□ ; r=\ ; r^ ; nj ■ □ 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 0 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 0 None Fresh soil 1,000 0 Numerous Largely gram-positive 1,000 O.OI Fewer Gram-negative 1,000 O.IO Few Gram-negative 1,000 1. 00 0 None Fresh milk 100 0 790 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 0 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 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 0 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 syginmm ooi aid ■s-nvajonnm ni NtOiaHiO^JAl QNV N3002J1IN lVI2i31DVg 1 1 1 1 1 \ / X \ / / v\ / / _ Vw" / vX / / \\ / / \ Y / "'A / >x. ( c^ S^ - ^^^^ ^ ^^^X v\'\ \^ \ \ ^>' \' 1 1 t 1 — 1 O ii < s^3ini"nnN 001 y3d stNvaonm^ ni aawnsNOD qidv ouMvimo o o o o o o o o <0 >£> ^ (M S"a3inmii^ ooi ^3d SkNvyonim ni a3tNnsN00 ss-ODmo METHODS OF MEASURING ANTIBIOTIC ACTIVITY 69 125 - / 100 M > 1- > \ O 5 50 z < - 1 / \ 25 I -^t ° 30' 36 42 48 54 60 66 72 INCUBATION PERIOD IN HOURS 84 96 Figure (962). Production of gliotoxin by Trichoderma. From Weindlinc by another. The most important methods at present in use are briefly summarized in the following pages. The Agar Plate-Dilution Method If an unknown antibiotic substance is tested, it is essential to employ several test organisms in order to throw light upon the selective activity of the substance on different bacteria. Nutrient agar media have usually been employed. Sterility is not absolutely essential for this method, al- though it is desirable. The unknown substance is diluted to various con- centrations (i, 0.3, 0.1, etc.j or I, 0.5, 0.25, etc.); these dilutions are added and thoroughly mixed with definite volumes (lo ml.) of sterile agar medium, melted and cooled to 42° to 45° C. The agar is allowed to solidify, and is streaked with three or four test bacteria, among the most common of which are E. coli, E. ty-phosa, Br. abortus, B. subtilis, S. aureus, M. lysodeikticus, and S. lutea. The age of the cultures ( 1 6 to 24 hours) is important. The plates are incubated at 28° or 37° C. for ^ g z cq -S o o O m O o O O o o o o o «^ O o o r<^ \/ /\ /\ \y /\ /\ o O o O o o o o o o o o o o O o ^<^ o o o o o rri rri «~o /\ r<^ o d~ ^\ /\ JT d d O o O o o o o O O o o „ o » r-| O o O o o r«-) o O o o o /^ \/ O m o o q /\ tri o o 2 d /\ 6 6 /\ 6 o" _Q /\ /\ /\ t^ /\ Q O Q Q o O O o Q Q o „ o „ Wh o o o o o o O o o o ro /\ hn o r<~i o o r^ r<^ o o y\ 3 re "^ i W) o o O rri O ^ 00000"00 0000ro/\0" o o <^ o- o o o O -^^ V O f^ o o O /^ - y\ r^ O tn o o o o o O O ro O o o o o <^. o O O r<-, O ro«~oc<^o O f^O O \> V V O O <^ O O <^ fo -f^ -n N/ /\ h 6 H ft^f^fJi 5 1-j H (^ 3 ^ re 3 i-J CO 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 - 1- 1.25 2 0 0 2 - ^^ - 0.8 < 0 "■"-l-..^ Q 1 1 1 1 t 1 "'^ n /I 0 .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- +++ 0 0 Paratyfhoid A + -hH- + -H- -H-f -K-l- Paratyfhoid B 0 + -F ++ -1^ 0 Shigella -H- 4-f -H- + ++ +f 0 Y bacillus -1- ^H- 4- ++ 0 -h E. coli -H-+ 4-H- -H- +-H- 0 0 C. difhtheriae -t-H- ++ + -H- + Ps. fyocyaneus 0 -H- -1- -H-+ 0 S. aureus + 0 0 H^-l- -f-F S. alius -h -1- 0 -H-+ -f- S. citreus 4-f+ 0 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 +++ +^ 0 0 From Franke and Ismet (294). 0 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 0 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 0 0 N. corallina 4-0 45 22 10 N. alba 4-0 25 0 0 M. rubrum 40 33 10 0 M. citreum 38 37 0 0 M. tuberculosis 8 10 0 0 M. smegmae 10 8 0 0 M.fhlei 20 25 0 0 Corynebacterium sp. 12 10 0 0 E. coli 0 0 0 0 S. aureus 25 19 0 0 M. ruber 35 28 0 0 B. mycoides 30 10 0 0 B. megatherium 33 5 0 0 B. mesentericus 30 2 0 0 B. subtilis 23 I 0 0 B. tumescens 22 0 0 0 Ps. fiuorescens 0 0 0 0 Ps. aeruginosa 0 0 0 0 P. vulgaris 0 0 0 0 S. marcescens 0 0 0 0 M. luieus 30 25 0 0 M. candicans 37 22 0 0 M. roseus 42 27 0 0 M. lysodeikticus 38 33 0 0 S. lutea 30 27 0 0 Az. vinelandii 3 0 0 0 Az. chroococcum 5 0 0 0 Rh. leguvmiosarum 0 0 0 0 Radiobacter 0 0 0 0 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 0 0 II 84.6 Potted soil, en- riched with E. coli 21 I 4.8 4 19.0 4 19.0 12 57.2 Potted soil, en- riched with mixtures of bacteria 15 12 80.0 2 13-3 0 0 I 6.7 Lake mud 9 3 33-3 4 44.4 0 0 2 22.2 Stable-manure compost 37 ' 2.7 20 54.0 4 10.8 12 324 Total 244 49 20.1 44 18.0 13 5-3 138 56.6 From Waksman, Horning, Welsch, and Woodruff (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 0 - -H- S. californicus + C 0 S. candidus -H- c,s ++ S. cellulosae + c 0 S. griseus (3326b) + c 0 S. lavendulae + c ++ S. reticuli + c 0 S. roseus + C + S. ruber + - 0 S. sap-ophyticus -H- c,s 0 S. scabies (3031) -1- c 0 Strefiomyces s^. (3069) -H- c 0 5. albus (G) -H- c,s 0 Streftomyces sp. (33 187) -K- c,s 0 N. gardneri 0 c -H- Micromonosfora sp. 0 - -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 0 S. halstedii + C 0 S. hominis ++ C,S 0 S. lifmanii + c 0 S. mtcroflavus + c 0 S. odortfer ++ - 0 S. fraecox + c 0 S. rutgersensis -H- C,S 0 S. samfsonii -H- C,S 0 S. scabies (3352) + - 0 5". scabies (302 1) -hH c 0 5. setonii -H- c,s 0 S. tetanusemus ++ c,s 0 S. coelicolor (3033) + Not tested Not tested Streftomyces^'p. (Lleske, No. 23) ++ c,s 0 Streftomyces sp. (Lieske, No. 25a) 4-f c 0 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 0 Ps. -fluorescens - 3 3 0 Br. abortus - 3 3 0 N. catarrhalis - 3 2 0 E. carotovora - 3 2 0 Sh. gallinarum - 2 2 o A . stutzeri - 2 I 0 H. fertussis - 3 0 0 Az. vinelandii - 0 0 0 S. cellulosae + 2 I 0 S. calif ornicus + 3 2 0 M. tuberculosis + 3 0 0 CI. welchii + 0 0 0 B. macerans + 3 o 0 B. megatherium + 0 0 0 B. folymyxa + 0 0 o B. mycoides + o 0 0 B. mesentericus + o 0 o B. cereus + o 0 0 B. subtilis I + o o 0 0 B. subtilis II + o 0 0 0 G. tetragena + 0 0 0 o S. lutea + o 0 o 0 Streptococci and staphylococci + 0 0 o 0 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 0 0 0 0 0 I B. mycoides 2 2 2 2 2 2 B. macerans 2 2 2 2 2 2 B. megatherium 0 0 0 0 I 2 B. folymyxa 0 0 2 2 2 2 B. cereus 2 2 2 2 2 2 M. lysodeikticus 0 0 0 I 2 2 S. muscae 0 0 0 I 2 2 S. lutea 0 0 0 0 I 2 A . aerogenes* 0 0 I 2 2 2 A . aero genes 0 0 0 Tr 2 2 E. coli-\ 0 0 0 0 2 2 E. colt (4348) 0 0 Tr I 2 2 5. marcescens 0 I 2 2 2 2 S. marcescens I I 2 2 2 2 Ps. fluorescensX 2 2 2 2 2 2 Sh. gallinarum 0 0 0 0 I 2 P. fseudotuberculosis 0 0 0 Tr 2 2 Br. abortus 0 0 0 0 2 2 S. cholerasuis 0 0 0 Tr 2 2 S. schottmillleri 0 0 0 I 2 2 S. abortivoequina 0 0 0 Tr 2 2 S. tyfhimurium 0 0 0 2 2 2 H. suis 0 0 0 2 2 2 H. influenzae 0 0 0 0 0 I Br. abortus 0 0 0 0 2 2 Az. agile 0 0 0 0 0 2 Az. vinelandii 0 0 0 0 0 2 Az. chroococcum 0 0 0 Tr 2 2 Az. indicum 0 0 0 2 2 2 M. fhlei 0 0 0 I 2 2 CI. butyricum\ 2 2 2 2 2 2 L. casei^ 0 0 0 2 2 2 S. albus 0 0 0 I 2 2 S. violaceus-ruber 0 0 5. lavendulae 0 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 0 20 20 600 A. fumigatus 20 100 0 300 150 800 A. fumigatus 20 300 0 300 60 800 A. fumigatus 84 100 0 600 300 >i ,000 A. fumigatus 84 300 0 300 100 >i ,000 P. luteum 1 08a 100 0 0 0 0 P. luteum 1 08a 300 0 0 20 10 P. notatum F 100 0 3 15 P. notatum F 700 10 10 >IOO P. notatum W 100 0 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 0.016 25,000 121 4.2 6.7 75 1.442 1,000,000 122 4-5 8.0 0 0.035 4,000 123 4.6 4-5 20 0.467 120,000 124 6.2 8.4 0 0.016 8,000 125 3.2 3-9 0 0.248 600 126 6.3 8.2 0 0.039 20,000 127 7.4 8.1 0 0.007 8,000 128 6.7 8.0 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. ■i)3innnikM asd siNvaomuM ni avons ~ivnais3a ^ O (D (M (\j rvj - - CO ^ o 1 1 1 1 1 1 Hd 1 o en TO r- vo tf^ ^ n ' 1 1 1 1 1 1 r fM f X o o o 1 i/ ' ! / / / \ <-/ / < ,?/ / -ij Eh i- ; w/ CO 5- ( v\ ^1 1/' ^ 2 8 5S a O 1- X,^X-^^^0 O^O < \ ^^ / D a z \ /\ ^\^ V-' A ^ X > \ ^- ■n 1- \ /■ ' \ / '' \ / / \ 1 1 1 ./ I \ I \ 1 \ 5; nj o 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 0 0 0 Actinomycin O.I 0.03 O.I Streptothricin 0 0 0 Clavacin 0.15 0.045 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- t ^ _> -^ ^ o rt T^ a. too 1 O i-i o ■h-i 2 W) a, TS bo 1 6 5 ^3 C Oh Oh e 6 ■1-' OS .;:; ^5 2 IS O & 6 ^ ^_ '^ w o 1 1 'S o o 1J^ 1 c 0^ 1 3 d. U ^ HJ 2 >JI o CIS OJ ^ § "a, ^ 1 6 S "o 3 "S i "o .s T3 ^ C C oT w w kH c o a, 3 5 'T3 C to o 'G re ^ rt a iH rS rt C ^ a -C J C o re ir .s .s ^ " w (U '35 T) 3 13 c 3 3 to 1 6 3 > 3 3 o g-s c -^ re ^_, iz o I 8 ^ u IJ re S I E : a; i g .s ^ u u u a, c o o .5 t« > > t— I re re U -u 73 .S OO re ^ 'Si re o O O •^ S ►> (U . o - O J3 O 13 .T ■" 3 o o 5 a O 3 " i g I- z pa' T. S .2 O ^ 2 •i ^ ■c & ^ 1 ^ 4) e o J2 > o flj 1 'S IS c > _8 a. E re 6 i 1 PL, 1 i ffi c (J ^ o 0 - •- _Q CJ & .t: > ^ B ^ .. — o ? .« Ph •« .5 i u Ml o 1 >-» o C u t- re S .^ • — .2 o c Cl, o c u S 2 ^ t: 6 05 T3 .- T 05 « 2 « o II r^ 1^0 • " o S ^ -S 'go § 2 > ^ 8 ex, I 6 -Q <-5 re c ci.2 ^ a ^ 5 i ^ j: o w 0 n3 -5 o JJ tT "^ ^ >^ JJ 2 u T3 0 i-i re 3 'S ^ _Q v; 13 D ^ ^ W) 3 -^ I^ 'e ;i Q PS c^ M ;i 1 0 ■0 N ci5 Si ^ .« ^ b re re M ■•^ J 're J {jjO & 'o, 1 c J^ c 0 .£ 5^ 0 «J __«j (U 0 3 5 s 13 0 3 ^ 3 3 ^ &fl 3 ■s 3 Dh >< 'g "o s 1 .S ;i a, g .S > a^ a,' ^ o 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, , 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 0 293 26.2 75 250 Shaken 6 0 231 17-3 100 300 Shaken 8 0 75 200 Shaken 12 0 142 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 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 = 0 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, _,o / f • t>» o//! o /T : ^ V) / /."^ P q! V \ ^ z o — ^ n llJ 2 ^ H \ k * z rs^ \ s - \^V^ 'x <. ^' CM H O ^\^^Nv '^- ^^ < o N. •^^ ^^ ^ N^;;50rt^^<-. .^ "2 1 o O) C0'~~'0'^ ^ ro(M — d~" ^Binnnm "ygd syoAiA^ns jo hhii^vooi —,o >0 Q> u _J n 2 1— 1 / Qj / o u CVi D- < L_ 1 i-^-h — ^1 "^1 1 1 1 — 1... 2i < o o o «3(\JC0'=l'O^^ i^ L_ 1 Xr-^-X' "?* 1 1 1 1 1 1 o OOO^-vOiO ^rofvl — c 3° ■ygxnmikN "aad s'yoAiA^ns jo nHiiavoon :S < a^ o ^ >- - _c o o 4J CL, o "^ c c ..s ;jq 4^ "- _c [o C o s o o s •3 CO M-l bJO 'e V, (N o •55 CJ OJ ^ _g oi a, j^ . u '^ >- 'e C3 <; ,C o W o z >^ '—' O '"' D- J2 JO oj o ,c >^ O o .*" O K N ^ rs o S .2 W _ o 15 '5 ^ o rt 1— 1 1) 1 1-1 3 -^ < _ct -C , c _C o o cw -! >-2 o ^3 ^—^ • S T3 C3 J^ CO O .s o s d 3 o ^ "S OJ 8 CO 3 5 a. § c o ^ c c c 1 c r3 a N 15 rv-\ CL, >• .y j~ u o c3 CO >^ 1— o "C -13 c 3 ■*i rt N U ?N 13 pj « ~~i J 3 •5 vl5 (S ►< OJ _c 3 C/2 M CO ex >-■ Pi ^ "^ ^ tin' =1 O c ^ 3 iZ en .2 'b 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) ■^ "V "\ \ : f\j >^ \ 1 N^ \ \ i I/) \, \ \ • _J V \ \ o I CD 1/1 in ^^ z 3 1- -J NA \ z (J V\ \ o < ^^ ' 1- CD < ^•^\. \ \ ^"^^ \ ^V, o ,o 1 1 1 1 1 \/''^''' l-l CO •£ ^ O) vO f^ o ^iinnim yad viii3j.Dvg do SNomikN ^ 0 -5 1 "7 tn \ uO Oi < I/) Xx ^1 00 ct; O I z 8 o o \ \ 1 °/' ° - 5 >- I \. ^ ,''' "^ Z o Q. ^v ' /' 1- < H. °' "5 lO (J 1- \.„y' __/ < ^ a^s**rt^'-^|J_ll_- ^ ^^^fi!^ sS^---- ' 1 o 1 1 1 o o O O O ' 5° o IT) O iT) O 01 o> f-. ^ .1 rn - aaxnnnit^ -yad viygiDva josNonniKN i3 aj 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 0 120 329,000 H4 0 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. 9 - Mi. ••••-fes. J5&=. ^ CONTROL e •••> ' '"\. ■-- — - - - 0.004 MG. " \ '"""- \ ooVMa— - ^ 7 _ \ 0! \ 1- J \ 0 -J 6 -1 " V i •%s \- °- b _ • < \ 5 u \ ' 5 4 _ . 0 \ u. 0 0 \ §3 - \ K O! < \ 0 *, 0 2 - *« - 1 J_ 1 1 1 \ 1 1 1 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 // <.^; ^/'> o /' ^ 125 y/i /'\^ / /A I/) // 1^ / ' UJ (- JQ UJ /, E 100 /'' _) // _l /'' 0 i u /' / m / ^ D ^ 75 _ A / Z >» / ~ / ^/ / / < / / a 3 Z 50 / J" O / / > / .0 X / / o / / / / ff y 25 - X-"" / / / y / / MIXTURE OF TYteOTHRICIN AND PROTAMINE ^ jy^^-^ ^ ^ . ) 15 30 45 60 TIME IN MINUTES 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 >^ O CO O " O s N e c^ iri tn ►J S " <: w " H S ^ H 5 fe N u-1 cl s i ^ ^ ffi Oh 6 - o o o o CIS rt O O 6 6 e 6 C i> C (U Or'-' Or'-' o h u h 6 s e 6 ^ n-j ^ n-i O • { ><^ ^ ? ^''^ } p of .^ oa 2; 0>^ ^ z Q 61 ^-^^'"" ' to o > - < u -^-' z i < Qi O 1 0.2 ^^—"'^ " ? /^-^'^'^ „v^9.--^ -^ l^ ?-'•■'" _0..4_M^.-- " CO — iij .'" .-— -* r^"-^' ^ 1 1 1 1 1 1 c\l rg o CO «o 'J- ry o ONIAIAanS 3011^ dO ^39lNnN vO ? ? ? 1 /•* 5 • ? : • 1 1 C\J • o! ,,.^' d! ° ^ x' J o ^ z 5| — >■ u 5 \ yr" < a I o; 1 ^i 1 n: ooZ 1- 1 o i J .J^ y UJ 2 >- 1- j (o"- i r ^ - ^ 1 H CM 1 1 1 1 1 1 (M O 00 >0 'J 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. 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L., Jr. Interfer- ence between the influenza viruses; the efi"ect of virus rendered non-infec- tive by ultraviolet radiation upon the multiplication of influenza viruses in the chick embryo. J. Exper. Med. 79:379-400 (1944). 1014. ZoBell, C. E. Bactericidal action of sea water. Proc. Soc. Exper. Biol. & Med. 34: 1 1 3-1 16 (1936). 1015. ZoRZOLi, G. Influenza dei filtrati di alcuni miceti sul Bacillo tubercolare umano e bovino. Ann. Inst. Carlo Forlanini 4: 208-220, 221-237 (i94o)' 1016. Zukerman, I., and Minkewitsch, I. Zur Frage des bakteriellen An- tagonismus. Wratschebnoje Delo, No. 7 (1925) ; abstract in Centralbl. f. BakterioL, I, Ref., 80:483-484 (1925). 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 0 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/. «« 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