuchungen in vitro uber die Art des Besredkaschen 
Antivirus. Zentralbl. f. Bakteriol., I, Or. 112:273-281 (1929). 

11. Alexandre, A., and Cacchi, R. Recherches sur quelques facteurs pro- 
bables determinant I'antagonisme entre le B. coll dans la phase "s" et le L. 
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113-134 (1940). 



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994. WoLLMAN, E. Action lytique des staphylocoques vivants sur les staphylo- 
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995. WoLLMAN, E. Recherchessur Tautolyse; lesautolysinesspecifiqucs. Compt. 
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996. WoLLMAN, E., and Reynals, F. D. Bacteriophage and autolysis. Compt. 
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INDEX OF MICROORGANISMS 



Absidia glauca, 248 

Ab. sfinosa, 252 
Achromobacter, 139 

A . lifolyticuni, qq 

A . stutzeri, 1 1 5 
Acrostalagmus, 60, 136 
Actinomyces, 40, 70, 102, 191, 224 
See also Strepomyces 

A. a/ bus, 107 

A. bovis, 72, 121 
Actinomycetaceae, 102 
Actinomycet-ales, 102 
Actinomycetes. See General Index 
A erobacter aero genes, 11, 13, 21, 34, 
44, 70, 81, 90, 94, 95> 96, 115' 
119, 152, 191, 192, 207 
Agrostis, 251 (Fig. 32) 
Algae, 6, 9, 54 
Alkali genes fecalis, no, 139 
Alternaria, 54, 60, 136, 139 

^. tenuis, 136 
Amebae, 9, 23, 146 
Anthrax organism. See General Index 
Argas, 149 
Ar mill aria, 136 
A scoria luTnbricoides, 27 
Ascomycetes, 54, 126, 138 
Aspergilli, 43, 126 
Asfergillus, 9, 48, 124, 135, 137, 142 

A. albus, 125, 135 

A. candidus, 130, 157 

A. clavatus, 124 (Fig. 12), 125, 126, 
134, 136, 157, 182, 183 

A. flavifes, 130 

A. fiavus, 67, 124, 125, 130, 131, 
132, 136, 157, 160, 181 

A. fumaricus, 126 

A. fumigatus, 67, 124 (Fig. 12), 
125, 126, 132, 133, 135, 158, 
159, 160, 183, 184, 244 

Al giganteus, 130, 132, 134, 157, 
181 



Asfergillus (cont.) 

A. nidulans, 1 30, 248 

A.niger, 51,94, 130, 136, 137, 138, 
153,186 

A. oryzae, 124, 130, 131, 185, 186 

A . -parasiticus, 130, 132, 157 

A. schiemannii, 126 

A. terreus, 126 
Azotobacter, 16, 43, 44, 109, 257 

Az. agile, 119 

Az. chroococcum, 16, 106, 119 

Az. indicum, 119 

Az. vinelandii, 1 06, 115, 119, 214 
(Fig. 26) 



Bacillus, 22, 81, 139 

B. anthracis, 52, 72, 80, 81, 83, 88, 

90, 94, 96, 97, 99, I 20, I 39, 209, 

221, 222 
B. brevis, 49, 53, 67, 68, 82, 83, 87, 

158, 159, 160, 167, 190, 262 
B. cereus, 13, 22, 60, 81, 94, 115, 

119, 192 
B. lentimorbus, 149 
B. macerans, 115, 119 
B. megatherium, 81, 82, 94, 106, 

115, 119, 120, 191, 192 
B. mesentericus, 22, 49, 53, 82, 83, 

84, 85, 86, 87, 106, 115, 139, 

140, 163, 164, 165, 187, 213, 

241, 263 
B. m.esentericus-vulgatus, 83, 85 
B. mucosus-cafsulatus, 80 
B. mycoides, 53, 59 (Fig. 5), 60, 70, 

82, 83, 84, 85, 94, 106, 108, 1 10, 

115, 117, 119, 126, 134, 139, 

140, 159, 191, 192, 195, 213, 

214, 219, 263 
B. mycoides-cytoliticus, 85, 86 
B. fetasites, 22 



332 



INDEX OF MICROORGANISMS 



Bacillus (cont.) 

B. folymyxa, 115, 119 

B. fofilliae, 149 

B. futrificus verrucosus, 1 00 

B. fyocyaneus. See Ps. aerugniosa 

B. ramosus, 250 (Fig. 31) 

B. simflex, 87, 139, 140, 157, 160, 
170, 253, 263 

B. subtilis, 22, 59, 60, 61, 69, 70, 
7i>73,74>75,8i,82, 83,84, 85, 
94, 106, 1 15, 1 16, 1 19, 126, 133, 
134, 139. 152, i59> i74> 181, 
187, 191, 192, 195, 205, 208, 
2i4(Fig. 26),2i5 (Fig. 27), 263 

B. suifestifer, 8 3 

B. thermofhilus, 8 3 

B. tumescens, 22, 106 

B. vulgatus, 94 
Bacterium, 139, 148, 250 (Fig. 31) 

B. acidi lacticiy 99 

B. aroideae, 146 

B. cazaubon, 148 

B. cocovenenans, 171 

B. efhestiae, 148 

B. gelechiae, 148 

B. lactis aeroge?ies, 99 

B. fyocyaneum. See Ps. aeruginosa 

B. solanacearum, 121 

B. termo, 223 

B. tyrogenes, 82 

B. violaceum, 5 i 
Bacteroides, 21 
Basidiomycetes, 54, 124 
Basisporium, 139 

B. gallarum, 1 3 8 
Beauveria, 139 

Blastomycoides der77ia(itidis, i 3 8 
Boofhilus bovis, 1 7 
Botrytis, 136 

5. tf//«, 136 

5. cinerea, 136 
Botulinus organism, 29 
Brucella, lOO, 202 

5r. abortus, 59, 69, 72, lOO, 115, 
119, 191, 215, 218, 243 



Brucella (cont.) 

Br. melitensis, 36, 72, 90, 98, 1 00, 

215 
Br. suis, 100 



C ef halo s for ium, 9, i 26 
CefhalotheciuTTi roseum, 136, 139 
Ceratostomella, 142 

C. «/w/, 78, 141, 249 
Cercomonas, 146 
Chaetomium, 125, 126, 185 

C-^. cochliodes, 130, 157, 185 
Chi or el la, 54, 157 
Chroniobacterium, 1 6 3 

CA. iodinum, 158, 165, 263 

CA. violaceum, 81, 94, 159 
Citromyces, 136 
Cladosforium, 9 
Clostridium acetohutylicum, 44 

C/. botulinum, 100, 226 

C/. butyricum, 100, 119, 219 

C/. chauvoei, 29, 89, 93 

C/. fall ax, 2 3 

C/. granulobacter-fectinovorum, 44 

C/. oedematiens, lOO, 230, 236 

C/. oedematis, 23, 72 

C/. ferfringens, lOO, 230 

C/. sefticum, 23, 72, 128, 230, 231 

C/. sforo genes, 90, lOO, 225, 226 

C/. tetani, 29, 72 

C/. welchii, 23, 72, lOO, 115, 151, 
191, 214, 215, 230 
Colletotrichum, 136, 256 

C. gloeosfcn'oides, i i 3 
Colfidium, 23 
Colfoda, 146 
Coniofhora cerebella, 137 
Corticium rolfsii, 255 

C sadakii, 255 
Corynebacteria, 98, 233 
CorynebacteriuTn, 106 

C. difhtheriae, ']!, 83, 86, 89, 90, 
92, 96, 198, 223, 238, 241 
Cryftochilum nigricans, 23 



INDEX OF MICROORGANISMS 



333 



Cunningha?nel la, 44, 45 
C. elegans, 136 



Dematiaceae, 137 
Dematium, 136 
Deuterofhoma, 136 
Dictyostileum discoideufn, 146 

D. mucoroides, 50 
Diplococci, 89, 90 
Diflococcus fneumojiiae, 120, 203, 

229, 238 
Dothiorella, 139 
Dysentery bacteria, 29-30 



Eberthella, 81 
E. typhi, 94 

E. typhosa, 32, 33, 34, 50, 61, 61 
69, 80, 81, 83, 85, 86, 89, 9O; 

95, 96, 98, 99, 107, 158, 185 

209, 223, 225 
Entamoeba histolytica, 27 
Erzvinia carotovora, 1 1 5 
Escherichia coli, 11, 21, 31, 32, 33, 34, 

44,47^50, 59> 61, 69, 71,72, 80 
81, 83, 84, 86, 89, 90, 92, 94, 95 

96, 97, 99, 100, 106, 107, 108 
115, 116, 119, 120, 125, 126 
i27> 133, 134, i74> i75> 181 
182, 191, 195, 196, 207, 208 

210, 211, 215, 217, 225, 236 
244 



FlavobacteriuTTi, 21, 34 

Fluorescent bacteria, 88-89, 93~95 

Friedlander's bacillus, 87 

Fungi Imperfecti, 54 

Fusarium, 9, 40, 60, 63, 105, 114, I 26, 
136, 138, 139, 146, 251, 255, 
256, 264 
Ff. conglutinans, 140 
F. culmorum, 247, 251, 255 
F. grarninearum, 248, 256 



Fusarium (cont.) 
F. lateritium, i 36 
F. lini, 251, 257 
F. main, 247 
F. moniliforme, i 3 8 
F. oxys forum cubense, 1 1 4 
F. sambucinum, 138 (Fig. 15) 
F. vasinfectum,, 137 



Gajfkya tetragena, 1 1 5 

Gambusia, 89 

Gas-gangrene organisms, 28-29, 131, 

230 
Gibber ella, 255 
Gleosforium, 60 
Gliocladium, 125, 126, 134, 136, 138, 

158, 184 
Gonococcus, 125, 226 
Gy?nnoascus, 134 



Helminthosforium, 54, 63, 136, 139, 
140, 250 (Fig. 31), 251, 255 

H. sativum, 88, 136, 137, 140, 248, 
251, 255, 256, 257 

H. teres, 136 
Hemophilus, 191, 202 

H . influenzae, 119 

H . pertussis, 1 1 5 

H. suis, 1 1 9 
Heterodera marioni, 150 
Hyphomycetes, 137 
Hypochnus centrifugus, 249 

H . sasakii, 249 



Klebsiella pneumoniae, 72, 90, 94, 99, 
207 



Lactobacillus acidophilus, 98, 1 00, 125 
L. bulgaricus, 52, 90, 98, 99, 1 00 
L. casei, 1 1 9 



334 



INDEX OF MICROORGANISMS 



Leish-mania, ij[.j 
Leftospira, 147 

L. icterohaemorrhagiaey 72 
Lucilia sericata^ i 5 i 



Macrofhomina fhaseoli, 138 
Melanosfora famfeana, 137 
Meningococci, 91, 127, 219, 239 
Metiingococcus, 226 
Micrococci, 89, 90, 98, 108 
Micrococcus, 33 

M. antibioticus , 98 

M. candicans, 21, 22, 106 

M. catarrhalis, 226 

M. ficwus, 94, 227 

M. luteus, 22, 106 

M. lysodeikticus, 61, 69, 70, 106, 
119, 195 

M. roseus, 106 

M. ruber, 106 

M. tetragenes, 98 
Micromonosfora, 102, 104, 105, iii, 
112, 158 

M. vulgaris, 102 (Fig. 10) 
Monascus, 136 
Monilia, 136 

M. albicans, 1 3 5 

M. jnictigena, 255 
Mucor, 9, 136 
Mucorales, 135 
Mycobacteriaceae, 102 
Mycobacterium, 102 

Af. citreum, 106 

Af. /"/^/^Z, 1 06, 119 

M. smegmae, 106 

M. tuberculosis, 23, 34, 35, 72, 83, 
85, 89, 90, 92, 96, 98, 99, 100, 

106, 107, 115, 124, 125, 133, 

139, 209, 227, 244 
My CO gone, 138 
Myxobacteria, 90 
Myxobacteriales, 99 
Myxobacterium, 60, 139 



'Neisseria, 81, 125, 191 

A'^. catarrhalis, 94, 115, 203 

N . flavus, 203 

N. gonorrhaeae, 72, 203, 233 

iV. intracellular, 203 

A'^. meningitiiis, 72, 120 
Neoaflectana glaseri, 148 
Nocardia, 102, 104, 108, i 1 1 

A'', ^/i-^, 1 06 

A'^, corallina, 1 06 

A'^. gardneri, 105, 112, 114, 120, 

I59> 175 
A'^. rubra, 106 



Oidium, 22 
Oikomonas, 146 

O. termo, 146 
Ofhiobolus, 40, 136, 139, 252 

O. graminis, 137, 247, 248, 253 

O. miyabeanus, 138 



Paratyphoid bacteria, 86, 90 
Pasteur ell a, 191 
F. avicida, 90 

?. />^j/w, 72, 86, 90, 91, 97, 99, 149 
P. fseudotuberculosis, 1 1 9 
Pectinofhora gossyfiella, 148 
Penicillium, 9,63, 124, 125, 130, 131, 

i35> 136, 137, 139. 142, 146, 

158, 185, 248 
P. africanum, 5 I 
P, chrysogenum, i 24 (Fig. 12), 125, 

126, 127, 130, 158, 159, 175, 

206, 262, 266 
P. citreo-roseum, i 30 
P. citrinum, 124 (Fig. 12), 125, 

157, 181 
P. claviforme, 124, 125, 126, 134, 

157, 182, 183 
P. cyclofium, 124, 125, 158 
P. expansum, 126, 134, 157, 182, 

251 (Fig. 33) 
P. funiculosum, i 26 



INDEX OF MICROORGANISMS 



335 



Pe/iicillium (cont.) 

P. luteum, 51, 126, 136 

P. luteum- fur fur ogenum, 126, 137 

P. notatum, 56, 67, 124 (Fig. 12), 
125, 126, 127, 128, 129, 130, 
158, 159, 160, 175, 176, 179, 
180, 195, 197, 206, 233, 237, 
259, 262, 266 

P. fatulum, 134, 157, 182 

P. fuberulum, 125, 158, 159, 181, 
185 

P. resticulosum, i 2 5 

P. sfinulosum, i 5 9 
Pestalozzia, 63 
Peziza, 1 3 5, 136 

P. sclerotiorum, 1 3 6 

P. trifoliorum, 136 
Pfeijferella mallei, 89 
Phoma terrestris, 247 
Phycomycetes, 54, 124, 126, 137 
Phymatotrichum, 2 5 i 

Ph. omnivorum, 251, 254, 255 
Pkytomonas, 81 

Ph. bozvlesii, 94 

Ph. tumefaciens, 90, 98 
Phytofhthora, 136, 139, 140 

Ph. cactorum, 249 

Ph. erythroseftica, 1 26 
Piftocefhalis, 137 
Plasmodiofhora brassicae, 247 
Plasmodium, 147 

Pneumococci, 90, 91, 98, 99, 105, 198, 
201, 204, 207, 216, 218, 219, 
239, 241, 243 
Pneumococcus, 72, 76, 86 
Polytoma uvella, 23 
Proteus vulgaris, 22, 72, 80, 85, 86, 92, 

99, 100, 106, 139, 140,236 
Psalliota camfestris, 138 
Pseuioeurotium zonatum, 138 (Fig. 

15) 

P seudomonas , 22, 33, 80 

Ps. aeruginosa, 33, 44, 49, 52, 67, 
72, 81, 86, 88, 89, 91, 92, 94, 
104, 105, 106, 107, 115, 117, 



120, 139, 159, 161, 162, 163, 
190, 221, 222, 223, 236, 263, 
265 

Ps. aviseftica, 99 

Ps. citri, 146, 251 

Ps. destructans, 250 

Ps. fuorescens, 10, 13, 22, 44, 45, 
53, 80, 81, 88, 89, 91, 94, 97, 
106, 115, 119, 121, 140, 141, 
191 

Ps. hyacinthi, 146 

Ps. juglcmdis, 139 

Ps. fhaseoli, 139 

Ps. futida, 22, 94 

Ps. fyocyaneus. See Ps. aeruginosa 

Ps. translucens, 139 
Pyronema, 16 

P. conjiuens, 141 
Pythium, 105, 136, 137, 139, 249, 255 

P. de Baryanum, 251 (Fig. 33) 

P. volutum, 251 (Fig. 32) 



Radiobacter, 106 
Rhizobium, 46, 109 

Rh. legU7ninosarum, 106 
Rh'^zoctonia, 40, 136, 139, 140, 185, 
215, 253, 254 (Fig. 34), 255, 
257,258 

R. solani, 87, 138, 252, 253, 258 
Rhizofus, 9, 44, 45, 48, 142 
Rhodococcus, 81 

R. cinnebareus, 94 

R. roseus, 94 



Saccharomyces, 139 

Sac. cereviseae, 91, 139 
Sac. ellifsoideus, 94 
Sac. marianus, 94 
Sac. fastorianus, 94 

Salmonella, 81, 192 
5. ahortivoequina, 119 
5. aertrycke, 191 
S. cholerasuis, 119, 192 



336 



INDEX OF MICROORGANISMS 



Sdmofiella (cont.) 
S. enteritidis, 2 2, 94 
S. gartnert, "jl 

S. faratypki, 72, 90, 97, 120 
S. fidlorum, 94 

5. schottmulleri, 96, 119, 191, 192 
5. suifestifer, 94 
S.tyfhi, 72, 73, 120 
5. tyfhimurium, 72, 119 
Sarcina, 21, 33, 99 
S. flava, 22 

5. /i^/-^^, 50, 59 (Fig, 5), 61, 69, 70, 
83, 94, 104, 106, 115, 119, 126, 
140, 151, 191, 192, 195 
S. ureae, 1 40 
Schrottnia, 60, 139 
5. arnericana, 138 
5. libertiana, 138, 255 
Sclerotium, 255 

5. oryzae sativae, 249 
S.rolfsii, 136, 137, 250 (Fig. 30) 
Serratia, 36, 93, 95 

S. marcescens, 53, 80, 81, 82, 88, 89, 
91, 93, 94, 104, 106, 115, 119, 
139, 140, 191 
Shiga bacillus, 30, 97 
Shigella, 86, 120 
5h. dysenteriae, 72 
Sh. gallinarum, 115, 119, 191 
Sh. faradysenteriae, 94 
Sficaria fur fur genes, 5 1 
Sforotrichum, 17 

Staphylococci, 82, 85, 89, 90, 91, 98, 
99, 105, 115, 200, 201, 202, 203, 
220, 224, 225, 227, 233, 235, 
236, 243 
Stafhylococcus, 182, 228 

5. albus, 22, 86, 92, 94, 96, 204, 228 
S. aureus, 59, 60, 69, 71, 72, 73, 75, 
76, 80, 86, 92, 94, 96, 100, 104, 
106, III, 120, 128, 133, 162, 
175, 181, 182, 191, 192, 202, 
203, 206, 207, 214 (Fig. 26), 
215, 216, 227, 229, 231, 233, 
235, 238 



Staphylococcus (cont.) 
S. citreus, 86, 94 
5. muscae, 119 
5. viridis, 86 
Sterigmatocystis, 136 
Stomoxys, 149 

Streptococci, 23, 72, 90, 91, 98, 105, 
107, 115, 157, 198, 201, 218, 
220, 224, 225, 227, 229, 233, 
235, 236, 237, 238, 239, 243 
Streftococcus agalactiae, 242 
5. cremoris, 98, 157 
S. dysgalactiae, 242 
S. enteritidis, 207 
5. faecal is, 203 

S. hemolyticus, 71, 86, 92, 165, 192, 
196, 204, 206, 216, 218, 232, 
238 
5. lactis, 90, 98, 128 
S. mastidis, 98 
5. viucosus, 86, 98 
5. pyogenes, 21, 22, 72, 77, 97, 104, 

120, 128, 203, 207, 228, 231, 

239 
5. salivarius, 203 
5. sefticemiae, 2 1 
5. therniophilus, lOO 
5. uberis, l^l 

S. viridans, 61, 72, 73, 128 
Strepto?nyces, 44, 45, 102 (Fig. 10), 

104, 105, 108, III, 112, 113, 

121, 214, 253 

5. albus, 105, 112, 113, 114, 119, 

120, 157 

S. albus, var. ochraleuceus, 1 1 2 
S. annul atus, 1 1 2 

5'. antibioticus, 59 (Fig. 5), 67, 1 02 
(Fig. 10), 105, 112, 114, 116, 

121, 157, 160, 171 
5. aurantlacus, 106 

5". aureus, i i 2 

5. bovis, I I 2 

5. californicus, 112, 115 

S. candidus, i i 2 

5. cellulosae, 112, 115 



INDEX OF MICROORGANISMS 



337 



Streptomyces (cont.) 






Tric/wdermay 9, 44, 45, 69, 125, 126, 


5. coelicolor, 1 1 3 






134, 138, 142, 184, 185, 215, 


5. cretaceus, 1 1 2 






248, 250 (Fig. 30), 253, 254 


5". f radii, 1 1 2 






(Fig. 34), 255 


S. globisforusy 106 






T. lignoruniy 136, 138 (Fig. 15), 


5. griseus, 106, 112, 113, 


114, 


^ ' 7> 


251, 252, 253, 255, 256 


159 






T. viridisy 252, 255 


5. halstedii, 1 1 3 






TrichomastriCy 23 


S. hominisy 1 1 3 






Trichomonas y 23 


S. lavendulae, 102 (Fig. 


10), 


105, 


Trie hot hecium roseum, 2 5 i 


112, 114, 116, 117, 


119, 


159. 


Tryfanosomdy 147 


i73> 174 






T. equiferduniy 227 


5. lifmaniiy 1 1 3 






Tylenchus Priticiy 150 


5. microflavus, 1 1 3 






Typhoid bacteria. See General Index 


S. odorifer, 1 1 3 






Tyrothrixy 82 


S. fraecoxy 105, 1 13, 121, 


, 122 






5. reticuliy 1 1 2 








5. roseusy 112 






Us til a go, 139 


5. rz^^r, 1 1 2 






U. avenae, 140 


5. rutgersensisy 1 1 3 






U. hordei, 140 


5. samfsoniiy 1 1 3 






C/. ««</(7, I 40 


5. safrophiticuSy 112 






U. reae, 1 40 


S. scabies y 105, 1 1 2, 113, 


121, 


122, 


U. zeae, 60, 136 


253 








5. setoniiy 1 1 3 








5. tetanusemuSy 1 1 3 






Verticillium, 136 


5. violaceusy 106, 1 10 






F. dahliaCy 146 


5. violaceus-ruber, 1 1 9 






Fi^no commay 36, 50, 72, 80, 81, 82, 


Streftomycetaecae-y 102 






83, 84, 85, 89, 90, 91, 94, 97, 


Streftothrix, 104 






98, 162 


Synchitrium endobioticuniy 7 


^47 




F. metchnikoviy 91, 92 



Tetramitus rostratuSy 2 3 
Thamnidium eleganSy 136 
Torula sfhaericCy 94 

r. suganiiy 136, 138, 185 
TorulofsiSy 136, 137 
Treponema palliday 234 



Y-bacillus, 86, 92 
Yeasts, ^^i? General Index 



ZygorhynchuSy 60 
Zygorsaccharomyces priorianuSy 94 



GENERAL INDEX 

See also Index of Microorganisms, page 331 



Acridine, 164, 230 
Acriflavine, 196 
Actinomyces lysozyme, 270 
Actinomycetes, 102-103 

antagonistic properties of, 1 04-1 14 
as antagonists, 102-123 

against actinomycetes, I 20-1 2 1 
against agents producing plant dis- 
eases, I 21-122 
against bacteria, 106, 108, 1 1 1- 

113, 120 
against fungi, 54, 11 3-1 14, 139, 
141-142 
in soil, 6, 8, 9, 109-1 12 
substances produced by, 54, 11 4-1 21, 
171-175 
in vivo activity of, 122-123 
Actinomycetin, 1 07-1 08, 114, 1 20, 
263, 270 
chemical and biological properties, 

157, 160, 161, 171 
effect on bacteria, 108 
effect on cells, 120, 199 
therapeutic value, 123 
toxicity, 161 
Actinomycin, 114-115, 270 
antiluminescent activity, 77 
chemical nature, 53, 157, 1 71-173 
compared with chemical substances, 

70 
compared with penicillin, streptothri- 

cin, and clavacin, 1 90-1 91 
compared with proactinomycin, i 20 
compared with tyrothricin, 206 
crystals. Fig. 18 (p. 1 70) 
differentiation of bacteria by, 220 
effect on bacteria. Fig. 5 (p. 59), 77, 
171, 206-207, 209-210, 211 
by species, 70, 115, 191 
effect on cells, 195 
effect on fungi, 78, 141 
pffect on soil bacteria, 21, 22 
in vivo activity, 122 
medium, 1 16 



Actinomycin {cont.) 

neutralizing agent, 219 
toxicity, 190, 206, 245 
Actinomycin A, 67, 160. See also Ac- 
tinomycin 
Actinomycin B, 67, 160, 171 
Actinomycosis, 28, 225 
Adaptive enzymes, 166 
Aerobic bacteria as antagonists, 99— lOO 
Agar diffusion method of measuring 

antibiotic activity, 73-75 
Agar method for testing antagonistic ac- 
tion, 64 
Agar plate-dilution method of measur- 
ing antibiotic activity, 69, 71 
Alfalfa, decomposition by microorgan- 
isms, 44-45 
Alfalfa-sick soils, 1 8 
Algae, 6, 9, 54 
Amebae, 9, 23, 146 
Anaerobic bacteria, 22, lOO 
Anaxogramic method of testing action 

of antagonists, 62 
Animal excreta. See Human and ani- 
mal wastes 
Animal pathogens, survival of, 28-32 
Animals, microscopic 
as antagonists, 145-146 
in soil, 5, 6 
Antagonism, 14, 54 
defined, 271 
types, 47, 49 
Antagonist, defined, 271 
Antagonistic action, 51-54, Fig. 5 (p. 

59) . . 

Antagonistic interrelationships among 

microorganisms, 38-41, 46-51 
Antagonistic microorganisms 

isolation and cultivation, 55-64 
production of antibiotic substances, 
51-54,64-79 
isolation and utilization of sub- 
stances, 78-79 



340 



GENERAL INDEX 



Antagonistic microorganisms (coni.) 
production of antibiotic substances 
(cont.) 
measurement of activity of sub- 
stances, 66- J J 
measurement of bactericidal action 

of substances, 77-78 
measurement of in vivo activity of 

substances, 78 
methods of growing organisms, 
64-66 
utilization for disease control, 223- 

226 
See also Actlnomycetes; Animals, mi- 
croscopic; Bacteria; Fungi 
Antagonistic substance, defined, 271 
Anthrax, 93, lOO 

survival of organism in soil, 18, 28, 

96 
treatment, 221, 222, 223 
use of organism in treating diseases 
in man, 224 
Antibacterial action, inhibition of, 197- 

199 
Antibiosis, 38-41, 271 
Antibiotic, defined, 271 
Antibiotic spectrum, defined, 271 
Antibiotic substances 

as means of differentiation of bac- 
teria, 219-220 
chemical nature. See Chemical na- 
ture of antibiotic substances 
classification, i 56-1 6 1 
compared with chemical antiseptics, 

189-195 
defined, 271 
Inhibition, 218-219 
mode of action, 67, 189-199, 212- 

217, 267, 268 
produced by actlnomycetes, 54, 114- 

121, 171-175 
produced by bacteria, 161-171 
produced by fungi, 175-185 
produced by yeasts, 63, 185-186, 

215 
production, 64-66 
properties, 1 89-1 90 
structural formulae, 164 
See also name of sfecific substance 
Anti-inhibitors, 198, 271 



Antiluminescent test for measuring anti- 
biotic activity, 76, 77 

Antiseptic snuff, penicillin in, 233 

Antiseptics, 189-195, 261 

Antivirus, 224 

Ants, fungi antagonistic to, 249 

Aromatic oils, as bacteriostatic agents, 
165 

Ascaris, 27 

Ascorbic acid, effect on actinomycin, 

219 

Aspergillic acid, 270 

antiluminescent activity, 77 
chemical nature, 53, 157, 160, 181 
effect on bacteria, 77, 125, 131 
in experimental infection with CI. 

ferfringens, 230 
toxicity, 161 

Aspergillin, 65, 157, 181 

Assay value, 74 

Associative interrelationships among mi- 
croorganisms, 42-45 

Autoantibiosis, 41 

Autolysis, 2 1 2 

Autophage, 96 

Autotoxins, 53 



Bacillus Tnesentericus filtrate, bacteri- 
cidal action of, 241 
Bacillus simplex izctor, 157, 160, 1 70, 

270 
Bacteria 

aerobic and anaerobic, 9, 99-1 01 
agents destructive to, 165, 207-213, 
215-217 
actlnomycetes, 106, 108, 1 1 1- 

113, 120 
insects, 1 49-1 50 
fungi, 124-135 
maggots, 151 
protozoa, 23, 143-146 
substance found in milk, 188 
substance produced by yeast, 215 
ticks, 149-150 
as antagonists, 80-101 

against agents producing plant dis- 
eases, 138 
against bacteria, 82-87, 88, 89-90 



GENERAL INDEX 



341 



Bacteria {cont.) 

as antagonists (coni.) 

against fungi, 63, 87-88, 139- 

142 
against insects, 148-149 
against protozoa, 143 
against viruses, i 5 2 
cocci, 97-99 

colon-typhoid group, 32-34, 95-97 
differentiation by means of anti- 
biotic substances, 219—220 
effect on tumors, 154-155 
fluorescent bacteria, 88-89, 93~95 
in fecal matter, 20-23, 84 
in soil, 6, 8, 9, 1 1, 247 
lysis of, 78, 82, 86, 200 
nonspore-forming bacteria, 88-99 
spore- forming bacteria, 82-88 
substances produced by, 161-171 
survival in soil, 28-36 
Bacterial agar plate method of isolating 
antagonistic microorganisms, 57- 
58 
Bacterial cell division, interference by 

antibiotic substances, 196 
Bactericidal, defined, 271 
Bactericidal action, methods of measur- 
ing, 77-7 8_ 
Bacterioantagonistic, defined, 271 
Bacteriolytic, defined, 271 
Bacteriophage, 96, 152, 153, 154, 201 
different from lysozyme, i 86 
relation to antibiotics, 2 1 3 
Bacteriostatic, defined, 271 
Bacteriostatic action, 66, 67 

inhibition of, 218-219 
Bacteriostatic and bactericidal agents. 
See Bacteria, agents destructive to 
Bacteriostatic spectrum, defined, 271 
Bacteriotherapy, 221, 225 
Bacterization, 257 
Barley infection, 251 
Biological conditioning, 196 
Biostatic complex, defined, 271 
Blackleg organism in soil, 18, 28, 29 
Blood cells, hemolysis of 

as test of antibiotic activity of tyroth- 

ricin, 76 
ty gramicidin, 239 
by tyrocidine, 239, 240 



Blood cells, hemolysis of {co?it.) 

by tyrothricin, 200-201, 239, 240 

Blood serum extract, inhibition of 
gramicidin by, 218-219 

Botulinus, 29, 226 

Bovine mastitis 

survival of organism, 30 
treatment with clavacin, 237 
treatment with gramicidin, 241-242 

Brucella organism, survival of, 36 

Bubonic plague organism, survival of, 
30 



Caecal bacteria, 149 
Cancer, rectal, and E. coli, 97 
Cationic detergent, 202 
Cattle tick, relation to Texas fever, i 7 
Cephalin, 218-219 

Chaetomin, 125, 157, 160, 185, 270 
Chemical composition of soils, 4-5 
Chemical nature of antibiotic sub- 
stances, 156-188 

classification of substances, 1 56-161 

microbial lysozyme, 186-187 

substances produced by actinomycetes, 
171-175 

substances produced by bacteria, 161- 
171 

substances produced by fungi, 175- 
185 

substances produced by yeasts, 185- 
186 
Chemist, problems for, 264-265 
Chemotherapy, 259, 261, 265-267 
Chlorellin, 54, 157, 270 
Chlororaphin, 160, 163, 164 
Cholera bacteria, survival of, 30, 36 
Citrinin, 130, 270 

chemical nature, 53, 157, 160, 161, 
181 

crystals, Fig. 18 (p. 1 70) 

effect on bacteria, 125, 131, 207 

structural formula, 164 

toxicity, 245 
Citrus canker, 251 
Clavacin, 182-183, 270 

antiluminescent activity, 77 

chemical nature, 157, 161 

compared with fumigacin, 192 



342 



GENERAL INDEX 



Clavacin (conf.) 

compared with penicillin, strepto- 
thricin, and actinomycin, 1 90-1 91 
effect on bacteria, 77, 125, 134, 161, 
195, 207, 208 
by species, 190, 191, 192 
effect on fungi, 141, 249 
production, 127, 134, 263-264 
structural formula, 164 
therapeutic value, 237 
toxicity, 245 
Clavatin, 134, 157, 270 
Claviformin, 125, 130, 134, 157, 183, 

270 
Clover-sick soils, i 8 
Cocci as antagonists, 97-99 
Coccidiosis organism, survival in soil, i 8 
Colds 

treatment with clavacin, 237 
treatment with penicillin, 233 
Collodion sac method of testing antago- 
nistic action, 62 
Colon-typhoid bacteria 
as antagonists, 95-97 
in manure, 21, 22 
in soil, 1 1, 32-34 
Competition among microorganisms, 

45-46 
Corylophilline, 158 
Cotton root rot, control of, 254 
Crowded plate method of isolating an- 
tagonistic microorganisms, 58 
Cultivation of antagonistic microorgan- 
isms, methods, 64-66 
Cup method of measuring antibiotic ac- 
tivity, 73-75 
Cytolytic bacteria, 86 



Damping-off disease, 28, 249, 250, 
255, 258 

Definitions of terms, 271-272 

Dehydrogenases, 76, 199, 211, 212, 
216 

Dermatitis, chronic, treatment with 
penicillin, 233 

Differentiation of bacteria by means 
of antibiotic substances, 219-220 

Dilution method of measuring anti- 
biotic activity, 69—73 



Diphtheria 

neutralization of toxin, 223 
survival of organism, 30 
treatment of carriers with filtrate of 
B. meseniericus, 241 
Diplococcin, 157, 270 
Direct antagonism, 47, 49 
Direct microscopic method of determin- 
ing abundance of microorganisms 
in soil, 7 
Direct soil inoculation method of iso- 
lating antagonistic microorganisms, 
58-59 
Disease control, 221-245 

of fecal-borne diseases in China, 27 
of plant diseases. See under Plants 
of soil-borne diseases, 14-15, 246- 

toxicity of antibiotic substances, 245 

use of antibiotic substances, 226-245 

use of microbial antagonists, 223- 

226 

Disinfectant spectrum, 193-194 

Disinfectants, chemical, compared with 

antibiotic substances, 189-195 
Double plate method of testing antago- 
nistic action, 62, 64 
Dutch elm disease, 141, 142, 249 
Dyes as bacteriostatic agents, 165 
Dysentery bacteria, survival of, 29-30 



E. coli 

and rectal cancer, 97 
antagonists of, 33-34 
influence of enrichment of soil with 

organism, 31 
survival of organism, 31, 32-34 
E. coli factor, 125, 130, 158, 270 
Enzyme action, inhibition by tyrothri- 

cin, 199-200 
Enzymes acting on polysaccharides, 1 60, 

165-167, 197, 216 
Equilibrium among soil microorgan- 
isms, 40 
Eye lesions, treatment with penicillin, 
233 



Fecal-borne diseases, control of, 27 



GENERAL INDEX 



343 



Fecal residues. See Human and animal 

wastes 
Fertilizer 

effect on microbial population, 8, 27 

use for disease control, 255 
Filter method of testing antagonistic 

action, 61-62 
Flavatin, 77, 157, i8l 
Flavicidin, 157, 181 
Flavicin, 67, 132, 135, 179, 181, 270 

chemical nature, 157, 160 

effect on bacteria, 125 

toxicity, 161 
Flax blight, 251 
Flax-sick soils, 1 40 
Fluorescent bacteria as antagonists, 88- 

89> 93-95 
Fluorescin, 162 

"Forced antagonism" method of isolat- 
ing antagonistic microorganisms, 
56, 59, 186 
Formulae, structural, of antibiotic sub- 
stances, 164 
Freudenreich's method of testing an- 
tagonistic action, 61 
Fumigacin, 67, 183-184, 270 
antiluminescent activity, 77 
chemical nature, 133, 158, 1 60 
compared with clavacin, 192 
crystals, 133, 158, Fig. 18 (p. 170) 
effect on bacteria, 77, 125, 134, 161, 
207 
by species, 133, 192 
effect on fungi, 141 
inactivation, 158 
medium, 135 
toxicity, 245 
Fumigatin, 67, 132-133, 182, 270 
chemical nature, 53, 133, 158, 1 60, 

161 
effect on bacteria, 125, 133 
structural formula, 164 
Fungi 

agents destructive to, 78, 137, 141, 
142 
actinomycetes, 54, 11 3-1 14, 139, 
141-142 
^ bacteria, 63, 87-88, 139-142 
substance produced by yeast, 63, 
215 



Fungi {cont.) 

as antagonists, 124-142 

against agents producing plant dis- 
eases, I 38 
against bacteria, 124-135 
against fungi, 63, 135-138 
against insects and other animal 
forms, 142, 148-150 
in fecal matter, 21, 23 
in soil, I, 6, 8, 9-10, 137, 247 
lysis of, 140 
pathogenic to plants, control of, 256- 

258 
relation to protozoa, 1 46 
substances produced by, 175-185 
Fungicidal, defined, 271 
Fungistatic, defined, 271 
Fungus infections 
of animals, 17 
of human skin, 243 



Gangrene, 28-29, 131, 225, 230 

Garbage disposal, 26 

Gigantic acid, 132, 157, 181, 270 

Gliotoxin, 69, 135, 184-185, 270 
antiluminescent activity, 77 
chemical nature, 53, 133, 158, 160 
compared with chemical substances, 

70 
crystals, 133, Fig. 18 (p. i 70) 
effect on bacteria, 77, 125, 195, 207 

by species, 70, 133 
effect on fungi, 138, 141 
medium, i 35 
toxicity, 245 

Glucose-dehydrogenase, 206 

Glucose-oxidase, 1 79, 197 

Gonorrhea, treatment with penicillin, 

233-234 
Gramicidin, 1 67-1 70, 270 
antiluminescent activity, 77 
chemical nature, 53, 158, 160, 161, 

216 
compared with chemical substances, 

70, 189 
compared with penicillin, 192, 203 
compared with tyrocidine, 192 
crystals. Fig. 18 (p. 170) 



344 



GENERAL INDEX 



Gramicidin (cont.) 

eflfect on bacteria, 77, 192, 195, 199- 
202, 206, 211, 216 
by species, 70, 203 
inhibition of, 198, 218-219 
mode of action, 196, 216 
therapeutic value, 239, 241-243 
toxicity, 239, 241, 245 
Gramidinic acid, 77 
Gram stain and sensitivity to antibiotic 
substances, 115, 157-159, 191, 
195 



Helvolic acid, 133, 158, 184, 245, 270 
Hemipyocyanin, 53, 78, 141, 160, 162, 

190 
Hemolysin production method of meas- 
uring antibiotic activity, 76 
Hemolytic action. See Blood cells, 

hemolysis of 
Hetero-antagonism, 48 
Heterotrophic bacteria in manure, 22 
Human and animal wastes, 19-37 
destruction of microorganisms, 27 
garbage, 26 

manure and fecal residues, 20-25 
composition and decomposition, 

23-25 
microbial population, 20-23, 84 
sewage, 25-26 
survival of pathogens, 27-36 
Humic acids, 4 
Humus compounds, effect on actino- 

mycin, 219 
a-Hydroxyphenazine, 162 



Implantation method of testing antago- 
nistic action, 62 

Inactivator, nontoxic, defined, 271 

Inactivators of viruses, 152-153 

Indirect antagonism, 47, 49 

Infections, wound. See Wound infec- 
tions 

Influenza organism, 30, 81, 153 

Inhibins, 143 

Inhibition 

of antibacterial action, 197-199 
of antibiotic action, 218-219 



Inhibitive substance, defined, 272 
Inhibitor, defined, 271, 272 
Inorganic constituents of soil, 5 
Insects 

activity of fungi against, 142 

bactericidal action of, 149-150 

control of, 147-150, 266 

in soil, 6, 9 
Interference phenomenon, 154 
Intestinal disturbances, treatment of, 

244 

In vivo activities of antibiotic sub- 
stances, methods of testing, 78 
lodinin, 165, 263, 270 
chemical nature, 53, 158 
effect on bacteria, 165 
inhibition of, 158, 198 
structural formula, 164 
Iso-antagonism, 48, 99, 213 
Isolation of antagonistic microorgan- 
isms, methods. See Methods of iso- 
lating antagonistic microorganisms 



Japanese beetle, control of, 1 48-1 50, 
266 



Ketones, 161 
Key-enzyme, 154 
Kojicacid, 164, 185, 270 



Lactenin, 156 

Lactic acid bacteria, lOO-lOl, 225 

Lauryl sulfate, 70, 77 

Leguminous plants, 258 

Leprosy, 17, 30 

Lipoids, 160, 161-165 

Liquid media for testing antagonistic 

action, 61-62 
Lysin of bacteria, 85 
Lysis 

of bacteria, 78, 82, 86, 200 

of fungi, 140 
Lysobacteria, 84 
Lysogenesis, defined, 272 
Lysozyme, 143, 156, 186-187, 270 

defined, 272 

effect on bacteria, 70, 161 



GENERAL INDEX 



345 



Lysozyme {cotit.) 

relation to bacteriophage, 213 

solubility, 160, 171 
Lytic action 

of actinomycetes, 104-109, 120-121 

of antibiotic substances, 212 



Maggots, bactericidal action of, 151 

Malarial parasites, 147 

Malignant tumors, 222 

Manure. See Human and animal 

wastes 
Mastitis. See Bovine mastitis 
Measurement of antibiotic activity, 
methods. See Methods of measur- 
ing antibiotic activity 
Mechanical separation method of deter- 
mining abundance of organisms in 
soil, 7 
Mechanism of antibiotic action. See un- 
der Antibiotic substances 
Medium, 61-64, 65 

effect of aeration on antibacterial ac- 
tivity of fungi, I 26 
staling, 41, 52 
Meningitis, treatment with penicillin, 

233,234 
Metabolic processes of cells, interfer- 
ence by antibiotic substances, 1 96 
Metabolic products, effect of, 61 
Methods of determining abundance of 

microorganisms in soil, 6-7 
Methods of growing organisms for pro- 
duction of antibiotic substances, 
64-66 
Methods of isolating antagonistic mi- 
croorganisms, 56-60, 78-79 
bacterial agar plate, 57-58 
crowded plate, 58 
direct soil inoculation, 58-59 
"forced antagonism," 56, 59, 186 
soil enrichment, 56-57 
Methods of measuring antibiotic ac- 
tivity, 66-77 
agar diffusion or "agar cup," 73-75 
agar plate-dilution, 69, 71 
antiluminescent test, 76 
interference with function, 76 
lysis of red blood cells, 76 



Methods of measuring antibiotic activity 
{cont.) 
serial dilution, 71, 73 
turbidimetric, 75-76 
Methods of measuring bactericidal ac- 
tion, 77-78 
Methods of testing antagonistic action 

of microorganisms, 60-64 
Methods of testing in vivo activity of 

antibiotic substances, 78 
Microbial cell, growth of, 13-14 
Microbiologist, problems for, 94, 262- 

264 
Micromonosporin, 114, 158, 171, 263, 

270 
Microorganisms 

in animal excreta, 20-23, 84 
in soil, 1-2, 6-10, II, 36-37, 247 
nutrition of, 12-13, 196 
See also Actinomycetes; Animals, mi- 
croscopic; Antagonistic microor- 
ganisms; Bacteria; Fungi; Mixed 
cultures 
Microscopic methods of determining 
abundance of microorganisms in 
soil, 7 
Milk 

bactericidal action, 188 
inhibition of gramicidin, 218-219 
pasteurized, 225 
Milky disease of larvae, 149 
Mixed culture inoculation for testing 

antagonistic action, 64 
Mixed cultures 

antagonistic interrelationships, 46-5 i 
associative interrelationships, 42-45 
competitive interrelationships, 45-46 
growth of microbial cells, 13-14, 38, 

81, 215-216 
mutualistic relationships, 14, 38-41, 

43 
nature, 41-42 
Mixed infections, 222, 223 
Mixed population. See Mixed cultures 
Mode of action of antibiotic substances. 

See under Antibiotic substances 
Morphology, 99, 2 1 3-2 1 5 
Much-lysin, 85 
Mucin, 219 
Mushroom fungi, 9 



346 



GENERAL INDEX 



Mutualistic relationships among micro- 
organisms, 14, 38-41, 43 
Mycoin, I 5 8 

Mycolysate, 55, 107, 224 
Mycophagy, 124 
Mycorrhizal fungi, 249 
Mytogenetic rays, 53 



Nematodes 

control of, 150-151 

in control of insect pests, 1 48-1 50 
Neocolysin, 224 

Nitrite production method for measur- 
ing activity of penicillin, 76 
Nitrogenous bases, 53 
Nitrogenous ring compounds, 53 
Nonspore-forming bacteria, 88-99 
Notatin, 67, 130, 270 

chemical nature, 158, 160, I 80 

effect on bacteria, 125 
Nutrition of microorganisms, 12-13, 
196 



Organic bases, 161 
Oxford unit, 74, 130 
Oxygen supply of soil, 3 
a-Oxyphenazine, 92, 93 



Para-amino-benzoic acid, eif ect on peni- 
cillin, 205-206 
Parasiticin, 132, 157, 270 
Parasitism, 1 4, 40 
Paratyphoid, 86, 90 
Partial disinfection of soil, 258 
Partial sterilization of soil, 145, 250 
Passive antagonism, 49 
Pathogenic organisms 

in fecal wastes, 23 

In soil, 14-15, 16-18, 27-36 
Patulin, 134, 157, 182-183, 237, 249, 

270 
Penatin, 125, I 30, 158, I 80, 270 
Penicidin, 125, 131, 158, 185, 270 
Penicillamine, 179 

Penicillic acid, 1 30, 1 81-182, 198, 
270 

chemical nature, 158, 160, 161 



Penicillic acid (cont.) 

effect on bacteria, 125, 131, 207 

structural formula, 164 
Penicillin, 67, 175-179, 260-261, 270 

animal experiments with, 128, 229- 

232 , 
antiluminescent activity, 77 
chemical nature, 53, 159, 177, 178- 

179 
compared with 

actinomycin, streptothricin, and 

clavacin, 1 90-1 91 
chemical substances, 70, i 89 
gramicidin, 192, 203 
sulfanilamide, 229, 232, 233, 261 
sulfathiazole, 204 
sulfonamide, 205, 227-230, 234 
crystals, frontispiece 
differentiation of bacteria by, 219- 

220 
effect on bacteria, 75, 77, 125, 195, 
198, 202-206, 211, 212, 234, 
259 
by species, 70, 72, 190, 191, 192, 

203, 204 
resistance of bacteria, 128, 228 
effect on cell morphology, 2 1 4-2 1 5 
effect on fungi, 141 
effect of ^-amino-benzoic acid and 

sulfapyridine, 205-206 
effect of reaction, 205 
effect on sulfanilamide, 205 
esters of, 1 79 

inactivation of, 175, 218, 229, 237 
in vivo activity, 226-228 
measurement, 71, 73, 76, 179 
medium, 65, 135 

mode of action, 202-206, 226-228 
production, 128-130, 175-179, 237 
stability, 205 

therapeutic value, 226-237, 261 
toxicity, 228-229, 245 
Penicillinase, 77, 175, 198, 218, 237 
Penicillin B, I 25, 1 30, I 58, 180, 270 
Penicillin-like substances, 179, i8l 
Penillic acid, 179 

Peptones as bacteriostatic agents, 165 
Phage in soil, 10 
Phenazine, 164 
Phenol, 66, 67, 70, 71, 77, 195 



GENERAL INDEX 



347 



Physical properties of soil, 2-4 
Physiologist, field for, 267-268 
Physiology of bacteria, effect of anti- 
biotic agents, 215-219 
Pigment formation and antagonism, 50- 

51 

Pigments, 53, 102-103, 160, 161-165 
Plants 

agents pathogenic to, 18, 246-247 
bactericidal action of juice, 188 
diseases of, control, 246-258 
by actinomycetes, i 21-122 
by antibiotic substances, 141 
by fungi, 138 

by use of antagonistic microorgan- 
isms, 1 21-122, 138, 248-249 
methods, 250-256 
of fungal diseases, 256-258 
influence on microbial population of 
soil, 7-8 
Plate culture method of determining 
abundance of microorganisms in 
soil, 7 
Pneumonia organism, survival, 30 
Poliomyelitis, 153-154 
Polypeptides, 53, 160, 1 67-1 71 
Polysaccharidases, 165-167, 243 
Potato scab, i 21-122, 253 
Proactinomycin, 175, 270 

chemical nature, 53, 159, 160, 161, 

171 

compared with actinomycin, i 20 

effect on bacteria, 120, 161 

toxicity, 158 
Prodigiosin, 53, 160 
Production of antibiotic substances, 
methods of growing organisms for, 
64-66 
Protamine, 217 
Protozoa 

in manure and urine, 23 

in soil, 6, 9 

relation to bacteria, 23, 143-146 

relation to fungi, 146 
Protozoan theory of soil fertility, 144 
Pseudomonas aeruginosa, antibacterial 

substances of, 1 61-163 
Puberulic acid, 159, 185, 270 
Pure cultures, i 3 
Pyocyanase, 67, 91-92, 162, 270 



Pyocyanase {cont.) 

antiluminescent activity, 77 

chemical nature, 53, 159, 160, 163 

effect on bacteria, 77, 190, 195 
by species, 70, 209 

practical value, 163, 221-222, 226 

toxicity, 245 
Pyocyaneus organism as antagonist, 89 
Pyocyanic acid, 162 
Pyocyanin, 67, 92-93, 270 

antiluminescent activity, 77 

chemical nature, 53, 159, 160, 163 

effect on bacteria, 77, 190, 195 
by species, 70 

effect on fungi, 141 

inhibitory action, 21 1 

isolation, 162 

structural formula, 164 

toxicity, 245 
Pyoxanthose, 162 
Pyrogenic substances, 178, 179, 228 



Quinones, 53, 102, 103, 161, 173 
classification, 182 
effect on bacteria, 195, 207, 211 
neutralization of iodinin, 198 



Radiations, 53 

Rectal cancer and £■. coli, 97 

Repressive antagonism, 49 

Rhizosphere, 5 

Rickettsiae, murine typhus, 232 

Root parasites. Fig. 2 (p. 42), 247, 248, 

249 
Rust spores, inhibition of, 1 40-1 41 



Saliva, antibacterial properties of, 27 

Saprophytic organisms in soil, 15-18 

Saprophytism, 40 

Sarcoma cells, 155 

Sea water, bactericidal action of, 34 

Seed inoculation, 256 

Selective culture method of determin- 
ing abundance of microorganisms 
in soil, 7 

Semisolid media for testing antagonistic 
action, 64 



348 



GENERAL INDEX 



Serial dilution method of measuring 

antibiotic action, 71,73 
Sewage, 22, 25-26 
Silkworms, destruction of, 148 
Simultaneous inoculation method of 
testing antagonistic action, 61, 62 
Skin diseases, 138, 243 
Smuts, bacteria antagonistic to, 139 
Soil 

actinomycetes in, 6, 8, 9, 109-1 1 2 
as culture medium, lo-l i 
as habitat of microorganisms, 1-2 
bacteria in, 6, 8, 9, 1 1, 247 
biological state, 5-6 
chemical composition, 4-5 
fungi in, I, 6, 8, 9-10, 137, 247 
heating, effect of, 144, 250 
microbial population. See Soil micro- 
organisms 
pathogens in, 14-15, 16-18, 27-36 
physical properties, 2-4 
saprophytes in, 15-18 
sterilization. See Sterilization of soil 
unsterilized, effect on plant growth. 
Fig. 34 (p. 254) 
Soil-borne diseases 

effects of actinomycetes, i 21-122 
effects of fungi, 138 
methods of control, 14-15, 246-258 
Soil enrichment method of isolating an- 
tagonistic microorganisms, 56-57 
Soil inoculation method of isolating an- 
tagonistic microorganisms, 58-59 
Soil microorganisms, 6-10, 36-37 
and nematodes, 150-151 
antagonism to plant pathogens, 248- 

249 
effect of bacteria, 3 i 
equilibrium, 40 
Solid media for testing antagonistic ac- 
tion, 62-64 
Space antagonism among microorgan- 
isms, 49-50 
Spinulosin, 67, 270 

chemical nature, 133, 159, 160, 161 
crystals, 133, 159 
effect on bacteria, 125, 132 
Spirocheticidal action of penicillin, 211, 
212, 234 



Spore-forming bacteria as antagonists, 

82-88 
Spore germination, 137, 141 
Spores, preparation of, 65-66 
Spot inoculation method of testing an- 
tagonistic action, 64 
Stable manures, 20-25 
Staling of medium, 41, 52 
Staphylococcus aureus infections, treat- 
ment with penicillin, 234-235 
Sterilization of soil 

effect on coliform bacteria, 1 1 
effect on germination of barley, 251 
effect on potato scab, 122 
partial, 145, 250, 258 
Strawberry root rot, 258 
Streptomycin, 174-175, 270 

chemical nature, 53, 159, 160, 161, 

171 
effect on bacteria, 117, 195, Fig. 27 

(p. 215) 
practical value, 123 
production, 117, 120 
toxicity, 122 
Streptothricin, 1 1 6-1 17, 118, 173- 
174, 270 
chemical nature, 53, 159, 160, 161, 

171 

compared with actinomycin, clavacin, 

and penicillin, 190-191 
differentiation of bacteria by, 220 
effect on bacteria, 195, 198 
by species, 70, 119, 191 
effect on fungi, 141-142 
therapeutic value, 123, 243, 244 
toxicity, 245 
Structural formulae of antibiotic sub- 
stances, 164 
Submerged growth, 176 
Subtilin, 159, 160, 270 
Successive inoculation method of test- 
ing antagonistic action, 61, 62 
Sulfanilamide 

antiluminescent activity, 77 
compared with penicillin, 229, 232, 

233, 261 
effect on bacteria, 70, 77, 189, 195, 

229, 261 
effect on penicillin, 205 



GENERAL INDEX 



349 



Sulfanilamide (cout.) 

inhibition of antibacterial action, 
198, 218, 219 

therapeutic value, 226 
Sulfapyridine, 205-206, 227 
Sulfathiazole, 204, 227, 229, 233 
Sulfhydryl groups, 197 
Sulfonamide 

compared with clavacin, 134 

compared with penicillin, 205, 227- 
230, 234 
Sulfur compounds, 53, 160 
Suppressor, defined, 271 
Surface tension, 197 
Symbiosis, 14, 38-41, 43 
Synergism, 40, 206 
Syphilis, treatment with penicillin, 234 



Tannic acid, effect on actinomycin, 219 
Temperature for growth of antagonists, 

Testing antagonistic action, methods. 
See Methods of testing antagonis- 
tic action 

Testing in vivo activity, methods, 78 

Tetanus organism, survival of, 29 

Texas fever, i 7 

Ticks, bactericidal action of, 1 49-1 50 

Tissue culture, 200 

Tolu-/>-quinone, 70, 77 

Toxicity of antibiotic substances, 122, 
i57-i59> 161, 245 

Toxin destruction, 223 

Toxin production, lOO 

Toxoflavin, 160, 170, 171, 270 

True antagonism, 47, 49 

Trypanosome parasites, 1 47 

Tubercle bacillus, 30, 34-36, 135 

Tuberculosis, treatment with extracts of 
A. fumigatus, 133, 244 

Tumors, 154-155, 222 

Turbidimetric method of measuring 
antibiotic activity, 75-76 

Typhoid organism, 90, 91, 213 
as antagonist, 97 
in soil, 29-30, 32-34, 80-81 

Typhus rickettsiae, 234 

Tyrbcidine, 67, 167-17 1, 270 



Tyrocidine (cout.) 

chemical nature, 53, 159, 160 
compared with gramicidin, 192 
crystals. Fig. 18 (p. 170) 
eifect on bacteria, 70, 192, 195, 206 
hemolytic effect of, 239, 240 
mode of action, 197, 199-202, 216 
therapeutic value, 230, 237-243 
toxicity, 245 

Tyrosinase, 171 

Tyrothricin, 68, 76, 87, 167-171, 270 
antiluminescent activity, 77 
chemical nature, 159 
compared with actinomycin, i 20 
compared with protamine, 217 
differentiation of bacteria by, 220 
effect on bacteria, 70, 77, 190, 206, 

217, 237-243 
effect on fungi, 141 
hemolytic effect of, 200-201, 239, 

240 
inhibition of, 219 
mode of action, 199-202 
therapeutic value, 230, 237-243 
toxicity, 245 



Udder infections. See Bovine mastitis 

Urinary infections, 234 

Urine, human, bacterial composition of, 

21 
Utilization of antibiotic substances. See 

Disease control 



Violacein, 159, 270 

Viruses 

antagonisms among, 153-154 
inactivators of, 152-153 

Vitamins, 12, 196, 219 



War-time surgery, 226 

Wastes, human and animal. See Human 
and animal wastes 

Water 

as culture medium, lO-ii 

as habitat of microorganisms, 1-2 

Wheat, diseases of, 247, 248, 251 



350 



GENERAL INDEX 



Wheat protein, antibiotic action of, 

187-188 
Worms, 6, 9 

Wound infections, 225, 265 
treatment, 232-233, 235, 236 



Xanthin oxidase, 197 



Yeasts 

action against sulfanilamide, 218 

in soil, 9 

substances produced by, 63, 185- 
186, 215 

utilization of, 225 
Yellow fever virus, 154