Advances in Applied Microbiology, Volume 18

ADVANCES

IN

Applied Microbiology VOLUME 78

CONTRIBUTORS TO THIS VOLUME

Martin Alexander J h o s BBrdy Jean-Marc Bollag J. S. Bond S . G. Bradley

John C. Godfrey Hiroshi Kawaguchi Kenneth E. Price

ADVANCES IN

Applied Microbiology Edited by D. PERLMAN School of Pharmacy The University of Wisconsin Madison, Wisconsin

VOLUME 18

@

1974

ACADEMIC PRESS, New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1974, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York,New

York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1

LIBRARY OF CONGRESS CATALOG CARDNUMBER: 59-13823 ISBN 0-12-002618-X PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS LIST OF CONTRIBUTORS ................................................

vii

Microbial Formation of Environmental Pollutants

MARTIN ALEXANDER I. Introduction .................................................. I1. Products of Pesticide Metabolism ................................ 111. Inorganic Nitrogen Compounds .................................. IV . Simple Carbon Compounds ..................................... V. Acidity and Mine Drainage ..................................... VI . Simple Sulfur Compounds ...................................... VII . Mercury ..................................................... VIII . Arsenic ...................................................... IX. Selenium and Tellurium ........................................ X . Oxygen ...................................................... XI . Nitrosamines .................................................. XI1. Other Carcinogens ............................................. XI11. Complex Human and Animal Toxins .............................. XIV . Other Phytotoxins ............................................. XV . Other Compounds with Odor and Taste ........................... XVI . Perspective ................................................... References ....................................................

1 2 13 28 34 37 43 46 47 49 50 53 56 60

62 63 64

Microbial Transformation of Pesticides

JEAN-MARCBOLLAG I. I1. I11. IV . V.

Introduction .................................................. 75 Mechanisms of Pesticide Transformation .......................... 77 Enzymatic Reactions in Pesticide Metabolism ...................... 81 Chemical Structure and Microbial Transformation Relationship . . . . . . . . 114 Conclusions ................................................... 122 References ................................................... 124

Taxonomic Criteria for Mycobacteria and Nocardiae

S . G. BRADLEYAND J . S. BOND I . Introduction .................................................. I1. Earlier Classification Schemes ................................... V

132 133

vi

CONTENTS

111. IV . V. VI . VII . VIII . IX . X.

Developing Classification Systems ................................ Differential Characters ......................................... Regulation of Metabolism ...................................... Degradation of IntracelluIar Proteins ............................. DNA Analyses ................................................ Definition of the Genera ........................................ Evaluation of Species .......................................... Concluding Remarks ........................................... References ....................................................

134 136 145 153 162 172 176 184 185

Effect of Structural Modifications on the Biological Properties of Aminoglycoside Antibiotics Containing 2-Deoxystreptamine

KENNETH E . PRICE.JOHN C . GODFREY. AND HIROSHI KAWACUCHI I . Introduction .................................................. I1. Relative Activity. Susceptibility to Enzymatic Inactivation. and Toxicity of Naturally Produced and Semisynthetic 2-Deoxystreptamine-Containing Antibiotics ................................................ I11 General Conclusions Regarding the Influence of Structural Variation on the Biological Properties of 2-DOS-Containing Compounds . . . . . . . . . . References ....................................................

.

191 217 283 299

Recent Developments of Antibiotic Research and Classification of Antibiotics According to Chemical Structure J ~ N O S BPRDY

I. I1. I11. IV . V.

Introduction .................................................. Antibiotic Research in the Past Decade ........................... Systematization of Antibiotics ................................... Classification of Antibiotics According to Chemical Structure . . . . . . . . . . Conclusions ................................................... References ....................................................

SUBJECTINDEX ..................................................... CONTENTSOF PREVIOUSVOLUMES......................................

309 310 336 345 397 402

407 411

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

MARTINALEXANDER,Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York ( 1 ) J ~ N O S B ~ R D YResearch ,

Institute for Pharmaceutical Chemistry, Budapest,

Hungary (309) JEAN-MARC BOLLAG,Laboratory of Soil Microbiology, Department of Agronomy, The Pennsylvania State University, University Park, Pennsylvania ( 7 5 )

J. S. BOND,Departments of Microbiology and Biochemistry, Virginia Commonwealth University, Richmond, Virginia ( 131) S. G. BRADLEY,Departments of Microbiology and Biochemistry, Virginia Commonwealth University, Richmond, Virginia ( 131) C . GODFREY,Bristol Laboratories, Division of Bristol-Myers Company, Syracuse, New York ( 1 9 1 )

JOHN

HIROSHIKAWAGUCHI, Bristol-Banyu Research Institute, Meguro, Tokyo, Japan (191)

KENNETH E . PRICE,Bristol Laboratories, Division of Brbtol-Myers Company, Syracuse, New York ( 1 9 1 )

vii

This Page Intentionally Left Blank

ADVANCES IN

Applied Microbiology VOLUME 78

This Page Intentionally Left Blank

Microbial Formation of Environmental Pollutants

MARTINALEXANDER Laboratory of Soil Microbiology. Department of Agronomy. Cornell Uniuersity. Ithaca. New York

I . Introduction .................................... I1. Products of Pesticide Metabolism . . . . . . . . . . . . . . . . . . I11. Inorganic Nitrogen Compounds .................... A . Ammonia ................................... B . Nitrate ..................................... C. Nitrite ..................................... D . Hydroxylamine .............................. E . Nitrogen Oxides ............................. IV. Simple Carbon Compounds ........................ A . Ethylene ................................... B . Carbon Monoxide ............................ C. Carbon Dioxide ............................. D. Organic Acids ............................... V. Acidity and Mine Drainage ....................... VI. Simple Sulfur Compounds ......................... A . Hydrogen Sulfide ............................ B . Sulfur Dioxide and Carbonyl Sulfide . . . . . . . . . . . . C. Volatile Organic Sulfur Compounds . . . . . . . . . . . . VII . Mercury ........................................ VIII . Arsenic ......................................... IX . Selenium and Tellurium .......................... X . Oxygen ........................................ XI . Nitrosamines .................................... XI1. Other Carcinogens ............................... XI11. Complex Human and Animal Toxins ................ XIV. Other Phytotoxins ................................ xv. Other Compounds with Odor and Taste . . . . . . . . . . . . . XVI . Perspective ..................................... References ......................................

.

I

1 2 13 13 16 20 23 25

28 28 29 31 33 34 37 37 40 41 43 46 47 49 50 53 56 60 62 63 64

Introduction

Probably since the first realization that microorganisms are important in communicable disease. individual species of bacteria and. later. viruses. protozoa. and fungi have been acknowledged as important to environmental pollution . At the present time in the technologically advanced countries. but not in most tropical regions. pollution by parasitic microorganisms is rarely of concern. Water pollution in these nations. more often than not. denotes not a water supply contaminated with bacteria. viruses. or protozoa able to parasitize humans but rather one containing unwanted chemicals. Yet. microorganisms are still causing pollution. sometimes to a modest but often to a serious extent. and 1

2

MARTIN ALEXANDER

the resulting deterioration of environmental quality arises not because the water supply contains a potential parasite able to grow within the body, but a free-living heterotroph or autotroph that proliferates in the water and there synthesizes undesirable or harmful metabolites. Moreover, the biogenesis of chemical pollutants not only affects water quality, but also brings about detrimental changes in soils and the atmosphere. The present discussion, of necessity, will only highlight some of the metabolites involved and will not delve in depth into the problem areas. It still should be obvious to the reader, however, that little environmental or microbiological research has been done on most facets of the microbial formation of polluting chemicals. 11.

Products of Pesticide Metabolism

Pesticides are essential in man’s arsenal of weapons to protect himself from certain communicable diseases and in his endeavors to provide himself with food and clothing. The enormous progress in the control of malaria and yellow fever, for example, is largely attributable to the effectiveness of insecticides in reducing the populations of insect vectors transmitting the protozoa and viruses. In the technologically advanced countries, the pest-control agents often make the difference between success and failure in food and fiber production and between making a profit or incurring a financial loss. In the developing countries of Latin America, Africa, and Asia, by contrast, regions where the food supply is often insufficient to meet the need of the local population, the use of pesticides may determine whether there is adequate food or widespread famine. The rapid increase in use of these substances in the developing countries is shown by the &-fold rise in pesticide usage in the cropped area of India in a 13-year period (Pradhan, 1970). Pesticides have gained prominence not only by virtue of their effectiveness in controlling insects, weeds, and plant pathogens, but because a few are significant environmental pollutants. All these compounds are toxic, or allegedly so, otherwise they would not have been employed in agriculture or public health, but the real or alleged toxicity at the levels used in the field is only to a narrow spectrum of organisms-a small or a large group of animals, rooted plants or microorganisms. Many of the chemicals themselves have little toxicity, however, even to the pest for whose control the substance is designed, and such compounds owe their effectiveness solely to their conversion to the actual toxicant in a process known as activation. Often the activation occurs in susceptible or closely related species, but the toxicant may also be generated microbiologically in certain environments, even when microorganisms are not the group of organisms whose control is sought. Activa-

MICROBIAL FORMATION OF ENVIRON.MENTAL POLLUTANTS

3

tion is evident among the organophosphorus insecticides, phenoxy alkanoic acid herbicides, and other pesticides. Other chemicals marketed as pesticides are indeed toxic, but they are, or can be, transformed microbiologically to new toxicants that act on species also inhibited by the original chemical. Hence, instead of the environment containing just the original pest-control agent, it may contain two or more. In addition, several pesticides that are active in their original forms are converted microbiologically to new inhibitors which act not on the same organisms succumbing to the parent compound, but instead on entirely different species; thus, a fungicide may be converted to a human carcinogen or to a phytotoxin. Excellent illustrations of how nontoxic chemicals are made into inhibitory products are found among the insecticidal phosphorothionates and thioethers. The former can be written as (RO),P(S)OX, where R is typically a short alkyl substituent and X can be any of a variety of groups. The latter have a thioether, -C-S-C--, in the molecule. Compounds with these structures typically have little toxicity, but they become extremely potent if the phosphorothionate is converted to the phosphate, R 0 2 P (O)OX, or the thioether-sulfur is oxidized to a sulfoxide, -C-S( 0)-C-, or sulfone, -C-S( 0,)-C--. The potential activity

I

of the product may be about 10,000-foldgreater than that of the precursor ( OBrien, 1960). In a typical study, Read ( 1971) applied the insecticides chlorfenvinphos and dyfonate to a soil, from which samples were taken at regular intervals for bioassay of their capacity to inhibit the larvae of a test insect. The toxicity of the treated samples was low at first, but the inhibitory effect increased with time as the chemicals were transformed and activated. Dimethoate, (CH,O),P( S)SCHrC(O)NHCH,, is a phosphorothionate that is activated in soil by an apparent oxidation to the corresponding oxygen analog (Bache and Lisk, 1966), although a precise role for microorganisms has yet to be established in this sequence. Several thioethers are converted in soil to the toxic agents, and of considerable importance is the fact that the hazardous product may be quite persistent and fail to succumb rapidly to biodegradation. This is well illustrated for the insecticide known as phorate, 0,O-diethyl S- ( ethylthiomethyl )phosphorodithioate, which is oxidized to the corresponding sulfoxide and sulfone.

4

MARTIN ALEXANDER

These products in turn persist for more than 16 weeks (Getzin and Shanks, 1970). A closely related insecticidal thioether is disulfoton, (C,H,O),P( S)SC,H,SC,H,a compound that, in its unaltered form, shows little toxicity, yet marked increases in toxicity ensue as the thioether-sulfur is oxidized to the sulfoxide and sulfone. Disulfoton sulfoxide and disulfoton sulfone persist for some time, so that soils containing them deleteriously affect insect inhabitants (Takase et al., 1971). A similar transformation is evident with 2-methyl-2- ( methylthio )propionaldehyde- ( 0-methylcarbamoyl ) oxime, an insecticidal, miticidal, and nematicidal agent sold under the name aldicarb or temik. This molecule is likewise converted to the corresponding sulfoxide and sulfone by oxidation of its sulfur. CH3

0

I1

1

CH~S-CC-CH=N-O-CC-NH-CH~ 0 CH3

T

CH 3s-

+

0

I /I C- CH=N-O-C-NH-CH I

3 -+

CHJ

0 CH,

t I CH,S-- C- CH =N1 1

0

II

0-C-NH-

CHa

(3)

0 CH3

The toxicity of this commercially important pesticide has been ascribed largely to the sulfoxide (Coppedge et al., 1967). Although such reactions are probably microbial, no definitive work exists to establish that the subterranean microflora is responsible for producing the insecticidal agents. Many of the synthetic compounds introduced or transported to soil or water neither are volatile nor are destroyed at appreciable rates by the resident communities, and thus they persist for months, years, or even decades. Some of these chemicals are transformed to long-lived products. The prolonged persistence of a toxicant is of special concern because the stress on susceptible populations is not soon relieved, and

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

5

the polluting or pesticidal agents continue to make their presence felt for long periods of time. Possible reasons for the failure of microorganisms to bring about rapid biodegradation and chemical structures that are refractory to attack have recently been reviewed (Alexander, 1973). Three chlorinated hydrocarbon insecticides-DDT, heptachlor, and aldrin-illustrate quite clearly the genesis of a new and durable toxicant, each of the parent molecules in these instances also persists for considerable time intervals. DDT, the widely known abbreviation for l,l,l-trichloro-2,2-bis ( p-chlorophenyl )ethane, controls a wide variety of insects important as vectors of microorganisms causing human disease and as pests affecting the food supply, but it is converted to DDD ( l,l-dichloro-2,2-bis ( p-chlorophenyl )ethane), also abbreviated TDE, which likewise is insecticidal. Indeed, DDD acts on a somewhat different spectrum of insects than its precursor, and it is marketed commercially because of its potency. That such a formation of one toxicant from a second is widespread is suggested by observations that the reaction, involving a simple reductive dechlorination, takes place in samples of soil (Parr et al., 1970), raw sewage (Pfaender and Alexander, 1972), marine waters (Patil et al., 1972), and estuarine sediments (Albone et al., 1972), among other habitats. A high percentage of the bacteria isolated from marine waters and raw sewage possesses the capacity of converting DDT to DDD, often in good yield, and a similar capacity presumably exists among isolates from other localities. The dechlorination probably involves a step in a cometabolic sequence, and some 20-75% of the bacteria from sewage and ocean waters in two studies appear able to cometabolize DDT (Patil et al., 1972 Pfaender and Alexander, 1973). Among the identified organisms effecting the requisite dechlorination are Aerobacter aerogenes, Escherichia coli, Klebsiella pneumoniae (Wedemeyer, 1966), Fusarium oxysporum ( Engst et al., 1967), Hydrogenomonas sp. (Pfaender and Alexander, 1972), Proteus vulgaris ( Barker et al., 1965), and Sacchnromyces ( Kallman and Andrews, 1963). Heptachlor ( 1,4,5,6,7,8,8-heptachloro-3a,4,7,7aetetrahydro-4,7-endomethanoindene ) is subject to epoxidation, and the resulting heptachlor epoxide has been found in studies of many different soils (Helling et al., 1971). The epoxide is reported to be the more toxic of the two compounds to animals, including insects, and it is possibly more toxic to humans, too. Heptachlor disappears slowly with time from treated land, but it is replaced by the epoxide, which endures for many years. Indeed, much of the parent substance is recovered in treated soils as the more toxic product (Murphy and Barthel, 1960; Wingo, 1966). The oxidation in soil probably is attributable to activities of the microflora, and a high percentage of the fungi, bacteria, and actinomycetes isolated

6

MARTIN ALEXANDER

from that environment is able to bring about the epoxidation (Miles et al., 1969). Chlorella pyrenoidosa also forms the epoxide in good yield ( Elsner et al., 1972). Aldrin ( 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-endo1,4-exo5,8-dimethanonaphthalene) is likewise subject to epoxidation in soil, and both the original pesticide and the product of epoxidation-known as dieldrin-are insecticidal. Dieldrin is also quite persistent, and it can be found many years after its first application to a field. Inasmuch as this conversion occurs in nonsterile, but not in sterilized, soil, the process is apparently microbial ( Lichtenstein and Schultz, 1960). The same transformation takes place in an aldrin-containing medium incubated with a soil inoculum (Lichtenstein et al., 1963). Species of Fusarium, Penicillium, Trichoderma, and actinomycetes (Tu et al., 1968) among others are able to synthesize the toxic epoxide. Dieldrin, in turn, can be metabolized to a still more toxic product, given the trivial name of photodieldrin, in samples of water and silt and by a large number of isolates from soil, water, silt, and the gastrointestinal tract ( Matsumura et al., 1970). Numerous other chlorinated molecules are subject to microbial modifications, and some of the metabolites that are generated are toxic and accumulate sometimes for short, sometimes for long periods of time. Included are not only insecticides, but also herbicides and fungicides, such as the chlorophenoxy alkanoic acid herbicides and the chlorophenol fungicides. A well known illustration of activation among members of these groups is the conversion of 2-( 2,4-dichlorophenoxy)ethyl sulfate to the phytotoxin 2,4-dichlorophenoxyacetic acid ( 2,4-D ) in soil. The former compound, which is commonly considered to be the herbicide, is in fact phytotoxic only after it is acted on microbiologically. Some evidence suggests that another inhibitor, 2- ( 2,Cdichlorophenoxy)ethanol, may be produced and accumulate during the transformation. The conversion of the substituted ethyl sulfate to the substituted ethanol can be brought about in culture by Bacillus cereus var. mycoides (Andus, 1953; Vlitos and King, 1953). The p-oxidation sequence for the metabolism of fatty acids serves as a novel means by which herbicides are produced by microbial populations. The compound sold as the herbicide 4- ( 2,4-dichlorophenoxy)butyric acid, 4-(2,4-DB), is itself innocuous at the rates typically used for weed control, but when applied to susceptible plants, the fatty acid portion of the molecule is subject to a p-oxidation sequence to yield the active principle, 2,4-D. Essentially the same reaction sequence is effected by microorganisms in the soil. Not only is the substituted butyric acid metabolized by p-oxidation but so too are the omega-substituted

7

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

2,4-dichlorophenoxy pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, and undecanoic acids. All such compounds having even-numbered fatty acids yield the herbicidal 2,4-D (Table I ) . Moreover, phenoxy compounds with odd numbers of carbons in the fatty acid moieties are metabolized to 2,4-dichlorophenol, presumably by p-oxidation to yield 2,4-dichlorophenoxyformic acid, which then undergoes decarboxylation. The identical @-oxidationpathway for the initial phases in the degradation of omega-substituted 2,4-dichlorophenoxyalkanoic acids occurs in axenic cultures of Nocardia coeliaca (Taylor and Wain, 1962). Moreover, analogous mechanisms are involved in the metabolism of unsubstituted and monochloro-substituted phenoxyalkanoic acids by cultures of Aspergillus niger, Nocardia opaca, and N . coeliaca (Faulkner and Woodcock, 1966; Taylor and Wain, 1962; Webley et al., 1958). Although appreciable accumulations of 2,4-dichlorophenol were not noted in early studies of soil amended with 2,4-D, which is one of the major phenoxy herbicides in agricultural practice, the phenol has been found as a product of the metabolism of 2,4-D in cultures of an Arthrobacter (Loos et al., 1967b) and in enzyme preparations derived from the bacterium (Loos TABLE I METABOLISM OF OMEGA-SUBSTITUTED PHENOXYALKANOIC ACIDSI N SOIL” Products detectedb

Substrate addedb Even-numbered acids 2,4-D butyric 2,4-D hexanoic 2,4-D octanoic 2,4-D decanoic Odd-numbered acids 2,4-D pentanoic 2,4-D heptanoic 2,4-11 nonanoic 2,4-D undecanoic

2,4Dichlorophenol

2,4-D acetic acid

2,4-1) propionic acid

+ + + + + + + +

2,4-D butyric acid

2,4-D pentanoic acid

c

+ + + + + + +

c

+ + +

From Gutenmann et al. (1964). The substrates and products are the omega-substituted 2,4-dichlorophenoxy alkanoic acids. The product listed in these combinations is the substrate.

8

MARTIN ALEXANDER

et al., 1967a), and it also can be formed by a strain of Flavobacterium incubated with 4-( 2,4-DB) ( MacRae et al., 1963). 2,4-Dichlorophenol is of especial significance since its formation from an actual or potential phytotoxin would lead to the appearance of a product with an entirely different spectrum of action than its precursor inasmuch as the phenol is not only antifungal to some degree, but it is also an antiviral agent (Ando et al., 1970) and is believed to be a sex pheromone for arthropods (Berger, 1972). Hence, a study was recently made of its possible formation both in a soil (Lima silt loam) and in fresh water-sediment collected from a rural stream. Both 2,4-D and 2,4,5-trichlorophenoxyaceticacid ( the herbicide known as 2,4,5-T) were added to initial concentrations of 50 ppm, and the soil and water were incubated under aerobic conditions. The data in Figs. 1 and 2 show that 2,4-D is converted to 2,4-dichlorophenoI and 2,4,5-T is transformed to its corresponding phenol, which is fungicidal, in both soil and water. The yields of the chlorophenols were consistently low, and neither persisted as long as the herbicides from which they were generated. Both metabolites were identified by gas chromatography and mass spectrometry. It is noteworthy that, although 2,4-D was destroyed quickly in the fresh water-sediment model ecosystem, an unidentified product appeared in the water on the sixth day of incubation, and it persisted for 12 weeks; thus, a pesticide that is readily susceptible to attack may give rise to a long-lived metabolite, which possibly may be an environmental pollutant ( K . W. Sharpee and M. Alexander, unpublished observations). The herbicide Zytron ( 0-2,4-dichlorophenyl O-methyl isopropyl phosphoramidothioate ) can also be converted to 2,4-dichlorophenol in soil. Although the phenol is toxic at concentrations greater than 10 ppm, its concentration in nature is not known to be high enough to have an antimicrobial effect. Zytron can be degraded, moreover, by Aspergillus clavatus (Fields and Hemphill, 1966). A chlorophenol used for the control of a multitude of pests is pentachlorophenol. This compound is dehalogenated in soil, apparently by microorganisms, to yield a variety of chlorinated metabolites, many of which have antifungal activity and which probably are toxic to other species as well. The products include 2,3,4,5-, 2,3,4,6-, and 2,3,5,6-tetrachloro-, 2,4,5-, and 2,3,5-trichloro-, 3,4- and 3,s-dichloro- and %monochlorophenols ( Ide et al., 1972). These phenols are characteristically resistant to microbial attack (Alexander and Aleem, 1961; MacRae and Alexander, 1965) and hence may cause prolonged pollution. Another environmental difficulty was recently encountered from the use of chlorinated phenols. Penta- and 2,3,4,6-tetrachlorophenolare applied as fungicides to freshly cut timber and hence they appear in saw-

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

9

DAYS

FIG. 1. Changes in the concentrations of 2,4-D and 2,4-dichlorophenol in a soil and in a fresh water-sediment model ecosystem.

dust and wood shavings. The problem came to light owing to the presence of such treated wood products in the poultry house, where the phenols are apparently methylated microbiologically to yield penta- and 2,3,4,6-tetrachloroanisole.The anisoles then appear in the chicken flesh and impart a musty taint to the chickens. The phenols are not methylated in autoclaved poultry litter, attesting to the role of microbial colonists. Such methylation reactions can be carried out in axenic cultures of Aspergillus, Penicillium, and Scopulariopsis (Curtis et al., 1972). The methyla-

10

MARTIN ALEXANDER

in a

tion is not restricted to the highly chlorinated phenols because even 2,4-dichlorophenol can be O-methylated by a strain of Arthrobacter to give 2,4-dichloroanisole ( Loos et al., 1967b). Other chlorinated pesticides are subject to enzymatic modification to yield possible environmental pollutants. An interesting case is the herbicide propanil ( 3',4'-dichloropropionanilide ) , which is transformed in soil to 3,4-dichloroaniline, and the latter then gives rise to 3,3',4,4'tetrachloroazobenzene. Formation of the azobenzene requires microorganisms since none is observed in sterilized soil receiving either propanil

MICROBIAL FORMATION OF ENVlRONMENTAL POLLUTANTS

11

or the dichloroaniline (Bartha and Pramer, 1970). The herbicide can be converted to the tetrachloroazobenzene in nutrient media or sterilized soil inoculated with a mixture of Penicillium piscurium and Geotrichum cundidum (Bordeleau and Bartha, 1971), while the azo compound can be formed from 3,4-dichloroaniline in Fusarium oxysporum cultures (Kaufman et al., 1972). The reason for potential environmental concern is that both 3,4-dichloroaniline and 3,3’,4,4’-tetrachloroazobenzene are mutagenic. It has been proposed that subterranean mutagens might alter the virulence of soil-borne pathogens so that they become more harmful to agricultural crops (Prasad, 1970). The chlorinated aromatic herbicide known as dichlobenil ( 2,6-dichlorobenzonitrile ) on occasion induces a leaf margin chlorosis in crops. The chlorosis is attributable not to the added chemical, but to the 2,6dichlorobenzamide generated from it. This synthesis of a phytotoxin with a somewhat different range of susceptible plants than its precursor phytotoxin results from microbial activity, and the new toxicant is apparently quite resistant to further enzymatic modification in nature (Verloop, 1972). It is also possible for microorganisms themselves to synthesize the herbicidal 2,6-dichlorobenzonitrile from the nonphytotoxic c~-amino-2,6-dichlorobenzaldoxime, a process occurring both in soil and in cultures of Pseudomonas putrefaciens ( Milborrow, 1963). Diuron, 3- ( 3,4-dichlorophenyl)-1,l-dimethylurea, is another popular herbicide. This compound is demethylated in soil to give rise to a new phytotoxin, 3-( 3,4-dichlorophenyl)-1-methylurea ( Dalton et al., 1966). Such dealkylations are common to microorganisms. A notable instance of a microbiologically induced altered spectrum of action, one which led to a significant change in agricultural practice, is evident in the case of pentachlorobenzyl alcohol. This compound was introduced into Japan for the control of the blast disease of rice. The chemical was effective and showed no phytotoxicity even when applied to the foliage of test plants at concentrations of 2000 to 10,000 ppm. Two years after its introduction, however, reports arrived of deleterious effects on tomato, melon, and cucumber plants resulting from its use. The symptoms were particularly severe if the plants were grown in soil containing compost made from rice plants receiving the fungicide the year before. Analysis of the rice straw inducing these changes demonstrated that it contained pentachloro- and 2,3,4,6- and/or 2,3,5,6-tetrachlorobenzoic acids, all of which are remarkably effective in suppressing the growth of leguminous and solanaceous plants. Laboratory trials then revealed that the same polychlorinated benzoates were formed in soil. The first phase in the chemical transformation involved the oxidation of pentachlorobenzyl alcohol to the corresponding benzoic acid. This oxidation did not occur and no phytotoxicity was evident in sterile soil

12

MARTIN ALEXANDER

receiving the alcohol, and bacteria, actinomycetes, and fungi in culture brought about by the same oxidation; hence, the microscopic community is implicated. As a result of this outcome, the production and marketing of these fungicide preparations terminated in 1968 (Ishida, 1972). Pentachloronitrobenzene, a fungicide applied to seed and soil for protection of crops against pathogens, is converted in soil, apparently by its residents, to pentachloroaniline. It also can be reduced to the aniline by fungi in culture. The product of the reduction then suppresses the growth of fungi and actinomycetes, although the aniline is less fungitoxic than the nitro compound (KO and Farley, 1969; Nakanishi and Oku, 1969). Thus, one antimicrobial agent can be generated from a second. Reactions in nature presumably brought about by microorganisms may convert antimicrobial compounds to phytotoxins. An excellent illustration is the partial dehalogenation of 2-chloro-6-( trichloromethyl ) pyridine, a chemical of practical valuc because it suppresses the nitrifying bacteria. By inhibiting the oxidation of ammonium to nitrate, it can prevent much of the loss of inorganic nitrogen from farm land since nitrate, but not ammonium, can be denitrified and the anion is readily washed through the soil. A recent report provides data showing that 2-chloro-6-(trichIoromethy1)pyridine is converted in soil to 6-chloropicolinic acid (Fig. 3 ) . The latter is more toxic than the former to tomatoes, cotton, sugar beets, and legumes (Geronimo et al., 1973). Chloral hydrate, a substance with herbicidal action, is converted rapidly in soil to trichloroacetic acid, the latter likewise being a useful herbicide. Microorganisms are reported to participate in the transformation ( Schutte and Stephan, 1969). The important fungicide thiram ( tetramethylthiuram disulfide ) is acted on, in part at least biologically, to yield products that are harmful to totally dissimilar organisms. For example, it is converted in the rumen, presumably by the indigenous microbiota, to CS, and probably H,S (Robbins and Kastelic, 1961). The former is fungicidal; the latter affects a broad range of organisms. CS, is also evolved from soil treated with thiram (Munnecke et al., 1962). As discussed below, thiram can be cleaved and nitrosated, the result being a carcinogen. A product of its biological reduction is dimethyldithiocarbamate, a reduction effected by Glomerella cingulata (Richards and Thorn, 1960) and a number of algae ( Lindahl, 1964) . Dimethyldithiocarbamate is likewise antifungal and is commercially marketed as such as the sodium salt, (CH,),NC(=

FIG.3. Conversion of 2-chloro-6- ( trichloromethyl )pyridine to 6-chloropicolinic acid.

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

13

S)SNa. The new fungicide in turn can be converted, in vitro at any rate, by Saccharomyces cerevisiae to two additional antimicrobial agents, one a complex of the substrate with a-aminobutyric acid, the second apparently with a-ketobutyric acid ( Kaars Sijpesteijn and Kaslander, 1964). Another fungicide shown to be modified in vitro to give a new toxicant is benlate, the methyl ester of 1-(butylcarbamoyl ) -2-benzimidazolecarbamic acid. It is metabolized by Saccharomyces pastorianus, the methyl ester of benzimidazole carbamic acid being one of the products. The latter compound is inhibitory to Neurospora crassa and Rhizoctonia solani (Clemons and Sisler, 1969). Diphenamide ( N,N-dimethyl 2,2-diphenylacetamide) is subject to microbial activation, too. This is shown by the normal development of a test plant in sterilized soil containing diphenamide and by its inhibition if sown in a nonsterile sample amended with the same chemical. A possible mechanism for the microbial activation is indicated by the capacity of Trichoderina viride and Aspergillus candidus to demethylate diphenamide to yield two phytotoxins in sequence: first, N-methyl 2,2diphenylacetamide; second, 2,2-diphenylacetamide. The inhibition absent in sterile, diphenamide-amended soil appears if the soil samples are inoculated with the fungi ( Kesner and Ries, 1967). Microbial hydrolysis of pesticides formulated as esters may release the active ingredient. Thus, an enzymatic hydrolysis by components of the microflora leads to cleavage of the pentachlorophenyl laurate present in treated fabrics in contact with soil, and free pentachlorophenol is generated ( Allsopp et al., 1970). Ill.

Inorganic Nitrogen Compounds

A. AMMONIA The relative contribution of microorganisms to the release of nitrogen compounds, other than N,, to the atmosphere has not been appreciated, or sometimes not even considered, by biologists. However, recent estimates have dramatically emphasized the role that terrestrial and marine populations play in determining the composition of the atmosphere. They also more adequately put in perspective the relative importance of microbiological as contrasted with human activities in generating nitrogenous gases. According to the estimates of Robinson and Robbins (1970), the major nitrogenous compound released to the atmosphere from the surface of the earth is ammonia, and essentially all of this ammonia originates from biological sources, presumably mainly by heterotrophic activity on land and in the oceans. As shown in Table 11, the ammonia-N emitted

14

MARTIN ALEXANDER

TABLE 11 ESTIMSTED ANNUAL GLOBALFORMATION OF VOL.4TlLE NITROGEN COMPOUNDS"

Compound and source

NHI, from biological activity NIIs, from combustion N20, from denitrification N02,bfrom microbial activity N02,b from combustion of coal, gasoline, oil, natural gas and othcr combustion processes and from petroleum refining a

Tons N emitted per year, X lo8 4900 3 ,6

410 1.50 16.1

From Robinson and Itobbins (1970). NO plus NO2, but expressed as NO2.

by living organisms is more than 8 times that of the nitrogen released as nitrogen oxides from all sources combined. It is interesting to compare these figures with those of Emery et al. (1955), who estimated that denitrification in the oceans produced 70 x lo6 metric tons of N2 per annum. Thus, biologically evolved ammonia is apparently the major source of nitrogen gas emitted to the atmosphere. Nevertheless, the regional contributions to these global changes are far from clear. Ammonia is not only an atmospheric pollutant, but its production below ground may adversely and seriously influence plant roots. Both seed germination and the growth of seedlings may suffer because of its presence. Indeed, some of the deleterious effects associated with the decomposition of nitrogen-rich plant remains near roots may be attributable to the mineralization of organic nitrogen and its release as ammonia, and concentrations in excess of about 10 ppm may be lethal. The injury appears to result not from the ammonium ion but from ammonia itself, a toxin whose level is governed by the prevailing pH of the surroundings (Megie et al., 1967). A toxicity of ammonia to the proliferation of Nitrobacter in soil is well known and comparatively frequent when the pH is above neutrality (Stojanovic and Alexander, 1958); similar inhibitions occur in waters receiving considerable nitrogen in a reduced form. Atmospheric ammonia can be absorbed by lakes, rivers, and streams, and this uptake may thereby create a new pollution problem because it would enrich the surface waters with a nutrient that can be, either as ammonium or-following nitrification-as nitrate, utilized by indigenous algae to initiate an unwanted bloom. In addition, the cost of treating public water supplies could increase owing to the reduction by ammonia of the disinfecting action of Cl,. Ammonia is generated during the decomposition of the native organic

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

15

matter of soil, the microbial hydrolysis of urea and the decay of animal manure in soil. Considerable attention has been given to its rate of loss, and many studies have demonstrated that ammonia release is governed by soil type, climatic conditions, the presence of vegetation, and the application of nitrogen fertilizers. Alkaline pH, warm temperatures, high rates of evaporation, and low cation exchange capacity of the soil favor its volatilization. Kim ( 1973), for example, found appreciable differences in the rate of ammonia loss in the field from soil in a pine and an oak forest and from a grassland soil of Korea; 3.41, 2.62, and 1.84 kg ammonia per hectare per week were evolved from the pine and oak forest soil and in the grassland, respectively, in the May to July sampling period. Makarov and Ignatova ( 1964) reported that 0.42 to 1.60 kg ammonia per hectare per week was generated from fallow soils in the U.S.S.R. in June. By contrast, MacRae and Ancajas (1970) found 0.12 to 1.21 mg ammonia-N loss per kilogram of soil in 7 weeks in a study of four flooded tropical soils under laboratory conditions. The gas probably originates largely from the mineralization of humus by the indigenous bacteria and fungi. Ammonia formation is appreciable and its volatilization can be marked when a field receives organic nitrogen compounds that are readily cleaved microbiologically. This is especially true of urea, a common fertilizer. Urea is hydrolyzed so rapidly by the ubiquitous and abundant urease-containing heterotrophs that ammonia accumulates in large amounts, and the rise in alkalinity accompanying the urease-catalyzed hydrolysis accentuates further the volatilization. If the urea is applied directly on the surface of the land, a frequent farm practice, there is insufficient time for the ammonia produced to react with the soil so that its loss to the atmosphere is much more marked than if it is introduced below the surface ( MacRae and Ancajas, 1970; Nommik, 1966). The loss is also appreciable in naturally alkaline conditions but is reduced in acid environments; nevertheless, the alkalinity created during the microbial hydrolysis may raise the pH around the urea fertilizer particles to an extent that considerable of the gas evolves. Given the many factors governing the volatilization of ammonia from urea fertilizers, a range of values is to be expected, but up to 10, 17, and even 70%of the urea-N has been reported in different investigations to be converted to ammonia that disappears from the soil (Khan and Haque, 1965; MacRae and Ancajas, 1970; Wagner and Smith, 1958). A new and major, albeit local, source of the metabolite is the cattle feedlot. Several reports show that much more ammonia is present in the air near feedlots than in air at some distance away. Sometimes the difference in concentration is 20 times or greater. A significant part of this nitrogen comes from the manure undergoing decomposition, but

16

MARTIN ALEXANDER

a large part of the nitrogen in cattle excreta is derived from urine, some 90% of which can be converted to ammonia and volatilized in a single week (Hansen, 1941; Hutchinson and Viets, 1969; Luebs et al., 1973). In one investigation, it was noted that enough ammonia was absorbed in a lake about 2 km from a large feedlot to increase its nitrogen content to a level above that potentially required for the initiation of a significant algal bloom ( Hutchinson and Viets, 1969). The gas is also evolved during the decomposition of plant remains in soil, the breakdown of sewage and other carbonaceous materials in water and during the reduction of nitrate. For example, one-quarter of the nitrogen in grass clippings may appear as ammonia in 2 weeks (Salt, 1965). The wastes of municipalities, septic tanks, and dairy and meat-packing plants may likewise add the same product to the environment. Some may be emitted during the reduction of nitrate under anaerobic conditions, though this mechanism of release does not seem to be too significant ( Woldendorp, 1965). Little is known about ammonia emission from natural bodies of water. Volatilization is favored when the p H is above neutrality, as is common in oceans and lakes. The p H may increase to higher than usual values as a result of the photosynthetic activity of algae and higher aquatic plants. Such circumstances enhance the rate of volatilization.

B. NITRATE The end product of the microbial degradation and mineralization of organic nitrogen in aerated environments is nitrate, and this anion is ubiquitous in the oceans, inland bodies of water, groundwaters, nnd soils. Natural organic substrates are attacked by one or more popdations of bacteria and fungi, and the nitrogen in them is ultimately released as ammonium. In locales where 0, is present and the pH is not too low, the nitrifiers oxidize the ammonium to nitrate, the conversion usually being nearly stoichiometric. In soil, nitrate but not ammonium is readily transported downward with percolating water to enter the groundwater. The anion in the groundwater may then be carried laterally to wells used for drinking purposes and surface waters. The intensification of agriculture, the ever-increasing tonnages of synthetic fertilizers, the rapid growth of large urban regions and the development of industrial centers have both provided a larger quantity of nitrogenous substrates for microbial utilization and also concentrated these substrates in a smaller area so that the levels of the final product, nitrate, in the locales of its formation often have approached a point where public concern has been aroused. A major causc of apprehension is methemoglobinemia, a disease of

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

17

human infants and livestock associated with nitrate. The occurrence of clinical methemoglobinemia in infants attributable to water supplies is generally correlated with the nitrate level in the water, most of such cases being associated with water supplies containing in excess of about 22 ppm nitrate-N (Committee on Nitrate Accumulation, 1972). A partial summary of reported cases and fatalities in the United States and Europe is presented in Table 111. A total of about 2000 human cases, many of which were fatal, have been linked with the drinking of nitrate-polluted water (Gruener and Shuval, 1970). The malady is typically found in infants less than 6 months of age. Outbreaks of methemoglobinemia have been reported sporadically in cattle, and sometimes 10-30% of the animals in a herd may die. In certain regions, several thousand head of cattle may be lost in a single year. The onset of the disease is rapid after the animal has consumed the nitrate, and death may ensue in less than an hour after symptoms first become evident ( Committee on Nitrate Accumulation, 1972). A second major cause of concern is eutrophication, the enrichment of natural waters with nutrients. Eutrophication is a natural process, but its rate is markedly increased by the discharge of wastes from municipalities, industry, food processing plants, and intensive agriculture. Enrichment with nitrate is important only if the nutrient element limiting algal biomass in a particular lake, river, or stream is nitrogen, and an inflow of nitrate into such a nitrogen-poor body of surface water could trigger an unwanted bloom. The massive growths of algae or higher plants is undesirable because they frequently result in fish mortalities, off-odors and tastes in drinking waters, odors offensive to nearby communities, higher costs for water treatment, impairment of transportation in inland waterways and decline in the recreational value of the lake or stream. Some of these problems arise directly from the enormous masses of vegetation and cell material, but some come about because of products emanating from the microbial decomposition of the biomass. TABLE I11 REPORTSO F HUMANMIOTHEMOGLOBINEMIA IN S E V l e R l L DIFFERI4;NT S U R V E Y S a

Region

Years

No. of cases

No. of fatalities

United States

1945-1950 1962-1966 1960-1969 1948-1964

ca. 278 40 ca. 10 ca. 1,000

39 0 0 ca. 80

Europe

From Committee on Nitrate Accumulation (1972).

18

MARTIN ALEXANDER

Public health agencies have established limits or standards because of the potential hazard of methemoglobinemia to infants, and both the U.S. Public Health Service and the World Health Organization recommend that water for human consumption should contain no more than 10 ppm of nitrate-N. It is sometimes stated that surface water with greater than 0.3 pprn nitrate-N may support excessive algal growths, provided that other essential nutrients for these organisms are also available. Other hazards from the consumption of nitrate-rich water, food, or feed have often been postulated, but the one getting increasing attention is the nitrosamine hazard. Carcinogenic nitrosamines can be formed from nitrate in the presence of appropriate amines. Indeed, it has even been reported that gastric cancer is correlated with the presence of nitrate-rich drinking water in an area of Colombia (Drasar and Hill, 1972). Nitrosamines will be considered below. An enormous amount of environmental monitoring attests to the increase in nitrate levels in certain rivers-for example, the Ohio, Delaware, and San Joaquin Rivers in the United States. Similarly, the nitrate concentrations in runoff waters in some watersheds are higher than known heretofore, and many surface waters contain more than 0.3 pprn of nitrate-N (Committee on Nitrate Accumulation, 1972). In Illinois, for example, all streams that have been regularly sampled since 1945 contain in excess of 0.3 ppm, and the concentration in some, and in the water reservoirs into which they run, exceeds 10 ppm. These high nitrate levels in Illinois are in areas where the soils are fertile, and hence a good part of the humus nitrogen might have been mineralized and then entered the water, and where fertilizers are extensively used (Harmeson et al., 1971). A recent survey found that 19 of the 969 water supply systems tested in the United States had nitrate in quantities exceeding the 10 pprn recommended limit, an occasional one containing up to 29 ppm, and that many groundwaters likewise exceeded the recommended maximum (Committee on Nitrate Accumulation, 1972). Similarly, most wells designed as sources of potable water in parts of the coastal plain of Israel have 10-20 ppm (Gruener and Shuval, 1970). It is clear, therefore, that microbial activities are generating a water pollutant in quantities that are no longer acceptable from the public health standpoint as well as in amounts that may promote nuisance blooms of algae. Human infants may also receive excessive nitrate in the foods they eat. In this instance, the microbial contribution to the genesis of the potential toxicant is through thc production in soil of nitrate, which is assimilated through the roots and accumulated in the above-ground portions of the plant. Beets, spinach, celery, and lettuce are prominent

hlICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

19

nitrate accumulators among the vegetable crops. Typical values for beets, radishes, lettuce, spinach, and celery are in the range of 0.09-0.84, 0.39-1.50, 0.02-1.06, 0.07-0.69, and 0.11-1.12% nitrate-N, respectively (Committee on Nitrate Accumulation, 1972; Lee et al., 1971). What are the primary nitrogen sources that are acted on microbiologically? Estimates of the quantities of the various substrates available to microorganisms suggest that the chief sources in the United States are synthetic fertilizers, the organic fraction of soil, animal and human excreta, and food processing wastes. The quantity coming from industry is unknown. For the year 1970 in the United States, 7.5, 4.2, and 1.2 million metric tons of nitrogen came from fertilizers and animal and human excreta, respectively, each of which to a large extent ultimately undergoes nitrification. A total of about 3.1 million metric tons of humusN, or about 40 kg of N per hectare per year for the total cropland, is mineralized, and much of this, too, is converted to nitrate. Wastes from dairy and poultry products, hides and leather, canneries, sugar refining and other food-processing operations contribute about 6.5 million tons of substrate-N for microbial utilization per annum. About two-thirds of the nitrogen in rainwater is in the ammonium form, and such precipitation contributes some 5.6 million tons to the soil (Committee on Nitrate Accumulation, 1972). Hence, the quantity of nitrogen available for microbial degradation and conversion to nitrate is enormous. Estimates that sizable quantities of nitrate are synthesized both from native soil organic matter and fertilizers are borne out by field studies. In considering such investigations, it is well to remember that, though much more nitrate is discharged from fertilized than unamended soil, only a portion of the total land mass receives fertilizer. The contribution of fertilizer nitrogen to nitrate accumulation probably has been accentuated in recent years by the enormously rapid increase in fertilizer use during the last two decades in the United States (Davis, 1973) and other technologically advanced countries, and the problems will probably begin to appear in the developing countries if projections of their greater reliance on synthetic fertilizers, such as those of Ewe11 (1972), are valid. Field studies of nitrate accumulation and movement in soil are numerous. These investigations show that small amounts accumulate in forest soils and enter adjacent waterways. In cropped land, the extent of nitrate accumulation and movement into groundwaters is extremely variable, and the quantity is dictated by climate, soil characteristics, presence and type of vegetation, and the quantity of fertilizer used. The nitrate-N level of the watertable in some fields may be as low as 0.1 ppm or greater than 40 ppm. Practices that improve soil fertility, in addition to fertilizers, generally increase the quantity of nitrate that may be formed and lost. High fertilization rates, however, generally lead to

20

MARTIN ALEXANDER

large nitrate accumulations, and a substantial part of the fertilizer-N in certain agricultural practices, which are often economically as well as environmentally unsound, may be lost as nitrate to the underlying water. A particularly acute problem of nitrate pollution arises in the livestock feedrot or in other practices that lead to confinement of large numbers of animals in small areas. Because most of the difficulties in handling large numbers of animals in confined areas have been overcome, farmers have recently adopted the practice of maintaining many cattle or chickens in feedlots or large poultry houses, whereas they formerly kept the animals in open land. More than 30,000 head of cattle or 250,000 chickens are not uncommon in a single operation. These feedlots or chicken houses thus receive excreta in copious amounts. A feedlot with 30,000 steers creates a nitrogenous waste equivalent in tonnage to a city of 250,000 people, and nitrogen in the droppings on a single large poultry farm may be equivalent to the nitrogen discharged by a city of 25,000. At the present time, few satisfactory and economical methods exist to treat and dispose of the manure coming from feedlots, and hence the nitrate load in the underlying soil has gone up markedly. Nitrate-N concentrations of greater than 10 ppm are common, and levels in excess of 50 ppm are not unknown (Stewart et ul,, 1967; Webber, 1971). The nitrate then moves with the underwater flow to pollute nearby wells, reducing the quality of the water to a point that it is no longer fit for human consumption. Even the application of sewage sludge to the land may lead to nitrate pollution. Land disposal of sewage is under active consideration, nevertheless, to prevent nitrogen contamination of surface waters. However, King and Morris (1972) demonstrated that such sludge, when much is added in a field, brings about an undesirably high nitrate level in the underlying soil.

C. NITRITE Nitrite is an intermediate in autotrophic nitrification and in the reduction of nitrate to ammonium for assimilatory purposes or to nitrogenous gases in a reaction sequence catalyzed by the denitrifying bacteria. As a rule, this anion does not accumulate in nature because the rates of its formation in both oxidative and reductive pathways are usually less than the rate of its further metabolism. Under certain circumstances, however, its synthesis during nitrate reduction or ammonium oxidation is more rapid than its further transformation, and it then accumulates. Because nitrite is quite toxic to humans, animals, and plants, this accumulation has attracted interest.

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

21

The early literature of animal toxicology contains many studies of the effects of nitrite on test animals. One of the most striking aspects of these investigations is the evidence showing that it is nitrite, not nitrate, that is the actual toxicant in methemoglobinemia. The more oxidized ion is absorbed and excreted readily by mammals, but certain conditions are conducive to the microbial reduction and accumulation of nitrite in the gastrointestinal tract. The nitrite thus formed by the gastroenteric microflora reacts rapidly with hemoglobin to form methemoglobin, and the capacity of the protein to combine with and then transport Oz is thereby lost (Gruener and Shuval, 1970). Thus, a microbial product, nitrite, is the real hazard of nitrate-induced methemoglobinemia in infants and ruminants. Nitrite also provokes headaches in certain people (Henderson and Raskin, 1972). Though this facet of its toxicity may explain why susceptible individuals get headaches after consuming frankfurters or other cured-meat products that are treated with nitrite, no data have yet been obtained to suggest that microorganisms are involved in the development of headaches. A possibly consequential source of nitrite is the nitrate-rich vegetable. In addition to the reactions that yield nitrite in the alimentary tract when such produce is consumed, nitrite may be formed in or on the plant tissues themselves. Keybets et u1. (1970) reported that in commercial spinach, which when fresh contains little or no nitrite, the nitrite level rose to 251 pprn in one week if the vegetable was stored at 20-23°C and to 78 pprn when stored 2 weeks at 4"C, a temperature comparable to that in many refrigerators. In an independent investigation, P. L. Minotti (unpublished observations) found nitrite in 50% of the bags of prepackaged fresh spinach that were purchased at weekly intervals for 20 weeks, the nitrite-N content ranging from 2 to 320 ppm on a dry-weight basis or 0.2 to 32 ppm on the basis of the fresh weight of the vegetable. After a one-week storage period in a refrigerator at SoC, 17 of the 20 weekly samples contained nitrite, the nitrite-N level ranging from 2 to 340 ppm on a dry-weight basis or 0.2 to 34 pprn on a fresh-weight basis. Of the 10 with no nitrite at the time of purchase, 9 had this anion produced during the storage at S0C, with 2 to 170 ppm being formed in the one week. Furthermore, relatively large amounts of the toxicant appeared in cans of spinach (which were free of nitrite) after they were opened and in samples of frozen spinach that were allowed to thaw if these samples were stored for several days at 8°C; in some instances, more than 750 ppm nitrite-N (on a dry-weight basis) appeared. Although some of the nitrite may be synthesized by the action of plant enzymes reducing the nitrate in the spinach, bacterial reduction

22

MARTIN ALEXANDER

is quite likely. Furthermore, the activity in the opened cans results from microbial colonization of the vegetable. Selenka ( 1971) also examined the possible role of bacteria in the formation of nitrite in baby foods. Fortunately, it is not common to store canned or frozen spinach after the container is opened, by contrast with the practice with fresh vegetables, but it is likely done in some households. This possible source of nitrite and the role of bacteria in the reduction clearly require evaluation. Nitrite is also inhibitory to the development of higher plants. For example, avocado and citrus are deleteriously affected by 5.0 ppm nitrite-N at pH 5.0, 10 ppm at pH 5.5, and 20 ppm at pH 6.0 (Curtis, 1949). Accumulations of these magnitudes are not uncommon in soil, although the accumulation during nitrification is significant only under alkaline conditions, where nitrite is least phytotoxic. Nitrite typically is prominent in neutral or alkaline soils receiving a variety of fertilizers when the fertilizer application rates are sufficient to produce high concentrations of ammonium-ammonia, Quantities as great as 90 ppm may appear, and the product sometimes persists for several months (Chapman and Liebig, 1952; Martin et al., 1942). In urea-treated soils, even if they are initially acid, the pH rises as ammonia is evolved immediately around the fertilizer particle, and the combination of high local pI4 and ammonia concentration also favors nitrite buildup; Wagner and Smith (1958), for example, reported 94 ppm nitrite-N produced from 500 ppm of urea-N added to a soil with an initial pH of 5.6. Nitrite appears also in a waste-treatment system now under active consideration for the prevention of nitrate pollution from the disposal of farm animal manure, a process in which the manure undergoes aerobic biodegradation and the ammonium thereby released is allowed to nitrify. The system is then made anaerobic in the presence of additional organic matter, so that the nitrate formed in the first phase is subject to denitrification. Not surprisingly, the nitrite level rises owing to the presence of considerable ammonium-ammonia and the high pH ( Prakasam. and Loehr, 1972). On the basis of studies of soil samples in the laboratory and pure cultures of the responsible bacteria, it appears that the nitrite level rises because Nitrobacter or a physiologically related autotroph is selectively inhibited at high pH by the ammonium-ammonia added or formed, the pH effect suggesting that the actual inhibitor is ammonia (Aleem and Alexander, 1960; Stojanovic and Alexander, 1958). Nitrite toxicity is sometimes a practical problem when flowers are germinated in composts containing soil that is steamed or otherwise heated. The affected plants are stunted and chlorotic and, in extreme cases, die. The phytotoxicity results from the appearance of nitrite in those composts that are alkaline and into which large amounts of nitrogenous organic materials are introduced (Birch and Eagle, 1969). The

lLiICROBIAL FORhfATION OF ENYIRONMENTAL POLLUTANTS

23

accumulation may be attributable to the selective suppression of nitriteoxidizing bacteria by ammonia so that the end product of Nitrosomonas metabolism is not oxidized further, but it is not clear whether heating is necessary for the production of the toxicant. High nitrite levels sometimes are evident in waterlogged soils, presumably being produced by the indigenous nitrate-reducing bacteria. According to Ford (1965), an important factor in the death of citrus roots under waterlogged conditions may be the nitrite thus synthesized.

D. HYDROXYLAMINE Several reports show or suggest that hydroxylamine, a potent mutagen, is present in natural bodies of water. For example, hydroxylamine was observed in the water of a lake in central Japan, the compound appearing at times of the year when 0, became deficient in lower regions of the lake. The nitrate concentration fell as a consequence of the activity of nitrate-reducing bacteria, and coinciding with the decline was an increase in both the nitrite and hydroxylamine concentrations ( Tanaka, 1953). Circumstantial evidence was provided recently that hydroxylamine occurs at all depths of a crater lake in Ethiopia (Baxter et aZ., 1973). No survey has yet been made from which it might be decided whether these occurrences are sporadic and limited to certain bodies of water or whether the compound is more widespread. It has been claimed that the mutagen was produced during the decomposition of Aphanizomenon flos-aquae in tanks and aquaria. Decay of the alga was accompanied by the death of fish inhabiting the tanks and aquaria, and Prescott (1948) proposed that hydroxylamine was the lethal agent. The mortality was not the result of 0, depletion resulting from growth of bacteria on the decomposing algae inasmuch as the 0, level did not fall appreciably. Unfortunately, neither quantitative data nor analytical methods were given so that the validity of the claim cannot be evaluated. Hydroxylamine also can be released, in oitro at any rate, during autotrophic nitrification. Thus, Nitrosomonas europaea produces up to 2.6 ppm of NH,OH-N from ammonium in the presence of the inhibitor hydrazine ( Yoshida and Alexander, 1964). Hydrazine is unquestionably a substance not commonly encountered in nature, and data suggesting that natural environmental stresses lead to such an accumulation are lacking. Another mechanism for hydroxylamine biosynthesis has been observed in axenic cultures and in samples of natural ecosystems. The activity seems to be a phase of heterotrophic rather than autotrophic nitrification. The first evidence for this type of reaction came from a study of a

24

MARTIN ALEXANDER

sewage-derived Arthrobacter strain that oxidized ammonium to hydroxylamine under normal growth conditions and at several pH values. The hydroxylamine-N reached a concentration of 15 ppm in growing cultures. By contrast, resting cells of the bacterium excreted as much as 60 ppm hydroxylamine-N/ when provided with ammonium, and they formed the same product from acetamide, glutamine, or glutamate but not from glycine or 1-aminoethanol. By contrast with the oxidation by N . europaea, the heterotrophic process requires a source of organic carbon, possibly because the ammonium needs to be bound first into an organic compound, which is then oxidized to the hydroxamate that is finally cleaved by the organism to give free hydroxylamine (Verstraete and Alexander, 1972a,b). A pattern of hydroxylamine formation from ammonium almost identical to that observed in cultures of Arthrobacter sp. has been noted in sewage, river water, and lake water amended with acetate or succinate, the maximum yield being 28.0, 3.1 and 0.1 ppm NH,OH-N in samples of these ecosystems, respectively. The pattern of release of other nitrogenous metabolites in sampIes of these waters (Fig. 4 ) was remarkably similar to that occurring in cultures of the sewage bacterium (Verstraete and Alexander, 1973). Because the process has been found in samples of several microbial habitats, the heterotrophic oxidation of ammonium to hydroxylamine may be widespread. It would be interesting to establish whether spontaneous mutations in natural habitats might not occasionally be attributable to mutagens, like hydroxylamine, excreted by microorganisms. 30 I.\\

/ \

\\NH,OH

I

$ -

20-

l

m

a

2-

I \

I

0

z

\

I

a

t

\\

/

W

U

y

/

\

I

\ \, NITRITE

I

10-

I

\\ O ' \

/O /

A/-

-t&.--A---A 1

0

CH,-FHOH-'

-._. NO\

I

MICROBIAL FORMATION OF ENVlRONMENTAL POLLUTANTS

25

E. NITROGEN OXIDES The nitrogen oxides are a highly important group of air pollutants, and atmospheric scientists and regulatory agencies have characterized the reactions of these oxides and their sources, and governmental agencies have sought means of reducing the quantity discharged in various combustion reactions. Yet, whether consideration is given only to NO and NO,, which are often of primary interest, or also to N,O, an examination of the estimates summarized in Table I1 leads one to conclude that the chief culprits on a global scale, though not in municipal or highly industrialized areas, are not automobiles or factories, but rather microorganisms. Bacteria make NO and not NO,, but the former gas is oxidized in the atmosphere to NO,. The values in Table I1 suggest that manmade emissions of these oxides represent only a small portion of the total, microorganisms possibly being more active by a factor of nine or more. Though N,O is not of prime concern relative to pollution, its formation will be discussed because of the similarity in factors affecting its synthesis and that of the other oxides and the greater body of information on N,O evolution. Many bacteria generate nitrogen oxides. More than 60 years ago, Beijerinck and Minkman (1910) reported that N,O was liberated by denitrifying bacteria supplied with nitrate or nitrite. Evolution of N O was also noted in early studies of the denitrifiers (Lebedev, 1911). Among the genera containing a few or many species able to reduce nitrate to gaseous products in culture are Achromobacter, Bacillus, Chromobacterium, Cytophaga, Micrococcus, Pseudomonas, Serratia, Thiobacillus, and Vibrio, although not all have been shown to evolve one or more oxides. Recent work has disclosed that nondenitrifying bacteria and fungi also make N,O; for example, Aspergillus flavus, Penicillium atrovenetum, Fusarium oxysporum, and F . solani from nitrite ( Bollag and Tung, 1972; Yoshida and Alexander, 1970), Bacillus subtilis, Escherichia coli, and Aerobacter aerogenes from nitrate and nitrite, and Nitrosomonas europaea under stress conditions from ammonium ( Yoshida and Alexander, 1970). Most bacteria using nitrate or nitrite as an alternate electron acceptor when 0, is unavailable produce N, as the major volatile nitrogenous product, but N,O is often found, frequently appearing before N, and then disappearing as N, is formed. Some bacteria generate large quantities of N,O, and at certain phases in the growth cycle, it may account for up to 67.5% of the composition of the gas phase (Kluyver and Verhoeven, 1954). Occasional cultures may be unable to reduce nitrate or nitrite to N,, and N,O may then be the final product of the reductive sequence, as with strains of Cytophaga (Cook, 1962) and Corynebacterium nephredii (Hart et al., 1965). NO is often

26

MARTIN ALEXANDER

detected, usually in small quantities, during the process of denitrification effected by growing cultures or resting cell suspensions of the active bacteria. However, the major product of nitrite reduction by Pseudornonas denitrificans may sometimes be NO ( Matsubura, 1971). The individual enzymatic steps in the biosynthesis of these gases and the sequence of their formation have been well characterized. After its formation from nitrate, nitrite is acted upon by nitrite reductase and is reduced to NO. This activity requires NADH or NADPH and a flavin (Radcliffe and Nicholas, 1968). Although some investigators felt that the next step also might be catalyzed by nitrite reductase, Payne et al. (1971) successfully separated nitrite and nitric oxidc reductase activities, the latter enzyme converting NO to N,O. For NO reduction, NADH or NADPH and flavins are also generally required (Najjar and Chung, 1956). The cofactors needed vary, however, in enzyme preparations from denitrifying bacteria. Furthermore, N,O may be produced enzymatically in a reaction involving both nitrite and hydroxylamine (Iwasaki and Matsubara, 1972), but the significance of this reaction NHzOH f HNOZ 4 NzO

+ 2H20

(4)

to the denitrifying bacteria, many of which are not known to make free hydroxylamine, remains unclear. The N,O, in turn, may be reduced to N, if it is not lost from the cultures or from the natural environment. Hence, the pathway for complete reduction, when it occurs, may be visualized to proceed usually as follows:

Many analyses have been performed of the emission of nitrogen oxides from soil. These were prompted initially by observations that N,O in the atmosphere was more concentrated near the earth's surface than at higher altitudes, the concentration gradient suggesting that atmospheric N,O originates from soil processes. These analyses have revealed that N20, NO, and NO, are discharged from the land mass to the atmosphere, the last compound presumably arising by a nonbiological oxidation of NO. On the basis of laboratory research with liquid media, it is generally believed that denitrification only occurs under anaerobiosis, but even a well aerated soil in the field contains anaerobic microenvironments, probably especially in the vicinity of readily available organic matter, and here the nitrate produced by aerobes may undergo denitrification. The work of Wijler and Delwiche (1954) using nitrate-amended alkaline soil maintained in a closed system, wherein gaseous products that otherwise would have escaped are further metabolized, revealed that

MICROBIAL FORMATION O F ENVIRONMENTAL POLLUTANTS

27

N,O appeared rapidly during denitrification and then disappeared with time as the N, level rose. The further reduction of N,O became less significant and,the N,O persisted longer in more acidic soils. NO was generated at the lower pH values, and it was almost equal to N,O production at pH of ca 4.9. The relative amounts of N,O and N, were also determined by soil moisture content, partial pressure of 02,and initial nitrate concentration. Because much of the N?O in an open system, as in the field, might be discharged to the atmosphere before it can be reduced further by the responsible bacteria, the relative losses of N,O in natural ecosystems may be appreciably higher than might be suggested from incubations in sealed containers. Similarly, NO appears to be reduced in soil so that its emission to the atmosphere may be greater than might be anticipated from tests conducted in sealed containers. Nommik (1956) reported that N,O was a more significant component, as compared to N,, in soils as the aggregate size and nitrate levels increased. He confirmed that in soil below pH 5.0, little or no N, was formed, the chief gas being N,O. However, little nitrate is produced in highly acid environments owing to the sensitivity of the autotrophic nitrifiers to low pH, so that an evaluation of tests with nitrateamended acid soils must be tempered with knowledge also of the factors affecting ammonium oxidation. Some quantitative values for rates of evolution of nitrogen oxides measured in the field are given in Table IV. NO may also originate not from an enzymatic reduction, but rather by a chemical decomposition of the nitrite formed microbiologically from nitrate or ammonium. NO, and small amounts of N,O are liberated nonbiologically when acidic soils are treated with nitrite, most of the NO, being made as a result of the spontaneous oxidation of the NO coming from the decomposition of nitrite (Nelson and Bremner, 1970). This is well illustrated in the study of Steen and Stojanovic (1971), TABLE IV EVOLUTION O F NITROGEN OXIDESFROM SOIL I N Gas

NO NO?* N20 NzO a

THE

FIELD

Recent fertilization

Gas evolved, as N (wlhalday)

Reference

NH4N03 (In pine forest) (In oak forest) (In grassland) NHaNO3 NaN03

0.2-0.4 9.1 5.2 8.3 2.7-7.2 10-280

Borisova ct al. (1972) Kim (1973) Kim (1973) Kim (1973) Borisova cf uZ. (1972) Burford and Stefanson (1973)

Presumably formed from NO.

28

MARTIN ALEXANDER

in which soil amended with urea released much of the added N as NO. Accompanying the hydrolysis of the urea was an accumulation of ammonia, the latter then inhibiting the last phase of nitrification so that nitrite built up; e.g., a concentration of 50 ppm nitrite-N developed in soil initially having 300 ppm urea-N. To confirm that the NO was from a nonenzymatic reaction, sterile soil samples were supplemented with ammonium and nitrite in concentrations equivalent to those occurring in the nonsterile soil at the time of maximum NO evolution, and the NO losses from these sterile samples were equivalent to those in the original test. Similarly, solutions with nitrite and urea or an ammonium salt volatilized NO in appreciable amounts. However, rates of NO production comparable to those reported by Steen and Stojanovic (1971), namely 8.9-35.5 kg of NO-N per hectare in 4 weeks, are far in excess of those noted in the field, and it is unlikely that such high nitrite concentrations are ever encountered except immediately after fertilization and in close proximity to the fertilizer itself. Much less is known about the genesis of nitrogen oxides in the sea, although N,O is formed in surface waters (Junge and Hahn, 1971). The finding of unexpectedly high N,O levels in regions of the central Pacific may indicate that the oceans, too, are a source of nitrogen oxides (Craig and Gordon, 1963). Biologically induced intoxications resulting from nitrogen oxides have occurred among farmers making silage from nitrate-rich plants. During the early phase of the fermentation of tissues of plants such as corn and oats, some of the excessive nitrate is converted to NO, which on exposure to air is oxidized to the toxic NO,. Poisonings and several deaths have ensued (Delaney et al., 1956; Lieb et al., 1956). Wang and Burris (1960) demonstrated that the abundance of NO in a field silo filled with corn may reach 9.7 volume percent, and the figure may be LIP to 47.2 volume percent in an artificial silo containing corn tissues. They suggested that enzymes of the plants as well as those of the fermentative bacteria are involved in making NO. IV.

Simple Carbon Compounds

A. E T H ~ E N E Ethylene has achieved considerable prominence as an atmospheric pollutant. It has also been extensively studied because of its importance in regulating various aspects of the physiology of higher plants. This gas at remarkably low concentrations may cause abscission of leaves, flowers, and fruits, accelerate plant senescence, enhance the degradation of chlorophyll, make certain flowers droop, increase respiration rate and

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

29

sugar content of plants, stimulate sprouting of bulbs and roots, inhibit growth of shoots and leaves, and hasten the ripening of unripe fruits. If present below ground, it can influence root elongation and the formation of lateral roots, thus potentially having a serious effect below as well as above ground. The recent investigations of Smith (1973) suggest that it may inhibit the proliferation of fungi in soil. Soils under anaerobiosis form considerable quantities of ethylene, and this evolution has a microbial origin because the process is largely abolished by sterilization procedures. The biogenesis of the alkene is inhibited by both 0, and nitrate, and the rate of its production in laboratory trials has been reported to range from 0.6 to 24 pg/kg of soil in a 10-day period. Ethane, propane, and propylene can also be generated in quantities up to a maximum of 0.6, 3.1, and 2.1 pg/kg in 10 days. The ethylene concentration in the soil varies with season of year, but the level is frequently sufficiently high to inhibit markedly the development of plant roots (Dowdell et al., 1972; Smith and Restall, 1971). The evolution is enhanced by addition of certain organic compounds to soil (Lynch, 1972). Although most studies emphasize the importance of the need for 0, deficiency in soil, the investigations of axenic cultures have centered on aerobes, particularly the fungi. Many fungi indeed can produce the gas. For example, Ilag and Curtis (1968) reported that species of Alternaria, Ascochyta, Aspergillus, Botrytis, Cephalosporium, Chaetomium, Dematium, Hansenula, Myrothecium, Neurospora, Penicillium, Schizophyllum, Sclerotinia, Scopulariopsis, Thamnidium, and Thielavia as well as selected soil actinomycetes are active. Individual cultures of Agaricus, Fusarium, Pyronema, and Verticillium also synthesize ethylene ( Hutchinson, 1973), and Penicillium digitatum has been a popular choice for physiological research on the in vitro process. Lynch (1972) proposed that Mucor hiemalis may be particularly important in ethylene evolution from certain soils. Among the bacteria, Pseudomonas solnnacearum can make the hydrocarbon, the organism in 20 ml of culture medium, for example, producing 0.35 mg ( Freebairn and Buddenhagen, 1964). B. CARBONMONOXIDE Most of the CO in the atmosphere is derived from combustion processes in the automobile engine, the burning of coal and fuel oil, industrial discharges, and the incineration of the solid wastes of human populations. Hence, man is chiefly responsible for the creation of this pollutant. Nevertheless, photosynthetic and heterotrophic microbial populations in the sea and on land may contribute to the genesis of this pollutant, too. Analysis of samples from Atlantic and Pacific Oceans reveal that the

30

MARTIN ALEXANDER

surface waters of both are supersaturated with respect to the partial pressure of CO in the air, with the concentration of dissolved CO sometimes being 15- to 75-fold greater than the atmospheric equilibrium values. Therefore, a transfer of this gas from the sea to the atmosphere must occur, and part of the air's store of CO must originate in the seas. It has been calculated, moreover, that the oceans thus emit some 9 X lo9 kg of CO per year, or about 5% of the total formed on the earth's surface. The production of CO is related to the presence of light since its formation in sea water follows a diurnal cycle, the maximum rate of evolution taking place in daylight (Lamontagne et al., 1971; Swinnerton et al., 1970). The light-affected evolution may reflect, in part, not a process brought about by photosynthetic organisms but rather a nonenzymatic photochemical reaction because CO, as well as ethylene, is liberated from sterile seawater into which is introduced organic products made by the phytoplankton (Wilson et al., 1970). On the other hand, the mechanism of formation may be largely biological because investigations by D. F. Wilson and his collaborators (cited by Lamontagne et al., 1971) demonstrated that, though this pollutant is indeed evolved from sterile sea water, most of the CO more likely is derived from the metabolic activity of marine organisms. The gas may come from the algae themselves or from the bacterial degradation of the phytoplankton and other carbonaceous substances in the surface waters. It has also been suggested that the actual annual rate of CO production is about 5 x 10" kg (Weinstock and Niki, 1971), which is far greater than the 2 x 10'l kg calculated to be released from combustion processes (Robbins et al., 1968), so that biological or other natural sources may be far more important than considered to be heretofore. In addition to marine environments, soil may be a source of CO. For example, Robinson (1930) observed that CO was a significant gas emitted from submerged soils incubated in the laboratory. He noted that 2.8-3.2% of the gas phase was composed of CO. More recently, Dobrovol'skiy et al. (1960) found small but still readily detectable quantities of the compound in the soil atmosphere. Experiments with individual cultures also attest to the activity of algae, bacteria, and fungi. One of the more interesting organisms is the giant kelp, Nereocystis, which Langdon (1917) reported to contain from 1.2 to 12.251: CO in the floater part that buoys up the alga. More recent work has verified that this kelp indeed gives rise to CO. The compound is made by other algae; e.g., by species of Egregia, lridia, Laminaria, Macrocystis, Rhodomela ( Loewus and Delwiche, 1963), Chlorella (Korotaev et al., 1964), and blue-green algae (Troxler and Dokos, 1973). Among the bacteria, species of Aerobacter, Alginomonas, Bacillus, Brevi-

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

31

bacterium, Escherichia, Lactobacillus, and Pseudomonas as well as Saccharomyces possess the identical capability (Junge et al., 1971, 1972). Bacillus cereus and Streptococcus mitis also form CO from several heme compounds provided that 0, is present; the gas apparently is derived from the a-methene bridge carbon of the porphyrin ring and 0, ( Engel et al., 1972). Strains of Alternaria, Aspergillus, Cephalosporium, Fusarium, Penicillum, and Pullularia likewise generate the toxicant, but with these fungi the substrates are flavonoids such as rutin and quercitin (Westlake et al., 1961). Fungal enzymes can apparently produce CO from an array of flavonoids (Padron et al., 1960).

C. CARB~N DIOXIDE Accepting the view that a pollutant is a substance that unfavorably alters the surroundings; whether it affects man, animals, or plants, even that ubiquitous microbial metabolite CO? must occasionally be deemed a pollutant. It may exert a detrimental effect in the two major ecosystems of the earth, water and soil; in soil by bringing about harmful effects on the physiology and development of plant roots, and in water by sometimes enhancing nuisance blooms of algae. The CO, evolved in microbial respiration and in the mineralization of organic matter may make up a significant portion of the gas phase of soil. The concentration, which is generally proportional to the amount of 0, consumed by the microflora, may range up to 6%of the soil atmosphere and be appreciably higher in poorly drained peats (Boynton, 1941; Kurlykova, 1962). Concentrations in excess of 40%by volume may be encountered in fields immediately above a stagnant water table. Such high levels are associated with the high solubility of CO, in soil water, rapid decomposition of organic materials, and the liberation of CO, as some of the water evaporates (Enoch and Dasberg, 1971). The high CO? levels can potentially cause considerable injury to roots growing in poorly drained or compacted soils or where much readily available organic matter is present and hence considerable heterotrophic metabolism. For example, the root growth of pea, bean, and vetch plants is totally inhibited if the air passed through the rooting medium contains 6.5%CO?, and even 1.5%CO, is harmful (Stolwijk and Thimann, 1957). The elongation of cotton roots likewise is decreased with increasing partial pressures of CO, in soil (Tackett and Pearson, 1964), and the exposure of raspberry roots to a gas phase containing 10%CO, prevents new roots and new shoots from developing (Rajappan and Boynton, 1960). CO, similarly reduces the absorption of water, K, N, P, Ca, and Mg by plants and even leads to the excretion of K from the roots (Chang and Loomis, 1945). Root growth and the concentrations of N, P, K,

32

MARTIN ALEXANDER

and B in roots and the levels of N, P, Ca, Mg, and Mn in the tops of citrus seedlings decreases as a result of exposure to this microbial product ( Labanauskas et al., 1971). Therefore, considering the levels observed in nature, it is quite probable that CO, frequently is involved in toxicity, especially where conditions of aeration, drainage, and organic debris favor the buildup of this ubiquitous yet frequently overlooked phytotoxin. The growing public awareness of eutrophication and the vested interests of industrial, agricultural, and other groups that may be discharging potential nutrients into waterways prompted an interesting debate on the possible importance of supplemental CO, in promoting multiplication of aquatic algae. If CO, were the limiting nutrient, then the discharge of nitrogen and phosphorus compounds into rivers, streams, and lakes presumably could nor be held responsible for the increasingly frequent appearance of unwanted blooms. The disagreements between the parties to the debate now appear to have been largely resolved, or so it seems for the moment, and the consensus is that the productivity of inland waters presently supporting only a sparse community of algae or higher plants, except in special and local circumstances, is not limited by the available CO, supply in the water. This CO, is that dissolved as bicarbonate, carbonate, carbonic acid, or CO, and that COL entering into the lake from atmospheric sources. The special circumstances cannot be ignored, however, because these waterways are of considerable significance to the people and communities in the vicinity. And it is in these localities that the microbial production of CO, in the water takes on particular importance. In acid waters, as in lakes adjacent to strip mines, little carbon in the form of dissolved CO,, carbonic acid, and bicarbonate is available for photosynthesis, and the growth rate of indigenous or invading algae will be slow and will be regulated to a large extent by the little CO, coming from the water-atmosphere equilibrium ( King, 1970). Similarly, algae will fare poorly in highly alkaline waters in which much of the carbon potentially available for photosynthesis is rendered unavailable by being removed as insoluble carbonates. Furthermore, primary production in bodies of water rich in phosphorus, nitrogen, and other nutrients and already supporting a dense bloom-one already probably unwanted and ofiensive-may be limited because the rate of entry of atmospheric CO, into the water is insufficient to meet the existing sizable demand. Under these conditions, the heterotrophic formation of CO, from organic substrates entering the environment might promote algal proliferation. In this context, it is appropriate to cite the laboratory evidence that the introduction of simple organic compounds into a mixed artificial community of bacteria and algae in a C0,-deficient medium does indeed

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

33

enhance growth of the algae. The bacteria degrade the sugar, polysaccharide, or organic acid provided to the mixture of populations, and the CO, thereby formed stimulates proliferation of algae like Anabaena, Anacystis, Chlamydomonas, Chlorella, Gloeotrichia, Phormidium, or Scenedesmus. The effect of the bacterial inoculum together with the carbon amendment can be reproduced simply by enriching the atmosphere with the gas (Boyd, 1972; Lange, 1971). One might assume, therefore, that bacteria-or fungi in some habitats-may generate CO, in sufficient amounts to be considered a water pollutant in those special circumstances where the available supply of inorganic carbon for photosynthesis is insufficient to meet the demand. This microbial product also may be of ecological significance because it is inhibitory to microbial populations in a number of ecosystems. The same is true of NHs, 02,H,S, organic acids, alcohols, and many complex organic compounds. The inhibition of one group of populations by products elaborated by a second has already been considered in detail (Alexander, 1971).

D. ORGANIC ACIDS Microbiologically produced organic substances sometimes reach levels that inhibit the growth of plants in the field or overcome their natural resistance to fungal infection. Many of these compounds undoubtedly have not yet been identified, but a few have turned out to be remarkably simple organic acids. In no instance, however, have the species responsible for the formation of these toxins in nature been characterized. Among the phytotoxic compounds that have been isolated from soils wherein microbial decomposition processes have led to an inhibition of plant growth are p-coumaric, ferulic, vanillic, p-hydroxybenzoic, syringic, and protocatechuic acids. The concentrations of the first two inhibitors in nature sometimes exceed 5 ppm. Some of the compounds inhibit seed germination as well, and several are especially toxic to seedlings ( Guenzi and McCalla, 1966; Hennequin and Juste, 1967). Lillaram ( 1970) found p-hydroxybenzoic and dihydroxystearic acid in soils and showed that they were toxic to Eleusine coracana. He proposed that these two metabolites might account for the poor growth of seedlings of E. coracana at sites containing the inhibitors. The microbial attack on crop residues that are allowed to decay in soil occasionally does harm to the development of higher plants. The inhibitors that are so formed include benzoic, phenylacetic, 3-phenylpropionic, and 4-phenylbutyric acids. These products of organic matter decay not only are directly phytotoxic but they also reduce the resistance of plants to microbial colonization so that a potential invader is more

34

MARTIN ALEXANDER

readily able to cause injury. Thus, application to the roots of cotton seedlings of an extract of decayed crop remains or of benzoic or 3-phenylpropionic acid increases the extent of rotting brought about the pathogenic fungus, Thielaviopsis basicola. A nearly avirulent clone of T . hasicola is also able to do considerable damage if the roots are exposed to 3-phenylpropionic acid or a fraction from the decayed tissues, suggesting that weak pathogens or possibly even innocuous microorganisms might parasitize plants coming into contact with products synthesized during decomposition processes ( Linderman and Tousson, 1968). In land that is flooded prior to the sowing of rice, growth of the seedlings sometimes is inhibited shortly after the water is added. The flooding leads to a rapid fall in the dissolved 0, content and the oxidation-reduction potential in the upper part of the soils, and coinciding with the onset of the inhibition is the appearance of organic acids that are presumably generated by indigenous bacteria utilizing readily available organic substances. Acetic, butyric, formic, lactic, succinic, and other aliphatic acids become evident, and some of these acids are distinctly deleterious to rice at low concentrations. Nevertheless, the organic acid level may be insufficient to account for the entire inhibition of growth, and other inhibitors are probably generated simultaneously and contribute to the injury (Takijima, 1964a,b). Flooding also results in the death of nematodes, and their decline is correlated with a rise in the level of butyric and propionic acids. When tested in vitro, butyric acid at concentrations found in soil rapidly kills the nematodes, and nontoxic levels of propionic acid increase the total toxicity associated with the acids present (Hollis and Rodriguez-Kabana, 1966). V.

Acidity and Mine Drainage

Major sources of microbiologically induced pollution are the sulfide ores. When left untouched by man, these ores bring about no deleterious changes in the surroundings. If exposed to the air, however, they undergo a series of alterations in which microorganisms play a crucial role, modifications leading to serious consequences in coal mining areas near the Appalachian mountain range and in other regions and in the Netherlands and other countries where soils were reclaimed from the sea for agriculture. The acid drainage from these coal mines often has a pH of less than 4.0 and contains large amounts of soluble sulfate and iron. For example, Carpenter and Herndon (1933) reported pH values of mine waters below 2.9 and sulfate concentrations from 3600 to 41,700, the water with the highest sulfate concentration having a pH of 1.4. Zinc, copper, and aluminum levels in the mine drainage rise to concentrations lethal to aquatic

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

35

life, and sometimes dangerously high levels of arsenic and cadmium are also found. In some instances, the pollution originating in the mine extends for many miles downstream, and large numbers of fish are destroyed (Turner, 1958). The aquatic communities may be completely upset, and many vascular plants and stream bed animals are eliminated. New groups of plants, such as cattails and mosses, and insects otherwise uncommon or unknown flourish, but the species diversity simultaneously declines (Harrison, 1958; Lackey, 1939). Wildlife may disappear from the polluted areas owing to their reluctance to drink the water, and the acid may be lethal to larvae and eggs of sensitive species of fish. Highly acid waters are unsuitable for household or industrial use or for livestock, and the paucity of desirable fish and wildlife and the unsightly iron hydroxide deposits in streams may reduce the value of the land. It has been estimated that 29,000 acres of impoundments and reservoirs and 4800 miles of streams are polluted by surface coal mining in the United States (Mackenthun, 1969). Metal will often be corroded, too, and even concrete structures may suffer damage. The highly acid waters have a disastrous effect on microorganisms. This may create serious problems if the biota affected is that involved in sewage purification. Indeed, high concentrations of such polluted waters will sterilize sewage (Carpenter and Herndon, 1933). Protozoan and algal communities are drastically altered, and species that are usually found only with difficulty assume prominence. The surviving microorganisms are typically acid tolerant, and fungi and yeasts may come to the forefront (Tuttle et al., 1968). Sulfur- and iron-oxidizing autotrophs proliferate extensively, and their numbers usually become extraordinarily large. The problem of acid drainage cannot be terminated by the simple expedient of ending mining operations, for the sulfuric acid and other unwanted products continue to be formed and transported in appreciable quantities to nearby surface waters. Components of coal mine spoils may also be acted on microbiologically to yield additional pollutants. The sulfides in the spoil materials are oxidized with the release of sulfuric acid and soluble forms of copper, zinc, and nickel in such amounts that plants may not establish themselves or will fare poorly on the spoil banks, and runoff from the spoil banks may be injurious to the biota of downhill land and adjacent streams ( Massey and Barnhisel, 1972). Land is sometimes reclaimed from the sea, and a common consequence of exposing previously underwater soil containing iron sulfides to 0, is the production of considerable acidity, soluble iron, and soluble cations. This type of environmental change has been a major problem in the Netherlands. Extreme acidity was likewise reported when sulfurrich soils along estuaries in South Carolina were drained, and the final

36

MARTIN ALEXANDER

p H fell to values of 2.0-3.0 and sometimes lower (Fleming and Alexander, 1961) . Drainage of sulfide-rich soils in mangrove swamps of Sierra Leone similarly resulted in the appearance of sulfuric acid, pH values of about 3.0, and soluble aluminum ( Hesse, 1961). The mining of sulfur may lead to pollution involving microorganisms, too. Soils adjacent to the mines sometimes acquire sufficient of the sulfur from dust that they become, as bacteria oxidize the element, extremely acid. As the pH falls to below 4.0, many plants fail to develop. In some instances, the p H drops to 1.5, in which case no higher plant will grow (Krol et al., 1972). Of local importance only but nonetheless quite fascinating are the hot acid soils in Yellowstone National Park, wherein sulfur-oxidizing Thiobacillus and Sulfolobus oxidize the elemental sulfur present to sulfuric acid ( Fliermans and Brock, 1972) . The precise role of chemoautotrophs in the production of acid waters from pyrite ores has aroused considerable interest and disagreement, The process is sometimes envisioned to involve three steps : the oxidation of the sulfide to sulfate, the conversion of the ferrous iron to the ferric state, and the formation and precipitation of ferric hydroxide (Brant and Moulton, 1960)

+ +

+

+ + +

(6) (7) (8) The final products formed from FeS, and 0, would then be Fe( O H ) and HrSO,. No question remains but that bacteria like Thiol7acillus ferrooxidans are able to oxidize both insoluble sulfides and iron, but the formation of the acid waters is not a simple, straightforward bacterial oxidation of pyrite, FeSL. The controversy has centered on delimiting those steps that are bacterial and those that are nonbiological. On the one hand, the bacteria are abundant in the acid waters, and they undoubtedly replicate at the expense of oxidizable inorganic substrates. hloreover, inoculation of pyrite with the bacteria enhances the rate of acid formation from the ore, as compared with the uninoculated sulfide. On the other hand, FeS2 is oxidized in the absence of microorganisms, albeit slowly (Smirnov, 1963). The bacteria may contribute to the transformation by oxidizing ferrous iron to the ferric state. 2FeS2 7 0 2 2H204 2FeS04 2112SOa 4FeS04 2HZSO4 0 , 4 2Fe2(S04)3 2H20 F C ~ ( S O ~ 6HJ0 ) ~ 4 2Fe(OH)3 3HzSOa

+ + 4H+

+ +

+ 2H20

($1) The ferric ions thus released enzymatically bring about a nonenzymatic oxidation of FeS,. 4Fe2+

14Fet3

0 2

4Fc3+

+ FeSz + 81120+ 15Fe2++ 2S04-2 + 16Hf

(10)

This then leads to the acidity. Singer and Stumm (1970) pointed out

37

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

that the rate-limiting step in pyrite oxidation and acid formation is ferrous oxidation, so it is here that the bacteria may play a critical role, by accelerating a reaction that is normally quite slow below pH 4.5. Walsh and Mitchell (1972) stated that T . ferrooxidans, a ferrous-iron oxidizer, is particularly active only below pH 3.5,and they suggested that an acid-tolerant Metallogenium, a filamentous iron oxidizer, initiates the process by markedly increasing the slow, spontaneous oxidation of ferrous ions in the pH range of 3.5-4.5,a process that brings about an increase in the acidity as the ferric ion is hydrated.

+ +

+

+ +

(11) (12) As the waters become more acid, Metallogenium dies out. However, T . ferrooxidans is now in an environment more close to its optimum pH, and it then carries on with the biogenesis of the ferric ion necessary for the next chemical step in the sequence. 3Fe+z j$O, 2H+ + 2Fe+2 H,O 2Fe+* 6 H 2 0--t 2Fe(OH)3 6H+

VI.

Simple Sulfur Compounds

A. HYDROGEN SULFIDE Hydrogen sulfide has long been noted for its potency, and it thus has been of interest to toxicologists and atmospheric and soil scientists. It is formed in many ecosystems, in numerous processes, and by uncounted species, and it is inhibitory to humans, animals, higher plants, and microorganisms. Surprisingly few quantitative data have been obtained on the rates of emission of H,S from terrestrial and aquatic environments. Robinson and Robbins (1970) estimated the relative contributions of various sulfur sources to the atmosphere (Table V ) . Their estimates are most surprising ESTIM.\TED

TABLE V COMPOUNDS

SOURCES O F SULFUR

Compound

H,S SO, Sulfate aerosols

I N THE

ATMOSPHERE^

Source Terrestrial emissions Marine emissions Industrial emissions Coal combustion Petroleum combustion and refining Smelting Sea spray

From Robinson and Robbins (1970).

Estimated emission (tons S/yr, X 10'9

70 30 3 51 14 8

44

38

MARTIN ALEXANDER

to a biologist, for they indicate that the quantity of biologically evolved H,S in terrestrial and marine emissions exceeds by a factor of 33 the H,S generated by industry. The amount is even appreciably greater than the total of H,S and SO, from all industrial and other nonbiological sources. Presumably this biologically produced H,S is that coming from the decomposition of organic materials on land and in water, including that originating in swamps, bogs, and tidal flats. Eriksson (1963) suggested that the decomposition of organic materials on the land area yielded 112 X lo6 tons of H,S each year to the atmosphere, and Marchesani et d. (1970) proposed that 0.07 ton of H,S was emitted per 1000 square miles each day from natural sources in the United States. Another view of the significance of the microflora comes from the work of Grey and Jensen (1972), who reported that the most important source of atmospheric sulfur, after industrial discharges, in the vicinity of Salt Lake City is the microflora inhabiting the lake- and riverbottom muds and marshes near Great Salt Lake. H,S may be formed in two ways by microorganisms: by the reduction of sulfate by Desulfovibrio and physiologically related bacteria and by cleavage of organic molecules. The sulfate reducers are ubiquitous in muds, swamps, and poorly drained soils, where they proliferate using sulfate as their terminal electron acceptor. These bacteria are, from an ecological standpoint, quite similar physiologically, and they are usually susceptible to acidity and to the presence of 0,; hence, they generally show little or no activity in natural environments containing dissolved 0, or having a low pH. An array of organic compounds can be converted to H,S by many heterotrophs in culture. Aerobes and anaerobes, thermophiles and psychrophiles, and bacteria, fungi, and actinomycetes cleave sulfur from organic molecules and release it as H,S. The substrates for such activities include proteins, polypeptides, glutathione, cystine, cysteine, homocysteine, and thioglycolate. Surprisingly, the enzymology of most of these cleavage reactions has all but been ignored with the exception of cysteine desulfhydrase, which catalyzes the conversion of the amino acid to pyruvic acid. HSCH&H(NH,)COOII

+ H20 + CH3C(O)COOH + 132s + NH3

(13)

Such sulfur-cleavage reactions are readily demonstrable both in culture and in samples from natural ecosystems to which are added the various substrates. Sulfide is generally formed slowly in soil, but the rate increases appreciably if organic materials are added. Chaudhry and Cornfield (1967) reported that up to 11, 20, and 54 ppm sulfide appeared in samples incubated in the laboratory for 3, 6, and 12 weeks, and Vamos (1959)

hlICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

39

detected 67 pprn one month after soil was flooded. Much of the sulfide reacts with iron in the soil and is precipitated as the iron sulfide, however, so that little is volatilized. On the other hand, in soils deficient in available iron and in other environments poor in cations that precipitate the sulfide, H,S is liberated to exert harmful effects on living organisms or to be volatilized to the overlying air. The rate of H,S evolution can be marked if sulfate is present and the environment becomes anaerobic. Such conversions in waterlogged soils, especially those used for rice cultivation, have been intensively investigated. The reduction is favored by readily utilizable constituents of plant remains which not only provide an energy source for Desulfovibrio but lead to a diminution in the supply of the 0, that is inhibitory to the sulfate reducers. Here, too, the extent to which free H,S is evolved is governed by the quantity of cations, especially iron, available to precipitate the sulfides generated ( Bloomfield, 1969). Certain saline soils are particularly rich in sulfate, and here sulfide accumulation may be appreciable. The reduction of the sulfate is accentuated in the rhizosphere, with accumulations sometimes being sufficient to induce death of plants (Jacq and Dommergues, 1970). H,S production may also be pronounced during the decomposition of organic refuse and the decay of algae in waters, and it is a common transformation in lake and ocean bottom sediments, swamps, bogs, marshes, estuarine waters, raw sewage, and industrial effluents. This common metabolite is a highly effective toxicant. Less than 0.1 ppm H,S in water has a dramatic effect on newly hatched fish fry, fry growth, and survival of fish eggs. This inhibition probably is encountered frequently in nature because the eggs and fry of many fishes are localized in a restricted area at or just above the mud-water interface (Smith and Oseid, 1972). The decline in nematode populations in soils that become flooded is also correlated with the rise in the H,S content of the environment, and the hypothesis that H,S is the actual lethal agent is supported by the observation that all nematodes are killed in laboratory tests when exposed to H,S at levels observed in flooded soils ( Rodriguez-Kabana et d,1965). H,S is also offensive, and it often contributes to the foul odors emanating from heaps of animal wastes. It may create odor problems in the vicinity of sewage treatment plants. For example, H,S emanating from a municipal stabilization pond during periods of faulty operation was carried by the wind and created a nuisance problem at resort hotels 1700 meters away. The level of H,S in the waste waters ranged from 10.7 to 24.8 ppni, it was at a concentration of 6.7-8.8 pprn in the air 15 meters from the pond, and sometimes at 0.36 ppm in the resort area 1700 meters downwind. A common standard for polluted air is in the

40

MARTIN ALEXANDER

vicinity of 0.1 ppm H2S, and the odor threshhold ranges from 0.13 to 1.0 pprn (Kendler and Donagi, 1970). Higher plants are remarkably sensitive to free H2S, and economic losses of some magnitude have been incurred because the roots of rice and fruit trees have come into contact with toxic quantities. Such inhibitory effects on rice have been reported in Japan and Europe, the H,Sexposed plants becoming wilted and having a reduced rate of water and inorganic nutrient uptake (Mitsui et al., 1954; Vamos, 1959). Root injury to citrus has also been associated with the microbial formation of H,S in poorly drained areas of Florida, and the toxicant appears as 0, deficiency is encountered during periods of waterlogging. That citrus roots are quite susceptible to injury, moreover, is indicated by reports that they are seriously affected in solution culture containing as little as 2-3 pprn H,S (Ford, 1973). The roots of avocado trees may also be affected. The high concentrations of H,S made under waterlogged conditions may directly kill the feeder roots, but at lower and nonlethal concentrations, the H,S-induced injury may predispose the roots to parasitism by the plant pathogen Phytophthora ( KIotz and SokoIoff, 1943).

B. SULFURDIOXIDE AND CARBONYL SULFIDE Microorganisms are capable of forming volatile sulfur compounds in addition to H,S. The evolution of one of these products, SO,, is largely known as an in vitro phenomenon, but the potential that it may be liberated in nature requires that some consideration be given to it. A sulfur metabolite whose formation in culture has been intensively explored is sulfite. Sulfite and bisulfite represent anionic forms in solution of the gas SO., much as carbonate and bicarbonate are anionic forms in solution of the gas CO,. Because of its marked solubility in water, part of the SO, would be removed from the atmosphere by the oceans, but if the partial pressure of SO, in the air is less than the equilibrium vapor pressure in the surface waters of the underlying sea, SO, might in fact be liberated to the atmosphere despite its solubility (Kellogg et al., 1972). In a similar fashion, SO1 would be lost from culture when the equilibrium vapor pressure in the liquid is greater than that in the gas phase above the medium. On this basis, it is noteworthy that several heterotrophs have been found to make SO, or, as it is often written, sulfite. This activity has been important in the wine industry because of the common use of SO,, and a number of reports document the capacity of species of Saccharomyces to synthesize sulfite or, as it is often written by these investigators, SO,. As much as 50 ppm is generated in culture, and cysteine, methionine, or sulfate may be the sulfur precursor (Minarik, 1972; Rankine and Pocock, 1969). Some of

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

41

the wine yeasts may, in fact, synthesize SO, from sulfate but be unable to reduce it further ( Dittrich and Staudenmayer, 1970). Microsporum gypseum, a dermatophyte, also excretes considerable sulfite when grown in media containing cystine ( Kunert, 1973). Sulfate is frequently formed in nature from organic compounds not by an inorganic pathway after H,S is cleaved but rather by an organic pathway. The latter sequence is brought about by heterotrophs and not Thiobacillus. This pathway is well illustrated by the conversion of cysteine to sulfate in soil samples, a transformation that involves the formation of cystine, cystine disulfoxide, and cysteine sulfinic acid prior to sulfate ( Freney, 1960). Cysteine sulfinic acid, HOOSCH,CH(NH,)COOH, could be a focal intermediate in the biogenesis of SO, in nature because it is acted on by a widespread enzyme to yield SO, (or sulfite) and alanine.

+

HOOSCHzCH(NH2)COOH -+ SO2 CHaCH(NH2)COOH SO2 H20-t HzS03

+

(14)

This enzymatic cleavage to yield SO, occurs in Neurospora crussa, Escherichia coli, and Alcaligenes faecalis ( Leinweber and Monty, 1961, 1965; Soda et al., 1964). SO, is also produced from the 3’-phosphoadenosine 5’-phosphosulfate generated in sulfate reduction. Carbonyl sulfide (S=C=O), a chemical that is toxic to the central nervous system and to fungi, is also of some interest. It is generated during the anaerobic decomposition of cattle manure, probably as a result of bacterial activity (Elliott and Travis, 1973), and it is also formed in soil from the pesticide known as nabam, disodium ethylenebisdithiocarbamate ( Moje et al., 1964). Axenic cultures of Trichomonas vaginalis (Gobert et al., 1971) and N . crassa (Somers et al., 1967) evolve the same compound, the latter fungus doing so from captan, another pesticide.

C. VOLATILE ORGANIC SULFURCOMPOUNDS Volatile metabolites containing sulfur are not restricted to inorganic compounds, for several simple products are liberated as bacteria or fungi proliferate in culture. Of these, methane thiol (methyl mercaptan) has received the greatest attention owing to the frequency of its occurrence, its powerful and offensive odor, and its phytotoxicity. It has been detected in paddy soils in concentrations up to 3.6 ppm, and the extent of its accumulation is generally affected by temperature (Asami and Takai, 1963) . It also apears in methionine-amended soil maintained under anaerobiosis (Francis et al., 1973). As evident from Table VI,

42

MARTIN ALEXANDER

TABLE V I VOLATILE SULFURCOMPOUNDS Pnonucm ~~~

Compound Methane thiol, CHaSII

~~

Organism Achrowiobacter starkeyi Pscudomonas sp. Schizophyllum breuicaulis Clostridium spp. Schizophyllum commune

Dimethyl sulfide, CH3SCH3

Ilimcthyl disulfide, CHISSCIII

Schizophyllum commune Schizophyllum brrvicaulis Arrobaclrr aerogencs Propionibaclrrium shrrmanii Achromobactar starkcyi Pseudomonas sp. Schizophyllum communc

Clostridium spp. Dimethyl trisulfide, CHiSSSCHi Pscudomonas putrcyaciens Ethane, propane, and butane thiols Clostridium letani

IN

CULTURE

~

S source

lief erenee

liuiz-Herrera and Starkcy (1970) Kallio and Larson Methionine (1955) Challenger and Me thionine Charlton (1947) Thioglycolate Labarre czt al. (1966) Challenger and Sulfate Charlton (1947) Challenger and Sulfate Charlton (1947) Challenger and Methionine Charlton (1947) Toan el al. (1965) Milk Dykstra el al. (1971) Milk Iluiz-Herrera and Methionine Rtarkey (1970) Kallio and Larson Methionhe (1955) Challenger and Sulfate Charlton (1947) Thioglyeolate Labarre rt al. (1966)

Methionine

Fish muscle

Miller et al. (1973)

Thioglyeolate

Labarre ct al. (1972)

fungi and bacteria can synthesize this mercaptan. It has been reported as a metabolite of actinomycetes and yeasts as well (Kadota and Ishida, 1972). Microbial processes give rise to other volatile sulfur compounds. For example, amended soils incubated in the laboratory under anerobiosis will give off dimethyl sulfide and dimethyl disulfide (Francis et al., 1973), and among the malodorous metabolites emitted from dairy manure are methane thiol, dimethyl sulfide, and diethyl sulfide, the dimethyl sulfide being one of the major contributors to the foul odor (White et al., 1971). Dimethyl sulfide is also present in sea water, and it has been suggested that this volatile substance may be a significant component of the sulfur cycle by virtue of its transfer from the oceans through the air and thence to the surface of the land (Lovelock et al., 1972). Methane thiol, dimethyl sulfide, and dimethyl disulfide, the

hlICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

43

latter two being evident in culture (Table VI), are released during the decomposition in soil of cruciferous plants, which are notably rich in sulfur compounds such as methionine and methyl cysteine sulfoxide (Lewis and Papavizas, 1970). Low concentrations of the three volatile compounds are markedly toxic to mycelial growth and germination of spores of fungi (Lewis and Papavizas, 1971). Ethane thiol, a product of Clostridium, has also been reported to be toxicant, inhibiting the ripening of the rice plant ( Inoue et al., 1955). VII.

Mercury

Environmental pollution with mercury has been proceeding for some time, but the problem only gained widespread recognition in the past two decades. In the period between 1953 and 1960, 116 people were poisoned irreversibly and 43 died from the consumption of fish contaminated with mercury originating in a vinyl chloride factory located in the Minamata Bay area of Japan. An episode essentially identical to the first, again linked with the consumption of fish contaminated with mercury discharged from a vinyl chloride factory, took place in Niigata, with 120 people suffering from the poisoning and five deaths. The same element was implicated in the drastic decrease in bird populations in Sweden, a decline apparently associated with the widespread use of methylmercury dicyandiamide, a fungicide. More recent and still more disastrous episodes of human poisoning have been reported. Microorganisms contribute to mercury poisoning because they are able to transform mercury compounds to yield metabolites, notably methylmercury, that are extremely hazardous to human health. These metabolites enter natural food chains and then become part of the human diet. The mercury that is available for microbial transformation is derived from both natural sources and activities of society. Much of the element is in the ore cinnabar, and waterways may receive the metal as Hg-containing soil subjected to erosion. In view of the natural cycle of circulation, release by human actions can be blamed for only part of the environmental issue, but man still uses much of the element in his own activities. For example, Saito (1972) gives the following figures for the uses of mercury in the United States in 1969 (in thousands of pounds): electrolytic chlorine, 1572; electrical apparatus, 1382; paints for antifouling and antimildew purposes, 739; catalysts, 221; agriculture, 204; and as a slimicide for pulp and paper making, 42. Mercury pollution of waterways is often linked with the chloralkali industry, a major source of pollution of aquatic habitats in industrialized nations. Metallic mercury is used in these factories in the manufacture of C12, whose daily production is enormous, and some of the mercury

44

MARTIN ALEXANDER

escapes to adjacent waters where it can be acted on by the aquatic microflora. The magnitude of this discharge can be appreciated from an estimate that the St. Claire River system has received 200,000 lb of mercury in a 20-year period (Wood, 1971). High concentrations of the element may still be found at the top of lake sediments and in aquatic organisms many years after a chloralkali factory discharging wastes into a lake ceases operation. The manufacture of vinyl chloride and urethane plastics typically results in mercury loss to waterways, and it is this kind of discharge that led to the tragic Minamata episode. The pulp and paper industry has consumed considerable amounts of phenylmercuric acetate as a slimicide, and the aryl mercury compound adheres to the particulate wastes entering a body of water and falls to the sediments. Divalent mercury also accumulates in aquatic sediments, where it exists in inorganic and possibly organic complexes. Agriculture contributes mercury to terrestrial habitats in the form of organic fungicides, particularly for seed protection. This mercury contaminates the soil, although only very locally, but the toxicant-treated seed may also be eaten by and poison birds feeding on the seed. From 29 to 82%of the fields planted to cereals in Europe very recently received seeds treated with mercurials, and Japan alone consumed 1600 metric tons of mercury compounds for agriculture ( Rissanen and Miettinen, 1972). Owing to the frequency of discharge of inorganic and organic mercury into inland bodies of water and its tendency to end up in the upper layers of bottom deposits, the behavior of mercury in freshwater sediments has attracted considerable attention. I t has been demonstrated that microorganisms in the sediments act on mercuric ions that may be present and give rise to monomethylmercury, CH,Hg+. This process has been verified as a consequence of microbial actions inasmuch as little or no methylation takes place in sterilized sediments. Dimethylmercury, CH,HgCH, may also be formed in the process (Jensen and Jernelov, 1969) . Phenylmercury and metallic mercury in industrial wastes can be converted to mercuric ions, which then may be methylated. The monomethylmercury moves from the sediments to the free water, but the dimethylmercury will tend to pass through the water and be discharged to the atmosphere (Jernelov, 1972). Some of the mercury will be discharged in water rich in organic matter, and anaerobic conditions thus will prevail. This may lead to the cation being precipitated as HgS. However, even this form of the element is subject to methylation ( Fagerstroni and Jernelov, 1971). The methylmercury then enters fish through the food chain, by direct passage through the gills, or both and accumulates in their tissues. For example, Gillespie (1972), using guppies as indicator organisms, found that methylmercury was prominent

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

45

in fish living in aquaria to which HgCl,, HgS, or metallic H g had been added. The alkyl mercury has also been found in fish derived from natural waters, and sometimes essentially all of the mercury in the tissues is present as methylmercury. Methylmercury stands out as an environmental hazard because it is more poisonous to man and other mammals than inorganic mercury cations and is excreted slowly. Neurological disorders are evident in dogs, cats, and rabbits having a daily intake of 0.4-1.0 mg of Hg per kilogram of body weight, and fish in certain fresh and coastal waters have such high concentrations of methylmercury that they have been condemned as unfit for human consumption. The poisoning episodes in Minamata and Niigata were traced to the presence of the alkylmercury in fish and shellfish that the people so sadly affected had eaten. The microbial metabolism of mercury is not restricted to aquatic ecosystems because soils likewise show transformations of this element, some or most of the processes probably being enzymatic. For example, appreciable amounts of methylmercury appear in soils treated with methylmercury dicyandiamide, and the former is absorbed through the roots and appears in beans, tomatoes, and millets grown on the treated soil (Bache et al., 1973). Direct evidence for microbial involvement in mercury transformations comes from the work of Kimura and Miller ( 1964), who found that metallic mercury vapor was emitted from natural soil treated with phenylmercuric acetate but little was volatilized from autoclaved soil. Furthermore, mercury is lost from soil, presumably in the metallic form, treated with a mixture of mercurous and mercuric chloride, a pesticide sometimes used to control turfgrass disease. The mechanism of this loss may be microbial (Gilmour and Miller, 1973). The metabolism of mercurials has also been explored with individual organisms or enzymes prepared from them. Thus, Clostridium cochlearium produces methylmercury from HgC12, HgO, and Hg( NO,) (Yamada and Tonomura, 1972), and Neurospom is capable of methylating mercuric ions (Landner, 1971). Bacteria can cleave the C-Hg bond in several organomercurials, and Furukawa et al. (1969) found that a Pseudomonas isolate volatilizes metallic mercury from phenylmercuric acetate, ethylmercuric phosphate, and methylmercuric chloride; the organic moiety is released as benzene, ethane, and methane, respectively. Other bacteria, by contrast, produce diphenylmercury from phenylmercuric acetate (Matsumura et al., 1971). A strain of E . coli, moreover, converts HgClr to elemental mercury (Summers and Silver, 1972), and an enzyme preparation from this bacterium catalyzes the conversion of HgC1, to a volatile product in the presence of NADPH (Komura et al., 1971). The enzyme system from a Pseudomonas strain, on the other hand, transforms phenylmercuric acetate, ethylmercuric phosphate,

46

MARTIN ALEXANDER

methylmercuric chloride as well as HgCl, to metallic mercury in the presence of either NADPH or NADH. The Hg-releasing enzyme is induced when the pseudomonad is grown in the presence of a number of mercurials ( Furukawa and Tonomura, 1972a,b). VIII.

Arsenic

Arsenic has a long and notorious history as a poison, and its reputation is surely amply deserved. It is toxic to humans, animals possessing central nervous systems, most higher plants, and certain lower organisms. Inorganic arsenic is often of environmental importance, and among the inorganic anions, arsenite is generally much more toxic to man, animals, plants, and insects than arsenate; that is, the +3 is more deleterious than the $5 oxidation state. The volatile trimethylarsine, ( C H , ) *As, is also a notable human toxicant. The potency of arsenic is evident from the occurrence of poisonings among people drinking water containing as little as 0.2 ppm As. This high potency has led the U.S. Public Health Service to establish 0.01 pprn as the recommended maximum for potable water supplies and 0.05 ppm as the maximum permissible level. Small amounts of arsenic occur in many natural materials. Soils often contain in the vicinity of 5 ppm, and trace quantities may be found in waters, although those used for drinking purposes in the United States rarely exceed 0.01 ppm. Arsenic compounds are sometimes employed in pigments and metal alloys and to color glass. Arsenite has been used for the control of aquatic vegetation, and arsenate has been widely employed to prevent plant growth along railroad tracks. Organic arsenicals are still now widely applied herbicides, and lead and calcium arsenates were common insecticides before 1960. Lead arsenate was once sprayed on fruit trees for so many years that young trees introduced into old orchards fared poorly, and still now, long after arsenate usage has terminated, these soils cannot be devoted to growing fruit trees. As pointed out by Pattison ( 1970), detergent formulations containing phosphates may have up to 70-80 ppm As, and wash water into which is introduced these heavily contaminated detergents may contain 0.15 pprn As. Microbial transformations of this element first became evident when human poisonings were reported in rooms containing wallpapers colored with arsenic-containing pigments. The pigment itself was not the lethal agent, but instead the wallpaper served as a support for the growth of fungi that liberated the volatile poison, trimethylarsine. This compound, and probably related metabolites, has a garlic odor to which the human nose is acutely sensitive. Arsenic-containing gases, usually

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

47

characterized as such by their garlic odor, are liberated by many fungi growing in media containing this element, e.g., by species of Fusarium, Aspergillus, Paecilomyces, Scopulariopsis ( Thom and Raper, 1932), and Lenzites (Merrill and French, 1964). It was left to Challenger (1951), working with Scopulariopsis hrevicaulis, to establish that the gas produced from trivalent arsenic was trimethylarsine. More recently, it has been noted that Candida humicola, Gliocladium roseum, and a strain of Penicillium can generate trimethylarsine from monomethylarsonate and dimethylarsinate, both pesticides. C . humicola also synthesizes the volatile product from arsenite and arsenate (Cox and Alexander, 1973). Arsenic volatilization is not restricted to fungi on wallpapers or in culture. It also occurs in soil. This volatilization was first shown in an arsenic-rich soil, where the process was extremely slow, and in a soil amended with calcium arsenate (Epps and Sturgis, 1939). A compound containing this element is also volatilized and a pungent odor is detected when an organic arsenic pesticide is added to soil, with a considerable portion of the applied chemical being lost to the atmosphere (Woolson and Kearney, 1973). In neither investigation was the product identified. The volatilization is not limited to fungi, moreover. Thus, a strain of Methanobacterium converts arsenate to dimethylarsine, and extracts of cells of Desulfovihrio synthesize a volatile, unidentified compound with a garlic odor from arsenate ( McBride and Wolfe, 1971). Alkylarsenicals are not rarities in nature either since methylarsonic and dimethylarsinic acids have been detected in lakes, ponds, wells, and saline water, accounting for less than one to 68%of the total arsenic present (Branian and Foreback, 1973). Pure cultures of several organisms are able to reduce arsenate to the more toxic arsenite or to oxidize trivalent arsenic to the pentavalent arsenate. The reduction is brought about by Chlorella (Blasco et al., 1972), Micrococcus sp., the yeast Pichia guillermondii (Bautista and Alexander, 1972), marine bacteria (Johnson, 1972), and extracts of Micrococcus lactilyticus cells (Woolfolk and Whiteley, 1962), while the oxidation occurs in cultures of pseudomonads (Turner, 1949) and in soil (Quastel and Scholefield,1953). IX.

Selenium and Tellurium

Selenium is markedly different from mercury and arsenic because, though several of its compounds are toxic, it is also an essential element for warm-blooded animals and possibly man. Of particular relevance to its possible role as a pollutant is the small margin of safety between that which is required and the level that is deleterious. Indeed, it is said to be the most toxic of the essential trace elements described to

48

MARTIN ALEXANDER

date. Because of the narrow safety margin, modifications induced microbiologically in the chemistry of selenium could have serious consequences, Consumption of selenium-rich plants has been a major problem in severaI regions. Toxicity has been reported to cattle, sheep, hogs, and horses, and people also have been adversely affected. Livestock feeding on such forage often suffer acute poisoning and die. Reduction in growth and egg laying among poultry is likewise a result of exposure to excessive selenium. Susceptibility to dental caries during the period of tooth development may also arise owing to excessive levels of the element. Either inorganic selenium salts or organic compounds present in higher plants may be implicated in the intoxication, but the potency varies with the compound, with selenite being more harmful to animals that selenate. As little as 4-6 mg Se/kg body weight of selenate or selenocysteine given to rats intraperitoneally is lethal, whereas 1.6 g Selkg body weight as dimethylselenide is the median lethal dose ( McConnell and Portman, 1952). On the other hand, 2 ppm of selenite in drinking water reduces growth, causes liver damage, and brings about early death of young rats (Anonymous, 1972). Microorganisms participate in the metabolism of this element in several ways. Although the process has not been studied, organic selenium compounds probably are converted to inorganic products by microbial populations since the plants that accumulate selenium contain Se-methylselenocysteine, selenocystathione, and other metabolites, and the element appears to be available to new plants growing in the area after the previous ones have died and decomposed on or in the soil. Even elemental selenium appears to undergo biological oxidation in soil inasmuch as the conversion of Sen to a more oxidized state is markedly reduced by the addition of toluene to soil (Geering et al., 1968). The uptake of the element by plants from soil amended with colloidal selenium (GisselNielsen and Bisbjerg, 1970) is also indicative of a subterranean transformation since it is probably the oxidized state that is assimilated. Definitive laboratory studies showing an oxidation are few in number, but Sapozhnikov ( 1937) reported that a photosynthetic purple sulfur bacterium oxidized elemental selenium to selenate. More recently, Torma and Habashi ( 1972) observed that ThiobacitZus jermoxiduns metabolized CuSe and formed amorphous elemental selenium. The ice sheets in Greenland contain selenium, and Weiss et al. (1971) suggested that these accumulations resulted from the liberation of volatile compounds in biological processes. It is not clear whether natural microfloras participate in changing the abundance of selenium in the atmosphere, but laboratory tests have shown that selenium can be volatilized from soil and that the emission is enhanced by organic matter additions and inhibited by autoclaving, both treatments indicating a

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

49

microbial role (Abu-Erreish et al., 1968). One of the gases has been identified as dimethylselenide, which is evolved from natural seleniferous soils or from samples to which inorganic salts of the element are added (A. J. Francis, J. M. Duxbury, and M. Alexander, unpublished observations). Dimethylselenide is also evolved from cultures in uitro. Thus, Aspergillus niger, Scopulariopsis breuicaulis (Challenger et al., 1954), Schizophyllum commune ( Challenger and Charlton, 1947), a strain of Penicillium (Fleming and Alexander, 1972), and coryneform bacteria (J. W. Doran and M. Alexander, unpublished observations) make the dialkyl metabolite from inorganic selenium compounds. Small amounts also appear from raw sewage when selenite is added (Fleming and Alexander, 1972 ) . A high percentage of the bacteria, actinomycetes, and fungi derived from soil, in culture at least, reduce selenate and selenite to the elemental form (Bautista and Alexander, 1972). The red end product is deposited in the cells or hyphae or sometimes in the medium. Colonies on agar media containing the anions often assume a brick-red color owing to the reduction. Although the reduction has been known in vitro for decades, the ecological significance, if any, of the transformation has yet to be defined. Tellurium is likewise a toxicant, but it differs from selenium in that it is not known to be required by any organism. Tellurite is the more toxic of the two common anions, and the minimum lethal dose when given orally to rats is reported as 31 mg of sodium tellurite and 56 mg of sodium tellurate per kilogram of body weight. The elemental form is relatively innocuous, and 1500 ppm only slightly affects animal growth ( Carlton and Kelly, 1967). In culture, S. breuicaulis, species of PeniciZliurn ( Bird and Challenger, 1939), and Schizophyllum cornmune ( Challenger and Charlton, 1947) synthesize dimethyl telluride, and fungi are active in forming the dimethyl derivative from TeCl,, H,TeO,, and H,TeO,, ( Fleming and Alexander, 1972). The reduction of oxides of the element has been observed in cultures of many organisms, the elemental tellurium generated imparting to colonies a black or dark gray coloration, and in this instance, too, the capacity is common to soil bacteria, actinomycetes, and fungi ( Bautista and Alexander, 1972). However, it has yet to be determined whether environmental contamination with tellurium is a reality and whether microorganisms modify the behavior of the element in natural ecosystems. X.

Oxygen

A most surprising microbiologically generated pollutant is 0,. During the day, algae produce considerable quantities of this gas, much of

50

MARTIN ALEXANDER

which probably escapes to the atmosphere. However, the content of dissolved 0, in the aquatic habitat also rises as the algal community carries out photosynthesis, and the water may become supersaturated. Excessively high levels of dissolved 0, may be toxic to fish, as indicated in a study of the death of fish in a lake and river in Wisconsin. The killing was associated with the appearance of a dense algal bloom dominated by Chlamydomonas. The surface layers of the water contained 30-32 ppm of 02,and the gas emboli in the gill capillaries and the gas bubbles in the subcutaneous tissues-which were the cause of the fish mortality-probably were derived from the O2 produced by the algae ( Woodbury, 1942). XI.

Nitrosamines

An enormous amount of research and a significant amount of time of analytical chemists and staff members of regulatory agencies are currently directed to the nitrosamines. The reason for the activity is that these compounds are known to be carcinogenic as well as teratogenic and mutagenic. Indeed, some of the N-nitroso compounds are carcinogenic in the parts-per-million range and probably at lower concentrations (Terracini et al., 1967), and a few are reported to be among the most potent of the recognized chemical mutagens (Fishbein et al., 1970). Secondary and tertiary amines that are potentially subject to N-nitrosation are widespread in regions inhabited or on organic substances utilized by microorganisms. Dimethyl-, diethyl-, and N-methyl-n-propylamines and secondary aromatic amines such as ephedrine, synephrine, hordenine, and N-methyltyramine are constituents of the tissues of higher plants (Bush et aE., 1970; Smith, 1971). Trimethylamine (FujiwaraArasaki and Mino, 1972) and dimethylamine (Rolle et al., 1971) are present in algae as well as Basidiomycetes (Smith, 1971). Manure contains diphenylamine (Bergstrom et al., 1972), and trimethylamine is frequently found in heaps of animal fecal matter (White et al., 1971). Many widely used pesticides are likewise amines, and some may themselves be nitrosated or converted to products that are prone to such a reaction. Nitrilotriacetic acid, a tertiary amine, was proposed and in some countries is now included as a component in detergent formulations, the quantity disseminated into waters reaching enormous tonnages per year. Synthetic compounds are available to microorganisms when they are applied or are transported to soil and water, and the amines in natural products can be acted upon microbiologically as higher plants or algae decay and as manure rots. Nitrosamines can be generated in the gastrointestinal tract provided that both the amine and either nitrite or nitrate are present. In some

MICROBIAL FORMATION O F ENVIRONMENTAL POLLUTANTS

51

instances, it is assumed that the bacterial inhabitants are the nitrosating agents, but the possibility that the reaction is nonbiological is often not excluded by the tests performed. Alam et al. (1971), for example, reported the formation of nitrosopiperidine from nitrate and piperidine in the rat stomach ( p H 3 . 4 4 . 3 ) ,small intestine ( p H 6.3-6.8) and gastric contents in vitro. Piperidine is present in food and may be formed from lysine by microorganisms. Similarly, the stomachs of rats given proline and nitrite have been observed to contain N-nitrosoproline ( Braunberg and Dailey, 1973), and those given methylurea or ethylurea and nitrite were shown to have methylnitrosourea or ethylnitrosourea. If, however, the stomach contents were neutralized with NaHCO, before providing the methylurea and nitrite, the nitrosamine was not evident (Mirvish and Chu, 1973). The consequence of the genesis of nitrosamines in the bodies of animals is suggested by reports that stomach tumors developed in rats and mice given morpholine, methylbenzylamine, N-methylaniline, ethylurea, methylurea as well as nitrite ( Sander, 1971). Rats fed aminopyrine or heptamethyleneimine and nitrite likewise developed malignant tumors ( Lijinsky et al., 1973). More direct evidence of a microbial involvement in N-nitrosation comes from the studies of Klubes et al. (1972), who incubated the bacteria-rich intestinal contents of rats with dimethylamine, nitrite, and glucose at pH 7.0 in the absence of 0,. They observed that dimethylnitrosamine was formed by contents of the cecum and large and small intestines. The conversion was less or the reaction was almost wholly abolished in the absence of glucose or in the presence of neomycin or high nitrite levels, supporting the view that micoorganisms are implicated. The finding of nitrosamine formation at a site colonized by bacteria or fungi does not necessarily show that microorganisms are nitrosating agents, Nitrosamines are synthesized from nitrite and secondary amines at low pH spontaneously or at neutral and sometimes alkaline pH values. Experiments with inhibitors or sterilized samples are not conclusive when the added precursors are tertiary amines or nitrate because the inhibitor or sterilizing treatment may only abolish the formation of the requisite immediate precursors, i.e., the secondary amine and nitrite. Even when the secondary amine and nitrite are present, moreover, a suppression or abolition of the reaction by inhibitors may reflect not an inhibition of putative nitrosating species but rather those which generate acid and thereby create conditions for spontaneous nitrosation. The intake by humans of either nitrosamines made outside the body or their formation in the gastrointestinal tract is deemed to pose a threat of cancer to the human population. Nevertheless, apart from the observation that nitrosamines are present in certain solanaceous plants almost

52

MARTIN ALEXANDER

totally unknown in man’s diet and their demonstration in some species of fish and processed meat products, essentially nothing is known about the possible entry into the human food chain of such potentially dangerous compounds. Nitrosamines generated in polluted waters, however, would be of direct concern if the compounds persisted as the water underwent biological purification or if they were retained or assimilated by fish or lower aquatic organisms on which the fish grazed. A nitrosamine produced in soil is of little toxicological consequence unless it enters groundwaters used for drinking purposes, moves laterally with eroding soil particles or groundwater to enter adjacent surface waters, is assimilated by plants used for food or feed, or is retained on the external surfaces of root crops. Therefore, should such products be formed in fresh water, sewage and soil, the threat they might pose cannot be dismissed readily, especially since the potential amine precursors are widespread and sometimes exist in high concentrations. Although the nitrite precursor is never added to natural environments and rarely is found in appreciable concentrations, the preceding discussion suggests that reasonable levels sometimes occur. Of direct relevance to a concern with potential microbial nitrosamine formation in natural habitats is the report that the number of deaths from stomach cancer in a town in England with a water supply containing nitrate at levels in excess of most public health standards is 32% greater for males and 62% greater for females than in towns having water supplies not polluted with nitrate ( Hill, 1972). The correlation between nitrate concentration in the water and stomach cancer may be fortuitous and, even if real, the responsible factor may not be a nitrosamine; however, such a correlation surely must be assessed further. One line of evidence that microorganisms may be able to form nitroso compounds in nature comes from studies of heterotrophic nitrification. Thus, in samples of raw sewage and river and lake water amended with a simple organic compound, ammonium can be oxidized microbiologically to a product characterized as l-nitrosoethanol ( Fig. 4), the yield sometimes reaching 59 ppm. This metabolite, which is a C- rather than an N-nitroso compound, is surprisingly resistant to biodegradation in river water and sewage (Verstraete and Alexander, 1973). Dimethylnitrosamine has been reported, however, to be generated in soils differing markedly in texture and pH. The N-nitroso compound was produced from tri- and dimethylamine in the presence of added nitrate or nitrite or that formed naturally in the samples, which were examined in laboratory trials. The yield was sometimes as high as 17 ppm of the nitrosamine, although it usually was lower. The same product was found in soil amended with the fungicide thiram (tetramethylthiuram disulfide). The levels of the various amines added were quite high, however ( Ayanaba et at., 1973a). Dimethylnitrosamine was also

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

53

made from trimethylamine in raw sewage at pH 5.0, 6.0, and 7.0, presumably after the tertiary amine was dealkylated. The nitroso compound also appeared in acid sewage into which thiram had been introduced. On the basis of tests with sterile samples, the nitrosation of the secondary amine, at least at neutral pH values, and the conversion of the tertiary to the secondary amine and nitrate to nitrite resulted from microbial metabolism ( Ayanaba and Alexander, 1973; Ayanaba et al., 1973b). Several microorganisms have been stated as being capable of synthesizing nitrosamines. Owing to the lack of information in some of the published papers on the changes or maintenance of pH in the culture medium during the full growth cycle, information necessary to assess whether nonenzymatic nitrosation occurred coincident with an increase in acidity of the medium, the validity of some of the reports must be accepted with some reservation. According to the study of Hawksworth and Hill (1971a), many bacteria are able to synthesize nitrosamines. They state that 5 of the 10 E. coli strains tested formed nitrosamines from diphenylamine, dimethylamine, diethylamine, piperidine, pyrrolidine, and N-methylaniline in a nitrate-containing medium at a pH greater than 6.5. Furthermore, 10%of the Clostridium strains, 12%of the Bacteroides strains, 40% of the strains of enterococci, and 18%of the isolates of bifidobacteria would nitrosate diphenylamine. They also observed that choline could be converted by some of the bacteria to dimethylamine and that proline was metabolized to pyrrolidine (Hawksworth and Hill, 1971b), both possible nitrosamine precursors. Sander ( 1968) showed that nitrosamines appeared in the growth medium of cultures of E. coli, Proteus vulgaris, and Serratia marcescens incubated with diphenylamine, di-n-propylamine or N-methylaniline. Dimethylnitrosamine also was noted to be generated when E. coli, Staphylococcus epidermidis, or Aspergillus oryzae hyphae were incubated with dimethylamine and nitrate or another bacterium was provided with trimethylamine and nitrite ( Ayanaba and Alexander, 1973). The only direct evidence for the existence of an enzyme capable of forming nitrosamines comes from a study of strains of Pseudomonas and Cryptococcus. Extracts of cells of both organisms formed N-nitrosodiphenylamine from diphenylamine and nitrite, and a soluble enzyme preparation from the yeast was also reported to synthesize dimethyl-, diethyl-, and di-n-propylnitrosamine from the corresponding amines and nitrite ( Ayanaba and Alexander, 1973). XII.

Other Carcinogens

It might be deemed somewhat esoteric for a role to be proposed for microorganisms in carcinogenesis, but one cannot but be impressed by the suggestion that environmental factors may be involved in the

54

MARTIN ALEXANDER

etiology of 80-90% of cancers in humans (Drasar and Hill, 1972). Considering the ubiquity of bacteria, fungi, and actinomycetes and the many substances they excrete in nature, it is not too farfetched to postulate that they are involved, directly or indirectly, in the induction of cancerous growths. One of the best cases for a widespread significance of microorganisms is in connection with cancer of the intestine. A surprising geographical distribution is evident in the incidence of cancer of the colon, the frequency being distinctly higher in the United States and Great Britain than in the sections of East Africa, Asia, and South America that have been surveyed. Genetic differences in the human population have been ruled out, but components of the diet, probably protein or fats, are implicated. Individuals in regions with high carcinoma frequency consume diets rich in animal protein and fat, whereas people in regions with low frequency have a diet that is largely vegetarian and contains little meat. This unique geographic distribution is not, on the basis of the available data, related to the presence of carcinogens in the cooked food itself, moreover. Therefore, it seems quite plausible to postulate that the diet makes its insidious effect felt by altering the composition of the bacterial community of the intestine. The different bacteria, in turn, have dissimilar substrates available to them as the foodstuffs pass through the alimentary tract, and the colon residents may then synthesize the carcinogens in situ. It is established, moreover, that steroids such as deoxycholic acid, cholenic acid, and apocholic acid are indeed carcinogens, and dehydronorcholene can be metabolized by intestinal bacteria to yield a carcinogen. The experimental findings obtained in support of this hypothesis do indeed disclose a difference in both bacteria and steroids in the intestinal contents of people residing in areas with low and high incidences of colon cancer. The bacterial differences are found in the densities of Bacteroides, aerobic streptococci, enterococci, lactobacilli, and yeasts, and the chemical differences are in the concentrations of acid and neutral steroids and urobilins (Aries et al., 1969; Hill and Aries, 1971; Hill et al., 1971). It has also been proposed that mycotoxins cause liver cancer in Asian countries, and several of these fungal metabolites will produce cancer in experimental animals, The carcinogens synthesized by fungi include the following: ( a ) ergot alkaloids, which will be discussed below; ( b ) aflatoxins, a group of compounds made by Aspergillus flavus and Aspergillus parasiticus and known to induce cancer in many organs; ( c ) sterigmatocystin, synthesized by Aspergillus versicolor, Aspergillus niduluns, and a species of Bipolaris and widely distributed in mold-contaminated foodstuffs of South Africa and Japan and reported in other regions also; ( d ) luteoskyrin and cyclochlorotine,elaborated by Penicillium islandicum,

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

55

which grows on “yellowed rice”; ( e ) patulin, produced by species of Penicillium, Aspergillus, and B yssochlamys; ( f ) penicillic acid, derived from strains of many species of Penicillium and Aspergillus, genera that are ubiquitous in soil and on moldy foods; and ( g ) griseofulvin, synthesized by a number of Penicillium species ( Enomoto and Saito, 1972). Carcinogenic aromatic hydrocarbons are widespread, and they have been detected in soils and natural waters. Their concentration in soil is commonly in the range of 0.1 to 1.0 pprn although lower levels are not unknown. They are also evident in inland surface waters, groundwater, sewage effluents, and in river bottom and marine sediments, sometimes in disturbingly large amounts. Their presence in these diverse environments is probably at least partially the result of the ability of photoautotrophs and heterotrophs to make them in waters, soils, and sediments. For example, carcinogenic polynuclear aromatic hydrocarbons exist in Chlorella uulgaris in concentrations up to 0.05 ppm, and 3,4benzpyrene, a member of this class of compounds, is in marine plankton in levels up to 0.40 ppm on a dry-weight basis (Andelman and Suess, 1970). Various bacteria accumulate up to 0.006 ppm ( Knorr and Schenck, 1968), whereas Scenedesmus obliquus contains 0.1 to 0.3 pprn of 3,4benzpyrene and 0.6 to 1.9 ppm of 3,4-benzofluoroanthene (Wagner and Siddiqi, 1973). The metabolism of cycasin (methylazoxymethanol-/3-D-glucoside) is a novel example of how a microflora can be involved in carcinogenesis. Cycasin, a constituent of the nut of the fern known as a cycad, is itself not active and shows no deleterious effects when fed to germfree rats. However, when given to conventional animals, members of the intestinal community hydrolyze the molecule to yield the free carcinogen methylazoxymethanol. CH,N(O) :NCHzOCeH1106

+ HzO

-+

CH,N(O) :NCHzOH

+ CeHizOe

(16)

Quite similar is the way by which bacteria release carcinogenic aromatic hydroxylamines. The liver contains enzymes that catalyze the N-hydroxylation of aromatic amines to give the corresponding and carcinogenic N-hydroxy compounds, but the latter are rapidly detoxified as they are conjugated with glucuronic acid. Upon entering the cecum and colon, however, the glucuronide is cleaved by the intestinal inhabitants to release the carcinogen. Because bacterial p-glucuronidase is absent from germfree animals, the glucuronides do not produce colon cancer in these animals ( Weisburger, 1971). A few other substances, in addition to those listed above, produced by microorganisms in culture act as carcinogens, but they are not known to be synthesized outside of laboratory media.

56

MARTIN ALEXANDER

XIII.

Complex Human and Animal Toxins

A surprisingly large number of complex toxic agents are produced by fungi and algae residing apart from the people or animals that are affected. The organisms, living as they do separate from the individuals suffering harm, are not parasites, and they do not cause communicable diseases. The substances the fungi and algae elaborate and which do the harm, insofar as they are known, are distinct chemical entities so that the compounds can indeed be deemed as environmental pollutants, rather than reflecting the actions of a parasite that does damage as it proliferates within the tissues of its host. Many types of compounds are involved, including peptides, alkaloids, unsaturated lactones, and complex aromatic molecules. The fungal toxins are typically but not invariably synthesized on grains and plant materials, while those of the algae are characteristically associated with aquatic ecosystems. Fortunately almost wholly forgotten now is ergotism and the havoc it wrought in Europe. The loss in human life was enormous. Barger ( 1931) documents the numerous outbreaks, the horrible development of the disease in humans, and the disastrous consequences of ergotism beginning with the year 857 and ending in the early part of this century. Individual episodes led to thousands and possibly tens of thousands of deaths, with occasional outbreaks causing more than 50% mortality in an affected region. Barger traces ergotism on an episode-by-episode basis, to the extent that historical records exist or have been maintained, for an entire millenium through what is now France, Switzerland, Germany, Czechoslovakia, Sweden, Norway, Denmark, Russia, Romania, Hungary, Italy, and England. The scientist intimately concerned with pollution from modern synthetic chemicals would do well to read how effective pollutants can be made by fungi and how utterly devastating their actions can be. Persons suffering from ergotism might lose toes, fingers, or whole limbs, their flesh often was eaten away, and amputation was often required-without, of course, benefit of anesthetics. Raging fever, convulsions, abortion, and mental derangements were also quite frequent. Ergotism is caused by species of Clauiceps that multiply and form sclerotia on a large number of grasses. The best known member of this group of fungi is Clauiceps purpurea. The Clauiceps sclerotia contain several substances injurious to man and animals, but it is their alkaloids, which are 3,4-substituted indole derivatives, that are of primary importance. Human ergotism was largely associated with the widespread use of rye as the major food cereal during the Middle Ages, and rye is particularly susceptible to infection by Clauiceps; hence, bread made from rye flour frequently contained the poison. Among the other grains,

MICRO5IAL FORMATION OF ENVIRONMENTAL POLLUTANTS

57

wheat, barley, and oats may be parasitized by the fungus, but such infestations were reasonably uncommon. Fortunately for those people consuming bread made from rye flour, infestations which were sufficiently severe to lead to widespread poisonings required an unusual coincidence of environmental conditions. The abrupt decline of ergotism in the nineteenth century is attributable to an understanding of the importance of the sclerotia to the intoxication, the cleaning of the grain, governmental warning programs, and replacement of much of the rye in the diet of Europeans with potatoes and maize. Government agencies have set standards on the permissible content of the sclerotia in grains, and current methods of grain cleaning allow these standards to be met easily. Nevertheless, human ergotism was reported in France and India in the 1950's. Ergotism remains as a problem of livestock raising, however, and significant livestock losses are incurred in a year favorable to Claviceps proliferation. Nervous disorders, abortion, paralysis, blindness, and often death are ascribed to heavy grazing on the infested grasses, and sheep and horses as well as cattle still on occasion succumb. Another human malady of staggering proportions is alimentary toxic aleukia. This noncommunicable disease arises from toxins of several species of Fusarium and Cladosporium as well as occasional strains of Alternaria, Mucor, and Penicillium. It was observed in what is now the Soviet Union in 1913 and then again in 1932, during which time entire families and sometimes whole villages were decimated. The disease was extensively studied when it appeared once more in the Orenburg and other districts of the U.S.S.R. in 1942, with a more severe outbreak in 1943 and a still higher incidence the following year. During this period of the Second World War, starvation or near-starvation was the rule in these districts, and the populace resorted to gathering grain that had fallen and been buried under the snow during the winter. The responsible fungi slowly developed on the grain in the winter but began to make the toxin during the spring thaw, at which time the people collected the overwintered grain. The disease incidence rate among residents in these localities was staggering, more than 10%of the people being afflicted and many dying (Joffe, 1971). The grains that support toxin production-millet, wheat, rye, oats, and buckwheat-are grown all over the world, yet the problem has not been reported outside the Soviet Union, probably because of the peculiar set of circumstances leading to toxin biosynthesis. Toxins of the mushrooms infrequently poison humans consuming these higher fungi. Benedict (1972) summarized the incidence of mushroom poisoning and reported 1980 cases in 40 years in Switzerland and 500 to 600 cases in a single year in Japan. About 5% of the poisonings in

58

MARTIN ALEXANDER

Switzerland led to death, and about 15 fatilities occurred in the one-year period in Japan. In the United States, 73 deaths attributable to eating mushrooms were recorded during a period of some 70 years. Among the mushrooms implicated in human disorders are Galerina sulciceps, Cortinarius orellanus, and species of Helvella, but of greatest importance is Amanita because of its potency and the high mortality rate associated with its consumption. Amanita species, mainly A. phalloides in Europe and A. vernu in North America, synthesize cyclopeptides that are responsible for more than 95%of the fatal cases of mushroom poisoning. The peptides are composed of several amino acids and have molecular weights of about 1000 ( Wieland, 1968). Extensive losses of turkeys resulting from the consumption of peanuts colonized by fungi led to the finding that Aspergillus jlavus, a common soil inhabitant, growing on the peanuts elaborated the highly toxic aflatoxins. Aflatoxins have also killed ducklings and chickens and have been responsible for disorders in pigs, cattle, and buffalo. Not only is A. flavus able to elaborate aflatoxins, but so too are Aspergillus parasiticus and probably strains of Penicillium, Mucor, and other Aspergillus species. The responsible fungi are widespread and they can make their hazardous metabolites on many food and feed products and in many countries where the prevailing temperatures are warm, the humidity is high and the facilities for harvesting, storing, and marketing remain primitive. Among the products in which aflatoxins have been detected are peanuts, peanut meal, peanut cake, peanut butter, cottonseed and the meal and cake prepared from it, Brazil nuts, rice, corn, and sweet potatoes (Detroy et al., 1971). Other fungi also have the capacity to generate chemicals of ecological importance. For example, Pithomyces chartarum colonizes the remains of plants lying in the field, and it may there produce a toxin known as sporidesmin. This compound, when consumed by sheep and cattle grazing on the plant remains, causes a facial eczema, a condition of some consequence in parts of New Zealand and Australia (Wright, 1968). Stachybotryotoxin, a partially characterized substance, is synthesized by Stachybotrys alternuns proliferating on straw, and use of this straw for roughage or bedding led to a large number of poisonings of horses in the U.S.S.R. in the 1930’s. Cattle fed with straw colonized by the same fungus likewise succumbed ( Forgacs, 1972). Zearalenone, an enantiomorph of 6- ( 10-hydroxy B-oxo-trans-l-undecenyl ) -p-resorcylic acid lactone, is made by Fusarium gramineum on maize that is harvested and stored while still quite moist, and it is responsible for an estrogenic syndrome in swine in which abortions may occur, the mammary glands enlarge, the vulva swells, and the testes of the male shrink (Mirocha et al., 1968). By contrast, corn still in the field may be colonized by

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

59

Penicillium rubrum or A. flams, and pigs and cattle consuming this corn suffer intoxication (Brook and White, 1966). Rhizoctonia leguminicola growing on certain legumes, notably red clover, may form l-acetoxy8-aminooctahydroindolizidine,a metabolite that provokes excessive salivation in cattle, which may then refuse further feed. In addition, more severe symptoms may develop (Aust et al., 1968). Strains of Aspergillus, Penicillium, and Mucor proliferating on sweet clover may synthesize 3,Y-methylenebis(4-hydroxycoumarin), a compound responsible for a fatal hemorrhagic disorder in cattle and sheep eating the moldy plants (Richards, 1972). Other toxins are produced on millet grain by Rhizopus nigricans and affect humans, on malt feed by Penicillium urticae and affect cattle, and on rye and barley by Penicillium viridicatum and cause a disease among swine (Bamburg et al., 1969). Just the outer surface of the problem of fungal toxins undoubtedly has been scratched, if for no other reason than that only toxins affecting humans and domesticated animals have been intensively investigated. It is to the algae that one turns for the chemically complex toxins generated in freshwater, marine, and brackish ecosystems. Algae causing mortalities of fish, livestock, waterfowl, and, on occasion, people are distributed in many aquatic ecosystems. More than 500 human deaths have been ascribed to algal poisons, and many thousands have fallen ill (Schwimmer and Schwimmer, 1964). The economic losses to fisheries, the shellfish industry, and owners of livestock have sometimes been quite severe. The poisons made by some algae are potent exotoxins that are excreted into the aquatic milieu, but others are endotoxins that are released to the water only following death and decomposition of the responsible species. Certain marine dinoflagellates may develop into sizable blooms that impart to the water a red or brown color, and these blooms often lead to mass mortalities of fish and other marine animals. Man may suffer severe illness or death should he consume shellfish, such as mussels or clams, that themselves have acquired the toxin-bearing dinoflagellate. The compounds that do the harm to humans eating the shellfish are commonly produced by Gonyautax species, such as G. catenella, G. tamarensis, or G. acatenella, but Pyrodinium phoneus sometimes makes shellfish hazardous as well. The poisons in these instances apparently do not affect the shellfish, but only the unfortunate person who feeds on them (Schantz, 1971). The toxin of G . catanella is a low-molecularweight compound, CloHl,N,0,.2HC1, that is given the trivial name saxitoxin. The minimum lethal dose is 9 rg/kg, a truly potent agent (Gentile, 1971). A bloom of G . tamurensis in the spring of 1968, moreover, not only led to illness of people who ate shellfish from the affected part of the sea near Great Britain, but proliferation of the alga was

60

MARTIN ALEXANDER

also correlated with the death of sea birds, pigeons, and invertebrates (Wood, 1968). Gymnodinium species have been responsible for the killing of thousands of tons of marine fish, and toxins of these dinoflagellates act on the nervous systems of fish, molluscs, and mammals, often causing their death. Prymnesium parvum, a chrysophyte residing and at times proliferating rapidly in brackish water, synthesizes an extracellular toxin that has led to the death of large numbers of fish in the Netherlands, Denmark, Israel, and elsewhere. The poison contains several phosphorus-containing components and is made up of 15 amino acids, fatty acids so far unidentified, and hexose sugars ( Shilo, 1971). Freshwater blue-green algae are responsible for numerous episodes of intoxication that have been recorded in many countries. The poisonings are linked with massive growths of the toxin-bearing organisms, which are chiefly species of Microcystis, Anabaena, and Aphanizomenon, with Anabaena flos-aquae and Microcystis aeruginosa as common offenders. Species of Nodularia, Coelosphaerium, and Gloeotrichia also affect animals in or consuming the water. Cattle in the thousands as well as sheep and horses drinking from bodies of water with the blue-greens have become sick and succumbed. Human gastroenteritis likewise has been attributed to heavy blooms in municipal water supplies, and allergic reactions have been documented among people. Wild birds, chickens, wild animals, and pets have all shown deleterious effects from one or another of these aquatic autotrophs, and fish mortalities have been observed, too. Several toxins appear to be responsible for the harm that is done, but only a few have been characterized; for instance, the active metabolite of M . aeruginosa is a cyclic polypeptide with a molecular weight of about 2600, whereas that of A. jlos-aquae is an alkaloid having a molecular weight of about 300 (Gentile, 1971; Gorham, 1964).

XIV.

Other Phytotoxins

The indigenous populations of soils continuously or intermittently receive fresh sources of organic nutrients from root excretions, falling leaves, and plant remains, and from these they make and undoubtedly excrete an array of compounds whose number has never been defined. Many of the products when extracted from soil, as well as characterized compounds or unfractionated excretions from laboratory-grown cultures, are phytotoxic and induce a diversity of symptoms on test plants. Investigations of these inhibitors in the greenhouse or laboratory frequently cannot be directly related to any actual problem, but evidence from fields

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

61

growing agronomic or horticultural crops or fruit trees shows that phytotoxicity induced by soil residents living in close proximity to plant roots is widespread and sometimes serious. Often these inhibitions are evident in poorly aerated or waterlogged soils, wherein the excreted metabolites do in fact accumulate because their further degradation is suppressed. On the other hand, some phytotoxins also are evident in soils that, from a macroecological standpoint, seem to be adequately oxygenated, hut they probably are formed and persist in an organic matter-rich microenvironment near the plant root where the microbial 0, demand exceeds the supply and where the root tissue is sufficiently close to allow the toxicant to cause damage. The antibiotic patulin is one such compound. Under certain conditions and when the soil is wet and crop residues remain, Penicillium urticae proliferates and patulin appears, This potent phytotoxin then does appreciable harm to nearby plants. P . urticae synthesizes this compound in culture, and soils deliberately inoculated with the fungus contain patulin and are deleterious to test plants (Norstadt and McCalla, 1968). Patulin formation may be implicated in a problem in old apple orchards or in nurseries where apple trees were previously grown. New trees introduced into these soils have shortened tap roots and grow poorly. Leachates from these soils produce the same symptoms when added to problem-free soil. An involvement of patulin is suggested by observations that the antibiotic is detectable in soils receiving residues of apple roots and that patulin is formed by a soil-derived strain of Penicillium expansum grown on the root residues (Borner, 1963a,b). Other types of molecules are formed in the subterranean ecosystem and may or do injure higher plants. Citrinin has been observed in soils containing Penicillium citrinum, and citrinin deliberately added to ihe soil or that apparently synthesized in organic matter-amended soils suppresses plant growth ( Skorobogatova and Mirchink, 1972). Acetaldehyde, furfuraldehyde, and isovaleraldehyde, all detrimental to plants, have been found in soils in which Chinese yams were showing symptoms of a disease known as black scurf, and the appearance of these aldehydes correlated with the occurrence of the condition in field-grown yams. The responsible organisms are unknown (Nishiniura et al., 1969). Certain diseases have even been attributed to the formation of HCN by terrestrial basidiomycetes ( Hutchinson, 1973), representatives of which liberate HCN in culture. Although most interest has been centered on the simpler molecules, Kimber (1973) recently demonstrated that a spectrum of substances made during the decomposition of straw are toxic, but the most effective products had molecular weights from 10,000 to 50,000. Several other phytotoxins have been discussed in preceding sections.

62

MARTIN ALEXANDER

XV.

Other Compounds with Odor and Taste

It should come as no surprise to microbiologists that microscopic organisms elaborate odors. To a few of our colleagues, it is a disturbing but nonetheless acknowledged fact that some species generate off -odors or foul smells. But the offensive, obnoxious, and occasionally vile smells-or sometimes off -tastes-frequently are highly objectionable to whoIe communities because they are not restricted to the laboratory but contaminate municipal water supplies or emanate from animal or human wastes discharged near suburban dwellings or resort hotels. Some of the odoriferous compounds have been considered above. Several microbial products, a few of which have been identified, impart odor and taste to drinking water. Particularly common metabolites are those causing the musty or earthy tastes observed in many potable water supplies derived from rivers and natural or artificial reservoirs. The chief organisms synthesizing these pollutants are algae and actinomycetes. Among the algae, it is largely blue-greens of the genera Anabaena, Aphanizomenon, and Oscillatoria that generate metabolites causing tastes and odors in waters, but Peridinium may also be an offender (Rebhun et al., 1971; Silvey et al., 1972). Blooms of these algae have been notable in reservoirs in southwestern United States and Israel. The odors of algae have been characterized quaintly, albeit descriptively, as fishy, earthy, geranium, nasturtium, candied violets, grassy, or smelling like a pigpen (Rohlich and Sarles, 1949). When an extensive bloom arises and at certain times of year, the odor may not be that of the algae but rather one attributable to bacterial products formed as the algae undergo decomposition, especially as the dissolved 0, level in the water falls to zero. Apart from the quaint adjectives used to describe the odors and tastes, the specific compounds made by the algae and bacteria have not been characterized. Actinomycetes, probably mainly Streptomyces, also impart earthy or musty odors to water supplies. For example, musty taste and odor characterized the water taken from the Cedar River in Iowa at a time when an unusually large number of actinomycetes was present (Morris et al., 1963). In these instances, the product commonly causing the earthy odor is geosmin, which is a typical streptomycete product in culture, too. Gerber ( 1968) identified geosmin as tran.s-l,lO-dimethyl-trans-9decalal. The smell of freshly turned soil is probably also that of geosmin or of related volatiles elaborated by streptomycetes. Collins et al. (1970) reported that 2-exo-hydroxy-2-methylbornaneis a second component of the streptomycete odor. Anaerobic bacteria are responsible for a highly localized but still quite disturbing problem of air pollution resulting from the existence of large

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

63

accumulations of cattle, hog, and poultry manure in proximity to suburban housing and to hotels in tourist resorts. Economic pressures and the need for efficient use of labor and equipment have forced U.S. farmers and feedlot managers to concentrate cattle, hogs, and poultry into high-density confinement units which, as pointed out above, may have many thousands of cattle or hundreds of thousands of chickens on a small parcel of land. The resulting enormous heaps of manure contain many bacterial nutrients, and the rapid microbial colonization of these piles quickly leads to a complete depletion of the 0, supply. With the onset of anaerobiosis, these populations make odors offensive to people living in the vicinity of the feedlot, swine barn, or poultry house. Some of the complaints, however, come not from the odor emanating from the site where the animals are but from the manure handling and disposal operations. Many of the compounds responsible for or contributing to the smell have been defined. The products from dairy animal wastes include HeS, methane thiol, dimethyl sulfide, diethyl sulfide, ethylamine, trimethylamine, propyl acetate, and n-butyl acetate (White et al., 1971). Ammonia, of course, is common. These compounds are readily dissipated to the atmosphere, but Mosier et al. (1973) were still able to identify methyl-, dimethyl-, ethyl-, n-propyl-, isopropyl-, n-butyl, and n-amylamines in the air over a large beef cattle feedlot. Among the volatiles emanating from poultry manure subjected to attack by anaerobic bacteria are H2S, methane thiol, ethane thiol, propane thiol, ammonia, indole, skatole, n- and isobutyric acids, and n- and isovaleric acids (Young et al., 1971). Such volatiles have highly offensive odors, and the odor threshold for some is remarkably low.

XVI.

Perspective

It should be obvious to the reader that waters, soils, and the atmosphere are being contaminated with products derived from microbial activities in natural ecosystems. Some of the metabolites may never reach concentrations that pose a threat to man, domesticated animals, agricultural crops, wildlife, or populations of other species. Others surely have already attained these levels in particular regions or in distinct habitats so that higher organisms occasionally suffer injury. It should also be obvious that the contribution of professional microbiologists to these areas of inquiry has been disturbingly small. Much of the information comes from entirely different disciplines. Yet, microbiologists can and should become more intimately involved with the various subject areas not only because the results they collect will allow society to overcome more readily some of its serious environmental prob-

64

MARTIN ALEXANDER

lems, but also because the data will permit a more full understanding of the behavior of microbial populations in nature. REFERENCES Abu-Erreish, G. M., Whitehead, E. I., and Olson, 0. E. (1968). Soil Sci. 106, 415420. Alam, B. S. Saporoschetz, I. B., and Epstein, S. S. (1971). Nature (London) 232, 199-200. Albone, E. S., Eglington, G., Evans, N. C., Hunter, J. M., and Rhead, M. M. (1972). Enoiron. Sci. Technol. 6, 914-919. Aleem, M. I. H., and Alexander, M. (1960). Appl. Microbiol. 8, 80-84. Alexander, M. ( 1971). “Microbial Ecology.” Wiley, New York. Alexander, M. ( 1973). Biotechnol. Bioeng. 15, 611-647. Alexander, M., and Aleem, M. I. H. ( 1961). J. Agr. Food Chem. 9,44-47. Allsopp, D., Hueck, H. J., Eggins, H. 0. W., and Lloyd, A. 0. (1970). I. Text. lnst. 61, 509-511. Andelman, J. B., and Suess, M. J. 1970). Bull. W.H.O.43, 479-508. Ando, K., Kato, A., and Suzuki, S. (1970). Biochem. Biophys. Res. Commun. 39, 1104-1 107. Anonymous. (1972). Food Cosmet. Toxicol. 10, 867-873. Aries, V., Crowther, J. S., Drasar, B. S., Hill, M. J., and Williams, R. E. 0. (1969). Gut 10, 334-335. Asami, T., and Takai, Y. (1963). Soil Sci. Plant Nutr. ( T o k y o ) 9, 65-69. Audus, L. J. (1953). Nature (London) 171, 523-524. Aust, S. D., Broquist, H. P., and Rinehart, K. L. (1968). Biotechnol. Bioeng. 10, 403-412. Ayanaba, A., and Alexander, M. (1973). Appl. Microbiol. 25, 862-868. Ayanaba, A., Verstraete, W., and Alexander, M. (1973a). Soil Sci. SOC. A m r . , Proc. 37, 565-568. Ayanaba, A., Verstraete, W., and Alexander, M. (1973b). J. Nut. Cancer lnst. 50, 811-813. Bache, C. A., and Lisk, D. J. ( 1966). J. Ass. Ofic. Anal. Chem. 49, 647-650. Bache, C. A,, Gutenmann, W. H., St. John, L. E., Sweet, R. D., Hatfield, H. H., and Lisk, D. J. (1973). J. Agr. Food Chem. 21, 607-613. Bamburg, J. R., Strong, F. M., and Smalley, E. B. (1969). J. Agr. Food Chem. 17, 443-450. Barger, G . (1931). “Ergot and Ergotism.” Gurney & Jackson, London. Barker, P. S., Morrison, F. O., and Whitaker, R. S. (1965). Nature (London) 205, 621-622. Bartha, R., and Pramer, D. (1970). Advan. Appl. Microbiol. 13, 317-341. Bautista, E. M., and Alexander, M. (1972). Soil Sci. SOC. Arner. Proc. 36, 918-920. Baxter, R. M., Wood, R. B., and Prosser, M. V. (1973). Limnol. Oceanogr. 18, 470-472. Beijerinck, M. W., and Minkman, D. C. J. (1910). Centralbl. Bakteriol., Parasitenk., Znfekstionskr. Hyg., Abt. 2 25, 30-63. Benedict, R. G. (1972). In “Microbial Toxins” ( S . Kadis, A. Ciegler, and S. J. Ajl, eds.), Vol. VIII, pp. 281-320. Academic Press, New York. Berger, R. S. (1972). Science 177, 704-705.

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

65

Bergstrom, P. D., Grant, D. W., and Morrison, S. M. ( 1972). Enoiron. Lett. 3, 151-157. Birch, P. D. W., and Eagle, D. J. (1969). J . Hort. Sci. 44, 321430. Bird, M. L., and Challenger, F. (1939). J. Chem. SOC., London pp. 163-168. Blasco, F., Robert, J.-J., and Gaudin, C. (1972). C. R . Acad. Sci., Ser. D 275, 1223-1126. Bloomfield, C. (1969). J . Soil Sci. 20, 207-221. Bollag, J.-M., and Tung, G. (1972). Soil Biol. G Biochem. 4, 271-276. Bordeleau, L. M., and Bartha, R. (1971). Soil Biol. G Biochem. 3, 281-284. Borisova, N. N., Burtseva, S. N., Rodionov, V. N., and Kirpaneva, 0. L. (1972). Sou. Soil Sci. pp. 540546. BGmer, H. (1963a). Phytopathol. Z. 48, 370-396. Borner, H. (1963b). Phytopathol. Z. 49, 1-28. Boyd, C. E. ( 1972). Weed Sci. 20,492497. Boynton, D. ( 1941). Cornell Unio. Agr. Exp. Sta. Bull. 763. Braman, R. S., and Foreback, C. C. (1973). Science 182, 1247-1249. Brant, R. A., and Moulton, E. Q. (1960). Ohio State Unio., Eng. Exp. Sta., Bull. 179. Braunberg, R. C., and Dailey, R. E. (1973). Proc. SOC. E x p . Biol. Med. 142, 993-996. Brook, P. J., and White, E. P. (1966). Annu. Reo. Phytopathol. 4, 171-194. Burford, J. R., and Stefanson, R. C. (1973). Soil Biol. G Biochem. 5, 133-141. Bush, L. P., Sims, J. L., and Atkinson, W. 0. (1970). Can. J. Plant Sci. 50, 289-294. Carlton, W. W., and Kelly, W. A. (1967). Toxicol. Appl. Pharmacol. 11, 203-214. Carpenter, L. V., and Herndon, L. K. (1933). W . Va. Unio. Eng. Erp. Sta., Res. Bull. 10. Challenger, F. (1951). Advan. Enzymol. 12, 429491. Challenger, F., and Charlton, P. T. ( 1947). J . Chem. SOC., London pp. 424429. Challenger, F., Lisle, D. B., and Dransfield, P. B. (1954). J . Chem. SOC., London pp. 1760-1771. Chang, H. T., and Loomis, W. E. (1945). Plant Physiol. 20, 221-232. Chapman, H. D., and Liebig, G. F. (1952). Soil Sci. SOC. Amer., Proc. 16, 276-282. Chaudhry, I. A., and Cornfield, A. H. ( 1967). J . Sci. Food Agr. 18, 38-40. Clemons, G. P., and Sisler, H. D. ( 1969). Phytopathology 59, 705-706. Collins, R. P., Knaak, L. E., and Soboslai, J. W. (1970). Lloydia 33, 199-200. Committee on Nitrate Accumulation. ( 1972). “Accumulation of Nitrate.” Nat. Acad. Sci., Washington, D.C. Cook, F. D. (1963). Abstr., Int. Congr. Microbiol., 8th, 1962 p. 55. Coppedge, J. R., Lindquist, D. A., Bull, D. L., and Dorough, H. W. (1967). J. Agr. Food Chem. 15,902-910. Cox, D. P., and Alexander, M. (1973). Bull. Enuiron. Contam. Toxic01. 9, 84-88. Craig, H., and Gordon, L. I. ( 1963). Geochim. Cosmochim. Acta 27, 949-955. Curtis, D. S. (1949). Soil Sci. 68, 441-450. Curtis, R. F., Land, D. G., Griffiths, N. M., Gee, M., Robinson, D., Peel, J. L., Dennis, C., and Gee, J. M. (1972). Nature (London) 235, 223-224. Dalton, R. L., Evans, A. W., and Rhodes, R. C. (1966). Weeds 14, 31-33 . Davis, V. W. (1973). In “Environmental Quality and Safety” (F. Coulston and F. Korte, eds. ), Vol. 2, pp. 260-270. Thieme, Stuttgart. Delaney, L. T., Schmidt, H. W., and Stroebel, C. F. (1956). PTOC.Stag Meet. Mayo Clin. 31, 189-198.

66

MARTIN ALEXANDER

Detroy, R. W., Lillehoj, E. B., and Ciegler, A. (1971). In “Microbial Toxins” (S. Kadis, A. Ciegler, and S. J. Ajl, eds.), Vol. VI, pp. 3-178. Academic Press, New York. Dittrich, H. H., and Staudenmayer, T. ( 1970). Zentralbl. Bakteriol., Parasitenk., Infektionskr. Hyg., Abt. 2 124, 113-118. Dobrovol’skiy, G. V., Bab’yeva, I. P., and Lobutev, A. P. (1960). Sou. Soil Sci. pp. 1181-1191, Dowdell, R. J., Smith, K. A., Crees, R., and Restall, S. W. F. ( 1972). Soil Biol. G Biochem. 4, 325-331. Drasar, B. S., and Hill, M. J. (1972). Amer. J. Clin. Nutr. 25, 1399-1404. Dykstra, G. J,, Drerup, D. L., Branen, A. L., and Keenan, T. W. (1971). J. Dairy Sci. 54, 168-172. Elliott, L. F., and Travis, T. A. ( 1973). Soil Sci. SOC.Amer., Proc. 37, 700-702. Elsner, S., Bieniek, D., Klein, W., and Korte, F. (1972). Chemosphere 1, 247-250. Emery, K. O., Orr, W. L., and Rittenberg, S. C. (1955). In “Essays in the Natural Sciences in Honor of Captain Allan Hancock,” pp. 299409. Univ. of Southern California Press, Los Angeles. Engel, R. R., Matsen, J. M., Chapman, S. S., and Schwartz, S. (1972). 1. Bacteriol. 112, 1310-1315. Engst, R., Kujawa, M., and Miiller, G. (1967). Nahrung 11, 401-404. Enoch, H., and Dasberg, S. (1971). Geoderma 6, 17-21. Enomoto, M., and Saito, M. (1972). Annu. Reu. Microbiol. 26, 279-312. Epps, E. A,, and Sturgis, M. B. (1939). Soil Sci. Soc. Amer., Proc. 4,215-218. Eriksson, E. (1963). J. Geophys. Res. 68, 40014008. Ewell, R. ( 1972). Chem. Technol. 2, 570-575. Fagerstrom, T., and Jernelov, A. ( 1971). Water Res. 5, 121-122. Faulkner, J. K., and Woodcock, D. (1966). J. Chem. Soc., C pp. 884-887. Fields, M. L., and Hemphill, D. D. (1966). Appl. Microbiol. 14, 724-731. Fishbein, L., Falk, H. L., and Flamm, W. G. (1970). “Chemical Mutagens: Environmental Effects on Biological Systems.” Academic Press, New York. Fleming, J. F., and Alexander, L. T. ( 1961). Soil S d . SOC. Amer., Roc. 25, 94-95. Fleming, R. W., and Alexander, M. (1972). Appl. Microbiol. 24, 424-429. Fliermans, C. B., and Brock, T. D. ( 1972). J. Bacteriol. 111, 343-350. Ford, H. W. (1965). Proc. A m r . S O C . Hort. Sci. 86,205-212. Ford, H. W. (1973). J. Amer. SOC. Hort. Sci. 98, 66-68. Forgacs, J. ( 1972). In “Microbial Toxins” (S. Kadis, A. Ciegler, and S. J. Ajl, eds. ), Vol. VIII, pp. 95-128. Academic Press, New York. Francis, A. J., Adamson, J., Duxbury, J. M., and Alexander, M. ( 1973). In “Modern Methods in the Study of Microbial Ecology” ( T . Rosswall, ed.), pp. 485-488. Swedish National Research Council, Stockholm. Freebairn, H. T., and Buddenhagen, I. W. (1964). Nature (London) 202,313414. Freney, J. R. (1960). Aust. J. Biol. Sci. 13, 387492. Fujiwara-Arasaki, T., and Mino, N. (1972). Proc. Int. Seaweed Symp., 7th, 1971 pp. 506-510. Furukawa, K., and Tonomura, K. (1972a). Agr. Biol. Chem. 36, 217-226. Furukawa, K., and Tonomura, K. (1972b). Agr. Biol. Chem. 36, 2441-2448. Furukawa, K., Suzuki, T., and Tonomura, K. (1969). Agr. Biol. Chem. 33, 128-130. Geering, H. R., Cary, E. E., Jones, L. H. P., and Allaway, W. H. (1968). Soil Sci. Soc. Amer., Proc. 32, 35-40. Gentile, J. H. (1971). In “Microbial Toxins” ( S . Kadis, A. Ciegler, and S. J. Ajl, eds. ), Vol. VII, pp. 27-66. Academic Press, New York.

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

67

Gerber, N. N. ( 1968). Tetrahedron Lett. 25, 2971-2974. Geronimo, J., Smith, L. L., Stockdale, G. D., and Goring, C. A. I. (1973). Agron. I. 65, 689-692. Getzin, L. W., and Shanks, C. H. ( 1970). J. Econ. Entomol. 63, 52-58. Cillespie, D. C. (1972). 1. Fish. Res. Bd. Can. 29, 1035-1041. Gihour, J. T., and Miller, M. S. (1973). I. Enuiron. Qual. 2, 145-148. Gissel-Nielsen, G., and Bisbjerg, B. ( 1970). Plant Soil 32, 382396. Gobert, N., Chaigneau, M., and Savel, J. (1971). C. R. Soc. B i d . 165,276-282. Gorham, P. R. ( 1964). In “Algae and Man” (D. F. Jackson, ed.), pp. 307-336. Plenum, New York. Grey, D. C., and Jensen, M. L. (1972). Science 177, 1099-1100. Gruener, N., and Shuval, H. I. (1970). In “Developments in Water Quality Research” ( H . I. Shuval, ed.), pp. 89-106. Ann Arbor-Humphrey Sci. Publ., Ann Arbor, Michigan. Guenzi, W. D., and McCalla, T. M. ( 1966). Soil Sci. SOC.Amer., PTOC.30,214-216. Gutenmann, W. H., Loos, M. A., Alexander, M., and Lisk, D. J. (1964). Soil Sci. SOC. Amer., PTOC. 28, 205-207. Hansen, F. ( 1941). Tidsskr. Planteaul 45, 401-419. Harmeson, R. H., Sollo, F. W., and Larson, T. E. ( 1971). 1. Amer. Water Works ASS.63, 303-310. Harrison, A. D. (1958). Int. Ver. Theor. Angew. Limnol., Verh. 13, 603-610. Hart, L. T., Larson, A. D., and McCleskey, C. S. (1965). 1. Bacten’ol. 89, 1104-1108. Hawksworth, G. M., and Hill, M. J. (1971a). Brit. 3. Cancer 25, 520-526. Hawksworth, G., and Hill, M. J. (1971b). Biochem. J. 122,28P-29P. Helling, C. S., Kearney, P. C., and Alexander, M. (1971). Adoan. Agron. 23, 147-240. Henderson, W. R., and Raskin, N. H. (1972). Lancet 2, 1162-1163. Hennequin, J.-R., and Juste, C. (1967). Ann. Agron. 18,545-569. Hesse, P. R. (1961). Plant Soil 14, 335-346. Hill, M. J. (1972). J. Med. Microbiol. 5, P14. Hill, M. J., and Aries, V . C. ( 1971). J. Pathol. 104, 129-139. Hill, M. J., Drasar, B. S., Aries, V., Crowther, J. S., Hawksworth, G., and Williams, R. E. 0. (1971). Lancet 1, 95-100. Hollis, J. P., and Rodriguez-Kabana, R. ( 1966). Phytopathology 56, 1015-1019. Hutchinson, G. L., and Viets, F. G. (1969). Science 166, 514-515. Hutchinson, S. A. (1973). Annu. Reu. Phytopathol. 11, 223-246. Ide, A., Niki, Y., Sakamoto, F., Watanabe, I., and Watanabe, H. (1972). Agr. Biol. Chem. 36, 1937-1944. Ilag, L., and Curtis, R. W. (1968). Science 159, 1357-1358. Inoue, H., Iwamoto, R., Fujii, H., and Imai, T. (1955). Abstr., Annu. Meet., SOC. Sci. Soil Manure, p. 9 (cited in Takai and Asami, 1962). Ishida, M. (1972). In “Environmental Toxicology of Pesticides” (F. M. Matsumura, G. M. Boush, and T. Misato, eds.), pp. 281-306. Academic Press, New York. Iwasaki, H., and Matsubara, T. (1972). J. Biochem. (Tokyo) 71, 645-652. Jacq, V., and Dommergues, Y. ( 1970 ) , Zentralbl. Bakteriol., Parasitenk., Infektionskr. Hyg., Abt. 2 125, 661-669. Jensen, S., and Jernelov, A. (1969). Nature (London) 223, 753-754. Jernelov, A. ( 1972). In “Environmental Mercury Contamination” ( R. Hartung and B. D. Dinman, eds.), pp. 167-172. Ann Arbor Sci. Publ., Ann Arbor, Michigan.

68

MARTIN ALEXANDER

Joffe, A. Z. (1971). In “Microbial Toxins” ( S . Kadis, A. Ciegler, and S. J. Ajl, eds.), Vol. VII, pp. 139-189. Academic Press, New York. Johnson, D. L. (1972). Nature (London) 2 4 0 , 4 4 4 5 . Junge, C., and Hahn, J. (1971). J. Geophys. Res. 76, 8143-8146. Junge, C., Seiler, W., Bock, R., Greese, K. D., and Radler, F. (1971). Naturwissensclzaften 58, 362363, Junge, C., Seiler, W., Schmidt, U., Bock, R., Greese, K. D., Radler, F., and Riiger, H. J. (1972). Naturwissenschaften 59, 514515. Kaars Sijpesteijn, A., and Kaslander, J. (1964). Outlook Agr. 4, 119-125. Kadota, H., and Ishida, Y . ( 1972). Annu. Reu. Microbiol. 26, 127-138. Kallio, R. E., and Larson, A. D. ( 1955). In “Amino Acid Metabolism” ( W . L). McElroy and H. B. Glass, eds.), pp. 616-631. Johns Hopkins Press, Baltimore, Maryland. Kallman, B. J., and Andrews, A. K. (1963). Science 141, 1050-1051. Kaufman, D. D., Plimmer, J. R., Iwan, J., and Klingebiel, U. I. ( 1972). 1. Agr. Food Chem. 20, 916-919. Kellogg, W. W., Cadle, R. D., Allen, E. R., Lazrus, A. L., and Martell, E. A. ( 1972 ) , Science 175, 587-596. Kendler, J., and Donagi, A. ( 1970). In “Developments in Water Quality Research” ( H . I. Shuval, ed.), pp. 241-249. Ann Arbor-Humphrey Sci. Publ., Ann Arbor, Michigan. Kesner, C. D., and Ries, S. K. (1967). Science 155, 210-211. Keybets, M. J. H., Groot, E. H., and Keller, G. H. M. (1970). Food Cosmet Toxicol. 8, 167-171. Khan, D. H., and Haque, M. Z. ( 1965). 1. Sci. Food Agr. 16,725-729. Kim, C . M. ( 1973). Soil Biol. 6 Biochem. 5, 163-166. Kimber, R. W. L. (1973). Plant Soil 38, 347-361. Kimura, Y., and Miller, V. L. ( 1964). J. Agr. Food Chern. 12, 253-257. King, D. L. (1970). J. Water Pollut. Contr. Fed. 42, 2035-2051. King, L. D., and Morris, H. D. ( 1972). J. Enuiron. Qual. 1, 442-446. Klotz, L. J., and Sokoloff, V. P. ( 1943). Calif. Avocado SOC. Yearb. 3 0 3 3 . Klubes, P., Cerna, I., Rabinowitz, A. D., and Jondorf, W. R. (1972). Food Cosnzet. Toxicol. 10, 757-767. Kluyver, A. J., and Verhoeven, W. (1954). Antonie van Leeuwenhoek; 1. Microbiol. Serol. 20, 241-262. Knorr, M., and Schenck, D. (1968). Arch. Hyg. Bakteriol. 152, 282-285. KO, W. H., and Farley, J. D. (1969). Phytopathology 59, 64-67. Komura, I., Funuba, T., and Izaki, K. ( 1971). J. Biochem (Tokyo) 70, 895-901. Korotaev, M. M., Kustov, V. V., Meleshko, G. I., Poddubnaya, L. T., and Shepelev, E. Ya. (1964). Prohl. Kosm. Biol. 3, 204-209. Krol, M., Maliszewska, W., and Siuta, J. ( 1972). Pol. 1. Soil Sci. 5, 25-33. Kunert, J. (1973). Z. Allg. Mikrobiol. 13, 489498. Kurlykova, M. V, ( 1962). Dokl. TSKHA (Timiryazev. Sel’skhokhoz. Akad.) 76, 63-68. Labanauskas, C. K., Stolzy, L. H., Klotz, L. J., and DeWolfe, T. A. (1971). Plant Soil 35, 337-346. Labarre, C., Chaigneau, M., Bory, J., and LeMoan, G. (1966). C. R. Acad. Sci., Ser. D 262, 2550-2553. Labarre, C., Chaigneau, M., and Bory, 1. ( 1972). C. R. Acad. Sci., Set. D 274, 2545-2548. Lackey, J. B. ( 1939). Pub. HeaZth Rep. 54, 740-746.

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

69

Lamontagne, R. A., Swinnerton, J. W., and Linnenbom, V. J. (1971). J. Geophys. Res. 76, 5117-5121. Landner, L. ( 1971). Nature (London) 230, 452454. Langdon, S. C. (1917). J. Amer. Chem. SOC. 39, 149-156. Lange, W. (1971). Can. J. Microbiol. 17, 303-314. Lebedev, A. J. (1911). Ber. Deut. Bot. Ges. 29,327-329. Lee, C. Y., Shallenberger, R. S., Downing, D. L., Stoewsand, G. S., and Peck, N. M. ( 1971). J. Sci. Food Agr. 22, 90-92. Leinweber, F.-J., and Monty, K. J. (1961). Biochem. Biophys. Res. Commun. 6, 355-358. Leinweber, F.-J., and Monty, K. J. (1965). 3. Biol. Chem. 240, 782-787. Lewis, J. A., and Papavizas, G. C. (1970). Soil Biol. & Biochem. 2, 239-246. Lewis, J. A,, and Papavizas, G. C. ( 1971). P ~ ~ t o ~ a ~ h 61, o l o208-214. g~ Lichtenstein, E. P., and Schulz, K. R. (1960). J. Econ. Entomol. 53, 192-197. Lichtenstein, E. P., Schulz, K. R., and Cowley, G. T. (1963). J. Econ. Entomol. 56, 485489. Lieb, J. M. P., Davis, W. N., Brown, T., and McQuiggan, M. (1956). Amer. J. Med. 24, 471-474. Lijinsky, W., Taylor, H. W., Snyder, C., and Nettesheim, P. (1973). Nature (London) 244, 176-178. Lillarani, N. T. ( 1970). Mysore J. Agr. Sci. 4, 69-75. Lindahl, P. E. B. ( 1964 ). Lantbrukshoegsk. Ann. 30,375-404. Linderman, R. C., and Tousson, T. A. (1968). Phytopathology 58, 1571-1574. Loewus, M. W., and Delwiche, C. C. (1963). Plant Physiol. 38, 371-374. Loos, M. A., Bollag, J.-M., and Alexander, M. (1967a). J. Agr. Food Chem. 15, 858-860. Loos, M. A,, Roberts, R. N., and Alexander, M. (196713). Can. J. Microbiol. 13, 691-699. Lovelock, J. E., Maggs, R. J., and Rasmussen, R. A. (1972). Nature (London) 237, 452-453. Luebs, R. E., Davis, K. R., and Laag, A. E. (1973). J. Enuiron. Qual. 2, 137-141. Lynch, J. M. (1972). Nature (London) 240,45-46. McBride, B. C., and Wolfe, R. S. (1971). Biochemistry 10, 4312-4317. McConnell, K. P., and Portman, 0. W. ( 1952). Proc. SOC. Exp. Biol. Med. 79, 230-23 I. Mackenthun, K. M. (1969). “The Practice of Water Pollution Biology.” U.S. Dept. Interior, Washington, D.C. MacRae, I. C., and Alexander, M. (1965). J. Agr. Food Chem. 13, 72-76. MacRae, I. C., and Ancajas, R. (1970). Plant Soil 33,97-103. MacRae, I. C., Alexander, M., and Rovira, A. D. ( 1963). J. Gen. Microbiol. 32, 69-76. Makarov, B. N., and Ignatova, V. P. (1964). Sou. Soil Sci. pp. 407-413. Marchesani, V. J., Towers, T., and Wohlers, H. C. (1970). J. Air PoZZut. Contr. Ass. 20, 19-22. Martin, W. P., Buehrer, T. E., and Caster, A. B. ( 1942). Soil Sci. SOC. Amer., Proc. 7, 223-228. Massey, H. F., and Barnhisel, R. I. (1972). SoiZ Sci. 113, 207-212. Matsubara, T. ( 1971).J. Biochem. (Tokyo) 69,991-1001. Matsumura, F., Patil, K. C., and Boush, C. M. (1970). Science 170, 1206-1207. Matsumura, F., Gotoh, Y., and Boush, G. M. (1971). Science 173, 49-51. Megie, C. A., Pearson, R. W., and Hiltbold, A. E. (1967). Agron. J. 59, 197-199.

70

MARTIN ALEXANDER

Merrill, W., and French, D. W. (1964). Proc. Minn. Acud. Sci. 31, 105-106. Milborrow, B. V. ( 1963). Biochem. J. 87, 255-258. Miles, J. R. W., Tu, C. M., and Harris, C. R. (1969). J. Econ. Entomol. 62, 1334-1338. Miller, A., Scanlan, R. A,, Lee, J. S., and Libbev, L. M. (1973). Appl. Microbiol. 26, 18-21. Minarik, E. ( 1972). Mitt. Rebe Wein, Obstbau Fruechteuerwert. (Klosterneuberg) 22, 245-252. Mirocha, C. J., Christensen, C. M., and Nelson, G. H. (1968). Biotechnol. Bioeng. 10, 469-482. Mirvish, S. S.,and Chu, C. (1973). J. Nut. Cancer Inst. 50, 745-750. Mitsui, S., Aso, S., Kumazawa, K., and Ishiwara, T. (1954). Trans. Int. Congr. Soil Sci. 5th, 1954 Vol. 2, pp. 364368. Moje, W., Munnecke, D. E., and Richardson, L. T. (1964). Nature (London) 202, 831-832. Morris, R. L., Dougherty, J. D., and Ronald, G. W. (1963). J. Amer. Water Works Ass. 55, 1380-1390. Mosier, A. R., Andre, C. E., and Viets, F. G. (1973). Enuiron. Sci. Technol. 7, 642-644. Munnecke, D. E., Domseh, K. H., and Eckert, J. W. (1962). Phytopathology 52, 1298-1306. Murphy, R. T., and Barthel, W. F. ( 1960). J. Agr. Food Chem. 8,442-445. Najjar, V. A., and Chung, C. W. (1956). In “Inorganic Nitrogen Meta1)olism” (W. D. McElroy and B. Glass, eds.), pp. 260-279. Johns Hopkins Press, Baltimore, Maryland. Nakanishi, T., and Oku, H. ( 1969). Phytopathology 59, 1761-1762. Nelson, D. W., and Bremner, J. M. (1970). Soil Biol. Biochem. 2, 203-215. Nishimura, S . , Hayashi, S., Chionishio, I., Ikuda, J., Sato, I., and Tanabe, K. (1969). Nippon Shokubutsu Byori Gakkaiho 35,286-293. Nommik, H. ( 1956). Acta Agr. Scand. 6, 195-228. Nommik, H. ( 1966). Acta Agr. Scand. 16, 147-154. Norstadt, F. A., and McCalla, T. M. (1968). Soil Sci. SOC. Amer., Proc. 32, 241-245. O’Brien, R. D. (1960). “Toxic Phosphorus Esters.” Academic Press, New York. Padron, J., Grist, K. L., Clark, J. B., and Wender, S. H. (1960). Biochem. Biophys. Res. Commun. 3, 412-416. Parr, J. F., Willis, G. H., and Smith, S. (1970). Soil Sci. 110, 306412. Patil, K. C., Matsumura, F., and Boush, G. M. (1972). Environ. Sci. Technol. 6, 629-632. Pattison, E. S . (1970). Science 170, 870. Payne, W. J., Riley, P. S., and Cox, C. D. ( 1971). J . Bacteriol. 106, 356-361. Pfaender, F. K., and Alexander, M. (1972). J. Agr. Food Chem. 20, 842-846. Pfaender, F. K., and Alexander, M. (1973). J. Agr. Food Chem. 21, 397-399. Pradhan, S. ( 1970). In “COST Working Group on Human Environment,” pp. 13-21. Bombay, India. Prakasam, T. B. S., and Loehr, R. C. (1972). Water Res. 6, 859-869. Prasad, I. (1970). Can. J. Microbiol. 16, 369-372. Prescott, G. W. (1948). Hydrobiologia 1, 1-13. Quastel, J. H., and Scholefield, P. G. ( 1953). Soil Sci. 75, 279-285. Radcliffe, B. C., and Nicholas, D. J. D. ( 1968). Biochim. Biophys. Acta 153, 545-554. Rajappan, P. V., and Boynton, D. ( 1960). Proc. Amer. SOC. Hort. Sci. 75, 402-406. Rankine, B. C., and Pocock, K. F. (1969). J. Sci. Food Agr. 20,104-109.

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

71

Read, D. C. ( 1971). J. Econ. Entomol. 64, 800-804. Rebhun, M., Fox, M. A,, and Sless, J. B. (1971). J. Amer. Water Works ASS. 63, 219-224. Richards, D. E. ( 1972). In “Microbial Toxins” ( S . Kadis, A. Ciegler, and S. J. Ajl, eds. ), Vol. VIII, pp. 3 4 5 . Academic Press, New York. Richards, L. T., and Thorn, G. D. (1960). Phytopathology 50, 651-652. Rissanen, K., and Miettinen, J. K. (1972). In “Mercury Contamination in Man and His Environment,” pp. 5-34. IAEA, Vienna. Robbins, R. C., and Kastelic, J. ( 1961). J. Agr. Food Chem. 9, 256-260. Robbins, R. C., Borg, K. M., and Robinson, E. (1968). J . Air Polht. Contr. ASS. 18, 106110. Robinson, E., and Robbins, R. C. (1970). In “Global Effects of Environmental Pollution” (S. F. Singer, ed.), pp. 50-64. Reidel Publ., Dordrecht, Netherlands. Robinson, W. 0. (1930). Soil Sci. 30, 197-217. Rodriguez-Kabana, R., Jordan, J. W., and Hollis, J. P. ( 1965). Science 148, 524-526. Rohlich, G. A., and Sarles, W. B. (1949). Taste Odor Contr. J . 18, 1-6. Rolle, I., Payer, R., and Soeder, C. J. (1971). Arch. Mikrobiol. 77, 185-195. Ruiz-Herrera, J., and Starkey, R. L. ( 1970). J. Bacteriol. 104, 1286-1293. Saito, N. (1972). In “Mercury Contamination in Man and His Environment,” pp. 35-42. IAEA, Vienna. Salt, P. D. ( 1965). Chern. Ind. (London) pp. 461462. Sander, J. (1968). Hoppe-Seyler’s Z. Physiol. Chem. 349, 429-432. Sander, J. ( 1971). Arzneim.-Forsch. 21, 1572-1580. Sapozhnikov, D. I. (1937). Mikrobiologiya 6, 643-644. Schantz, E. J. ( 1971). In “Microbial Toxins” ( S . Kadis, A. Ciegler, and S. J. Ajl, eds.), Vol. VII, pp. 3-26. Academic Press, New York. Schiitte, H. R., and Stephan, U. (1969). Z. Pflanzenernaehr. Bodenk. 123,212-219. Schwimmer, D., and Schwimmer, M. ( 1964). In “Algae and Man” ( D . F. Jackson, ed.), pp. 368-412. Plenum, New York. Selenka, F. ( 1971). Zentralbl. Bakteriol., Parasitenk., Infektionskr. H y g . , Abt. 1 : Orig., Reihe B 155, 58-69. Shilo, M. (1971). In “Microbial Toxins” (S. Kadis, A. Ciegler, and S. J. Ajl, eds.), Vol. VII, pp. 67-103. Academic Press, New York. Silvey, J. K., Henley, 13. E., and Wyatt, J. T. (1972). J. Amer. Water Works Ass. 64, 35-39. Singer, P. C., and Stumm, W. (1970). Science 167, 1121-1123. Skorobogatova, R. A., and Mirchink, T. G. (1972). Biol. Nauki 15, 114-117. Smirnov, V. V. ( 1963). Mikrobiologiya 32, 695-699. Smith, A. M. ( 1973). Nature (London) 246,311-313. Smith, K. A., and Restall, S. W. F. ( 1971). J. Soil Sci. 22, 430-443. Smith, L. L., and Oseid, D. M. (1972). Water Res. 6, 711-720. Smith, T. A. (1971). Biol. Reo. Cambridge Phil. SOC. 46, 201-241. Soda, K., Novogrodsky, A., and Meister, A. (1964). Biochemistry 3, 1450-1454. Somers, E., Richmond, D. V., and Pickard, J. A. (1967). Nature (London) 215, 214. Steen, W. C., and Stojanovic, B. J. (1971). Soil Sci. Soc. Amer., Proc. 35, 277-282. Stewart, B. A., Yiets, F. G., Hutchinson, G. L., and Kemper, W. D. (1967). Enuiron. Sci. Technol.. 1, 736-739. Stojanovic, B. J., and Alexander, M. (1958). Soil Sci. 86, 208-215. Stolwijk, J. A. J., and Thimann, K. V. (1957). Plant Physiol. 32, 513-520. Summers, A. O., and Silver, S. (1972). J . Bacteriol. 112, 1228-1236.

72

MARTIN ALEXANDER

Swinnerton, J. W., Linnenbom, V. J., and Lamontagne, R. A. (1970). Science 167, 984-986. Tackett, J. L., and Pearson, R. W. ( 1964). Soil Sci. Soc. Amer., Proc. 28, 741-743. Takai, Y., and Asami, T. ( 1962). Soil Sci. Plant Nutr. (Tokyo) 8, 132-136. Takase, I., Tsuda, H., and Yoshimoto, Y. (1971). Nippon Oyo Dobutsu Konchu Gakkai-Shi 15, 63-69. Takijima, Y. (1964a). Soil Sci. Plant Nutr. (Tokyo) 10, 204-211. Takijima, Y. (1964b). Soil Sci. Plant Nutr. ( T o k y o ) 10,212-219. Tanaka, M. ( 1953). Nature (London) 171, 1160-1161. Taylor, H. F., and Wain, R. L. (1962). Proy. Roy. Soc., Ser. B 156, 172-186. Terracini, B., Magee, P. N., and Barnes, J. M. ( 1967). Brit. J. Cancer 21, 559-565. Thom, C., and Raper, K. B. (1932). Science 76,548-550. Toan, T. T., Bassette, R., and Claydon, T. J. (1965). J. Dairy Sci. 48, 1174-1178. Torma, A. E., and Habashi, F. (1972). Can. J. Microbiol. 18, 1780-1781. Troxler, R. F., and Dokos, J. M. ( 1973). Plant Physiol. 51, 72-75. Tu, C. M., Miles, J. R. W., and Harris, C. R. (1968). Life Sci. 7, 311-322. Turner, A. W. (1949). Nature (London) 164, 76-77. Turner, W. R. ( 1958). Progr. Fish. Cult. 20,45-46. Tuttle, J. H., Randles, C. I., and Dugan, P. R. ( 1968). J. Bacteriol. 95, 1495-1503. Vamos, R. (1959). Plant Soil 11, 65-77. Verloop, A. ( 1972). Residue Rev. 43, 55-103. Verstraete, W., and Alexander, M. ( 1972a). J . Bacteriol. 110, 955-961. Verstraete, W., and Alexander, M. ( 1972b). J. Bacteriol. 110, 962-967. Verstraete, W., and Alexander, M. (1973). Enuiron. Sci. Technol. 7, 3942. Vlitos, A. J., and King, L. J. (1953). Nature (London) 171, 523. Wagner, G. H., and Smith, G . E. (1958). Soil Sci. 85, 125-129. Wagner, K.-H., and Siddiqi, 1. ( 1973). Naturwissenschaften 60, 109-1 10. Walsh, F., and Mitchell, R. (1972). Enuiron. Sci. Technol. 6, 809-812. Wang, L. C., and Burris, R. H. ( 1960). J. Agr. Food Chem. 8, 239-242. Webber, L. R. (1971). In “Identification and Measurement of Environmental Pollutants” (B. Westley, ed.), pp. 110-114. National Research Council of Canada, Ottawa. Webley, D. M., Duff, R. B., and Farmer, V. C. (1958). J . Gen. Microbiol. 18, 733-746. Wedemeyer, G. ( 1966). Science 152, 647. Weinstock, B., and Niki, €1. ( 1971). Science 176, 290-292. Weisburger, J. H. ( 1971). Cancer 28, 60-70. Weiss, H. V., Koide, M., and Goldberg, E. D. (1971). Science 172, 261-263. Westlake, D. W. S., Roxburgh, J. M., and Talbot, G. ( 1961). Nature (London) 189, 510-511. White, R. K., Taiganides, E. P., and Cole, G. D. (1971). In “Livestock Waste Management and Pollution Abatement,” pp. 110-113. Amer. SOC.Agr. Eng., St. Joseph, Missouri. Wieland, T. ( 1968). Science 159, 946-952. Wijler, J., and Delwiche, C. C. (1954). Plant Soil 5, 155-169. Wilson, D. F., Swinnerton, J. W., and Lamontagne, R. A. (1970). Science 168, 1577-1579. Wingo, C W. (1966). Mo. Agr. Exp. Sta., Res. Bull. 914. Woldendorp, J. W. (1965). Ann. Inst. Pasteur, Paris, Suppl. pp. 316-327. Wood, J. M. (1971). Aduan. Enuiron. Sci. Technol. 2, 39-56. Wood, P. C. (1968). Nature (London) 220,21.

MICROBIAL FORMATION OF ENVIRONMENTAL POLLUTANTS

73

Woodbury, L. A. (1942). Trans. Amer. Fish. SOC. 71, 112-117. Woolfolk, C. A., and Whiteley, H. R. (1962). J . Bacteriol. 84, 647-658. Woolson, E. A., and Keamey, P. C. ( 1973). Enuiron. Sci. Technot. 7 , 47-50. Wright, D. E. (1968). Annu. Rev. Microbiol. 22, 269-282. Yamada, M., and Tonomura, K. (1972). Hakko Kogaku Zasshi 50, 159-166. Yoshida, T., and Alexander, M. (1964). Can. J . Microbiol. 10, 923-926. Yoshida, T., and Alexander, M. ( 1970). Soil Sci. SOC. Amer., Proc. 34,880-882. Young, R. J., Dondero, N. C., Ludington, D. C., and Loehr, R. C. (1971). I n “Identification and Measurement of Environmental Pollutants ( B. Westley, ed. ), pp. 98-104. National Research Council of Canada, Ottawa.

This Page Intentionally Left Blank

Microbial Transformation of Pesticides

JEAN-MARC BOLLAG Laboratory of Soil Microbiology, Department of Agronomy, The Pennsylvania State Uniuersity, University Park, Pennsylvania

I. Introduction ...................................... 11. Mechanisms of Pesticide Transformation .............. A. Pesticide as a Nutrient Source ................... B. Cometabolism ................................ C. Conjugate Formation .......................... D. Microbial Accumulation of Pesticides ............ 111. Enzymatic Reactions in Pesticide Metabolism .......... A. Oxidative Reactions ............................ B. Reductions ................................... C . Hydrolysis ................................... D. Dehalogenation Mechanisms .................... E. Synthetic Reactions ........................... IV. Chemical Structure and Microbial Transformation Relationship ...................................... A. Microbial Transformation of Pesticidal Groups .... B. Effect of Various Substitutions on Biodegradability . . C. Molecular Recalcitrance and Pesticide Transformation V. Conclusions ..................................... References .......................................

I.

75 77 78 78 80 80 81 81

97 99 103 109 114 115 119 121 122 124

Introduction

The fate of applied xenobiotic compounds, such as pesticides, in the environment is of great importance, since disappearance, persistence, or partial transformation of such a compound determines its usefulness or its potential hazardous effect. There may be chemical and physical factors that influence the fate of a pesticide, but the least predictable transformation is usually caused by microorganisms. Because these compounds have become an integral part of our economy, there can be no question that considerable effort must be expended in order to gain an understanding of the mechanisms of pesticide transformations. There is a need to know the actual biochemical reactions involved in pesticide metabolism, since this can give a basis for the understanding of their short or long persistence in a natural environment and can also contribute to the clarification of the relationship between chemical structure and susceptibility to probable microbial transformations. For this purpose it is necessary to investigate the metabolic reactions and the enzyme systems and to isolate and identify the resulting products in laboratory experiments. The information derived from such studies can serve as a signpost for subsequent investigations in a natural ecosystem which 75

76

JEAN-MARC BOLLAG

is usually too complex as a primary research medium for conclusive results. The soil, for instance, which can be considered as the most complex microbial habitat and an area where pesticides or their derivatives are deposited either by direct application or by decaying foliage, possesses many characteristics for an efficient chemical or physical attack on a molecule and for its removal by adsorption, leaching, volatilization or photodecomposition. On the other hand, the soil simultaneously offers, in many instances, the necessary prerequisites for the proliferation of a vast variety of living organisms. Consequently, it is very difficult to discriminate in a soil environment between microbial, chemical, and physical factors contributing to the removal or transformation of a pesticidal molecule. It was also stated that breakdown products of biological and physicochemical activity in soil are often similar because the reagents-oxygen, water, and nucleophiles-are the same in each instance (Crosby and Li, 1969). Photolysis, for instance, can cause the hydrolysis of esters and amides, dealkylation of amines, and other effects, and the same reaction can be initiated by enzymatic activity resulting in identical “products.” Biological transformation of xenobiotic compounds, for example, pesticides in a soil, freshwater, or estuarine ecosystem, appears to be caused primarily by bacteria, actinomycetes, and fungi. It has to be emphasized that much less attention in research has been devoted to the possible interference of the microfauna and algae, and this subject is only partially covered in this review. Initial research on microbial pesticide metabolism was characterized solely by isolating organisms capable of using a compound as their only source of both carbon and energy, but later it was realized that other mechanisms, such as cometabolic transformation, conjugation reactions, or the mere accumulation of a pesticide within a microbe, are important factors of microbial interference. The complete degradation of a pesticidal molecule to its inorganic parts or its fragmentation into components that can be further used in an oxidative cycle, like the Krebs cycle, removes its potential toxicity completely from the environment. The mechanisms which cause only partial change or temporary removal do not eliminate the potential hazard of an applied chemical or its transformation product in nature. There are several review articles that include aspects on the microbial degradation of pesticides ( Menzie, 1969; Alexander, 1969; Helling et a,?., 1971) and numerous reviews that cover microbial attacks on pesticidal classes or related topics, some of which are referred to in this article. In this review it was attempted to assess the probable transformation capabilities of pesticides by microorganisms and to characterize

MICROBIAL TRANSFORMATION OF PESTICIDES

77

the specific enzymatic reactions that are part of the metabolism for the major known groups of pesticides. II.

Mechanisms of Pesticide Transformation

If a pesticide is exposed to a microbial species, there are four major possibilities for its transformation or inactivation by the organism: (1) the pesticide can serve as a substrate for growth and energy; ( 2 ) the xenobiotic compound can undergo “cometabolism,” i.e., microorganisms transform it, but cannot derive energy for growth from it; ( 3 ) the entire pesticidal molecule or an intermediate of it can be conjugated with naturally occurring compounds; and (4) the pesticide is incorporated and accumulates within the organism. It is self-evident that in many cases the transformation of a pesticide does not occur by only one type of mechanism during its exposure to one organism or to a whole microflora under natural conditions. In addition, a specific compound can be metabolized by various pathways in the environment; consequently, different products can result from the same initial material. All microbial transformations are caused by enzymes, and since all the applied pesticides are foreign materials, possessing a molecular configuration that may not occur in nature, it is understandable that many of the enzymes catalyzing a certain reaction are induced. This often causes an initial lag period until metabolic activity can be determined. Although many enzymes are induced, the transformations that they catalyze are usually reactions also encountered in the metabolism of natural substances. However, it is difficult to predict which molecular change can be expected by a specific microbe, since each group of organisms, even various strains of one genus, can alter a selected molecule differently. For example, the insecticide carbaryll was hydroxylated by different species of Penicillium on the ring, at the side chain, or not at all, respectively ( Bollag and Liu, 1972a); Fusarium muniliforme dealkylated the ethyl group of the herbicide atrazine, while F . roseum showed a stronger activity in the removal of the isopropyl substitution (Kaufman and Blake, 1970); and DDT was metabolized to TDE and a dicofol-like compound by Trichoderma uiride, while variants of the same species produced DDA or DDE ( Matsumura and Boush, 1968). Nevertheless, a certain mode of biological attack can be anticipated on the basis of the molecular structure of the pesticide, and the knowledge acquired should help to foresee such a transformation (Section 111). Cosinion and chemical designation of pesticides referred to in this text are listed in Table IV.

78

JEAN-MARC BOLLAG

AS A. PESTICIDE

A

NUTRIENTSOURCE

From a practical point of view the complete microbial breakdown of an organic molecule to its inorganic components is the desired mechanism if one is interested in avoiding the persistence of a potentially hazardous compound in the environment. As elaborated in laboratory experiments, numerous organic pesticides can serve as the sole carbon or energy source for growth and proliferation of certain microorganisms. If a pesticide can be used in such a way, it is degraded and fragmented to compounds that can be channeled into known oxidative cycles such as the Krebs cycle, and thus the organism can derive all the necessary energy . In order to determine whether a pesticide can serve as the only carbon source needed for growth, the general experimental approach proceeds by an enrichment culture technique. After isolation of the surviving microorganisms, the pesticide is added to a basal salts medium and further observations are usually performed with a pure culture. However, it was also shown that in certain cases a pesticide can be decomposed only in the presence of two different microbial species (Gunner and Zuckerman, 1968). It appears to be an obvious conclusion that in a natural ecosystem like the soil, with an abundance of various microbes, there is an even greater possibility for the single or combined transformation and complete use of a specific pesticide by the microbial population.

B. COMETABOLISM The phenomenon that a microorganism can transform a chemical without deriving energy to support its growth is a relatively recent observation, and its detection and significance is related to the modern use of xenobiotic compounds in various environments. Foster (1962) used the expression “co-oxidation,” Jensen ( 1963 ) suggested the term “cometabolism,” and Ruiz-Herrera and Starkey ( 1969) designated this process as “co-dissimilation,” but although all designations try to express the same thought, it appears that cometabolism is the most general term, and therefore it will be used in this review. The potential importance of cometabolism for the transformation of pesticides was first pointed out by Alexander ( 1967), and the findings of many investigations can now be explained by this process (Table I ) (Horvath, 1972). Cometabolism generally does not result in extensive degradation of a pesticidal molecule, but it can cause a reduction, elimination or probably increase of toxicity in the environment. However, it was also demonstrated that different microorganisms can degrade a certain pesticide

79

MICROBIAL TRANSFORMATION OF PESTICIDES

TABLE I PESTICIDES SUBJECTTO COMETABOLISM A N D ACCUMULATED PRODUCTS Substrate Chlorobenzilate

Product

4,4’-l)ichlorobenzophenone Chloroneb 2,5-Dichloro-4methoxyphenol DDT p,p-Dichlorodiphenylmethane 3,t5-I>ichlorocatechol 3,5-Dichloro-%hy(metabolite of droxymuconic 2,4-D, 2,4,5-T, semialdehyde and 2,3,6-TBA) p,p’-Diehlorodiphen- p-Chlorophenyl aceylmethane (metahtic acid olite of D D T ) 3-Nitrophenol Nitro hydroquinone 2,4,.i-T 2,3,6-TBA

Organism

R hodotorula gracilis Fusarium sp. Aerobacter aerogenes Achromobacter sp.

Reference Miyasaki et a1 (1970) Wiese and Vargas (1973) Wedemeyer (1967) Horvath (1970b)

Hydrogemonas sp.

Focht and AIexander (1971)

Flavobacterium sp.

Raymond and Alexander ( 1971) Horvath (1970a) Horvath (1971)

3,5-L>ichlorocatechol Brevibacterium sp. 3,S-l)ichlorocatechol Brevibacterium sp.

considerably by subsequent cometabolic attack; for instance, the herbicides 2,4,5-trichlorophenoxyacetateand 2,3,6-trichlorobenzoate are converted by a cometabolic oxidation to 3,5-dichlorocatechol by a Breuibacterium sp. (Horvath, 1970a,1971), whereas an Achromobacter sp. was capable of cooxidizing the resulting 3,5-dichlorocatechol to 3,5-dichloro2-hydroxymuconic semialdehyde ( Horvath, 1970b). It was also speculated that cometabolism could account for complete mineralization of a chemical if a carbon and energy source were supplied to mixed microbial populations in the form of a biodegradable analog of the chemical under investigation ( Horvath, 1972). The process of cometabolism is effected by bacteria as well as actinomycetes and fungi, and therefore it can be assumed that its occurrence is widespread in natural ecosystems. Many observations of microbial transformations that could not be understood, since the microbes did not derive any energy or nutritional use from it, are now interpretable by this mode of metabolism. The process of cometabolism, especially as a factor in the microbial transformation of pesticides, requires still further intensive study for a clear understanding of its actual cause, its transformation capabilities as related to the structure of chemicals, and the extent to which such a transformation forms a more or less persistent compound. Although it is difficult to demonstrate unequivocally that microbial cometabolism also occurs under natural conditions,

80

JEAN-MARC BOLLAG

there is little doubt that it takes place, and therefore the ecological importance of this biological reaction has to be fully explored. C. CONJUCATE FORMATION

Whereas the biotransformations described previously include the attack of the original molecule, conjugation reactions are syntheses by which a pesticide, or any of its metabolites, is combined with naturally occurring compounds, generally amino acids or carbohydrates. The formation of a conjugate usually makes the molecule more polar, and therefore more water- and less lipid-soluble. Conjugations of pesticides and other xenobiotic chemicals are common and frequent reactions in all higher organisms, but they have not been found to a similar extent in microorganisms. It is not likely that this observation is an experimental oversight, but it could present a metabolic characteristic of microbes, especially bacteria. Nevertheless, this mode of transformation requires further exploration, since a pesticide, or an intermediate of it, is only bound to another molecule. A conjugated compound can easily be cleaved again, and the released chemicals can subsequently exert a toxic influence. The herbicide amitrole, for instance, was coupled with alanine by Escherichia coli and subsequently incorporated into cellular protein ( Williams et al., 1965). This conjugation reaction occurred presumably since the conjugated metabolite showed structural similarity to histidine and functioned as an analog of histidine. Various examples of conjugate formation are described under Section II1,E.

D. MICROBIALACCUMULATION OF PESTICIDES The possibility that pesticides are incorporated into microorganisms by an active or passive accumulation mechanism provokes special concern, since the microbial interference means-as in a conjugation reaction-only temporary removal of a toxic compound. Most observations of pesticide accumulation within the cells were registered with chlorinated hydrocarbons like DDT, dieldrin, aldrin, and heptachlor. Mycelia of actinomycetes and fungi added to soil containing dieldrin, DDT, and pentachloronitrobenzene accumulated these compounds to levels above ambient concentrations (KO and Lockwood, 1968). This observation also was confirmed with specific bacteria, actinomycetes, and fungi in culture solutions containing DDT and dieldrin (Chacko and Lockwood, 1967). In various studies it was found that not only live bacterial cells, but also autoclaved cells, show a similar uptake of pesticides, which appears to indicate that an actual metabolic factor is not involved in the accumulation process. Johnson and Kennedy (1973) found that the accumulation

MICROBIAL TRANSFORMATION OF PESTICIDES

81

rate of DDT and methoxychlor by autoclaved cells was greater than that for the living bacteria; for instance, after autoclaving the cells of Aerobacter aerogenes, the uptake of methoxychlor was double the amount absorbed by living cells. They suggested that the molecular polarity and lipid solubility influences the retention of the organochlorine insecticides by the bacterial cells. Experiments with yeast, Sacchuromyces cerevisiae, also showed that the adsorption capacity for lindane and dieldrin increased after boiling of the organism, and that the two insecticides could be removed by washing with fresh water (Voerman and Tammes, 1969). Adsorption and concentration of the insecticide aldrin was determined for floc-forming bacteria which were isolated from Lake Erie, and it was suggested that the adsorption capacity of flocculent bacteria might even be evaluated for removal of pesticides in an aqueous environment ( Leshniowsky et al., 1970). Since aquatic microorganisms and plankton in freshwater and marine environments are an important nutrient source for a broad spectrum of aquatic filter-feeding organisms, their accumulation of pesticides can constitute a hazardous link in the food chain to fish and higher vertebrates. Therefore, the findings of extensive biomagnification by these organisms has to provoke considerable concern. A marine diatom, Cylindrotheca closterium, adsorbed and concentrated DDT up to approximately 200-fold from its culture medium containing 0.1 ppm of the insecticide ( Keil and Priester, 1969). Likewise, it was found that cultures of the blue-green alga Anacystis nidulans, the green alga Scenedesmus obliquus, the flagellate Euglena gracilis, and the two ciliates Paramecium bursaria and P . multimicronucleatum concentrated DDT and parathion after exposure for 7 days at a rate of 100 to 964 and 50 to 116 times, respectively (Gregory et al., 1969). Ill.

Enzymatic Reactions in Pesticide Metabolism

Following is an attempt to classify the enzymatic reactions into groups that cover the majority of biotransformations which pesticides undergo. However, it is clear that all attempts to categorize natural processes have their shortcomings; therefore, this should be considered as a trial to assort the essential enzymatic characteristics for easier evaluation.

A. OXLDATIVE REACTIONS 1 . Hydroxylation Introduction of a hydroxyl group to a pesticide is a frequent primary transformation of a molecule resulting in the formation of a compound which can be biologically more reactive, often more polar, and conse-

82

JEAN-MARC BOLLAG

quently more soluble in water. Enzymes that catalyze this reaction have been variously termed “hydroxylases,” “monooxygenases,” or mixed-function oxidases. For plants and animals, the insertion of a hydroxyl group into a compound often provides a center at which conjugation can occur, but this is rarely the purpose of microbial hydroxylations. Hydroxylation can occur with aliphatic as well as aromatic compounds, and often it constitutes only a step in a more complex reaction; for instance, dealkylation reactions proceed via a hydroxylated intermediate, which is, however, in many cases an unstable compound. All studies in which microbial hydroxylation of pesticides was investigated in more detail claim that the reaction takes place only in the presence of oxygen and the reduced form of nicotinamide adenine dinucleotide phosphate ( NADPH ) , or nicotinamide adenine dinucleotide (NADH), indicating that the process is catalyzed by a mixed-function oxidase, whereby the molecular oxygen is apparently incorporated without intermediate water formation. Like most other processes of xenobiotic compounds, more detailed studies on the mechanism of hydroxylation have been performed with mammalian liver microsomal systems ( DaIy, 1971). Hydroxylation of aromatic pesticides is an important step as a tool for introducing polar groups into the molecule as well as a prerequisite for further degradation by ring cleavage. Most observations on hydroxylations at various positions and with different microorganisms were made with phenoxyalkanoate pesticides which probably constitute the group of herbicides whose microbial degradation was most thoroughly studied. While 2,4-D was hydroxylated to the 6-hydroxy derivative by a Pseudomoms sp. (Evans et al., 1971b), the fungus Aspergillus niger produced essentially 2,4-dichloro-5-hydroxyphenoxyacetic acid and, to a lesser extent, 2,5-dichloro-4-hydroxy0-CH,COOH

HO

/ Pseudomonas sp.

Cl

Asperffillus niger OH

83

MICROBIAL TRANSFORMATION OF PESTICIDES

phenoxyacetic acid ( Faulkner and Woodcock, 1964; 1965). The latter product indicates a shift of a chlorine atom coupled with the replacement of a hydroxyl group which will be considered further as a NIH-shift under epoxidation reactions. The herbicide MCPA was transformed similarly to 2,4-D, resulting in the production of the 6-hydroxy (Evans et al., 1963) and the 5-hydroxy derivative (Faulkner and Woodcock, 1964) by a Pseudomonm sp. and A. niger, respectively. Hydroxylation appears to be a common mode of attack by A. niger on phenoxyacetic acids. From 4-chlorophenoxyacetic acid it was possible to isolate 4-chloro-2-hydroxy- and 4-chloro-3-hydroxyphenoxyaceticacid, and the exposure of phenoxyacetic and 2-chlorophenoxyacetic acid to A. niger resulted in the formation of all possible hydroxy derivatives ( Faulkner and Woodcock, 1961; Clifford and Woodcock, 1964). Chlorinated phenoxyalkanoic acids undergo cleavage of the ether linkage by the metabolic activity of various bacterial species resulting in the corresponding phenol. Bollag et al. (1968a) isolated a soluble enzyme preparation from a soil Arthrobacter sp. which converted 2,4-dichlorophenol and 4-chlorophenol to 3,5-dichlorocatechol and 4-ch1orocatecho1, respectively. The enzyme involved appears to be a mixed-function oxidase, since both oxygen and NADPH were required for the hydroxylation reaction. Oxidation of an aromatic amine group can be initiated by N-hydroxylation, which appears to be a major pathway for the oxidation of chlorinated anilines by Fusarium oxysporum. p-Chloroaniline, which constitutes an intermediate of several herbicides, was hydroxylated to pchlorophenylhydroxylamine, which could accumulate temporarily in the growth medium of the fungus up to 76%of the amount theoretically possible ( Kaufman et al., 1973). p-Chlorophenylhydroxylamine was subsequently metabolized to p-chloronitrosobenzene and p-chloronitrobenzene.

+-+Q-Q NH,

NHOH

NO

NO*

c1

c1

c1

c1

The insecticide carbaryl was oxidized by hydroxylation at different positions; from the growth medium of the fungus Gliocladium roseum, it was possible to isolate and identify 1-naphthyl N-hydroxymethyl carbamate as well as 4-hydroxy- and 5-hydroxy-1-naphthyl methylcarbamate, which indicated side chain and ring hydroxylation, respectively ( Liu and Bollag, 1971a).

84

JEAN-MARC BOLLAG

O H I1 I 0 - C -N-CH3

I

O H II I 0- C-N-

O H II I 0-C-N-CH,

/

OH

&J 4-d CH,OH

I:':

0- C-N-CH,

@ OH

Soil fungi were tested in relation to their ability to hydroxylate carbaryl, and it was found that hydroxylation in the side chain or in the positions on the aromatic ring varied qualitatively as well as quantitatively with various fungal species (Bollag and Liu, 1972a). An isolated Mucor species, for example, accumulated essentially ring-hydroxylated products, whereas Aspergillus terreus hydroxylated mostly on the side chain. Considerable differences could be detected even within one genus, where different species hydroxylated at different positions or showed no activity at all. Wallnofer et al. (1972), investigating the metabolism of the systemic fungicide 2,5-dimethyl-3-furancarboxylicacid anilide, found that the pesticide ( 60 pmoles/liter ) was hydroxylated by Rhizopus japonicus to 2-hydroxymethyl-5-methyl- and 2-methyl-5-hydroxymethyl-3-furancarboxylic acid anilide at a ratio of 23 and 12 pmoles/liter, respectively. The formed metabolites were not further degraded by the fungus.

nNH;)-J CH,OH

CH3

2. Dealkylation Numerous pesticides, such as phenylureas, acylanilides, carbamates, s-triazines possess alkyl moieties which very often present active groups

85

MICROBIAL TRANSFORMATION OF PESTICIDES

producing a desired toxic influence. Therefore, dealkylation reactions are of great importance since they are a first step in the detoxication of pesticides and alkyl groups of side chains are frequently the first target of microbial attack. Most of our knowledge on the mechanism of dealkylation originates from studies performed with the microsomal fraction of liver (Gram, 1971), and only a few reports exist on the dealkylating activity of pesticides by microbial enzymes. Usually it is assumed that a dealkylation reaction results in the dealkylated product and an aldehyde: R-X-CHZ-R'

-+

R-X

+

R'-CHO

X can represent an N or 0 atom, and the dealkylation can produce an amine or an alcohol, respectively. Both N- and O-dealkylation are catalyzed by a mixed-function oxidase requiring a reduced nicotinamide nucleotide as a hydrogen donator. a. N-Dealkylation. The mechanism of n-dealkylation is not yet clear. The question arises especially around the possible formation of N-oxide as an intermediate, and studies with microsomal liver systems are not conclusive (Gram, 1971). However, in several cases it was possible to isolate an N-hydroxylated intermediate, which in turn can be metabolized to the dealkylated product. For example, carbaryl is transformed by the fungus Aspergillus terreus to l-naphthyl N-hydroxymethyl carbamate and subsequently to l-naphthyl carbamate ( Liu and Bollag, 1971b). o n

11 I 0 - C -N-CH,

O H II I 0 -C-N--CH,OH

0 II O-C-NH,

--& -fJ$

The N-hydroxymethyl intermediate is also chemically degraded to l-naphthyl carbamate, .but the study gave evidence that, through the additional biological activity, the formation of the dealkylated product was increased considerably. In many other cases of N-dealkylation it was suspected that an N-hydroxyalkyl intermediate might be formed, but these compounds are often chemically unstable and decompose further to the dealkylated product. Hydroxylation of the methyl group in the side chain of carbaryl was also observed with many other soil fungi (Liu and Bollag, 1971a; Bollag and Liu, 1972a), and it can be assumed that further transformation of the 1-naphthyl N-hydroxymethyl carbamate results in the dealkylated product. A carbamate-related amide, the herbicide diphenamid, was stepwise dealkylated by Trichoderma viride and Aspergillus candidus to N-methyl

86

JEAN-MARC BOLLAG

2,2-diphenylacetamide and, subsequently, to 2,2-diphenylacetamide ( Kesner and Ries, 1967) :

Po -qo II

6

,CH,

HC-C-N,

CH,

II

6

HC-C-N’

CH,

H ‘

~

Po HC-C-N, II

0

,H

H

An interesting observation was that diphenamid, the applied herbicide, is less toxic to the target plants than the two dealkylated metabolites. N-Dealkylation has been demonstrated to be a major reaction in the metabolism of dimethyl-substituted phenylureas ( Geissbuhler, 1969). In liquid cultures of mixed soil bacteria, Geissbiihler et al. (1963) isolated and identified 3-(4-chlorophenoxy )phenyl-l-methylurea and 3- ( 4-chlorophenoxy ) phenyhrea from chloroxuron:

/ Similar patterns of dealkylation were also reported for other phenylureas: diuron is stepwise dealkylated in soil to 3-( 3,4-dichlorophenyl) -1methylurea and 3,4-dichlorophenylurea ( Dalton et al., 1966); monolinuron and linuran were dealkylated by an Aspergillus niger sp. (Borner, 1967); metobromuron was converted by the fungus Talaromyces wortmanii to 1-(p-bromophenyl ) -3-methoxyurea and 1-(p-bromophenyl ) 3-methylurea, indicating a dealkylation and dealkoxylation reaction, and subsequently to p-bromophenylurea (Tweedy et al., 1970a); and Rhizoctonia solani metabolized chlorbromuron to the demethylated product ( Weinberger and Bollag, 1972). Wallnofer et al. ( 1973) also found that Rhizopus japonica was active in demethylation of phenylurea herbicides; however, buturon did not lose the N-methyl, but the N-butyryl ( l-methyl-2-propynyl) group, resulting in the formation of 3- (p-chlorophenyl ) -1-methylurea. s-Triazine herbicides are metabolized by soil microorganisms, and the removal of the alkyl side chains appears to be the primary mode of attack. In pure culture studies, simazine was dealkylated by Aspergillus and the ring porfumigatus to 2-chloro-4-ethylamino-6-amino-s-triazine,

MICROBIAL TRANSFORMATION OF PESTICIDES

87

tion of the molecule remained intact (Kearney et al., 1965); attempts to isolate a cell-free preparation from the mycelium were not successful. The N-dealkylation of atrazine to either 2-chloro-4-amino-6-ethylaminos-triazine or 2-chloro-4-an~ino-6-isopropyl-amino-s-triazinewas shown by 12 different soil fungi ( Kaufman and Blake, 1970) : c1 I

All the fungi investigated were able to dealkylate the herbicide by either alkylamino group, but the removal of the ethyl side chain or the isopropyl group by various fungal species was quantitatively different. Aspergillus fumigatus, for example, removed essentially the ethyl moiety, whereas Rhizopus stolonifer metabolized the isopropyl group more readily. In these experiments there was no evidence that the ring of the s-triazine molecule was cleaved. It is noteworthy that dealkylation of s-triazines does not necessarily mean reduction in their herbicidal or phytotoxic activity (Knuesli et al., 1969). Additional pesticides which undergo dealkylation as an initial degradation reaction include : the dipyridyl herbicide paraquat, which was apparently demethylated by an unidentified bacterium ( Funderburk and Bozarth, 1967); trifluralin, which was dealkylated by removal of a propyl group by A . niger (Funderburk et al., 1967); and dinitramine, which is degraded to the dealkylated chemical by a cell extract from A. fumigatus in the presence of NADPH and ferrous ions (Laanio et al., 1973). b. O-Dealkylation. The removal of a methyl or another alkyl group from an oxygen atom functioning as a linkage to the other molecular moiety can be considered as an ether cleavage or an O-dealkylation reaction. Although the enzymatic mechanism for cleavage of the oxygen from the hydrocarbon appears to be very similar in all investigated reactions, in this review O-dealkylation as the removal of an alkyl group was distinguished from cleavage of the ether linkage as the separation of another hydrocarbon from oxygen.

88

JEAN-MARC B O U A G

Axelrod (1956) first demonstrated the enzymatic cleavage of methoxylated compounds by microsomal preparations from rat liver; both oxygen and NADPH were necessary for the conversion of anisole to phenol and formaldehyde. Methoxylated aromatic compounds are demethylated by soil fungi; 0-,m-, and p-methoxybenzoic acid were converted to the corresponding hydroxybenzoic acids and veratric acid was demethylated to vanillic acid by species of Hormodendrum, Haplographium, and Penicillium ( Henderson, 1957). Cell-free preparations from Pseudomonas fluorescens, capable of converting vanillate to protocatechuate and formaldehyde, were first obtained by Cartwright and Smith (1967), and it was established that oxygen, reduced nicotinamide nucleotides, and reduced glutathione are required for the demethylation reaction: COOH

Q -

COOH

.+

HCHO

OCH,

OH

OH

However, it was not possible to clarify whether the protocatechuate was directly formed from vanillate or indirectly via p-hydroxybenzoate. The 0-dealkylases, induced growth of species of P. flu0rescen.s and Nocardia on 4-alkoxybenzoates, specifically attack the ether linkage and are indifferent to the nature of the alkyl group which, itself, determines reaction rates (Cartwright et al., 1971). Enzymes of an Arthrobacter sp. also converted vanillate to protocatechuate and m-methoxybenzoate to m-hydroxybenzoate with the simultaneous formation of formaldehyde (Raymond and Alexander, 1972). The 0-demethylase system from P . testosteroni was shown to be composed of at least two protein fractions (Ribbons, 1971), and an enzyme extract from P. puticla was further resolved and three protein components were purified ( Bernhardt et al., 1971). Stenersen (1969) found that the insecticide bromophos was oxidized by a double-dealkylation to the bisdemethylated bromophos by the fungi Alternaria tenius and Trichoderma lignorum: CHJO,,/s c1 HO S cH3>odBr HO o

- x d'-

0-Dealkylation was also found with anisole, whose chlorinated derivatives were occasionally found as intermediates during the breakdown of chlorinated phenoxyacetic acids; replacement cultures of Aspergillus niger oxidized anisole to phenol (Bocks et al., 1964). Mycelia of Rhizoctonia solani and some other fungi converted the ( Hock and Sissoil fungicide chloroneb to 2,5-dichloro-4-methoxyphenol

89

MICROBIAL TRANSFORMATION OF PESTICIDES

ler, 1969), and in some cases a further dealkylation to 2,5-dichlorohydroquinone was observed ( Wiese and Vargas, 1973):

@

-.p"

>cl@cl

c1 OCH,

OCH,

OH

Boothroyd et al. (1961) found that different fungi demethylate the fungicide griseofulvin at different methoxy groups attached to the molecule. Botrytis allii formed the 2'-demethylgriseofulvin, Cercospora rnelonis produced the 6-demethylgriseofulvin, and Microsporum canis generated 4-demethylgriseofulvin.

3. Cbauage of Ether Linkage As outlined under 0-dealkylation reactions, which can also be considered as a cleavage of an ether linkage, it appears that the ether cleavage enzymes investigated are quite versatile in the range of substrates they can oxidize. This can probably be explained by the natural abundance of methoxylated compounds in connection with lignin and other organic material in soil. In view of this, it is not very surprising that many pesticides containing ether linkages, like 2,4-D, related phenoxyacetates, and dicamba, are relatively easily metabolized. The cleavage of an ether linkage is considered to be a reaction caused by a mixed function oxidase insofar as all detailed studies revealed that reduced pyridine nucleotides and molecular oxygen are required. A large number of microorganisms capable of metabolizing phenoxyalkanoic herbicides produce the corresponding phenols as intermediates (Loos, 1969). This transformation could also be shown with cell-free extracts from an Arthrobacter sp. (Bollag et al., 1967; Loos et al., 1967a). In order to clarify the mechanism of ether cleavage, Helling et al. (1968), using phenoxy-'*O acetic acid, demonstrated that the cell-free extract of a MCPA-grown Arthrobacter sp. catalyzed, in the presence of oxygen, the cleavage between the aliphatic side chain and the ether-oxygen atom as indicated by the complete retention of lXOin the phenol molecule. Two mechanisms are apparently involved which cause the cleavage of the ether linkage: (1) a reductive reaction observed with higher phenoxyacetic acids ( MacRae and Alexander, 1963) and ( 2 ) an oxidative reaction which was established with 2,4-D and MCPA (Tiedje and Alexander, 1969; Gamar and Gaunt, 1971). MacRae and Alexander (1963) reported that a Flavobacterium sp. caused the cleavage of the ether linkage of ,-linked (omega-linked) 2,4-dichlorophenoxyalkyl carboxylic acids from the propionic to the

90

JEAN-MARC BOLLAC

undecanoic homolog. The cleavage apparently resulted in the production of a phenol and the fatty acid corresponding to the aliphatic moiety: O-(CH,),-COOH

61'

+

\

CH,-(CH,),-COOH

\

c1

c1

A soluble enzyme preparation from an ATthTobacteT sp. catalyzed the cleavage of the ether linkage of 2,4-D resulting in the formation of 2,4-dichlorophenol and alanine (Tiedje and Alexander, 1969). S'ince acetate and glycolate were not attacked by the cell-free extract, but glyoxylate was rapidly metabolized, it was suggested that glyoxylate was the initial product, and its further transformation resulted in the production of alanine. A similar investigation was performed by Gamar and Gaunt (1971) using MCPA as the substrate and a crude extract prepared from a Pseudomonas sp. grown on a basal medium with MCPA as sole source of carbon. MCPA was oxidized by the cell-free preparation in the presence of a reduced nicotinamide nucleotide (NADH or NADPH) to 2-methyl-4-chlorophenol and glyoxylic acid: 0-CH,COOH

Q.'" c1

4

2

H

3

+

CHO-CCOOH

c1

4. Oxidation of Aromatic Ring Numerous pesticides are cyclic compounds, and consequently their complete biodegradation can be achieved only after cleavage of the ring. Many microorganisms have the ability to oxidize aromatic substances and can use the resulting aliphatic compounds as substrates in the intermediary metabolism. Intensive studies contributed to a quite clear understanding of the general catabolic pathways of aromatic molecules, and many microbial enzymes involved in this reaction sequence could be isolated and characterized (Dagley, 1971; Stanier and Ornston, 1973). It is generally accepted that dihydroxylation is a prerequisite for enzymatic cleavage of the benzene ring. Ring fission can be brought about by dioxygenases through three different pathways depending upon the distribution of the hydroxyl groups; catechol, for example, which possesses hydroxyls on adjacent carbons, can be oxidized by ortho or meta

MICROBIAL TRANSFORMATION OF PESTICIDES

91

cleavage forming cis,cis-muconic acid or a-hydroxymuconic semialdehyde, respectively :

ortho cleavage

metu cleavage

A third pathway can occur with para-dihydric phenols; the ring of gentisic acid, for example, can be cleaved between the hydroxyl and the adjacent carboxyl group, resulting in the formation of maleylpyruvic acid:

qcmy OH

OH

B g - C O O H

OH

Aromatic pesticides-and also other xenobiotic compounds-are usually distinguished by multiple and variable substitutions on the ring or different ring formations, causing a molecular structure which often cannot be easily attacked. Therefore, each pesticide possessing a cyclic structure has to be investigated independently to determine whether ring fission can be provoked by microbial activity. Evans (1969) and Chapman ( 1972) summarized known bacterial pathways of various phenolic compounds, but detailed knowledge on the microbial ring cleavage of aromatic pesticides is quite scarce, and only fragmentary knowledge exists on the fate of products after ring cleavage. However, one group of pesticides, the chlorinated phenoxyalkanoic herbicides, were intensively studied in relation to ring cleavage, and the fate of the chlorinated catechols was followed and elaborated with enzyme preparations from different bacteria. Cell-free extracts from an Arthrobacter sp. metabolized 3,5-dichlorocatechol and 4-chlorocatechol, degradation products of 2,4-D and 4-~hlorophenoxyacetate,to ring fission products which retained the halogens (Bollag et al., 1968a). By dilution of the cell-free extract it was possible to achieve the accumulation of the muconic acids. Tiedje et al. (1969) identified cis,cis3-chloro- and cis,cis-2,4-dichloromuconic acid from 4-chloro-and 3,5-dichlorocatechol,respectively :

?H

c1

c1

92

JEAN-MARC BOLLAG

The formation of the cis&-muconic acids implies an ortho-fission mechanism. Extracts of the bacterium also converted catechol, 3- and 4-methylcatechol to the corresponding muconic acids. Evans et al. (1971b) also described the conversion of 3,5-dichlorocatechol, suspecting that they had found cis&-muconic acid in the culture medium of a Pseudomonas sp., but another Pseudomonus strain apparently produced a-chloromuconate, and therefore, they concluded that, in the latter case, dechlorination at the p-position has taken place at some stage before ring cleavage. The intermediate muconic acid of MCPA was tentatively identified as &,cis-y-chloro-a-methylmuconate as shown in the reaction sequence of a cell-free system from a Pseudomonas sp. (Gaunt and Evans, 1971); the ring-fission enzyme required Fe2+or Fe3+ and reduced glutathione €or activity, as do many other catechol oxygenases. A meta-cleaving oxygenase, a catechol 1,6-oxygenase, from an Achromobacter sp. was active on methylated and chlorinated catechols ( Horvath, 1970b). 3-Methylcatechol, 4-ch1orocatecho1, and 3,5-dichlorocatechol were oxidized to 2-hydroxy-3-methylmuconic semialdehyde, 4-chloro-2-hydroxymuconic semialdehyde, and 3,5-dichloro-2-hydroxymuconic semialdehyde, respectively:

-

OHC H W

T CHS

&OH c1

~

oF : c1

HOOC c1

c1

c1

It is of interest to emphasize that halogen or alkyl substitutions of the aromatic compound were not released prior to the ring cleavage, and therefore, the fate of the resulting ring-fission products have to be followed further if one is concerned with their toxic impact. An important practical question is related to the microbial degradation of DDT which is considered to be of the most persistent pesticides in the environment. Whereas several microbes were found to be capable of dehalogenating DDT to DDD and also DDE (see Section III,D), there is still no clear knowledge as to the extent to which the cleavage of the aromatic rings of DDT or its metabolites takes place. Focht and Alexander (1971) isolated a Hydrogemonas sp. from sewage effluent capable of cleaving the ring of DDT analogs. Cell suspensions of the bacterium, which were grown on diphenylmethane, did not metabolize DDT and p,p’-dichlorobenzophenone, but they did transform the corre-

MICROBIAL TRANSFORMA'MON OF PESTICIDES

93

sponding monochloro and nonchlorinated compounds. They concluded that the presence of the para-chlorine substitution on the phenyl rings and the substitution of carbonyl or trichloromethyl group on the carbon atom binding the two phenyl groups inhibited the metabolism of DDT or analogs by the Hydrogemonas sp. However, one of the benzene rings of diphenylmethane, p,p'-dichlorodiphenylmethane, and 1,l-diphenyl2,2,2-trichloroethane was cleaved as indicated with the formation of phenylacetic, p-chlorophenylacetic, and 2-phenyl-3,3,3-trichloropropionic acid, respectively:

Q

c1 I

-0 6-

CH,-COOH I

c1

Q HC-CQ,

CH,(CCI$COOH

-0 I

-

In a subsequent investigation it was found that high protein concentrations in bacterial extracts of a Hydrogemom sp. caused the transformation of DDT, DDD, and other products under anaerobic conditions. If whole cells were added and aerobic conditions were provided, subsequently, the formation of p-chlorophenylacetic acid could be determined (Pfaender and Alexander, 1972). This observation infers that one microbe is capable of attacking DDT and causing its degradation to a single chlorinated benzene compound. Gunner and Zuckerman ( 1968) described the microbial degradation of the pyrimidyl ring of the insecticide diazinon in the presence of two microorganisms. When a Streptomyces sp. or an Arthrobacter sp. were incubated individually with diazinon, the pyrimidyl ring was not attacked, but the two microbes together cleaved and metabolized the ring structure. The ring-fission mechanism of several important groups of pesticides by microorganisms, if it actually occurs, is still very obscure and requires a lot of experimental work for its clarification. There are only indicative

94

JEAN-MARC BOLLAG

data that the rings of s-triazine herbicides ( Kaufman and Kearney, 1970), the substituted anilines which are intermediates from numerous pesticides (Chisaka and Kearney, 1970), and 1-naphthol (Bollag and Liu, 1972b), resulting from the bicyclic ring of certain methylcarbamate insecticides, are cleaved and further metabolized. Most information leading to the assumption that ring cleavage takes place results from experiments with radiolabeled pesticides whereby it was possible to trap "CO,. In the case of s-triazines, for example, evolution of IfC02has been reported from microbial systems treated with 14C-ring-labeleds-triazines, but in nearly all cases, only up to 4% of the applied herbicide evolved as ' C O , ( Kaufman and Kearney, 1970).

5. p-Oxidation This reaction was found especially in the oxidation of long-chain phenoxyalkanoate herbicides. p-oxidation of an aliphatic side chain proceeds by the stepwise removal of two-carbon fragments from a fatty acid, and the shortened acid can then be further oxidized. Bacteria (Taylor and Wain, 1962), actinomycetes (Webley et al., 1957), and fungi (Byrde and Woodcock, 1957) can metabolize w-phenoxyalkanoic acids by p-oxidation. Oxidation of 2,4-dichlorophenoxyalkanoicacids, for instance, with an even number of carbon atoms in the side chain results in the formation of 2,4-dichlorophenoxyacetate whereas acids with an odd number of carbon atoms are converted to 2,4-dichlorophenol (Loos, 1969) : CH2-CHz-CH,-CHa+CH2-COOH I

;

@

-

CHa-CHa+CH,I 0

COOH

qcl

c1

C1

J

CH,-COOH I 0

c1

ecl CHz-CHaI

C1

\

CH,-fCH,-COOH

CHa- CH,- COOH I

I

__t

\

c1

-

6-" c1

MICROBIAL TRANSFORMATION OF PESTICIDES

95

The mechanism of p-oxidation of phenoxyalkanoate herbicides was first established with pure cultures, but subsequently, the same kind of metabolism was also shown to occur in natural soil (Gutenmann et al., 1964). 6. Epoxidation

Addition of an oxygen atom to a double bond represents an epoxidation reaction, which is now recognized as a common process for the metabolism of xenobiotic compounds. In recent years it became clear that this is a widespread process in the metabolism of aromatic substances. Arene oxides are intermediates which are transformed enzymatically or nonenzymatically to dihydrodiols and ( pre ) mercapturic acids and cause considerable concern, since, as metabolic intermediates, they are capable of initiating tissue necrosis and carcinogenesis (Daly et al., 1972). Arene oxide, lP-naphthalene oxide, has been identified in a biological system as the obligatory intermediate in the formation of naphthols from naphthalene (Jerina et al., 1969). Monooxygenases of bacteria and fungi can introduce a hydroxyl group to an aromatic compound accompanied by migration of an original substituent. This phenomenon, termed NIH-shift, proceeds via the intermediary formation of an arene oxide. Guroff et al. (1966) found that halogen substituents may be displaced from carbon-4 to carbon-3 when phenylalanine is hydroxylated by Pseudomonas:

c1

OH

Although it was shown that various microorganisms cause the NIH-shift, its importance in the microbial degradation of pesticides has yet to be clearly established. However, one example is well known: Faulkner and Woodcock (1964, 1965) found that Aspergillus niger metabolized the herbicide 2,4-D to 2,4-dichloro-5-hydroxy-and 2,5-dichloro-4-hydroxyphenoxyacetic acid:

b"

OCH,-COOH

\

C1

OCH,-COOH

OCH,-COOH

HOf q\C 1

c1

+.

c1QC1\ OH

96

JEAN-MARC BOLLAG

The latter product generated by the fungal activity shows the shift of a chlorine atom which was coupled with the introduction of a hydroxyl group in its place. When the herbicide MCPA, in which the chlorine at carbon-2 is substituted by a methyl group, was exposed to the same fungus, hydroxylation occurred, but the NIH-shift was not observed ( Faulkner and Woodcock, 1965). An enzyme system from Pseudomonm oleovorans was shown to catalyze the epoxidation of alkenes; 1,7-octadiene was converted to both 7,8-epoxy-l-octene and 1,2-7,8-diepoxyoctane, whereas l-octene was oxidized to both 7-octenel-01 and 1,2-epoxyoctane (May and Abbott, 1973). It appeared that the enzymatic epoxidation reaction is mechanistically similar to the reaction causing the methyl group hydroxylation of alkanes and fatty acids. Both enzyme systems are composed of three protein components ( reductase, rubredoxin, and hydroxylase) and require molecular oxygen and NADH (NADPH cannot be substituted) for activity. Several cyclodiene insecticides, such as aldrin, isodrin, and heptachlor, can -undergo epoxidation by various microorganisms yielding products with increased toxicity in the environment. Korte et al. (1962) demonstrated that the fungi Aspergillus niger, A. fEavus, Penicillium notatum, and P . chrysogenum converted aldrin to dieldrin:

“pJJ c1

c1

o* l c -

dl

c1 Cl

I

c1

Ninety-two different strains of bacteria, actinomycetes, and fungi isolated from soil were tested for their ability to transform aldrin, and most of them could epoxidize the original pesticide dieldrin (Tu et al., 1968). The most active fungal isolate, a Fusarium sp., oxidized about 9% of the added aldrin to dieldrin during the 6-week incubation period. A similar epoxidation of a chlorinated hydrocarbon containing an isolated double bond was observed with the insecticide heptachlor, which was oxidized to heptachlor epoxide (Miles et al., 1969). Thirty-five of 47 fungi, and 26 of 45 bacteria and actinomycetes isolated from soil produced the epoxide; the greatest activity was shown by a Nocardia sp. which epoxidized 6%of the applied heptachlor. Another epoxidation reaction occurred after an initial chemical hydrolysis of heptachlor to l-hydroxychlordene. The chemical intermediate product was converted to l-hydroxy-2,3-epoxychlordeneby 43 of 47 fungi, but only 4 of the 45 bacteria and actinomycetes tested showed activity.

hlICROBIAL TRANSFORMATION OF PESTICIDES

97

7. Sulfoxidation This reaction consists of the oxidation of divalent sulfur to the sulfoxide and, sometimes, to the sulfone: > S + >SO + >SO2, but evidence from detailed studies was received only from experiments with microsomal enzymes from plant and animal systems. Sulfoxidation of pesticides in soil was attributed to biological transformation with aldicarb ( Coppedge et al., 1967), phorate (Getzin and Chapman, 1960), and the s-triazine prometryne (Plimmer and Kearney, 1969), but no specific microorganisms were isolated capable of sulfoxidizing the various pesticides. Ahmed and Casida ( 1958), investigating the metabolism of organophosphorus insecticides, determined that the green alga Chlorella pyrenoidosa and the yeast Torulopsis utilis oxidized phorate to its respective sulfoxide, but slowly converted this product to the phosphorothiolate sulfoxide with little formation of the salfide or sulfone.

B. REDUC~IONS Several groups of pesticides are subject to reduction, but this reaction is usually less common than oxidation in the transformation of xenobiotic compounds. The reduction of the nitro group to amine has been found during the metabolism of various pesticides by the activity of different bacteria and fungi. It is anticipated that the reduction takes place in stages involving the intermediate formation of a nitroso and hydroxyamino group : R-NO,

R-N=O

i

--t

R-NHOH

-+

R-NHZ

In experiments with Escherichia coli, Saz and Slie (1954) found that various organic nitro compounds are reduced in the presence of cysteine to the corresponding arylamines. 2,4-Dinitrophenol, which is used in fungicidal preparations, was reduced by Fusarium oxysporum in a liquid basal medium to 2-amino-4-nitrophenol and 4-amino-2-nitrophenol ( Madhosingh, 1961) . Formation of the 4-amino-2-nitrophenol compound appeared to be favored in acid cultures, whereas the 2-amino isomer dominated at a higher pH value. The degradative pathway of the herbicide DNOC by a pseudomonad isolated from a garden soil was followed by isolation of intermediates from growing cultures, cell suspensions, and cell-free extracts, and the reactions sequence was determined as : 3,5-dinitro-o-cresol+ 3-amino-5nitro-o-cresol + 3-methyl-5-nitrocatechol -+ 3-methyl-5-aminocatechol + 2,3,5-trihydroxytoluene (Tewfik and Evans, 1966). Hamdi and Tewfik (1970) also determined that DNOC is reduced to 3-aminod-nitro-ocresol by Rhizobium leguminosarum.

98

JEAN-MARC BOLLAG

The fungicide pentachloronitrobenzene ( PCNB ) is easily reduced in culture solution by various actinomycetes and fungi to the corresponding aniline (Chacko et al., 1966; Nakanishi and Oku, 1969), and the same transformation was observed in soil (KO and Farley, 1969).

-

cl@ c1

cl@cl

' c1

c1

c1

c1

c1

Lichtenstein and Schulz (1964) found that the organophosphorus insecticide parathion was metabolized in soil either by hydrolysis or by reduction to its amino, form, apparently depending 0x1 populations of soil microorganisms. Since Neuberg and Welde (1914) showed that in the presence of yeast nitrobenzene was reduced to aniline, Lichtenstein and Schulz also tested the effect of yeast on the metabolism of parathion and found it responsible for the reduction of the insecticide to aminoparathion; bacteria apparently did not participate in this reduction. Likewise, the reduction of a nitro to an amino group occurred during the metabolism of the organosphosphorus insecticide Sumithion ( Fenitrothion) by Bacillus subtilis (Miyamoto et al., 1966); experiments with washed cell suspensions of B . subtilis indicated that the nitro group of various phosphorothioates was reduced under aerobic as well as under anaerobic conditions. Various chlorinated hydrocarbons are transformed by reductive dehalogenation processes, but these reactions will be considered in Section II1,D. Frequently, it is also observed that reduction is a process which can produce a center for conjugation, and such a pathway was found in various metal-containing agricultural pesticides. In the case of arsene-containing compounds, it was found that a strain of Methanobacterium reduced arsenate ( 5 + ) to dimethylarsine ( 3 - ) under anaerobic conditions, whereas reduction and methylation reductions occurred intermittently (Fig. 1); in these experiments, methylcobalamin served as methyl donor of choice ( McBride and Wolfe, 1971). The sequence of reactions involved the reduction of arsenate (5+, arsenic valency ) to arsenite (3+ ). This intermediate was methylated to methylarsonic acid, subsequently reduced and methylated to dimethylarsinic acid ( I f ) , and finally further reduced to form the dimethylarsine ( 3 - ) . This reaction could also be shown with cell extract from the Methanobacterium, but adenosine triphosphate and hydrogen had to be added.

99

MICROBIAL TRANSFORhlATION OF PESTICIDES

-

OH I HO-As5+OH

7%

As3+-OH I1 0

II

0 Arsenate

HO-AS3"OH I1

0

Arsenite

Methylarsonic acid

J ?H3 As3--CH,

y

3

HO--As'+-CH, II

I

n

0

Dimethy larsine

Dimethylarsinic acid

FIG.1. Metabolic transformation of arsenate by a Methanobacterium sp.

C. HYDROLYSIS Hydrolysis is a reaction type that can be initiated enzymatically or chemically, and therefore, it may be difficult in some cases to determine the true origin. Generally, it can be assumed that hydrolysis converts a lipophilic compound into a hydrophilic, water-soluble substance. In the microbial breakdown of pesticides, hydrolytic enzymes include amidases, esterases, nitrilases, and phosphatases which yield an acid on one side and an alcohol or amine on the other: I

-C-C-N-

Ester

I l l -C-C-0-C-

Nitrile

-C-C=N

I

0

I

I

I

-

R

Amide

I

I I

-

0 I II -C-C-OH

I

I I

0 II

-C-C-OH

+

H-N-

+

HO-C-

I I I

0

I II -C-C-NH, I

--C-C-OH

I

I

0 It

f

NH,

Carbamates can be considered simultaneously as esters and amides and represent special cases insofar as the intermediate carbamic and carbonic acids are unstable compounds and degrade spontaneously with the liberation of CO,. However, it is evident that, independent of an esterase or an amidase reaction, the end products are the same. Hydrolysis appears to be the major reaction for acylanilide herbicides causing the cleavage of the C-N bond and the release of the side chain. Two species of Penicillium and one species of Pululluria isolated

100

JEAN-MARC BOLLAG

from soil were capable of hydrolyzing the herbicide karsil (Sharabi and Bordeleau, 1969), whereas 3,4-dichloroaniline and 2-methylvaleric acid were identified as intermediates. A cell-free extract was prepared from a Penicillium sp., and the specificity of the partially purified acylamidase was tested on various anilides and structurally related compounds. Activity was enhanced with increasing chain length, up to fourcarbon compounds. Substitution of the N-acyl group or the phenyl ring also influenced the enzyme activity. A phenylurea, diuron, and a phenylcarbamate herbicide, CIPC, were not attacked by the enzyme preparation. An acylamidase which hydrolyzed propanil to 3,4-dichloroaniline and propionic acid was also isolated from the mycelium of Fusarium solani (Lanzilotta and Pramer, 1970). Other acylanilides such as dicryl and karsil as well as phenylureas (monuron and fenuron) appeared to be unaffected by this enzyme system. Extensive studies were performed on the enzymatic hydrolysis of the phenylcarbamate chlorpropham ( CIPC ) with partially purified cell-free preparations obtained from a strain of Pseudomonas striata isolated from soil enrichment cultures (Kearney, 1965). An enzyme, purified by ammonium sulfate precipitation and column chromatography by gradient elution on DEAE-celluIose, catalyzes the hydrolysis of CIPC to 3-chloroaniline, carbon dioxide, and isopropyl alcohol:

However, it is not clear whether the enzymatic cleavage proceeds by hydrolysis of the ether linkage or of the amide bond or both, since the initial products produced by both reactions would be unstable. It is interesting to observe that this enzyme exhibits a broad substrate specificity, since a large number of structurally related phenylcarbamates and acylanilides were hydrolyzed, but ureas and methylcarbamates were not metabolized. The metabolism of urea herbicides was studied and ehcidated especially with Bacillus sphaericus, which was isolated from soil treated with monolinuron. Whole cells or cell-free extracts of B. sphaericus ( Wallnofer, 1969; Wallnofer and Bader, 1970) degraded various N’-methoxyphenylurea compounds by releasing CO, from the ureido portion of the molecule and leaving the corresponding aniline moieties as well as an unidentified product. The cell-free extract hydrolyzed the N’-methoxyphenylurea compounds monolinuron, linuron, chlorbromuron, and metobromuron, but the N’,N’-

MICROBIAL TRANSFORMATION OF PESTICIDES

101

dimethylphenylureas monuron, diuron, buturon, and fluometuron were not attacked (Engelhardt et al., 1971). However, there was no clear explanation as to why the decomposition of urea appears to be specific for the methoxy-substituted phenylureas. Thirteen acylanilides, which are used partially as herbicides or fungicides, were hydrolyzed by the cell-free extract at a rate at least 10 times higher than that of the methoxy-substituted phenylureas. In all these studies cell-free extract activity was found only if the enzyme preparation was induced after growth on the herbicide of choice, namely, linuron. Engelhart et al. (1971, 1972) also clarified the hydrolytic pathway of linuron. After incubation with extracts of B. sphaericus, it was possible to identify 3,4-dichloroaniline and CO, as well as N,O-dimethylhydroxylamine by characterization of its dinitrophenyl derivative. This leads to the general conclusion that phenylamide compounds are hydrolyzed to the corresponding anilines and acids, but the acid moiety formed during the composition is dissociated rapidly to the alkylalkoxyamine and CO,. The proposed reaction sequence for linuron is as follows:

c=o I

I

C1

Cl

L in u r 0 n

3,4-Dichloroaniline

N , 0-Dimethylhydroxylamine

Another mechanism of phenylurea degradation was proposed in the review of Geissbiihler (1969) from experiments with soil samples, where stepwise dealkylation of dimethyl-substituted phenylureas would precede hydrolysis. N-Lauroyl-1-valine, an amino acid derivative used as a pesticide with a preventive effect against rice blast, was metabolized by Pseudomonas aeruginosa. Since one product was identified as lauric acid, it was suggested that cleavage of the N-acyl linkage occurred, resulting in the formation of lauric acid and valine, but the latter compound was not detected because it might have been metabolized rapidly after its release ( Shida et al., 1973). Nitrile hydrolysis was shown when sterile and nonsterile soil treated

102

JEAN-MARC BOLLAG

with ioxynil were compared. No breakdown of the herbicide was detectable in the sterile soil, but in the nonsterile soil ioxynil was converted to 3,5-diiodo-4-hydroxybenzoicacid, with 3,5-diiodo-4-hydroxybenzamide as an intermediate product: CN

I

CONH,

COOH I

OH

OH

I

1

OH

Hydrolytic degradation also constitutes one of the major reactions in the metabolism of organophosphorus insecticides which contain either the P=O (phosphate) or the P=S ( phosphorothioate) groupings. It must be stressed that phosphorus esters are also easily susceptible to catalytic cleavage induced by nitrogenous compounds like amino acids and by heavy metal ions. Consequently, it has to be expected that nonenzymatic hydrolysis of phosphorus occurs easily in soil and other habitats. Mounter et al. (1955) demonstrated the presence in freeze-dried bacterial cells of a phosphatase which hydrolyzed dialkylfluorophosphates with the release of fluoride ions, and Mounter and Tuck (1956) showed that Escherichia coli and PropionihacteTium pentasaceum hydrolyze paraoxon, TEPP, and the diethyl, diisopropyl, and di-n-butyl fluorophosphates. Ahmed and Casida (1958) concluded that Pseudomonas fluorescens hydrolyzed Phorate ( Thimet ) without a subsequent oxidation reaction since, after incubation with the microbes, the residual organophosphate recovered partitioned completely into hexane from an acetone-water mixture. The soil fungus Trichoderma &ride and a Pseudomonas sp. degraded malathion, presumably by hydrolysis of the ester groups, to various carboxylic acid derivatives ( Matsumura and Boush, 1966). The insecticide trichlorfon ( Dipterex) was metabolized in a culture medium by AspeTgillus niger, Penicillium notatum, and a Fusarium sp. to hydrolytic products; one was identified as O-methyl-2,2,2-trichloro1-hydroxyethylphosphonic acid, and a second metabolite was tentatively acid ( Zayed et al., identified as 2,2,2-trichloro-l-hydroxyethylphosphonic 1965) : 0 OH C1 It I I CH,O-P-CH-C-C1 I I OCH, C1

0 II

+

OH I

C1 I

HO-P-CH-C-CI I I OCH, C1

-

0 OH CI I1 I I HO-P-CH-C-C1 I

OH

I

c1

Conversion of carbaryl to 1-naphthol also appears to be a hydrolytic reaction generated by various soil microorganisms, but the simultaneous chemical hydrolysis of the insecticide makes it difficult to establish to what extent the reaction is biological or chemical ( Bollag and Liu, 1971).

hIICROBIAL TRANSFORMATION OF PESTICIDES

103

The partial conversion of dieldrin by a hydrolytic reaction was shown in laboratory experiments. Matsumura and Boush ( 1967) isolated several species of Pseudomonas and Bacillus from soil samples, and they suggested, on the basis of an identical R, value with an authentic compound, that 6,7-trans-dihydroxydihydroaIdrin( aldrin diol ) might be a major product, whereas Wedemeyer (1968) came to a similar conclusion with the bacterium Aerobacter amogenes after various chromatographic analyses. c1 c1 I

c1

Cl

Although there are numerous reports with plants and animals which describe enzymatic hydrolysis of chlorotriazines to the hydroxy derivative, there is only one report claiming that atrazine is transformed by a microorganism, by the fungus Fusarium roseum, to its corresponding hydroxy analog (Couch et al., 1965).

MECHANISMS D. DEHALOGENATION Halogenated aliphatic and aromatic pesticides are widely used, and therefore, they are of increasing importance in the environment. The major metabolic problem which they pose relates to the question of the stability of the carbon-bound halogen. The transformation of the organic halogen to an inorganic form can usually be considered as a detoxication reaction, but if the metabolism does not involve the release of the halogen, the resulting intermediate may cause concern in the various ecosystems. The possible biological attack on halogenated compounds varies widely, and it is clear that the carbon-halogen bond and the number of halogen substitutions, as well as other structural features of the molecule, determine the metabolic fate of such a compound; therefore, one might expect different enzymatic mechanisms. The microbial enzymes which catalyze the removal of the halogen from the organic molecule were divided into three groups (Table 11): ( 1 ) hydrolytic dehalogenation, in which a hydroxyl group replaces the halogen atom; ( 2 ) reductive dehalogenation, where halogens are exchanged with hydrogen; and ( 3 ) dehydrohalogenation, in which both hydrogen and chlorine are removed from the molecule with the resultant formation of a double bond. The dehalogenating enzymes involved in these reactions are not clearly characterized, and therefore the division into various dehalogenating

TABLE I1 DEHALOQENATION MECHANISMS OF PESTICIDES OR THEIRMETABOLIC INTERMEDIATES BY MICROORQANISMS Schematic reaction

Example

Refer en ces

Fluoroacetate + glycolic acid 3-Bromopropanol 3-hydroxypropionic acid 2-Hydroxyphenoxyacetic acid 4 Zchlorophenoxyacetic acid 3-Chlorobenzoic acid 3-hydroxybenzoic acid

Goldman (1965) Tonomura et al. (1965) Castro and Bartnicki (1965) Faulkner and Woodcock (1961)

2-Fluorobenzoic acid + catechol

Goldman et al. (1967)

DDT + D D D (TDE)

Kallman and Andrews (1963), Mendel and Walton (1966), Chacko el al. (1966), Wedemeyer (1966, 1967), Johnson et al. (1967), Plimmer et al. (1968), Braunberg and Beck (1968), Matsumwa and Boush (1968), French and Hoopingarner (1970) Miles et al. (1969)

1. Hydrolytic dehalogenation

RCHz(halogen) RCHzOH

-+

-+

Halogen

on

-+

2. Reductive dehalogenation RC(ha1ogen)s -+ RCH(ha1ogen)2

Heptachlor -+ chlordene 3. Dehydrohalogenation RCHJ3(halogen)a -+ RCH =C(halogen)z

DDT-t DDE

Lindane -+ y-pentachlorocyclohexene -pChloro-a-methylmuconic acid + r-carboxymethylene-a-methyl-Aaflbutenolide

Johnston et al. (1972)

Stenersen (1965), Mendel and Walton (1966), Guenzi and Beard (1967), Matsumura and Boush (1968), Chacko et al. (1966), Johnson et al. (1967), Langlois (1967), Braunberg and Beck (1968) Yule et al. (1967) Gaunt and Evans (1971)

105

MICROBIAL TRANSFORMATION OF PESTICIDES

TABLE I1 (Continued) Schematic reaction

Example

References

cis, cis-3-Chloromuconic acid + (4-carboxymetliylene but-%enolide) -+ maleylactic acid eis,cis-2,4-Dichloromuconic acid -+ (2-chloro-4-carboxymethylene but-2enolide) --+ chloromaleylacetic acid pyruvic acid Dalapon 3-Chloropropionic acid ---f acrylic acid Ethylene dibromide -+ ethylene

Bollag et al. (1968b), Tiedje et al. (1969), Evans et al. (1971a),

---$ -+

Bollag et al. (1968b), Tiedje et al. (1969), Evans et al. (1971b)

Kearney et al. (1964) Bollag and Alexander (1971) Castro and Belser (1968)

mechanisms should be considered only as an attempt to categorize the observed microbial removal of the halogens.

1. Hydrolytic Dehalogenution In this reaction the halogen is replaced by a hydroxyl group, but the specific enzymatic mechanism involved was not elaborated in the case of microbial pesticide metabolism. The introduced hydroxyl group can be generated from water as demonstrated by experiments in lSO enriched water (Goldman and Milne, 1966), or the oxygen of the hydroxyl group can originate from the reduction of molecular oxygen by the catalytic activity of a NADPH-dependent hydroxylase ( Kaufman et aE., 1962). Aliphatic compounds containing C atoms bearing only one halogen will be transformed to alcohols. The halidohydrolases catalyze relatively simple reactions in which a halogen at the 2-position of a short-chain fatty acid is replaced by a hydroxyl group. An enzyme of this kind was found in a soil Pseudomonas sp, ( Goldman, 1965) which catalyzes chloro-, fluoro-, and iodoacetate: Hal. CH2COO-

+ HO-

+

HOCH2COO-

+ Hal.-

Fluoroacetate, on which the bacteria were grown, was the preferred substrate, and chloride and iodide are released from their substrates at only 15 and 0.53, respectively, of the rate of fluoride release. Castro and Bartnicki (1965) found that a pseudomonad grown in a medium containing 3-bromopropanol also replaced the halide with a hydroxyl group, and they isolated and identified 3-hydroxypropionic acid.

106

JEAN-MARC BOLLAG

Dehalogenation of aromatic compounds by microorganisms usually occurs after ring cleavage at it is described later under “dehydrohalogenation” in the metabolism of various chlorinated phenoxyacetic acids. However, it was also found that a halogen can be directly replaced on a benzene ring by a hydroxyl group. Faulkner and Woodcock (1961) observed that 2-chlorophenoxyacetic acid is converted to 2-hydroxyphenoxyacetic acid by Aspergillus niger, and Johnston et al. (1972) determined that a Pseudomonas sp. transformed 3-chlorobenzoic acid to 3-hydroxybenzoic acid. There are no investigations reported on the microbial enzymes performing this reaction, and therefore, it is not possible to make comparisons with the microsomal hydroxylating system of rat liver which is capable of converting both 4-chloro- and 4-fluoroaniline to 4-hydroxyaniline ( Daly et al., 1968). Goldman et al. (1967) concluded that dicarboxylation and defluorination occurs simultaneously when 2-fluorobenzoate is converted to catechol by a pseudomonad. Further support for this conclusion was obtained when the reaction was carried out in an atmosphere containing 50% laOzand 50%‘*Ox, since the catechol produced contained either 2 atoms of le0or 2 atoms of l8O, indicating a one-step reaction ( Milne et al., 1968).

2. Reductive Dehalogenation This mechanism of halogen removal has been demonstrated with numerous microorganisms in the conversion of DDT to DDD (TDE) : c1

c1

dl

Ci DDT

DDD (TDE)

The process was first described with microorganisms in studies with yeast (Kallman and Andrews, 1963), and subsequentIy with Escherichia coli (Mendel and Walton, 1966), Proteus vulgaris (Barker et al., 1965), Serratia marcescens ( Stenersen, 1965), soil actinomycetes, namely Nocardia erythropolis and five species of Streptomyces (Chacko et al., 1966), Aerobacter aerogenes ( Mendel et al., 1967), plant pathogenic and saprophytic bacteria (Johnson et al., 1967), and in soil samples under anaerobic conditions (Guenzi and Beard, 1967). Most of the studies provide evidence that anaerobic conditions favor reductive dechlorination over competing reactions. Wedemeyer (1966) isolated a cell-free system from Aerobacter

MICROBIAL TRANSFORMATION OF PESTICIDES

107

aerogenes which catalyzed, anaerobically, the reduction of DDT to DDD. Since the addition of 0.001 M cyanide or carbon monoxide completely inhibited the conversion, Wedemeyer suggested that reduced cytochrome oxidase is probably responsible for the reductive dechlorination. French and Hoopingarner ( 1970) obtained membrane fractions from Escherichia coli which also produced DDD under anaerobic conditions if flavine adenine dinucleotide (FAD) was added. The cytoplasmic factor, alone or in the presence of boiled membrane fraction, was completely inactive. Plimmer et al. (1968) demonstrated conclusively with deuterated DDT that DDD is the result of a direct reductive dechlorination which does not involve the formation of DDE as an intermediary metabolite. Retention of the deuterium atom in DDD excluded the possibility of dehydrohalogenation and subsequent reduction. One pathway of degradation of the insecticide heptachlor is caused by bacteria and actinomycetes and apparently proceeds by reductive dechlorination resulting in the formation of chlordene as an intermediate ( Miles et al., 1969). 3. Dehydrohalogenution

The simultaneous removal of a hydrogen and halogen was found especially with chlorinated hydrocarbon insecticides. The formation of DDE from DDT is perhaps the most familiar reaction in this group of pesticides :

DDT

DDE

In comparative studies of soil samples kept under anaerobic and aerobic conditions, it was found that DDT is rapidly converted to DDD in the absence of oxygen, whereas the transformation to DDE occurred aerobically and at a slow rate (Guenzi and Beard, 1968). In several cases the formation of TDE was accompanied, even anaerobically, by DDE, but on a much smaller scale (Stenersen, 1965; Mendel and Walton, 1966; Guenzi and Beard, 1967). Matsumura and Boush (1968) found that the majority of variants of the soil fungus Trichoderm viride produced TDE, while some variants produced only DDE; this indicates that different enzyme systems causing the degradation of DDT exist even among variants of the same species.

108

JEAN-MARC BOLLAG

Soil microorganisms appear to be responsible for the breakdown of lindane, and the isolation of 7-pentachlorocyclohexeneimplies a dehydrochlorination reaction (Yule et al., 1967). In the metabolism of chlorophenoxyacetates by soil pseudomonads it was shown that, after ring fission of 4-chloro-substitued phenoxyacetates, the formed chloromuconic acid derivative was lactonized by dehydrochlorination. For example, an enzyme preparation from a Pseudomonm sp. catalyzed the transformation of y-chloro-a-methylmuconic acid, an intermediate of MCPA, to 7-carboxymethylene-a-methyl-a"e-butenolide (Gaunt and Evans, 1971) :

Cl

The lactonizing enzyme, functioning simukaneously as a dehydrochlorinase, required Mn'+ or Mg2+ as cofactors and was stimulated by Fez+ and Co". Therefore, Gaunt and Evans (1971) concluded that it bears no resemblance to the DDT-dehydrohalogenating enzyme investigated by Lipke and Kearns (1959)) which had no cofactor requirement and also differed in other tests. The observation that a chloride is removed from a benzene ring oply after ring cleavage and the formation of a muconic acid was also established in the bacterial metabolism of 2,4-D and 4-chlorophenoxyacetic acid (Bollag et al., 196813; Tiedje et al., 1969; Evans et aZ., 1971a,b). Castro and Belser ( 1968) established that soil-water cultures dehydrohalogenated nematocidal soil fumigants. Ethylene dibromide was converted almost quantitatively to ethylene in sterilized soil which was inoculated with soil suspensions: BrCH2CH2Br .+ CHz=CHz

+ 2Br'

and meso- and dl-2,3-dibromobutane were transformed by soil-water suspensions to bromine and butene: B r q B r CH, meso

-

H&./--..CH,

109

MICROBIAL TRANSFORMATION OF PESTICIDES

Kearney et al. (1964) reported the isolation and partial purification of an enzyme from an Arthrobacter sp. that removed chlorine from dalapon resulting in the formation of pyruvic acid, and this reaction should probably also be categorized under dehydrohalogenation. It was not possible to isolate intermediates from this system and to make a clear conclusion concerning the mechanism involved, but it was proposed that 2-chloroacrylate and 2-chloro-2-hydroxypropionate are unstable intermediary products :

c1 I

CH,-C-COOH c I1

-I

C&=C-COOH dl

+

I

c1

-

0 ll CH,-C-COOH

The partially purified enzyme had its greatest activity on dalapon with less activity on 2-chloropropionate, dichloroacetate, and 2,2-dichlorobutyrate. No activity was detected on any p-chloro-substituted aliphatic acid. This observation differs from the isolated enzyme system of Micrococcus denitrificans which dehalogenates chlorinated aliphatic acids only with the halogen in the P-position (Bollag and Alexander, 1971). 3- and 4-Carbon acids-even unsaturated compounds like acrylic or crotonic acid-were dechlorinated only if the halide was in the p-position, but the dehalogenating enzyme system failed on the chlorinated acetic acids and on all other aliphatic acids with halogens solely on the @-carbon. There was more evidence that the enzyme preparation from M . denitrificans dechlorinated 2-chloropropionic acid via acrylic acid and not via 3-hydroxypropionic acid, indicating that the halogen is removed by dehydrohalogenation.

E. SYNTHETICREACTIONS In this category of enzyme reactions, the formation of a conjugate or condensate during pesticide metabolism is considered. A conjugation reaction implies the coupling of a pesticide, or an intermediate thereof, to an endogenous substrate resulting in the formation of, for example, methylated or acetylated compounds, amino acid conjugates, or glycosides, while the formation of a condensate implies the enzymatic condensation of a pesticide or an intermediate thereof. Williams (1971) stated that synthetic reactions require a source of energy that is usually supplied via adenosine triphosphate ( ATP ) . Several methylation reactions have been observed in the microbial metabolism of pesticides. A few examples are known in which O-methylation of chlorinated phenolic compounds took place (Fig. 2), and the

qcl

110

JEAN-MARC BOLLAG

cl$

\

c1

c1

c1

___)

\

c1

c1

cl+

c1

\

c1

c1

FIG.2. 0-methylation of 2,4-dichlorophenol, pentachlorophenol, and 2,5-dichloro4-methoxyphenol by various microorganisms.

methylation of heavy metals which are used as pesticides deserves special attention. Loos et al. ( 1967b) isolated 2,4-dichloroanisole during the metabolism of 2,4-D in the growth medium of an Arthrobacter sp., and they suspected that it might have been produced by an enzymatic 0-methylation of 2,4-dichlorophenol. Cserjesi and Johnson ( 1972) found that pentachlorophenol, a substance of fungicidal and various other pesticidal activities, can be methylated by the fungus Trichoderma viride in a growth medium, and the resulting product was identified by melting point determination and infrared spectroscopy as pentachloroanisole. The fungicide chloroneb was demethylated by numerous microorganisms to 2,5-dichloro-4-methoxyphenol,but some of the same microbes, especially Trichoderma viride and Mucor ramunnianus, could also reverse the reaction and methylate the dealkylated fungicide ( Wiese and Vargas, 1973). Both reactions could be shown to be independent if the two compounds were amended to a liquid basal medium. In addition, it was observed that some fungi could both methylate and demethylate 2,5-dichloro-4-methoxyphenol to produce chloroneb and 2,s-dichlorohydroquinone, respectively. A noteworthy observation relates to the finding that pentachlorothioanisole was a product during the metabolism of pentachloronitrobenzene by various species of Fusariurn oxysporurn ( Nakanishi and Oku, 1969). No enzymatic studies were reported on the methylation of the chlorinated phenol pesticides, and it can only be an assumption that the enzymes are comparable to the 0-methyltransferases which use S-adenosylmethionine as a methyl donor (Axelrod, 1971). Mercury fungicides that can cause serious poisoning effects have come under critical examination. Jernelov (1969) showed in a review that metallic mercury is oxidized chemically, but the subsequent conversion

MICROBIAL TRANSFORMATION OF PESTICIDES

111

of divalent inorganic mercury to methylmercury and dimethylmercury is caused by microorganisms : Hg

Chemical

Hgt2

Biologiral

CH3Hgf2

Biological

CHsHgCHS

Evidence for microbial methylation of mercury was presented by Wood et al. (1968)) who demonstrated that extracts of a Methanobacterium strain transferred the methyl group from methylcobalamin ( Co3+) to Hg2+.Yamada and Tonomura (1972) reported the methylation reaction of mercury in a pure culture of Clostridium cochlearium which was isolated from soil. McBride and Wolfe (1971) showed that under anaerobic conditions the same organisms also synthesize dimethylarsine from a variety of arsenic derivatives; adenosine triphosphate and hydrogen were found to be essential for this reaction with cell-free extract. In these studies it was also established that selenium and tellurium are readily methylated by a Methanobacterium sp. However, it should be pointed out that the transfer of methyl groups from Co3+to Hg2+may also occur as a nonenzymatic process (Imura et al., 1971; Bertilisson and Neujahr, 1971)) but it is enhanced by anaerobic conditions and by increasing numbers of bacteria capable of synthesizing alkylcobalamins ( Lezius and Barker, 1965; Wood and Wolfe, 1966). The formation of an arsenic gas compound generated by a fungus was already observed in the last century (Gosio, 1893), but only forty years later it was possible to correctly identify the volatile compound as trimethylarsine ( Challenger et aZ., 1933). Challenger described extensively in his review (1945) the ability of Scopulariopsis breuicaulis to methylate organic and inorganic forms of arsenic and other metalloids. Challenger et al. (1954) partially established the mechanism of methylation of arsenic. Arsenic-metabolizing microorganisms were isolated from soil and sewage and tentatively identified as Candida humicola, Gliocladium roseum, and a Penicillium species; these fungi formed trimethylarsine gas from monomethylarsonic acid and dimethylarsinic acid (COXand Alexander, 1973). An acetylation reaction in the metabolism of phenylurea herbicides was observed by Tweedy et al. (1970a,b). In many mammalian species acetylation is a common conjugation reaction especially for foreign aromatic amines ( Weber, 1971), but there is !ittle knowledge involving microorganisms using this process in pesticide transformation. The fungi Talaromyces wortmanii and Fusarium oxysporum metabolize metobromuron by demethylation and demethoxylation with the apparent, subseiuent acetylation of the aniline intermediate. p-Bromoaniline was not found as an intermediate, but it can be assumed that acetylation of the aniline is a fast process; consequently, it does not accumulate in

112

JEAN-MARC BOLLAG

the culture medium. If p-bromoaniline was used as a substrate, it was completely acetylated to p-bromoacetanilide by the two fungi tested as well as by a Bacillus sp. and Chlorella vulgaris (Tweedy et al., 1970a).

Likewise, p-chloroaniline was converted to p-chloroacetanilide in the growth medium of Fusarium oxysporum, but only approximately 3%of the parent aniline was acetylated (Kaufman et al., 1973). One report states that formylation of aniline in soil was detected as a transformation process ( Kearney and Plimmer, 1972); 3,4-dichloroformylanilide was identified as a product of 3,4-dichloroaniline, but the possible participation of microorganisms in this reaction was not examined. Conjugation reactions of sulfhydryl-containing compounds, with natural metabolites such as amino acids, has been shown to take place in vitro. Kaars Sijpesteijn et al. (1962), studying the transformation of dithiocarbamate fungicides, showed that when cell suspensions of various microorganisms were incubated with the sodium salt of dimethyldithiocarbamate, the compound was converted to 7 - ( dimethylthiocarbamoythio ) -a-aminobutyric acid and the corresponding keto acid:

(CH,),: N * C . S (CH,), II

.CO .COOH

S

The studies were performed with washed cell suspensions of Saccharomyces cerevisiae, Hansenula anomala, and Escherichia coli as well as mycelial pellets of Glomerella cingulata, Aspergillus niger, and Cladosporium cucumerinum; all these microorganisms produced at least one conjugate from dimethyldithiocarbamate. A sulfhydryl-oxidizing enzyme system which catalyzes the conversion of dithiocarbamates to the corresponding disuIfides was isolated from the cuIture filtrate of PiricuZaria oryzae and Polyporus versicolor (Neufeld et al., 1958). For instance, sodium diethyldithiocarbamate was oxidized by atmospheric oxygen to tetraethylthiuram disulfide: 2(CZHs)2N-C-SH

II

S

+ 3402

-+

(CZH~)~N-C-S-S-C-N(C~H~)~

II

S

I1

S

+ H2O

MICROBIAL TRANSFORMATION OF PESTICIDES

113

There is some concern related to aniline-based herbicides like phenylureas, phenylcarbamates, and acylanilides whose aniline intermediate product can be polymerized to an azo-derivative, a group of compounds with possible carcinogenic effects in animals (Weisburger and Weisburger, 1966). Bartha and Pramer (1967) reported first that soil treated with propanil, 3’,4’-dichloropropionanilide, produced as a major metabolite 3,3’,4,4’-tetrachloroazobenzene (Fig. 3, B ) . The synthesis of the azo compound was a result of microbial activity, since the condensation product was not detected in sterilized soil that received propanil or 3,4-dichloroaniline. Bartha et al. (1968) studied the ability of aniline and mono- and dichlorinated anilines to form azo compounds; aniline did not form a condensation product, but all monochloro- and some dichloroanilines were transformed to their corresponding dichloro- and tetrachloroazobenzenes. Of particular interest are reports describing the formation of asymmetric as well as symmetric azo compounds which are the result of different anilines added simultaneously to soil (Bartha, 1969; Kearney et al., 1969). Hybridization, between different substituted anilines released from propanil and solan, produced two symmetrically formed azobenzenes, 3,3’,4,4‘-tetrachloroazobenzene ( Fig. 3, B ) and 3,3‘-dichloro-4,4’-dimethylazobenzene, as well as the hybrid, 3,3‘,4-trichloro-4’methylazobenzene ( Bartha, 1969). Condensation of halogenated anilines can be further complicated as

Cl (C)

FIG.3. Formation of azobenzenes from 3,4-dichloroaniline.

114

JEAN-MARC BOLLAG

illustrated by the isolation of 1,3-bis( 3,4-dichlorophenyl)triazene ( Fig. 3, D), which apparently arises from the reaction of 3,4-dichloroaniline with nitrite to form an intermediate diazonium cation, which subsequently reacts with another molecule of free aniline to produce the triazene ( Plimmer et al., 1970). Another aniline condensation product is 4-( 3,4-dichloroanilino)-3,3’,4’-trichloroazobenzene ( Fig. 3, C ) which resulted from the addition of another 3,4-dichloroaniline molecule to the previously formed azobenzene ( Linke, 1970). A report by Daniels and Saunders (1953) described the synthesis of 4,4’-dichloroazobenzene from monochloroaniline by a peroxidase; therefore, it was assumed that an analogous enzyme system is also active in the soil. Bartha et al. (1968) demonstrated that there was a considerable similarity in the azobenzene condensation of various anilines by the selected soil and a horseradish peroxidase. It was also possible to extract peroxidase from soil which would catalyze the conversion of chloroanilines to chloroazobenzenes after addition of H,O, ( Bartha and Bordeleau, 1969). A pathway of chloroazobenzene formation was proposed by Bordeleau et al. (1972). They concluded from their studies that an initial attack of peroxidase produced a free chloroanilino radical, which was transformed to another labile intermediate, chlorophenylhydroxylamine, which condensed spontaneously with excess chloroaniline and formed chloroazobenzene. This reaction sequence was suggested as the main pathway, although another pathway could also be anticipated. IV.

Chemical Structure and Microbial Transformation Relationship

The concern over the persistence of a pesticide, or a derivative of it, and the related probable toxic hazard in the environment evoked much speculation on the ability of microbial organisms to transform such a xenobiotic compound. It is self-evident that an experimental approach contributes much to elucidate this problem, but the innumerable possibilities of chemical structures to which a microorganism can be exposed makes it unreal to test all possible transformations of each compound under the various conditions. Therefore, it is desirable to know the possible avenues of microbial attack in relation to specific molecular configuration. Acquaintance with enzymatic reactions in the metabolism of investigated pesticides or other compounds helps to anticipate certain chemical changes; on the other hand, it has been recognized that factors like cellular permeation of the chemical and its steric and electronic characteristics influence microbial activity. A new approach, combining chemical reactivity with substituent parameters and understanding of the multiconditional character of structure-activity or structure-degrad-

MICROBIAL TRANSFORMATION OF PESTICIDES

115

ability relationships using regression analysis, was initiated and developed by Hansch and other authors (Hansch, 1969, 1971; Verloop, 1972). It was shown that hydrophobic, steric, and electronic factors could be used to formulate mathematical structure-activity relationships, and the value of the equations has been tested on numerous bioactive compounds. Publications related to the application of the Hansch approach have been numerous in recent years (Verloop, 1972), but specific investigations on the relationships between microbial enzymes and transformation of a chemical structure have not yet been elaborated. From practical experience and from laboratory tests some general conclusions can be made concerning the probable microbial transformation of certain groups of pesticides and the possible enzymatic attack of certain linkages or substitutions.

A. MICROBIALTRANSFORMATION OF PESTICIDAL GROUPS There have been relatively few systematic studies designed to obtain data relating the chemical structure of pesticides to their probable microbial transformation. Most investigations in the past focused only on those microorganisms that could be isolated and were able to use the pesticidal molecules as a source of carbon, nitrogen, phosphorus, sulfur, or energy, or as a combination of these. The phenomenon of cometabolism, for instance, was not taken into consideration. It must be emphasized that laboratory conditions, using axenic cultures, do not permit the observation of the combined activity of various microbial species, as it has to be anticipated in an ecosystem with its inherent microflora. The same is true if metabolism would occur by auxotrophic microorganisms that require for their proliferation specific conditions that are not provided in general screening experiments. Consequently, investigations on the structure-biodegradability relationship have to be critically evaluated, but indications in the laboratory often have been shown to be compatible with the applied experience in pesticide use. In addition, the knowledge acquired on the biochemical or enzymatic processes-as outlined in Section III-provides the actual reactions that occur during the transformation of individual chemical groups of pesticides. From these results it is possible to establish a relationship to the persistence of the various pesticidal groups (Table 111). Pesticides which are attacked initially by a hydrolytic mechanism, like phenylcarbamates or organophosphates, are relatively short-lived in soil, whereas pesticidal groups which are dealkylated by a primary reaction appear to be more persistent. Halogenated alkanoic acids undergo dehalogenation and persist a relatively short time, but haloge-

TABLE 111 PERSISTENCE OF PESTICIDES IN SOILA N D ENZYMATIC TRANSFORMATION REACTIONS Enzymatic transformations by microorganisms Example

Pesticidal group Herbicides Halogenated alkanoic acids

c1 0 I II €I -C-C & -OH I

c1

Period of persistence in soil

2 to 8 Weeks

Primary reactions

Subsequent reactions

Dehalogenation

Dalapon

5'

s-Triazines

18 Months

N-Dealkylation, dehalogenation

Deamination

4-18 Weeks

poxidation, cleavage of ether linkage, ring hy droxylation

Ring hydroxylation, ring cleavage

4-15 Months

N-Dealkylation, hydrolysis

Hydrolysis, acetylation, condensation reaction

Simazine

Phenoxyalkanoic acids

O--CH,--COOH QC'

Cl

2.4-D

Phenylureas

H O I II N-C-N,

,CH,

Cl

Linuron

F*

0

Phenylcarbamates

H

O I It N-C-0-C

H,CHS

7 Weeks

Ester hydrolysis

Hydroxylation, ring cleavage

'CH,

dl

-zB

CIPC

Acylanilides

'::: N-c-cH&H,

Hydrolysis

Condensation reaction, acetylation

8 $

Cl

Propanil

Benzoic acids

> 6 Weeks

bC1

0

Decarboxylation, dehalogenation

Y

NH*

c1

Amiben

(Continued)

w F a,

TABLE I11 (Continued) Enzymatic transformations by microorganisms Pesticidal group

Example

Period of persistence in soil

Primary reactions

Subsequent reactions ~ ~

~

Insecticides Organophosphates

Ring cleavage

Diazinon

Halogenated hydrocarbons

>9 Years

Epoxidation, dehalogenation

Hydrolysis

2-8 Weeks

Side-chain hydroxylation, ring hydroxylation, hydrolysis

Hydrolysis, ring cleavage

Sulfoxidation

Hydrolysis

Heptachlor

Methylcarbamates

Carbaryl

Fungicides Thiocarbamates

yL HsC--S -C -C=N-O-C-N-CH, I 1 I/ I H,C H O H Temik

_

_

MICROBIAL TRANSFORMATION OF PESTICIDES

119

nated hydrocarbons with a more complex molecular configuration, like heptachlor, lindane, or DDT, persist for extended time periods. Generally, it was found that certain linkages in pesticides are readily susceptible to cleavage, and the rate at which these linkages cleave depend on the characteristics of the remaining molecule. Such observations indicate some general trends in the biodegradability of pesticide groups, but it must be stressed that numerous environmental conditions can interfere in the availability of a pesticide to microbial attack.

B. EFFECTOF VARIOUSSUBSTITUTIONS ON BIODEGRADABILITY Minor alterations in the structure of pesticide molecules frequently cause a drastic change in the susceptibility of such compounds to biotransformation. Introduction of polar groups, such as OH, COOH, NO2, and others, often affords microbial systems a site of attack, while others such as halogen or alkyl substitutions make a molecule more resistant. The rate of a reaction is also strongly influenced by steric and electronic factors of other atoms in the molecule. Generally, it can be stated that the type, the number, and the position of substitutions affect the rate of microbial decomposition of organic compounds. Which of these three factors is most influential depends upon the organic compounds studied. Investigations on this topic related to pesticide degradation were concerned especially with the influence of halogens in aliphatic acid herbicides and with the effect of substitution in the benzene ring. Experiments using sewage microorganisms, for instance, confirmed that unsubstituted aliphatic acids are degraded readily, but the rate of decomposition was much slower with substituted acids as substrates ( Dias and Alexander, 1971). A single halogen substitution, particularly if on the a-carbon, makes the molecule less susceptible to attack, and dihalogenated compounds were even more resistant to biodegradation. Jensen (1959) found that strains of Trichoderma viride, Clonostachys sp., and Acrostalagmus sp. degraded monochloroacetate more rapidly than dichloroacetate, but trichloroacetate was not attacked by these fungi. Many similar studies also indicated that increasing the number of halogen substitutions increased the resistance of a molecule to biodegradation. It was also shown that the rate of decomposition depends on the specific halogen substituent, i.e. chlorine, bromine, fluorine, or iodine, but no general conclusion concerning the possible attack by various organisms could be drawn ( Hirsch and Alexander, 1960). Introduction of substituents on a benzene ring influences its degradation considerably, and since many pesticides have an aromatic ring as an essential part of the molecule, its substitution is a determining factor for resistance to biodegradation. Systematic surveys of the effect of

120

JEAN-MARC BOLLAG

chemical structure on the microbial degradation of substituted benzenes have their shortcomings, since specific test conditions have to be selected. Results from various studies-with compounds that were not necessarily pesticidal-showed a certain agreement and trend related to the influence of the position of the substituent on the aromatic ring and its chemical nature. Kameda et al. (1957) found that not one of 34 soil pseudomonads was capable of degrading meta isomers of nitro-, amino-, and methoxybenzoates, but some could use the corresponding ortho- and para-substituted molecule. Likewise, Alexander and Lustigman ( 1966) determined that, in studies with a mixed soil microflora using an ultraviolet spectrophotometric assay to follow the destruction of aromaticity, meta isomer substitutions of various groups were almost invariably degraded more slowly than the ortho- or para-substituted analogs. In experiments with different monosubstituted compounds, they showed that phenol and benzoate were degraded rapidly, aniline and anisole were attacked less readily, and benzenesulfonate and especially nitrobenzene appeared to be most resistant to microbial transformation. With aromatic compounds containing two substituents, carboxyl and phenolic hydroxyl groups favored microbial degradation of the molecule while other groups, such as chloro, nitro, and sulfonate substitutions, reduced rates of metabolism. It should be pointed out that these experiments were performed while the test compound was supplied as the sole carbon source and cometabolic transformations were not considered. The effect of the chemical structure on the persistence of chlorinated phenoxyalkanoate herbicides was summarized by Alexander ( 1965b). He concluded that the type of linkage of the aliphatic acid to the ring, and the position-in this case not the number of chlorines-determines the persistence of these pesticides. Compounds containing a chlorine in the meta position are not metabolized to a significant extent, and substances which have the ring linked to the aliphatic side chain at the alpha position are more resistant to degradation. Similar observations were made with chlorophenols which have fungicidal activity. Also, CartWright and Cain (1959) reported that organisms could easily be isolated for growth on 0- and p-nitrobenzoic acids but with difficulty on the meta-substituted derivative. On the other hand it was determined that microbial degradation of chlorinated N-phenylcarbamates in soil perfusion studies was more rapid if substitution occurred at the meta-position than with the ortho- or para- substituted compounds ( Kaufman, 1966). However, Kearney ( 1967), studying the influence of physicochemical properties of phenylcarbamates that influence hydrolysis by a microbial enzyme, found that the isopropyl ester of p-nitrophenyl carbamate is hydrolyzed considerably faster than the corresponding meta compound. In addition, it was deter-

MICROBIAL TRANSFORMATION OF PESTICIDES

121

mined that reaction rates of hydrolysis decreased with the following meta-.substituents on the ring: NO, > CH,CO > CI > CH,O > H. The size of the molecule also has an effect on enzymatic hydrolysis as indicated by the faster degradation of the isopropyl ester of phenyl as compared to the 2-naphthylcarbamic acid. J.-M. Bollag, N. M. Henninger, and B. Bollag (unpublished data, 1973) found that the fungus Rhixoctonia solani metabolized chlorinated and brominated anilines most rapidly if the halogen was substituted in the para position; mta-substituted anilines were transformed slower and a substitution in the orthoposition proved to be most resistant to fungal attack. Studies on the persistence of DDT with a Hydrogenomonas sp. revealed that especially the para-chloro substitution and substituents on the methylene-carbon governed the resistance of the DDT molecule to microbial metabolism (Focht and Alexander, 1970). Many other examples of specific investigations which indicate a change of resistance to microbial transformation by a simple substitution in a pesticidal molecule could be cited, but much more research needs to be done, hopefully also by use of the Hansch approach, to be able to generalize in clearer terms the effect of small molecular alterations in a specific chemical group. No rules that are generally applicable can yet be identified, but certain trends could be established that influence biodegradability. It can also be stated that it is not possible to find a relationship between the effects of chemical structure of pesticides on toxicity to a target organism and the effects of a molecule on its microbial degradability. This problem has to be evaluated for each pesticidal group or even each single compound; this indicates the complexity for developing from theoretical considerations and experimental data a useful and practical pesticide. AND PESTICIDE TRANSFORMATION C. MOLECULARRECALCITRANCE

Alexander (1965a) attributed two main causes to the recalcitrance of chemicals: ( a ) environmental conditions not conducive to microbial ability to change a certain molecule and ( b ) the structural configuration of a compound, which makes it either totally or partially resistant to biodegradation under all circumstances. Whereas the first parameter for a compound as nonbiodegradable is generally accepted, the second cause cited often arouses criticism if the definition is considered from a basic scientific point of view. While Alexander (1965a) states that “every biologically synthesized organic molecule doubtlessly will, under some set of circumstances, be destroyed by one or several species,” he doubts that all synthetic organic compounds, which are increasingly produced

122

JEAN-MARC BOLLAG

and discharged into the various ecosystems, can be biodegraded. There is no doubt that, for instance, certain pesticidal groups are difficult to attack by microorganisms, and they-or a derivative of them-may persist for a considerable length of time in the environment. Although in several cases it appeared that a pesticide is nontransformable biologically, this finding had to be corrected when on-going research discovered that a pesticidal molecule could be metabolized, at least partially, under certain environmental conditions and by specific organisms. Are there really synthetic compounds that cannot be altered by microorganisms after mutational or nongenetic adaptation if there is a need for it? Presently, for instance, the question of possible biological transformation of synthetic polymers with a high molecular weight is yet unresolved, and it presents a justified practical concern related to the pollution of the environment. It appears that whether there are synthetic chemicals intrinsically resistant to biological degradation raises an academic question whose answer may, with synthesis of new chemicals, always be delayed.

V.

Conclusions

Microbial and biochemical processes affecting the fate and behavior of pesticides have been investigated essentially in model systems using isolated microbial cultures or enzyme systems. This appears-with the presently available techniques-to be the only feasible approach, if one is interested in elaborating the mechanism of pesticide transformation, clarifying the actual microbial activity by isolation and identification of intermediates, and establishing the rates at which these processes occur. With respect to pollution, the transformation of a xenobiotic compound should not be a matter of conjecture, since it is important to know the fate of the original pesticide as well as the resulting transformation products. The clarification of the extent to which microorganisms interfere and transform introduced chemicals or their decomposition products should help in determining the potential hazard of their use. It is also necessary to keep in mind that under various conditions or in different ecosystems, a chemical can be transformed by different metabolic pathways or organisms and consequently, the resulting product can vary. The knowledge of enzymatic reactions in the metabolism of pesticides or their identified intermediates should contribute to understanding transformation possibilities of newly developed compounds. This general problem, which is related to the molecular configuration and the resistance to microbial attack, needs far more research for pertinent and applicable conclusions, i.e., for the development of new pesticides which

123

MICROBIAL TRANSFORMATION OF PESTICIDES

have the desired toxic activity and are simultaneously susceptible to adequate microbial metabolism. One must be aware of the difficulty in extrapolating from experiments and results obtained in vitro to the complex environment of a natural habitat. Isolated organisms which alter a pesticide in pure culture conditions are not necessarily those responsible for its transformation in uiuo. However, it appears that basic laboratory studies are a prerequisite for establishing the possibilities of microbial pesticide transformations in a natural environment. PESTICIDlC3

Common or trade name Aldicarb Aldrin Amit,role Atrazine Brornophos Buturon Carbaryl Chlorbromuron Chlorobeneilate Chloroneb Chloroxuron Chlorpropham CIPC 2,4-1) I1a1apo n DDA 1IIIII (TIIE) 1lDE IIDT Iliazinon Dicamba Ilicofol ilicryl Dieldrin Dinitramine Diphenamid Dipterex Diuron

TABLE I V MENTIONICD I N 'PHI', TEXTAND THEIR CHEMICAL

I)ESIGNATION

Chemical designation 2-Methyl,-2-(methylthio)propionaldehyde0-(methylcarbamoy1)oxime 1,2,3,4,10,l0-Hexachloro-1,4,4aJ5,8,8a-hexahydro-1,4-endo,eso-5,8diniethanonaphthalene %Amino- 1,2,4-tariazole 2-Chloro-4-(ethylamino)-6- (isopropy1amino)-s-triazine 0-(4-Bromo-2,5-dichlorophenyl) 0,O-dimethylphosphorothioate 3-(p-Chlorophenyl)- 1-methyl- 1-( 1-methyl-2-propyny1)urea 1-Naphthyl N-methylcarbamate 3- (3-Chloro-4-bromophenyl)-l-methoxy-l-methylurea Ethyl 4,4'-dichlorobenzilate 1,4-Dichloro-2,5-dimethoxybenzene 3-[4-(p-Chlorophenoxy)phenyl]-l, 1-dimethylurea See CIPC Isopropyl N-(3-chlorophenyl) carbamate 2,4-Dichlorophenoxyacetic acid 2,2-Dichloropropionic acid 2,2-Bis(p-chlorophenyl)aceticacid 2,2-Bis(p-chlorophenyI)- 1,l-dichloroetharie 2,2-Bis (p-chloropheny1)- 1,1-dichloroethene 2,2-Biu (p-chloropheny1)-1, 1,1-trichloroethane 0,O-Diethyl 0-(2-isopropy1-4-methyl-6-pyrimidinyl) phosphorothioate 3,6-L)ichloro-o-anisic acid 1,l-bis (p-~hlorophenyl)-2,2,2-trichloroethanol N-(3,4-IIichlorophenyl) methacrylamide 1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahy~ro1,4-endo,exo-5,8-dimethanonaphthalene N3,N~-r)iethyl-2,4-dinitro-6-trifluoromethyl-m-phenylenediamine N,N-Dimethyl 2,2-diphenylacetamide See Trichlorfon 3. (3,4-lXchlorophenyl)-1,l-dimethylurea (Continued)

124

JEAN-MARC BOLLAG

TABLE I V (Continued) Common or trade name

DNOC Fenitrothion Fenuron Fluometuron Griseofulvin Heptachlor Ioxynil Isodrin Karsil Lindane Linuron Malathion MCPA Methoxychlor Metobromuron Monolinuron Monuron Par aoxon Paraquat Parathion PCNR Phorate Prometryne Propanil Simazine Solan Sumithion 2,4,5-T 2,3,6-TBA TDE Temik TEPP Thimet Trichlorfon Trifluralin

Chemical designation 3,5-Dinitro-o-cresol See Sumithion l,l-Dimethyl-3-phenylurea l,l-I~imethyl-3-(~,or,a-trifluoro-m-tolyl)urea 7-Chloro-4: 6 :2’-trimethoxy-6’-methylgris-2’-ene-3 :4’-dione 1,4,5,6,7,R,8-Heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene 4-Hydroxy-3,5-diiodobenzonitrile 1,2,3,4,10,10-Hexachloro1,4,4a,.5,8,8a-hexahydi-ol,Cendo,endo5,s-dimethanonaphthalene N-(3,4-lXchlorophenyl) 2-methylpentanamide y- 1,2,3,4,.~,6-Hexschlorocyclohexane 3-(3,4-I~ichlorophenyl)-l-methoxy-l-methylurea 0,O- Dimethyl S-bis (carboethox y)ethyl phosphorodi thioate 4-Chloro-Zmethylphenoxyacetic acid 2,2-Ris(p-methoxyphenyl)-l, 1,l-trichloroethane 3- (p-Bromopheny1)- 1-methoxy- 1-methylurea 3-(p-Chlorophenyl)-l-melhoxy-1-methylurea 3-(p-Chlorophenyl)-l,1-dimethylurea 0,O-diethyl-0-p-nitrophenyl phosphate 1,l’-Dimethyl 4,4’-bipyridinium salt 0,O-Diethyl 0-p-nitrophenyl phosphorothioate Pentachloronitrobenzene 0,O-Diethyl S-(ethylthiomethyl) phosphorodithioate 2,4-Bis (isopropylamino)-6-methylthio-s-triazine 3‘,4’-Dichloropropionanilide ZChloro-4,6-bis(ethylarnino)-s-triazine N-(3-Chloro-4-methylphenyl)-2-methylpentanamide 0,O-Dimethyl 0-(3-methyl-4-nitrophenyl)phosphorothionate 2,4,5-Trichlorophenoxyacetic acid 2,3,6-Trichlorobenzoic acid See D D D 2-Methyl-2-(mrthylthio) propionaldehyde-l 0-(methylcarbamoyl) oxime Tetraethyl pyrophosphate See Phorate 0,O-Dimethyl 2,2,2-trichloro- 1-hydroxyethyl phosphonate ol,a,a-Trifluoro-~,6-dinitro-N,~-dipropyl-p-toluidine

REFERENCES Ahmed, M. K., and Casida, J. E. (1958). J. Econ. Entomol. 51,59. Alexander, M. (1965a). Aduan. Appl. Microbiol. 7, 35. Alexander, M. ( 196513). Soil Sci. SOC. A m r . , Proc. 29, 1. Alexander, M. (1967). In “Agriculture and the Quality of Our Environment,” Publ. No. 85, pp. 331-342. Amer. Ass. Advan. Sci., Washington, D.C. Alexander, M. (1969). In “Soil Biology,” pp. 209-240. UNESCO, Paris. Alexander, M., and Lustigman, B. K. ( 1966). J. Agr. Food Chem. 14,410. Axelrod, J. (1956). Biochem. J. 63, 634.

MICROBIAL TRANSFORMATION OF PESTICIDES

125

Axelrod, J. ( 1971). In “Handbuch der experimentellen Pharmakologie” ( B. B. Brodie and J. R. Gillette, eds.), Vol. 28, Part 11, pp. 609-619. Springer-Verlag, Berlin and New York. Barker, P. S., Morrison, F. O., and Whitaker, R. S. (1965). Nature (London) 205, 621. Bartha, R. (1969). Science 166, 1299. Bartha, R., and Bordeleau, L. M. (1969). Soil Biol. G Biochem. 1, 139. Bartha, R., and Pramer, D. (1967). Science 156, 1617. Bartha, R., Linke, H. A. B., and Pramer, D. (1968). Science 161, 582. Bernhardt, F.-H., Ruf, H. H., Staudinger, H., and Ulrich, V. ( 1971). Hoppe Seyler’s 2. Physiol. Chem. 352, 1091. Bertilisson, L., and Neujahr, H. Y. ( 1971). Biochemistry 10, 2805. Bocks, S. M., Lindsay-Smith, J. R., and Norman, R. 0. C. (1964). Nature (London) 201, 398. Bollag, J.-M., and Alexander, M. (1971). Soil Biol. G Biochem. 3, 91. Bollag, J.-M., and Liu, S.-Y. ( 1971). Soil Bio2. 6 Biochem. 3, 337. Bollag, J.-M., and Liu, S.-Y. ( 1972a). Nature (London) 236, 177. Bollag, J.-M., an,d Liu, S.-Y. (1972b). Can. J. Microbiol. 18, 1113. Bollag, J.-M., Helling, C. S., and Alexander, M. (1967). Appl. Microbiol. 15, 1393. Bollag, J.-M., Helling, C. S., and Alexander, M. (1968a). J. Agr. Food Chem. 16, 826. Bollag, J.-M., Briggs, G. G., Dawson, J. E., and Alexander, M. ( 196813). J . Agr. Food Chem. 16, 829. Boothroyd, B., Napier, E. J,, and Somerfield, G. A. (1961). Biochem. J. 80, 34. Bordeleau, L. M., Rosen, J. D., and Bartha, R. (1972). J. Agr. Food Chem. 20, 573. Mrner, H. ( 1967). Z. Pflan~enkr.(Pflanzenpathol.) Pflanzenschutz 74, 135. Braunberg, R. C., and Beck, V. (1968). J . Agr. Food Chem. 16, 451. Byrde, R. J. W., and Woodcock, D. ( 1957). Bioclzep. J. 65, 682. Cartwright, N. J., and Cain, R. B. (1959). Biochem. J. 71, 248. Cartwright, N. J., and Smith, A. R. W. (1967). Biochem J. 102, 826. Cartwright, N. J., Holdom, K. S., and Broadbent, D. A. (1971). Microbios 3, 113. Castro, C. E., and Bartnicki, E. W. (1965). Biochim. Biophys. Acta 100, 384. Castro, C. E., and Belser, N. 0. (1968). Enuiron. Sci. Techno2. 2, 779. Chacko, C . I., and Lockwood, J. L. ( 1967). Can. J. Microbial. 13, 1123. Chacko, C. I., Lockwood, J. L., and Zabik, M. (1966). Science 154, 893. Challenger, F. ( 1945). Chem. Reu. 36, 315. Challenger, F., Higginbottom, C., and Ellis, L. (1933). J. Chem. SOC. London p. 95: Challenger, F., Lisle, D., and Dransfield, P. (1954). J. Chem. SOC., London p. 1760. Chapman, P. J. (1972). I n “Degradation of Synthetic Organic Molecules in the Biosphere,” pp. 17-55. Nat. Acad. Sci. Washington, D.C. Chisaka, H., and Kearney, P. C. (1970). J . Agr. Food Chem. 18,854. Clifford, D. R., and Woodcock, D. (1964). Nature (London) 203, 763. Coppedge, J. R., Lindquist, D. A., Bull, D. L., and Dorough, H. W. ( 1967). J . Agr. Food Chem. 15,902. Couch, R. W., Gramlich, J. V., Davis, D. E., and Funderburk, H. H. ( 1965). Proc. S . Weed Conf. 18, 623.

126

JEAN-MARC BOLLAG

Cox, D. P., and Alexander, M. (1973). Bull. Enuiron. Contam. Toxicol. 9, 84.

Crosbi, D. G., and Li, M.-Y. ( 1969). In “Degradation of Herbicides” (P. C. Kearney and D. D. Kaufman, eds.), pp. 321363. Dekker, New York. Cserjesi, A. J., and Johnson, E. L. (1972). Can. J . Microbiol. 18, 45. Dagley, S . (1971). Adoan. Microbial Physiol. 6, 1. Dalton, R. L., Evans, A. W., and Rhodes, R. C. (1966). Weeds 14, 31. Daly, J. ( 1971). I n “Handbuch der experimentellen Pharmakologie” (B. B. Brodie and J. R. Gillette, eds.), Vol. 28, Part 11, pp. 285-311. Springer Verlag, Berlin and New York. Daly, J. W., Guroff, G., Udenfriend, S., and Witkop, B. (1968). Biochem. Pharmucol. 17, 31. Daly, J. W., Jerina, D. M., and Witkop, B. ( 1972). Experientia 28, 1129. Daniels, D. G. H., and Saunders, B. C. (1953). J . Chem. SOC., London p. 822. Dias, F. F., and Alexander, M. ( 1971). Appl. Microbiol. 22, 114. Engelhardt, G., Wallnofer, P. R., and Plapp, R. (1971). Appl. Microbiol. 22, 284. Engelhardt, G., Wallniifer, P. R., and Plapp, R. (1972). Appl. Microbiol. 23, 664. Evans, W. C. (1969). In “Fermentation Advances” (D. Perlman, ed.), pp. 649-687. Academic Press, New York. Evans, W. C., Gaunt, J. K., and Davies, J. I. (1963). Abstr. Commun., Int. Congr. Biochem., 5th, 1961. p. 364. Evans, W. C., Smith, B. S. W., Moss, P., and Femley, H. N. (1971a). Biochem. J . 122, 509. Evans, W. C., Smith, B. S. W., Femley, H. N., and Davies, J. I. (1971b). Biochem. J . 122, 543. Faulkner, J. K., and Woodcock, D. (1961). J. Chem. Soc., London p. 5397. Faulkner, J. K., and Woodcock, D. ( 1964). Nature (London) 203, 865. Faulkner, J. K., and Woodcock, D. (1965). J . Chem. Soc., London p. 1187. Focht, D. D., and Alexander, M. ( 1970). Science 170, 91. Focht, D. D., and Alexander, M. (1971). J . Agr. Food Chem. 19, 20. Foster, J. W. (1962). In “The Oxygenases” ( 0 .Hayaishi, ed.),pp. 241-271. Academic Press, New York. French, A. L., and Hoopingamer, R. A. (1970). J . Econ. Entomol. 63, 756. Funderburk, H. H., and Bozarth, G. A. ( 1967). J . Agr. Food Chem. 15,563. Funderburk, H. €I., Schultz, D. P., Negi, N. S., Rodriguez-Kabana, R., and CurI, E. A. ( 1967). Proc. S. Weed Conf. 20, 389. Gamar, Y., and Gaunt, J. K. ( 1971). Biochem. J . 122, 527. Gaunt, J. K., and Evans, W. C. (1971). Biochem. J . 122,533. Geissbuhler, H. (1969). In “Degradation of Herbicides” ( P . C. Kearney and D. D. Kaufman, eds.), pp. 79-111. Dekker, New York. Geissbiihler, H., Haselbach, C., Aebi, H., and Ebner, L. (1963). Weed Res. 3, 277. Getzin, L. W., and Chapman, R. K. ( 1960). J . Econ. Entomol. 53, 47. Goldman, P. (1965). J . Biol. Chem. 240, 3434. Goldman, P., and Milne, G. W. A. (1966). J. Biol. Chem. 241, 5557. Goldman, P., Milne, G. W. A., and Pignataro, M. T. (1967). Arch. Biochem. Biophys. 118, 178. Gosio, B. (1893). Arch. Ital. Biol. 18, 253. Gram, T. E. ( 1971). In “Handbuch der experimentellen Pharmakologie” (B. B. Brodie and J. R. Gillette, eds.), Vol. 28, Part 11, pp. 334-348. Springer-Verlag, Berlin and New York. Gregory, W. W., Reed, J .K., and Priester, L. E. (1969). J . Protozoal. 16, 69.

MICROBIAL TRANSFORMATION OF PESTICIDES

127

Guenzi, W. D., and Beard, W. E. ( 1967). Science 156, 116. Guenzi, W. D., and Beard, W. E. (1968). Soil Sci. SOC. Amer., Proc. 32, 522. Cunner, H. B., and Zuckerman, B. M. (1968). Nature (London) 217, 1183. Curoff, G., Kondo, K., and Daly, J. (1966). Biochem. Biophys. Res. Commun. 25, 622. Guroff, G., Daly, J. W., Jerina, D. M., Renson, J., Witkop, B., and Udenfriend, S. (1967). Science 157, 1524. Gutenmann, W. H., Loos, M. A., Alexander, M., and Lisk, D. J. (1964). Soil Sci. SOC. Amer., Proc. 28, 205. Hamdi, Y. A., and Tewfik. M. S. (1970). Soil Biol. G Biochem. 2, 163. Hansch, C. (1969). Accounts Chem. Res. 2, 232. Hansch, C. ( 1971). In “Drug Design” ( E . J. Ariens, ed.), Vol. 1, pp. 271-342. Academic Press, New York. Helling, C. S., Bollag, J.-M., and Dawson, J. E. ( 1968). J . Agr. Food Chem. 16, 538. Helling, C. S., Kearney, P. C., and Alexander, M. (1971). Adoun. Agron. 23, 147. Henderson, M. E. K. (1957). J. Gen. Microbiol. 18,686. Hirsch, P., and Alexander, M. (1960). Can. J . Microbiol. 6, 241. Hock, W. K., and Sisler, H. D. (1969). J . Agr. Food Chem. 17, 123. Horvath, R. S. ( 1970a). Bull. Enuiron. Contum. Toxicol. 5, 537. Horvath, R. S. (1970b). Biochem. 3. 119,871. Horvath, R. S. (1971). J . Agr. Food Chem. 19, 291. Horvath, R. S. (1972). Bacteriol. Reu. 36, 146. Imura, N., Sukegawa, E., Pan, S.-K., Nagao, K., Kim, J.-Y.,Kwan, T., and Ukita, T. (1971). Science 172, 1248. Jensen, H. L. (1959). Actu Agr. Scund. 9, 421. Jensen, H. L. (1963). Actu Agr. Scund. 13, 404. Jerina, D., Daly, J., Witkop, B., Zaltzman-Nirenberg, P., and Udenfriend, S. (1969). Biochemistry 9, 147. Jemelov, A. ( 1969). In “Chemical Fallout: Current Research on Persistent Pesticides” (M. W. Miller and G. G. Berg, eds.), pp. 68-74. Thomas, Springfield, Illinois. Johnson, B. T., and Kennedy, J. 0.(1973). Appl. Microbiol. 26, 66. Johnson, B. T., Goodman, R. N., and Goldberg, H. S. ( 1967). Science 157,560. Johnston, H. W., Briggs, G. G., and Alexander, M. (1972). Soil Biol. G Biochem. 4, 187. Kaars Sijpesteijn, A., Kaslander, J., and van der Kerk, G. J. M. (1962). Biochim. Biophys. Actu 62, 587. Kallman, B. J., and Andrews, A. K. ( 1963). Science 141, 1050. Kameda, Y., Toyoura, E., and Kimura, Y. (1957). Kanuzawa Duiguku Yukugukubu Kenkyu Nempo 7, 37. Kaufman, D. D. (1966). In “Pesticides and Their Effects on Soils and Water,” Symp. Pap., Spec. Publ. No. 8, pp. 85-94. Soil Sci. SOC.Amer., Inc., Madison, Wisc. Kaufman, D. D., and Blake, J. (1970). Soil. Biol. G Biochem. 2, 73. Kaufman, D. D., and Kearney, P. C. (1970). Residue Reu. 32,235. Kaufman, D. D., Plimmer, J. R., and Klingebiel, U. I. (1973). J. Agr. Food Chem. 21, 127. Kaufman, S., Bridgers, W. F., Eisenberg, F., and Friedman, S. ( 1962). Biochem. Biophys. Res. Commun. 9, 497. Kearney, P. C. (1965). J . Agr. Food Chem. 13,561. Kearney, P. C. ( 1967). J . Agr. Food Chern. 15,568. Kearney, P. C., and Plimmer, J. R. (1972). J . Agr. Food Chem. 20,584. Kearney, P. C., Kaufman, D. D., and Beall, M. L. (1964). Biochem. Biophys. Res. Commnun. 14, 29.

128

JEAN-MARC BOLLAG

Kearney, P. C., Kaufman, D. D., and Sheets, T. J. (1965). J . Agr. Food Chem. 17, 1418. Kearney, P . C., Plimmer, J. R., and Guardia, F. B. (1969). J . Agr. Food Chem. 17, 1418. Keil, J. E., and Priester, L. E. ( 1969). Bull. Enuiron. Contam. Toxicol. 4, 169. Kesner, C. D., and Ries, S. K. ( 1967 ). Science 155, 210. Knuesli, E., Berrer, D., Dupuis, G., and Esser, H. (1969). In “Degradation of Herbicides” (P. C. Kearney and D. D. Kaufman, eds.), pp. 51-78. Dekker, New York. KO, W. H., and Farley, J. 0.(1969). Phytopathology 59, 64. KO, W. H., and Lockwood, J. L. (1968). Can. J . Microbiol. 14, 1075. Korte, F., Ludwig, G., and Vogel, J. (1962). Justus Liebigs Ann. Chem. 656, 135. Laanio, T. L., Kearney, P. C., and Kaufman, D. D. ( 1973). Pestic. Biochem. Physiol. 3, 271. Langlois, B. E. (1967). J . Dairy Sci. 50, 1168. Lanzilotta, R. P., and Pramer, D. (1970). Appl. Microbiol. 19, 307. Leshniowsky, W. O., Dugan, P. R., Pfister, R. M., Frea, J. I., and Randles, C. 1. (1970). Science 169, 993. Lezius, A. G., and Barker, H. A. (1965). Biochemistry 4, 510. Lichtenstein, E. P., and Schulz, K. R. (1964). J . Econ. Entomol. 57, 618. Linke, H. A. B. ( 1970). Naturwissenschaften 57, 307. Lipke, H., and Kearns, C. W. (1959). J . Biol. Chem. 234, 2129. Liu, S.-Y., and Bollag, J.-M. (1971a). J . Agr. Food Chem. 19, 487. Liu, S.-Y., and Bollag, J.-M. (1971b). Pestic. Biochem. Physiol. 1, 366. Loos, M. A. ( 1969). In “Degradation of Herbicides” (P. C . Kearney and D. D. Kaufman, eds.), pp. 1-49. Dekker, New York. Loos, M. A., Bollag, J.-M., and Alexander, M. (1967a). J . Agr. Food Chem. 15, 858. Loos, M. A., Roberts, R. N., and Alexander, M. (1967b). Can. j . Microbiol. 13, 691. McBride, B. C., and Wolfe, R. S. (1971). Biochemistry 10, 4312. MacRae, I. C., and Alexander, M. (1963). J . Bacterial. 86, 1231. Madhosingh, C. (1961). Can. 1. Microbiol. 7, 553. Matsumura, F., and Boush, G. M. (1966). Science 153, 1278. Matsumura, F., and Boush, G. M. (1967). Science 156, 959. Matsumura, F., and Boush, G. M. (1968). J . Econ. Entomol. 61, 610. May, S. W., and Abbott, B. J. (1973). J . Biol. Chem. 248, 1725. Mendel, J. L., and Walton, M. S. (1966). Science 151, 1527. Mendel, J. L., Klein, A. K., Chen, J. T., and Walton, M, S. (1967). J . Ass. Ofic. Anal. Chem. 50, 897. Menzie, C. M. (1969). US.,Fish Wildl. Sew., Spec. Sci. Rep; Wildl. 127. Miles, J. R. W., Tu, C. M., and Harris, C. R. (1969). J. Econ. Entomol. 62, 1334. Milne, G. W. A., Goldman, P., and Holtzman, J. L. (1968). J . Biol. Chem. 243, 5374. Miyamoto, J., Kitagawa, K., and Sato, Y. (1966). Jap. J . Exp. Med. 36, 211. Miyazaki, S., Boush, M., and Matsumura, F. (1970). 1. Agr. Food Chem. 18, 87. Mounter, L. A., and Tuck, K. D. (1956). J. B i d . Chem. 221, 537.

MICROBIAL TRANSFORMATION OF PESTICIDES

129

Mounter, L. A., Baxter, R. F., and Chanutin, A. (1955). J . Biol. Chem. 215, 699. Nakanishi, T., and Oku, H. (1969). Phytopathology 59, 1761. Neuberg, C., and Welde, E. (1914). Biochem. Z. 60, 472. Neufeld, H. A,, Green, L. F., Latterell, F. M., and Weintraub, R. L. (1958). J . Biol. Chern. 232, 1093. Pfaender, F. K., and Alexander, M. (1972). J . Agr. Food Chem. 20, 842. Plimmer, J. R., and Kearney, P. C. (1969). Abstr., 158th Meet., Amer. Chem. SOC. Paper A-30. Plimmer, J. R., Kearney, P. C., and von Endt, D. W. (1968). J. Agr. Food Chem. 16, 594. Pfimmer,, J. R., Kearney, P. C., Chisaka, H., Yount, J. B., and Klingebiel, U. I. (1970). J. Agr. Food Chem. 18, 859. Raymond, D. D., and Alexander, M. (1972). Pestic. Biochem. Physiol. 2, 270. Raymond, D. G . M., and Alexander, M. (1971). Pestic. Biochem. Physiol. 1, 123. Ribbons, D. W. (1971). FEBS Lett. 12, 161. Ruiz-Herrera, J., and Starkey, R. L. (1969). J . Bacteriol. 99, 544. Saz, A. K., and She, R. B. (1954). Arch. Biochem. Biophys. 51, 5. Sharabi, N. El-D., and Bordeleau, L. M. (1969). Appl. Microbiol. 18, 369. Shida, T., Honima, Y., and Misato, T. (1973). Agr. Biol. Chem. 37, 1027. Stanier, R. Y., and Ornston, L. N. (1973). Advan. MicrobioZ. PhysioE. 9, 89. Stenersen, J. (1969). Bull. Enuiron. Contam. Toxicol. 4, 104. Stenersen, J. H. V. ( 1965). Nature (London) 207, 660. Taylor, H. F., and Wain, R. L. (1962). Proc. Roy. SOC., Ser. 156, 172. Tewfik, M. S., and Evans, W. C. (1966). Biochem. J . 99, 31p. Tiedje, J. M., and Alexander, M. (1969). J. Agr. Food Chem. 17, 1080. Tiedje, J. M., Duxbury, J. M., Alexander, M., and Dawson, J. E. (1969). J. Ag?. Food Chem. 17, 1021. Tonomura, K., Futai, F., Tanabe, O., and Yamaoka, T. (1965). Agr. Biol. Chem. 29, 124. Tu, C . M., Miles, J. R. W., and Harris, C. R. (1968). Life Sci. 7, 311. Tweedy, B. G., Loeppky, C., and Ross, J. A. (1970a). J . Agr. Food Chem. 18, 851. Tweedy, B. G., Loeppky, C., and Ross, J. A. ( 1970b). Science 168,482. Verloop, A. (1972). In “Drug Design” ( E . J. Ariens, ed.), Vol. 3, pp. 133-187. Academic Press, New York. Voerman, S . , and Tammes, P. M. L. (1969). Bull. Enoiron. Contam. Toxicol. 4 , 271. Wallnofer, P. (1969). Weed Res. 9, 333. Wallnofer, P. R., and Bader, J. (1970). App!. Microbiol. 19, 714. Wallnofer, P. R., Koniger, M., Safe, S., and Hutzinger, 0. (1972). J . Agr. Food Chem. 20, 20. Wallnofer, P. R., Safe, S., and Hutzinger, 0. (1973). Pest. Biochem. Physiol. 3, 253. Weber, W. W. (1971). In “Handbuch der experimentellen Pharmakologie” (B. B. Brodie and J. R. Gillette, eds.), Vol. 28, Part 11, pp. 564483. Springer-Verlag, Berlin and New York. Webley, D. M., Duff, R. B., and Farmer, V. C. (1957). Nature (London) 179, 1130. Wedemeyer, G. ( 1966). Science 152, 647. Wedemeyer, G . (1967). Appl. Microbiol. 15, 569.

130

JEAN-MARC BOLLAG

Wedemeyer, G. (1968). AppZ. Microbiol. IS, 661. Weinberger, M., and Bollag, J.-M. (1972). Appl. Microbiol. 24,750. Weisburger, J. H., and Weisburger, E. K. (1966). Clzern. Eng. News 44, 124. Wiese, M. V., and Vargas, J. M. ( 1973). Pestic. Biochem. Physiol. 3, 214. Williams, A. K., Cox, S. T., and Eagon, R. G . (1965). Biochem. Biophys. Kes. Commzm. 18, 250. Williams, R. T. ( 1971). In “Handbuch der experimentellen Pharmakologie” (B. B. Brodie and J. R. Gillette, eds.), Vol. 28, Part 11, pp. 226-242. Springer-Verlag, Berlin and New York. Wood, J. M., and Wolfe, R. S. (1966). 1. Bacterial. 92, 696. Wood, J. M., Kennedy, F. S., and Rosen, C. G . (1968). Nature (London) 220, 173. Yamada, M., and Tonomura, K. (1972). Hakko Kogaku Zasshi 50, 159. Yule, W. N., Chiba, M., and Morley, H. V. (1967). J. Agr. Food Chem. 15, 1000. Zaki, M. A., Taylor, H. F., and Wain, R. L. (1967). Ann. Appl. Biol. 59, 481. Zayed, S. M. A. D., Mostafa, I. Y., and Hassan, A. (1965). Arch. Mikrobiol. 51, 118.

Taxonomic Criteria for Mycobacteria and Nocardiae

S . G. BRADLEY AND J . S . BOND Departments of Microbiology and Biochemistry Virginia Commonwealth Uniuersity. Richmond. Virginia

I. Introduction .................................... I1. Earlier Classification 'Schemes ..................... I11. Developing Classification Systems .................. A . Cardinal Classification ........................ B. Numerical Taxonomy ......................... C . Natural Classification ......................... IV. Differential Characters ............................ A. Acid-Fast Staining ........................... B. Chemical Composition ........................ C. Bacteriophage Typing ........................ D . Bacteriocin Typing .......................... E . Cell Wall Composition ........................ F. Lipid Composition ........................... G . Serological Analyses .......................... V. Regulation of Metabolism ......................... A . Regulation in Actinomycetes .................. B. Catabolism in Mycobacterium .................. C. Catabolism in Nocardia ....................... VI . Degradation of Intracellular Proteins ................ A. Rates of Protein Degradation . . . . . . . . . . . . . . . . . . B . Half-lives of Enzymes ........................ C. Effects of Metabolic Conditions ................ D . Mechanism of Protein Degradation ............. E . Biological Significance ........................ VII . DNA Analyses .................................. A . DNA :DNA Association ....................... B. Optical Association .......................... C . Phylogenetic Implications ..................... D . Neutral Mutations ........................... VIII . Definition of the Genera .......................... A. Mycobacterium Lehmann et Neumann 1896 ..... B . Nocurdia Trevisan 1889 ....................... C . Actinomudura Lechevalier et Lechevalier 1970 ... D . Proactinomyces Jensen 1931 ................... E . Oerskouia Prauser, Lechevalier et Lechevalier 1970 IX. Evaluation of Species ............................ A . Species of Nocurdia .......................... B . Species of Proactinomyces ..................... C. Species of Mycobacterium ..................... X. Concluding Remarks ............................. References ...................................... 131

132 133 134 134 135 136 136 136 137 138 141 141 143 144 145 147 147 149 153 153 156 157 159 160 162 162 165 167 171 172 172 173 174 174 175 176 176 178 181 184 185

132

9. G. BRADLEY AND J. S. BOND

1. Introduction

The taxonomy of the actinomycetes constitutes a notorious example of bad systematics. Different taxonomists have given the same organism different names. Other taxonomists have given different organisms the same name. The actinomycete literature is difficult or impossible to follow because we do not know what organisms the authors were studying (Gottlieb, 1960). Taxonomy is the study of the bases, principles, procedures, and rules for classifying organisms. There are three distinct aspects of taxonomy: classification, nomenclature, and identification. Nomenclature is the naming of the populations defined and delineated by classification and, in the present context, is governed by the rules of the International Code of Nomenclature of Bacteria. Classification is the arranging of organisms into related groups. Identification is the assigning of an unknown strain to a particular taxonomic group. If the unknown culture is sufficiently similar to a previously defined group, it is given the same name as the established group. If the organism is not reasonably like an established taxon, it must be classified rather than identified. There is a logical sequence of events in taxonomy: classification, nomenclature, and identification. Although the basic unit in taxonomy is the species, the only tangible unit is the strain or individual. In practice a species is an abstraction that refers to a collection of strains that share many characteristics. Ideally a species is an easily recognizable taxon based upon distinctive morphological and physiological characteristics that are described qualitatively rather than quantitatively. Biochemical properties, for example, are most often characteristics used in describing strains but are not usually diagnostic of a species. Pigment production, in instances in which the pigment is a unique chemical structure, is generally strain specific, but this property has diagnostic value when most of the isolants assigned to a species by other criteria are found to produce the same pigment or its congener. A species should not be proposed on the basis of the study of a single isolant; instead a large number of diagnostically similar strains obtained from geographically distributed sources should be compared. It is our opinion that the species concept should be based upon an analysis of a representative portion of the natural population. A species concept should not be based upon the differences that exist among microorganisms, because differences can be found in a single strain and its progeny. Rather a species should be based upon similarities that tend to unite groups of related strains by means of characteristics that appear relevant to the particular group of organisms being studied (Luedemann, 1971 ) .

MYCOBACTERIA AND NOCARDIAE

II.

133

Earlier Classification Schemes

A number of systems of classification for the actinomycetes have been promulgated. The main differences are nomenclatural rather than of substance, and are based upon interpretations of priorities and descriptions from the early days of research on the actinomycetes. The taxonomic keys developed by Waksman and Henrici ( 1944), Krasil'nikov (1966), and Baldacci (1947) have been used widely. Baldacci designated the microaerophilic actinomycetes as Cohnistreptothrix; the aerobic actinomycetes lacking sporulating aerial hyphae, as Proactinomyces; and the aerobic actinomycetes with sporogenous aerial hyphae, as Actinomy ces. Negroni (1953) proposed the following scheme: Family Mycobacteriaceae Chester genus Mycobacterium Lehmann et Neumann type species M . tuberculosis ( Schroeter ) genus Corynebacterium Lehmann et Neumann type species C . diphtheriae (Fliigge) genus Propionibacterium Orla-Jensen type species P. freudenreichii van Niel Family Proactinomycetaceae Lehmann et Neumann emend. Waksman et Umbreit genus Proactinomyces Jensen type species P . agrestis Gray et Thornton genus Cohnistreptothrix Pinoy type species C . israeli Kruse Family Actinomycetaceae Buchanan genus Actinomyces Harz type species A. albus Rossi-Doria Family Micromonosporaceae Krasil'nikov genus Micromonospora grskov type species M . chalcea Foulerton Redaelli and Piantoni (1953) proposed the following scheme: Actinomyces: aerobic filamentous actinomycetes fragmenting only rarely; forms aerial growth; includes Streptomyces and Actinomadura Proactinomyces: aerobic filamentous actinomycetes that fragment frequently, scant to no aerial growth; includes Nocardia asteroides Cohnistreptothrix: anaerobic or microaerophilic actinomycetes that regularly fragment; no aerial growth; equivalent to Actinomyces in the sense of Bergey's manual (Breed et aZ., 1957) Mycobacterium: aerobic, acid-fast actinomycetes that do not form mycelia or form only rudimentary mycelia; no aerial growth; includes M . tuberculosis

134

S. G . BRADLEY AND

J. S. BOND

Krasil'nilov (1W)proposed the following scheme: Order Actinomycetales Buchanan Family Actinomycetaceae Buchanan 1918 genus Actinomyces Harz 1877 (in the sense of Streptomyces albus) genus Proactinomyces (Lehmann et Haag) Jensen 1934 genus M ycobacterium Lehmann et Neumann 1896 genus Mycococcus Krasil'nikov 1938 Ill.

Developing Classification Systems

A. CARDINAL CLASSIFICATION In the early period of the development of bacteriology as a science, only a few characteristics of bacteria could be studied. Bacteria, therefore, were arranged into groups based upon morphological features, staining properties, and a few physiological tests, e.g., gelatin liquefaction. As bacteriology progressed, the number of recognizable attributes of bacterial cells and populations increased. Certain characteristics, such as production of spores and the presence and arrangement of flagella, were chosen as key or cardinal characteristics because it was thought that they were stable determinative features of a taxon. These selections constituted a priori subjective weighting of the key characteristics. The number of key characteristics used by bacterial taxonomists is very small compared with the number of attributes that can be determined. Cardinal characteristics are used to generate dichotomous keys. In dichotomous keys, there is a series of contrasting paired statements, each one leading into another pair of contrasting statements. The differential statements should be based upon features that are easily and reliably measured. Key characters should have a low mutation rate or be determined by multiple genes (loci). Simple dichotomous keys lead to erroneous conclusions when any one attribute is variable or misread. For this reason, dichotomous keys have been partially replaced by diagnostic tables that contain the results of several tests done on a spectrum of genera or species. Conclusions based upon these tables are not seriously limited by variations in a single response, but the tables are somewhat cumbersome to use unless the microbiologist has the expertise to select the appropriate table based upon preliminary tests. This is usually possible in the clinical laboratory where selective media and differential media are used and a circumscribed range of genera and species is of concern. Diagnostic tables have restricted utility in identifying bacteria from soil, fresh water, or marine samples.

MYCOBACTERIA AND NOCARDIAE

135

B. NUMERICAL TAXONOMY In the early phase of microbiology, the taxonomist had to rely on the few characteristics that could be readily scored. Later some characteristics were considered more important or reliable than other features. These attributes were used to develop the dichotomous keys discussed previously. Alternatively microorganisms can be classified by the Adansonian approach. The principles of Adansonian taxonomy are: ( 1) it is based upon as many characters as possible, ( 2 ) each character is given equal weight, ( 3 ) the overall or phenetic similarity between each pair of strains is calculated from the number of characters that they have in common, and (4)organisms that share many characteristics are grouped together (Sneath, 1970). In Adansonian taxonomy, it is essential that a large number of organisms be examined with respect to a large number of characters. Accordingly, computers are needed to process the data. It is essential therefore that all information be collected such that it can be coded into a format suitable for automated data processing. The only acceptable characters are unit characters, in which two or more states or responses cannot be subdivided logically. Once all unit characters have been scored and recorded for a pair of organisms, the numerical coefficient is calculated. Two coefficients are widely used: the similarity coefficient (Cs) and the matching coefficient (em).

cs = 100a/(n + b ) ;

Cm

= 100(a

+ c ) / ( a f b + c)

where a = the number of shared positive responses; b = the number of unlike responses; c = the number of shared negative responses. The justification for selecting the similarity coefficients is that there are large numbers of shared negative responses that could give high Cm values for artifactual reasons, e.g., failure to grow or a slow growth rate. The logic for selecting the matching coefficient is that it is not always possible to determine what is a positive response and what is a negative response, e.g., susceptibility to an antibiotic. The most controversial aspect of numerical taxonomy has been the principle that all characters shall be given equal importance or weight. Actually there is no objective way of assessing the weight of a character, so there is no practical alternative to this principle. The test battery for numerical taxonomy should reflect a wide range of an organism’s genetic potential. At least 60 differentiating unit characters must be determined. Unit characters that have no differentiating value in the study population must be excluded from the data used to calculate Cs or Cm.

136

S. G . BRADLEY AND J . S. BOND

C. NATURAL CLASSIFICATION The term natural classification is used to describe arrangements of closely related microorganisms based upon overall genetic similarity. A natural classification presumably reflects evolutionary closeness. Since there is little, if any, direct evidence on how microorganisms have evolved, phylogenetic classifications are largely based upon personal interpretations of selected data. In conventional systematics, and even in numerical taxonomy, only a small portion of an organisms’ attributes is considered. The more completely a group of microorganisms has been characterized, the more reliable are the judgments about relationships. Although reasonably useful taxonomic systems have been devised on the bases of cardinal features on the one hand, or overall phenotypic similarity using unweighted characters on the other hand, similarity between the genetic determinants themselves seems the most appropriate criterion for setting up a microbial classification. The genetic potential (genotype) of a microbe is encoded in the linear order of the four nucleotides in its deoxyribonucleic acid (DNA). These sequences are translated into colinear sequences of amino acids in structural and catalytic proteins which directly or indirectly constitute the phenotype of the cell. Accordingly, evolutionary divergence from a common ancestor proceeds as the progeny accumulate nucleotide substitutions in their DNA. Recent studies strongly indicate that remnants of an organism’s evolutionary history are retained, inscribed in the DNA. Because of an increased understanding of the molecular architecture of DNA, an evolutionary approach to bacterial classification, long hindered by the lack of an adequate fossil record, is however now feasible. It should be noted that there are no definitive guidelines by which to construct taxa based upon DNA nucleotide homology. Moreover, there is no established reference ancestral type from which nucleotide divergence radiated during evolution. In fact it is not possible to distinguish absolutely between recently transferred nucleotide sequences (by genetic recombination) and conserved ancestral nucleotide sequences. IV.

Differential Characters

A. ACID-FAST STAINING The historically diagnostic characteristic of the mycobacteria is that they are acid-fast, but this property is not always sufficient to distinguish them from strains of nocardiae and corynebacteria. According to Goodfellow (1972) the acid-fast character is not a good one because it is

MYCOBACTEIUA AND NOCARDIAE

137

dependent on the age of the culture, the composition of the growth medium and the length of time since the strain was isolated. It is recognized, for example, that acid-fastness can be enhanced by growth in media such as Middlebrook 7H10 agar supplemented with 1%glycerol (Beaman and Burnside, 1973). Moreover, the members of the genus Nocardia are heterogeneous with respect to acid-fastness. Some strains of Nocardia are reported to be strongly acid fast (Georg et al., 1916) whereas other strains are readily decolorized with acid-alcohol ( Gordon and Mihm, 1959a). Significantly, Fisher and Barksdale ( 1973) have demonstrated that acid-fast staining of the leprosy bacterium can be removed by extracting the cells with pyridine. Cells of strains unequivocally identified as Mycobacterium retain the acid-fast stain, whether or not the dried smears have been extracted with pyridine at room temperature for 4 hours. Beaman and Burnside (1974) further demonstrated that the acid-fastness of all members of the genus Nocardia can be removed by extraction with pyridine whereas members of the genus Mycobacterium are unaffected with respect to this property. Acidfast staining of dried smears, one set after pyridine extraction and one set without pyridine extraction, seems to constitute a useful method to distinguish between “variably acid-fast” nocardiae and cultivable mycobacteria. Smears are air dried and fixed for 30 minutes in Kellenberger buffer at pH 6.5. The smears are rinsed in deionized water and dried. Duplicate sets of slides are either extracted in fresh pyridine at room temperature for 4 hours or remain unextracted. All slides are stained by the Kinyoun acid-fast method using 1%concentrated HCl in 70%ethanol as the decolorizing agent. The smears are counterstained for 30 seconds with aqueous methylene blue. Pyridine removes the acid-fastness of all members of the genus Nocardia examined but the mycobacteria are not visibly affected by pyridine. Pyridine extraction seems to provide a simple method by which acid-fast nocardiae may be distinguished from most cultivable mycobacteria ( Beaman and Burnside, 1973). B. CHEMICAL COMPOSITION The actinomycete cell is about 85%water, 11-12% organic matter and 1 4 %inorganic substances. Protein constitutes 5291%of the total organic matter; nucleic acids make up 5 1 0 %of the total organic matter. Lipid and carbohydrate contents vary markedly with species and cultural conditions. Streptomycetes usually contain 1-102 lipid, and mycobacteria contain &25% lipid, with respect to the total organic matter. The carbohydrate content of the streptomycete is 3845%whereas that of the mycobacteria is 2 5 %of the total organic matter (Kwapinski and Seeliger,

138

S. G . BRADLEY AND J . S. BOND

1964) , A red pigment ( 1,6-phenazinedio1-5,10-dioxide) ; and orange pigment ( 1,6-phenazinediol-5-oxide ) , and a yellow pigment ( 1,6-phenazinediol ) constitute a family of three phenazines found in Streptomyces thioluteus, Actinomadura dassonvillei, and Streptosporangium amethystogenes; therefore the occurrence of these three phenazines does not seem to have much taxonomic value. Similarly Serratia marcescens produces prodigiosin; Streptomyces longisporus Tuber, Actinomadura madurue, and Actiomadura pelletieri produce red pigments with the methoxytripyrrole nucleus of prodigiosin ( H. A. Lechevalier et ul., 1971). H. A. Lechevalier et al. (1971) have concluded that antibiotic production is even less useful than pigment production in the speciation of actinomycetes. Arai et al. (1963) have used the infrared spectrum of whole cells as a tool for differentiating between Nocardia and Mycobacterium. Washed cells are lyophilized, desiccated further over P,O, and mixed with KBr powder. Tablets are prepared and the absorption pattern determined over the range of 950 cm-' to 3000 cm-' (wave number). Arai and his co-workers concluded that they could differentiate between Nocardia and Mycobacterium by this method.

C. BACTERIOPHAGE TYPING Lysis of bacteria by bacteriophage is one means of identifying closely related organisms. Sensitivity to actinophages has been used as an index of relationships among actinomycetes (Bradley, 1968). Most actinophages are able to attack numerous species .within a genus (Anderson and Bradley, 1961). Because attempts to identify bacteria by serological techniques have frequently failed to provide the definitive identification necessary for epidemiologic studies, bacteriophage typing has sometimes been used when reliable phage-typing preparations are available. Phage for typing may be propagated in the laboratory by a variety of methods. Although propagation in broth is simple, this method does not give as high titered lysates as soft agar methods. The titer of a particular phage lysate is a function of the phage being propagated, the host, and the conditions used. Many difficulties beset the use of undiluted phage lysates in phage typing, therefore a stock preparation is usually diluted to give a routine test dilution. This step is necessary because the lysate may contain antibiotics, defective phages, or bacteriocins. Moreover phage may adsorb to and kill a bacterium but be unable to replicate in the putative host. One of the principal applications of bacterial viruses has been to develop diagnostic schemes for pathogenic bacteria. Enrichment techniques have formed the standard technique for isolating bacteriophage from fomites, soil, or other sources. However, the volumes of medium

MYCOBACTERIA AND NOCARDIAE

139

required and the time consumed by such methods are very demanding, especially in situations in which organisms such as M. tuberculosis are involved. Membrane filters may be used to isolate bacterial viruses (Grant, 1973). Bacteria from a logarithmic growth phase culture are deposited aseptically on a membrane filter. A second membrane is placed over the cell layer, and the “sandwich is placed in a membrane filter holder. The putative bacteriophage source is homogenized if necessary, centrifuged to remove microscopic particulate matter, and filtered through a conventional bacteriological filter. This filtrate is slowly sieved through the bacterial “sandwich.” In instances where the bacteriophage source is viscous (e.g., sputum) a mucolytic agent may be required to reduce viscosity and improve the flow rate. The bacterial layer is then washed into appropriate medium for the putative host and incubated at the optimum growth temperature. It is usually advantageous to introduce additional host from a logarithmic phase culture to facilitate phage propagation. The resulting phage lysate is clarified, filtered, and assayed by orthodox techniques. Phage typing is a relatively simple procedure in which an agar plate is inoculated with sufficient bacterial culture to give a confluent growth (Bradley and Jones, 1968). Next a drop of a “routine test dilution” of each typing phage is placed at a predetermined position on the agar surface. After incubation, the degree of lysis produced by each phage is recorded and the phage type is determined from published patterns. It should be obvious that cultures from clinical specimens may contain mixtures of strains; unless the strains are separated and tested individually, a nontypable reaction may be observed. The practice of transferring from confluent growth what appears to be a pure primary or secondary culture of a clinical isolant, rather than from an isolated colony, is imprudent. The bacterial indicator lawn may be obtained by (1) flooding the agar with a sample from a broth culture, or ( 2 ) mixing an inoculum into an overlay medium, or (3) spreading a smaller inoculum over the agar surface with a sterile bent glass rod. The excess fluid in the inoculum should be removed and the agar surface should appear dry before proceeding to the next step. The phage-typing reagent may be applied with a loop, a pipette, or a syringe and needle. Multiple inoculating devices that use loops, capillary tubes, or solid cylinders have been manufactured to facilitate this step (Bradley, 1968). The relationships of bacterial strains to one another can be deduced from host-range patterns using monovalent or polyvalent phages (Jones and Bradley, 1962). In one method of analysis, results of phage tests may be considered all-or-none phenomena, and the classification constructed by overlapping viral susceptibilities. Additionally, quantitative as well as qualitative aspects of phage susceptibility can be used in developing

140

S. G . BRADLEY AND J. S. BOND

a classification. Phage typing, especially with polyvalent phages must be done with care and understanding. For example, the relative power of a bacteriophage to produce plaques on a particular putative host is dependent upon the propagating strain used in preparing the stock lysate. Such host modification of virulence may represent nonhereditary alteration of the virus or selection for phage mutants (Manion et aZ., 1964). Numerous technical faults may make scoring of phage typing assays difficult. A dense inoculative suspension of the test bacterium may obscure plaque formation in the confluent bacterial growth. To date mycobacteriophage susceptibility has not been maximally utilized as a taxonomic tool. Bacteriophages able to infect nocardiae and mycobacteria are ubiquitous; they may be found in soil, water, feces, sewage or the bacterial culture itself. Although actinophages are generally referred to as lytic or temperate, it must be remembered that lytic phages isolated from the soil may be able to lysogenize appropriate recipients and that temperate phages are virulent for numerous indicator strains ( Bradley and Jones, 1968). Bacterial viruses attack only closely related hosts, therefore, susceptibility to particular bacteriophages can be used to speciate certain bacteria (Bradley and Anderson, 1958). Using these methods, Anderson and Bradley noted the intermediate position of madurae-like organisms. Their observations are consistent with the later proposal to establish a genus Actinomadura. Mycobacteriophage 33D obtained from a lysogenic atypical nonphotochromogenic member of Runyon’s Group I11 did not lyse any of 11 tested strains of BCG but did lyse 5 strains of mycobacteria from bovine infections and 19 strains of M . tuberculosis. Mycobacteriophage phage DSBA lysed all 35 strains of M . bovis and M . tuberculosis (Buraczewska et al., 1971). In addition, Manion et aZ. (1964) concluded that ( 1 ) some farcinica cultures are mycobacteria whereas others are nocardiae; ( 2 ) the rhodochrous complex is distinct from N . asteroides group; and ( 3 ) M . smegmatis, M . butyricurn, M . friburgensis, and M . ranae (U.S.A. strains) are synonyms. Viral susceptibility can be easily determined both qualitatively and quantitatively on a large scale. By use of available multiple-inoculating instruments, several hundred assays can be set up in one day by a single worker; usually the tests can be scored the next day. By analyzing the data, with or without the aid of a computer, general relationships among actinomycetes can be found. Affinities inferred by this method reflect phylogeny and evolution as accurately as any other system in use today. Although phage-testing can reliably identify strains and is useful for recognition of species, genera, and families, we do not advocate that host range should be used as the sole basis for establishing a species (Jones and Bradley, 1962).

MYCOBACTERIA AND NOCARDIAE

141

D. BACTERIOCIN TYPING Mycobacteriocins are a class of antibiotics that act only on strains of the same or closely related species. The mycobacteriocins are composed of proteins. These antibiotic-like agents kill susceptible mycobacteria after first adsorbing onto specific receptor sites on the cell surface. Mycobacteriocins and mycobacteriophage both appear to adsorb to similar receptors. Although mycobacteriocinogenic strains possess the stable genetic potentiality to produce a mycobacteriocin, they do not do so all the time. Some mycobacteriocinogenic strains can be induced to produce mycobacteriocins by treatment with ultraviolet light or mitomycin C. The cells which actually produce bacteriocins die in the process. Accordingly, there is a striking parallel between the behavior of mycobacteriocinogenic strains and lysogenic strains ( Bradley, 1968). Bacteriocinogeny is a stable genetic character and production of bacteriocin can be used for typing of bacteria. Takeya and Tokiwa (1972) classified several rapidly growing mycobacteria by using a simple technique to detect mycobacteriocin activity. All strains designated as M . abscessus and M . runyonii displayed the same response pattern as the putative mycobacteriocins. This agrees with other methods that indicate that these two taxa should be reduced to synonymy (as M . abscessus or M . chelonei).

E. CELLWALLCOMPOSITION The glycan moiety of peptidoglycan is remarkably uniform. It is usually made up of alternating ,8-1,4-linked N-acetylglucosamine and Nacetylmuramic acid residues. In the mycobacteria and in Nocardia kirouani, muramic acid does not occur as N-acetyl but as the N-glycolyl derivative. In these organisms, the amino group in position 2 is not substituted by an acetyl group (-COCH,) but by a glycolyl group (-COCH,OH ) ( Azuma et al., 1970). The mycobacteria, nocardiae, actinomadurae, and pseudonocardiae contain only alanine, glutamate, and meso-diaminopimelic acid in their peptidoglycan. Although the chemical composition of these cell walls have been determined only qualitatively, it seems likely that the peptidoglycan is cross-linked in a direct way (Fig. 1). Actinomadurae differ from mycobacteria, nocardiae, and pseudonocardiae in that the former do not contain arabinogalactan in their walls (Table I ) . The cell walls of mycobacteria always contain the amino acids alanine, glutamate, and meso-diaminopimelic acid and the monosaccharides arabinose and galactose. The peptide subunits of the peptidoglycan of M . smegmatis and M . tuberculosis are directly cross-linked,

142

S. G . BRADLEY AND J. S. BOND

-G-M-G-

D-G~u-NH, I

I

I

D-Ala

m-DAP-

t

NHZ

FIG. 1. Portion of the primary structure of a directly cross-linked peptidoglycan. G = glucosamine; M = muramic acid; L-ALA = alanine; D-GLU = glutamic acid; m-DAP = meso-diaminopimelic acid.

and both the a-carboxyl group of D-glutamate and the carboxyl group of meso-diaminopimelic acid that are not involved in a peptide linkage are amidated. In the peptidoglycan of M . tuberculosis H37Rv a small portion of the D-glutamate residues is not amidated but is substituted by glycine. In general the cell wall peptidoglycan of Nocardia and Pseudonocardia are the same as that of the mycobacteria. In some Nocardia, however, meso-diaminopimelic acid is replaced by L,L-diaminopimelic acid and no arabinose and galactose are present in the cell walls. The taxonomic position of these species (N. alba, N. flaua, N. gardneri, N. mesenterica, N . rubra, and N. salmonicolor), however, is uncertain (Schleifer and Kandler, 1972). One should note that in the studies of H. A. Lechevalier TABLE I DIAGNOSTIC COMPONENTS OF ACTINOMYCETE CELL WALLS Genus

Diagnostic constituents

Actinomadlira Mycobacterium Nocardia Oerskovia Streptomyces

meso-DAPO, sometimes maduroseb meso-DAP, arabinose, galactose meso-DAP, arabinose, galactose Lysine, aspartate LDAP, glycine

a

Diaminopimelic acid.

* 3-O-Methyl-~-galactose.

MYCOBACTERIA AND NOCARDIAE

143

et al. (1971) no attempt is made to differentiate between meso- and D-diaminopimelic acid ( DAP ) . The study of cell wall composition has furnished a convenient way of delineating the boundaries between the nonsporulating streptomycetes that form soft colonies and the typical nocardiae and between the typical streptomycetes and the nocardiae with aerial hyphae. The streptomycetes contain GDAP and the nocardiae contain meso-DAP. The isomers can be distinguished by paper chromatography of whole cell hydrolyzates. M. P. Lechevalier and Lechevalier (1970) have concluded that all the strains in a genus should have the same diagnostic constituents (Table I ) . This means that the genus Nocardia in the sense of Bergey (Breed et al., 1957) is heterogeneous. Their concern was shared by actinomycetologists, who debated whether the mycetoma-producing species “madurae” and “pelletieri” should be placed in the genus Streptomyces or the genus Nocardia. Accordingly, H. A. Lechevalier and Lechevalier ( 1970 ) established the genus Actinomadura to harbor “mudurae,” “pelletieri,” and “dassonuillei.” However, cell wall composition seems to be a practical taxonomic criterion only if major constituents are taken into account. M. P. Lechevalier and Lechevalier (1970) have pointed out that different organisms may have the same major constituents in their cell wall, e.g., Actinomadura mudurae and Dermatophilus congolensis.

F. LIPIDCOMPOSITION Mycolic acids may be described as long-chain p-hydroxy acids having a long alkyl branch in the a-position. Mycolic acids occur with or without oxygen functions (ketone, methoxyl, or carboxyl) in addition to the p-hydroxyl acid system. Cyclopropane rings, double bonds and methyl branches may be present or absent. Strains of M . tuberculosis produce methoxymycolic acid whereas M . smegmatis and M . phlei characteristically produce a variety of unsaturated cyclopropane and methylbranched mycolic acids. Isoniazid appears to act on mycobacteria by inhibiting either directly or indirectly the synthesis of mycolic acids. Lipid composition is of assistance in the recognition of the genera Mycobacterium, Nocurdia, and Corynebacterium. All three genera contain meso-DAP, arabinose, and galactose. Associated with the cell walls of these three genera are the complex a-branched, p-hydroxylated mycolic acids. There are three rather distinct types of mycolic acids: ( a ) those with carbon skeletons of about 80 carbon atoms associated with strains of Mycobacterium (mycolic acids stricto sensu); ( b ) those with skeletons of about 50 carbons found in strains of Nocardia (nocardomycolic acids); and ( c ) those with smaller skeletons of about 30 atoms

144

S. G . BRADLEY AND J. S. BOND

of carbon, which seem to be associated with certain strains of Corynebacterium (corynomycolic acid) (H. A. Lechevalier et al., 1971). H. A. Lechevalier et al. have devised a way to distinguish among these three kinds of mycolic acids. In their method, purified methylated mycolic acids are injected into a gas chromatograph where they are pyrolyzed into a fatty ester moiety and an aldehyde moiety. The true mycolic acid molecules split to release unbranched saturated fatty esters having 24-26 carbon atoms whereas the nocardomycolic acids and corynomycolic acids release C , , to C , , fatty esters. The pyrolysis fragments are identified according to their retention times, using authentic samples of the fatty esters and aldehydes as standards. The nocardomycolic acids can be distinguished from the corynomycolic acids by the size of the aldehyde moiety formed on pyroIysis. The aldehydres from nocardomycolic acids are rather large (C,?,to C:$*) and are eluted much less readily from the gas chromatograph than the smaller aldehydes (C,, to C I R )from corynomycolic acids. G. SEROLOGICAL ANALYSES

Nocardia and Mycobacterium have been studied with serological methods such as agglutination, complement fixation, sensitin tests, immunodiffusion, and immunoelectrophoresis. From these studies it has been established that various species of Nocardia and Mycobacterium have antigens in common (Castelnuovo et al., 1968; Kwapinski, 1970). However, it has also been possible to differentiate among strains within each genus (Castelnuovo et al., 1964; Magnusson and Mariat, 1968). In an extensive serological study of nocardia by comparative immunodiffusion against antimycobacterial sera, Ridell and Norlin ( 1973) found that cultures labeled Mycobacterium pellegrino and Mycobacteriurn rhodochrous did not react with the antimycobacterial sera to a larger extent than any other nocardial strain. Ridell and Norlin (1973) also found that cultures labeled Nocardia farcinica were heterogeneous; five out of eight were seroIogicaIly related to the mycobacteria. Castelnuovo et al. ( 1964) utilizing antigenic relationships and phage susceptibility concluded that strains designated as N . corallina, N . rubra, M . rhodochrous, and M . pellegrino belong to a common group. Moreover, these strains were considered more closely related to the Nocardia than to the genus Mycobacterium. The antigenic relationships between the cell sap (plasm) of a large number of actinomycetes was determined by Kwapinski ( 196613). He found the following serogroups: (1) M. tuberculosis and M . bouis; ( 2 ) M. avium and M . kansasii; (3) M . balnei (or M . murinum); ( 4 ) B4. smegmatis (and “rhodochrous”), and ( 5 ) M. fortuitum. Moreover, the

MYCOBACTERIA AND NOCARDIAE

145

antigenic relationships between the antigens present in the culture filtrates of a large number of actinomycetes was determined by Kwapinski (1966a). He found the following serogroups: ( 1 ) M . tuberculosis and M . bouis; ( 2 ) Group 2 scotochromogenic mycobacteria; ( 3 ) M . kansasii; ( 4 ) M . smegmatis and M . phbi; ( 5 ) M . smegmatis, M . plilei and M . marinum; and ( 6 ) M . fortuitum, “rhodochrous” and M . avium. Reznikov and Dawson (1973) suggest that the M . avium-M. intracellulare complex contains numerous serotypes, perhaps more than SO. Magnusson and Mariat ( 1968 ) have used delayed-type skin reactions on guinea pigs to compare selected actinomycetes with N . farcinica ATCC 3318 and N . asteroides ATCC 19247. They concluded that N . farcinica and N . asteroides are two distinct species. It is important to note that three of the strains that they assigned to N . farcinica were originally isolated from cattle with farcy in Africa. These cultures are considered true mycobacteria by most workers, including us. As discussed elsewhere in this review, the cell walls of mycobacteria and nocardiae both contain arabinogalactan, and this polymer is probably responsible for the serological cross-reactions of these genera ( Cummins, 1954).

V.

Regulation of Metabolism

Not all of the genotypic potential of an organism is expressed at any one time. Phenotypic expression is controlled and regulated by several different processes such as activation or inhibition of enzyme activity and regulation of enzyme concentrations. Enzymatic activity may be inhibited by the end product of a pathway. In feedback inhibition, the end product acts on the first enzyme unique to the biosynthesis of the end product. The catalytic site of an enzyme susceptible to end-product inhibition is different from the region that binds the end product. Such enzymes are called allosteric enzymes, Feedback inhibition is generally reversible, that is, the bound end product can dissociate from the enzyme, thereby restoring enzymatic activity. Feedback inhibition allows a cell to consume precursors and energy from a metabolic pool at a rate essential for efficient biosynthesis, but no faster. Allosteric inhibition must be distinguished from competitive inhibition, in which a substrate and an inhibitor compete for the same catalytic site on the enzyme. Metabolic activity is also controlled at the transcriptional level. Genes do not continuously make mRNA but are switched on or off by binding or dissociation of repressors. Classically, the structural genes controlling a biosynthetic pathway are repressed when the product is supplied exogenously. Repression must be clearly differentiated from end-product inhibition; repression affects enzyme synthesis, whereas allosteric inhibition affects enzyme action. The genes subject to repression have a com-

146

S. G .

BRADLEY AND J . S. BOND

plex organization. The region to which DNA-dependent RNA polymerase attaches is the promoter region. The region that determines whether or not the RNA polymerase will make mRNA is the operator. Contiguous to these two controlling regions are the corresponding structural genes which may code for a few or many enzymes. The overall genetic unit, which consists of the promoter, operator, and structural genes, has been designated the operon. In repression, a regulatory gene, which is usually some distance away from the operon, produces an inactive or unstable repressor. The aporepressor (or inactive repressor) must react with the anabolic product or a derivative of it (the corepressor) to make an active, stable repressor that can bind to the operator. When active repressor is bound to the operator, the RNA polymerase is prevented from transcribing the DNA. When the appropriate anabolic product is not provided exogenously, the aporepressor cannot bind to the operator and the RNA polymerase is able to direct the synthesis of new mRNA. Repression may regulate catabolic as well as anabolic processes. Many bacteria can utilize particular nutrients (e.g., amino acids) as either precursors or energy sources. If an alternative energy source is supplied (e.g., glucose), the genes controlling the biosynthesis of enzymes that degrade the precursor substrate are repressed. This effect has been termed glucose repression or catabolite repression. Catabolite repression, like end-product repression, affects enzyme synthesis and not enzyme activity. Catabolite repression is specific and presumably analogous to end-product repression in biosynthetic pathways. Exogenously supplied metabolites may increase the amount of enzyme produced; such enzymes are said to be induced. Induction and repression are merely different manifestations of a similar control mechanism. In induction, the structural genes controlling a catabolic pathway are repressed unless the appropriate substrate is added. This is so because the regulatory gene normally produces an active stable repressor that binds to the operator region, thereby preventing the RNA polymerase from transcribing the DNA. Added inducer, or a derivative of it, must react with the active stable repressor to convert it to an inactive form that cannot bind to the operator region. After the active repressor is removed or destroyed, the RNA polymerase is able to direct the synthesis of new mRNA. It should be noted that an inducer may be, but is not necessarily, a substrate for the induced enzyme. Moreover, a substrate for the induced enzyme may be, but is not necessarily, an inducer. Constitutive enzymes are those whose concentrations do not fluctuate appreciably under a variety of growth conditions. An induced enzyme system may become constitutive by mutation in either the operator region or the regulatory gene. A mutation that alters the operator in such a way that it can no longer bind the active repressor will result in constitu-

MYCOBACTERIA A N D NOCARDIAE

147

tive enzyme formation. Alternatively, a mutation in the regulatory gene that prevents repressor synthesis or makes the repressor unable to react with the corresponding inducer (or its derivative) will lead to constitutive enzyme formation. Similar mutations in the operator region or regulatory gene controlling an anabolic pathway will result in derepression of the pathway. Such derepressed mutants, which overproduce an end product, are used extensively in the fermentation industry to produce amino acids and vitamins. A. REGULATION IN ACTINOMYCETES Many mycobacteria and some nocardiae are intensely pigmented when grown in the light but are pale or colorless when grown in the dark. Colonial morphology of mycobacteria and nocardiae is altered by the addition of malachite green to the culture medium. Three are myriads of examples of nonhereditary alterations in the phenotype of an organism (Bradley, 1970). A potential substrate may induce the production of a new enzyme; alternatively, an externally supplied amino acid may repress the synthesis of its biosynthetic enzymes. Repression does not always involve obviously related metabolites. Glucose, for example, inhibits the synthesis of a number of catabolic enzymes. Because many of the immediate products of catabolism frequently inhibit synthesis of a particular enzyme, the more general term catabolite repression is preferable to glucose repression. Moreover, in some actinomycetes, amino acids catabolically repress glucose utilization. The phenotype of a cell is also affected by less well characterized factors. At elevated temperatures, some enzymes whose essential products can be exogenously supplied are denatured. Such microbes therefore are nutritionally dependent on exogenous growth factors at elevated temperatures but are nutritionally sufficient at lower incubation temperatures. Moreover, exogenous factors may prolong or shorten the biological half-lives of cellular structural and enzymatic proteins. The ultimate consequences of regulation of the rates of protein degradation are similar, but in the reverse direction, to effects on protein biosynthesis. It is obvious that a microorganisni may manifest a variety of attributes depending upon cultural conditions. In taxonomic studies where it is necessary to compare phenotypes, constant controlled cultural conditions are essential, but practically never achieved.

B. CATABOLISM IN Mycobacterium Glycerol is the primary carbon source employed in the cultivation of mycobacteria although they can also use glucose (Ramakrishnan et al., 1972). M . phEei cells grown on glycerol medium and glucose medium

148

S . G. BRADLEY AND J. S. BOND

have identical growth rates as measured by protein or DNA content, but glycerol-grown cells have more mass per unit volume than glucosegrown cells. The increased weight of glycerol-grown cells is attributable to an increased lipid and polysaccharide content. The uptake and utilization of glucose and glycerol by M . phlei are different. The rates of glycerol uptake, respiration and assimilation are saturated at a low substrate concentration, whereas those for glucose do not show saturation even at high substrate concentrations. It has been proposed that glycerol, at least in M . smegmatis, is phosphorylated and the resulting glycerol 3-phosphate converted subsequently to pyruvate. The key enzymes of both the glycolytic and the hexose monophosphate pathways are both present in glucose-grown M . tuberculosis. Glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are not detected in glycerol grown cells, indicating that glycolysis is predominant over the hexose monophosphate shunt. In glycerol-grown M . phlei however, key enzymes of both the glycolytic and hexose monophosphate shunt pathways are found. Although mycobacteria seem to prefer glucose or glycerol for a carbon source, they are generally able to use myo-inositol, D-mannitol, mannose, fructose, and ethylene glycol. It is probable that cells do not maintain alternative pathways merely as optional activities; rather they seem to have different functions although they may replace each other when necessary. In general, glycolysis ( i.e., the Embden-Meyerhof-Parnas ( EMP ) pathway) generates NADH for energy whereas the hexose monophosphate shunt generates pentose phosphate for biosynthesis. Acetone-dried mycobacterial cells are able to oxidize all the intermediates of the tricarboxylic acid cycle. The respiratory chain in M . phlei is illustrated in Fig. 2. Moreover, many of the enzymes of the tricarboxylic acid cycle have been purified from extracts of M . tuberculosis. Resting cells of mycobacteria grown on glucose or glycerol usually do not oxidize most of the tricarboxylic acid intermediates, presumably because of restrictive permeability. Intact cells of M . smegmatis, grown on fumarate and acetate, however, oxidize these intermediates. In addiSubstrate-NAD

~ F l a v o p r o t e i n

FIG. 2. The respiratory chain in Mycobacterium phki. KeH = naphthoquinone vitamin KoH; b, cl, c, a, and a3 = cytochromes.

MYCOBACTERIA AND NOCARDIAE

149

tion, carbon dioxide is fixed into malonate by M . avium. The glyoxylate bypass pathway is operative in extracts of M . tuberculosis. Intermediates of the tricarboxylic acid cycle are continuously drawn off and used as precursors of amino acids. The tricarboxylic acid intermediates, therefore, must be continuously replenished if the cycle is to continue functioning. This usually happens by the carboxylation of pyruvate to yield oxaloacetate. However, as mentioned above, many mycobacteria are able to utilize acetate as the sole carbon source. If the tricarboxylic acid cycle is to continue to function under these conditions, there must be another means of replenishing the tricarboxylic acid cycle intermediates. The glyoxylate bypass meets this need. A number of amino acids are effectively taken up by mycobacterial cells and promote growth: alanine, glutamate, asparagine, and aspartate. The uptake of L-glutamate in M . avium follows Michaelis-Menten kinetics whereas the uptake of D-glutamate in M . avium and M . smegmatis proceeds by passive diffusion. The uptake of D-alanine in M . smegmatis is an active process displaying saturation kinetics characteristic of an enzymatic process whereas the uptake of L-alanine, L-glutamine, and D- and L-valine takes place by both active and passive processes. The passive process is sensitive to sulfhydryl blocking agents and shows competition among structurally related amino acids, indicating that the passive process is a facilitated diffusion. Asparagine is the preferred source of nitrogen for growth of mycobacteria and also serves as a carbon source for growth. Washed cells of M . tuberculosis deamidate asparagine to aspartic acid and ammonia. In some mycobacteria, there is an inducible asparaginase and a constitutive asparaginase which differ in pH optima, inhibition by L-aspartate and sensitivity to general enzyme inhibitors. In addition to asparaginase, there is an aspartotransferase which catalyzes the transfer of the amino group of asparagine to hydroxylamine, forming aspartohydroxamic acid. Glutamine can substitute for asparagine as a nitrogen source for growth of mycobacteria, though the cellular yield is less. Washed cells of M . tuberculosis and M . smegmatis grown in Sauton’s medium oxidize glutamate after a lag period but when the culture is grown in a modified medium containing glutamate, there is no lag. It has been proposed that the lag is due to induction of a glutamate transport system. IN Nocardia C. CATABOLISM

Cerb6n and Ortigoza-Perado (1968) reported that the uptake of D-glUCOSe and D-mannose by N . asteroides is dependent on an optimal phosphate concentration. They further indicated that glucose uptake is by facilitated diffusion, while the system for mannose requires energy.

150

S.

G.

BRADLEY AND J . S. BOND

In addition, Calmes and Deal (1972) showed that glycerol uptake in N . asteroides is constitutive and carrier-mediated at substrate concentrations less than 80 p M . They did not determine whether glycerol uptake is energy dependent. However, at glycerol concentrations above SO pM, glycerol is accumulated by passive diffusion. Brown and Clark (1961) first reported evidence !or the tricarboxylic acid cycle in N . C O T ~ ~ M Subsequently, . Cain et al. (1968) were able to show the conversion of succinate to citrate, and the presence of succinate dehydrogenase, fumarase, malate dehydrogenase, and citrate synthetase in N . eythropolis. Evidence for the presence of the EmbdenMeyerhof-Parnas pathway in N . coralha (Brown and Reda, 1967), the pentose cycle in N . coraZZina an N . opaca (Brown and Clark, 1966; Probst and Schlegel, 1973) and the Entner-Doudoroff pathway (Fig. 3) in N . opaca (Probst and Schlegel, 1973) have aIso been reported. Rann and Cain (1969) and Probst and Schlegel (1973) reported that certain catabolic enzymes are inducible in N . opaca. Rann and Cain showed that both hydroxybenzoate and benzoate eIicit the induction of all the enzymes of both the protocatechuate and catechol pathways although the two pathways are biochemically quite distinct. The induction pattern indicates coordinate induction of the different enzymes in the pathway. Probst and Schlegel ( 1973) reported that 6-phosphogluconate dehydrogenase and 2-keto-3-deoxy-6-phosphogluconate aldolase, two key enzymes in the Entner-Doudoroff pathway, are induced by fructose or glucose in the growth medium. They indicated that these two enzymes are repressed by hydrogen gas. Specific catabolic systems for oxidation of asparagine, mannitol, and succinate in five strains of N . erythropolis, gluconate in four strains and glycerol in two strains are inducible as measured manometrically. Gluconate- and glucose-metabolizing enzymes in one strain and enzymes responsible for glycerol oxidation in another strain of N . erythropolis are considered constitutive because the ability of the cells to oxidize Ihese substrates remains essentially at a constant level regardless of ATP

G LU C 0S E

G LUC 0S E - 6 - PH 0s PHAT E I

FIG.3. The Entner-Doudoroff pathway.

MYCOBACTERIA AND NOCARDIAE

151

the composition of the growth medium. It should be noted that asparagine, gluconate, and succinate support good growth of all five strains, whereas glucose supports good growth of only one strain, glycerol of two strains and mannitol of three strains. The enzymatic activities of N . erythropolis grown in peptone-yeast exiract medium necessary to oxidize asparagine vary with cultural age, indicating temporal control in the utilization of asparagine. The precise mode of this control remains to be determined. It is possible that these variations reflect changes in the ability of the substrate to penetrate the cells. Alternatively, it is possible that accumulation or leakage of an inducer may be the determinative factor. Glucose uptake activity by cells of N . erythropolis is considered constitutive because the rate of uptake of this hexose is essentially the same regardless of the composition of the medium. This uptake activity is specific in that D-2-deoxyglucosedoes not inhibit glucose uptake. Glucose uptake activity is saturable and is energy dependent in that 1 mM 2,4dinitrophenol, sodium azide, or KCN inhibits the rate of uptake. Accordingly, glucose uptake in N . erythropolis is by active transport. The apparent K , for glucose uptake is 250 ,AM. Glycerol uptake by induced and uninduced N . erythropolis cells is saturable; but when cells are induced in glycerol, the rate of uptake is increased 4-fold. These data indicate that there is an inducible and a constitutive component in the glycerol transport system. Glycerol uptake is carrier mediated. Uptake of succinate by induced cells of N . erythropolis is by active transport. The apparent K,, fot succinate uptake is 40 p M . Similarly, uptake of mannitol in N . erythropolis is inducible and carrier mediated. The addition of succinate to a medium containing mannitol as the nutritional inducer does not completely inhibit the induction of mannitol uptake activity by succinate-grown or glucose-grown cells. Succinate does, however, reduce the extent of induction of the mannitol transport system. Hexokinase, glucose-6-phosphate dehydrogenase ( N ADP+-linked) , membrane-bound glucose dehydrogenase, and mannitol dehydrogenase ( NAD+-linked) activities are inducible in N . erythropolis. Glucose is thought to be dissimilated through the Embden-Meyerhof-Parnas pathway in glucose-grown N . erythropolis because fructose-1,6-diphosphate aldolase and hexokinase activities were higher in glucose-grown cells than in asparagine-grown cells. In addition, glucose may be degraded through the oxidation of glucose in gluconate by the induced membranebound glucose dehydrogenase and further dissimilated through the constitutive hexose monophosphate shunt because high activities of constitutive 6-phosphogluconate dehydrogenase, the first key enzyme in the pathway have been detected. Gluconate is thought to be dissimilated in

152

S. G. BRADLEY AND J . S. BOND

gluconate grown cells by the hexose monophosphate shunt and by the Embden-Meyerhof-Parnas pathway although to a lesser extent when compared to the dissimilation of glucose by glucose-grown cells (Fig.

4). Mannitol may be degraded in mannitol-grown cells by conversion to fructose by mannitol dehydrogenase ( NAD+-linked) and then further dissimilated by the Embden-Meyerhof-Parnas pathway. In addition, some of the intermediates of mannitol catabolism are probably dissimilated through the hexose monophosphate shunt because the activity of glucose6-phosphate dehydrogenase is high. In N . erythropolis, both the Embden-Meyerhof-Parnas pathway and the hexose monophosphate shunt are operative and the latter is constitutive. The failure of gluconate to induce 2-keto-3-deoxy-6-phosphogluconatealdolase indicates that the Entner-Doudoroff pathway is not intact in N . erythropolis. However, in N . opaca, both 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6phosphogluconate aldolase are induced by gluconate and fructose, indicating the presence of a functional Entner-Doudoroff pathway. In N . erythropolis, the substrate serving as the sole carbon source does not invariably lead to the complete repression of the utilization of other substrates. In fact, some substrates induce or derepress catabolic enzymes other than those required for their utilization. In N . erythropolis,

-

GLUCONATE

GLUCOSE

-

6-PHOSPHO GLUCONATE

/\

ETHANOL

I

GLUCOSE 6-PHOSPHATE

PYRUVATE

...-..'.

MANNITOL

I

b

FRUCTOSE

1

FRUCT SE 1.6DIPHOSPHATE

/*

c *.%. DIHYDROXYTRICARBOXYLIC 3-PHOSPHOACETONE ACID CYCLE GLYCERALDEHYDE PHOSPHATE

f

i

/I SUCCINATE

=,.

.' \.

.'

\,

ASPA RAGI NE

i

GLYCEROL

FIG.4. Proposed metabolic pathways for Nocardia erythropolis ( syn. Proactiptomyces opucus) . Solid arrows: demonstrated biotransformations; dashed arrows: proposed biotransformations.

MYCOBACTERIA AND NOCARDIAE

153

glucose derepresses mannitol utilization as determined by manometric, transport, and enzyme studies whereas succinate somewhat reduces the induction of the mannitol catabolic system. Catabolic repression has not been demonstrated in N . erythropolis. VI.

Degradation of lntracellular Proteins

The regulation of cellular metabolism is dependent on the amount or concentration of specific enzymes and their activities. The role of protein degradation in determining the concentrations and activity of enzymes in cells has been a relatively neglected area of study. In the last 10 years, however, a good deal of evidence has accumulated, demonstrating that intracellular protein degradation plays an important role in determining the steady-state concentrations of many enzymes as well as fluctuations of those concentrations. In both prokaryotic and eukaryotic cells, the process seems to be particularly important for the adaptation of cells to changing environmental conditions, protein modification, and the elimination of abnormal or nonfunctional proteins. The literature on turnover of intracellular proteins in bacteria has been reviewed recently by Pine (1972) and in animal cells by Rechcigl (1971) and Schimke and Doyle ( 1970). A. RATESOF PROTEINDEGRADATION

It is established that the proteins of mammalian cells are continually being degraded and replaced by synthesis ( Schimke, 1969). This “dynamic state” was recognized by Schoenheimer (1942) in the 1940’s but not until recently has the extent of this degradative process or the heterogeneity of degradation rates for individual enzymes been appreciated. The vast majority of work on the half-lives of individual proteins and cellular components has been done with rat liver. The life-span of an adult liver cell has been estimated at 160-400 days (Schimke, 1969). The mean half-lives of proteins in cell fractions (Arias et al., 1969), however, are as follows: homogenate, 3.3 days; nuclear, 5.1 days; mitochondrial, 6.8 days; lysosomal, 7.1 days; microsomal, 3.0 days; supernatant, 5.1 days; endoplasmic reticulum ( smooth or rough), 2.0 days; plasma membrane, 1.8 days. This means, for example, that in a cell that “lives” 160 days, half of the proteins associated with the plasma membrane are replaced by new proteins in 1.8 days. The half-lives of these proteins are estimated by pulse-labeling rats with ~-arginine-guanidino-l~C. Liver homogenates are prepared from different rats at various times (e.g., 1, 3, 5, 7 days) after the injection of the label. The homogenates are fractionated and the counts per milligram

154

S. G. BRADLEY AND J. S. BOND

of protein pIotted vs time on semilogarithmic graphs to estimate halflives. The arginine-guanidino-'"C label has been selected for studies of protein turnover in rat liver because arginase activity in this tissue is high and prevents extensive reutilization of arginine after protein degradation. Other amino acids ( e.g., leucine-14C) are reincorporated into protein to a greater extent than arginine, and reincorporation results in underestimates of half-lives. Since radioisotopic labeling, with subsequent determination of decay of label, is the major technique used to estimate half-lives, this is a very important consideration. Even arginine, however, may be reincorporated. Arginine resulting from protein degradation may not mix with other pools of amino acids or with compartments containing arginase. The cellular site of protein degradation is unknown at this time. It has been suggested (Walter, 1960) that amino acids resulting from degradation of proteins are more readily incorporated into new proteins than those coming into the cell or in other pools. If this is the case, the above estimates of half-lives of cellular components are low and protein turnover is even more extensive than present estimates. Even assuming that this is not the case, the data on the turnover of cellular components are impressive. The protein concentration of the adult liver cell does not change, but the existing pool of proteins is in a constant and rapid flux. The continual turnover of protein in animal cells has often been contrasted to a lack of demonstrable turnover in growing bacteria. However, it is now becoming apparent that there is a continuous process of protein degradation during exponential growth of bacteria as well as in starved, lag phase, stationary phase, and sporulating bacteria (Fig. 5 )* The occurrence of protein turnover in nongrowing populations of Escherichia co2i (no net synthesis of protein) has been demonstrated in starved mutant strains with specific amino acid requirements (Mandelstam, 1958) . The incorporation of glycine-''C into protein is measured to assess synthesis; the release of leucine-"C from prelabeled proteins is used to estimate protein degradation. The results show that starved bacteria release more than three times as much labeled leucine as the growing cultures. In the nongrowing bacteria the rate of protein synthesis is approximately equal to the rate of protein breakdown (4-58 per hour). Rates of proteolysis in starved bacteria (nitrogen, glucose, or required amino acid starvation), such as in Bacillus cereus (Urba, 1959) and Pseudomoms saccharophila ( Young and Klein, 1967) and various strains of Escherichia coli (Nath and Koch, 1971), are generally found to be in the range of 2-6.5% per hour. From these values, a mean rate constant for degradation can be calculated, and consequently a mean half-life for proteins in bacteria can be determined. Expressing protein turnover

155

MYCOBACTERIA AND NOCARDIAE

0

3

2

I

HOURS

FIG.5. Protein degradation in Nocardia erythropolis ( syn. Proactinomyces opacus). Release of leucine-"C from radiolabeled cells grown in asparagine medium containing leucine-'C was measured after subculture to fresh asparagine medium (0-0) or buffer (.------a). ( R . H. Pang, J. S. Bond, and S.G. Bradley, unpublished data).

data in terms of half-life rather than percentage degraded per hour has some advantages. For instance, the extent of the turnover can be more easily thought of in terms of the lifetime of the cell. Also, since most of the data on turnover of individual proteins and cellular components are expressed in terms of half-life, this expression of new data will facilitate comparisons with the literature. Protein degradation is a first-order process (Berlin and Schimke, 1965) and thus the rate of degradation of a protein ( P ) can be described by the equation -[d(P)]/dt

=

k(P)

where k is the rate constant for degradation. Rearranging and integrating: -[d(P)I/(P) = k dt -In (1') = kt C when t = 0, C = -ln(Po)

+

where (Po)is the protein concentration at zero time. -ln(P) = kt - ln(P0) ln(P0) - In(P) = kt In[(Po)/(P)I = kt 2.3 log [(Po)/(P)]= kt

156

S. G. BRADLEY A N D J . S. BOND

If 5%of the protein is degraded per hour, then 2.3 log [(Po)/(0.95Po)l= k (1 hr) k = 0.05 hr-1

Half-life ( tl,z) is related to k: t i / z = 0.69/k

In this example, then fIl2 = 0.69/0.05 hr-I t 1 / 2= 13.8 hours

This then is an estimate of the mean half-life of proteins in nongrowing bacteria where the rate of degradation proceeds at 5% per hour. For 2%per hour, it would be 37.5 hours; for 6.5%, 10.35 hours. It should be noted, however, that individual proteins have vastly different half-lives. Since it has been estimated that 70%of bacterial cell protein “cannot be decomposed under any conditions” (Pine, 1972) (or at least they turn over very slowly), some proteins must turn over extremely rapidly in order to achieve these average half-lives.

B. HALF-LIVES OF ENZYMES The heterogeneity of half-lives of individual enzymes is striking (Table 11). In liver, the half-lives of enzymes that have been measured, range from 11 minutes (ornithine decarboxylase) to 20 days (NAD glycohydrolase). The data also reveal that there is no clear-cut correlation between the half-life of an enzyme and the cellular fraction in which TABLE I1

In Vivo HALF-LIVES OF SOME ENZYMES Enzyme

Half-lifea

Ornithine decarboxylase 6-Aminolevulinate synthetase Tyrosine aminotransferase Tryptophan oxygenase Glucokinase Catalase Alanine aminotransferase Arginase NAD glycohydrolase

11 Min 70 Min 1.5 Hr 2.5 Hr 12 Hr 1 Day 3 Days 4.5 Days 20 Days

~

These values are estimates of half-lives in r a t liver taken from a more complete list compiled by Rechcigl (1971).

MYCOBACTERIA AND NOCARDIAE

157

the enzyme is found (Schimke et al., 1968). In this regard, the half-life of the bulk of mitochondria1 protein of rat liver has been estimated at 8.5 to 10 days, while cytochrome b,, associated with the outer mitochondrial membrane has a half-life of 4 5 days and 6-aminolevulinic acid synthetase with a half-life of 60-74 minutes is also present in mitochondria. Enzymes associated with the endoplasmic reticulum, such as NADP-cytochrome c reductase (half-life = 3 days) and cytochrome b, (half-life = 5 days), also turn over at different rates, as do soluble enzymes in the cell cytoplasm (e.g., tryptophan oxygenase, half-life = 2.5 hours, and arginase, half-life = 4-5 days). Peroxisomal enzymes, however, may be an exception to this rule. Poole (1969) has presented data showing that the protein components of peroxisomes have similar turnover rates, suggesting that peroxisomes are destroyed as whole particles. Muscle proteins have, as a general rule, longer half-lives than liver proteins. The mean half-life of liver proteins from young male rats, for instance, has been estimated at about 1 day while the estimate for muscle proteins was about 5 days (Garlick, 1969). Comparisons of the halflives of specific enzymes in the two tissues also show this to be true. Thus alanine aminotransferase in liver has a half-life of 3 days, and in muscle it is 20 days (Segal et al., 1969); cytochrome c in liver has a half-life of 6 days as compared to 32 days in muscle (Terjung et al., 1973). In the case of alanine aminotransferase, the enzyme has been purified from these two tissues and found to be identical by a number of criteria ( electrophoretically, immunochemically, and in pH, salt, and heat stability). Thus, the differences in in vivo half-life must be due to the different cellular environments. C. EFFECTS OF METABOLICCONDITIONS

Degradation of protein in growing Escherichia coli has now been measured in several laboratories (Nath and Koch, 1971; Pine, 1970; Goldberg, 1972a). It is agreed that it is a normal and continuous process in growing cells, and it appears to occur at a somewhat slower rate than in starved cells. Pine (1970) has estimated that 2 5 3 %of the protein is degraded per hour (mean half-life about 23.5 hours). This is in the lower range of rates found in starved bacterial cells but may be an underestimate if reincorporation of labeled amino acids is efficient. During continuous exponential growth, a very rapid turnover of approximately 5%of the labeled protein occurs. This release of amino acids is seen within 45 seconds and is thought to represent processes involving modifications of proteins. These reactions involve cleaving off certain

158

S. G . BRADLEY AND J . S. BOND

amino acids, or amino acid sequences, which are not present on the active, completed protein; for example, the removal of N-terminal methionine residues of many proteins ( Sarimo and Pine, 1969). A group of “rapidly degrading protein” (half-lives up to an hour) is observed in growing cells and starved cells (estimated at 1-7% of the protein). These proteins are degraded in a first-order process as usual. It has been proposed that these proteins represent abnormal proteins ( malformed, nonfunctional, or incomplete) which are more susceptible to proteolysis than normal (functional) proteins. In support of this proposal, are experiments showing that incorporation of puromycin or various amino acid analogs into proteins results in increased proteolysis in E . coli (Goldberg, 1972a). These proteins are degraded at similar rates in growing and nongrowing cells. The idea that normal proteins in bacterial cells turn over at relatively rapid rates (in growing cultures) has not really been tested, however. During starvation, approximately 35% of the proteins synthesized are unstable (half-lives = 3 minutes) and it has been suggested that this is due to the formation of incomplete peptide fragments (molecular weights below 20,000) which are vulnerable to proteolytic attack ( Brunschede and Bremer, 1971). This is consistent with the findings that p-galactosidase synthesis is drastically reduced (rate of synthesis is reduced to 0.7%)during starvation, but not due to reduced synthesis of p-galactosidase messenger RNA. The enzyme is stable once completed but the synthesis of peptide fragments could explain the marked reduction of enzyme produced when growing cells are starved. There is a group of proteins (estimated at 20-30s of the total) which appears to be susceptible to proteolysis under conditions of starvation but not in time of plenty (Nath and Koch, 1971). Protein inhibitors ( phenyl methane sulfonyl fluoride or p-toluenesulfonyl fluoride) can inhibit this increased degradation in starving E . coli whereas these same inhibitors do not inhibit degradation of abnormal proteins (Prouty and Goldberg, 1972). On the basis of these findings, two discrete proteolytic systems have been proposed: a system present in growing and nongrowing cells mainly active in hydrolysis of abnormal or incomplete proteins and an additional system, “serine-protease,” activated during starvation to permit new enzyme synthesis. The half-lives of proteins can and do change under different metabolic conditions. Drugs, hormones, ligands for specific enzymes and diet are among the factors that can affect degradation rates. For instance, phenobarbital reduces the rate of degradation of cytochrome b, and NADPHcytochrome c reductase while the half-lives of other microsomal proteins are not affected (Kuriyama et al., 1969; Arias et al., 1969). Insulin inhibits proteolysis in perfused livers (Mortimore and Mordon, 1970)

MYCOBACTERIA AND NOCARDIAE

159

whereas glucagon stimulates the process (Mallette et al., 1969). Administration of tryptophan or a-methyltryptophan stabilizes tryptophan oxygenase in vitro and in vivo (Schimke et al., 1965) and iron decreases the degradation of ferritin ( Drysdale and Munro, 1966).

D. MECHANISMOF PROTEIN DEGRADATION The mechanisms or machinery responsible for degradation of intracellular proteins is as yet unknown. Thus, the site of degradation, proteases involved and initiating events are not well characterized or understood. At least three types of proteolytic systems have been proposed for the process in eukaryotic cells, and one or more of these may be active in vivo. One type includes neutral proteases, demonstrated in the brain, lens, and liver mitochondria. A second type includes acid cathepsins associated with lysosomes. There is little doubt that these cathepsins are active in some type of intracellular digestion, specifically in cellular autophagy (bulk segregation and digestion of portions of the cell cytoplasm) and in the digestion of exogenous proteins that are taken into cells by pinocytosis. Nevertheless, there still remains a question as to whether they play a significant role in the degradation of intracellular proteins under normal physiological conditions. A third type includes specific degradative enzymes for different proteins or groups of proteins. Significantly, a protease specific for pyridoxal enzymes has been reported in rat small intestine and skeletal muscle. Other intracellular proteases may exist. It is possible for instance, that proteases are short-lived in vivo and detectable only when their synthesis is stimulated or their degradation inhibited. Proteases may also exist ordinarily in inactive forms (as chymotrypsin and trypsin do in the pancreas ) . Also, there are numerous well characterized protease inhibitors that have been isolated from microorganisms ( Umezawa, 1972) as well as from plants and animals (Kassell, 1970). Their function is unknown at present, but the widespread occurrence of these inhibitors might indicate that they act as regulators of proteolysis in vivo. Another aspect of the process of intracellular protein degradation that remains unresolved concerns the question of why some proteins are degraded more rapidly than others. It appears that many factors may share in determining in vivo half-lives of proteins. Among the factors that have been suggested are (1) molecular weight; larger proteins are generally degraded more rapidly than smaller ones (Dice et al., 1973); ( 2 ) susceptibility to proteolysis (consequence of peptide bonds that are exposed and conformation); enzymes with short in vivo half-lives are more readily inactivated by proteases in vitro (Bond, 1971); proteins with short in vivo half-lives are more readily digested by proteases

160

5. G. BRADLEY AND J . S. BOND

in vitro (Goldberg, 1972b) ; ( 3 ) conformation of protein as regulated by coenzymes, substrates or other ligands (Litwack and Rosenfield, 1973; Bond, 1973); ( 4 ) susceptibility to lysosomal degradation; lysosomes may act as sieves for intracellular proteins (Haider and Segal, 1972); ( 5 ) presence of specific proteases in cells [specific for an enzyme (Kenney, 1967) or for certain groups of enzymes, such as pyridoxal-phosphate enzymes (Kominami et al., 1972)l; ( 6 ) degree of attachment to membranes or chromatin; e.g., nuclear proteins appear to be much more vulnerable to proteolysis after disassociation from chromatin ( Dice and Schimke, 1973). In addition to these factors, chemical modifications of enzymes (e.g., phosphorylation, carboxylation, acetylation, glucosylation) may be determinants in the initiation of degradation (Grisolia and Hood, 1972). These processes are known to alter protein configurations and therefore have the potential to alter rates of degradation.

E. BIOLOGICALSIGNIFICANCE The process of intracellular protein degradation is involved in the regulation of enzyme and protein content of cells and in the ability of cells to adapt or adjust to various environmental conditions. For example, mammalian cells, of constant size, are continually being exposed to different nutrients in the extracellular fluid (after meals, exercise, hormonal secretion). They must be able to adapt to new situations, and sometimes this requires new enzyme synthesis. A fairly static cell population must be able to degrade or otherwise dispose of some of its protein components in order to synthesize others. For many of the intracellular enzymes there are good reasons to believe that the disposal is accomplished by intracellular degradation ( Schimke, 1969). A population of growing and dividing cells may dilute out unnecessary or defective proteins in successive generations of daughter cells, but this mode of regulation of the quantity or quality of proteins (enzymes) is not available to long-lived cells of constant size. In cells which are deprived of required amino acids, protein degradation again is important for adaptation. In E . coli when no net synthesis of protein is occurring (e.g., during amino acid starvation), p-galactosidase can be induced, indicating that net synthesis of this particular type of protein does occur (Mandelstam, 1958). This is possible only by virtue of the degradation of other proteins which allows sufficient flow of material through amino acid pools to enable cells to synthesize inducible enzymes. In eukaryotic cells where there is continual degradation of all components of the cell, the steady-state level of an enzyme is dependent on

MYCOBACTERIA AND NOCARDIAE

161

its rate of degradation (or its first-order rate constant for degradation, k) and its rate of synthesis. If two enzymes are synthesized at the same rate, the one with the smaller rate constant for degradation (longer half-life) will have a higher steady-state level. The rate constant for degradation also is a factor in determining how rapidly the concentration of that enzyme will fluctuate in response to inducers or other stimuli (such as protein synthesis inhibitors ) . Enzymes with short half-lives (large k) will display a rapid drop in concentration in response to a protein synthesis inhibitor and can be induced rapidly in response to an increase in synthesis (Berlin and Schimke, 1965). Enzymes with longer half-lives show much more sluggish responses to changes in synthesis. Thus, protein degradation not only influences steady-state levels of enzymes but also the rate of change of enzyme concentration from one steady-state level to another. Protein degradation appears also to serve a salvage or part of a restoration function. This is best documented in prokaryotes. Thus, when amino acid analogs are incorporated into protein or nonfunctional enzymes are produced or incomplete protein fragments are formed, increases in the rate of protein catabolism are observed. The degradative process is a mechanism to dispose of defective proteins. Nonfunctional proteins have the potential to retard optimal functioning of cells. Protein catabolism may actually be a most important function in normal growing cells. Despite the remarkable apparent accuracy of the protein synthesizing system, mistakes in the amino acid sequence of proteins may occur. These mistakes may or may not affect the activity or stability of proteins, but if they are detrimental, the process of protein degradation provides one mechanism to remove the defective molecules. Another important function of this catabolic process may be reclamation of proteins inactivated by environmental stimuli ( Dinman, 1972). For instance, proteins may be inactivated by irradiation, heavy metal contamination, or environmental pollutants. The process of intracellular degradation seems to be of fundamental importance to living organisms, and yet the mechanisms involved are not understood at all. One possible approach to deciphering mechanisms and functions of the process would be to use specific protease inhibitors as biochemical tools. The numerous well described, naturally occurring inhibitors produced by microorganisms, particularly the actinomycetes (Umezawa, 1972), are potentially useful for both basic research and clinically related studies. One protease inhibitor, pepstatin, isolated from filtrates of actinomycetes, is already used clinically in the treatment of stomach or duodenal ulcers. Pepstatin is a specific and powerful inhibitor of pepsin and is used as an extracellular protease inhibitor. Intracellular proteases have been implicated in the growth of cancer (Troll

1.62

S. G. BRADLEY AND J . S. BOND

et al., 1970), and hence, the screening of inhibitors for these enzymes might well prove to be a very fruitful area of research. VII.

DNA Analyses

Genetic relatedness can best be measured by comparing the homology of genetic macromolecules from different strains. Genetic homology is defined in biological terms as the similarity between sequences of loci, but in biophysical terms it is the similarity between nucleotide sequences within DNA molecules. The Watson and Crick model of double-stranded DNA predicts that the guanine ( G ) content of a DNA molecule equals its cytosine content ( C ) ; likewise, the adenine ( A ) and thymine ( T ) contents are equal. However, the mole percent of guanine cytosine [that is, %GC= ( G + C ) l O O / ( A + T + G f C ) ] may vary from one species to another. Closely related organisms have very similar nucleotide sequences and therefore very similar GC ratios. Similarity in %GC does not necessarily indicate genoniic similarity but, unlike %GCof DNA from two organisms, establishes that they are not identical. The relationships among selected actinomycetes, with special emphasis on mycobacteria, nocardiae, and streptomycetes have been determined, based upon the nucleotide composition of their DNA. The mycobacterial and nocardial DNA preparations contain 61-694: GC (Tarnok et al., 1967; Wayne and Gross, 1968). The streptomycetes constitute a homogeneous group whose DNA contains between 69 and 74%GC.

+

A. DNA: DNA ASSOCIATION Because the phenotype of an organism is determined by the nucleotide sequence of its DNA, a comparison of the nucleotide sequences of DNA preparations from two organisms should give a definitive evaluation of their relatedness. Unfortunately, complete direct sequence analysis of a DNA molecule is not possible at this time. The complementary nature of the DNA double helix, however, can be used to circumvent these technical difficulties. It has been established that the two strands of the DNA helix can be separated and specifically associated or annealed. The phenomenon of association of complementary strands of DNA provides a powerful tool for exploring the relationships among microorganisms. Investigators analyzing DNA preparations with 60-70% GC encountered many problems with earlier hybridization methods ( Yamaguchi, 1967). Accordingly, my colleagues and I have modified the method of Warnaar and Cohen (1966) for quantitative assay of association between denatured DNA fixed to nitrocellulose membrane filters

MYCOBACTERIA AND NOCARDIAE

163

and free, denatured DNA, We have used this modified technique to assess, on a molecular level, the relationships among representatives of many genera of actinomycetes, in particular Mycobacterium, Nocardia, and Streptomyces. The relationships among these organisms are of particular interest because of their special relevance for industry, medicine, and agriculture. Moreover, the taxonomy of the actinomycetes remains a subject for active study and debate. The rate of association of complementary single-stranded DNA molecules is dependent upon a number of experimental conditions: ( 1 ) it is proportional to the square root of the molecular weight of the single stranded DNA; ( 2 ) it is slightly faster for DNA with a high %GCthan in DNA with a low %GC; ( 3 ) it is dependent upon the salt concentration of the diluent; ( 4 ) it is inversely proportional to the viscosity of the diluent; ( 5 ) it is dependent on the concentration of the DNA; and ( 6 ) it is dependent upon the purity of the DNA preparation. The rate of association of complementary single-stranded DNA is independent of pH between 5 and 9 at high salt concentrations. Impurities in actinomycete DNA preparations may interfere with studies on nucleotide composition or the kinetics and extent of association by single-stranded DNA. Wayne and Gross (1968) noted that one of their mycobacterial DNA preparations contained a substance that prevented determination of the nucleotide composition. Impurities in DNA preparations are indicated by ( a ) opalescence of the DNA solution; ( b ) an A2B,j/A.'I0ratio less than 2.0; ( c ) an A2G5/A230 ratio less than 2.2; ( d ) RNA detected by the orcinol assay (Hatcher and Goldstein, 1969); or ( e ) protein detected by the Lowry assay (Lowry et al., 1951). Hill et al. (1972) have used agar-gel diffusion of a DNA preparation against concanavalin A to detect contaminating polysaccharides. The agar gel contained 8.5 gm NaCl, 0.12 gm K2HP04,5 gm sodium azide, and 1 gm Ionagar No. 2 (Colabs Inc., Glenwood, Illinois) in 1 liter of distilled water. The final pH was adjusted to 7.6 with 1 NaOH. Wells (ca. 3 mm in diameter) were cut in a circular pattern around the center well (ca. 1 cm between each well). The center well was filled with concanavalin A (2.5 or 5.0 mg/ml); the peripheral wells were filled with the DNA sample (ca. 500 pg/ml). Precipitation bands indicate the presence of contaminating carbohydrate (Fig. 6, Tables 111 and IV). Both Hill et al. (1972) and we have detected carbohydrate in DNA preparations prepared from M . kansasii and M . tuberculosis. The arabinogalactan isolated from the cell wall of M. bovis reacted to form a precipitate with concanavalin A. Inhibitor studies indicate that concanavalin A reacts with arabinogalactan by interacting with the C-2, C-3, and C-5 hydroxyl groups of the a-D-arabinofuranosyl residues situated at the ends of the polysaccharide chains. Moreover,

164

5. G . BRADLEY AND J . S. BOND

FIG.6. Demonstration of carbohydrate contamiaating DNA preparations. Bands of precipitation (indicated by arrows) are formed between the center well containing 0.2 ml of 2.5 mg concanavalin A per milliliter and certain peripheral wells filled with DNA samples that contain contaminating carbohydrate. TABLE I11 DNA PREPARATIONS CONTAINING CONTAMINATING CARBOHYDRATICS

Actinomadura madurae 606 Actinoplanes philippinensis 367 Mycobacterium bovis BCG M . jarcinica 436 M . flauescens 624 M . fortuitum 471 M . intracellulare 475

'

M . kansasii 476 M . marinum 437 M . parafinicum 507 M . smegmatis 405 Nocardia cauiae 319 N. conuoluta 512 N . corallina 324, 333, 335, 339, 513

I

the concanavalin A molecule is able to combine with the unmodified hydroxyl groups at the C-3, C-4, and C-6 positions of a-D-glucopyranosyl residues ( glycogen, dextran, amylopectin) , the epimeric a-D-mannopyra-

MYCOBACTERIA AND NOCAHDIAE

165

TABLE IV DNA PREPARATIONS FREEOF CONTAMINATING CARBOHYDRATES Actinomadura madurae 302 Actinomyces discofoliatus A . israelii A . odontolyticus W1047 A . suis 852 Actinoplanes philip pinensis 367 Actinop ycnidium caerulium 449 h’scherichia coli CSH-2 Micrococcus lysodeiklicus 418 Micromonospora sp. 401 Mycobacterium avium 573 M . bovis Tm 410 M . farcinica 378, 526 M . jortuiturn 471 M . marinum 437 M . phlei 435 M . smegmatis 403, 406, 433, 477 M . tuberculosis H37Ra Mycococcus sp. 3-54 Myxococcus xanthus Nocardia asteroides 300, 323, 334, 421, 571 N . blackwellii 509 N . brasiliensis 301, 473 N . coeliaca 520

N . corallina 336, 338 N . erythropolis 305, 340, 398, 439, 446, 456, 457, 458, 469, 474 N . jarcinica 330, 420, 462 N . globerula 472, 508 N . gypsoides 515 N . opaca 331, 332, 510 N . paraguayensis 303 N . phenoltolerans 514 Nocardia sp. 304, 466, 467, 468 N . transvalensis 516 Proteus mirnbilis Pm5 Pseudomonas aeruginosa 14502 Slreptomyces albus 355, 1685 S. aureojaciens 349 S. cinnamomeus 350 S. coelicolor 348, 352 S. erythreus S. griseiis 1, 104, 360 S. rimosus 400 S. venezuelae 13 S. violaceoruber 1, 16, 199, 307, 357, 358 StreptospoTangium roseum 345

nosy1 residues (yeast, mannans, and phosphomannans), and the C-3, C-4, C-6 (or possibly C-1, C-3, and C-4) hydroxyl groups of the p-D-fructofuranosyl residues in levans (Goldstein and Mesaki, 1970). In order to free DNA preparations of contaminating carbohydrate, DNA samples prepared by the Marmur technique (Marmur, 1961) are treated with a-amylase and p-amylase (200 pglml, final concentration, Nutritional Biochemicals Corp., Cleveland, Ohio) for 1 hour at 37OC. The amylase-treated DNA solution is deproteinized with chloroform; after clarification by centrifugation, the DNA is precipitated with ethanol, harvested, and dissolved in saline-citrate diluent. The DNA is precipitated with 2-ethoxyethanol, harvested, and dissolved in saline-citrate diluent. These added steps usually remove contaminating carbohydrate.

B. OPTICALASSOCIATION The degree of association between denatured DNA samples of diverse origins can be determined quantitatively from association rates of the individual DNA preparations and their mixture. It should be noted that one of the most convenient ways to measure percent association is the

S. G . BRADLEY AND J. S. BOND

166

absorbance at 260 nm. Because dissociated DNA absorbs more ultraviolet light than associated DNA, monitoring the decrease in absorbance with time will provide the necessary information on the degree of association. It is expected that DNA association, a process that involves the collision of two complementary strands, follows second-order kinetics : -dC/dt

kCiCz

or -dC/dt

=

kC2

where C is the concentration of denatured DNA in terms of moles of nucleotide per liter and k is the second-order rate constant. Experimentally, equal amounts of C, and C? are used for hybridization studies and all nucleotides in single-stranded DNA are estimated, so that C, C, = C. The kinetics of the reaction may also be described by the equation

+

dC/dt

-

k(Co - C S ) ~

or the integrated form l/(Co

- C,)

=

kt + A

or

l/(Co - Ca) = kt

+ 1/Co

where C, is the initial nucleotide concentration of the denatured DNA in moles per liter, C, is the nucleotide concentration of the associated DNA in moles per liter, and A is the constant of integration (equal to l/CO). Accordingly, the association of duplex molecules is a function of the initial concentration of each DNA species and the time of incubation. Rearranging the last equation: k = ( l / t )[C,/C, (C, - C3)]. Because C, = C, C, C, = C C, k = (l/t[C, - C)/C,C]. Based upon this equation, Britten and Kohne (19SS) introduced the acronym “Cot”: Cot = l / k [ C o - C ) / C ] or C , / C , = k Cot+ 1, which has the units (moles per liter) (seconds). A Cot value is readily calculated using the numerically equivalent statement Cot = $4 (A,,,) (incubation time in hours), where A?,, is the initial absorbance of the DNA solution. To evaluate critically whether renaturation is proceeding by second-order kinetics, C,/C is plotted against time. Experimentally, measurements should be made during the early stages of nucleotide association when CBis ca. 0 and t is ca. 0. This plot should generate a straight line (Fig. 7 ) ; moreover, the calculated second-order rate constant ( k) should be the same regardless of the initial reactant concentration. Optically measured DNA association has a number of desirable features for determining relatedness among actinomycetes: ( a ) no radiolabeled

+ +

+

167

MYCOBACTERIA AND NOCARDIAE

%-A,

2.0-

0

2

6

4

HOURS

FIG. 7. Kinetics of DNA association. Sheared and denatured DNA of Nocardia erythropolis (syn. Proactinomyces opacus) ( O),of Corynebaderium sp. ATCC 184 ( .), and an equal mixture of the two preparations associated according t o second-order kinetics. These two strains show essentially complete nucleotide homology.

DNA is required; ( b ) high association temperatures can be used without the complication of leaching of the immobilized DNA from the immobilizing menstruum; and ( c ) absorbance accurately measures associated sequences per se whereas radioassay methods measure not only associated sequences, but also unassociated ( single-stranded ) loops and free ends. The percent homology is calculated from the optical measurements using the expression: % homology = 100 100 (Cot % A Cot B - 2 Cot % mix)/( Cot % A Cot 1/2 B ) . This equation is derived from one developed by Seidler and Mandel (1971). Representative data for optically measured association of DNA from M . avium and M . intruceZZuZure with DNA from selected mycobacteria are presented in Table V.

+

+

+

C. PHYLOGENETIC IMPLICATIONS In order to make taxonomic inferences based upon nucleic acid association data, the extent of DNA nucleotide sequences held in common between a test DNA and a reference DNA must be compared. The degree of shared sequences becomes a quantitative index of relatedness. However, DNA samples from related organisms usually contain identical

S. G. BRADLEY AND J . S. BOND

168

TABLE V ASSOCIATION OF DNA FROM Mycobacterium avium 573 AND M . inti.acellulare 475 WITH DNA FROM SELECTED MYCOBACTERIA

% homology with Test DNA from

573

M . avium 573 M . bovis 545 M . farcinica 406 M . farcinica 420 M . farcinica 436 M . farcinica 479 M . Jlavescens 624 M . fortuitum 471 M . intracellulare 475 M . kansasii 476 M . marinum 437 M . parafinicum 507 M . phlei 435 M . smegmatis 403 M . smegmatis 433 M . smegmatis 461 M . smegmatis 477 M . tuberculosis 546

100 27 i-3 28 8 24 f 3 16 4 14 f 6 0 0 48 f 6 0 0 7*3 9 f 3 14 f 4 7 0 14 f 4 18 f 3

a

+

*

475 48 7 6 16 6 6

f6 + 2 f 6 f6 f 6 f 6 0

NDa 100 18

+6 0

30 3 4 5 6 4 4

f7 f f f f + +

3 4 5 6 4 5

ND, not done.

nucleotide sequences, a spectrum of partially matched sequences and totally dissimilar sequences; therefore, it is not possible to arrive at a single value that defines the absolute relatedness of one organism to another. This apparent complication actually provides a basis for inferring phylogenetic relationships among organisms. The exactness of base pairing, as well as the extent of association between single-stranded DNA preparations from different strains, can be measured experimentally. In one method used extensively, association is allowed to proceed at two different incubation temperatures. At the higher temperature, only well matched sequences should form duplexes. At the lower temperature, partially matched and exactly matched sequences should form duplexes (Table VI). In our laboratory, we find it adequate and convenient to use two incubation temperatures, one exacting and one nonexacting. Usually the nonexacting incubation temperature is set at 3OoC to 35OC less than the T , of the reference DNA and the exacting temperature a 15OC to 2OoC less than the T , of the reference DNA. The percent association relative to the homologous reac-

169

MYCOBACTERlA AND NOCARDIAE

TABLE VI EFFECT OF NUCLEOTIDE ASSOCIATION TEMPERATURE UPON THE CALCULATED SIMILARITY BETWEEN MIXEDDNA SAMPLES FROM MYCOBACTERIA % Homology at a A Tm of Mixed DNA samples from

M . avium 573 and M . intracellulare 475 M . bovis BCG and M . tuberculosis H37Ra N . rubra 434 and N . rubra 513 0

20°C

25°C

30°C

35°C

12

25

51

0

98

100

100

82

82

75

NDa

ND

ND, not done.

tion is determined for each incubation temperature. The ratio of (the relative binding at the exacting temperature) / (the relative binding at the nonexacting temperature) has been designated the Divergence Index ( D I ) . Divergence index values are useful gauging the presence or absence of closely related genetic material. A ratio close to 1.0 indicates that all the sequences that bind the reference DNA are almost identical to it whereas a DI value approaching 0.0 indicates that the test DNA possesses almost no regions of identity with the reference DNA but may possess many similar nucleotide sequences. There appears to be a correlation between AT,,,, and DI. Divergence index values can be interpreted at the molecular level in terms of the distribution of nucleotide divergence. We refer to the nucleotide divergence occurring more or less randomly distributed throughout the genome as dispersed divergence. This is in contrast to localized divergence or localized conservation, where changes occur in specific regions only. The duplexes formed during exacting incubation conditions are only those of closely matched sequences ( b ) whereas duplexes formed during nonexacting conditions are composed of both incompletely matched ( a ) and closely matched ( b ) sequences. Symbolically DI equals b/ ( a b ) . The values of a and b are subject to the following limitations: a b < 100 or a b = 100; a $- b > 0 or a b = 0; b < a b or b = a $- b. The total number of sequences available to react is a h c = 100, where c is the percent of DNA sequence unable to anneal because the nucleotide sequences in the mobile DNA and the immobilized DNA are extensively mismatched. From a graphical presentation of data of this type, certain proposals

+

+

+

+ + +

+

S. G . BRADLEY AND J . S. BOND

170

......... ...... ...... I

-........._

"

EXACTING

BINDING

FIG.8. Patterns of nucleotide divergence and the effects of different distributions on DNA association results. The reference nucleotide sequences and their exact complement are denoted by a solid line; partially matched sequences by a dashed line and mismatched sequences by a dotted line.

about the mechanism by which genetic diversity arose can be developed (Fig. 8). The grid formed by coordinates DI [i.e., b / (a b ) ] versus Sb [i.e. 100b/( a b c ) ] can be bisected by a line from the origin to the coordinates DI = 1 and %b= 100. This diagonal has been referred to as the line of maximum divergence. The line of maximum divergence is generated by setting a b = 100, or by setting c = 0. No experimental points should fall below the line of maximum divergence because b cannot be greater than a b. Experimental points having coordinates near DI = 1 and Xb = 100 are generated by DNA preparations which associate exactly; i.e., a and c are very small. Experimental points having coordinates between DI = 0 to 1 and Xb = 0 are generated by DNA preparations which form very few duplexes, i.e., a and b are very small. Coordinates DI = 0.5 and X b = 50 reflect DNA preparations which lack c and consist of an equal number of DNA fragments of type a and type b, i.e., are capable of forming equal amounts of exactly matched and partially matched duplexes. Coordinates DI = 0.8 and Xb = 20 would reflect a DNA preparation in which c is large and a is small. When DNA samples from selected actinomycetes are analyzed and plotted in this way, the experimental values generate a line which ap-

+

+ +

+ +

MYCOBACTERIA AND NOCARDIAE

171

proximately corresponds to the line of maximum divergence. These results indicate that genetic diversity in the actinomycetes is primarily due to dispersed nucleotide divergence. Accordingly, random mutation is the probable mechanism by which genetic diversity has arisen in this group of microorganisms. Conversely these results indicate that gene transfer involving unique nucleotide sequences is not a major factor in the evolution of the actinomycetes. Moreover, there is no evidence that particular regions of the actinomycete genome are unusually susceptible to mutation, thereby creating extensive localized nucleotide divergence. D. NEUTRAL MUTATIONS

A basic premise in DNA association analysis is that the ability to form specific DNA duplexes with samples from two organisms constitutes evidence for relatedness. Conversely the lack of specific hybrid duplex formation is interpreted to indicate some degree of unrelatedness. That base mispairing indicates phenotypic dissimilarity, however, is not absolutely established. It is conceivable that phenetically similar organisms might possess substantial amounts of genomic diversity. In such instances, there would be a significant degree of base mispairing during DNA association. King and Jukes (1969) have proposed that there may be random neutral genetic mutations that have no effect upon the competitive fitness of the organism. Wright ( 1966) has suggested that neutral mutations can become fixed as evolutionary changes through the action of genetic drift or by selection for neutrality. Kimura and Ohta (1971) went further and proposed that many mutational events are selectively neutral. This proposition has aroused much controversy. Johnson ( 1972) has compared published experimental data with mathematically developed predictions; Johnson concluded that the data contradicted the neutral mutation hypothesis. Yamazaki and Maruyama ( 1973) extended Johnson’s treatment. They arrived at a theoretical statement similar to Johnson’s but concluded that their expectations and the data were consistent with the neutral mutation hypothesis. A molecular basis for neutral mutations can be proposed. It is well established that there is more than one codon for each amino acid (Bradley, 1966). Kimura ( 1968) examined the codon dictionary and suggested that in the 61 amino acid specifying codons, there are 549 possible single-base substitutions, one-fourth of these ( 134) being substitutions to synonymous codons. hforeover, of the three positions in a codon, mutations occurring in the first two positions usually cause amino acid substitutions and are frequently eliminated by natural selection

172

S. G.

BRADLEY AND J. S. BOND

whereas mutations in the third position are often selectively neutral and would be maintained (King and Jukes, 1969). It is possible, therefore, that substantial nucleotide divergence has accumulated within a species with little change in overt phenotype. It is conceivable that nucleotide divergence as a result of neutral mutations might proceed rapidly. VIII.

Definition of t h e Genera

A. Mycobacterium LEHMANN ET NEUMANN1896 Classically the mycobacteria are aerobic to microaerophilic acid-fast actinomycetes that generally occur as rods but occasionally appear as branched filaments or even in a mycelial form. The members of the genus are gram-positive, nonmotile, do not form spores, conidia, or aerial hyphae, produce acid from sugars oxidatively, and are usually catalase positive. Mycobacteria have an unusually high lipid content, which includes the characteristic long-chained mycolic acids. The genus contains rapid and slowly growing strains together with taxa that do not grow in uitro. At suitable incubation temperatures, the rapidly growing strains achieve maximum growth within 7 days, but the slow growing strains require 2-6 weeks. Attempts to construct reliable classification schemes for this genus are hampered by the difficulties in comparing strains having diverse growth and metabolic rates. Accordingly, there is the danger of grouping strains together not because they share a number of characteristics, but because of only one characteristic, similar rates of growth. Mycobacterium lepraernurium, the etiologic agent of murine leprosy, is a possible example of incorrect classification on account of its restricted capability to grow in conventional laboratory media. Based upon morphological studies, UyedP ( 1964a,b) has concluded that this bacterium should not be considered a member of the genus Mycobacterium but should be placed in a new genus related to Nocardia. However, based upon the chemical composition of the cell wall, M . lepraemurium is correctly assigned to the genus Mycobacterium (Cummins et al., 1967; Draper, 1971). Azuma et al. (1973) have identified five components of the cell wall of M . lepraemurium; they are ( a ) arabinose mycolate, ( b ) mycolic acids, ( c ) alaninyl glutaminyl diaminopimelyl alanine, ( d ) N-acetylglucosaminyl-/?-1,4-N-glycolylmuramic acid, and ( e ) arabinogalactan. The DNA nucleotide composition of the mycobacteria varies between 64 and 70%GC. The type species of this genus is M . tuberculosis H37Rv (Kubica et al., 1972a). Strain H37Rv of M . tuberculosis (Zopf) Lehmann et Neumann is typical of the species in growth characteristics, drug susceptibility, and biochemical activity. This strain agrees well with Koch's original description of the tubercle bacilIus. The well documented history

MYCOBACTEFUA AND NOCARDIAE

173

of strain H37Rv, its world-wide distribution and long use by bacteriologists make it most appropriate as the neotype of the species M . tuberculosis.

B. Nocardia TREVISAN 1889 Classically members of the genus Nocardia produce mycelia which ultimately fragment into rods or coccoid bodies or both. These germinate forming branching hyphae 0.5-1 pm in diameter. The nocardiae are considered obligate aerobes, gsam-positive, occasionally slightly acid-fast, and nonmotile. The colonies of nocardiae are similar in gross appearance to those of mycobacteria; however, aerial mycelium is usually produced. The extent of the aerial mycelium may range from sparse and invisible to the unaided eye to completely covering the substrate mycelium with a white nap. Short chains of arthrospores may be found on the aerial hyphae, but endospores are not produced. Aesculin, allantoin, and urea are hydrolyzed, nitrate is reduced to nitrite, and a brown water-soluble exopigment is formed on rich media. Nocardiae are resistant to lysozyme, form acid from glucose, fructose, and glycerol oxidatively, and can utilize acetate, n-butyrate, malate, propionate, pyruvate, succinate, and paraffin as sole sources of carbon for energy and growth. The cell walls contain meso-DAP, arabinose, and galactose. Nocardiae contain lipid LCN-A and nocardomycolic acids. Numerous nocardiae have been isolated as agents of disease in man and other animals. The reservoir for pathogenic nocardiae is thought to be the soil, and the disease is not thought to be directly transmissible from animal to animal or to man. The DNA nucleotide composition of the nocardiae varies between 65 and 70%GC. There is considerable confusion over the status of the type species of the genus Nocardia. Magnusson and Mariat (1968) prefer N . farcinica, which they relate to the etiologic agent of bovine farcy. It has been pointed out that strain ATCC 3318, which is reputed to be derived from Nocards original isolant of N. farcinica, is not the same as strain NCTC 4524 which is purported to be a duplicate of ATCC 3318 (H. A. Lechevalier et al., 1971). Strain ATCC 3318 is a nocardia in the sense of N . asteroides whereas strain NCTC 4524 is a mycobacterium. Indeed, the strains of actinomycetes isolated from recent cases of bovine farcy in Africa are really strains of Mycobacterium. We support the opinion of Lechevalier et al. (1973) that N . farcinica is a nomen dubium and therefore concur that N . asteroides strain ATCC 19247 should be accepted as the neotype culture for this species and genus (Sneath and Skerman, 1966). In addition to the types species, N . asteroides ( Eppinger) Blanchard, the genus Nocardia includes N . brasiliensis

174

S. G. BRADLEY AND J. S. BOND

( Lindenberg) Castellani et Chlamers and N . caviae (Erikson) Gordon et Mihm. ET LECHEVALIER 1970 C. Actinomadura LECHEVALIER

Members of the genus Actinomadura are aerobic gram-positive nonacid-fast actinomycetes that form a branched substrate mycelium with a sparse to abundant aerial mycelium. Chains of arthrospores may be borne on the aerial hyphae, often as short lateral branches of 5-15 spores. The mycelium of actinomadurae does not contain lipid LCN-A; their cell walls contain meso-DAP. The nucleotide composition of their DNA is 72-77% GC. H. A. Lechevalier and Lechevalier (1970) included three species in the genus; they are A. dassonvillei, A. madurae, and A. pelletieri. D. Proactinomyces

JENSEN

1931

Gordon and Mihm (1959b) consolidated a large number of similar strains that they had received under a plethora of generic and specific names into a taxon that they designated tentatively as Mycobacterium rhodochrous. Jones and Bradley ( 1962), however, concluded that the “rhodochrous complex” is more closely related to the genus Nocardia than to the genus Mycobacterium. Moreover, Bradley (1971) demonstrated that the “rhodochrous complex” is heterogeneous, containing at least two genospecies. The nomenclature and classification of the organisms designated as M. rhodochrous by Gordon (1966) remains confused ( Cross and Goodfellow, 1973). For example, Stottmeier and Molloy ( 1973) reported that members of the “rhodochrous complex” produced a milky turbidity on Middlebrook 7H10 agar supplemented with 1%ethylene glycol whereas none of 37 tested mycobacteria and only 2 of 19 tested nocardiae degraded the added ethylene glycol. It is not clear from their paper, but it is probable that members of both N . erythropolis and N. rubra were included in the group designated M . rhodochrous. It should be noted that contrary to the assertion of Stottmeier and Molloy, a culture designated as M . rhodochrous has been implicated in human disease (Tsukamura et al., 1970). The numerical taxonomic evidence of Goodfellow et al. (1972) indicates that the “rhodochrous complex” constitutes a recognizable taxon clearly separate from, and of a rank equal to, the genera Nocardia and Mycobacterium. Jensen ( 1931) described a group of soil actinomycetes that formed unstable mycelia, produced soft colonies on agar, and were partially acid-fast. He proposed the name Proactinomyces for this genus. We propose that Proactinomyces Jensen 1931be recognized as the correct generic designation for the “rhodochrous complex.”

MYCOBACTERIA AND NOCARDIAE

175

Members of the genus Proactinomyces are aerobic gram-positive actinomycetes that are pleomorphic but may form a primary mycelium that soon fragments into irregular elements. On synthetic nutrient medium, proactinomycetes produced a well developed unseptate mycelium during the first day of growth. Mycelial filaments are long or short, densely or sparsely branched, more or less straight or strongly twisted. Their thickness varies widely, depending on the species: in some the diameter of the mycelial filament is 0.5 pm, in others 1.0-1.2 pm, but in most it is 0.7-0.8 pm. After 3-5 days and occasionally even earlier, numerous cross walls arise inside the proactinomycetic mycelium, and the filaments soon fragment into short rods 1.5 to 3pm and subsequently into cocci. When inoculated into a fresh nutrient medium, the rod-shaped and coccoid cells give rise to a mycelium. The organisms are nonmotile, may be partially acid-fast by traditional staining, and do not produce aerial mycelium. Colonies can be rough, smooth, or mucoid, mycobacterium-like and are usually pigmented buff, pink, orange, or red although colorless variants occur. Proactinomycetes develop well on many nutrient media. They assimilate proteins, carbohydrates, fats, paraffin, and wax. Moreover they form acid from glucose, fructose, maltose, sucrose, mannitol, and sorbitol oxidatively and utilize n-butyrate, malate, propionate, pyruvate, succinate, and acetate as sole sources of carbon and energy. Proactinomycetes are sensitive to lysozyme, methyl violet, pyronin, and benzylpenicillin and grow well at 10OC. Their cell walls contain mesoDAP, arabinose, and galactose. Proactinomycetes contain nocardomycolic acids but preliminary data indicate that they are different from those of N . asteroides. Proactinomycetes contain lipid LCN-A but the R, value may be lower than that of lipid LCN-A from N . asteroides. Many strains of proactinomycetes require thiamine for growth. The proactinomycetes are widely distributed in soil ( Krasil’nikov, 1966). The nucleotide composition of their DNA varies between 61 and 68%GC. The genus PTOactinomyces contains two distinct species, P. corallinus ( Goodfellow’s subgroup 14 C ) and P. opacus (Goodfellow’s subgroup 14 D ) . We are designating P . corallinus as the type species. LECHEVALIER ET LECHEVALIER 1970 E. Oerskouia PRAUSER, The oerskoviae are aerobic gram-positive actinomycetes that form a primary mycelium which quickly fragments into motile rodlike elements. The motile rods are monotrichous when small (0.4 x 1.1 pm) but peritrichous when long. They germinate to form extensively branched hyphae 0.5 pm in diameter. Oerskoviae are not acid-fast, do not form spores, and produce yellow colonies that lack aerial hyphae. Oerskovia strains are fermentatively active, hydrolyze many organic compounds, and uti-

176

S. G. BRADLEY AND J . S . BOND

lize a wide range of carbon sources for energy and growth. They have a cell wall type VI (lysine instead of DAP); aspartic acid may or may not be present. Oerskouia cells contain major amounts of galactose but no lipid LCN-A. The nucleotide composition of their DNA is 71-75% GC. These organisms are readily isolated from soil samples. Because these motile actinomycetes are quite different from nocardiae, Prauser et al. (1970) proposed that the genus Oerskouia be established to harbor “grskov’s motile nocardia.” Jones and Bradley ( 1964b) had shown earlier that “grskov’s motile nocardia” was distinct from true nocardiae or members of the “rhodochrous complex.” The type species is 0. turbata.

IX.

Evaluation of Species

A. SPECIESOF Nocardia Goodfellow (1971) studied a collection of 283 nocardioform bacteria by means of 241 tests which included many biochemical, physiological, and nutritional characters. His results were subjected to computer analysis, using two different coefficients of association. A few strains were grouped together on the basis of the simple matching coefficient because of shared negative responses that were excluded by the similarity coefficient. Using the matching coefficient, 15 clusters were formed whereas 18 clusters were generated using the similarity coefficient. In each analysis, the seven major clusters corresponded to ( a ) Nocardia asteroides, ( b ) N . cauiae, ( c ) N . brasiliensis, ( d ) Actinomadura madurae, ( e ) A. pelktieri, ( f ) Oerskovia turbata, and ( g) Proactinomyces sp. Goodfellow’s data revealed many new characters which could be weighted for identification purposes. The clusters and subgroups generated in Goodfellow’s study are readily differentiable from one another by several unrelated phenotypic features. In Goodfellow’s study, the N . asteroides cluster contained 5 subgroups. Previously, Tsukamura ( 1969) had concluded that the N . asteroides cluster was heterogeneous, containing a subgroup that he called N . farcinica and a subgroup called N . asteroides. Unfortunately it is not possible to equate Goodfellow’s subgroups with Tsukamura’s subgroups. It should be noted that Goodfellow’s N . asteroides cluster includes a subgroup designated 1C whose members are mycobacteria. Apparently the formation of this cluster has been influenced by factors such as growth rate rather than biological relatedness. Berd (1973) studied 65 cultures of “ N . asteroides” by a battery of morphological, physiological, and biochemical tests. His strains could be divided into two well defined subgroups, one typified by ATCC 3318 and the other typified by ATCC 19247. His study also included five African isolants, designated as N .

MYCOBACTERIA AND NOCARDIAE

177

farcinica. Berd correctly identified these five strains as mycobacteria. Berd supports Tsukamura’s subgrouping of the N . asteroides complex but considers the selection of the epithet N . farcinica for group B unfortunate. Tsukamura’s group B includes ATCC 3318, which has been referred to as N . farcinica. Bradley (1973) also concluded that cultures labeled N. asteroides constitute at least two species based upon DNA :DNA association analyses. 1. Nocardia asteroides ( Eppinger ) Blanchard

N . asteroides initially develops an extensive mycelium; later the hyphae fragment into rods and cocci 0.5 pm in diameter. The cells are weakly acid-fast by conventional staining procedures. The colonies are beige, yellow, orange, pink, or red; they may be smooth, shiny, granular, or wrinkled and of a doughy consistency or friable. Microscopically N . asteroides forms sparse to abundant, short to long and straight, branching or twisted aerial hyphae. Macroscopically a white coating of aerial growth generally covers the colonial surface. The aerial hyphae may fragment into arthrospores. The organism grows well on most media, but casein, gelatin, and tyrosine are not degraded. Acid is produced from glucose and glycerol. Acetate, malate, propionate, pyruvate, succinate, or testosterone can be used as the carbon source. In general, strains of N . asteroides can be identified by the following features; they form macroscopically sparse visible aerial mycelium, do not degrade casein, tyrosine, or xanthine (Gordon and Mihm, 1962a). N . asteroides is pathogenic for man, guinea pigs, and rabbits. The neotype culture for N. asteroides is ATCC 19247. 2. Nocardia brasiliensis ( Lindenberg) Castellani et Chalmers N. brasiliensis forms a primary mycelium that may fragment into rods. A few strains are weakly acid-fast ’and most colonies are beige or pale yellow. The colonies are flat and spreading or wrinkled and heaped-up. Microscopically, N . brasiliensis forms sparse to abundant, short to long and straight to gnarled aerial hyphae. Macroscopically the aerial hyphae may or may not be visible. Arthrospores are rarely formed by the aerial mycelium. The organism grows well on most media; casein, gelatin, guanine, hypoxanthine, and tyrosine are degraded. Acid is produced from galactose, glucose, glycerol, inositol, mannitol, and mannose. Acetate, citrate, malate, propionate, pyruvate, and succinate can be used as the sole carbon source. Monoethanolamine or serine can serve as the sole nitrogen source. Nitrate is slowly reduced to nitrite. In general, strains of N . brasiliensis degrade casein and tyrosine but not xanthine (Gordon and Mihm, 1959a). N . brasiliensis is one of the etiologic agents of actinomycotic mycetoma. The neotype culture for N . brasiliensis is ATCC 19296.

178

S. G. BRADLEY AND J . S. BOND

3. Nocardia caviae (Erickson) Gordon and Mihm N . caviae forms a primary mycelium that fragments into short filaments and rods. The cells are variably acid-fast by conventional staining procedures. The colonies vary from cream to tan to peach-colored; they may be flat and spreading or wrinkled and heaped up. The aerial mycelium cannot be seen by the unaided eye. Some cultures produce a tan soluble pigment. The organism grows well on most media; guanine, hypoxanthine, and xanthine are degraded. Acid is produced from glucose, glycerol, inositol, maltose, and mannitol. Acetate, malate, propionate, pyruvate, succinate, and testosterone can be used as the sole carbon source. Monoethanolamine or serine can serve as the sole nitrogen source. Nitrate is reduced to nitrite. In general, a nocardial strain that degrades xanthine but not casein or tyrosine may be presumed to belong to N . caviae (Gordon and Mihm, 1962b). N . caviae is pathogenic for man and the guinea pig. The neotype culture for N . caviae is ATCC 14629. N . caviae is a later synonym for N . otitidis-caviarum Snijders.

B. SPECIESOF Proactinomyces Extensive numerical taxonomic studies have established that the strains in the “rhodochrous complex” form a taxon which occupies a rank equivalent to nocardiae, actinomadurae, mycobacteria, and oerskoviae ( Goodfellow, 1971; Goodfellow et al., 1972). Jones and Bradley (1964a) had previously shown that the “rhodochrous complex” could be differentiated from the true nocardiae typified by N . asteroidm. Tsukamura (1971a) has proposed that the “rhodochrous complex” be included in a new genus Gordona. This genus is distinguished from nocardiae by the absence of mycelium, ability to form acid from mannose, positive nitrate reduction and ability to utilize sucrose as a sole carbon source. G. bronchalis was designated as the type species. Because Jensenia canicruria is generally considered a member of the “rhodochrous complex,” and if this group is to be considered a new genus, Gordona is a later synonym for the earlier name Jensenia. Jensenia, in turn, is a later synonym for Proactinomyces. Bradley ( 1971) concluded that the “rhodochrous complex” consists of at least two distinct species, one typified by N . erythropolis and the other by N . rubra; DNA from the former species contains 6143% GC whereas than from the latter species contains 6648%GC. Similarly, Goodfellow (1971) noted that cluster 14 contains two major subgroups: subgroup 14 C corresponds to N . rubra and N . cornllina whereas subgroup 14 D corresponds to the taxa N . erythropolis and Jensenia canicruria. We have attempted to identify simple tests and growth responses that will facilitate delineation of these two taxa. The following physio-

MYCOBACTERIA AND NOCARDIAE

179

logical tests did not help delineate between the “rubra-corallina” subgroup and the “erythropolis-canicruria”subgroup: growth on citrate, fructose, glucose, glucose plus 0.0005%crystal violet, glucose plus 0.02% NaN3, leucine, malate, mannitol, mannose, sorbitol, or succinate; failure to grow on adenine, alanine, arabinose, caffeine, cholate, cholesterol, cytosine, deoxycholate, deoxyribonucleate, galactose, glutamine, guanine, lactose, maltose, p-aminobenzoate, proline, rhamnose, sorbose, sucrose, tryptophan, xanthine, or xylose. Responses characteristic of strains, but not species, were obtained with growth on acetate, ethanol, glucose plus 0.1%phenol, or thymine. Responses that are helpful in differentiating between the “erythropolis-canicruria” subgroup and the “rubra-corallina” subgroup are growth on asparagine, arginine, gluconate, glutamate, glycerol, phenylalanine, tyrosine or uracil, and urease production. Adams et al. (1970) have established that N . erythropolis and J . canicruria are synonyms. This conclusion is consistent with the demonstration of genetic recombination between mutants of N. erythropolis and J . canicruria ( Adams and Bradley, 1963). Bradley and Huitron extended this synonymy to include strains designated N . erythropolis, J . canicruria, N . coeliaca, and N. opaca 765A, but not N . globerula 472. However, Goodfellow (1971) placed N . coeliaca ATCC 13181 in cluster 12, which is related to cluster 14 at less than the 70%level of similarity. We recommend that the type species for the taxon including strains designated as Jensenia canicruria, Nocardia erythropolis, Proactinomyces erythropolis, N . coeliaca, and P. opacus be Proactinomyces opacus (den Dooren de Jong) Jensen 1932 and that the type culture be strain 439 of Bradley and Huitron ( 1973). We recommend that the type species for the taxon including strains designated as N . rubra and N . corallina be Proactinomyces corallinus (Bergey et al.) Jensen 1932 and that the neotype culture be ATCC 4273. Based upon growth habit, acid-fast staining, physiological tests and phage susceptibility, Juhasz and Banicke ( 1965) concluded that the correct generic name for the specific epithet “pellegrino” is Nocardia. N . pellegrino is included in Goodfellow’s “rhodochrous complex” as a member of subgroup 14A. Accordingly, there are three possible additional species within the genus Proactinomyces; they are P. globerulus, P. pellegrino, and P. thamnopheos (nee: M . thamnopheos) (M. P. Lechevalier et al., 1971). 1. Proactinomyces corallinus (Bergey et al.) Jensen P. corallinus does not produce aerial hyphae on any nutrient medium. The colonies are pigmented red, but yellow and white variants occur. Pigment is not released into the medium; the pigment is poorly soluble

180

S. G . BRADLEY AND J. S. BOND

in alcohol, ether, and acetic acid; it is readily soluble in chloroform and belongs to the carotenoid family. During the early stages of growth, filaments grow into nonseptate mycelia; after 2-3 days numerous septa develop, and the entire mycelium breaks up into short rod-shaped and coccoid elements. Upon inoculation into fresh nutrient substrate, the coccoid elements germinate, giving rise to a mycelium. The cells are gram-positive and weakly acid-fast. The colonies are rough or smooth and have a doughlike consistency. A colony adheres at its base to the agar; part of the mycelium grows into the substrate. Cultures of this species grow well on synthetic medium although thiamine is sometimes required. Sugars, fatty acids, fats, and amino acids are utilized as sources of carbon ( Krasil'nikov, 1966). P . corallinus strains produce urease and can use tyrosine as a sole carbon source. The nucleotide content of their DNA is 66-681%GC. The type culture of P . coraZZinus is ATCC 4273. 2. Proactinomyces opacus (den Dooren de Jong) Jensen

P . opacus does not produce aerial hyphae on any nutrient medium. The colonies are pigmented pale pink, but cream-colored and white variants occur. Pigment is not released into the medium; the pigment is poorly soluble in alcohol, ether, and acetic acid; it is readily soluble in chloroform and belongs to the carotenoid family. During the early stages of growth, filaments grow into nonseptate mycelia; after 1-2 days numerous septa develop and the entire mycelium breaks up into short rod-shaped and coccoid elements. Upon inoculation into fresh medium, the coccoid bodies germinate, giving rise to a mycelium. The cells are gram-positive and are not acid-fast. The colonies are rough or smooth and may have a pastelike consistency or may be mucoid. A colony adheres at its base to the agar; part of the mycelium grows into the substrate. Cultures of this species grow well on synthetic medium supplemented with thiamine. Sugars, fats, fatty acids, and amino acids are utilized as a source of carbon. P . opacus strains do not produce urease and can use arginine, asparagine, gluconate, glutamate, glycerol, phenylalanine, and uracil as a sole carbon source. The nucleotide content of their DNA is 61-63% GC. The type culture of P. opacus is strain 439 of Bradley and Huitron ( 1973). 3. Proactinomyces globerulus Reed P . globerulus does not produce aerial hyphae. Its colonies are pale pink; the pigment is not water soluble but is readily soluble in chloroform. The cultures form a transitory mycelium which rapidly fragments into short rods and coccoid elements. The cells are gram-positive and are not acid-fast. The colonies are flat and spreading or smooth and pastelike.

MYCOBACTERIA AND NOCARDIAE

181

P . globerulus can use fructose, glucose, phenylalanine, sorbitol, starch, and trehalose as a sole carbon source; it cannot degrade tyrosine. The nucleotide content of its DNA is 62%GC. The type culture of P . globerulus is ATCC 9356. C . SPECIESOF Mycobacterium It is widely acknowledged that the taxonomy of the genus Mycobacterium is unsatisfactory. The species are largely defined on a single morphological character. In addition, many cultures are evidently mislabeled, so great care must be taken to check them against the original description and the type culture (Sneath, 1970). Moreover, in comparing strains, it is difficult to compensate for differential growth rates. I t has been repeatedly observed that particular slow growing strains- are scored as giving a negative response but give positive responses upon longer incubation. When slow growing strains and fast growing strains are compared by numerical analyses, the slow growing strains are usually clustered together, based upon their negative responses. In order to compensate for variation due solely to growth rate, Sneath (1968) has proposed that responses be considered in terms of vigor and pattern.

1 . Synonymy The etiologic agent of bovine tuberculosis has been considered at various times as a species or as a subspecies in the genus Mycobacterium. It is generally agreed that the “bovis” organism is closely related to M . tuberculosis Juhlin (1967) concluded that M . tuberculosis can be reliably differentiated from M . bovis strains, including strain BCG, by a variety of physiological tests. The scientific name M . bovis was not validly published until 1970 (Karlson and Lessel, 1970). Tsukamura ( 1966) using Adansonian classification techniques and Bradley ( 1972) using DNA :DNA association studies concluded that M . bovis constituted a subspecies of M . tuberculosis. Similarly Gangadharam and Droubi (1973) could not distinguish between M . tuberculosis and M . boois by susceptibility to p-nitrobenzoic acid. Other examples of synonymy within the genus Mycobacterium include: (1) M . abscessus = M . borstelense = M . runyonii = M . piscium = M . chelonei (Cross and Goodfellow, 1973); ( 2 ) M . marinum = M . platypoecilus = M . balnei (Silcox and David, 1971); (3) M. smegmatis = M . butyricum M . lacticola = M . ranae (i.e., mislabeled cultures in the United States) (Gordon and Mihm, 1959b); (4) M . vaccae = M . parafortuitum = M . diernhoferi = M . aurum (Goodfellow et al., 1972); and (5) M . fortuitum = M . minetti = M . peregrinum = M . salmoniphilum (Kubica et al., 197213). Stanford and Gunthorpe ( 1969) have docu-

182

S. G . BRADLEY AND J. S. BOND

mented that M . fortuitum da Costa Cruz 1938 is a later synonym of M. ranae Bergey et al., 1923. However, Runyon (1972) has proposed that the species epithet fortuitum be conserved in preference to ranae because the American Type Culture Collection distributed world-wide for many years a culture designated M . r a m but which was in reality M. smegmatis and because M . fortuitum is universally known in the public health and medical communities and literature whereas M . ranae is an unfamiliar name.

2. Classification Schemes One of the widely used systems for grouping acid-fast bacteria isolated in clinical laboratories was devised by Runyon (1959). He proposed the following groups based upon pigment production and growth rate; ( 1 ) M . tuberculosis; ( 2 ) photochromogenic strains that grow slowly and produce a yellow pigment only when exposed to light-group I; ( 3 ) scotochromogenic strains that grow slowly and form an orangeyellow pigment both in the light and dark-group 11; ( 4 ) nonchromogenic strains that grow slowly, do not produce pigment or niacin, have smooth colonies, and are resistant to isoniazid-group 111; and ( 5 ) fast growing strains-group IV. Cross and Goodfellow (1973) proposed a similar but more extensive grouping: ( a ) M . leprae and other noncultivable species (e.g., M . lepraemurium). It is conceivable that M . leprae should not be included in the genus Mycohacterium; ( b ) M. tuberculosis (also M . bovis and M . microti) ; ( c ) photochromogenic mycobacteria (e.g., M . kansasii and M . marinum); ( d ) slowly growing scotochromogenic mycobacteria (e.g., M . scrofuluceum or M . marianum, M . gordonae, and M . flavescens); ( e ) nonchromogenic slow-growing niacin-negative mycobacteria ( M . avium, M . intracellulare, M . terrae, M . gastri, and M ulcerans); ( f ) rapidly growing nonpigmented mycobacteria ( M . fortuitum, M . abscessus, M . smegmatis, M . diernhoferi, and M . chitae) ; and ( g ) rapidly growing scotochromogenic mycobacteria ( e.g., M . gilvum, M. obuense, M . thermoresistibile, M . vaccae, M . duualii, M . flauescens, M . phlei, and M . rhodesiae). Wayne et al. (1971) carried out a cooperative numerical taxonomic analysis of slowly growing scotochromogenic mycobacteria of Runyon’s group 11. These workers defined four clusters; three of the clusters corresponded to named species ( M . flauescens, M . gordonae, and M . scrofukceum) . The fourth cluster may represent a new species. Kubica (1973) has formulated a simplified taxonomic scheme that facilitates identification of most clinically important mycobacteria recovered from man. The scheme is based upon growth rate and 11 other simple tests selected from an extensive numerical analysis that evaluated 82 taxonomic characters. A total of 1250 cultures out of 1420 cultures

MYCOBACTERIA AND NOCARDIAE

183

examined were correctly identified by the abbreviated scheme, and only

7 of the potentially pathogenic strains were incorrectly identified as nonpathogens. The following characteristics were selected for the abbreviated scheme: niacin production; nitrate reduction; catalase production; photochromogenicity; Tween hydrolysis; tellurite reduction; NaCl tolerance; aryl sulfatase activity, and growth on MacConkey medium. The groups generated by Kubica's scheme are: group I-M. kansasii and M . marinum; group II-M. scrofulaceum, M . gordonae, and M . f i v e s cens; group III-M. xenopi, M . avium, M . intracellulare, M . gastri, M . terrae, and M . trivab; group IV-M. fortuitum, M . vaccae, M . abscessus, M . smegmatis, and M . phlei; and the principal human pathogens M . tuberculosis, M . bovis, and M . africanum.

3. Unsolued Problems

A number of unresolved taxonomic dilemmas still confront the mycobacteriologist. These include: (1)are M . avium, M . intracellulare, and M . scrofuhceum synonymous? Tsukamura and Mizumo ( 1968) combined these three taxa, but we believe that they are distinct. Tsukamura (1971b) also reduced M . gastri and M . nonchromogenicum to subspecies of M . avium. ( 2 ) Newly proposed species which have not been studied by most mycobacteriologists, and therefore have not been confirmed as unique, include M . obuense (Tsukamura and Mizuno, 1971) and M . farcinica (Bradley, 1972). (3) Tsukamura ( 1971b) has reduced M . abscessus to a subspecies of M . fortuitum. (4)On the other hand, Tsukamura (1970) has proposed that the genus Mycobacterium be split into two genera: Mycobacterium and Mycomycobacterium. Mycobacterium would include M . tuberculosis (type species), M . kansasii, M . avium, M . thermoresistibile, and M . marinum. Mycomycobacterium would include M . smegmatis (type species), M . borstelense, M . abscessus, M . fortuitum, M . phlei, and M . aurum. Although the genus Mycobacterium may merit splitting at a later time, this division does not seem to have been adequately justified. (5) Is M . microti Reed, 1957 a synonym for M . tuberculosis subspecies bouis? ( 6 ) The Judicial Commission in 1973 conserved the epithet M . marianum and rejected M . scrofulaceum. Because of the confusion that exists between M . marinum and M . marianum, we have elected to use M . scrofulaceum in this review and advocate that the Judicial Commission reconsider their decision. 4. List of Species

We believe that the following taxa are reasonable species of the genus Mycobacterium: M . abscessus Moore and Frerichs 1953; M . a v i u m Chester 1901; M . flavescens Bojalil, Cerbbn and Trujillo 1962; M . Fortuitum da Costa Cruz 1938; M . gastri Wayne 1966; M . gordonae Bojalil,

184

S. G. BRADLEY AND J. S. BOND

Cerbbn and Trupillo 1962; M . intracellulare ( Cuttino and McCabe) Runyon 1965; M . kansasii Hauduroy 1955; M . leprae (Hansen) Lehmann and Neumann 1896; M . lepraemurium Marchoux and Sore1 1912; M . marinum Aronson 1926; M . nonchromogenicum Tsukamura 1965; M . paratuberculosis Bergey, Harrison, Breed, Hammer and Huntoon 1923; M. phlei Lehmann and Neumann 1899; M . scrofulaceum Prissick and Masson 1956; M . smegmtis (Trevisan) Lehmann and Neumann 1899; M . terrae Wayne 1966; M . trivale Kubica 1970; M . tuberculosis (Zopf) Lehmann and Neumann 1896; M . ulcerans MacCallum 1950; M . vaccae Bonicke and Juhasz 1964; and M . xempi Schwabacher 1959.

5. Mycobacterium tuberculosis M . tuberculosis is an obligate aerobe that has relatively simple nutritional requirements. It will grow in synthetic media containing acetate or glycerol as the sole carbon source and ammonium as the sole nitrogen source. Since it shows a marked nutritional preference for lipids, egg yolk is often a constituent of the enriched media used in diagnostic work, e.g., in Lowenstein-Jensen’s medium. M . tuberculosis is relatively resistant to alkali and phenol. M . tuberculosis forms rough, yellowish colonies after several weeks of incubation and produces niacin. M. tuberculosis forms a characteristic glycolipid, called the “cord factor,” which is 6,6’-dimycolytrehalose. Cord factor is thought to be responsible for the characteristic serpetine intertwining of chains of M. tuberculosis cells and is considered a determinative factor in pathogenicity. M . tuberculosis is pathogenic for man, rabbits, guinea pigs, and mice. The cells are rod-shaped, curved, or club-shaped; they are gram-positive and acidfast. The cells vary in length from 1 to 10 pm and are 0.8 pm in diameter. The nucleotide composition of the DNA is 64% GC. The type culture of the species is M . tuberculosis H37Rv. X.

Concluding Remarks

Since 1970, the nocardioform bacteria have been assigned to one of three genera: Nocardia, Oerskovia, and Actinomadura. Members of the genus Nocardia form somewhat persistent mycelia, produce firm colonies on agar, and are partially acid-fast by conventional staining techniques. Members of the genus Actinomadura form persistent mycelia, produce firm colonies on agar, but are not acid-fast. Oerskoviae form unstable mycelia, produce soft colonies on agar, and are not acid-fast. These three taxa are remarkably similar to three of the four groups proposd by Jensen (1932, 1953). Jensen’s fourth group was described as forming unstable mycelia, producing soft colonies on agar, and partially acid-fast. He assigned Nocardia corallina and Nocardia opaca to this group, which

MYCOBACTENA AND NOCARDIAE

185

he called Proactinomyces. It is obvious that the “rhodochrous” complex is synonymous with Jensen’s partially acid-fast a-forms. Accordingly, we propose that Proactinomyces Jensen 1931 be recognized as the correct generic designation for the “rhodochrous” complex. The generic name Proactinomyces is legitimate and has been validly published ( Lessel, 1960). Jensen (1931) did not designate the type species of Proactinomyces; Hauduroy et at. subsequently proposed that the type species of Proactinomyces be P . agrestis, but this proposal was not validly published (Buchanan et al., 1966). Moreover, the epithet “agrestis” is illegitimate. Accordingly, we are designating Proactinomyces corallinus ( Bergey et al.) Jensen 1932 as the type species of the genus and ATCC-4273 as the neotype culture. The genus Proactinomyces also includes the species P. opacus (den Dooren de Jong ) Jensen 1932 which encompasses strains designated as Jensenia canicruria, Proactinomyces erythropolis, and Nocardia erythropolis. The type culture is strain 439 of Bradley and Huitron ( 1973). Strain 439 is a descendant of strain 765A (Gordon and Mihm, 1959b) and presumably is a descendant of den Dooren de Jong’s original isolant. A third species is P. gloherulus Reed 1939; the type culture is ATCC-9356. The genus probably includes organisms previously designated as M . pellegrino and M . tharnnophlos. We propose that the genera Nocardia and Proactinomyces be included in the same family. The name Proactinomycetaceae Lehmann et Neumann 1927 seems appropriate for the family to harbor the genera Nocardia and Proactinomyces. ACKNOWLEDGMENT The new data presented in this review were generated in research supported by research grants AI-09097 and AI-09098 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service.

REFERENCES Adams, J. N., and Bradley, S. C . (1963). Science 140, 1393-1394. Adarns, M. M., Adams, J. N., and Brownell, C . H. (1970). Int. J. Syst. Bacteriol. 20, 133-147. Anderson, D. L., and Bradley, S. G. ( 1961). Antirnicrob. Ag. Chemother. p p . 898-903. Arai, T . , Kuroda, S., and Koyama, Y. (1963). J. Gen. Appl. Microbial. 9, 119-136. Arias, I., Doyle, D., and Schimke, R. T. ( 1969). J. Biol. Chem. 244, 3303-3315. Azuma, I., Thomas, D. W., Adam, A., Ghuysen, J. M., Bonaly, R., Petit, J. F., and Lederer, E. (1970). Biochim. Biophys. Actu 208, 444-451. Azuma, I., Yarnamura, Y., Tanaka, Y,, Kohsaka, K., Mori, T., and Itoh, T. (1973). J . Bacteriol. 113, 515-518.

186

S. G. BRADLEY A N D J. S . BOND

Baldacci, E. ( 1947). Mycopathologia 4, 60-84. Beaman, B. L., and Burnside, J. (1973). Appl. Microbiol. 26, 426428. Berd, D. (1973). Amer. Reu. Resp. Dis. 108, 909-917. Berlin, C. M., and Schinike, R. T . (1965). Mol. Pharmacol. 1, 149-156. Bond, J. S. ( 1971). Biochem. Biophys. Res. Commun. 43,333-339. Bond, J. S. (1973). Biochim. Biophys. Acta 327, 157-165. Bradley, S. G. (1966). Aduan. Appl. Microbiol. 8, 29-59. Bradley, S. G. (1968). Aduan. Appl. Microbiol. 10, 101-135. Bradley, S. G. ( 1970). In “Host-Virus Relationships in Mycobacterium Nocurdiu and Actinomyces” ( S . E. Juhasz and G. Plummer, eds.), pp. 3-14. Thomas, Springfield, Illinois. Bradley, S. G. ( 1971), Aduan. Front. Plant Sci. 28, 349-362. Bradley, S. G. ( 1972). Amer. Reu. Resp. Dis. 106, 122-124. Bradley, S. G. (1973). J. Bacteriol. 113, 645-651. Bradley, S. G., and Anderson, D. L. (1958). Science 128, 413414. Bradley, S. G., and Huitron, M. E. (1973). Deuelop. Ind. Microbiol. 14, 189-199. Bradley, S. G., and Jones, L. A. (1988). Progr. Ind. Microbiol. 7 , 43-75. Breed, R. S., Murray, E. G. D., and Smith, N. R., eds. (1957). “Bergey’s Manual of Determinative Bacteriology,” 7th ed. Williams & Wilkins, Baltimore, Maryland. Britten, R. J., and Kohne, D. E. (1968). Science 161,529-540. Brown, O., and Clark, J. B. ( 1961). Proc. Okla. Acacl. Sci. 41, 94. Brown, O., and Clark, J. B. (1966). Proc. SOC. Exp. Biol. Mecl. 122, 887-890. Brown, O., and Reda, S. ( 1967). J. Gen. Microbiol. 47, 199-205. Brunscliede, H., and Bremer, H. ( 1971). J. Mol. Biol. 57, 35-57. Buchanan, R. E., Holt, J. G., and Lessel, E. F., Jr., eds. (1966). “Index Bergeyana.” Williams & Wilkins, Baltimore, Maryland. Buraczewska, M., Manowska, W., and Rdultowska, H. (1971). Amer. Reu. Resp. Dis. 103, 116117. Cain, R. B., Tranter, E. K., and Daroh, J. A. (1968). Biochem. J. 106,211-227. Calmes, R., and Deal, S. J. (1972). Can. J. MicrobioE. 18, 1703-1708. Castelnuovo, G., Bellezza, G., Duncan, M. E., and Asselineau, J. (1964). Ann. Inst. Pasteur, Paris 144, 139-147. Castelnuovo, G., Bellezza, G., Giuliani, H. J., and Asselineau, J. ( 1968). Ann. Inst. Pasteur, Paris 144, 139-147. C e r b h , J., and Ortigoza-Perado, J. (1968). J. Bacterial. 95, 350-354. Cross, T., and Goodfellow, M. ( 1973). In “Actinomycetabs: Characteristics and Practical Importance” (G. Sykes and F. A. Skinner, eds.), pp. 11-112. Academic Press, New York. Cummins, C. S. (1954). Brit. J. Erp. Pathol. 35, 166-180. Cummins, C. S., Atfield, G., Rees, R. J. W., and Valentine, R. C. (1967). 1. Gen. Microbiol. 47, 377-384. Dice, J. F., and Schimke, R. T. (1973). Arch. Biochem. Biophys. 158, 97-105. Dice, J. F., Dehlinger, P. J., and Schinike, R. T. (1973). J. B i d . Chem. 248, 42204228. Dinman, B. D. ( 1972). Science 175, 495-497. Draper, P. (1971). J. Cen. Microbiol. 69, 313-324. Drysdale, J. W., and Munro, H. N. (1966). J. Biol. Chem. 241, 3630-3637. Fisher, C. A., and Barksdale, L. (1973). J. Bacteriol. 133, 1389-1399. Gangadharam, P. R., and Droubi, A. J. (1973). Amer. Rev. Resp. Dis. 108, 143-146. Garlick, P. J. ( 1969). Nature (London) 223, 61-62. Georg, L. K., Ajello, L., McDurmont, C., and Hosty, T. S. (1961). Amer. Reu. Resp. Dis. 84, 337-347.

MYCOBACTEHIA AND NOCARDIAE

187

Goldberg, A. L. (1972a). PTOC.Nut. Acad. Sci. US.69, 422426. Goldberg, A. L. ( 1972b). Proc. Nut. Acad. Sci. US.69, 2640-2644. Goldstein, I. J., and Mesaki, A. (1970). J. Bacteriol. 103,422425. Goodfellow, M. (1971). J. Gen. Microhiol. 69, 33-80. Goodfellow, M. ( 1972). “Numerical Taxonomy of Actinomycetales.” Univ. de LOS Andes, Ediciones del Centro De Microscopia Electronica, Merida, Venezuela. Goodfellow, M., Fleming, A., and Sackin, M. J. (1972). Int. J. Syst. Bacteriol. 22, 81-98. Gordon, R. E., (1966). J. Gen. Microbiol. 43, 329-343. Gordon, R. E., and Mihm, J. M. (1959a). J. Gen. Microbiol. 20, 129-135. Gordon, R. E., and Mihm, J. M. ( 1959b). J. Gen. Microbiol. 21, 736-748. Gordon, R. E., and Mihm, J. M. (1962a). J. Gen. Microbiol. 27, 1-10. Gordon, R. E., and Mihm, J. M. 1962b). Ann. N.Y. Acad. Sci. 98, 628-636. Gottlieb, D. ( 1960). “The Actinomycetes-Challenge to the Taxonomist.” University of Maryland, College Park. Grant, J. (1973). American Laboratory, October, 1973, pp. 17-23. Int. Sci. Commun. Inc., Green Farms, Connecticut. Grisolia, S., and Hood, W. (1972). In “Biochemical Regulatory Mechanisms in Eukaryotic Cells” ( E . Kun and S. Grisolia, eds.), pp. 137-203. Wiley (Interscience), New York. Haider, M., and Segal, H. L. (1972). Arch. Biochem. Biophys. 148, 228-237. Hatcher, D. W., and Goldstein, G. (1969). Anal. Biochem. 31, 42-50. Hill, E. B., Wayne, L. G., and Gross, W. M. ( 1972). J. Bacterial. 112, 1033-1039. Jensen, H. L. (1931). PTOC.Linn. SOC.N S W . 56, 345-370. Jensen, H. L. ( 1932). Proc. Linn. SOC.N.S.W. 57, 364-376. Jensen, H. L. ( 1953). In “Actinomycetales: Morphology, Biology and Systematics” ( E. Baldacci and P. Redaelli, eds. ), pp. 69-88. Fondazione Enianuele Paterno, Rome. Johnson, G. B. (1972). Nature (London), New Biol. 237, 170-171. Jones, L. A., and Bradley, S. G. ( 1962). Develop. Ind. Microhiol. 3,257-264. Jones, L. A., and Bradley, S. G. (1964a). Mycologia 61, 505-513. Jones, L. A,, and Bradley, S . G. (1964b). Develop. Ind. Microbiol. 5, 267-272. Juhasz, S. E., and Bonicke, R. (1965). Beitr. Klin. Erjorsch. Tuberk. Lungenkr. 130, 155-169. Juhlin, I. (1967). Acta Pathol. Microhiol. Scand. 70, Suppl. 189, 1-120. Karlson, A. G., and Lessel, E. F., Jr. (1970). Int. J. Syst. Bacteriol. 20, 273-282. Kassel, B. (1970). In “Methods in Enzymology” ( G . E. Perlmann and L. Lorand, eds.), Vol. 19, pp. 839-906. Academic Press, New York. Kenney, F. T. (1967). Science 156, 525-528. Kiniura, M. (1968). Genet. Res. 11, 247-269. Kimura, M., and Ohta, T. ( 1971). Nature ( London ) 229,467-469. King, J. L., and Jukes, T. H. (1969). Science 164,788-798. Kominami, E., Kobayashi, K., Kominami, S., and Katunuma, N. (1972). J. Biol. Chem. 247, 6848-6855. Krasil’nikov, N. A. ( 1966). “Keys to Actinomycetales.” Israel Program Sci. Transl., Jerusalem. Kubica, G. P. ( 1973). Amer. Rev. Resp. Dis. 107,9-21. Kubica, G. P., Kim, T. H., and Dunbar, F. P. (1972a). Int. J. Syst. Bacterial. 22, 99-106. Kubica, G. P., Baess, I. Gordon, R. E., Jenkins, P. A,, Kwapinski, J. B. G., McDurmont, C., Pattyn, S. R., Saito, H. Silcox, V., Stanford, J. L., Takeya, K., and Tsukamura, M. (197213). 1. Gen. Microbiol. 73, 55-70.

188

S. G .

BRADLEY AND J.

S. BOND

Kuriyama, Y., Omura, T., Siekevitz, P., and Palade, G. E. (1969). 1. Biol. Chem. 244, 2017-2026. Kwapinski, J. B. G. ( 1966a). Zentralbl. Bakteriol., Parasitenk., Infektionskr. H y g . 200, 80-88. Kwapinski, J. B. G. ( 1966b). Zentralbl. Bakteriol., Parasitenk, Infektionskr. H y g . 200, 380-387. Kwapinski, J. B. G. (1970). In ‘The Actinomycetales” ( H . Prauser, ed.), pp. 345-369. Fischer, Jena. Kwapinski, J. B. G., and Seeliger, H. P. R. (1964). Zentralbl. Bakteriol., Parasitenk., Infektionskr. H y g . , Abt. 1:Orig. 195, 13-240. Lechevalier, H. A,, and Lechevalier, M. P. ( 1970). In “The Actinomycetales” (H. Prauser, ed. ), pp. 393-405. Fischer, Jena. Lechevalier, H. A., Lechevalier, M. P., and Gerber, N. N. ( 1971). Aduan. Appl. Microbiol. 14, 47-72. Lechevalier, M. P., and Lechevalier, H. A. (1970). In “The Actinomycetales” ( H . Prauser, ed), pp. 311-316. Fischer, Jena. Lechevalier, M. P., Horan, A. C., and Lechevalier, H. (1971). J. Bacteriol. 105, 313-318. Lechevalier, M. P., Lechevalier, H., and Horan, A . C. (1973). Can. J. Microbiol. 19, 965-972. Lessel, E. F., Jr. (1960). Int. Bull. Bacteriol. Nomench. Taxonom. 10, 87-192. Litwack, G., and Rosenfield, S. (1973). Biochern. Biophys. Res. Commun. 52, 181-188. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. Luedemann, G. M. (1971). Trans. N.Y. Acad. Sci. [2] 33,207-218. Magnusson, M., and Mariat, F. (1968). J. Gen. Microbiol. 51, 151-158. Mallette, L. E. E., Exton, J. H., and Park, C. R. (1969). J. Biol. Chem. 244, 5713-5728. Mandelstam, J. (1958). Biochem. 1. 69, 110-119. Manion, R. E., Bradley, S. G., Zinneman, H. H., and Hall, W. H. (1964). J. Bacteriol. 87, 1056-1059. Marmur, J. (1961). J. Mol. Biol. 3, 208-218. Mortimore, G. E., and Mordon, C. E. (1970). J. Bio2. Chem. 245,2375-2383. Nath, K., and Koch, A. L. (1971). 1. Biol. C h m . 246, 69566967. Negroni, P. ( 1953). In “Actinomycetales: Morphology, Biology and Systematics” ( E . Baldacci and P. RedaeIli, eds.), pp. 13-19. Fondazione Emanuele Paterno, Rome. Pine, M. J. (1970). J. Bacteriol. 103, 207-215. Pine, M. J. (1972). Annu. Reu. Microbiol. 26, 103-126. Poole, B. (1969). Ann. N.Y. Acad. Sci. 168, 229-243. Prauser, J., Lechevalier, M. P., and Lechevalier, H. (1970). Appl. Microhiol. 19, 534. Probst, I., and Schlegel, H. G. (1973). Arch. Mikrobiol. 88, 317-330. Prouty, W. F., and Goldberg, A. L. (1972). J. Biol. Chem. 247, 3341-3352. Ramakrishnan, T,,Murthy, P. S., and Gopinathan, K. P. (1972). Bacteriol. Reu. 36, 65-108. Rann, D. L., and Cain, R. B. (1969). Biochem. I. 114, 77p. Rechcigl, M. ( 1971). In “Enzyme Synthesis and Degradation in Mammalian Systems,” ( M. Rechcigl, ed. ) pp. 236410. Univ. Park Press, Baltimore, Maryland. Redaelli, P. and Piantoni, L. ( 1953). In “Actinomycetales: Morphology, Biology

MYCOBACTEFUA AND NOCARDIAE

189

and Systematics” ( E . Baldacci and P. Redaelli, eds.), pp. 191-210. Fondazione Emanuele Paterno, Rome. Reznikov, M. and Dawson, D. J. (1973). Appl. Microbiol. 26, 470-473. Ridell, M., and Norlin ( 1973). 1. Bacteriol. 113, 1-7. Runyon, E. H. (1959). Med. Clin. N. Amer. 43,273-290. Runyon, E. H. (1972).Int. 1. Syst. Bacteriol. 22, 50-51. Sarimo, S. S., and Pine, M. J. (1969). J. Bacteriol. 98, 368374, Schimke, R. T. (1969). Curr. Top. Cell. Regul. 1, 77-124. Schimke, R. T., and Doyle, D. (1970). Annu. Reu. Biochem. 39, 929-976. Schimke, R. T., Sweeney, E. W., and Berlin, C. M. (1965). J. Biol. Chem. 240, 46094620. Schimke, R. T., Ganschow, R., Doyle, D., and Arias, I. (1968). Fed. Proc., Fed. A m r . SOC. Exp. Biol. 27, 1223-1230. Schleifer, K. H., and Kandler, 0. ( 1972). Bacteriol. Reu. 36, 407-477. Schoenheimer, R. ( 1942). “The Dynamic State of Body Constituents.” Harvard Univ. Press, Cambridge, Massachusetts. Segal, H. L., Matsuzawa, T., Haider, M., and Abraham, G. J. (1969). Biochem. Biophys. Res. Commun. 36, 764-770. Seidler, R. J., and Mandel, M. ( 1971). J. Bacteriol. 106, 608-614. Silcox, V. A,, and David, H. L. (1971). Appl. Microbiol. 21, 327-334. Sneath, P. H. A. (1968). J. Gen. Microbiol. 54, 1-11. Sneath, P. H. A. (1970). In “The Actinomycetales” ( H . Prauser, ed.), pp. 371-377. Fischer, Jena. Sneath, P. H. A., and Skerman, V. B. D. (1966). lnt. J. Syst. Bacteriol. 16, 1-133. Stanford, J. I+ and Gunthorpe, W. J. (1969). J. Bucteriol. 98, 375-383. Stottmeier, K D., and Molloy, M. E. (1973). Appl. Microbiol. 26, 213-214. Takeya, K., and Tokiwa, H. (1972). lnt. J . Syst. Bacteriol. 22, 178-180. Tarnok, I., Rohrscheidt, E., and Bonicke, R. (1967). Rms. Patol. Apparato Resp. 17, 3-16. Terjung, R. L., Winder, W. W., Baldwin, K. M., and Holloszy, J. 0. ( 1973). J. Biol. Chem. 248, 7404-7406. Troll, W., Klassen, A., and Janoff, A. (1970). Science 169, 1211-1213. Tsukamura, M. (1966). J. Gen. Microbiol. 45, 253-273. Tsukamura, M. (1969). J . Gen. Microbiol. 56, 265-287. Tsukamura, M. (1970). Jap. J. Microbiol. 14, 187-195. Tsukamura, M. (1971a). J. Gen. Microbiol. 68, 15-26. Tsukamura, M. (1971b). Jap. J. Tuberc. Chest Dis. 17, 18-30. Tsukamura, M., and Mizuno, S . (1968). Jap. J. Microbiol. 12, 371-384. Tsukamura, M., and Mizuno, S. (19711. 1. Gen. Microbiol. 68, 129-134. Tsukamura, M., Inagaki, H., and Kondo, H. ( 1970). Kekkuku 45, 153-157. Umezawa, H. (1972). “Enzyme Inhibitors of Microbial Origin.” Univ. of Tokyo Press, Tokyo. Urba, R. C. (1959). Biochem. J. 71, 513-518. UyedL, S. ( 1964a). Lepro (Tokyo) 33,265-271. UyedP, S. (1964b). Lepro (Tokyo) 33,272-275. Waksman, S. A., and Henrici, A. T. ( 1943). J. Bacteriol. 46, 337-341. Walter, H. ( 1960). Nature (London) 188, 643-645. Wamaar, S. O., and Cohen, J. A. (1966). Biochem. Biophys. Res. Commun. 24, 554-558. Wayne, L. G., and Cross, W. M. (1968). J. Bacteriol. 96, 1915-1919.

190

S. G. BRADLEY AND J . S. BOND

Wayne, L. G., Dietz, T. M., Gernez-Rieux, C., Jenkins, P. A., Kappler, W., Kubica, G. P., Kwapinski, J. B. G., Meissner, G., Pattyn, S. R., Runyon, E. H., Schroder, K. H., Silcox, V. A,, Tacquet, A., Tsukamura, M., and Wolinsky, E. ( 1971). 1. Gen. Microbiol. 66, 255-271. Wright, S. (1966). Proc. Nut. Acud. Sci. U.S.55, 1074-1081. Yamaguchi, T. (1967). J. Gen. Appl. Microbiol. 13, 63-71. Yamazaki, T., and Maruyama, T. (1973). Nature ( L o n d o n ) New Bid. 245, 140-141. Young, H. L., and Klein, H. P. ( 1967). J . Bacteriol. 93, 830-834.

Effect of Structural Modifications on the Biological Properties of Aminoglycoside Antibiotics Containing 2-Deoxystreptamine

KENNETHE. PRICEAND JOHNC . GODFREY Bristol Laboratories, Diuision of Bristol-Myers Company, Syracuse, New York AND

HIROSHIKAWACUCHI

Bristol-Banyu Research Institute, Meguro, Tokyo, Japan I. Introduction ...................................... A. Discovery and Characterization of Streptomycin, a Streptidine-Containing Aminoglycoside .......... B. General Characteristics of Commercially Available 2-Deoxystreptamine-Containing Aminoglycosides ... 11. Relative Activity, Susceptibility to Enzymatic Inactivation, and Toxicity of Naturally Produced and Semisynthetic 2-Deoxystreptamine-Containing Antibiotics ............ A. 4-Substituted-2-Deoxystreptamines ............... B. 5-Substituted-2-DOS’s ( Hygromycin B and Related Antibiotics ) ....................... C. 6-Glycosyl-Substituted-2-DOS’s . . . . . . . . . . . . . . . . . . D. 4,5-Disubstituted-2-DOS’s ...................... E. 4,6-Disubstituted-2-DOS’s ...................... 111. General Conclusions Regarding the Influence of Structural Variation on the Biological Properties of 2-DOS-Containing Compounds ...................... References .......................................

I.

191 192 199

217 219 228 228 228 252

283 299

Introduction

In view of the fact that streptomycin has been in clinical use since 1946, neomycin since 1950, kanamycin since 1958, paromomycin since 1960, and gentamicin since 1962, it seems remarkable that meaningful progress in unraveling the relationships between structure and activity of these aminoglycoside antibiotics has occurred only during the last 6 to 7 years. Studies in preceding years were not entirely devoid of progress, however, since it has been shown in the case of streptomycin that removal of both amidino groups ( I ) ,blockage of amino functions (I), or reductive amination of the aldehyde group (2,3) eliminate activity, whereas reduction to dihydrostreptomycin ( 4 ) , substitution of one amidino group with a carbamoyl moiety as in bluensomycin ( 5 ) , or demethylation of the N-methylamine ( 6 ) does not eliminate nor improve activity. During this same period (prior to 1967), limited chemical and biosynthetic modifications of the other above-listed antibiotics, all of which 191

192

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

contain a 2-deoxystreptamine (2-DOS) moiety, were attempted, but as will be shown in the present report, not a single improved product resulted. However, since 1967 the state of knowledge of structure-activity relationships among aminoglycoside antibiotics has increased very rapidly. The events that initiated this new era were discoveries made during that year and the next that the resistance of certain gram-negative bacteria to streptomycin and kanamycin was mediated by transferable R-factors (7-10). From the time of these discoveries up to the present, there has been increasing evidence that this type of resistance mechanism (enzymatic inactivation) is the principal one not only for streptomycin, but also for the 2-DOS-containing aminoglycoside antibiotics. R-factormediated resistance, in which inactivation occurs by means of phosphorylating, adenylylating, or acetylating enzymes, has been reported to occur in strains of Staphylococcus uureus, members of the Enterobacteriaceae family, and Pseudomonas aeruginosu (11). The realization that the spectrum of microorganisms susceptible to the aminoglycosides could be significantly broadened if derivatives refractory to the action of such enzymes were obtained, has led to an increased effort to find new enzyme-resistant antibiotics from natural sources, i.e., through soil screening programs. In addition, recognition of the potential utility of new agents resistant to inactivation has initiated an intensive effort to produce them through chemical modification procedures. There is a major difference, however, in the degree of sophistication found in the programs that are presently under way and those that were carried out in previous years. This difference is primarily due to the large volume of structure-activity information that has become available as a result of identification of the sites of enzymatic attack, the nature of the inactivation products produced, and especially, the determination of those structural features that affect susceptibility to inactivation. The present report is an attempt to summarize the current status of knowledge about structure-activity relationships of 2-DOS-containing aminoglycosides and to give a report on some of the recent advances in this field that are likely to result in development of clinically superior aminoglycoside antibiotics. A. DISCOVERY AND CHARACTERIZATION OF STREPTOMYCIN, A STREPTIDINE-CONTAINING AMINOGLYCOSIDE The discovery of streptomycin ( S M ) , the first of the clinically useful aminoglycoside antibiotics, was reported by Waksman and his collaborators in 1944 (12). This water-soluble basic compound was produced by an actinomycete that had been isolated from a manure-contaminated soil sample.The producing organism was ultimately classified as a strain

193

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

of Streptomyces griseus. Isolation of the antibiotic in relatively pure form was reported in the following year by Carter and his colleagues ( 1 3 ) ,who utilized charcoal adsorption followed by elution with methanolic hydrogen chloride. Exposure of the eluate to alumina chromatography afforded fractions with a maximal potency of 900 units/mg (14).Although many investigators have contributed to the structure determination of SM ( I ) , the complete structure of the antibiotic was first proposed by Kuehl and his co-workers ( 1 5 ) , a prediction subsequently confirmed by the same group (16). Waksman and Schatz ( 1 7 ) and numerous other investigators have demonstrated that SM is active against a broad spectrum of gram-negative and gram-positive bacteria, including Mycobacterium tuberculosis, but has no significant inhibitory effects against viruses (18),fungi (IQ),or the protozoan Entamoeba histolytica ( 2 0 ) .

I I

I HO

1”

I II

H,N\

C=NH

NH 1

Streptobiosamine

-NH-C

II

Streptidine i 1

Streptomycin (1)

A brief summary of data showing the spectrum of activity of SM against clinically important bacteria as reported by Garrod and O’Grady (21) is presented in Table I. These results show clearly that SM has a remarkable range of effectiveness against gram-negative bacteria, other than P . Qeruginosa and certain strains of Proteus sp., and that it also possesses some activity against many gram-positive organisms. Thus, as noted by Garrod and OGrady ( 2 1 ) ,Waksman was eminently successful in his calculated effort to find an antibiotic which was inhibitory for many of those organisms which :ire generally refractory to the action of the naturally produced penicillins. The mode of inhibitory action of SM for bacteria has been exhaustively examined, with the result that multiple effects on cellular processes have been found. The finding that the antibiotic inhibits protein synthesis in broken-cell preparations from SM-sensitive bacteria but not from resis-

194

KENNETH E.

PRICE,JOHN c. GODFREY, AND

HIROSHI KAWAGUCHI

TABLE I SENSITIVITY OF BACTERIA TO STREPTOMYCIN

Gram-negative bacteriaa Escherichia coli Klebsiella aerogenes Kle bsiella pneumoniae Proteus sp. Pseudomonas aeruginosa Salmonella typhi Salmonella paratyphi Salmonella sp. Shigella sonnei Shigella jlexneri Neisseria gonorrhoeae

Gram-positive bacteriau 2-4 2 1 4- >256 16-64 8-16

Staphylococcus aureus Streptococcus p yogenes Streptococcus pneumoniae Streptococcus faecalis Clostridium sp. Mycobacterium tuberculosis

MICb (&/ml) 2 32 64 64- > 256 > 128 0.5

4-8 4-16 2-4 2-8 4

a The results given are those obtained with sensitive strains which have not previously been in contact with streptomycin. Resistant variants are common with all species. Data of Garrod and O’Grady (27). Reprinted with permission from “Antibiotic and Chemotherapy,” 3rd ed., p. 98. Livingstone, Edinburgh (1968). * Minimum inhihitory concentrations.

tant ones (22) induced Spotts and Stanier (23) to investigate the site of binding of the antibiotic by sensitive, resistant, and SM-dependent cells. Kesults of these studies suggested that SM interacts with bacterial ribosomes. In subsequent studies (24,25), the site of antibiotic action was demonstrated to be 30 S ribosomal subunits. Traub and his colleagues (26) and Staehelin and Meselson (27) further pinpointed the site of SM sensitivity by demonstrating that it was located in the 23 S core particles of the 30 S ribosomal subunits. The binding of SM to this site, as previously noted, results in a multiplicity of effects including inhibition of correct protein synthesis, the occurrence of misreading (incorporation of a “wrong” amino acid in the peptide chain), inhibition of chain extension, polysome breakdown ( 28), and finally, accumulation of SM-bound monosomes (29). It is now believed that the primary biochemical lesion ( inhibition of protein synthesis and/or synthesis of faulty protein) is induced by attachment of an SM molecule to a nucleotide of a coding triplet in a one-to-one ratio. Other degradative processes such as permeability changes and impairment of respiration may occur prior to cell death (21). For a more detailed discussion of the mode of inhibitory action of SM on “susceptible” bacterial cells, see the excellent review by Gale et al. (30), A major handicap possessed by SM came to light soon after its initial clinical application. Finland and his co-workers (31) observed 8 thera-

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

195

peutic failures in 12 patients receiving the drug for treatment of urinary tract infections. In each case, the lack of efficacy seemed to be attributable to rapid emergence of bacterial populations possessing resistance to SM. An examination of organisms subjected to several in vitro transfers in the presence of sublethal concentrations of the compound revealed that a significant number of cells which possessed high-level SM resistance were usually present in any sizable culture population (32,33). Subsequent laboratory studies conducted by a multitude of investigators have demonstrated that strains of many bacterial species, including M . tuberculosis, are comprised of such mixed populations ( 2 1 ) . Cells in these cultures can acquire high-level SM resistance through a single mutational event that influences the properties of a specific ribosomal protein (11). These altered ribosomes either fail to bind the antibiotic or cause configurational changes that do not permit the drug to exert its characteristic inhibitory effects ( 30). The true role that such mutants play in causing a lack of responsiveness to SM therapy has not been clearly established. However, presumptive evidence indicates that resistance of this type may be of significance in enterococcal strains of clinical origin. Moellering et al. ( 3 4 ) found strains having two different levels of resistance to SM. The majority of SM-refractory strains possessed low-level resistance [minimum inhibitory concentration ( MIC ) of 63-500 ,.tg/ml] and responded synergistically to mixtures of penicillin and SM. The finding that SM uptake by these organisms was significantly enhanced when they were exposed to penicillin suggests that they possessed a permeability barrier to SM that could be overcome, at least in part, by cell wall inhibitors. Strains with highlevel resistance (MIC of >500 pg/ml), however, did not respond synergistically to the combination of antibiotics. Subsequent experiments by Zimmerman and co-workers (35) showed that a spontaneous mutant of an enterococcal strain with low-level resistance that had been isolated from a blood culture of a patient with bacterial endocarditis had highlevel resistance that was ribosomally mediated. The authors suggested that the high-level resistance found in the strains of clinical origin that had been isolated by Moellering et al. ( 3 4 ) could also be attributed to ribosomal resistance. A second investigation involving clinical isolates was conducted by Tseng et al. ( 3 6 ) , who studied a group of 200 P . aeruginosa strains. These investigators found a number of isolates with low-level and a few with high level resistance to SM. The former strains, which were characterized by a diminished ability to take up the antibiotic, had ribosomes that were fully sensitive to SM. However, one of the strains with high-level resistance (MIC of 20,000 pglml) reportedly possessed ribosomes that were resistant to the action of SM. Thus, while it appears that bacteria with high-level (ribosomal) resistance to SM

196

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWACUCHI

are readily selected in laboratory experiments, they may actually be relatively rare in strains from clinical sources. On the other hand, variants with low-level resistance due to permeability barriers can be isolated with somewhat greater frequency. Such strains tend to be cross-resistant with 2-DOS-containing antibiotics whereas those having ribosomal resistance are not ( 3 7 ) . Overall, however, based on evidence obtained in the last few years, there is probably little doubt but that the most imporant mechanism of S resistance among organisms isolated from clinical sources is that involving inactivation of the antibiotic by R-factor-mediated enzymes. H. Umezawa and his collaborators ( 9 ) discovered that an R’ Escherichia co2i strain could inactivate SM by an enzymatic process that required ATP. Yamada et al. (38) and H. Umezawa et al. ( 9 ) showed that the site of inactivation by the enzyme, an adenylyltransferase, was the C-3’-hydroxyl group (present in the N-methylglucosamine moiety of the antibiotic). The absolute identity of the inactivated product, SM adenylylate (II), was provided by Takasawa et al. (39) and Harwood and Smith (40). Since it is not capable of binding to SM-sensitive ribosomes, it is completely devoid of antibiotic activity ( 4 1 ) .

Adenylic acid

13

Streptomycin adenylate

(TI)

A second transferable R-factor that mediates enzymatic inactivation of SM was reported by Ozanne et al. ( 1 0 ) .This enzyme phosphorylates the same hydroxyI group as that attacked by the above-described adenylyl-

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

197

transferase. The product, SM phosphate (111), does not bind to ribosomes of SM-sensitive cells, and thus has no antibiotic activity (41). Although the phosphotransferase cannot utilize spectinomycin ( IV) as a substrate, the adenylylating enzyme inactivates both this antibiotic and SM (10). Neither enzyme has any effect on 2-DOS-containing aminoglycosides, but as shown in Table I1 (6,41a), both will inactivate a number of SM-like compounds ( 1 1 , 4 2 ) . 0 I1

HO-P-OH I H

O

G

NHCH, 1'

HO

0

0

HO

Streptomycin phosphate

(rn)

NHCH, H,CHN Spectinomycin

(W

The finding that bioactive compounds like streptomycin B (mannosylstreptomycin) are not inactivated by either one of the episomally mediated enzymes strongly emphasizes the potential value of modifying aminoglycosides chemically so that they still retain antibiotic activity but are not susceptible to the action of inactivating enzymes. In the case of SM, the significance of this type of resistance mechanism cannot be overestimated since the enzymes, particularly the phosphotransferase, are widely distributed among clinical isolates (9,10,43-46), and in fact are so prevalent that they are directly responsible for a significant diminution in the utilization of SM in the clinic. At the present time, the antibi-

TABLE I1 SUSCEPTIBILITY TO ENZYMATIC INACTIVATION AND RELATIVE BIOPOTENCY OF STREPTOMYCIN AND VAEIOUSDERIVATIVES

Inactivated by

No.

R*

Compound name

RI

R3

R4

R5

NH

I/

SM-ATa

SM-PTa

Biopotencyb

(SM = 100)

Streptomycin (SM)

NHCNHZ NH

CHO

CH,

H

H

+

+

100

(V)

Dihydrostreptomycin

NHCNH2 NH

CH,OH

CHI

H

H

+

+

100

NHCNH2

CHzOH

CH,

CH,

H

-

-t-

4

(W

N-methyldihydrostreptomycin

/I

N-dernethyldihydrostreptomycin Streptomycin B

(1x1

Bluensomy cin'

II

NHCNH? 0

/I

OCNHp

CHzOH

H

H

H

-

+

CHO

CHa

H

Mannose

-

-

12.5

CHZOH

CH,

H

H

+

+

NTd

SM-AT = Streptomycin adenylyltransferase, SM-PT

* Data from Heding and Lutzen (6).

b

X

9

E 12.5

NH

(VIII)

"2

P

NH NHCNH2

8 z

II

(VII)

2 P

(1)

I1

Y

=

streptomycin phosphotransferase.

Bluensomycin was originally known as glebomycin ( 4 1 ~ ) . N T = not tested.

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

199

otic, which is not orally absorbed, is used primarily for parenteral treatment of tuberculosis (always in combination with other agents because of the heteroresistant nature of mycobacterial populations), plague, and tularemia ( 21 ) . The acute intravenous LD,, of a highly purified lot of SM for mice was 300 mg/kg ( 4 7 ) , a value some 4 to 5 times lower than that found when the drug was administered to the same species by the subcutaneous route. In man, the compound produces both vestibular and auditory toxicity, the former occurring at somewhat lower dosage than the latter. Doses of 2 gm routinely administered to adults as a single daily dose almost invariably produce vestibular disturbances; the incidence of vertigo following 1-gm doses is very much reduced, and it is virtually absent with 0.5-gm doses ( 4 8 ) . Despite this dose-toxicity relationship, there is still a general lack of predictability regarding the SM dosages and regimens that induce eighth-nerve toxicity, even though the total dosage and state of the patient’s renal function are certainly factors that contribute to toxicity ( 2 1 ) . For example, among 22 patients with eighth nerve damage that were studied by Cawthorne and Ranger (49), 21 had vestibular damage and 3, a partial loss of hearing. None of these individuals had received more than 20 gm of SM, and 12 had received only 12 gm or less. Obviously total dosage was not a significant factor in these cases. SM has also been shown to have the capability to produce neuromuscular blockade ( 5 0 ) . The compound is so weakly active in this regard, however, that the likelihood of observing this phenomenon in patients receiving the drug at recommended dosage is quite remote. Dihydrostreptomycin also affects the eighth nerve and can cause neuromuscular blockade. While it is less likely than SM to cause vestibular damage, it has significantly greater potential to induce hearing loss ( 2 1 ) . CHARACTERISTICS OF COMMERCIALLY AVAILABLE B. GENERAL 2-DEOXYSTREPTAMINE-CONTAINING AMINOGLYCOSIDES

The first 2-DOS-containing antibiotics discovered were the neomycins. The producing organism was isolated in 1949 from soil by Waksman and Lechevalier (51 ) and was subsequently given the designation Streptomyces fradiae. The partially purified antibiotic preparation appeared to hold particular promise at that time because of its potent antimycobacterial activity and overall antimicrobial spectrum. The configurational studies necessary to establish the structures of the major antibiotics produced in the “neomycin” fermentation were finally completed by Rinehart and his co-workers in 1962 ( 5 2 ) . This occurred subsequent to the structural assignment of the weakly active agent, neamine or neomy-

200

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWACUCHI

cin A ( X ) , which occurs in the neomycin fermentation and is also obtained as a degradation product of the other neomycins (53). The two major antibiotics in the fermentation were neomycin B (XI) and neomycin C (XII) which differ from each other only in a single asymmetric center at C-5 in the neosamine moiety of neobiosamine. Commercial neomycin is a mixture of the comparably active compounds, neomycin B and C, the former comprising 85-901 of the mixture (54). Framycetin, originally isolated from a strain of Streptomyces Zauendulae (55), was subsequently proved to be identical to neomycin B ( 5 4 ) . Minor antibiotics produced in fermentations of S . fradiae and designated as neomycins D, E, and F, have just been shown to be members of the paromomycin family and will be discussed below. Production of neomycin B by a species of Micromonospora (strain 59-683) has been described by Wagman et al. (55a) in a recent pulication. This is the first report of neomycin production by a nonstreptomycete.

2,6-Diarninodideoxyglucose /

/

/

HO

/

/ Neornycin A (nearnine)

(X)

Paromomycin, an antibiotic closely related to the neomycins, was discovered in 1956 (56). The producing organism was a soil isolate given the designation, Streptomyces rimosus forma paromomycinus. Laboratory studies by Coffey et al. (57) indicated that the antibiotic obtained from broth filtrates had marked and broadspectrum antibiotic activity against gram-positive and gram-negative bacteria as well as acid-fast organisms. The compound readily cured experimental infections in mice when administered subcutaneously, but was relatively ineffective by the oral route. In contrast to SM and the neomycins, paromomycin was found to be orally active against the protozoan, E. histolytica, in both in uitro and in viuo tests. The gross structure of this antibiotic, paromomycin I (XIII), was established by Haskell and his co-workers (58) concurrently with this group’s structural elucidation of two of its degradation fragments, paromamine and paromobiosamine (59). An isomeric analog,

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

I

OH

201

Neobiosamine

(XI) R = CH,NH,, Rl= H (B) (XII) R = H, R, = CH,NH, (C) Neomycins B and C

paromomycin I1 (XIV), has since been isolated by Rinehart et al. ( 5 2 ) . This compound differs from paromomycin I only in that it contains neobiosamine C instead of neobiosamine B ( 5 4 ) . The identity of paromomycin and the antibiotics catenulin, hydroxymycin, and aminosidin has been reported by Schillings and Schaffner ( 60). Zygomycin A, another aminoglycoside antibiotic, has also been shown to be identical to paromomycin (61). Finally, neomycins D, E, and F, which have been found in minute quantities in the fermentation broths of the neomycin-producing culture, S . fradiae, were recently shown to be indistinguishable from paromamine, paromomycin I, and paromomycin 11, respectively. The compounds were found in <1% amounts in commercial preparations of neomycin by use of ion-exclusion chromatography and gas-liquid chromatographic procedures ( 62). In view of the general structural similarities between the neomycins and the paromomycins, which differ only in the nature of the substituent at C-6' in the glucosamine moiety (amino in the case of neomycins, hydroxyl in the case of paromomyciiis), it is not surprising that this group of antibiotics are coproduced by the S. jradiae culture. The first report of isolation of the kanamycins was made by H.Umeizawa et al. ( 6 3 ) in 1957. These authors also described the kanamycin-

202

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

1'

I

Paromamine.

Neobiosamine

(XIII) R = CH,NH,, R, = H (I) (XI")R = H, R, = CH,NH, (11) Paromomycins I and I1

producing strain to which they assigned the name Streptomyces kanunyceticus. Chromatographic studies with broth filtrates obtained from fermentations of this organism revealed that there were always two, and occasionally three, bioactive components present. The structure proof of the major one, kanamycin A (XV), was independently provided by Cron et al. ( 6 4 ) and Maeda et al. (65). It is noteworthy that this compound, in contrast to the previously discussed neomycins and paromomycins, is a 4,6- rather than a 4,5-disubstituted-2-DOS antibiotic. Kanamycin A was totally synthesized by S. Umezawa and his co-investigators (66). The earliest report on its antimicrobial properties was published by Takeuchi et al. (67). The antibiotic was found to inhibit both gram-negative and gram-positive bacteria and, as was found with the other aminoglycoside antibiotics, was markedly active against mycobacterial species. Subsequent investigations have resulted in structural assignments for the other components of the kanamycin complex. The complete structural determination of kanamycin B (XVI) was ultimately provided by Hichens and Rinehart (68) and Ito et al. ( 6 9 ) , although Schmitz and his associates ( 7 0 ) had identified the major cyclic moieties of the antibiotic in 1958, 5 to 6 years earlier. Total synthesis of kanamycin B was

203

MODIFICATION OF AMINOCLYCOSIDE ANTIBIOTICS

achieved by S . Umezawa et a2. ( 7 1 ) . The remaining kanamycin, designated as C (XVII), produced 2-DOS, kanosamine, and 2-amino-2-deoxyglucose upon hydrolysis ( 7 2 ) .Stereochemical assignments were provided by Hichens and Rinehart ( 6 8 ) . This antibiotic’s total synthesis was also accomplished by S . Umezawa and his co-workers ( 7 3 ) .While both kanamycin B and kanamycin C have the same broad spectrum of antibacterial activity as kanamycin A, the intrinsic activity of the former is about twice that of kanamycin A while the activity of the latter is significantly less than that of kanamycin A.

OH

/!

Kmosamine

i I

(XV) R = OH, R, = NH, (A) (XVI) R = R, = NH, (B) (XVn)R = NH, , R, = OH (C)

Kanamycins A, B, and C

The most recently marketed aminoglycoside antibiotic is gentamicin C which is a mixture of closely related antibiotics. The first isolation of the gentamicin complex was described in 1963 by Weinstein and his colleagues from the Schering Corporation ( 7 4 ) .Two gentamicin-producing cultures have been described; one is Micromonospora purpurea; the second, Micromonospora echinospora ( 7 5 ) .The gentamicin C complex is a mixture of three closely related antibiotics, gentamicin C, (XVIII), C,, (XIX), and C, ( X X ) , the gross structures of which have been proposed by Cooper et al. ( 7 6 ) . All are characterized by having the unusual sugar, garosamine, in glycosidic linkage with the C-6 hydroxyl of

204

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

2-DOS. Commercial preparations of gentamicin C reportedly are comprised of 28.3%of C1, 34.6%of C,%,and 37.1%of Ce ( 7 7 ) . Weinstein et uZ. (75) reported that gentamicin C, like the other aminoglycoside antibiotics, has only weak activity against streptococci, but is highly active against other gram-positive bacteria, including M . tuberculosis. Moreover, it is appreciably more active than the previously described aminoglycosides against gram-negative bacteria, and of even greater significance, demonstrates a marked inhibitory effect against most strains

of P. aeruginosa.

I

I -

i

.

g

c

H

3

5"

1"

/

Garosamine

0

I

I

I

I I

(XVIII) R = R, = CH3 (C,) (XIX)R = R, = H (Cia) (XX)R = CH,, R, = H (C,) Gentamicins C , , Cla, and C,

Several other antibiotics that are closely related to members of the gentamicin C complex are coproduced as minor components in the gentamicin fermentation, These include gentamicin A (XXI), whose gross structure was established by Maehr and Schaffner (78), and gentamicin B (XXII), whose structural assignment was given by Weinstein et a2. ( 79). Gentamicin A contains the unusual sugar, C-4-demethylgarosamine, instead of garosamine, which is common to all other known gentamicins. An additional pair of coproduced members of the complex has recently been isolated and the compounds' structures determined (80).

MODIFICATION OF AM1;NOGLYCOSIDE ANTIBIOTICS

205

Gentamicin B, (XXIII) is identical to gentamicin B except that it has a C-CH, at the @-position while gentamicin X (XXIV) is similar in some respects to gentamicin A, but differs in that its C-6 moiety is garosamine rather than C-4-demethylgarosamine.

0

(XXI) R, (XXII) R, (XXIII) Rl (XXIV) R,

& = OH, = OH, ri, = H. & = MI,, = OH, % = CH,, & = NH,, = NH,, = H, & = OH, = NH,, R, = H.

R4 = H

(A) R4 = CH, (B) R., = CH, (B,) R4 = CH, (X)

Gentamicins A, B, B, , and X

1 . Spectrum of Antimicrobial Activity

The commercially available 2-DOS-containing antibiotics, neomycin ( a mixture of B and C ) , framycetin (neomycin B ) , kanamycin A, paromomycin I, and gentamicin C ( a mixture of C,, C,,, and C2),have antimicrobial spectra that are generally similiar to that of SM. Garrod and O’Grady (21) have compared the in vitro activity of these compounds against a variety of gram-positive and gram-negative organisms. Results of this study are presented in Table 111. All antibiotics displayed excellent inhibitory action against S. aureus strains with the activity of gentamicin > neomycin > kanamycin = paromomycin > streptomycin, while Streptococcus faecalis, another grampositive species, was quite resistant to all the agents. Various members of the gram-negative Enterobacteriaceae family ( E . coli, Klebsiella sp., Proteus sp., Shigella sp., etc.) have a remarkably uniform degree of susceptibility to the action of each of these antibiotics, gentamicin once again proving to be the most potent compound. Finally, in the case of P . aeruginosa strains, only gentamicin was significantly inhibitory for this species, thus providing the one true deviation in spectrum ob-

MEAN^ MINIMUM INHIBITORY Organism

Staphylococcus aureus Streptococcus jaecali Escherichia coli Klebsiella sp. Aerobacter sp. Proteus mirabilis Proteus vulgaris Proteus morgania Proteus rettgeri Pseudomonas aeruginosa Salmonella sp. Shigella sp.

TABLE I11 CONCENTRATIONS (MIC)

No. of strains

Streptomycin

29 32 22 20 10

2

Neomycin

7: R

(/.IG/ML)

OF

Z

AMINOGLYCOSIDE .4NTIBlOTICS

Kanamycin

Framycetinb

1 32 4 2 2 4 4 4 2 128 2 4

0 .3

Paromomycin

21 p Gentamicin

3

64

64

8

8 2 2 8 4 8

4 4

8

6 6 10

4

7

4

31

32 16 8

14

17

0 ..?I

8

8 32 2 8

64

8 2 2 8 4

8 8 32 2 8

1 64 8 2

2 8 4 4 4

512 2 8

0.125 8 1 1 0.5 2 1

1 1 4 1

2

a Tests mainly of recent isolates a t Hammersmith Hospital by plate dilution method with %fold differences. Means are of log, of MIC to the nearest log,. In the series, tested strains showing a clearly abnormal degree of resistance (sometimes following treatment with the antibiotic) were omitted from these calculations. Data of Garrod and O’Grady (21). Reprinted with permission from “Antibiotic and Chemotherapy,” 3rd ed., p. 98. Livingstone, Edinburgh (1968). b Neomycin €3.

ij R

U

0

3:

z

n 0

m m 4

P

B

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

207

served among this group of aminoglycosides. As previously noted, all are markedly inhibitory for strains of M . tuberculosis.

2. Mode of Action The mechanism of inhibitory action of the 2-DOS-containing aminoglycosides is “superficially” similar to that of streptomycin in that they all inhibit protein synthesis in sensitive bacteria (30). Furthermore, it has been unequivocally established that this suppressive effect occurs, just as with SM, as a result of an interaction of the antibiotic and the 30 S ribosomal subunit (81). Benveniste and Davies (82) compared the relative ability to inhibit protein synthesis of various 2-DOS-containing antibiotics by measuring suppression of R17 phage RNA-directed polypeptide synthesis. They found that gentamicin C,, gentamicin C , , and kanamycin A had essentially comparable suppressive ability, with all inhibiting synthesis by approximately 50%when tested at a 10 pglml concentration. Neomycin B and C were more active since 10 pglml caused 80 and 72%inhibition, respectively, while paromomycin’s capacity to inhibit was intermediate to the others (65%suppression). These antibiotics also cause miscoding, but differ from SM in that the degree of their activity varies according to the ratio of the number of ribosomal units bound per unit of drug. This suggested to Weisblum and Davies (81) that 2-DOS-containing drugs may interact at multiple sites on the ribosome. Interestingly, the presence of 2-DOS or streptidine in the molecule appears to be necessary to produce misreading, since aminocyclitol antibiotics, such as spectinomycin and kasugamycin which lack 2-DOS, react with 30 S ribosomes to inhibit protein synthesis but do not cause misreading ( 83,84).

3. Resistance Mechanisms and Their Significance Little information is available concerning the mechanism by which “acquired” resistance occurs among 2-DOS-containing aminoglycosides. It is known, however, that strains “trained” in the laboratory to show antibiotic resistance of this type are generally cross-resistant to other members of the aminoglycoside family (21 ). In the case of SM, however, the degree of cross-resistance observed between it and the 2-DOS-containing antibiotics is generally of a lower order of magnitude (see Table IV) . It seems likely that this type of resistance stems from selection of cell populations that have reduced permeability to aminoglycoside antibiotics. The occurrence of ribosomal resistance to 2-DOS-containing antibiotics may be even more rare than has been found in the case of SM. This could account for the difficulty one has in obtaining mutants that develop single-step high-level resistance to these drugs in in vitro tests ( 3 0 ) .

208

KENNETH E. PRICE, JOHN

c R O S S - ~ ~ F S I S r l ‘ A N C EAMONG

c.

GODFREY, AND HIROSHI KAWAGUCHI

TABLE IV ANTrnIoTICs

OF THI”,

NEOMYCIN GROUP“

Indexb of increase in resistance to Antibiotic to which habituated

Streptomycin

Neomycin

Framycetin

Streptomycin Neom ycin Framycetin Kanamycin Paromom ycin

10 6 6 6 6

5

4

5

5

8 8 9 8

8 8 9 8

8 9 10 8

8 10 9 9

Kanamycin

Paromomycin

a Data from Garrod and O’Grady (21). Reprinted with permission from “Antibiotic and Chemot,herapy,” 3rd ed., p. 98. Livingstone, Edinburgh (1968). For example, 8 = increased 28-fold (i.e., 256-fold).

However, mutants in which the genes that specify resistance are chromosomal and code for altered ribosomes, have been isolated in the laboratory. Masukawa et al. (85,86), for example, have utilized kanamycinresistant ribosomes obtained from an E . coli strain to demonstrate that the site of the antibiotic’s inhibitory action is the 23 S core of 30 S ribosomes, just as has been found for SM. The only observation (87) indicating that altered ribosomes are responsible for resistance of clinical strains to a 2-DOS-containing antibiotic ( gentamicin) is somewhat clouded by the fact that the organisms in question ( P . aeruginosa) have been subsequently found to produce an enzyme that inactivates the antibiotic (88). Enzymatic inactivation of the 2-DOS-containing aminoglycosides, as is the case with SM, is the principal mechanism of resistance among naturally occurring clinical and nonclinical isolates. A given enzyme may have the capacity to inactivate more than one antibiotic and a particular antibiotic may be inactivated by more than one enzyme ( 11 ) . I n addition to the SM-inactivating enzymes previously discussed, there are at present eight different bacterial enzymes that are capable of inactivating aminoglycosides. Table V gives enzyme designations and abbreviations, the list of some of the antibiotics that act as substrates for them, and the sites where they attack; also compiled are the known enzymeproducing species with an indication as to which have been shown to be donors or recipients of plasmids that mediate enzymatic inactivation. The summary in Table V (88-114) includes information about naturally produced antibiotics, such as tobramycin, ribostamycin, butirosins, and lividomycins, as well as the commercially available antibiotics that have been the major subject of discussion in this section. The inclusion

TABLE

v :P R I N C I P A L

SUBSTRATES, SITES

Enzyme designation

OF

ATTACK,A N D MEDIATOR TRANSFERABILITY OF AMINOGLYCOSIDE-~N.~CTIVATING ENZYMES

Substrates (see Fig. 1)

Sites of attack

Neomycin-kanamycin phosphotransferaser (NPTI)

Neomycins, paromomycins, ribostamycin, lividomycins, kanamycins

C-3’-OH in the 4-substituent of 2-DOS and the 5”-OH in the ribosyl

Neomycin-kanamycin phosphotransferaseII (NPTII)

Neomycins, paromomycins, ribostamycin, butirosins, kanamycins

C-3’-0H in the 4-substituent of 2-DOS

Lividomycin phosphotransferase (LPT) Gentamicin nucleotidyltransferase (GNT)

Lividom y cins

C-5”-OH in the 5-0-ribosyl

Gentamicins, kanamycins, tobramycin

moiety of lividomycin C-2”-OH in the &substituent of 2-DOS

Kanamycin acetyltransferase (KAT)

Neomycins, kanamycins h B, tobraniyoiu, genC2, ribotamicins C1, stamycin, butirosins Gentamicins, tobramycin,b kanamycin B*

moiety of lividomycin

Gentamicin acetyltransferaseI (GATI) Gentamicin acetyltransferaseII (GATII)

Gentamicin acetyltransferaseIII (GATIII)

+

+

Neomycins,b paromomycins,b kanamycins B C,b ribostamycin,b tobramycin, gentamicins, butirosins,b lividomycinsb Neomycins, paromomycins, ribostamycin, kanamycins, tobramycin, gentamicins, lividom ycins

+

C-6‘-NHz in the 4-substituent of 2-DOS C-3-NH2 of 2-.DOS moiety of gentamicins and tobramycin C-2’-NH2 of the 4-substituent of 2-DOS

C-3-NH, of 2-DOS

Enzyme-producing organisms (transferability)

Escherichia coli (+),a Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus E . coli (+),Klebsiella pneumoniae (+),P . aeruginosa, E. cloacae, S. aureus P. aeruginosa

References 89-95

9.8, 96-98

99

E. coli (+),K . pneumoniae (+),F. cloacae, P. aeruganosa E . coli (+), P. aeruginosa, E . cloacae, E. aerogenes

92, 100-10.4

P. aeruginosa, K . pneumoniae ( +),E . coli (+)

92, 109-113

PTovidencia stuartii

92, 113, l l S a

P. aeruginosa

88, 114

a (+) indicates that transfer of resistance between the indicated organism and another member of the Enterobacteriaceae family has been demonstrated. These compounds are relatively poor substrates. Note added in proof: New enzyme designations were recommended on June 5 , 1974 (Bratislava,Czech.) by a committee of the Plasmid Nomenclature Group: NPTI changed to APH(3‘)-I, aminoglycoside phosphotransferase (3’)-I, NPTII to APH(3’)-II, LPT to APH(5”), GNT to ANT(2“), aminoglycoside nucleotidyltransferase (2”), KAT to AAC(6’), aminoglycosideacetyltransferase (6’), GAT1 to AAC(3)-I, GAT11 to AAC(2’), GAT111 to AAC(3)-II.

210

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

of data on this first group of compounds is essential in order to fully and definitively characterize those enzymes that are capable of inactivating 2-DOS-containing aminoglycosides. The structures of representative antibiotics in the series discussed in Table V are presented in Fig. 1. The phosphorylating enzymes probably have the widest distribution and are the most prevalent ones among clinically important organisms (92). NPTI, which is an R-factor mediated enzyme whose transferability has been demonstrated in E . coli, causes O-phosphorylation at the 3’-OH group of neomycins B [compound (XXV) in Fig. 11 and C, paromomycins I (XXVI) and 11, ribostamycin (XXVII), and kanamycins A, B (XXVIII), and C. As might be expected, it cannot utilize tobramycin (XXIX) or members of the gentamicin C complex, C,,C,, ( X X X ) , and C,, as substrates since they lack a hydroxyl group at the critical site. Less obvious was the finding that NPT, does not interact with butirosins A and B (XXXI). Since butirosin B differs from ribostamycin, which is a good substitute for the enzyme, only in that it has an L-(-)-y-aminoa-hydroxybutyramide group rather than a primary amine at C-1 of 2-DOS, it is obvious that the presence of this unusual substituent on 2-DOS somehow interferes with the action of the enzyme. As will be shown subsequently (Section II,E), the introduction of this amino acid into the 2-DOS moiety of aminoglycosides other than butirosins also effectively blocks phosphorylation by NPT,. Additional substrates for NPT, are lividomycins A and B (XXXII). Their high degree of susceptibility to inactivation by the enzyme could not have been predicted since they lack a 3‘-hydroxyl group. However, it has now become clear that the site of phosphorylation in these compounds is the 5”-OH of ribose, a substituent shown in molecular models to be in close proximity to C-3 of the aminohexose moiety that is linked to 2-DOS at C-4. A second enzyme, which also inactivates by phosphorylating the 3’-hydroxyl group of susceptible aminoglycosides, can be differentiated from NPT, on the basis of its substrate profile. The production of this enzyme, which has been designated as NPTII, is under the control of an extrachromosomal gene whose transferability has been demonstrated in E. coli and Klebsiella pneumoniae strains. In addition to these species, the enzyme has been shown to be produced by strains of Enterobacter doacae, P. aeruginosa, and S. aureus (29). NPTII,, like NPTI, markedly inhibits the antibacterial action of neomycins, paromycins, ribostamycin, and the kanamycins. However, in contrast to NPTI, it readily reacts with the 3’-OH of the butirosins and is incapable of phosphorylating the hydroxyl at C-5 in the ribosyl moiety of lividomycin. Lividomycin phosphotransferase ( LPT ) is an enzyme whose production is regulated by a gene found in a P . aeruginosa strain (Tl-13). On the basis of preliminary data, it appears that the enzyme inactivates

MODIFICATION OF AMINQGLYCOSIDE ANTIBIOTICS

211

lividomycins via phosphorylation of the ribose 5”-OH but utilizes neither kanamycins nor neomycins as substrates. The incidence of this type of enzymatic inactivation among clinical isolates and the nature of the gene that mediates its production are not yet known. Gentamicin nucleotidyltransferase ( GNT) was first identified in a group of R+ K . pneumoniae strains that were responsible for a series of hospital-centered infections. The mechanism of gentamicin C inactivation was found to involve adenylylation of the 2”-OH group of garosamine, an amino sugar that is present in all members of the “C” complex. The mediating plasmid can be readily transferred to E . coli K12 in conjugation experiments. In a cell-free system containing an aminoglycoside antibiotic and one of several nucleotides, the enzyme obtained from one such R+ E . coli strain (JR66/677) was found to produce not only the adenylylate of the antibiotic, but the inosinate and guanylate as well. This mechanism of resistance, i.e., nucleotidylation, which occurs with relatively low frequency in clinical isolates, affects only aminoglycosides having aminohexose moieties that are glycosidically linked at the 6-position of 2-DOS. Thus, kanamycins, tobramycin, and the gentamicins are good substrates whereas those antibiotics that possess a 5-0-pentosyl moiety ( neomycins, paromomycins, ribostamycin, butirosins, and lividomycins ) are not. Enzymatic inactivation resulting from N-acetylation of gentamicin’s amino groups is probably the primary mechanism by which clinical isolates resist this antibiotic’s inhibitory action. One such acetylating enzyme, designated as kanamycin acetyltransferase ( KAT) , was originally detected in an E. coli strain. Isolation of the inactivated kanamycin product revealed that it was a mono-N-acetyl derivative and that the site of acetylation was the 6’-NH, group (present in the glucosamine moiety linked at C-4 of 2-DOS) of kanamycin A. All 2-DOS-containing aminoglycosides that have a primary amine at C-6’ will act as substrates for the enzyme, Antibiotics like kanamycin C, the paromomycins, and the lividomycins which have a hydroxyl substituent at C-6’, and gentamicin C1, which has an N-methylamino group at this position, are not substrates for the enzyme. The production of KAT has been shown to be regulated by a transferable plasmid in E. coli strains. The nature of the gene controlling KAT production in P. aeruginosa has not yet been established. Interestingly, in whole-cell preparations, the phenotypic expression of the KAT gene, i.e., the increase in resistance of cells to inhibition by those antibiotics which act as substrates, varies markedly depending upon whether it is present in E. coli or P . aeruginosa. In the former, the enzyme-producing cells are only slightly less susceptible to inhibition than wild-type E. coli strains, whereas cells of the latter display resistance that is markedly greater than that of non-enzyme-producing strains.

212

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI KAT

Neomycln B

(xxv) KAT

I

Paromomycln I ( W I )

,?+* HO

OH

Rlbostamycin

(xxvn)

Kanamyeln B (xxv111)

FIG. 1. Structures of representative aminoglycosides for which the sites of attack by inactivating enzymes have been identified. NPTI = neomycin-kanamycin phosphotransferaser; NPTII = neomycin-kanamycin phosphotransferase11; LPT = lividomycin phosphotransferase; GNT = gentamicin nucleotidyltransferase; KAT = kanamycin acetyltransferase; GATI = gentamicin acetyltransferasei; GATII = gentamicin acetyltransferaseIL;GAT111 = gentamicin acetyltransferaseIII.

KAT

I

KAT

?H

,

CH3

GNT' (0)

HO

OH

Butirosin B (XXXII

Lividomycin B

(XXXII)

FIG.1 (Continued)

214

KENNETH E. PRICE, JOHN c . GODPREY, AND HiROSHI K A ~ A G U C H I

Recently, however, Haas and Davies (115) have reported the isolation of a laboratory variant of the KAT-carrying E . coli strain (W677/R5) which has marked resistance to those aminoglycosides that have a 6’-NH2 function. The crude enzyme obtained from this strain by osmotic shock can be utilized in conjunction with lC-labeled acetyl coenzyme A to assay the concentration of aminoglycosides present in serum samples. GATI, or gentamicin acetyltransferaseI, acts on a relatively limited range of substrates. This R-factor-mediated enzyme has been shown to be transferable in Enterobacteriaceae species but not in P . aeruginosa. However, evidence available up to this time suggests that it is probably the major mechanism involved in resistance of the latter species to gentamicin. GATI, whose site of attack is the C-3-NH, group of 2-DOS, has a high degree of affinity for all members of the gentamicin C complex and a measurable, but lower one, for tobramycin. Slight reactivity with kanamycin B has also been reported, but no other aminoglycoside acts as a substrate for the enzyme. Although strains possessing this enzyme have marked resistance to gentamicin C, they are fully susceptible to tobramycin and the other aminoglycoside antibiotics. Another gentamicin acetylating enzyme has been given the designation GAT,,. This enzyme, which was originally detected in a strain of Providencia stuartii, may also be present in strains of Proteus rettgeri (115~). It acetylates the C-2-amino group of the aminohexose that is glycosidically linked with 2-DOS at C-4. This enzyme has an unusual substrate profile since it affects the activity of all the naturally produced aminoglycosides except kanamycin A, gentamicin B, and gentamicin B,, all of which lack an amino group at C-2’. Thus far, no information is available as to whether this type of resistance is associated with the bacterial chromosome or with a transferabIe plasmid. Recently, a third acetyltransferase, GATIII, that is capable of inactivating gentamicin has been identified. This enzyme, whose regulatory gene has not been characterized, utilizes all naturally produced aminoglycosides except butirosins as substrates. Its site of attack is the same as that of GATI, i.e., the C-3-amino group of 2-DOS. Thus far, it has been found only in P . aeruginosa. It is obvious from the summary in Table V that aminoglycoside-inactivating capability is widely distributed among clinical isolates with strains of S . aureus, P. aeruginosa, P . stuartii, E . coli, K . pneumoninue, and Enterobacter sp. all having been shown to be enzyme producers. Furthermore, it is likely that an enzymatic mechanism of resistance is also responsible for the failure of certain strains of Serratia (116,117) and indole-positive Proteus (118) to be inhibited by aminoglycosides. In view of the many recent reports describing the involvement of aminoglycoside-resistant strains in hospital-centered outbreaks (l19-125), there seems to be little

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

215

question but that continued utilization of the currently available aminoglycoside antibiotics will exert selective pressures that favor the transfer of episomally mediated resistance. If so, this is likely to result in an even sharper rise in the incidence of hospital-centered epidemics that involve antibiotic-resistance organisms. The potential seriousness of this problem has stimulated the pharmaceutical industry to attempt to prepare enzyme-resistant aminoglycoside antibiotics by means of chemical modification procedures.

4. Nature of Toxicity ~ O TMammals The principal types of toxicity associated with aminoglycoside usage are impairment of the vestibular or auditory functions of the eighth cranial nerve and necrosis of the kidney tubules ( 1 2 6 ) . As can be seen in Table VI, each antibiotic possesses a characteristic toxicity profile ( 127-131 ). Examination of the toxicity indices for streptomycin reveals that it has much greater potential to cause vestibular than auditory or renal damage. Conversely, dihydrostreptomycin tends to produce significantly greater impairment of auditory than vestibular function. Like streptomycin, however, it has a limited potential to cause nephrotoxicity. Unfortunately, the auditory damage produced by this drug is frequently delayed and irreversible, with the result that it is not utilized by parenteral routes of administration. Neomycin produces little vestibular toxicity, but does offer a genuine threat to patients because of its pronounced tendency to cause auditory and renal damage. In fact, the danger of producing such toxicity is so great that the compound is no longer recommended for parenteral use. Kanamycin, like neomycin, has a greater tendency to produce auditory toxicity than vestibular toxicity. However, its potential for inducing impairment of renal function is considerably less than that of the neomycins. Gentamicin and tobramycin behave similarly in that they each have a detrimental effect on both vestibular and auditory functions of the eighth nerve and also have the potential to cause significant damage to the renal tubules. As noted for SM (Section IA), the incidence of toxicity found with 2-DOS-containing aminoglycoside antibiotics is markedly influenced by the dosage size, the duration of treatment, the state of the patient’s renal function prior to initiation of therapy and finally, to individual idiosyncrasy. There is little question, however, that the toxic potential of these compounds is directly related to their peak concentrations in serum since this establishes the maximum level of antibiotic that will be presented to the kidney tubules and to the perilymph. Thus, results

TABLE VI TOXICOLOGICAL AND PHARMACOLOGICAL PROPERTIES OF VARIOUS AMINOGLYCOSIDES Maximum recommended parenteral dose in man (gm /day)

Relative toxicity indexa Antibiotic

Vestibular

Streptomycin Dihydrostreptomycin Neomycin Kanamycin A Gentamicin C Tobramycin

4+

I+ I+

1+

3+ 2+

Auditory

(A)

Renal

1+

-

2.0

4+

-

4+

NUP NUP

1+ 2+ 2+

1.5 0.35 0 . 3Sd

4+ 2+ 2+ 2+

+

m

v

Maximum safe peak serum level in man k4/mu

Acute IV LD50 in mice (mg/kg) (B)

Ratio (A)/(B)

50 NUP

30ob 20ob 43 280 79 80

6.66 5.34 4.43 4.38

NUP 40 12 12d

E0 m

M

4 + = High potential for producing indicated type of toxicity; 1 = low potential. bLDso for streptomycin from Florey et al. (47), that for dihydrostreptomycin from Christensen (196),and t h a t for gentamicin C from Waite and Weinstein (136~). All others were determined at Bristol Laboratories. c NUP, not used parenterally. d These values are estimates since tobramycin is still undergoing pharmacological and clinical investigation in man (194, 135). 0

0

m z

MODIFICATION OF AMINOCLYCOSIDE ANTIBIOTICS

217

of toxicological studies in animals, along with clinical experience in man, have helped to define each antibiotic’s maximum “safe” peak serum concentration. Official guidelines have been developed on the basis of these values which specifically indicate the maximum daily dosage of antibiotic that is acceptable for parenteral use. In the case of SM, it is 2 gm; for kanamycin, 1.5 gm; for both gentamicin C (132,133) and tobramycin (134,135), 0.35 gm. Thus, on a straight weight basis, the toxic potential of SM is slightly less than that of kanamycin while that of gentamicin and tobramycin is 3- to 4-fold greater (see Table VI) (47, 1 3 4 1 3 6 ~ ) . It is of interest that precisely this same relationship is obtained when acute intravenous LD,,’s of the compounds for mice are examined. The net result, therefore, is that the ratio between the maximum recommended daily dose in man and the acute IV LD,, in mice is quite similar for each of the four antibiotics.

II.

Relative Activity, Susceptibility to Enzymatic Inactivation, and Toxicity of Naturally Produced and Semisynthetic 2-Deoxystreptamine-ContainingAntibiotics

Consideration will now be given to examining the relationships between the structures of a number of naturally produced and semisynthetic aminoglycoside antibiotics and ( 1 ) their intrinsic antimicrobial activity, ( 2 ) the breadth of their antimicrobial spectrum, and ( 3 ) their inherent toxicity for mammals. In examining the relationships between structure and activity, one parameter considered will be the relative capability of the compounds to inhibit protein synthesis in a cell-free system when used at a standard concentration (10 pg/ml). The data utilized will be those of Benveniste and Davies (82), who measured the relative percentage inhibition of R17 phage RNA-directed polypeptide synthesis produced by a number of aminoglycosides. Intrinsic antimicrobial potency will also be estimated by utilizing MIC results obtained with several representative aminoglycoside-sensitive bacterial species in %fold serial dilution assays. The gram-positive species chosen was S. aureus, the MIC results generally being acquired from tests in which strain “209P or the comparably sensitive strain “Smith was used. In the few remaining cases, data obtained with other wild-type, aminoglycoside-sensitive S. aureus strains were employed. One of the gram-negative species used in these activity comparisons was E . coli. Data obtained with this species is considered to be representative of data one would find for others in the Enterobacteriaceae family since, as noted in Table 111, there is a remarkable

218

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

uniformity of response to each aminoglycoside antibiotic displayed by most members of this family. The data used in almost every instance were obtained with strain K12. In cases where data for this strain were not available, those obtained with another aminoglycoside-sensitive E. coli strain were utilized. The other gram-negative species for which comparative in vitro susceptibility test results were examined was P . aeruginosa. Strains used as a source of MIC data were either A9843, one of two comparably sensitive strains, A3 or D15, or another wild-type strain which gives a characteristic response to known aminoglycosides. Since broth dilution tests were employed in some instances and agar dilution tests in others with different media and bacterial strains being utilized in the tests, meaningful comparisons of potency cannot be made on the basis of actual MIC values. However, if one compares the activity of a new derivative to that of a known agent and then expresses the relative potency of each in terms of a given standard, the values obtained will not only be considerably more reliable, but also will allow for a greater appreciation of potency differences. The standards chosen for this investigation were neamine for simple 4- or 6-substituted-2-DOS derivatives and commercially available gentamicin C for the diglycosyl-substituted-2-DOS derivatives. The latter standard displays high-level activity against all three of the test species. In those cases where the activity of the test compound against all organisms was less than one-hundredth that of the standard, the compound was not considered to have antibiotic activity. This interpretation was necessary since even a trace amount of contamination with the parent antibiotic could account for activity of this order. The antimicrobial spectrum of most aminoglycosides against wild-type organisms is generally quite similar except that some are active against P . aeruginosa whereas others are not. However, other variations in the spectrum of activity of the aminoglycosides have been noted in tests involving clinical isolates. These occur primarily as a result of differences in the antibiotics’ susceptibility to inactivation by enzymes, with those that are least affected having the broadest antimicrobial spectra. The information provided in the tables indicates whether an antibiotic’s activity is significantly reduced in standard susceptibility tests using intact organisms capable of producing a given enzyme. Consideration is not given to those cases where a compound can serve as a substrate for an enzyme in cell-free systems but is not inactivated in tests with whole cells. Since some of the compounds investigated were not tested against all the enzyme-producing organisms, it was necessary in many cases to make a prediction as to whether a given antibiotic would or would not be inactivated by them. Such predictions or guesses were generally “educated” ones, since a compound obviously could not be inactivated if

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

219

the functional group at the target site was absent or if a blocking group was present at that site. Information showing that a particular response was “predicted rather than established by testing is clearly indicated in the tables by encircling the signs: @ meaning inactivation is predicted and 0meaning that resistance to inactivation is predicted. The relationship between a compound’s structure and its inherent toxicity for the mammalian host will also be considered when appropriate data are available. As noted in the preceding section (I,B,4), there appears to be a definite relationship between the maximum safe therapeutic dosage of a compound in man and its acute intravenous LD,, for mice. A. 4-SUBSTITUTED-2-DEOXYSTREPTAMINES As can be seen in Tables VII-X, derivatives of 2-DOS in which the 4-substituent is an amino sugar generally have antimicrobial activity although 2-DOS itself does not. However, with once exception (apramycin ) , the activity of these compounds is considerably weaker than that of the 4,5- and 4,6-diglycosyl-substituted-2-DOSantibiotics.

1. Neamine and Related Compounds a. Fragments of Naturally Occurring Antibiotics Differing from Neamine in the 4-Substituent of 2-Deoxystreptamine (2-DOS). Neamine or neomycin A, as previously noted, is a naturally occurring degradation product of the other neomycins (53). As can be seen in Table VII (82, 137-145), this compound is an antibiotic in its own right, displaying activity in Mueller-Hinton Broth (Difco) against S. aureus Smith (MIC = 6.3 pg/ml) and E. coli K12 (MIC = 12.5 pg/ml), but not against P . aeruginosa A9843 (MIC = >lo0 pg/ml). The activity of this standard against the first two organisms is approximately 50-fold less than that of gentamicin C, the standard used for 4,5- and 4,6-disubstitued 2-DOS compounds. Neamine has no measurable inhibitory effect against P . aeruginosa and, at a minimum, is 200-fold less active than gentamicin against members of this species. Neamine’s lack of activity against this organism can probably be attributed to its high degree of susceptibility to inactivation by NPTI and NPTII, phosphorylating enzymes that are commonly found in wild-type strains of P . aeruginosa. Neamine is also inactivated by acetyltransferases that acylate is C6’-amino (KAT), C-2’amino ( GATIr),and C-3-amino ( GAT,,,) group. The second compound shown has been assigned the designation, kanamine, and differs from neamine only in that it has a hydroxyl rather than amino group at C-2 of the 4-substituent of 2-DOS. Kanamine, which is a degradation product of kanamycin A, has activity only one-

TABLE VII BIOLOGICAL PROPERTIES OF FRAGMENTS OF NATURALLY OCCURRING ANTIBIOTICSDIFFERING FROM NEAMINE IN T H E 4-SUBSTITUENT

O F 2-DEOXYSTREPTAMINE

n

Rz

Compound Neamine (neomycin A) Kanamine Paromamine Paromamine 3'-phosphate Nebramine (tobramine) Lividamine Gentamine CI.

Inhibition of protein R I synthesis

Relative activity against selected wild-type organisms S.

E. coli

l ( 1 2 . 5 ) 1(>100) 51 0.25 0.03 51

NHz OH NHz

on OH OH

OH OH OH

NHz NHz OH

-

l(6.3). 0.13 0.03

NHz

OPOJHZ

OH

OH

-

... . . ...... .

NH2 NHz NHz

H H

OH OH

NHz

on

30

1

-

0.13

H

H

NHt

32

37

-

P.aeluginoso

aureus

1

,

Inactivated by indicated enzyme NPT

NPT

I

I1

+ + +

+ + +

* Data from Bristol Laboratories

GAT

KAT

I

I1

GAT 111

-

+ +

-

+

+

-

-

-

@

References

BL.683, 197

@ c

158

@

139, 140

. . . . . . . . . . . . . . . . ,Not a n antibiotic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89, 141

0.5

>8

-

<0.06

NTd

-

0.5

>8

In parentheses: minimum inhibitory concentration (pg/ml) determined in Mueller-Hinton Broth. or Bristol-Banyu Research Institute. Encircled signs = predicted response. d NT, not tested.

a

GAT GNT

-

-

+ -

+

-

-

++ +

+

+

+

BL, 82, ?42 BL. 143, 144 BL,82,137,145

MODIFICATION OF AMINOGLYOOSIDE ANTIBIOTICS

221

fourth to one-eighth that of neamine against S. aureus and E . coli. Thus elimination of an amino group from the 4-substituent markedly suppresses the intrinsic activity of neamine. Paromamine, another naturally produced antibiotic, is a degradation product of the paromomycins. It differs from neamine in that its C-6’-substituent is a hydroxyl rather than amino group (59,62). Elimination of the C-6’-amino group from the 4-substituent suppresses activity to an even greater extent than does removal of the C-2’-amine, since MIC‘s against both S. aureus and E . coli are at least 32-fold higher than those of neamine. Paromamine’s spectrum of activity, based on its response to inactivating enzymes, is quite similar to that of neamine, except that it is not inactivated by KAT. Exposure of paromamine, a compound totally synthesized by S. Umezawa et al. ( 1 3 9 ) , to a cell-free enzyme preparation from E . coli J5 Rll-2, a producer of NPTI, yields paromamine 3’-phosphate, which has no antibiotic activity ( 1 4 6 ) . Nebramine (tobramine), which has not yet been isolated from natural sources, is a degradation product of tobramycin, an antibiotic formerly designated as nebramycin factor 6 ( 1 4 2 ) . This compound is a 3’-deoxy analog of neamine that has similar ability to suppress protein synthesis and has comparable inhibitory effects against S. aureus and E . coli. However, in contrast to neamine, it has marked antipseudomonal activity, a property almost certainly attributable to its inability to act as a substrate for the phosphorylating enzymes NPT, and NPT,,. Lividamine ( 3'-deoxyparamamine ) differs structurally from tobramine in that it has a hydroxyl rather than amino group at C-6’. This compound has not been isolated from fermentation broths, but was originally obtained as an acid methanolysis degradation product of lividomycin A ( 1 4 3 ) . A lividamine sample prepared at Bristol Laboratories was found, like its C-6’-hydroxyl-containing congener, paromamine, to have activity considerably weaker than that of neamine. This once again demonstrates that compounds have an extremely low level of activity when their C-6’substituent is hydroxyl rather than amino. However, the presence of this hydroxyl group and the absence of one at C-3’ do significantly affect lividamine’s response to inactivating enzymes since, in contrast to neamine, it is refractory to the action of NPTI, NPTIr, and KAT. Gentamine C,, ( 3’,4’,-dideoxyneamine ), a methanolysis-derived fragment of gentamicin C,, (76), has antimicrobial potency similar to that of tobramine (3’-deoxyneamine). This is exemplified both by the degree to which they interfere with protein synthesis and by their level of inhibitory activity against the three test organisms. The antibiotics also respond similarly to the inactivating effects of bacterial enzymes. MIC values are not available for gentamine C, and C,, which differ, respectively, from gentamine C,, in that their C-6’ groups are

222

KENNETH E . PRICE, J O H N C. GODFREY, AND HIROSHI KAWACUCHI

-CH( CH,)NHCH, and -CH( CH,)NH, rather than -CH,NH,. However, since the parent compounds (which differ from these fragments only in that they have the amino sugar, garosamine, in glycosidic linkage at the 6-position of 2-DOS) have virtually identical activity ( 1 4 7 ) , it is likely that the three gentamines also have comparable potency. A degradation product of the gentamicin-like antibiotic sisomicin has been designated sisamine. The antimicrobial activity of this methanolysisderived fragment, which is 4-0-( 3’,4’-dideoxyhex-4-enose)-3-DOS ( 148 ) has not been reported. It is predictable, however, relative to neamine, that it will have good antipseudomonal activity and be highly refractory to the action of phosphorylating enzymes. b. Fragments of Naturally Occurring Antibiotics Differing from Neamine in the 2-DOS Moiety. The activities of several fragments of naturally occurring antibiotics which vary from neamine in the 2-DOS moiety are presented in Table VIII (137,149-151~).The first two compounds are hybrimycins A, and B,. These are hydrolysis products of the biosynthetically prepared antibiotics hybrimycin A and hybrimycin B (149). The compounds differ from neamine in that they contain, respectively, streptamine and 2-epi-streptamine moieties rather than 2-DOS. Hybrimycin A, is one-fourth to one-eighth as active as neamine and considerably more potent than the 2-epi-streptamine-containing hybrimycin, B,. It is predicted that they will act as substrates for both phosphorylating and acetylating enzymes. Butirosamine is the fragment of butirosin A or B obtained after hydrolytic cleavage and removal of the pentose moieties of these antibiotics. As previously noted, the C-l-amino group in the 2-DOS portion of the butirosins has been acylated with the unusual amino acid, L( - ) -y-aminoa-hydroxybutyric acid ( L-AHBA), which is also called S-4-amino2-hydroxybutyric acid. The antimicrobial potency of butirosamine against S . aureus and E . coli is several fold greater than that of neamine. Moreover, the compounds activity against P . aeruginosa is markedly greater than that of the standard agent. This broadening in spectrum is probably attributable to the fact that butirosamine is not susceptible to attack by the phosphorylating enzyme, N I T I . However, since the compound is inactivated, as are the parent butirosins, by NPTII, it appears that this enzyme plays a lesser role than NPT, in determining the susceptibility of P. aeruginosa to antibiotics. The observed increases in potency and resistance to enzymatic inactivation possessed by butirosamine can undoubtedly be attributed to the presence of the C-l-aminoacyl group, which presumably interferes with enzyme action through steric hindrance of the sites of enzymatic attack. c. Derivatives of Neamine Obtained by Chemical or Enzymatic Modification. Data on the final group of compounds in this series are sum-

BIOLOGICAL PROPERTIES

OF

TABLE VIII FRAGMENTS O F NATUR.4LLY OCCURRING ANTIBIOTICSDIFFERING FROM NEAMINE IN THE 2-DEOXYSTREPTAMINE MOIETY

I no

NH-R,

..

B

E

Relative activity against selected wild-type organisms

Compound

RI

Neamine H HybrimycinAa H Hybrimycin Ba H Butirosamine L-AHBA~

Rz H OH

RJ

RI

H

O H OH OH OH

H

H OH

H

H

P. aeru-

S. aureus

E. coli

ginosa

l 0.13 0.03

1 0.25 <0.25

NTb NT

2

1

2

Data from Bristol Laboratories or Bristol-Banyu Research Institute.

* NT. not tested. c

d

Encircled signs = predicted response. AHBA. 7-aminwa-hydroxybutyryl.

>8

Inactivated by indicated enzymes NPT I

NPT

I1

GNT

KAT

GAT I

GAT I1

GAT 111

Acute i.v. LDno in mice

+

+

-

+

180

BL,a 157

@

8

+

-

149

EI

-

@

CB 8

-

CB

@ @

+

@

-

+

@<

260

149 BL,i50,151, 151a

-

+

CB

e

@

References

224

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

marized in Table IX (137,145,151-154). These are all chemically modified derivatives of neamine that are not components of naturally produced antibiotics. The first of these, 3’-methoxyneamine, is not an antibiotic, i.e., its activity is less than one-hundredth that of neamine. No activity data have been published for 3’-epi-paromamine.Although it seems likely that this compound cannot be phosphorylated at the C3’-position because of its unnatural configuration, it is not possible to predict what effect epimerization of the 3’-OH will have on its antibiotic activity. Data obtained with the next compound shows that introduction of a methoxy group at the C4’-position markedly suppresses activity and offers little or no protection against phosphorylation by NPTI and NPTII. A marked enhancement of neamine’s intrinsic activity is found when the compound is deoxygenated at the C-3’- and C-4‘-positions and acylated with L-AHBA at the C-1-amino group of the 2-DOS moiety. In addition to having greatly increased potency, the compound is resistant to the inactivating effects of the phosphotransferases and all of the acetyltransferases except KAT and GAT,, The tetradeoxy derivative of neamine is less active than neamine itself, but does have activity against a broader spectrum of microorganisms, including P. aeruginosa. Activity against the latter is undoubtedly due to deoxygenation at the C-3’-position which prevents inactivation via phosphorylation. The last compound in Table IX has a 4-aminodeoxyglycose moiety that is glycosidically linked at the 4-position of 2-DOS. The fact that this compound is not an antibiotic indicates that the site of the amino group in the 4-0-aminoglucose substituent of 2-DOS is critical insofar as antimicrobial activity is concerned. A biosynthetically prepared compound, which is the only known example of a neamine derivative that has been substituted with a hexose at the C-5-position of 2-DOS, was described by Endo and Perlman ( 155). They induced transglycosidation by use of a mixture comprised of an enzyme ( clarase), neamine, and maltose. ResuIts of in vitro susceptibility tests indicate that glycosylation causes a 2- to 4-fold enhancement of neamine’s antimicrobial potency. It is predicted that this antibiotic will give essentially the same response to inactivating enzymes as neamine. One question that arises, however, concerns the susceptibility of the compound to GNT ( gentamicin nucleotidyltransferase), an enzyme acting at the 2”-OH of hexoses that are glycosidically linked at the C-6-position of 2-DOS, but having no effect against 5-0-pentosyl-substituted neamines.

2. Apramycin (NebPamycin Factor 2 ) This 4-substituted-2-DOS compound is an extremely potent antibiotic relative to neamine and its structural analogs. It was one of eight water-

TABLE IX BIOLOGICAL PROPERTIES OF DER~VATIVES OF NEAMINE OBTAINED B Y CHEMICAL OR ENZYMATIC MODIFICATION

Relative activity against selected wild-type organisms

RI

Compound

RZ

H OH Neamine I‘-Methoxynearnine H OH 3’-eptParomamine H OH 4’-Methoxyneamine H OH 3’,4’-Dideoxy butirwmine L-AHBAc OH 3’,4’,5,6Tetra deoxyneamine H H 44-(C’-AminodeoxyH OH D-glucose)-Z-DOS ~

6

RJ

S.

Ra

Rs

Rs

aurew

E.coli

P. aeru- NPT NPT ginosa I I1 GNT KAT

+

+

+

+

-

GAT I

Acute i.v. GAT GAT LDso I1 111 inmice References

+

+

+

eb

OH on NH= I 1 1 -t 180 OCHa OH NHz ................................. .Notanantibiotic.. ................................ H OH OH .................................. No data available................................. OH OCHI NH1 0.03 0.01 51 63 -

OH N H ~ H OH NHp H OH NH2 OH OH

RI

Rb

Inactivated by indicated enzyme

NHz

H

OH NHz

H

H

H

NHz

2

4

>

H

NHz

H

H

H

NHz

0.5

0.5

>8

OH

OH

H

NH,

OH

OH ~

~

~~

Data from Bristol Laboratories or Bristol-Banyu Research Institute. Encircled sign = predicted response. AHBA = r-aminmz-hydroxybutyryl.

8

-

-

.................................

-

-

-

-

+

-

+

-

@ @

@

63

-

BL,O137 159 i5S 152

146,161,154

57BL

.Notanantibiotic..................................

145

226

KENNETH E. PRICE, JOHN C. GODFREY, AND HlROSHI KAWAGUCHI

soluble basic antibiotics isolated from fermentation broths of the soil isolate Streptomyces tenebrarius (156). Members of this complex of antibiotics, originally called nebramycins, were designated as “factors” 1, l’, 2, 3,4, 5, 5’, and 6 on the basis of their behavior in chromatographic systems. Factors 2, 4, and 5’ proved to be the major components in the fermentation and factors 1, l’, and 3, minor ones (157). Strain selection studies yielded an isolate that produces factor 2 (apramycin) as the only major component of the fermentation (156). The structure of apramycin was elucidated by OConnor and Lam ( 1 5 8 ) . Biological data obtained with this compound and one of its chemically modified analogs are shown in Table X (137,158,155). It can be seen that relative to neamine, apramycin is 8- to 16-fold more active against neamine-sensitive organisms and >50-fold more active against P. aeruginosa. In fact, its overall potency is only about 4-fold less than that of gentamicin (see Table XIII) and, because of its unique 4-O-aminooctosyl component, its structural configuration is such that it apparently cannot be recognized by any of the known aminoglycoside-inactivating enzymes. Its acute toxicity for mice is approximately two-thirds that of neamine and about one-fourth that of gentamincin (see Table XIII) . Replacement of the terminal 4-aminoglucose moiety with a methyl substituent yields a compound having a similar antimicrobial spectrum, but which, on the average, is some 4-fold less active than apramycin itself (158).

B. 5-SUBSTITUTED-2-DOS’S ( HYGROMYCIN B AND RELATEDANTIBIOTICS) Hygromycin B is an antibiotic that is coproduced with hygromycin in Streptomyces hygroscopicus fermentations. Its isolation was accomplished by chromatography on a cation-exchange resin, adsorption on carbon, and countercurrent distribution procedures ( 160). The compound was found to have antimicrobial as well as antiprotozoal and anthelmintic . structure was independently elucidated in 1970 by activity ( 1 6 0 ~ ) Its Neuss and his co-workers at Eli Lilly and Co. (161) and by Shoji and Nakagawa of Shionogi and Co., Ltd., Osaka, Japan (162). The latter investigators found that hygromycin B was being coproduced with the closely related antibiotic, A-396-1, by a strain of Streptoverticillium eurocidicus ( subsequently named Streptomyces eurocidicus ) . Their structural analysis indicated that both antibiotics have unusual “spiro” linkages between a hexose and an aminoheptose. Kondo and his co-workers ( 163) had previously reported the discovery of another structurally similar antibiotic to which they had given the designation, destomycin A. This compound is produced concurrently with destomycin B in fermentations of Streptomyces rimofaciens. A comparison of hygromycin B with

TABLE X BIOLOG~CAL PROPERTIES O F APRAMYCIN (NEBRAMYCIN FACTOR 2)

Relative activity against selected wild-type organisms

Compound Neamine Apramycin No. 1 a

b

R (See Table VII for structure) u-4-Aminodeoxyglucose CHI

S . aureus

E. coli

1 16 -4b

1 8 -2s

Inactivated by indicated enzyme

P. aeru-

NPT

NPT

ginosa

I

I1

GNT

KAT

1

+

+-

-

+-

>50

->12.5b

Data from Bristol Laboratories or Bristol-Banyu Research Institute. Activity estimated.

-

-

-

-

-

D

Acute i.v.

GAT

GAT

GAT

LDao

I

I1

111

inmice

-

+-

+ -

-

H

References

$ 8

180 280

-

BL,* 157 158, 159 168

2 2

228

KENNETH E . PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

these antibiotics indicated that the antimicrobial properties of destomycin A are closely related to those of hygromycin B, whereas those of destomycin B are not. The precise structural relationships between hygromycin B, the abovelisted hygromycin B-like antibiotics, and still another member of the class, SS-56c, were ultimately provided by Inouye and his co-workers ( 164). The antimicrobial activity of the latter compound, which is also produced by a strain of S. eurocidicus, has been compared with that of the other antibiotics in Table XI (137,163-165). These data indicate that the four compounds have activity against S. aureus and E. coli that is about one-fourth to one-eighth that of neamine but in contrast to neamine, give a measurable inhibitory effect against P. aeruginosa. No experimental data are available to show the response of these compounds to inactivating enzymes. It is likely, however, owing to the unusual nature of the substituent linked at the C-5-position of 2-DOS or streptamine (SS-56C), that these compounds in most cases will be refractory to enzymatic inactivation. It is possible that all these closely reIated antibiotics are highly toxic since the acute intravenous LD,, of one of them (destomycin A ) is only 5-10 mg/kg, a dosage some 20- to 40-fold lower than that found for neamine.

c.

6-GLYCOSYL-S~ST~TED-2-DOS's

The structures of the only examples of 6-substituted-2-DOS compounds known at this time are given in Table XI1 (139,166-168). The first three compounds, which are products of chemical synthesis, do not possess significant antibiotic activity. The last compound, garamine, is a fragment of sisomicin that was obtained by chemical degradation of that naturally produced antibiotic. It too was found to have extremely weak antimicrobial activity.

I. Ribostamycin and Butirosins a. Naturally Occurring Antibiotics. Ribostamycin was isolated from fermentation filtrates of a soil actinomycete that was given the designation Streptomyces ribosidificus ( 169). In addition, it has been synthe. structure sized by condensation of neamine and D-ribose ( 1 6 9 ~ )The of this new antibiotic, which was originally called SF-733, was first published in 1970 by Akita and his co-workers ( 170). Data in Table XI11 (82,136~,150,169,171-173) show that ribostamycin not only inhibits protein synthesis to a significantly greater extent than

TABLE XI BIOLOGICAL PROPERTIES OF HYGROMYCIN B A N D RELATED ANTIBIOTICS

HO

$

NH-R,

Relative activity against selected wild-type organisms

Compound Neamine Hygromycin B Destomycin A A-39&1 SS-56C

RI

R2

S. R3

(See Table V I I for structure) H H CHj H CHJ H H H H

H

OH

H

aurew

1

O.O@ 0.06 0.25 0.13

Inactivated by indicated enzyme

P . aeru-

NPT

NPT

E. eoli

ginosa

I

I1

1 0.135 0.13 0.25 0.13

NT

1

>4 >2

b

+e

e e e

GNT

KAT

GAT I

+ - + c e e e e e e e e e e e e e e e e

GAT I1

GAT 111

+e

+ e e e e

e e e

Acute i.V. LDro in mice

180

-

5-10

-

-

References

BL.0157 165-166

165-165 164 164

Data from Bristol Laboratories or Bristol-Banyu Research Institute. Potency values for these organisms extimated to be similar t o those of destomycin A. = Encircled sign = predicted response. a

b

M M

(D

230

KENNETH E. PRICE, JOHN C. GODFREY, A N D HIROSHI KAWAGUCHI

TABLE X I 1 6-GLYCOSYL-SUBSTITUTED-2-DEOXYSTREPTAMINES

6-0-(a- or b-Aminodeoxy-D-glucosy1)2-DOS 6-0- (4,6-l>iaminodeoxyglucosy1)-2-

nos

6-0- (D-Glucosaminy1)-%DOS Garamine a

OH

OH

H

NHz

CH?OH

BL,'166

OH

OH

H

NHI

CHzNHz

BL, 166

NHz OH

OH NHCHJ

H

OH

OH CHI

CHzOH H

139 167, 168

Data from Bristol Laboratories or Bristol-Banyu Research Institute.

neamine (65% vs. 37%),but has, in fact, inhibitory action equal to or greater than that of gentamicin C. However, despite its high level of suppressive activity in the cell-free protein synthesis assay, the compound is only one-fourth to one-sixteenth as effective as gentamicin in suppressing bacterial growth in serial dilution susceptibility tests. Even so, the compound is significantly more inhibitory ( 5- to 10-fold) than neamine, a change in activity attributable to the presence of the 5-0-ribosyl moiety in the molecule. Ribostamycin is highly susceptible to the action of the phosphorylating enzymes, NPTI and N U I I , and thus is not inhibitory for P. aemginosa strains. It is, however, resistant to the action of GNT and GATI, enzymes that readily inactivate gentamicin, and thus possesses an enzymatic susceptibility profile that is identical to that of neamine. The acute toxicity of this new antibiotic is about onefourth that of gentamicin and two-thirds that of neamine, whose intravenous LD,, for mice is 180 mg/kg. Butirosins A and B differ from each other structurally only in that the former has a 5-0-xylosyl and the latter, a 5-0-ribosyl moiety. These

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

231

antibiotics were independently discovered by workers at Parke, Davis and Co. (174) and at the Bristol-Banyu Research Institute (BBRI) in Tokyo ( 150). The producing organism in each instance was a bacterial strain classified as Bacillus circulans. These were the first reports of an aminoglycoside antibiotic being produced by a member of the order Eubacteriales as all other antibiotics of this type have been isolated from streptomycetes, organisms assigned to the order Actinomycetales. The butirosins are close structural relatives of ribostamycin from which they differ only by virtue of the fact that they have the unusual amino acid ( L-AHBA), in acyl linkage acid, L- ( -) -7-amino-a-hydroxybutyric with the C-1-amino group of 2-DOS (172,175-177). This finding was also the first of its kind since none of the previously reported 2-DOS-containing aminoglycoside antibiotics possess an amino acid moiety. Introduction of L-AHBA at the C-1-amino group of ribostamycin by chemical acylation yielded semisynthetic butirosin B (178). Data in Table XI11 show that the butirosins’ ability to inhibit protein synthesis is comparable to that of ribostamycin. The three antibiotics also have an essentially identical level of inhibitory action against many bacterial strains. S. uureus, for example, is inhibited by the compounds at concentrations 8-16 times that required for gentamicin C. They possess relatively greater activity against E . coli, each being about one-fourth as active as gentamicin. The butirosins, unlike ribostamycin, were found to have significant antipseudomonal activity. Examination of the enzyme susceptibility profile of these new agents provides a probable basis for their broader spectrum of antibacterial activity relative to that of ribostamy-cin. The amino acid moiety, L-AHBA, not only confers resistance to inactivation caused by NPTI, a phosphorylating enzyme, but to GAT111 as well. The latter enzyme acetylates both gentamicin and ribostamycin at the C-3-amino group of 2-DOS. Finally, results of acute toxicity tests indicate that the butirosins have a toxic potential less than one-half that of ribostamycin and one-eighth that of gentamicin C. The workers at BBRI, who had given the designation Bu-1709Al and A,, respectively, to butirosins A and B, subsequently determined the structures of two other bioactive components produced in fermentation broths concurrently with butirosins A and B (173). The new antibiotics, Bu-1709El and E2, differ structurally from A, and A,, respectively, only in that they have a hydroxyl rather than an amino group at C-6’. In vitro susceptibility test data indicate that the antibacterial potencies of Bu-1709El and E2 are virtually identical, each being about one-eighth as active as butirosin A or B. These results are consistent with those presented in Table VII, where it was shown that neamine,

BIOLOGICAL PROPERTIES

OF RIBOSTAMYCIN,

TABLE XI11 BUTJROSINS, .4ND THEIR NATURALLY OCCURRING

R,

Compound Gentamicin c Ribastamycin Butirosin A Butirosin B Bu-1709 El Bu-17W L Bu-1975 Ci Bu-1975 Cz 0

b c

R1

Rz

Rs

R.

(See Table XXXVI for structure) H L-AHBA L-AHBA L-AE~BA bAHBA L-MBA I-AHBA

OH OH OH OH OH H H

NU1

NHz NH, OH OH NH, NHz

H OH H OH H OH

H

OH

H OH H OH H OH

Value is for gentamicin CP. In parentheses, minimum inhibitory ooncentntien (&mi)

-

-

-

w

to

RELATIVES

0

OH

8

Relative activity against selected wild-type organisms Inhibition of protein P.wsynthesis S.aureua E. coli ginosa 550 65 72 72

to

1 (0.13)b l ( 0 . 2 5 ) 0.25 0.06 0.13 0.25 0.13 0.25 0.01 0.03 0.01 0.03 0.13 0.5 0.13 0.25

determined in MueUer-Hinton Broth. Data from Bristol hboratories or Bristol-Banyu Research Inetitute.

l(0.5) <0.01 0.13 0.13 0.03 0.03 0.25 0.25

Inactivated by indicated enzyme

NPT I

NPT I1

-

-

-

+

-

-

-

-

+

-

-

-

-

GNT

KAT

GAT GAT I I1

GAT I11

+ + + + +

+ + - + - + + - + - + - + - + - + - + _

_ -

+ +

-

+ + -

+ +

-

Acute i.v. LDso inmice 79 260 580 >580 890

-

520

-

4

References B b 8.2*136a BL, 8.2.150.169 BL, 8.2,17.2-17S BL, 8.9.171-175 BL, 175 BL,1Y3 BL BL

MODIFICATION OF AMINOCLYCOSIDE ANTIBIOTICS

233

which has C8’-amino group, is considerably more active than paromamine, which has a C-6’-hydroxyl group. The response of Bu-1709E1 and Ez to inactivating enzymes is much like that of the butirosins except that they are not susceptible to acetylation of the C6’-amino group by the enzyme, KAT. Finally, the acute toxicity of Bu-1709E1 was found to be appreciably less than that of the corresponding butirosin. The last two compounds in Table XI11 are a pair of butirosin-like antibiotics that were recently discovered and characterized at BBRI. These two agents, which have been given the designation, Bu-1975C1 and Bu-1975Cz, are water-soluble aminoglycoside antibiotics that are produced, like the butirosins, by a strain of B. circulans (179). These new congeners of the butirosins are characterized by the fact that each lacks a hydroxyl group at the C-4‘-position. Their antibacterial activity against wild-type bacterial strains is like that of the butirosins, although the new agents may be slightly more active against P. aeruginosa. Surprisingly, loss of the C-.l’-hydroxyl seems to have modified the antibiotics sufficiently to protect them from being inactivated to any appreciable extent by NPTrI, a phosphorylating enzyme whose site of attack is the neighboring 3’-hydroxyl. This could account for the enhanced antipseudomonal activity displayed by these compounds since most strains of this species produce NPTIr. The inability of this enzyme to inactivate the two Bu-1975 antibiotics not only extends their spectrum to include additional pseudomonads, but also to staphylococcal and Enterobacteriaceae strains that are resistant to other aminoglycosides by virtue of this mechanism ( 1 8 0 ) . Deoxygenation at the C-4’ position does not seem to have any influence on the toxic potential of the compounds for mice since the intravenous LD,, for Bu-l975C, was quite similar to that of its oxygenated congener, butirosin A. b. Modifications in the 2-DOS Moiety. i. Acylation of the C-1-amino group. The remarkable reduction in susceptibility to enzymes conferred upon the butirosins by the presence of an L-AHBA substituent at the C-l-amino group prompted investigators at both Parke, Davis (171) and BBRI ( 1 5 0 ) to acylate this amino group with other amino acids. The Parke, Davis workers deacylated a 4 : 1 mixture of butirosins A and B and assumed that the resulting mixture contained 4 parts 5-O-xylosylneamine and 1 part ribostamycin (5-O-ribosylneamine), while at BBRI, pure butirosin A was cleaved by mild alkaline hydrolysis to yield 5-O-xylosylneamine. The procedures for synthesizing new acylated derivatives from these AHBA-free preparations have been described by Haskell et al. (171) and Tsukiura et al. ( 1 5 0 ) . Table XIV compares the activity of ribostamycin and its chemically prepared isomer 5-O-xylosylneamine. It can be seen that the two antibi-

BIOLOGICAL PROPERTIES

OF

TABLE XI\’ RIBOSTAMYCIN DERIVATIVES MODIFIED IN THE 5-0-PENTOSE P E N T O S E AND 2-DEOXYSTREPTBMINE MOIETIES

AND IN

BOTHT H E

k, on Relative activity’ against selected wild-tsDe oraanisms -

__

R,

Compound

H Ribostamycin H 5-O-Xylos ylneamine Butirosin B L-COCHCHzCHzNHz I

Rz

Rr

8.a w e u s

E. coli

H OH H

OH H

0.06 0.06 0.13

0.25 0.25 0.25

OH

Inactivated by indicated enzyme

P.,aerugmoaa

NPT I

NPT I1

GNT

KAT

<0.01 <0.01 0.13

+ + -

+ + +

-

+ f f

GAT I

GAT I1

-

-k

f

+

GAT I11 Reference

+

f

-

150 150 160

-

e* e e e e e e+ e

-

+ + + e +

-

@

-

@

-

@

+ +

-

No. I

DL-COCHCHZCHZNH~ H

OH

0.06

0.13

0.06

-

+

-

No. 2

AH DL-COCHZCHCHZNHZ H

OH

0.01

0.13

0.01

-

No. 3

H

OH

0.01

0.03

0.01

-

No. 4

NH2 L-COCHCHZNHZ

H,OH

OH,H

0.13

0.25

0.13

e

No. 5

AH L-COCH(CH~)~NHZ

H

OH

0.06

0.13

0.06

-

+ + e +

-

OH L-COCH(CHZ)ZNH~

OH,H

0.01

0.06

NTC

@

@

H

OH

0.01

0.01


+

H

OH

0.01

0.03

0.01

-

+ +

I

I

AH

No. 6

L - C O C H ( C H Z ) ~ N H Z H,OH

No. 7

AH COCHzNHn

No. 8

NH

fI

COCH(CHdaNHCNH3

I

-

-

NHz R

All activities are relative to that of gentamicin C.

6

Encircled signs = predicted response.

0

NT, not tested.

-

8

150

160

150 171 150

CB

e e

150

e+

0

150

171

236

KENNETH E. PRICE, JOHN

c. GODFREY,

AND HmosHI KAWAGUCHI

otics have identical antibacterial activity and that they respond in the same way to inactivating enzymes. As previously noted, butirosin A, which possesses L-AHBA in acyl linkage at the C-1-amino group of 5-O-xylosylneamine, has good activity against P. aerugirwsa and is resistant to several enzymes that act on 5-0-xylosylneamine. Derivative No. 1, which has been acylated with racemic AHBA, is somewhat less active than butirosin A suggesting that the D-epimer is relatively less active than the L. Compound No. 2, although also a racemic mixture, has such poor activity that there is little doubt but that the shift of the hydroxyl group from the a- to the p-carbon is detrimental to antibiotic activity. No change in response to enzyme-inactivating activity is thought to occur, however. The importance of the a-hydroxyl group is again underscored by results obtained with ribostamycin derivative No. 3, a compound in which this substituent has been replaced with an amino group. This new hydroxyl-free acyl group yields a compound which, relative to butirosin A, has approximately 8-fold less activity, although it apparently can still interfere with the action of inactivating enzymes. The next three derivatives (Nos. 4-6) show the effect of varying the number of carbons in the acyl group. The propionyl analog, No. 4, is virtually as active as AHBA itself. Furthermore, in view of its good antipseudomonal activity, it obviously interferes with the phosphorylating enzyme, NPTI, just as does AHBA. Compounds Nos. 5 and 6 are, respectively, the C-1-N-valeryl and caproyl analogs of butirosin A. The former is about one-half as active as butirosin A while the caproic acidcontaining analog is minimally inhibitory for the test organisms. It is thought that the latter derivative is unable to block the site of attack of NPTI although unequivocal evidence for this is not available. The acyl groups possessed by the last two derivatives are the natural amino acids, glycine (No. 7 ) and arginine (No. 8 ) . The activity of each is only one-fourth to one-eighth that of butirosin A. This is predictable since neither possesses the a-hydroxyl group that appears to be essential for good activity. In contrast to most of the other acylated compounds examined, the one containing glycine does not appear to be refractory to the action of the phosphorylating enzyme. NPTI. ii. Other modifications. DeFuria and Claridge (182) described two new antibiotics that were produced in culture broths of a B. circulans mutant which could produce butirosins only when exogenous 2-DOS was added late in the course of the fermentation. The new agents were obtained when streptamine or streptidine, rather than 2-DOS, were utilized to supplement the fermentation medium. A sample of the first anti-

MODIFICATION O F AMINOGLYCOSIDE ANTIBIOTICS

237

biotic, which was isolated by preparative chromatography, was partially characterized and its antimicrobial spectrum was determined. This streptamine addition product was found to be as active as ribostamycin and the butirosins against S. aureus and E . coli, but had little or no activity against P. aeruginosa. Despite its lack of inhibitory action against members of this species, the compound was not inactivated to any significant extent by the phosphorylating enzyme, NPT,. The second antibiotic detected has not yet been characterized but may be a streptidine-containing analog of ribostamycin and/or 5-O-xylosylneamine. Similar experiments by Kojima and Satoh (183) with a 2-DOS requiring mutant of S. ribosidificus resulted in the biosynthesis of three new derivatives when appropriate aminocyclitols were used to supplement the culture medium. The new antibiotics were presumed to be the C-1-Nmethyl, the streptarnine, and the epi-streptamine analogs of ribostamycin. All had less bioactivity than ribostamycin. Addition of neamine and 3’,4’dideoxyneamine to fermentation broths of the same mutant strain resulted in the production of ribostamycin and 3’,4’-dideoxyribostamycin, respectively. Kojima and his colleagues ( 1 8 4 ) also found that the ribostamycinproducing culture, S. ribosidificus, could be induced to produce a bioconversion product of ribostamycin if the fermentation were supplemented with D-xylose as the only carbon source. The product obtained was an N-carboxymethyl derivative. The position of the carboxymethyl group was unequivocally shown by means of nuclear magnetic resonance (NMR) studies to be at the C-3-amino group ( 1 8 4 ~ ) The . authors did not include any antibiotic susceptibility test results in their reports. A second inactive metabolite of ribostamycin that is produced by S. ribosidificus has been identified by the same workers as C-3-acetylribostamycin (184a). c. Modifications in the 4-Substituent. i. N-Acylation. Several derivatives of ribostamycin having an acylated C-g’-amino group were prepared by Tsukiura and his co-workers (150). Acyl groups utilized include glycyl, y-aminobutyryl, L-AHBA, and L-lysyl. None of these derivatives had significant antimicrobial activity. ii. Deoxygenation. Since antibiotics like tobramycin and Bu-1975, which lack, respectively, the C-3’- and C4’-hydroxyl group of the 4-O-diaminoglucose substituent of 2-DOS, possess a high level of antibacterial activity and are resistant to inactivation by phosphorylation, deoxygenated derivatives of ribostamycin and butirosin B have been synthesized in an effort to modify their potency and spectrum. Table XV shows data obtained with several such derivatives. The first derivative, 3‘-deoxyribostamycin, is slightly more inhibitory

BIOLOGIC~L PROPERTIES

TABLE XV BUTIROSIN DERIVATIVES MODIFIEDB Y

OF ~ ~ I B O S T A M Y C I NLND

DEOXYGENATION

I N THE 4-SUBSTITUENT OF 2-DEOXYSTREPTAMINE

3‘

1’

-NH-R,

R,

0

OH

n

Relative activity= against selected wild-type organisms

Compound

RI

Rz

Ribostamycin H OH 3’-Deoxyribostamycin H H 3’-Deoxy-5-O-xylosylneamine H H 4‘-Deoxy-.SO-xylosylneamine H OH 3',4'-Dideox yribostamycin H H Butirosin B L-AHBA OH 3’,4’-DideoxybutiL-AHB.~ H rosin B

NPT

NPT

GAT

GAT

GAT

9

Rd

Rs S. aureus E. coli

ginosa

I

I1

GNT

KAT

I

I1

111

OH

H H

OH OH

0.06

0.25

+

+

-

+

+

260

150, 186

0.5

-

-

-

+

0.13

<0.01 0.25

@

-

185a

OH O H

H

0.06

0.125

-

H

OH

H

0.13

0.5

H OH

H

0.06 0.13

0.13 0.25

0.06 0.13

-

H

OH OH

H

H

OH

0.25

0.5

0.13

OH

All activities are relative to that of gentamicin C. Encircled sign = predicted response. c Bristol Laboratories or Bristol-Banyu Research Institute. 0

P. aeru-

Acute i.v. LDso

R3

~~

6

Inactivated by indicated enzyme

~~

0.06

+ +


+

+

+

-

-

-

+

-

-

-

-

+ +

-

B

+ +

+ +

@

E

-

-

-

b

)

CB @

in mice References

-

CB @

-

a m j;

BLc 280

BL

@

@

-

185, 145 171, 172

@

e

-

145, 186

0

B 2> 7c

w

5

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

239

than the parent compound for S. aureus and E. coli and is at least 25-fold more active against P . aeruginosa. The latter effect is undoubtedly due to the fact that the compound cannot be phosphorylated at the 3‘-hydroxyl group. Although NPTr does not act at this deoxygenated site, it can phosphorylate the 5’’-hydroxyl group in the ribose moiety, just as has been observed with lividomycin (Table V). Preliminary studies indicate, however, that NPT, will preferentially phosphorylate the C3’-hydroxyl if it is present, just as was found in experiments involving 3’,4’-dideoxyribostamycin and ribostamycin ( 146). Deoxygenation at C-3’ protects ribostamycin from inactivation by NPTII since the enzyme is capable of attacking at this site only. The second derivative, 3’-deoxy-5-O-xylosylneamine, has a spectrum that is generally similar to that of 3’-deoxyribostamycin, although the activity of the former is some 2- to 4-fold less than that of the latter. Results obtained with 4’-deoxy-5-O-xylosylneamine show that this derivative is slightly more active than ribostamycin against S. aureus and E . coli but, like the latter compound, has no activity against P . aeruginosa. The new deoxygenated derivative shows the same profile of susceptibility to inactivating enzymes as ribostamycin and 5-0-xylosylneamine. The activity of the 3’,4’-dideoxy derivative of ribostamycin against the three test organisms is somewhat less than that of 3’-deoxyribostamycin. Both compounds can apparently be phosphorylated by NPTI at their C-5’’-hydroxyl group (146,185). The last new derivative in Table XV (145,150,171,172,185,186) is 3’,4’dideoxybutirosin B which was synthesized by Ikeda and his colleagues (186). This compound’s activity was comparable to or slightly better than that of the fully oxygenated parent compound. No improvement in activity against P. aeruginosa was observed although the new derivative is refractory to the inhibitory effects of NPT,,. It has been found, however, that strains of Enterobacteriaceae which produce this enzyme are susceptible to the deoxygenated compound, but not to butirosin B (180). iii. Other modifications. Ribostamycin 3’-phosphate and butirosin A 3’-phosphate have been isolated after exposure of the parent compounds to partially purified phosphorylating enzymes ( 146). The first product could be obtained with either NPTI or NPTII, while the second was produced only by NPT,,. Neither compound has significant antibiotic activity (146,151) . d. Modifications in the 5-Substituent. A number of analogs of ribostamycin, its isomer 5-O-xylosylneamine, and butirosin A that have modifications in the C-5-hydroxyl group of the 5-0-ribosyl or 5-0-xylosyl moiety have been prepared. Data obtained with these compounds are shown in Table XVI (150,l71,172,187,188). Deoxygenation at C-5” of 5-0-

K 0

TABLE XVI BIOLOGICAL PROPERTIES

OF

RIBOSTAMYCIN AND BUTIROSIN DERIVATIVES MODIFIEDAT

THE C-5”-POSITION

HO 2:

Ho

NH,

3m

1‘

n Q

0

Relative activity0 against selected aild-type organisms

Inactivated by indicated enzyme

A,cute 1.V.

Compound

RI

Rihostamycin H H 5-0-xylosylneamine 5-0- (5-Deoxysylosy1)neamine H 5-0- (5-Aminodeoxyxylosy1)neamine H 5”-.4minodeoxyrib~ stamycin H 5-0-(5-ChlorodeoxyH xylosyl)neamine Butirosin A L-AHBA 5”-DeoxybutirosinA L-AHBA 5”-Aminodeoxybutirosin A L-AHBA 0

b c

P.,aerugznosa

R2

RJ

Ri

S. aureus

E. coli

H

OH H

OH

OH

0 06 0.06

0 25 0.25

<0.01

OH

<0.01

H

0.01

0.03

<0.01

OH

H H

NH,

0.06

0.13

<0.01

H

OH

NHz

0.06

0.13

<0.01

OH

C1

OH

H H H

OH

OH

H

OH

OH

NPT I

NPT

I1

GNT

+

+ +

-

+

+ + +

+

+ +

-

-

LDso

KAT

GAT I

GAT I1

GAT 111

inmice

+ +

-

+ +

+ +

260 280

BL,bf60 150

+ + +

-

CI+ 63

@ @

-

BL BL

@

fB

-

BL

8

580

4E

. . . , . . . . , . _ _ __ ._ _. .. . . . _ . _ .__ _ . . N o t a n a n t i b i o t i c . . . . . _ _ ... . . . . . _ .. . . . . . . . . . . . . .

H

0.13 0.03

0.25 0.03

NH2

0.13

0.25

All activities are relative to that of gentamicin C. Data from Bristol Laboratories and Bristol-Banyu Research Institute. Encircled sign = predicted response.

0.13 <0.01

0.125

-

-

+

-

+

-

+

-

+ + +

. ..

References

-

+ +

-

-

-

BL BL. 171, 172 BL

-

63

0

280

BL,187, 188

U

D

F3

F %

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

241

xylosylneamine reduced antibacterial potency by 4- to 8-fold, while substitution of an amino group for the 5”-hydroxyl of this compound or ribostamycin caused, at most, only a slight loss in antibacterial activity. Neither of the above-cited modifications caused a change in the pattern of enzymatic inactivation. Chlorination at the same site produced a compound essentially devoid of antibacterial activity, In the butirosin series, deoxygenation at C-5” again caused a marked decrease in activity, while amination of butirosin A did not significantly affect potency. The preparation and bioactivity of the 5”-amino derivative of butirosin B was recently described by Culbertson et al. ( 1 8 8 ) . These workers found this compound somewhat less active than the corresponding derivative of.butirosin A. Neither modification influenced susceptibility to enzymatic inactivation. e. Modifications in Both 4- and 5-Substituents. Several compounds that were chemically modified in the 4-substituent of 2-DOS and also at the C-5-OH of the 5-O-pentosyl moiety have been examined in in vitro susceptibility tests. Results are presented in Table XVII. The trideoxygenated ribostamycin derivative was less active against E. coli than either ribostamycin or its 3’,4’-dideoxy derivative. It also had considerably less activity than the dideoxygenated compound against P. aeruginosa. Thus, the fact that there is an overall loss in activity when the 5”-hydroxyl is reduced is completely consistent with results obtained for previously described antibiotics such as ribostamycin and butirosin A ( see Table XVI ) . The substitution of hydroxyl for amino at C-6’, along with amination at the 5-hydroxyl of the xylosyl moiety yielded a compound, 5-0-(5aminodeoxyxylosyl) paromamine, that has very weak activity. This is to be expected in view of the fact that paromamine with its C-6’-OH has very poor antimicrobial activity relative to that of neamine, its C-6’NH2 analog. 2. Neomycins, Paromomycins, and Lividomycins a. Naturally Occurring Antibiotics: Neomycins B and C,Paromomycins I and ZZ, and Lividomycins A and B. These antibiotics bear a close structural relationship to ribostamycin, differing principally in that they have a mono- or disaccharide in glycosidic linkage with C-3 of the ribosyl moiety of ribostamycin or ribostamycin-like compounds. As can be seen in Table XVIII (82,136a,189-193), the neomycins are more potent inhibitors of protein synthesis than gentamicin. However, the comparably potent neomycin isomers possess activity only one-half to one-fourth that of gentamicin against S. aureus, E. coli, and P. aeruginosa strains in serial dilution sensitivity tests. The susceptibility of neomycin B and

TABLE XVII O F RIBOSTAMYCIN DERIVATIVES MODIFIED I N BOTH THE 4BIOLOGICAL PROPERTIES ~-SUBSTITUENTS OF 2-DEOXYSTREPTAMINE

R,

OH

Relative activitya against selected wild-type organisms

GNT

+

-

0.06

+ +

-

-

<0.01

-

-

~ 0 . 0 1 <0.01

+

+

0.25

0.01

OH

0.06

0.13

OH

H

0.06

0.06

H

NHz

0.01

RJ

R,

Rs

Rb S. aureus

Ribostamycin 3'.4'-Dideoxyribostamycin 3',4',5"-Trideox yribostamyein 5-0-(SAminodeoxyxylosy1)paromamine

OH

OH

NHz

H

OH

OH

H

H

NHz

H

OH

H

H

NHz

H

OH

OH

OH

OH

a

I1

0.06

RZ

b

NPT

P.aeruginosa

RI

All activities are relative to that of gentamiein C. Data from Bristol Laboratories and Bristol-Banyu Research Institute. Encircled symbols = predicted response.

Inactivated by indicated enzyme

NPT I

E. coli

Compound

AND

GAT I

GAT

-

+ +

-

+ + +

-

630

63

145, 185

-

-

-

4-

+

BL31@

KAT

I1

GAT I11

+

References BL,bf50

+ 1 8 5

TABLE XVIII O F NATURALLY OCCURRING NEOMYCINS, PAROMOMYCINS, A N D LNIDOMYCINS BIOLOGICAL PROPERTIES

I

OH

Relative activity a g a k t Inhibi- selected wild-type organmms

__

Inactivated by indicated enzyme

Acute

t.i"" of

Compound

RI

RZ

Q?

R4

,v

protein

Rj

synthesis S.aureus E.coli

P.,neruginoso

a

(See Table XXXVI for structure) OH NHz H H CHzNHi OH NHz H CHzNIIz H €I CHzNHz OH OH H OH OH H CHZNHZ H

55

OH H H

OH Mannose OH Mannose OH H

H

H H

CHzNHz CHzNHz CHzNHz

1 0.25 0.25

0.5 0.5

-

1 0.25 0.25 0.06 0.06

0.13 0.06


-

0.13 0.06 0.13

0.25 0.13 0.13

<0.01 0.06 0.13

80

72 65

Data from Bristol Laboratories or B r s t k B a n G Reaearch Institute.

NPT I1

GAT

-

++ ++

-

4-

-

LPT

GNT KAT

I

GAT I1

GAT .LD& I11 mmice

References

~

~

Gentamicin C Neomycin B Neomycin C Paromomyein1 ParomomycinII 4"'-Mannosylparomomycin I Lividomycjn A Lividomyein B

NPT I

1

<0.01

++ + + + + +

-

-

f

+

+ -

-

+

++ +--

-

-

-

+ 4- 4+ +f ++

79 24 44 160 174

+ +

280

+

+

+

+ f

-

140

BLa89.2.36~1 BL:88 BL,82 BL 89 189 BL:I& 191 293

BL'198.295 BL:189

244

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

neomycin C to the action of inactivating enzymes appears to be identica1 to that of ribostamycin. Acute intravenous LD,, values show that the neomycins are two to three times as toxic for mice as gentamicin. Paromomycin I inhibits protein synthesis more effectively than gentamicin, but suppresses bacterial growth to a much lesser extent th‘an this standard agent. This result, along with those previously found with ribostamycin, the butirosins, and the neomycins indicates that there is a relatively poor correlation between the “quantitative” ability of a compound to inhibit protein synthesis in a cell-free system and its ability to inhibit growth and multiplication of intact cells. Obviously, factors other than the compound’s ability to antagonize the ribosomal site of protein synthesis must be involved in those tests in which the interaction between the antibiotic and whole cells is measured. Since paromomycin I has a C-6’-OH rather than the C-6’-NH2 group that is characteristic of the neomycins, it is not affected by the acetylating enzyme, KAT. However, its responses to all other enzymes are similar to those of the neomycins. Paromomycin 11, which is the C-5”’-CH,NH2 epimer of paromomycin I, is a structural analog of neomycin C, differing only in the C-6’-position where a hydroxyl group is present. This compound has low activity, just as does paromomycin I, against the three test strains. Thus, as has been noted previously with other 2-DOS-containing aminoglycosides, compounds with a C-6’-hydroxyl group are considerably less potent than those having an amino substituent at this position. Also in agreement with previously observed results is the finding that 6’-OH-containing compounds are less toxic for mice than their 6‘NH2 counterparts. The next compound in Table XVIII, 4”’-mannosylparomomycin I, while somewhat less active than the parent compound, has a spectrum of activity based on its susceptibility to enzymatic inactivation that appears to be identical to that found for paromomycin I. Lividomycin A is identical to 4”’-mannosyIparomomycin I except that it lacks a hydroxyl group at C-3’. Lividomycin B, on the other hand, is the 3’-deoxy analog of paromomycin I. The two lividomycins are coproduced with 4”‘-mannosylparomomycin I and paromomycin I in fermentations of Streptomyces liuidus, a culture that was isolated and described by Oda et al. ( 1 9 4 ) . In subsequent studies, the biologically active components of this fermentation were isolated and their structures elucidated ( 143,191,192, 195,196). The lividomycins, in contrast to the paromomycins, possess significant antipseudomonal activity which can probably be attributed to the fact that they lack a 3’-hydroxyl group and thus cannot be phosphorylated at that site. On the other hand, as was found with 3’-deoxyribostamycin and 3’,4‘-dideoxyribostamycin, they are capable

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

245

of being phosphorylated at the 5”-OH group of the 5-O-ribosyl moiety of 2-DOS (97,197). Although NPT, has the ability to act at the C-5”-OH, NPTI, does not. Recently, evidence has been uncovered by H. Umezawa (99) which indicates that P . aeruginosa TI-13 produces a new enzyme, lividomycin phosphotransferase ( LPT), which selectively phosphorylates the 5”-hydroxyl of the lividomycins. Paromomycin I, lividomycin A, and lividomycin B have comparable activity against S. aureus and E. coli. It is of interest that the latter compound has been prepared by chemical conversion of lividomycin A (198). The lividomycins differ from paromomycin in that they are significantly more active against P . aeruginosa. They also differ from paromomycin in the extent of their susceptibility to enzymatic inactivation since they are refractory to the action of NPT,, and susceptible to LPT. The acute intravenous LD,, of the lividomycins are similar to those of the paromomycins and considerably higher than those of the neomycins. b. Modifications in the 2-DOS Moiety. i. Hydroxylation. Shier et al. (199,200) isolated a mutant of the neomycin-producing culture, S. fradiae, which was incapable of synthesizing antibiotics in the absence of an added aminocyclitol. A mixture of two new antibiotics designated as hybrimycins A, and A? were produced when the added aminocyclitol was streptamine. Similarly, hybrimycins B, and B, were obtained when 2-epi-streptamine was utilized as a precursor. Finally, a third pair of hybrimycins (C, and C,) were produced upon addition of streptamine to a mutant of the paromomycin-producing culture, S. rimosus forma paromomycinus. The antimicrobial properties of these compounds are compared in Table XIX ( 199,201) . It is apparent from the results obtained in cell-free protein synthesis assays (20 pg/ml concentrations of the antibiotics were used in these tests ) and in serial dilution antibiotic susceptibility tests that introduction of a hydroxyl group having an equatorial configuration into the 2-position of 2-DOS (hybrimycin A,) does not significantly affect activity as compared to that of the natural antibiotic, neomycin B. The C-2-hydroxy analog of neomycin C also proved to be a highly active antibiotic. Hybrimycins B, and B?, on the other hand, in which the C-2-hydroxyl is in the axial configuration, are considerably less potent, both as suppressors of protein synthesis and as inhibitors of bacterial growth. The response, where it has been determined, of all four of these biosynthetically produced antibiotics to inactivating enzymes is like that of neomycin B. The streptamine-containing congeners of the paromomycins, hybrimycins C, and C,, possess activity equal to that of hybrimycins B, and B, and about one-half that of paromomycin I (see Table XVIII).

TABLE XIX BIOLOGICAL PROPERTIES OF THE HYBRIMYCINS

1'

m

n

OH HO

p*ToH

4

s,,,

"'

R4

0 Relative activity* against selected wild-type organisms

Compound

RI

RZ

IL

R4

Rj

Nwmycin B HybrimycinAI HybrimycinAz HybrimycinBL HybrimycinBz HybrimycinC' HybrimycinCz

H H H OH OH

H OH OH H H OH OH

NHz NHz NHz NHz NHz OH OH

H H CHINHI

CHtNHp CHzNHz H CHINHZ H CHzNHz H

H

H

H

CHzNHz H CHzNHz

Data from Davies (201).

b

AU activities are relative to that of gentamicin C.

Data from Bristol Labpratories or Bristol-Banyu Research Institute.

d

Enclrcled sign = predicted response. NT, not tested.

Inhibition of protein synthesis0 S . a u r m

89 88

89 30 20

-

0.25 0.13 0.13

0.06 0.06

0.06 0.03

E.wZi

P..aeruuinosa

0.5

0.25 0.25

0.25

0.13 0.01 0.01

0.5

0.13 0.13 0.13 0.13

NTb NT

Inactivated by indicated enzyme NPT

I

+ +

4-

+ +

f +

NPT I1

f @d

@

@

@ @ CB

GNT

KAT

-

f f f

-

f

f

--

GAT I

-

GAT I1

GAT

111

References

2 8

+ @

BLp199.201 19.MO1

@

@

@

@ @

CB

19*m 19*201 19*.%01

19*201 19*201

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

247

ii. N-Acylation. In view of the marked broadening of the antimicrobial spectrum of ribostamycin that is conferred by the presence of a C-1-NAHBA substituent as is found in butirosin B, it was considered of interest to determine what effect this group would have on the activities and antimicrobial spectra of neomycins, paromomycins, and lividomycins that had been acylated with it. Results are summarized in Table XX (202). The activity of the C-1-N-AHBA neomycins against S . aureus and E. coli was about the same as that of the parent neomycins. However, the presence of this group markedly influenced activity against P . aeruginosa. MIC values obtained with the substituted derivatives were equal to those of gentamicin and about 4-fold lower than those obtained with the parent compounds. This same trend was observed with paromomycin C-1-N-AHBA since its activity against S . aureus and E . coli was like that of paromomycin I itself, while the inhibitory effect against P . aeruginosa was at least 20 times greater than that of the parent compound. The improved antipseudomonal activity noted above can probably be attributed to the increased resistance to inactivation by NPTI that is conferred upon the compounds when they are acylated with L-AHBA. The activity of the acylated lividomycin derivatives against the three test organisms did not differ appreciably from that of lividomycin A or B. Nevertheless, the spectrum of the acylated compounds is broader since the C-1-N-AHBA group prevents NPTI from phosphorylating the C-5”-OH of the lividomycins. The effect that this acyl group has on the action of the new phosphorylating enzyme, LPT, is not known. c. Modifications in the 4-Substituent. Since aminoglycosides with a C6’-NH2 group generally have greater intrinsic antimicrobial activity than those with a C-6’-OH group, S. Umezawa and his co-workers (203) considered it of interest to prepare lividomycins having an amino group at C-6’. Results obtained with two such derivatives prepared by these workers and one other C-6’ modified derivative synthesized by Watanabe et al. (203a) are shown in Table XXI. The first semisynthetic compound, C-6’-aminodeoxylividomycin B (C-3’-deoxyneomycin B), is 2- to 4-fold more potent than lividomycin B itself which, of course, has a hydroxyl group at C-6’. Actually the activity of the C8’-amino compound is quite similar to that of its fully oxygenated congener, neomycin B. The second compound is identical to the first except that it has been N-methylated at C-6’. This compounds activity is very similar to that of C-6’-aminodeoxylividomycin. However, since it is not subject to inactivation by the acetylating enzyme, KAT, it has a somewhat broader spectrum than the compound with the primary amine at C-6’. The derivative alkylated at the C8’-amino group with a 2-hydroxyethyl substituent

TABLE XX BIOLOGICAL PROPERTIES OF C-~-N-~AMINO-~HYDROXYBUTYRAMIDES OF NEOMYCIN, PAROMOMYCIN, AND LIVIDOMYCIN

-0-

5"

R,

0 Relative.activity again& selected wdd-type organisms Compound

RI

Rz

Gentamicin C NeomycinB-1-N-AHBA NeamycinGl-N-AHBA Paromomycin I-I-N-AHBA Lividomycin El-N-AHBA Lividomycin A-1-N-AHBA

OH OH OH H H

NHz NHz OH OH OH

,Data

Fl4

Rs

(See Table XXVI for structure)

H H H H Mannose

H CHzNHz H H H

from Bristol Laboratoriesor ~ r i s t o l - ~ m y~ue s e a c hInstitute.

* @= Encircled signs = predicted response. response not predictable. c

R?

CHINHZ H CHINHI CHzNHz CHzNHz

1 0.13 0.13 0.13 0.13 0 06

Inactivated by indicated enzyme

ginma

NPT I

1 0.25 0.5 0.25

1 1 1 0.25

-

0.06 0.13

0.13 0.13

-

S.ourew E . 4

P,aeru-

-

-

NPT

I1

-

+ +

+

1

LPT

GNT

KAT

GAT I

-

+-

++ +

+-

-

-

8' _ -

-

_

-

_

-

-

-

GAT I1

GAT 111 References

+@ " B L+ B L m @

@

G B L BL BL BL,202

0

8

BIOLOGICAL PROPERTIES

OF

TABLE XXI NEOMYCIN B, LIVIDOMYCIN B,

AND THEIR

ANALOQS

C-6'-MODIFIED

'\ 3

Relative activity" a g a e t selected wild-type organisms Compound NeomycinB Lividomyein B 6'-Aminodeoxylividomycin B (3'deoxyn~mycinB) 6'-Methylammodeoxylividomycin B 6'-hxy-6'-(Z-hydroxyethylamino) lividomyein B (.

b c

Rz

R1 OH H H

S.aurew

E.coli

P..aerugsnasa

NHI OH NHz

0.25 0.13 0.25

0.25

0.25 0.13 0.5

H

NHCHi

0.25

0.25

0.25

H

NHCHzCHzOH

0.25

0.25

0.13

AU activitiea are relative to that of gentamicin C.

Data from Bristol Laboratories or Bristal-Banyu Research Institute. Encircled sign = predicted response.

0.5 0.13

Inactivated by indicated enryme

NPT I

NPT I1

LPT

GNT

+ ++ + +

f

-

-

-

-

+f +

-

4-

-

-

KAT +

+-

-

GAT I +

-

GAT I1 +

+

B

GAT 111 L

+

References b

BL,189

$'

@

1466,mS,mSa

$

@

ms,msa

@

@

%So

B ~ O L O G I C h t PltOPERTIEfi O F

TAHLE XXIl N-BENZYL A N D N-(MODIFIED-J~EKZYL) DERIVATmES

O F hTNOMYCIN

B

NH--R

Relative activitya against selected wild-type organisms

Compound

Neomycin B

No, 1

R

E

S. aureun

E . aoli

0.25

0.5

0.01

0.01

P.,oerugtnosa 0.25

<0.01

Inactivated by indicated enzyme

NP IT

NzT

GNT

KAT

GAT I

CAT I1

GAT 111

References

+

+

-

+

-

4-

+

$00

G3b

@

-

€3

-

8

9

8008

No. 2

...................................

Not anantibiotic ...................................

$06

No. 3

...................................

Not anantibiotic ...................................

206

Cl'

No. 4

--\

C)/-m

................................

. . .Not an antibiotic.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

206

OCHj No. 5

...................................

206

Not an antibiotic.. ................................

206

25 1

Not anantibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a All activities are b Encircled sign =

relative to that of gentamicin C . predicted response.

252

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

had activity generally comparable to that of 6’-methylaminodeoxylividomycin B. Its enzyme susceptibility profile is also like that of the latter compound. d. Modifications in the 5-Substituent. Several lividomycins have been chemically modified at the C-5-hydroxyl of their 5-0-ribosyl moiety. In the case of some of these compounds, the goal was to prepare semisynthetic antibiotics that could not be inactivated by phosphorylation of the C-5”-hydroxyl. The C-5” deoxygenated derivatives of both lividomycin A (204) and lividomycin B (189),as has previously been found with ribostamycin and the butirosins (see Table XVI), were virtually devoid of antibacterial activity and, for the purposes of this paper, are not considered to be antibiotics. Results obtained with the aminated derivative (C-5”amino) of lividomycin A were similar (204). The final compound in this group is lividomycin A-S’-phosphate. Although prepared by chemical synthesis, this compound is considered to be identical to that produced by exposure of lividomycin A to a mixture containing ATP and a crude enzyme from the NPT, producing E . coli strain K12 ML1410 R-81, or the enzyme from the LPT producing organism, P. aeruginosa TI-13 ( 205). As expected, this C-5”-phosphorylated compound was devoid of activity. e. Modifications in 2-DOS and Both 4- and 5-Suhstituents. Shier and Rinehart ( 206) prepared a series of hexa-N-benzylneomycins in order to determine whether the increased lipophilicity of such compounds, relative to the parent compound, neomycin B, would endow them with superior antimicrobial properties. Results are summarized in Table XXII. All six derivatives proved to have markedly reduced antibacterial potency, the only measurable activity being produced by derivative No. 1. The activity of the other five compounds, all of which are derivatives with N-(substituted-benzyl) moieties, is so low that they are not considered to be antibiotics. Compound No. 1 had minimal activity against S. aweus and E . coli and none against P . aeruginosa. It is predicted that this compound will not be susceptible to inactivation by KAT, GATII, or GATIII, enzymes that readily inactivate the parent compound, neomycin B.

E. ~,~-DIsuBSTITUTED-~-DOS’S 1. Kanamycins

a. Naturally Occurring Antibiotics: Kanamycins A, B, C, and Nebramycins, Including Tobramycin. The 4,6-diglycosyl-substituted-2-DOS

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

253

antibiotics are generally less effective inhibitors of protein synthesis than the 4,Ei-disubstituted compounds. The neomycins and paromomycin I (see Table XVIII), for example, cause inhibition ranging from 65 to 801, whereas values for gentamicin, tobramycin, and several of the kanamycins range from 50 to 58%.On the other hand, in serial dilution susceptibility tests, the 4,6-disubstituted antibiotics are as active or more active than the 4,5-disubstituted compounds. This once again supports the previously made observation that there is not a particularly good correlation between the degree of suppression of protein synthesis caused by an antibiotic in a cell-free system and the extent to which it inhibits bacterial growth. Antibacterial test results obtained with a number of naturally produced 4,6-disubstituted-2-DOS-containing aminoglycoside antibiotics are presented in Table XXIII. Kanamycin A, which differs from kanamycin B by virtue of the fact that it has a hydroxyl rather than an amino group at C-2’, inhibits protein synthesis to only a slightly lesser degree than the latter. However, in serial dilution susceptibility tests, the order of magnitude of the difference between the twd compounds is much greater, kanamycin B being at least 2-fold more active than kanamycin A. Unfortunately, the higher potency of kanamycin B does not endow it with a superior therapeutic index since its acute toxicity is also proportionately greater. Both kanamycins are quite active against S. aureus and E . coli but are almost without effect on P . aeruginosa. The lack of efficacy against this organism is almost certainly attributable to the compounds’ high degree of susceptibility to the inactivating enzymes, NPT, and NPTII. Interestingly, kanamycin B is capable of being inactivated by all the known enzymes except GATI, which is specific for certain gentamicins, and LPT, an enzyme that may act solely on lividomycins. Kanamycin C, which has a hydroxyl at C-6’ rather than the amino group of kanamycins A and B, is a much weaker antibiotic than the other two. Its activity against S . aureus and E . coli is about one-sixteenth that of gentamicin as compared to one-fourth for kanamycin A and one-half for kanamycin B. Surprisingly, its acute toxicity is greater than that of kanamycin A, perhaps due to the presence of the amino group at C-2’. Except for KAT, kanamycin C is susceptible to the same enzymes as kanamycin B. Nebramycin factor 4 is one of the several antibiotics present in fermentations of the light-sensitive soil organism, S. tenebrarius (207). One of the factors (No. 2 ) is apramycin, (see Section II,A,2), while a second factor, No. 5, is identical to kanamycin B ( 2 0 7 ~ As ) . can be seen in Table XXIII, factor 4 is a close structural relative to kanamycin B, differing only in that its C-6” substituent is carbamoyl rather than hydroxyl

RrOLOGilCAL PROPERTIES OF THE

€% rp

TABLE XXIII KANAMYCINS, THE NEBMAMYCINS, AND T OB Il AM YC IN

m

HOH n

0

0--%,

Rehtive activity against Inhibi- selected wild-type organisms

tiuu of

Compound ~~

R1

It*

ns

R4

protein synthesis S.curow E . coli

l'.,aeru-

ginosu

Inactivated hy indicated enzyme

HPT I

NPT I1

GNT filT

GAT GAT I I1

c

Acute

8

1.v.

GAT LDso 111 in mice

Reference8

~

Gentamicjn C Kanamyrin A Kanamycin B Kanamycin C Nebramycin factor 4 Ncbramycin firclor 5' Tobramycin (faclot 6) Kanamycin A-8'-N-acctvl Kanamyem B-W-N-acctyl Kanamycin A-3'-phwphate 0

b

@OH

NH2

NH,

NHz Nlh NHZ

OH

NHP OH

Data from Bristnl tboratories o r BrisW-Bauyu Resparch Institute.

Detrrmined at Eli LiUy and Co.

55 50 58

3Q

-

1

1

0.25

0.25 0.5 0.06

0.5 0.06 0.25

0.5 0.5

1 0.01 0.03


-

++ + -t

-

+ + -k

-

-f-

+

+ + + + -t4- +4- -b+

+

-

-

--

+

+

-

+

,t

7

++ -b

-+ -+ -t +

9

280

13%

225 2100

2 115 1 2 + f + + 7 9 n .................................. .Not 80 antibiotic.. ............................... 14 .................................. .Kot nu mtibbtc.................................. ~.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . not an lotibiotis~.................................

-1~

55

1 1

~

ELa 1,9680

EL,b

BL, 82 BL 82

RL: 168 EL BL,82

82,115 8% 119 141

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

255

( 2 0 7 ~ )Although . its activity and profile of susceptibility to inactivating enzymes are virtually identical to those of kanamycin B, it does appear to be somewhat less toxic. However, it should be noted that the LD,, of factor 4 was obtained in a laboratory (159) where values generally tend to be somewhat higher than those obtained at BL, the source of the LD,, values for all other compounds. A recent report shows that factor 5 (kanamycin B ) is not a primary product of the S . tenebram’us fermentation but is actually a hydrolysis product of factor 4 (207a). Factor 5’ is one of the major components in the S . tenebrarius fermentation ( 1 5 7 ) . It bears the same relationship to tobramycin ( nebramycin factor 6 ) that factor 4 bears to kanamycin B, i.e., factor 5’ has a carbamoyl rather than the hydroxyl function that is present in tobramycin at C-6”. The latter compound apparently occurs as a result of acidor base-catalyzed hydrolysis of factor 5’ ( 2 0 7 4 . The actual structure proof for factor 6 or tobramycin was given by Koch and Rhoades in 1971 (141 ), who found that it was a C3’-deoxy derivative of kanamycin B. This compound was shown by Takagi et al. (208) to be amenable to preparation by chemical synthesis. These investigators produced tobramycin by selectively dehydroxylating kanamycin B at the C-3’-position. The antimicrobial potency of nebramycin factor 5’ and tobramycin proved to be greater, relative to gentamicin C, than that of any of the antibiotics thus far examined, Activity of the two compounds against S . aureus and E . coli was comparable to that of the standard compound, while their activity against P . aeruginosa exceeded that of gentamicin by approximately 2-fold. Both compounds are resistant to phosphorylation since they lack the 3’-OH group but are capable of being inactivated, just as is kanamycin B, by GNT ( gentamicin nucleotidyltransferase), GATII, GATrrI, and KAT. The acute toxicity of tobramycin is slightly greater than that of factor 5’ and equal to that of gentamicin C. The remaining compounds in Table XXIII are enzymatically produced derivatives of kanamycins A and 13. The C-6’-N-acetyl derivatives obtained by exposure of the antibiotics to KAT, have little or no ability to inhibit protein synthesis and display no measurable activity against the three test organisms. The C3’-phosphate of kanamycin A which was prepared by use of NPTI, is similarly devoid of antibacterial activity. b. Modifications in the 2-DOS Moiety. i. N-Acylation of Kanamycin A. Kawaguchi and his co-workers (209) acylated kanamycin A’s C-1-NH, group with L-AHBA and a series of related acyl groups to determine whether such derivatives would acquire any of the desirable properties that introduction of L-AHBA at that same position confers upon ribostamycin. Results obtained with a representative group of compounds are presented in Table XXIV ( 2 1 0 ) . Compound No. 1, C-l-N-a-hydroxyglutarylkanamycin A, has markedly

TABLE XXIV BIOLOGICAL PROPERTIES OF DERIVATIVES OF KANAMYCIN A MODIFIEDI N

1

THE 2-DEOXYSTREPTAMINE

MOIETY

NH--R,

HO*

6"

5"

0

OH 0 0

Relative activitya against selected wild-type organisms

Inactivated by indicated enzyme GAT GAT

NPT

GAT

NPT

pinosa

I

11

GNT

KAT

I

I1

III

in mice

--

-

+-

280

-

-

Ri

RI

S. awew

E. coli

Kanamycin A No. 1

H COCH(CH2)zCOzH

H H

0.25 0.01

0.25

0.01 0.03

+-

+ -

+ -

++

No. 2

H

0.01 0.01

0.03

No. 3

I OH CO(CHz)sNHz COCH(CHz)zNHr

0.01 0.03

-

-

--

++

No. 4

NHz COCH (CHz)zNHz

H

.............................

, N o t a n antibiotic.

c1 COCO(CHd2NH2 D-AHBA

H H

..............................

- + + .Not an antibiotic.. ............................

Compound

H

0.03

0.03

I

No. 5 No. 6 No. 7 (Amikacin or BB-K 8) No. 8 No. 9 No. 10 a 5

I

L-AHBA COCHJ

H H

H H

COCHJ

L-AHBA

0.06

0.25

0.13

0.25

0.5

0.5

.............................

............................. .............................

AU activities are relative to that of gentamicin C. Data from Bristol Laboratories or Bristol-Banyu Research Institute.

Not an antibiotic.

-

-

-

-

.Not a n antibiotic. .Not a n antibiotic.

-

s

qcute LDFQ l.v.

P.aeru-

r2

References

+

2

-

BLb BL

8

-

BL

BL

5

........

-

BL

.............................

-

BL BL, 210

-

............................. .............................

300 23000 915

235

BL, 210 BL BL

BL. 210

P

x

r"

%

-

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

257

less intrinsic activity (against S. aureus and E. coti) than the parent compound, but does have greater activity against P . aeruginosa as a result of this acyl group’s suppressive effect on the action of phosphorylating enzymes. Results obtained with compounds Nos. 2 and 3 which have, respectively, y-aminobutyryl and a,y-diaminobutyryl acyl substituents, are similar to those found with compound No. 1. Compound No. 4, y-amino-a-chlorobutyrylkanamycinA, and compound No. 5, y-aminocY-ketobutyrylkanamycinA, have no measurable antibiotic activity. Compound No. 6, kanamycin A acylated with the D-epimer of AHBA, was more active than any of the preceding compounds, but still significantly less active than the kanamycin A derivative acylated with L-AHBA (compound NO. 7 ) . The latter compound, known as BB-K 8 and recently assigned the generic name amikacin, is one-fourth as active as gentamicin against S. aureus and about one-half as active as that standard compound against E . coli and P. aeruginosa. This derivative, in contrast to other previously described, nonkanamycin A antibiotics that have C-1-N-acyl groups, is susceptible only to KAT, which acetylates the C-6’-NH2 group. Thus, amikacin is virtually as active as gentamicin, but is markedly more resistant to enzymatic inactivation than this reference compound. Furthermore, its acute toxicity for mice is like that of kanamycin, and thus is only one-fourth that of gentamicin. Although the compounds are not listed in Table XXIV, antibiotics obtained when kanamycin A was acylated with p-amino-a-hydroxypropionic acid (BL, 211) or w-amino-a-hydroxyvalericacid ( BL) were found to be almost as active as arnikacin. However, increasing the aliphatic chain to more than five carbons proved to be detrimental to activity. Thus, it can be concluded for kanamycin A, just as was observed with the 4,5-disubstituted-2-DOS-containing antibiotics, that the structural characteristics of the C-1-N-acyl derivatives giving maximal activity are extremely specific. The amino acid used for acylation must contain no less than three and no more than five carbons; it must have an a-hydroxyl group in the L-configuration; and it must have an w-amino group. However, the requirements for an acyl group that can block enzymatic inactivation appear to be considerably less rigid. The next two compounds in Table M I V are the C-1-N-acetyl and C-3-N-acetyl derivatives of kanamycin A. Neither are antibiotics since they have less than one-hundredth the activity of the reference compound, gentamicin C. Kanamycin A-3-N-acetyl is the product obtained when the antibiotic is exposed to the inactivating enzyme, GAT,,,. Interestingly, compounds obtained after acetylation of either the C-lor C-3-amino group have markedly reduced toxicity compared to kanamycin A itself. The final compound in Table XXIV is the C-3-N-AHBA derivative

258

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

of kanamycin. Although a superior compound was obtained when L-AHBA was used to acylate the C-1-NH, group of 2-DOS, acylation of the C-3-NH2 group of 2-DOS (compound No. 10) results in a compound devoid of antibiotic activity. In contrast to the N-acetylated derivative, the acute toxicity of compound No. 10 is actually slightly greater than that of amikacin, the C-1-N-AHBA derivative. In addition to kanamycin A-l-N-acetyl (compound No. S ) , a number of other biologically inactive kanamycin derivatives have been prepared at BL by acylation of kanamycin’s C-l-amine. Acyl groups utilized included eight different substituted-benzoyls, as well as cyclobutanecarbonyl, and acethydroxamoyl. Inactive C-3-acylated derivatives of kanamycin other than kanamycin A-3-N-acetyl (compound No. 9 ) and 3-N-AHBA (compound No. 10) which were prepared at BL included 4-chlorobutyryl and six different substituted-benzoyls. Finally, as expected, two derivatives of kanamycin A that were acylated at both the C-l- and C-3-amines with acetyl or p-toluenesulfonyl are inactive. ii. N-Acylation of Other Antibiotics. In view of the improved antibiotic activity obtained when kanamycin A was acylated at the C-1-NH, group with L-AHBA, the effect of acylating other kanamycin-like aminoglycosides with this group has been investigated. Results are given in Table

xxv.

N-Acylation of kanamycin B with L-AHBA had little effect on the resulting compounds activity against S. aureus and E . coli, but did bring about a remarkable improvement in its antipseudomonal activity. This improvement in the compound’s antimicrobial spectrum is undoubtedly a consequence of kanamycin B-1-N-AHBA’s significantly increased resistance to the action of inactivating enzymes. Similar results were obtained when the acyl group was L- ( - ) -p-amino-a-hydroxypropionyl (211). A beneficial effect was also observed as a result of L-AHBA acylation of kanamycin C. However, the modified antibiotic still has weak antimicrobial activity much like that of the parent compound. The new derivative, however, was found to serve as a substrate for only one inactivating enzyme, GATIr. Tobramycin, in contrast to what has been observed with all previously discussed antibiotics, lost potency when acylated with L-AHBA.However, the spectrum of activity of the new compound, based on its susceptibility to enzymatic inactivation, was broadened relative to that of the parent compound. c. Modifications in the 4-Substituent. i. N-Acylation. Each member of a series of eight kanamycin A derivatives prepared by acylation of the C-6’-amino group proved to be biologically inactive. Similar results

TABLE XXV BIOLOGICAL PROPERTIES OF C-1-N-AHBA DERNATNES O F KANAMYCINS B AND C,

AND

TOBRAMYCIN

0 L,

0

B

Relative activity" against selected wild-type organisms

E. coli

P. aeru-

NPT

uinoea

I

NPT I1

+

+

+

+

Compound

Ri

Rz

RE

S. aureue

Kanamycin B Kanamycin B-1-AHBA Kanamycin C Kanamycin C-1-AHBA Tobramycin Tobramycin-1-AHBA

H L-AHBA H I,-AHBA

OH OH OH OH

NHz NHz

0.5

0.5

0.03

0.25

0.5

0.5

OH

0.06

0.06

OH NH2 NHz

0.13

0.06

0

H

K

L-AHBA

H

1

1

0.25

0.5

AII activities are relative to that of gentamicin C. Data from Bristol Laboratories or Bristol-Banyu Rerrearcb Institute.

<0.01 0.13 2 0.5

8

Inactivated by indicated enzyme

-

-

GNT

KAT

-

+ -

+ +

-

-

+ +

-

-

+

+ -

-

*2:

GAT

GAT

I

I1

GAT I11

References

-

+ + ++ + +

+ ++ -

BL' BL BL BL BL BL

--

2 d

260

KENNETH E. PRICE, JOHN c. GODFREY, AND HIROSHI KAWAGUCHI

were obtained with 10 kanamycin B derivatives. These 18 compounds were prepared at BL, at BBRI, or by Lemieux ( 2 1 2 ) . The only ones that will be singled out for discussion are several of the acetyl and L-AHBA derivatives. Kanamycin A-6’-N-acetyl is a substance that is produced naturally by the action of the enzyme, KAT. Its acute intravenous LD,, for mice is >3000 mg/kg compared to 280 mglkg for kanamycin A. The C-6’-N-AHBA derivatives of kanamycin A and kanamycin B were among the compounds tested and found inactive. The acute intravenous LD,, of the kanamycin A derivative is 1158 mglkg. Thus, in the case of both the C-6’-N-acetyl and the C-6’-N-AHBA derivative, the loss in activity against microbes paralleled the loss in toxicity for mammals. A single example of a kanamycin-like antibiotic acylated at the 2’amino group was prepared by Kondo and his co-workers ( 2 1 3 ) . These investigators found that 3‘,4‘-dideoxykanamycin B-C-2’-N-AHBA was a minimally active antibiotic whose potency was one-thirtieth or less that of the standard, gentamicin C. ii. N-Alkylation. Mono-N-methylation of kanamycin A or B at the C6’-amino group yielded compounds (see Table XXVI) (145,214) whose activities are very similar to those of the parent compounds. As expected, these alkylated derivatives were no longer susceptible to inactivation by the enzyme KAT. When larger alkyl substituents such as cyclooctylmethyl or 3-methylpentyl were introduced at C-6’ of kanamycin A, activity was eliminated ( 2 1 5 ) . Another kanamycin A derivative that was N,N-dialkylated with hydroxyethyl groups at the C-6’-amine had no antibiotic activity ( 2 1 2 ) . iii. Deoxygenation. Derivatives of kanamycin A and B deoxygenated in the 4-glycosyl group of 2-DOS have been prepared. Results of biological testing are presented in Table XXVII (145,216,217). The first compound, 3’-deoxykanamycin A, is as active as the parent compound against S . aureus and E. coli and %-fold more active against P . aeruginosa. The extent of the improvement in antipseudomonal activity, which is attributable to the compounds resistance to phosphorylation, is only slightly less than that observed with the naturally produced analog, 3’-deoxykanamycin B ( tobramycin ) . The 4’-deoxy derivative, which retained full kanamycin A activity against S . aureus and E . coli, is only slightly more effective against P. aeruginosa than the parent compound. Although it is susceptible to the inactivating effects of NPTr, preliminary evidence suggests that it is only partially inactivated by NPTII, which may account for its increased antipseudomonal activity. The potency of the next derivative, the 3’,4‘-dideoxygenated analog of kanamycin A is the same as that of the 3‘-deoxygenated compound. Deoxygenation of kanamycin B to give the 3‘,4’-dideoxy derivative yields a compound that is about one-half as active as gentamicin against

TABLE XXVI

BIOLOGICAL PROPERTIES

O F 6'-N-METEYLKANAMYCINS

A

AND

B

&-NH

V

z

Relative activity0 against selected wild-type organisms

mU r

Inactivated by indicated enzyme

Z

P . aeru-

Compound

RI

Rz

S. aureuu

E. coli

Kanamycin A 6'4"MethylkanamyoinA Kanamycin B 6'4"Methylkanamycin B

OH OH NHz NHz

H CHJ H

0.25 0.25 0.5 0.5

0.25 0.25 0.5 0.5

0

6

CHJ

All activities are relative to that of gentamioin C. Data from Bristol Laboratories or Bristol-Banyu Research Institute.

ginosa

0.01 <0.01 0.03 0.01

NPT I

NPT I1

+ + + +

+ +

+ +

GNT

KAT

GAT I

+

++-

-

f

-If

GAT I1

-

++

GAT

111

+ ++ f

References BLb 146, 614

BL

BL

ri m

ij

TABLE XXVII BIOLOGICAL PROPERTIES

OF

KANAMYCINS DEOXYGENATED IN THE

s: N

4-SUBSTITUENT O F 2-DEOXYSTREPTAMINE

m HO

NH*

n 0

0 Relative activity0 against selected wild-tvDe .. organisms Compound

RI

Rz

Kanamycin A 3'-Deoxykanamycin A

OH OH

mycin A 3'.4'-Dideoxykanamycin A KanamycinB 3',4'-Dideoxykanamycin B KanamycinC G'-Deoxykanamycin C a

0.01

+

+

0.25

-

0.13

0.06

+

0.25 0.5

0.25 0.5

0.25 0.03

+

+

-

+ +

0.5 0.06

0.5 0.06

0.5 <0.01

-

-

+

S. aureus

E. coli

OH

OH

NHz

0.25

0.25

H

OH

NHz

0.25

0.13

OH

OH

H

NHz

0.13

OH NHz

H OH

H OH

NHz NHz

NHz

H

OH

H OH

NH2 OH

OH

OH

NHz ~~~

~~~

~

~~~~

All activities are relative to that of gentamicin C. Research Institute.

* Data from Bristol Laboratories or Bristol-Banyu = Encircled sign = predicted response.

&wsa

-

i-

-

@

+

GNT

+

+

+

+

KAT

+

+

+ ++ + -

GAT I

GAT I1

GAT I11

Acute i.v. LDIO inmioe

-

-

-

+

280

@c

-

-

-

--

-

+ + +

@

$ +

+

. . . . . . . . . , . . . . . . . . . . . . . . . . . . . .Notanantibiotic.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

H ~

Inactivated by indicated enzyme

I

Ra

NH2

-NPT

NPT I1

R3

4'-Deox ykana-

P. aeru-

-

References BLb BL, 216 BL

132

71 225

BL,BI7 BL

-

BL

MODIFICATION O F AMINOGLYCOSIDE ANTIBIOTICS

263

all the test organisms. Its response to inactivating enzymes is also much like that of the standard agent except that it is effective against organisms which produce the gentamicin-inactivating enzyme, GAT,. The final compound in the table is a kanamycin C derivative that has been deoxygenated at C-6’. It does not possess significant antimicrobial activity. iv. Other. Table XXVII (145,218,219) shows results obtained with miscellaneous kanamycin A and kanamycin B derivatives that have been modified at various sites in the 4-substituent of 2-DOS. Compound No. 1 is kanamycin A-C-3’-OCH3. This modification was made with the expectation that it would block phosphorylation at that site. Unfortunately, the compound was found to be devoid of antibacterial activity. Kanamycin A was also modified at the C3’-position by substitution of an amine for the hydroxyl. One of the two derivatives prepared, No. 2, had the neighboring C-2’-hydroxyl in the usual equatorial configuration while the other (No. 3) had this hydroxyl in the axial configuration. No biological data have been reported for these compounds. Compound No. 4, a C-6’-N-methyl analog of kanamycin A, was prepared in order to prevent acetylation at C-6’ by KAT. This was achieved without an appreciable loss in antimicrobial potency. Compound No. 5 is also a C-6’-N-methyl derivative, but one that had been prepared from 3’,4’-dideoxykanamycin A. Although the activity of this N-methyl compound was reduced by 2-fold relative to its parent, inactivation by KAT was blocked. A related derivative, C-6’-N-methyl-3’,4’-dideoxykanamycin B (compound No. 6 ) , had activity comparable to that of the nonmethylated compound (see Table XXVII) and was resistant to inactivation by KAT. The final compounds 4-substituent on 2-DOS is an unusual sugar since it has an olefinic portion ( A ~ - ~ It) .possesses very weak antibacterial activity but is not susceptible to being phosphorylated. d . Modifications in the 6-Substituent. i. C-3” modifications. A variety of derivatives of kanamycin A that have been modified at the C-3”-position have been prepared. Results obtained with these compounds are presented in Table XXIX. Kanamycin A-C-3”-N-acetyl is devoid of antibacterial activity, This derivative has not been demonstrated to be an inactivation product of bacterial acetyltransferases. The probability is great that at some time in the future, an enzyme will be detected which acts at this site. Acute toxicities of the C-S”-N-acetyl derivative and the following compound (No. 2), a C-3”-N-AHBA analog of kanamycin A which also lacks antibacterial activity, are much lower than that of the biologically active parent compound. The C-3”-N-AHBA analog of kanamycin B was also shown to lack antibacterial activity (213), but no acute toxicity data have been reported for it.

TABLE XXVIII BIOLOGICakL PROPEKTIJ3S O F K A N A M Y C I N S W I T H V A H I O U S ?vfODIFICATIONS I N THE; 4 - S U B S T I T U E N T

OF 2 - 1 ) E O X Y S T H G P T A M I N E

0 Relative activity0 against selected wild-type organisms Compound

RI

Ri

RJ

R4

S. aureus

P.,aeru-

E. coEi

otnosa

Inactivated by indicated enzyme NPT

I

NPT I1

GNT

~~

Kanamycin A Kanamycin B No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 0

b

OH NU2

on

OH

on

OH

NHI NHI

OH OH OH H

OH

OCHJ

OH

OH H

OH epi-OH OH NHz NFlz

H

OH

H

A3'

H H

H

H H CHI CHI CHs €1

KAT

GAT I

GAT I1

GAT 111

++ ++ ++ ++ -- +- ++ . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .Not a n antibiotic. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 0.25 0.5

0.25 0.5

.......... ._ _ . _ . , .. _. . . . . 0.25 0.25 0.5 0.03

AU activities are relative to that of gentamicin C. Data from Bnsiol Laboratories and Rristol-Banvu Research I n s t i t u t e . Encircled siKn = predicted response.

0.25 0.13 0.5 0 06

References

~~~~

0.01 0.03

. .Not a n antibiotic.. . . . . . . . . . . . . . . . . . . . . . . .. . . . . .

. . . . . . . . . . . Not a n antibiotic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . <0.01 @ c 0.13 @ 0.5 @

+

0.03

-

+-

+ ++

- e l

a

BLS

BL

,918

919

119

21 4

146 31 4 145

TABLE XXIX MODIFIED AT BIOLOGICAL PROPERTIES O F K ANAMYCIN A DERIVATIVES

THE

C-3" POSITION

k lr-%oH B *"

I

P

0

Relative activitya against selected wild-tvue ". orranisms

-

Compound

R

S. aureua

E. coli

Kanamycin A

NH2

0.25

0.25

No. No. No. No. No. No. No.

1 2 3 4 5 6

7

0

II

NH-CCHI NH-AHBA NH-CHa N(CHd2 NH---CH?CH~C~HS NH-CHt-pyrid-4-yl NH-CHz-cyclooctyl

ginosa

0.01

............................... ............................... 0.25

0.25

<0.01

0.03

0.13

<0.01

..............................

..............................

...............................

.All activities are relative to that of gentamicin C. Data from Bristol Laboratories or Bristol-Banyu Research Institute. c Encircled sign = predicted response.

b

Inactivated bv indicated enavme

Acute 1.V.

P. aeru-

NPT I

NPT I1

GNT

+

+

+

LDso in mice

KAT

GAT I

GAT I1

GAT I11

+

-

-

+

280

Notanantibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Not an antibiotic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

>5000 1470

+ +. + + +, CB + . N o t an antibiotic.. . . . . . . . . . C B C

-

-

+ +

. N o t an antibiotic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-

Notanantibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-

References BLS BL BL BL BL BL BL BL

266

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

Compound No. 3 is a C-3”-N-methyl derivative of kanamycin A. The activity of this compound is indistinguishable from that of kanamycin A itself. Its response to inactivating enzymes is probably like that of the parent compound although it is not known whether substituents other than a primary amine at C-3” can influence the action of the enzyme, GNT, whose site of attack is the neighboring C-2”-hydroxyl. Dimethylation of the C-3”-amine (compound No. 4 ) yields a biologically inactive derivative. Thus, the C-3” substituent cannot be acylated or fully alkylated if the compound is to retain antimicrobial activity. It is clear from results obtained with compounds Nos. 5-7, that monoN-alkylation of the C-3”-amine with bulky alkyl substituents also reduces or eliminates biological activity. ii. C-6” modifications. Surprisingly, as can be seen in Table XXX (171,212,220,221 ), deoxygenation at C-6” yields a derivative with activity at least comparable to that of kanamycin A. These two compounds have identical acute intravenous LD,,,’s for mice. The corresponding kanamycin B analog has been prepared at BBRI and found to have antibacterial activity identical to that of kanamycin B ( 179). Compound No. 2 has an N-methylcarbamoyl substituent at C-6” while compound No. 3 has an N-phenylcarbamoyl group at that position. The first derivative, like C3’-deoxykanamycin B ( tobramycin ) and its C-6”carbamoyl derivative ( nebramycin factor 5’, Table XXIII ), has virtually the same activity as the parent compound itself. Compound No. 3, however, possesses no antimicrobial activity. Another C-6”-substituted kanamycin A derivative that gives full biological potency is the C-6”-tetrazol-2-yl (compound No. 4 ) . Substituted tetrazoles, however, such as the 5-methyl and 5-ethyl, possess respectively, approximately one-fourth and one-half the activity found with the unsubstituted heterocycle (212). Compound No. 5, which has an amino group substituted for the C-6”hydroxyl, has only one-eighth the antimicrobial potency of the parent compound. Despite this reduction in activity, the derivative proved to be significantly more toxic than kanamycin A. This is only the second instance noted thus far where a compound with weak antibacterial potency has been found to be highly toxic for mice. The next derivative (No. 6 ) where a secondary amine was formed at C-6” by N-arylation has such low activity that it is not considered to be a true antibiotic. Kanamycin A derivatives, in which C-6” has been modified to give a carboxyl group (No. 7 ) , an ethyl ester of this acid (No. 8 ) , or the corresponding amide (No. 9 ) , were prepared by Kobayashi et al. (221 ). All these modified kanamycins have considerably less antibacterial POtency than the parent compound.

TABLE XXX BIOLOGICAL PROPERTIES OF KANAMYCIN A DERIVATIVES MODIFIED AT

THE

C-6" POSITION

0

*1

*x I4

z

0

0

0

r

*

Relative activitya against selected n,ild-type organisms Compound

R

Kanamycin A No. 1 No. 2 No. 3 No. 4 No. 5 No. fi NO. 7 No. 8 No. 9

CHzOH CH3 CH~OCONHCHJ CHzOCONHCeHs 2- tetrazol-2-yl) C H LHINHz C H1NH-(2,4-dinitrophenyl) COzH COzCzH; CONHz ~~~

~

(1

b 0

S. auieus

E . eoEi

P. aeruginosa

Inactivated by indicated enzyme

NPT I

NPT I1

GNT

KAT

++ ++ ++ ++ + + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N o t a n antibiotic. . . . . . . . . 0.01 0.25 0.25 ++ ++ +@ < ++ <0.01 0.03 0.03 . . . . . . . . . . . . . . . . . . . . . . .. . .Not an antibiotic.. . . . . . . . 0.25 0.25

0.13

0.03 0.03

<0.01

0.25 0.5 0.25

0.06

0.06

0.01

~

All activities are relative to that of gentamicin C. Data from Bristol Laboratories or Bristol-Banyu Research Institute. Encircled sign = predicted response.

0.01 0.01 <0.01

<0.01 <0.01 <0.01

+ + +

+ + +

@ @ @

@ @ @

GAT I

GAT I1

GAT 111

-- ++ - + ............ -- ++ ............ -

@ @ @

A+e l.v. LDsa inmice 280 280

72

cl 0

z m

References BLb BL BL BL BL. 212 BL. 620 BL. 212

221

171 171

+

z

268

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

In addition to the above-discussed C-6” modified compounds, two monosaccharides have been glycosidically linked to kanamycin A at the C-6”-position. The sugars studied were glucose ( 2 2 2 ) , for which some question still exists as to whether it is linked as an a-glucosyl moiety at C-6” or at C-4”, and 2-aminodeoxyglucose (212). Each of these derivatives was about one-eighth as active as kanamycin A. e. Modifications in 2-DOS and the 4-Substituent. Each compound in this series, which was derived from kanamycin A or B, had had at least one of its amino groups in the 4-substituent of 2-DOS or in 2-DOS itself, acylated with L-AHBA. Results obtained in biological tests are presented in Table XXXI where amikacin, the C-1-N-AHBA derivative of kanamycin A, has been used as a control. Compound No. 1, a 3’,4’-dideoxy derivative of kanamycin B acylated at the C-1-amine with L-AHBA,has essentially the same potency as amikacin, but differs from that compound in that it is susceptible to GAT,,. Acylation of the C-3-NH, group of 3’,4’-dideoxykanamycin B with L-AHBA, as in compounds Nos. 2 and 4, eliminates activity. The detrimental effect on activity observed here is identical to that found when kanamycin A was acylated at the C-3-amine with L-AHBA (compound No. 10, Table XXIV ) . Compound No. 3, a 3’,4’-dideoxykanamycin B derivative acylated at both the C-1- and C-e’-amines had relatively good activity against S. aureus and E . coli but not P . aeruginosa. This finding is somewhat surprising in view of the fact that the C-g’-N-AHBA derivative of 3’,4‘dideoxykanamycin B had very poor activity against all three test organisms (see Section 1I.E.l.c.i). Kojima and Satoh (183) prepared two derivatives of the kanamycins by addition of new aminocyclitols to the fermentation broths of a S. kanamyceticus mutant that required 2-DOS for antibiotic production. Addition of C-1-N-methyl-2-DOS and streptamine surprisingly resulted in the formation of 6’-deamino-6’-hydroxyl-1-N-methylkanamycin -6-0-( 3-aminodeoxy-c~-~-g~ucopyranosy~ )and 4-0- ( ~-~-glucopyranosyl) 2-epi-streptamine rather than the expected 1-N-methylkanamycin A and 2-epi-hydroxykanamycin A. f. Modifications in 2-DOS and the 6-Substituent. i. N-Alkylation. Compounds in which the C-1- and C-3-amino groups of 2-DOS and the C3”-amine of its 6-substituent were N-alkylated with various substituents have been prepared at BL. Results of biological testing are presented in Table XXXII. The first derivative ( No. 1) , after N-alkylation with 3-methylbutyl groups, has activity so poor that it cannot be classified as an antibiotic. The next three compounds (Nos. 2 4 ) , which have respectively, cyclohexylmethyl, cydooctylmethyl, and benzyl groups, are weakIy active

TABLE XXXI

BIOLOGICAL PROPERTIES

OF

AMIKACINANALOGSMODIFIEDI N

T H E 4-SUBSTITUENT OF 2-DEOXYSTREPTAMINE

0

cl

0 Relative activity0 against selected wild-type organisms P.ae+uCompound

RI

Rz

R3

Ra

Ra

S. aureus

E. coli

ginosa

i

Inactivated by indicated enzyme NPT I

NPT

I1

GNT

XAT

GAT

GAT

I

I1

+-

GAT I11

2:

References

$

g 0

Amikacin No.1 No. 2 No.3 No. 4 a

b

L-AHBA L-AHBA H L-AHBA H

H H L-AHBA

H L-AHBA

OH NHp NHI NH-AHBA NH-AHBA

OH H

H H H

OH H H

H H

- + - + - + Not anantibiotic ..............................

0.25 0.25

0.5

0.5 0.25

-

-

0.5

0.25

0.13

0.03

-

-

.............................. ..............................

All activities are relative to that of gentamicin C. Data from Bristol Laboratories or Bristol-Banyu Research Institute.

-

-

-

-

-

-

Notanantibiotic

+

-

..............................

2 BL?.??10 146,813 22.9 81.9

815

$

TABLE XXXII

BIOLOGICAL PROPERTIES

OF

KANAMYCIN A DERIV.4TlVES

h~IJLTIALKYLATED AT

c-1, c-3, AND c-3"NITROGENS

H,N

Relative actirityu agai-t selected wild-type organisms

Compound

Kanamyoin A No. 1 No. 2

No. 3 No. 4

a t. c

R

H -CHtCH2CH(CHdz - C H r - o

S . aweus

E. coli

NPT

I

NPT 11

GNT

KAT

GAT I

GAT I1

GAT 111

+ + +. . . Notanantibiotic ................................

0.25

0.25

0.01

f

<0.01

0.01

0.01

-

-

0.

0.03

0.06

0.06

-

-

o

+

References BL, BL

0.01

0.01

0.01

-

-

@

+

+

-

g G?

-

-

BL

-

-

-

BI,

-

-

-

BL

F

J--H (?c-

AIL sctivitiea are relative to that of gentamiein C. D ta from Bristol Laboratories or Bristol-Banyu Research Institute. = Response not predictable.

6

P.,asrupnoaa

-5 > 3

Inactivated by indicatcd enzyme

+

2-4m

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

27 1

antibiotics, compound No. 3 having the highest potency. It is of interest that any given compound in this group has essentially equivalent activity against each of the three test organisms. The presence of an N-alkyl or an N-aralkyl substituent at the C-1-amine probably accounts for the broadened spectrum, relative to kanamycin A, possessed by these derivatives. Although the action of the phosphorylating enzymes and some acetylating enzymes appear to be blocked by N-alkylation, it is difficult to predict whether alkylation of the C-3”amine will interfere with GNT, whose site of action is the neighboring C-2”-hydroxyl. ii. Amikacin modified at C-6”. Data are available for a single amikacin derivative that was modified at C-6’’ (Table XXXIII). This compound, which contains an amine at that position, has biological properties identical to those of its kanamycin A congener (see Section 1I.E.l.d.ii); i.e., it is only one-eighth as active as the parent compound but is nevertheless much more toxic for mice. g. Modificationsin the 4- and 6-Substituents. None of the five examples in which modifications were made in both the 4- and 6-substituents of 2-DOS possess sufficient antimicrobial activity to be designated as an antibiotic. However, since this very fact is important with respect to structure-activity relationships, the structures of these compounds are presented in Table XXXIV (82,138,212,213,223). The first compound, which is 4,6-diglucosyl-2-DOS, not only lacks activity in in vitro susceptibility tests, but also fails to inhibit protein synthesis in a cell-free system. The next compound (No. 2 ) is 4,6-di-(2-aminodeoxyglucosyl)-2-DOS, and the third, which also has a 2-amino group in both the 4- and 6-glycosyl moieties, differs from compound No. 2 in that the C-Cposition is inverted in both sugars. Compound No. 4 also contains two glucosamine moieties (C-6-aminodeoxyglucose) in glycosidic linkage with the 4- and 6-positions of 2-DOS. This derivative was shown by Benveniste and Davies (82) to be incapable of inhibiting protein synthesis. The final compound has (2’,6’diamino ) -2’,3’,4’,6’-tetradeoxyglucose as the 4-substituent of 2-DOS and a C-3”-N-AHBA-glucose moiety as the 6-substituent. Since it is known that kanamycin C-A-3”-N-AHBA (see compound No. 2, Table XXIX) is devoid of activity, it is not surprising that derivative No. 5 in this table also lacks activity. The finding that none of the above-listed compounds act as antibiotics makes it very clear that the 4- and/or 6-sugar not only must have amine functions but must have them at specific sites. h. Modifications in 2-DOS and the 4- and 6-Substituents. Fujii and his co-workers ( 2 2 4 ) prepared a series of 16 tetra-N-aralkyl derivatives of kanamycin A. Although all compounds had some antimicrobial activity, five of them were significantly more active than the others. Results

TABLE XXXIII BIOLOGICAL PROPERTIES OF AMIKACIN MODIFIED AT

THE

C-6" POSITION

How Relative activity. against selected wild-type organisms

R

S. aureus

E. colt

ginosa

NPT I

NPT I1

GNT

EAT

GAT I

GAT I1

GAT

Compound

I11

Acute i.v. LDso in mice

Amikacin No. 1

OH NH,

0.25 0.06

0.5 0.03

0.5 0.06

-

-

-

+ +

-

-

-

-

300 72

P. aeru-

a

Inactivated by indicated enzyme

All activities are relative to that of gentamicin C . Data from Bht.01 Laboratories or Bristol-Banyu Resesrch Institute.

References BLa

BL

ANALOGS O F KANAMYCIN MODIFIED IN

TABLE XXXIV 4- AND 6-SUBSTITUENTS

BOTH THE

OF 2-DEOXYSTREPTAYINE

8

Compound

R1

RZ

RJ

R4

R5

R6

R7

Rs

Ra

Rio

Kanamycin A Kanamycin B No. 1 No. 2 No. 3 No. 4 No. 5

OH NHz OH NH2 NHZ OH NH2

OH OH OH OH OH OH H

OH OH OH OH H OH H

H H H H

NHz

OH OH OH NHz NHl OH OH

NH2 NHZ

H H H H OH H H

OH OH OH

OH OH OH OH OH NH, OH

a

OH H H

NHz OH OH OH NHI

NHl

Data from Bristol Laboratories or Bristol-Banyu Research Institute.

OH OH OH OH NH-ABHA

OH H OH OH

References

*z

BL. BL

cl

82.138, 623

rn

If2 31.8 82

I13

Fi

274

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

obtained with these five compounds, along with those found with several tetra-N-alkylated derivatives prepared at BL, are shown in Table XXXV. The alkylating groups used for the first five compounds, all of which possess an aromatic moiety, endowed the derivatives with a broader spectrum of activity than the parent compound based upon their increased resistance to phosphorylating and acetylating enzymes. However, tetra-N-alkylation with these groups yielded derivatives with activity only one-fourth that of the parent compound, kanamycin A. In addition, P . aeruginosa was even less susceptible than S . aureus and E . coli to these compounds. Derivative No. 6, which is an analog of kanamycin A that has been tetra-N-alkylated with 3-methylbutyl groups, has low activity ( about one-eighth that of kanamycin A ) , but does possess a disproportionately high degree of effectiveness against P . aeruginosa. Results with the tetraN-cyclohexyl (No. 7) and tetra-N-cyclooctyl (No. 8 ) derivatives were virtually identical. Thus, the data in this table suggest that the lack of aromaticity in N-alkyl groups is in some way related to their relatively high potency against P . aeruginosa.

2. Gentamicins a. Naturally Occurring Antibiotics Gentamicins A, B, B1, C1, C,,, C,, X, Sisomicin, Verdamicin, and Antibiotic G418. As previously noted in Section I,B, fermentation of M. purpurea and M . echinospora may each yield up to seven different gentamicins. The activities of these antibiotics are compared in Table XXXVI ( 78,82,136a,150,225-228). Gentamicin A differs from the other gentamicins in one very significant respect. It is the only one of these antibiotics that does not have the aminohexose, garosamine, in glycosidic linkage at the C-6-position of 2-DOS. It has instead an aminopentose, 4-demethylgarosamine, linked at that position. Furthermore, in contrast to the three members of the gentamicin C complex which are present in the commercially available preparation, gentamicin A has hydroxyl groups present at C-3’, C-4’, and C-6’ of the 4-substituent of 2-DOS. The net result of these differences is that gentamicin A is only one-fourth to one-eighth as active against S . aureus and E . coli as the “ C complex and is virtually devoid of antipseudomonal activity. The latter property probably stems from the compound’s high susceptibility to the phosphotransferases NPTI and NPT,,. Gentamicin A also differs from the several members of the gentamicin C complex in that it cannot be inactivated by KAT because of its hydroxyl group at C-6’. The observation that gentamicin A serves as a rather poor substrate for GAT1 is significant since it strongly suggests that only aminoglycosides having a garosamine moiety will be affected by this enzyme, Finally, gentamicin A has been shown to inhibit

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

275

protein synthesis to only a moderate extent and to have relatively low toxicity for mice. Gentamicin X is structurally identical to gentamicin A, except that it has a garosamine moiety linked at the 6-position of 2-DOS. Its activity against S. aureus is somewhat greater than that of gentamicin A, but is comparable against the other organisms. Gentamicin X is susceptible to GAT1 since it has a garosamine moiety, but like gentamicin A, resists inactivation by KAT. The former appears to be somewhat more toxic than the latter. Gentamicin B is similar in some respects to X but has an amino rather than a hydroxyl group at C-6’, while the opposite relationship prevails at C-2’. Its overall activity against the test organisms is greater than that of gentamicins A and X and its acute intravenous toxicity is about the same as that of X. It is susceptible to all the inactivating enzymes except GATII, which acts only on compounds that have an amine present at C-2’. Gentamicin B1, another close structural relative of gentamicin B, differs from it only in that it has a C-CH, at the 6’-position. The presence of this substituent does not seem to significantly influence the compound’s antibacterial potency, spectrum, or acute toxicity. It should be noted that all four of the preceding compounds possess interesting antiprotozoal properties and that several also have significant anthelmintic activity. For example, gentamicins A and X are some 20to 50-fold more inhibitory for Trichomonas vaginalis than gentamicin C. Gentamicins B and B, possess relatively weaker, but nevertheless significant, activity against this organism. Gentamicin X was also found to have a marked inhibitory action in in vitro tests against still another protozoan, E. histolytica. Finally, in mouse infections caused by the helminth Syphacia obvelata, both gentamicins B and X proved to be therapeutically efficacious, although the former was some 10-fold more effective than the latter. The antiprotozoal and anthelmintic activity of these 2-DOS-containing compounds is not unique among antibiotics of this class since paromomycin (see Section 1,B) and hygromycin B (see Section 1I.B) are utilized in man and animals for treatment of parasitic diseases. The activities of gentamicins C , , C,,, and C, are very similar, as are their structures. The one site at which structural variations occur is the C-6’-position. Gentamicin C, has both N-CHs and C-CH, substituents, gentamicin C,, has a primary amine only, while gentamicin C, has both a primary amine and a C-CH, substituent. The potency of C,, appears to be slightly greater than that of C, based on the percentage inhibition of protein synthesis obtained in a cell-free system, but C,, demonstrates approximately the same ability to inhibit growth of the

TABLE XXXV BIOLOGICAL PROPERTIES OF KANAMYCIN A DERNATIVES ALKYLATED AT ALL NITROGENS NH-R

by-+

NH-R

N

4

0)

Relative activity. against selected wild-type organisms Compound Kansmycin A

No. 1

R

n -CHzCHKH¶-

8

S . aureua

E. coli

0.25

0.25

0.06

0.06

P . aeruginosa

0.01

<0.01

Inactivated by indicated enzyme

NPT

NPT

+

+

t

-

-

8"

I

I1

GNT

GAT

GAT

GAT

i-

-

-

+

BLb

-

-

-

-

664

K4T

I

I1

I11

References

0

<0.01

-

-

0.03

0.01

-

-

0.06

0.06

<0.01

-

-

No. 5

0.06

0.03

<0.01

-

-

No. 6

0.03

0.03

0.03

-

-

No. 7

0.03

0.03

0.03

-

-

No. 8

0.06

0.06

0.03

-

-

0.06

0.06

No. 3

0.06

No. 4

--CHzCHXH-

No. 2

a b c

-411 activities are relative to that of gentamicin C. Data from Bristol Laboratories or Bristol-Banyu Research Institute.

0

=

Response not predictable.

TABLE XXXVI BIOLOGICAL PROPERTIES OF THE NATURALLY OCCURRINGGENTAMICINS

n

Compound

RI

Rz

I(J

R4

Rs

Rs

NHz NHI OH OH NH2 NH, NHz NHz N Ha NH.

OH OH OH OH

OH OH OH OH H H H

H

OH OH NH2 NH2 NBC& NHz NH, NHz NHz

H CHI CHJ CH3

Gentamicin C Gentamicin A Gentamicin X Gentamicin B Gentamicin BI Gentamicin CI Gentamicin CI. Gentamicin Cz Simicin Verdamicin G418

H

H H H

H

OH

Ad' A4'

OH

H H CHI CH3 H CHJ H CH3 OH

CH.

Relative act'kity ag+t Inhibi- selected wild-type organisms tion of protein P.,ocru- NPT synthesis S.aumra 8.coli ~~nosa I

30 -

CH3 CHI

CHI CH3 CH3 CHI

-

37 55

-

56

1 (0.13)- i(0.25) 0.13 0.25 0.5 n.13 0.25 0.25 0.13 0.25 0.5 1 1 1 1 1 1 2

In parentheses, mmimum inhibitory concentration in d m l , determined in Mueller-Hinton Broth. Uata from Bristol Laboratories or BrEtol-Banyu Kesearch Institute. Deteroilned by Weinstem d d.(225a). d Determined at Schering Corp. (226,. Encucled symbols = predict4 response. a

b 0

l(0.5) 0.01 0.01

0.06 0.03 I

1 0.5 2 1 0.06

3 Inactivated by indicated enzyme NPT GAT GAT I1 GNT EAT I I1

-

-

$-

f

+ +

4-

-

+ ++f+ ++4- +4+ f ++ ++ + + +- +- 4-+ + - + -

-

-

4-

-

+ f

Acute

++

++4- +-- +++ +4- ++ ff f + + +

@ 4-

+

= @

b

1.v.

GAT LDso I11 inmice

+

79

332' 225r 228d 23Od

88 70 7 0 34

@ @ -

References

BL,*lSEa,150

78,82,225,2%5a

225,226 295,226' 225,226' BL,82 BL.82 B L BL.82,m 2E7U

228

5

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

279

test organisms as C, and C,. The patterns of response to enzymatic inactivation are similar, although the C-6’-N-CH3 substituent of gentamicin C, blocks the action of KAT. All the compounds are comparably toxic for mice when administered as a single dose by the intravenous route. The detection of sisomicin, also known as antibiotic 6640, in fermentation filtrates of Micromonospora inyoensis, a new species of Micromonospora, was reported by Weinstein et al. ( 2 2 7 ) . In the same series of papers (229,230), a description was given of the fermentation procedures used to produce the compound, the methods employed for isolating it, and its biological properties. Sisomicin was found by Cooper and his co-workers ( 1 4 8 ) in 1971 to be a dehydro derivative of gentamicin Cia. Mass spectral methods revealed that the site of unsaturation was at C-4’ in the purpurosamine ring. Sisomicin is more active than members of the gentamicin C complex against certain gram-negative organisms, particularly members of the Enterobacteriaceae family and P . aeruginosa, but comparably active against S . aureus. Its pattern of response to inactivating enzymes is identical to that of gentamicin Cia. The superior antimicrobial activity of sisomicin is essentially negated by its 2-fold higher degree of toxicity. A structural analog of sisomicin, known as verdamicin, that also contains an unsaturated sugar was found to be produced by yet another Micromonosporu sp., M . grisea ( 2 2 7 ) . Structural studies proved that verdamicin is a dehydro analog of gentamicin C,; i.e., it has a C-CH, substituent at C-6’ (231 ) . Its activity is generally comparable to that of gentamicin (perhaps slightly more active), but it is less active than sisomicin. Its antimicrobial spectrum does not differ from that of the other two compounds. The final antibiotic tested in Table XXXVI has been given the designation G418. Just as with the other gentamicins and gentamicin-related compounds, it is produced by a species of Micromonospora, M . rhodorangea ( 2 3 2 ) . Its isolation, physical and chemical characteristics ( 2 3 3 ) , as well as its biological properties have been described ( 2 2 8 ) . It is reportedly active against E. histolytica, T. vaginalis, S . obuelata, and Hymenolopsis nana in both in vitro and in vivo test systems. This appears to be the first 2-DOS-containing antibiotic that has activity against the last-mentioned helminth. As far as antibacterial activity is concerned, the compound appears to be about one-fourth as active as gentamicin C against S . aureus and E . coli and about one-sixteenth as active against P . aeruginosa. G418 has garosamine as the C-6-substituent of 2-DOS but differs significantly from all the previously described antibiotics in that it has a heptopyranose moiety at C-4 of 2-DOS ( 2 3 4 ) . h. Modifications in the 2-DOS Moiety. Table XXXVII shows data

280

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

obtained when gentamicin C1, C,,, and C, were each acylated at the C-1-NH, with L-AHBA. Acylation of C,, and C, had little effect on their potency against S. aureus and E . coli. However, this modification did cause a slight reduction in antipseudomonal activity. The potency of C-1-N-AHBA gentamicin C1, on the other hand, was markedly poorer than that of the parent compound against all three of the test organisms. Although the expected increase in resistance to inactivating enzymes following acylation with L-AHBA did occur, it is of interest that this acyl group did not interfere with the action of GATI, an enzyme that acetylates the C-3-amino group of the gentamicins. In addition to the compounds listed in Table XXXVII, another series modified in the 2-DOS moiety at the CS-hydroxyl has been prepared from gentamicin C,, (235). Among substituents introduced were methanesulfonyl, methoxyl, and carbamoyl. Although specific data are not available, all three derivatives were reported to retain activity against S . aureus, P . aeruginosa, E . coli, and a number of other Enterobacteriacae species. c . Modifications in the 6-Substituent. The structures of a group of gentamicin C,-related compounds are shown in Table XXXVIII (235-237). Derivatives Nos. 1-8 which have been modified at C-2”, are described as having antimicrobial activity (235), but no biological data were reported. Compound No. 9 was synthesized by Daniels and his co-workers ( 2 3 6 ) by means of a condensation reaction involving gentamine C , , and thus may be considered as a hybrid between gentamicin C, and a C-2”-deoxykanamycin. It was found to be essentially devoid of activity which the authors considered surprising in view of the “interesting antibacterial activity found for the related 2”-deoxygentamicin C2.” No biological data are available at present for the latter compound. The last derivative (No. l o ) , C4”-deoxygentamicin C , , was synthesized by Mallams et al. (237), who found it to have little, if any, inhibitory effect on bacteria indicating that the C4”-hydroxyl group is essential for activity in the gentamicins. A single gentamicin derivative with modifications in both 2-DOS and its 6-substituent has been described (235). The compound, which is 2”,5-dicarbamoylgentarnicin C,,, reportedly has antibacterial activity although no specific data have been published. d . Other Modifications. Cooper et al. (238) have prepared the 1,2‘,3,6’tetra-N-benzyl derivative of gentamicin Cia. Although this new compound could no longer serve as a substrate for any of the N-acetyl transferases, it was essentially devoid of antibiotic activity. Thus, it was similar in this respect to the fully N-acetylated derivative of gentamicin studied by Weinstein et al. (239). These investigators found that acetylation suppressed all antibacterial effects, but at the same time virtually elimi-

TABLE XXXVII BIOLOGICAL PROPERTIES OF C-1-N-AHBA DERIVATIVES OF

HO

THE

GENTAMICINS

NH-R,

I

OH

E

9

8r

0 Relative activityo against selected wild-type organisms

Compound

RI

Rz

R3

S. aureus

Gentamicin C I

H

CH3 CHj CHI CHJ H H

CH3 CH3

0.5 0.13

No. 1

L-AHBS

Gentamicin Cz No. 2 Gentamicin Cro No. 3

H L-AHBA H L-AHBA

E. cola

P.aenrginosa

NPT I

NPT I1

-

-

H

1

0.25 1

1 0.06 0.5

H

1

1

0.13

H H

1 0.5

1 1

1

1

Inactivated b y indicated enzyme

0.5

-

-

-

GNT

KAT

t-

-

-

+ + -

+ + + +

GAT I

GAT 11

GAT 111

+ + + + + +

+ + + + + +

+

ec

+

e e

+

References

z

5 m,

0

BLb BL BL BL BL BL

All activities are relative t o that of gentamicin C.

* Data from Bristol Laboratories or Bristol-Banyu Research Institute. Encircled sign = predicted response.

M

E

282

KENNETH E.

PRICE,

JOHN C.

GODFREY,

AND HIROSHI KAWAGUCHI

TABLE XXXVIII GENTAMICINS MODIFIEDI N THE G.4ROSAMINE MOIETY

Compound Gentamiein C1 No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10

Ri

Rz

H OH H OCONHR7’ -Carbon ylOH H OSO2CH3 H H H H NHz NHz €I H 0R, H H OH H

R3

R,

115

R6

References

CH3 CH3 CH3 CH3 CH, CH3 CHI CH3 CH3 H CH3

OH OH OH OH

CI-13 CH, CH3 CH3

H

BLa

H

236 236

OH

H H CH3 H CH3 H CH3 H OH CHzOH CHI H

OH OH OH OH

H H

H H

CH3 CH3

235 235 235 236 236

235 236 237

Data from Bristol Laboratories or Bristol-Banyu Research Institute. It7 = lower alkyl.

mated toxicity for mice with intravenous LD,, values rising from 75 mg/kg for gentamicin C , , to >8000 mg/kg for the fully N-acetylated derivative. It was also observed that there was concurrent elimination of neurotoxicity along with the marked reduction in acute lethality. Similar results were obtained with neomycin and its multiacetylated derivative. Here thc LD,” increased from 40 to 275 mg/kg. The authors proposed, since they were able to demonstrate that the compounds could be recovered in unmodified form from the urine of cats, that the “molecular pharmacology” of aminoglycoside antibiotics was “specifically related to the amino groups.”

MODIFICATION OF AMINOCLYCOSIDE ANTIBIOTICS

283

Ill.

General Conclusions Regarding the Influence of Structural Variation on the Biological Properties of 2-DOS-Containing Cornpounds

Since 2-DOS has no antibiotic activity itself, it must be substituted with one or more glycosidic moieties in order to acquire antimicrobial properties. Introduction of various aminohexoses at the 6-position of 2-DOS via glycosidic linkage yields compounds that are still devoid of activity. However, derivatives such as hygromycin B,, in which 2-DOS has been substituted at C-5, do have antibacterial activity, albeit weak. When 2-DOS is substituted at the 4-position with appropriate glycosides, the resulting compounds are antibiotics whose activities range from weak to highly potent. Thus, with the exception of apramycin, which is the only compound that falls into the latter category, highly active antibiotics can be obtained only when 2-DOS has been substituted at both its 4- and 5- or 4- and 6-positions. The requirement that there be an amino function in the 4-substituent in order to have activity is an absolute one, just as is the necessity to have a primary or secondary amine at C-3 of 2-DOS. A basic function is also required at the C-1 site of 2-DOS. However, instead of a simple amino group, the substituent may be an o-amino-a-hydroxy acid that is three to five carbons in length and linked to C-1 of 2-DOS through an amide bond. Evidence that the pentosyl substituent at C-5 of 2-DOS need not have an amino group is given by the fact that compounds having a xylosyl or ribosyl moiety at that position are fairly potent antibiotics. In fact, introduction of an amino group at the C-5-position of these pentoses yields compounds whose activity is no greater than and frequently less than that of the unmodified compound. On the other hand, antimicrobial activity is significantly enhanced for those compounds which have a 2,6-diaminoglucose moiety in glycosidic linkage with the C-3-hydroxyl of ribose, The CH,NH, substituent at C-5 of the diaminoglucose moiety in the case of the lividomycins, neomycin B, and paromomycin I is in the axial position, while in neomycin C and paromomycin 11, it has the equatorial configuration. The assignment of configuration here is based upon the stereochemistry of the C-5 to C-6 linkage. Finally, there are several compounds in which another hexose (a-mannose) is linked to C-4 of the just discussed 2,6-diaminohexose. The presence of this additional glycosyl moiety causes at best a minimal reduction in antimicrobial activity. Among 4,6-disubstituted-2-DOS-containing antibiotics such as the kanamycins and gentamicins, the site of the amino group( s ) in the C-6 moiety may be even more critical for activity than the number and

284

KENNETH E . PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

location of amino groups in the 4-substituent. It has been found, for example, in a kanamycin C analog, that when the 6-glycosyl substituent of 2-DOS is 2-aminoglucose, the compound is devoid of activity. Similarly, when both the 4- and 6-glycosyl moieties of 2-DOS have C-&amino groups, the compound is not an antibiotic even though the 4-substituent is the same as that found in kanamycin A. Thus, to achieve maximum activity with the kanamycins, it is essential to have a C-3-amino group in the sugar which is linked at C-6 of 2-DOS. The presence of a second amine ( at C-6”) in addition to the one at the (2-3’’ yields a compound whose activity is substantially lower. Conversion of the C-3” primary amine to a secondary one (NHCH,) does not affect activity significantly, whereas N,N-dimethylation eliminates all activity. Although there are not enough examples among the gentamicins to unequivocally demonstrate that C-3” is the key site for the amine function in the C-6-glycosyl moiety of 2-DOS, it is known that the presence of an additional amino group at C-2” in that moiety negates all antimicrabial effects. A comparison of the activities of gentamicin B and kanamycin A, which have the same C-4-substituent, suggests that the C-6 moiety of the gentamicins ( garosamine ) confers anti-pseudomonal activity that is 2- to 3-fold higher than that conferred by the C-6-substituent of the kanamycins (3-aminoglucose). It is unlikely that this difference is due to the fact that garosamine’s amine is a secondary one (NHCH,) whereas the amine of the corresponding group in the kanamycins is a primary one. Consideration must also be given to the role played by the amines that are present in the C-4-substituent of 2-DOS. Virtually all 2-DOS-containing aminoglycoside antibiotics possess a C-2’-amino group in their C-4-substituent. The only exceptions are kanamycin A and gentamicins B and B1,all of which have a hydroxyl group at C-2’. In every case, these compounds are less active than related derivatives (like kanamycin B) which do have an amino group at C-2’. Of even greater significance as far as activity is concerned is the amino function present at C-6 of the 4-glycosyl group of 2-DOS in antibiotics such as ribostamycin, neomycins B and c, butirosins A and B, kanamycins A and B, and gentamicins B, B,, C,, C,,, C,,sisomycin, verdamycin, and G418. Paromomycins I and 11, for example, which are congeners of neomycins B and C, have a hydroxyl group at C-6’ and are 2- to 4fold less active. Similarly, Bu-1709 El and Ez (C-6’-hydroxy compounds) are about 8-fold less active than the corresponding aminecontaining compounds, butirosins A and B. Lividomycin A and B are another pair of 4,5-disubstituted-2-DOS antibiotics that have a C-6’hydroxyl in the C-4 moiety of 2-DOS. The potency of these compounds is also relatively low.

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

285

Among the 4,6-disubstituted-2-DOS-containing aminoglycosides, kanamycin A and B are considerably more active than their .C-6’-hydroxylcontaining congener, kanamycin C. Two of the gentamicins ( A and X) also have C8’-hydroxyls. However, there are no closely related C-6’amino-containing relatives with which their activities can be compared. The one representative of the kanamycins in which the single amine of the C-4-substituent is located at C-4’ has no antibiotic activity. To summarize then, requirements for high activity among 4,S-diglycosyl-substituted-2-DOS derivatives are the presence of amino groups at C-1 and C-3 of 2-DOS, as well as at C-2’ and C-6’ of the 4-substituent. Activity can be further increased by the presence of 2,6-diaminoglucose that is in glycosidic linkage with C-3” of the C-5-0-pentose moiety. Thus there appears to be a generally positive relationship between degree of potency and the total number of amino groups in the molecule. High potency in the 4,6-disubstituted-2-DOS series is found for those derivatives that have C-3”-NH2 or C3”-NHCH, in the C-6 moiety, C-land C-3-amino groups in 2-DOS and finally, amino groups at both C-2’ and C-6’ in the 4-glycosyl substituent. Again, at least in respect to the C-4-substituent7 the greater the number of amino groups, the greater the activity. It should be further noted that acylation of any of the above-cited amines except the one at C-1 of 2-DOS eliminates activity. Mono-Nalkylation with a small alkyl group, e.g., methyl, usually has a minimal effect on activity. However, in cases where all amines have been N-alkylated, there appears to be a cumulative effect resulting in a weakly active antibiotic. Mono-N-alkylation of any amine with a bulky substituent or N,N-dialkylation of any sort yields an inactive compound. The next point to be considered is the extent of the contribution to antimicrobial potency made by the hydroxyl groups of the 2-DOS-containing aminoglycosides. Considering 2-DOS itself first, it has been established that the simultaneous presence of glycosides at the 4,s- or 5,6-positions is essential for high potency. Examples where substitutions were made at all three of the hydroxyls of 2-DOS involved gentamicin C,, which had, in addition to the usual glycosides at the 4- and 6-positions, either a methanesulfonyl, methoxyl, or carbamoyl group at C-5. Although these compounds are reportedly active, detailed MIC data have not been published. In those cases where a hydroxyl group was introduced by biosynthetic means at C-2 of 2-DOS, streptamine and epi-streptamine analogs of ribostamycin and neomycins B and C were obtained. The neomycin derivatives which contain streptamine ( C-2-hydroxyl in the equatorial configuration) have activity comparable to that of the parent neomycins, while the epi-streptamine derivatives, which have the hydroxyl in axial configu-

286

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI W W A G U C H I

ration, have activity one-fourth or less that of the unsubstituted compounds. No activity data were given for the ribostamycin derivatives. The importance of the hydroxyls present in the pentoses linked at the Cd-position of 4,5-disubstituted-2-DOS antibiotics will now be considered. No modifications of the C-2”-hydroxyl have been reported. In the case of the C-3”-hydroxyl of ribose, the only variation involves formation of a glycosidic link with a 2,6-diaminoglucose moiety. It is also of interest, in connection with the unsubstituted C-3”-hydroxyl, that its configuration has no influence on activity since both xylose- and ribose-containing derivatives have equal potency. In contrast to the other hydroxyls, those at C-5” of xylose and ribose have undergone numerous modifications. The response to these changes is generally identical for both pentoses. For example, deoxygenation is extremely detrimental to activity since it causes a 4- to &fold loss in potency relative to that of the parent compound. The single chlorodeoxy derivative tested was completely devoid of activity while aminodeoxy compounds have activity that is usually equivalent to, but occasionally less than that of the unsubstituted antibiotics. Finally, the only reported modification in the hydroxyls of the 2,6diaminoglucoside moiety glycosidically linked at C-3” of ribose is that at C-4”’ which involves condensation with mannose. This modification causes only a slight reduction in activity. The contribution to activity made by the hydroxyl groups present in the C-6-glycosyl substituent of 2-DOS will be considered next. Modifications involving the hydroxy1 groups of the C-6 moiety of 2-DOS have been made at C-2”, C-4”, and C-6”. In the case of C-2” modifications of kanamycins, the single example involves introduction of an amino group at that position in a kanamycin C derivative which has a C-3”deamino-C-3”-hydroxyl in the C-6 moiety of 2-DOS. Since compounds lacking a C3”-amino in the C-6 moiety are not active, it is not possible to assess the affect created by introduction of an amine at C-2”. The one representative having a modification at C-4” is the deoxy derivative of gentamicin C,. Deoxygenation eliminated antimicrobial effects showing that this or some other substituent is essential for activity. All other compounds having changes at C-2” of the C-6 moiety are derivatives of gentamicin C,. Modifications included deoxygenation, amination, and alkoxylation, among others. Although reportedly active, no quantitative data on these derivatives’ antimicrobial effects are available. Changes in the C-6”-hydroxyl of the C-6 moiety were confined to the kanamycins. Deoxygenation at this position has little affect on activity. This is in marked contrast to the antagonistic effect on activity that is induced when this same modification was made at C-5” of the

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

287

CS-pentose moiety of 4,5-disubstituted-2-DOS antibiotics. Other modifications at C-6” having a minimal effect on kanamycin activity include the introduction of a carbamoyl (nebramycin factor 4 and 5’) or an N-tetrazol-2-yl moiety. Introduction of a methylcarbamoyl substituent reduced activity by 2-fold, while a phenylcarbamoyl group eliminated activity. Compounds in which the 6”-carbon in the 6-moiety has been oxidized to carboxyl, as well as those where this acid function was esterified or converted to the carboxamide, have activity only one-eighth that of the parent compound, kanamycin A. Similarly, C6”-deoxyamino derivatives of kanamycin A and amikacin have poor activity relative to the unsubstituted compounds. The final glycosyl moieties to be considered in respect to the function of their hydroxyl groups are those linked at C-4 of 2-DOS. As previously noted, replacement of the C-2’-hydroxyl of kanamycin A with an amino group results in a 2-fold increase in activity. No other substitutions have been made at that position. The effect of deoxygenation at C-3’ is a very significant one. In the case of neamine, paromomycins (which now can be classed as lividomycins), and kanamycin A, the effect on activity is negligible. However, the potency of the C-3’-deoxy derivative of ribostamycin and kanamycin B (tobramycin) is increased by 2- to 4-fold over that of the parent compound. Introduction of a methoxyl group at C-3’ completeiy eliminated activity, both in the case of neamine and kanamycin A. Derivatives which have been deoxygenated at both C-3’ and C-4’ have intrinsic activity comparable to or slightly greater than that of the parent compounds in the case of neamine, kanamycin A, kanamycin B, ribostamycin, and the butirosins. A derivative of kanamycin B, in which the aminoglucose moiety linked at C-4 with 2-DOS is unsaturated has activity about one-sixteenth that of kanamycin B itself. ( ) are also found among the gentamicins. Unsaturated derivatives ( Sisomicin and verdamicin, which have this modification, are the analogs of gentamicin C,, and gentamicin Cp, respectively. The intrinsic activity of sisomicin is about 2-fold greater than that of C1, while verdamicin’s potency is essentially comparable to that of gentamicin C,. The final site to be considered is the 6’-carbon in the C-4 substituent of 2-DOS. As observed previously, amination of this carbon invariably improves activity at least by 2-fold, and in some cases by as much as 4to 8-fold. The extreme importance of the type of substituent present at this site is clearly indicated by the fact that the C6’-deoxy derivative is completely devoid of activity. Compounds possessing a C-methyl at C-6’ in addition to a C8’-NH, or C6’-NHCH3 are as active as those having only the primary or the secondary amine at that position.

288

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

Thus, in summarizing the contribution that hydroxyl groups per se make to aminoglycoside activity, it appears that all of those examined can be modified in some manner and still retain antibiotic activity. In 2-DOS, activity is enhanced when glycosidic bonds are formed with the 4,5- or 4,6-hydroxyls. Furthermore, 4,6-diglycosyl-2-DOS antibiotics can have new substituents introduced at C-5 without eliminating antibiotic activity. Changes in the C-5- and C-6-glycosyl moieties of 2-DOS have essentially been confined to the C-5”-hydroxyl of the 5-0-pentoses and to the C-2”- and C-6”-hydroxyls of the C-6-substituent of 2-DOS. In all instances, modification of one of these hydroxyls by means of deoxygenation, esterification, or amination may yield a compound whose potency is at least comparable to that of the unsubstituted parent. The hydroxyls having the greatest effect on antimicrobial activity when modified are those present in the C-4-glycosidic moiety of 2-DOS. Although deoxygenation of C-3’ and/or C-4’ may not cause a significant change in biopotency, there are instances where this modification of C-3’ yields compounds with enhanced activity. The hydroxyl found at C-2’ is an extremely important one, since amination at this site causes a minimum 2-fold increase in activity. The hydroxyl of greatest significance, however, is that located at C-6’. Deoxygenation completely eliminates antimicrobial activity, whereas amination of C-6’ can increase biopotency by as much as 4-to 8-fold. The role which chemical modification of naturally occurring antibiotics plays in changing their response to inactivating enzymes will now be considered. Only those derivatives that have retained significant antibacterial activity are included in the discussion. Ribostamycin is susceptible to the 0-phosphorylating enzymes, NPTI and NPTII, as well as the N-acetylating enzymes, KAT, GATII, and GATIII, which act, respectively, at C-6’, C-2’, and C-3. In an effort to prevent phosphorylation of this compound at C-3’, the primary target site for both NPTI and NPTII, several deoxygenated derivatives have been prepared. Both the 3‘- and 3’,4’-deoxy analogs conferred resistance to NPTII, but not to NPTI. The reason for this has been uncovered in recent studies in which it was demonstrated that when ribostamycin has been deoxygenated at C-3’, NPTI will then phosphorylate the 5”hydroxyl of ribose. This finding has led to the preparation of 3’,4’,5’’trideoxyribostamycin. This compound‘s intrinsic activity is somewhat lower than that of ribostamycin; but it is, nevertheless, completely refractory to both NPTI and NPTII. Deoxygenation of 5-0-xylosylneamine at C-4’ caused no change in the compound’s susceptibility to enzymes. Butirosin B is a naturally occurring derivative of ribostamycin in which the C-1-amine has been acylated with L- ( - ) -7-amino-a-hydroxybutyric

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

289

acid. This unusual amino acid, presumably through a mechanism involving steric hindrance of the sites critical for enzyme attachment, prevents phosphorylation by NPTI, but not by NPTII. In addition, it blocks acetylation of the C-3-amino group of 2-DOS by the enzyme, GAT,,,. Interestingly, the naturally occurring antibiotic, Bu-1975 (4'-deoxybutirosin) , is not activated by NPTII. Most of the above cited modifications which have been found to extend the spectrum of ribostamycin can be utilized with equal success in the case of the neomycins and paromomycins. The latter compounds, of course, are not affected by the acetylating enzyme, KAT, since they have a hydroxyl group at C-6’. The lividomycins, which are actually 3’-deoxyparomomycins, are inactivated by NPT, and a newly described enzyme, lividomycin phosphotransferase ( LPT), through phosphorylation of the C-5”-hydroxyl. They are also susceptible to the N-acetylating enzymes, GAT, and GAT,,,. However, acylation of the C-l-amino group of these compounds by L-AHBA yields derivatives which are subject to inactivation only by GAT,, and possibly LPT. Those chemical modifications considered to be of the greatest value in reducing the enzymatic susceptibility of 4,6-diglycosyl-2-DOS antibiotics will now be considered. Deoxygenation at both C-3’ and C-4’ has been carried out with kanamycin A and kanamycin B, the latter derivative being designated as DKB, in order to prevent phosphorylation of the C-3’-hydroxyl. The resulting compounds proved to be completely refractory to both NPTI and NPTII, just as is the naturally occurring antibiotic, 3’-deoxykanamycin B ( tobramycin) , Similar results were obtained with the 3’-deoxy analog of kanamycin A. The deoxygenated derivatives of kanamycin A and B are still susceptible to gentamicin nucleotidyltransferase ( GNT ) which acts at the 2”-hydroxyl, as well as to the acetylating enzymes, KAT, GATII, and GAT,,,. Of course, the inactivating action of KAT can be blocked in the kanamycins by monoN-alkylation at the C-6’-amine with a methyl group. Introduction of an L-AHBA acyl group at the C-l-amine of the 2-DOS moiety of the kanamycins causes an even more striking change in their susceptibility to enzymes than that obtained by other modifications. The L-AHBA derivative of kanamycin A, known as BB-K 8 or amikacin, is refractory to all of the known enzymes except KAT. The corresponding kanamycin B derivative, although almost as resistant, can be inactivated by GAT11 as well as KAT. Acylation of the C-l-amino groups of the various gentamicin C antibiotics with L-AHBA yields compounds which, although still quite active, have lower potency against P. aeruginosa than the unsubstituted parent compound. On the other hand, this modification does bring about a

290

KENNETH E. PRICE, JOHN

c.

GODFREY,

AND

HmosnI

KAWAGUCHI

significant change in the response of the compounds to inactivating enzymes since it makes them refractory to GNT and GATIII. They are still susceptible to the action of GAT11 and GATI, the latter being an enzyme which selectively acetylates the gentamicins. The failure of L-AHBA acylation to prevent inactivation by GAT1 is somewhat surprising since the enzyme’s site of attack is C-3, the same as that of GATIII, an enzyme whose inactivating effects are blocked by the L-AHBA acyl group. The gentamicin C antibiotics cannot be phosphorylated since they do not have hydroxyls at C-3’ and C-4’. It should also be noted that gentamicin C1,which has an N-CH, substituent at C-6’, is not acetylated by KAT, whereas the other members of the C complex are. Changes, such as deoxygenation and amination, have been made at C-2” in an effort to block nucleotidylation by GNT. The extent to which these changes affect the activity of the gentamicins is not known. The significance attached to development of a new semisynthetic, enzyme-resistant antibiotic like amikacin cannot be overemphasized. This is illustrated clearly in Table XXXIX where the antimicrobial activity of amikacin has been compared with that of nine other aminoglycoside antibiotics. All were tested against a collection of 163 clinical isolates, each of which possesses resistance to one or more aminoglycosides. Assurance that the data obtained in the study were not merely the end result of conditions that exist within a localized setting is given by the fact that the isolates examined were obtained from many and diverse sources. In fact, there were on the average only about two to three resistant strains obtained from any given source. The 10 antibiotics listed in the table fall into four different categories when considered in terms of their effectiveness against the 163 clinical isolates. The least active group was comprised of ribostamycin, neomycin B, and paromomycin, each of which inhibited less than 10%of the test organisms. All these antibiotics are fully susceptible to inactivation by NPTI, NPTII, and GATIII, and two of them, ribostamycin and neomycin, can be inactivated by KAT. Members of the next group of antibiotics, which includes lividomycin A, butirosin B, and kanamycin A, each inhibited about 2016 of the isolates. Lividomycin A is susceptible to NPTI and GATIII, and butirosin to NPTII and KAT, while both are inactivated by GATII. Kanamycin A, although a good substrate for NPTI, NPTIr, GNT, KAT, and GATIII, is nut susceptible to the action of GATII. The third group contains three 4,6-diglycosyl-substituted-2-DOS antibiotics that have been deoxygenated at c-3’or at both c-3’and c-4’. These antibiotics, tobramycin, DKB ( 3’,4’-dideoxykanamycin B ) , and gentamicin C, inhibited 33 to 41% of the 163 test organisms. None of

TABLE X X X I X TO VARIOUS ANTIBIOTICS OF 163 AMINOGLYCOSIDE-RESISTANT CLINICALISOLATES COMPARATIVE SUSCEPTIBILITY Percentage of strains susceptible"

Organism

NeoLividoNo. of Ribosta- mycin Paromo- mycin strains mycin B mycin A

Butirosin

Escherichia coli Enterobacter sp. Klebsiella pneumoniae Proteus sp. Providencia stuartii Serratia marcescens Salmonella sp. Herellea sp. Pseudomonas aeruginosa Staphylococcus aureus

21 15 13 18 25 19 3 3 39 7 163

10 13 0 0 0 32 0 66 0 0

10 13 0 0 0 32 0 33 3 0

15 13 0 0 0 32 0 66 0 0

43 33 54 0 0 32 0 33 8 0

29 33 34 0 0 42 100 66 8 0

% susceptible*

100

4

7

8

19

22

Kanamycin A

DKB

Gentamicin C 48 53 30 6 36 68 100 33 18 100

90 93 100 94 100 93 100 100

0

33 62 100

43 53 23 0 8 21 100 33 54 100

100

3*

18

41

33

36

91

g

5 0

0 39 60 16 0 100

0

Tobramycin 43 53 23 22 36 16 100

Amikacin

87

a The susceptibility cutoff point (SCP) for Amikacin (BB-K S), kanamycin A, butirosin B, lividomycin A, paromomycin I, and ribostamycin is 20 figlml. The SCP for gentamicin C, tobramycin, D K B (3',4'-dideoxykanamycin B), and neomycin B is 8 pglml. Number of strains susceptible/total number tested equals % susceptible.

$ 32

2 p

2

g

:

292

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

these compounds can be phosphorylated, but all are susceptible to inactivation by GNT, KAT, GATIr, and GATIlI. Gentamicin C can also be inactivated by GATI. The final compound, amikacin, a C-1-N-AHBA analog of kanamycin A, is in a class by itself since it inhibited 148 or 91% of the 163 test organisms. This antibiotic acts as a substrate for only one enzyme, kanamycin acetyltransferase ( KAT ) . These results suggest that there is a definite relationship between the breadth of the antimicrobial spectrum of these antibiotics and their susceptibility to inactivation by enzymes. Presumptive evidence that the strains listed in Table XXXIX are indeed enzyme-producers has been presented by Price and his co-workers ( QZ),who found in preliminary studies that a minimum of 60% of them have the ability to produce one or more inactivating enzymes. Acute IV LD,,,’s have been obtained in mice for a relatively small number of the aminoglycosides discussed in this review, but data are available for most of the therapeutically important compounds, some of their close relatives, and in a few cases, their principal degradation products. To the extent that information is available, an attempt has been made to determine the influence of structural variations upon toxicity. Preliminary examipation of the data shows that toxicity is intrinsically associated with neamine. This compound, which has an intravenous LD,, of 180 mglkg, is an important component of most 2-DOS-containing aminoglycoside antibiotics. All derivatives of neamine were compared with it on an equimolar basis to determine their relative potential to produce lethality. Modifications which appeared to have little or no effect on neamine’s toxicity are listed and discussed below. 1. Introduction into neamine of a C-5-0-xylosyl or C-5-O-ribosyl moiety. LD,, data obtained with both 5-O-xylosylneamine and ribostamycin ( 5-O-ribosylneamine ) indicate that neither pentose has any effect on the toxicity of neamine. 2. Introduction into neamine of a C-6-0-( 3-aminoglucose ) moiety. In view of the fact that there is an amino group present at C-3” in the C-6-0-glycosyl moiety of kanamycin B, and since it is known that amines make a significant contribution to the toxicity possessed by aminoglycoside antibiotics, it is somewhat surprising that this modification does not enhance toxicity. 3. Deoxygenation at C-4’ in the C-4-0-glycosyl substituent of 2-DOS. This modification failed to influence toxicity as indicated by the fact that 4’-deoxy-5-0-xylosylneamine has toxicity equal to that of the parent compound and that Bu-1975C1 (4’-deoxybutirosin B ) has an LD,, comparable to that of its oxygenated congener. 4. Deoxygenation at C-6/’ in the C-6-0-glycosyl substituent of 2-DOS.

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

293

Acute toxicities of kanamycin A and its C-6”-deoxy derivative were identical. 5. Acylation with L-AHBA at the C-l- or C-3-amine of 4,6-diglycosylsubstituted 2-DOS derivatives, Amikacin, the C-1-N-AHBA derivative of kanamycin A, has an acute intravenous LD,, that is comparable to that of the parent compound, On the other hand, the LD,, of the C-3-N-AHBA derivative of kanamycin A is somewhat lower than that of the parent (235 mg/kg vs. 280 mg/kg) even though one might expect it to be higher in view of the fact that the molecular weight of the derivative is higher. However, the extent of this difference is not great enough to warrant assuming that acylation at this site causes a true enhancement in toxicity. The second group of modifications to be considered are those that have been found to increase the toxicity of neamine. 1. Presence of 2,6-diaminoglucose in glycosidic linkage with C-3” of C-5-0-ribosyl-substituted neamine. All six of the compounds in which the C-5-substituent of 2-DOS is comprised, at least in part, of a 2,6-diaminoglucose moiety were found to have significantly greater toxicity than neamine. The compounds include neomycins B and C which are each about 10 times more toxic than neamine when compared to it on an equimolar basis, The remaining compounds are paromomycins I and I1 and lividomycins A and B, which, although possessing other structural features that tend to decrease toxicity, are nevertheless at least 2-fold more toxic than neamine. 2. Substitution of an amine for hydroxyl at C-5” in a C-5-O-pentosyl moiety or at C-6” in a C-6-0-(3-aminoglucose) moiety. The single representative having an aminated C-5” group is 5”-aminodeoxybutirosin A. This compound‘s toxicity was approximately twice that of butirosin A. There are two 4,6-disubstituted-2-DOS antibiotics in which C-6” has been aminated. These are 6”-aminodeoxykanamycin A and 6”-aminodeoxyamikacin, each of which is about 4-fold more toxic than its parent compound. 3. Deoxygenation at C-3’ in the C-4-0-glycosyl substituent of 2-DOS. Most derivatives that have been deoxygenated at C-3’ are significantly more toxic than their oxygenated counterparts. For example, 3’,4’,5,6tetradeoxyneamine is &fold more toxic than neamine while 3’-deoxykanamycin B (tobramycin) and 3’,4’-dideoxykanamycin B (DKB) are each about 2-fold more toxic than kanamycin B. Although the corresponding oxygenated analogs are not available for direct comparison with gentamicins C,, C,,, and Cp, these 3’,4’-dideoxy compounds have a degree of toxicity that is comparable to that of tobramycin and DKB and considerably greater than that of fully oyxgenated gentamicins such as A, X, B, and B,.

294

KENNETH E. PRICE, JOHN

c.

GODFREY, AND HIROSHI ILAWAGUCHI

4. Unsaturation at C-4’ in the C-4-0-glycosyl substituent of 2-DOS. The only representative of this type for which an LD,, is available is sisomicin. This compounds toxicity is some 2-fold greater than that of its saturated counterpart, gentamicin C,. The third and last series of modifications are those that tend to reduce the intrinsic toxicity of neamine. 1. Replacement of the amine with a hydroxyl group at C-6’ in the C-4-0-glycosyl substituent of 2-DOS. Among those compounds considered to be antibiotics, this modification is one of the major ones leading to a reduction in neamine toxicity. Paromomycin I and 11, which have a hydroxyl group at C-6’, are 4- to 6-fold less toxic than the corresponding C-6‘-amino-containing compounds, neomycins B and C. The lividomycins, which also have a hydroxyl group at C-6’, have toxicity comparable to that of the paromomycins. This same pattern is maintained for Bu-1709E1,the C-6’-hydroxy analog of butirosin A which, as expected, is less toxic than the parent compound. Another example is provided by kanamycin C, which is less toxic than kanamycin B, an analog that differs from kanamycin C only in that it has a C-6’-amino group. 2. Replacement of the amine with a hydroxyl group at C-2’ in the C-4-0-glycosyl substituent of 2-DOS. The best example of this type of modification is provided by kanamycin A, which has a hydroxyl group at C-2’ and an amino group at C-6’. It is only about one-half as toxic as kanamycin B, a compound having amino groups at both positions, and is also less toxic than kanamycin C, which has an amino group at C-2’ but a hydroxyl rather than amine at C-6’. Two members of the gentamicin series;B and Bi, also have a C-2’-hydroxyl and a C-6’amine. They are somewhat more toxic than kanamycin A, presumably because their C-6-0-glycosyl substituent is garosamine rather than 3-aminoglucose. 3. Substitution of a carbamoyl moiety for hydroxyl at C-6” in the C-6-0-glycosyl moiety of 2-DOS. The two derivatives having this modification are produced in the nebramycin fermentation and are called factors 4 and 5’. Both the first compound, which is an analog of kanamycin B, and the second, an analog of tobramycin, are significantly less toxic than their respective parent compounds. 4. Introduction of L-AHBA via acylation of the C-l-amine in the 2-DOS moiety of 4,5-diglycosyl-containing antibiotics. Butirosin A, butirosin B, and Bu-1975C1 each have acute toxicity for mice that is significantly lower ( a t least 2-fold) than that of neamine. Since it has been shown that introduction of a pentosyl moiety at C-5 of neamine does not influence toxicity, the decreased tendency to cause lethality must be attributable to the presence of the L-AHBA acyl group at the C-l-amine of 2-DOS.

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

295

TABLE XL TOXICITY INDEXES A N D RELATIVE ANTIMICROBIAL ACTIVITIESOF VARIOUS AMINOGLYCOSIDE ANTIBIOTICS Ratio of expected LD60 Compound Neamine Ribostamycin 4’-Deoxy-5-xylosylneamine 5-O-X ylosylneamine Butirosin A Butirosin B 5”-Aminodeoxybutirosin A Bu-1975Ci Bu-1709Ei Neomycin B Neomycin C Paromomycin I Paromomycin I1 Lividomycin A Lividomycin B Kanamycin A 6”-Deoxykanamycin A 6”-Aminodeoxykanamycin A 1-N-Acetylkanamycin A 1-N-AHBA kanamycin A (amikacin) 6”-Aminodeoxyamikacin 3-N- Acetylkanamycin A 3-N-AHBA kanamycin A 6’-N-Acetylkanamycin A 6‘-N-AHBA kanamycin A 3”-N-Acetylkanamycin A 3”-N-AHBA kanamycin A Kanamycin B 3’-Deoxykanamycin B (tobramycin) 3’-Deoxy-6”-carbamoy1kanamycin B (nebramycin factor 5’) 6”-C arb amoylkanam y cin B (nebramy cin factor 4) 3’,4’-Dideoxykanamycin B (DKB) Kanamycin C Gentamicin C complex Gentamicin A Gentamicin X Gentamicin B Gentamicin B1 Gentamicin C1 Gentamicin C,, Gentamicin CI Sisomicin

MIC“ relative to gentamicin C complex

actual LD60

S. aureus

E . coli

1.00 0.977 0.875 0.875 0.534 0,534 1.11 0.579 0.349 14.33 7.82 2.16 1.98 1.52 2.39 0.968 0.936 3.75 1.00 1.09 4.53 0.321 1.39 0.098 0.282 0.059 0.222 2.05 3.30

0.06 0.13 0.06 0.13 0.13 0.13 0.13 0.01 0.25 0.25 0.06 0.06 0.06 0.13 0.25 0.25 0.03

0.25 0.06 0 0 0 0 0 0 0.50 1.00

0.25 0.50 0.25 0.25 0.50 0.25 0.50 0.03 0.50 0.50 0.13 0.06 0.13 0.13 0.25 0.50 0.03 0 0.50 0.03 0 0 0 0 0 0 0.50 1.00

2.48 1.34

1.00 0.25

0.50 0.50

3.55 1.20 3.28 0.789 1.20 1.18 1.20 3.03 3.59 3.70 7.59

0.50 0.06 1.00 0.13 0.50 0.25 0.13 0.50 1.00 1.00 1.00

0.50 0.06 1.00 0.25 0.13 0.25 0.25 1.00 1.00 1.00 2.00

Ob

a MIC = minimum inhibitory concentrations. *Relative activity value of <0.01 is considered to be zero for the purpose of analysis.

296

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

5. Acetylation of the C-1-, C-3-, C-6’-, or C-S”-amine. Kanamycin A has been acetylated at each of the indicated amino groups yielding antimicrobially inert and essentially nontoxic compounds in each case. 6. Acylation with L-AHBA of the C-6’ or C-S”-amine. Kanamycin A derivatives containing L-AHBA as an acyl group at the C-6’- or C-3”amine do not have significant antimicrobial activity or toxicity for mice. The final question to be examined is whether there is a significant correlation between a compound‘s toxic potential for mice and its intrinsic antimicrobial activity. Calculation of each compound’s toxicity relative to that of neamine (corrected for the difference in molecular weights) gives a toxicity index. Indexes for the above-discussed 4,5and 4,6-diglycosyl-2-DOS antibiotics were all derived by the method shown below for ribostamycin. Example: Neamine’s molecular weight = 322 and its observed LD,, = 180 mglkg; ribostamycin’s molecular weight = 454 and its observed LD,, = 260 mglkg. If x = the calculated or expected toxicity of ribostamycin, then x t 180 mg/kg = 454 t 322 and x = 254 mglkg. A comparison of 254 mglkg with the experimentally obtained value of 260 mglkg gives 0.977 (254 t 260), indicating that ribostamycin is only 97.7%as toxic as expected. Although in the example given, the difference between expected and observed values is minimal, it can be seen in Table XL that, for certain compounds, differences of a fairly high magnitude are obtained. The toxicity indexes listed here have been plotted vs. their antistaphylococcal activity (relative to that of the gentamicin C complex) in Fig. 2. It can be seen that, although there is a definite relationship ( P = <0.05) between the compounds’ acute toxicity for mice and their ability to inhibit growth of S. uureus, the fit of the line is poor ( T = 0.38) because some of the 41 compounds have toxicity that is disproportionately high relative to their antimicrobial activity. These exceptions are 3-N-AHBA kanamycin A, 6”-aminodeoxykanamycin A, 6”-aminodeoxyamikacin, the neomycins, and to a lesser extent, the paromomycins and lividomycins. In the case of the neomycin-like antibiotics, their high toxicity can probably be attributed to the presence of the 2,6-diaminoglucose moiety that is linked with the 5-O-ribosyl substituent of 2-DOS. The extremely high toxicity observed with the C-6’-amine-containing neomycins B and C is apparently offset in the case of the paromomycins and lividomycins, at least to some extent, by the presence of a hydroxyl group at C-6’. Introduction of an amino group at C-6’’ results in kanamycin A and amikacin derivatives which possess a 3,6-diaminoglucose moiety linked at C-6 of 2-DOS. This diamino sugar, like the one glycosidically linked

297

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

X

/

1 X

/

1

/

/ /

@5

$4

/

@3

2

X

/

82 X X X

&

L

01 -1

X

X

0.1

/

/ .

0.2

0.3

7-

0.5 0:s 0:7 0.4 ACTIVITY AGAINST S. AUREUS

0.0

v

0.9

1.0

FIG. 2. This plot shows the relationship between intravenous toxicity indexes (all values relative to that of neamine) and inhibitory activities against S. uureus (relative to that of gentamicin C ) for various aminoglycoside antibiotics. For the purpose of analysis, all activity values of <0.01 were considered to equal zero. Point 1 is 3-N-AHBA kanamycin A; 2, lividomycin A; 3, paromomycin 11; 4, paromomycin I; 5, lividomycin B; 6, 6”-aminodeoxykanamycin A; 7, 6”-aminodeoxyamikacin; 8, neomycin C ; and 9, neomycin B (toxicity index = 14.33).

with ribose in the neomycins, confers a high level of toxicity upon those compounds which contain it. The remaining derivative, a C-3-N-AHBA derivative of kanamycin A, although relatively toxic, has no measurable antimicrobial activity. NO explanation is available that would account for the wide divergence that occurs between these two properties in this compound. If one disregards these nine highly toxic derivatives, the correlation between the two variables is excellent ( P =
298

KENNETH E. PRICE, JOHN

c. GODFREY,

AND HIROSHI KAWAGUCHI

/ I

/ALL

POIHTS

(‘=

0461

/

2’0

ACTIVITY AGAINST E COLI

FIG.3. This plot shows the relationship between intravenous toxicity indexes (all values relative to that of neamine) and inhibitory activities against E . coli (relative to that of gentamicin C ) for various aminoglycoside antibiotics. For the purpose of analysis, all activity values of <0.01 were considered to equal zero. Point 1 is 3-N-AHBA kanamycin A; 2, lividomycin A; 3, paromomycin 11; 4, paromomycin I; 5, lividomycin B; 6, 6-aminodeoxykanamycin A; 7, 6”-aminodeoxyamikacin; 8, neomycin C; and 9, neomycin B (toxicity index = 14.33).

with S . aureus, since T = 0.46. Exclusion of the same nine compounds from the analysis establishes that there is a very clear-cut relationship ( P =
MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

299

REFERENCES 1. Polglase, W. J. (1965). Nature (London) 206, 298. 2. Treffers, H. P., and Alexander, D. C. (1956). Antibiot. Ann. p. 802. 3. Treffers, H. P., Belser, N. O., and Alexander, D. C. (1953). Antihiot. Ann. p. 594. 4. Flaks, J. G., Cox, E. C., and White, J. R. (1962). Biochem. Biophys. Res. Commun. 7 , 385. 5. Bannister, B., and Argoudelis, A. B. (1963). J. Amer. Chem. SOC. 85, 234. 6. Heding, H., and Lutzen, 0. ( 1972). J. Antibiot. 25, 287. 7. Umezawa, H. Okanishi, M., Utahara, R., Maeda, K., and Kondo, S. (1967). J . Antibiot., Ser. A 20, 136. 8. Umezawa, H., Okanishi, M., Kondo, S., Hamada, K., Utahara, R., Maeda, K., and Mitsuhashi, S . (1967). Science 157, 1559. 9. Umezawa, H., Takasawa, S., Okanishi, M., and Utahara, R. (1968). J . Antibiot. 21, 81.

10. Ozanne, B., Benveniste, R., Tipper, D., and Davies, J. (1969). J. BucterioZ. 100, 1144. 11. Benveniste, R., and Davies, J. (1973). Ann. Reo. Biochem. 42, 471. 12. Schatz, A., Bugie, E., and Waksman, S. A. (1944). Proc. SOC. Ezp. B i d . Med. 55, 66. 13. Carter, H. E., Clark, R. K,., Jr., Dickman, S. R., Loo, Y. H., Skell, P. S., and Strong, W. A. (1945). J. Biol. Chem. 160, 337. 14. Vander Brook, M. J., Wiek, A. N., DeVries, W. H., Harris, R., and Cartland, G. F. (1946). J . Biol. Chem. 165, 463. 15. Kuehl, F. A., Peck, R. L., Hoffhine, C. E., Jr., Peel, E. W., and Folkers, K. (1947). J. Amer. Chem. Sac. 69, 1234. 16. Kuehl, F. A., Peck, R. L., Hoffhine, C. E., Jr., and Folkers, K. (1948). J. Amer. Chem. SOC. 70, 2325. 17. Waksman, S. A., and Schatz, A. (1945). J . Amer. Phurm. Ass. 34, 273. 18. Rake, G., and Hamre, D. M. (1946). Fed. Proc., 5, 253. 19. Littman, M. L. (1947). J. BacteTiol. 54, 399. 20. Spingarn, C. L., and Edelman, M. H. (1947). J. Purasitol. 33, 416. 21. Garrod, L. P., and OGrady, F. (1968). “Antibiotic and Chemotherapy,” 3rd ed., p. 98. Livingstone, Edinburgh. 22. Erdos, T., and Ulman, A. (1960). Nature (London) 185, 100. 23. Spotts, C. R., and Stanier, R. Y. (1961). h’ature (London) 192, 633. 24. Cox, E. C., White, J. R., and Flaks, J. G. (1964). Proc. Nut. Acad. Sci. U.S. 51, 703. 25. Davies, J. E. (1964). Proc. Nut. Acad. Sci. U S . 51, 659. 26. Traub, P., Hosokawa, K., and Nomura, M. (1966). J. Mol. Biol. 19, 211. 27. Staehelin, T., and Meselson, M. ( 1966). J. Mol. Biol. 19, 207. 28. Modolell, J., and Davis, B. D. ( 1969). Nature (London) 224, 345. 29. Luzzatto, L., Apirion, D., and Schlessinger, D. (1969). J . Mol. Biol. 42, 315. 30. Gale, E. G., Cundliffe, E., Reynolds, P. E., Richmond, M. H., and Waring, M. J. ( 1973). “The Molecular Basis of Antibiotic Action,” p. 278. Wiley, New York. 31.. Finland, M., Murray, R., Harris, H. W., Kilham, L., and Meads, M. (1946). J. Amer. Med. Ass. 132, 16. 32. Murray, R., Kilham, L., Wilcox, C., and Finland, M. (1946). Proc. SOC. Ezp. Biol. Med. 63, 470.

300

KENNETH E. PRICE, AND JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

33. Miller, C. P., and Bohnhoff, M. (1946). J. AWT. Med. Ass. 130, 485. 34. Moellering, R., Weinberg, A,, Zimmerman, R., and Wennersten, C. ( 1970). Clin. Res. 18, 445. 35. Zimmerman, R. A., Moellering, R. C., Jr., and Weinberg, A. N. ( 1971). J . Bacteriol. 105, 873. 36. Tseng, J. T., Bryan, L. E., and Van Den Elzen, H. M. (1972). Antimicrob. Ag. Chemother. 2, 136. 37. Davies, J. (1971). J. Infec. Dis. 124, Suppl., S7. 38. Yamada, T., Tipper, D., and Davies, J. ( 1968). Nature ( London) 219,288. 39. Takasawa, S., Utahara, R., Okanishi, M., Maeda, K., and Umezawa, H. (1968). J. Antibiot. 21, 477. 40. Harwood, J. H., and Smith, D. H. ( 1969). J. Bacteriol. 97, 1262. 41. Yamada, T., Kvitek, K., and Davies, J. (1970). PTOC.Int. Congr. Chemother., 6th, 1969 B2, p. 562. 41a. Okanishi, M., Koshiyama, H., Ohmori, T., Matsuzaki, M., Ohashi, S., and Kawaguchi, H. (1962). J. Antibiot., Ser. A 15, 7. 42. Davies, J. (1968). Antimicrob. Ag. Chemother. p. 297. 43. Umezawa, H., Okanishi, M., Kondo, S., Hamada, K., and Utahara, R. (1967). Science 157, 1559. 44. Doi, O., Ogura, M., Tanaka, N., and Uniezawa, H. (1968). Appl. Microbiol. 16, 1276. 45. Doi, O., Miganioto, M., Tanaka, N., and Umezawa, H. (1968). Appl. Microbiol. 16, 1282. 46. Okanishi, M., Kondo, S., Utahara, R., and Umezawa, H. (1968). J. Antibiot. 21, 13. 47. Florey, H. W., Chain, E., Heatley, N. G., Jennings, M. A., Sanders, A. G., Abraham, E. P., and Florey, M. E. (1949). “Antibiotics,” Vol. 11, p. 1401. Oxford Univ. Press, London and New York. 48. Bignall, J. R., Crofton, J. W., and Thomas, J. A. B. (1951). Brit. Med. J . 1, 554. 49. Cawthorne, T., and Ranger, D. ( 1957). Brit. Mecl. J. 1, 1444. 50. Lullman, H., and Reuter, H. ( 1960). Antibiot. Chemother. (Basel) 1, 375. 51. Waksman, S. A,, and Lechevalier, H. A. (1949). Science 109,305. 52. Rinehart, K. L., Jr., Hichens, M., Argoudelis, A. D., Chilton, W. S., Carter, H. E., Georgiadis, M. P., Schaffner, C. P., and Schillings, R. T. (1962). J. Amer. Chem. Soc. 84, 3218. 53. Carter, H. E., Dyer, J. R., Shaw, P. D., Rinehart, K. L., Jr., and Hichens, M. (1961).J. Amer. Chem. SOC. 83, 3723. 54. Rinehart, K. L., Jr. (1961). “The Neomycins and Related Antibiotics,” p. 1. Wiley, New York. 55. Decaris; L. J. (1953). Ann. Pharm. FT. 2, 44. 55a. Wagman, G. H., Marquez, J. A., Walkins, P. D., Bailey, J. V., Gentile, F., and Weinstein, M. J. (1973). J . Antibiot. 26, 732. 56. Frohardt, R. P., Haskell, T. H., Ehrlich, J., and Knudsen, M. P. (1956). Belgian Patent 547,976. 57. Coffey, G. L., Anderson, L. E., Fisher, M. W., Galbraith, M. M., Hillegas, A. B., Kohberger, D. L., Thompson, P. E., Weston, K. S., and Ehrlich, J. (1959). J. Antibiot. Chemother. 9, 730. 58. Haskell, T. H., French, J. C., and Bartz, Q. R. (1959). J. Amer. Chem. SOC. 81, 3482. 59. Haskell, T. H., French, J. C., and Bartz, Q. R. (1959). J. Amer. Chem. SOC.81, 3480.

MODIFICATION OF AMINOGLYCOSlDE ANTIBIOTICS

301

60. Schillings, R. T., and Shaffner, C. P. (1962). Antimicrob. Ag. Chemother. p. 274. 61. Horii, S. ( 1 6 2 ) . J. Antibiot., Ser. A 15, 187. 62. Hessler, E. J., Jahnke, H. K., Robertson, J. H., Tsuji, K., Rinehart, K. L., Jr., and Shier, W. T. (1970). J. Antibiot. 23, 464. 63. Umezawa, H., Ueda, M., Maeda, K., Yagashita, K., Kondo, S., Okami, Y., Utahara, R., Osato, Y., Nitta, K., and Takeuchi, T. (1957). 3. Antibiot. Ser. A 10, 181. 64. Cron, M. J., Evans, D. L., Palermiti, F. M., Whitehead, D. F., Hooper, I. R., Chu, P., and Lemieux, R. U. (1958). 3. Amer. Chem. SOC. 80,4741. 65. Maeda, K., Murase, M., Mawatari, H., and Umezawa, H. (1958). J . Antibiot., Ser. A 11, 163. 66. Umezawa, S., Tatsuta, K., and Koto, S. (1969). Bull. Chem. Soc. Jup. 42, 533. 67. Takeuchi, T., Hikiji, T., Nitta, K., Yamazaki, S., Abe, S., Takayama, H., and Umezawa, H. (1957). J. Antibiot., Ser. A 10, 107. 68. Hichens, M., and Rinehart, K. L., Jr. ( 1963). 3. Amer. Chem. SOC. 85, 1547. 69. Ito, T., Nishio, M., and Ogawa, H. (1964). 3. Antibiot., Ser. A 17, 189. 70. Schmitz, H., Fardig, 0. B., O'Herron, F. A., Rousche, M. A., and Hooper, I. R. (1958). J. Amer. Chem. SOC. 80, 2912. 71. Umezawa, S., Koto, S., Tatsuta, K., Hineno, H., Nishimura, Y., and Tsumura, T. (1969). Bull. Chem. SOC. Jap. 42, 537. 72. Murase, M. ( 1961). J. Antibiot., Ser. A 14, 367. 73. Umezawa, S., Koto, S., Tatsuta, K., and Tsumura, T. (1969). Bull. Chem. Soc. Jap. 42, 529. 74. Weinstein, M. J., Leudemann, G. M., Oden, E. M., Wagman, C. H., Rosselet, J. P., Marquez, J. A., Coniglio, C. T., Charney, W., Herzog, H. L., and Black, J. ( 1963). 3. Med. Chem. 6, 463. 75. Weinstein, M. J,, Leudemann, G. M., Oden, E. M., and Wagman, G. H. (1964). Antimicrob. Ag. Chemother. p. 1. 76. Cooper, D. J,, Marigliano, H. M., Yudis, M. D., and Traubel, T. (1969). 1. lnfec. Dis. 119, 342. 77. Wilson, W. L., Richard, G., and Hughes, D. W. (1973). 3. Phurm. Sci. 62, 282. 78. Maehr, H., and Schaffner, C. P. ( 1967). 3. Amer. Chem. SOC. 89, 6787. 79. Weinstein, J,, Cooper, D. J,, and Daniels, P. J. L. (1972). Abstr. 12th ICAAC Atlantic City, N . J . p. 9. 80. Cooper, D. J., Waitz, J. A., Counelis, M., and Weinstein, J. (1972). Belgian Patent 768, 796. 81. Weisblum, B., and Davies, J. (1968). Bucteriol. Rev. 32, 493. 82. Benveniste, R., and Davies, J. (1973). Antimicrob. Ag. Chemother. 4, 402. 83. Davies, J., Anderson, P., and Davis, B. D. (1965). Science 149, 1096. 84. Tanaka, N., Yoshida, Y., Sashikata, K., Yamaguchi, H., and Umezawa, H. (1966). J. Antibiot. 19, 65. 85. Masukawa, H., Tanaka, N., and Umezawa, H. (1968). 3. Antibiot. 21, 517. 86. Masukawa, H. (1969). J. Antibiot. 22, 612. 87. Tanaka, N. (1970). 3. Antibiot. 23, 469. 88. Davies, J. (personal communication). 89. Kondo, S., Okanishi, M., Utahara, R., Maeda, K., and Umezawa, H. ( 1968). 3. Antibiot. 21, 22. 90. Doi, O., Ogura, M., Tanaka, N., and Umezawa, H. (1968a). Appl. Microbiol. IS, 1276.

302

KENNETH

E. PRICE, JOHN

c.

GODFREY, AND HIROSHI KAWAGUCHI

91. Doi, O., Miyamoto, M., Tanaka, N., and Umezawa, H. ( 196813). Appl. Microbiol. 16, 1282. 92. Price, K. E., Pursiano, T. A., DeFuria, M. D., and Wright, G. E. (1974). Antimicrob. Ag. Chemother. 5, 143. 93. Kondo, S., Yamamoto, H., Naganawa, H., and Umezawa, H. (1972). J. Antibiot. 25, 483. 94. Umezawa, H., Doi, O., Ogura, M., Kondo, S., and Tanaka, N. (1968). I. Antibiot. 21, 154. 95. Umezawa, H., Yamamoto, H., Yagisawa, M., Kondo, S., and Takeuchi, T., and Chabbert, Y. A. ( 1973). J . Antibiot. 26, 407. 96. Brzezinska, M., and Davies, J. (1972). Abstr. 12th ICAAC, Atluntic City, N.J. p. 84. 97. Yagisawa, M., Yamamoto, H., Naganawa, H., Kondo, S., Takeuchi, T., and Umezawa, H. (1972). J . Antibiot. 25, 748. 98. Brzezinska, M., and Davies, J. ( 1973). Antimicrob. Ag. Chemother. 3, 266. 99. Umezawa, H. (personal communication). 100. Martin, C. M., Ikari, N. S., Zimmerman, J., and Waitz, J. A. (1971). J. Infec. Dis. 124, Suppl., S24. 101. Benveniste, R., and Davies, J. (1971). FEBS Lett. 14,293. 102. Naganawa, H., Yagisawa, M., Kondo, S., Takeuchi, T., and Umezawa, H. (1971). 1. Antibiot. 24, 913. 103. Witchitz, J. L., and Chabbert, Y. A. (1971). Ann. Inst. Pusteur, Paris 121, 733. 104. Yagisawa, M., Naganawa, H., Kondo, S., Takeuchi, T., and Umezawa, H. (1972). J. Antibiot. 25, 492. 105. Okanishi, M., Kondo, S., Suzuki, Y., Okamoto, S., and Umezawa, H. (1967). J. Antibiot., Ser. A 20, 132. 106. Umezawa, H., Okanishi, M., Utahara, R., Maeda, K., and Kondo, S. (1967). J. Antibiot., Ser A 20, 136. 107. Benveniste, R., and Davies, J. ( 1971). Biochemistry 10, 1787. 108. Yamamoto, H., Yagasawa, M., Naganawa, H., Kondo, S., Takeuchi, T., and Umezawa, H. (1972). J. Antibiot. 25, 746. 109. Brzezinska, M., Benveniste, R., Davies, J., Daniels, P. J. L., and Weinstein, J. (1972). Biochemistry 11, 761. 110. Witchitz, J. L. ( 1972). J. Antibiot. 25, 622. 111. Umezawa, H., Yagasawa, M., Matsuhashi, Y., Naganawa, H., Tamamoto, H., Kondo, S., Takeuchi, T., and Chabbert, Y. A. (1973). I. Antibiot. 26, 612. 112. LeGoffic, F., and Moreau, N. (1973). F E B S Lett. 29,289. 113. Benveniste, R., and Davies, 1. (1973). Proc. Nut. Acud. Sci. US.70, 2276. 113a. Chevereau, M., Daniels, P,-J. L., Davies, J., and LeGoffic, F. ( 1974). Biochem. 13. 598. 114. Daniels, P. ( personal communication). 115. Haas, M. J,, and Davies, J. (1973).Antimicrob. Ag. Chemother. 4, 497. 116. Wilfert, J. N., Barrett, F. F., Ewing, W. H., Finland, M., and Kass, E. H. (1970). Appl. Microbiol. 19, 345. 117. Oberhofer, T. R., and Hajkowski, R. (1970). Amer. J. Clin. Puthol. 54, 726. 118. Adler, J. L., Burke, J. P., Martin, D. F., and Finland, M. (1971). Ann. Intern. Med. 75, 517. 119. Curreri, P. W., Bruck, H. M., Lindberg, R. B., Mason, A. D., and Pruitt, B. A. (1973). Ann. Surg. 177, 133. 120. Snelling, C. F. T., Ronald, A. R., Cates, C. Y., and Forsythe, W. C. (1971). J. Infec. Dis. 124, Suppl., S26.

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

303

121. Snelling, C. F. T., Ronald, A. R., Kernahan, D. A., Waters, W. R., and Vistnes, L. M. ( 1970). Antimicrob. Ag. Chemother. p. 380. 122. Eisenach, K. D., Reber, R. M., Eitzman, D. V., and Baer, H. (1972). Pediatrics 50, 395. 123. Overturf, G., Paik, G., and Wilkins, J. (1973). Abstr. 13th ICAAC, Washington, D.C. No. 182. 124. Kluge, R., Standiford, H., Tatem, B., Young, V., Greene, W., Schimpff, S., Calia, F., and Hornick, R. (1973). Abstr. 13th ICAAC, Washington, D.C. No. 189. 125. Schulman, J. A., Terry, P. M., and Hough, C. E. (1971). J. Infec. Dis. 124, Suppl., S18. 126. Waitz, J. A,, Moss, E. L., Jr., and Weinstein, M. J. (1971). J. Infec. Dis. 124, Suppl., S18. 127. Brummett, R. E., Himes, D., Saine, B., and Vernon, J. (1972). Arch. Otolaryngol. 96, 505. 128. Waitz, J. A. (personal communication). 129. Hawkins, J. E., Jr., Johnsson, L. G., and Aran, J. M. (1969). J. Infec. Dis. 119, 417. 130. Erlanson, P., and Lundgren, A. (1964). Acta Med. Scand. 176, 147. 131. Welles, J. S.., Emmerson, J. L., Gibson, W. R., Nickander, R., Owen, N. V., and Anderson, R. C. (1973). Toricol. Appl. Pharmacol. 25, 398. 131a. Bristol Laboratories (unpublished data). 132. “Physicians’ Desk Reference.” (1973). 27th ed. Med. Econ. Co., Oradell, New Jersey. 133. “AMA Drug Evaluations.” (1973). 2nd ed. Pub. Sciences Group, Inc. Acton, Massachusetts. 134. Rosenblatt, J. E., and Cortez, L. M. (1973). Abstr. 13th ICAAC, Washington, D.C. No. 127. 135. Regamey, C., Gordon, R. C., and Kirby, W. M. M. (1973). Clin. P h a m c o l . Ther. 14, 396. 136. Christensen, H. E., ed. (1972). “The Toxic Substances List.” U.S. Dept. of Health, Education and Welfare, Washington, D.C. 136a. Waitz, J. A,, and Weinstein, M. (1970). Med. J. Aust., Suppl. p. 19. 137. Umezawa, S., Tsuchiya, T. and Jikihara, T. (1971). J. Antibiot. 24, 711. 138. Murase, M., Ito, T., Fukatsu, S., and Umezawa, H. (1970). “Progress in Antimicrobial and Anticancer Chemotherapy,” Vol. 11, p. 1098. Univ. of Tokyo Press, Tokyo. 139. Umezawa, S., Miyazawa, T., and Tsuchiya, T. (1972). J. Antibiot. 25, 530. 140. Heifetz, C. (personal communication). 141. Umezawa, H. (1968). Asian Med. J. 11, 291. 142. Koch, K. F., and Rhoades, J. A. (1971). Antimicrob. Ag. Chemother. p. 309. 143. Oda, T., Mori, T., and Kyotani, Y. (1971). J. Antibiot. 24, 503. 144. Kobayashi, F., Yamaguchi, M., and Mitsuhashi, S. (1972). Antimicrob. Ag. Chemother. 1, 17. 145. Umezawa, S. ( 1972). Personal communication. 146. Umezawa, H., Yagisawa, M., Yamamoto, H., Kondo, S., Hamada, M., Takeuchi, T., and Chabbert, Y. A. (1973). Jap. Antibiot. Res. Ass., 187th Meet. Paper No. 3. 147. Weinstein, M. J., Wagman, G. H., Oden, E. M., and Marquez, J. A. (1967). J. Bacteriol. 94, 789.

304

KENNETH E. PRICE, JOHN

c.

GODFREY,

AND HIROSHI KAWAGUCHI

148. Cooper, D. J., Jaret, R. S., and Reimann, H. (1971). Chem. Commun. p. 285. 149. Shier, W. T., Rinehart, K. L., Jr,, and Gottlieb, D. (1970). J. Antibiot. 23,

51. 150. Tsukiura, H., Fujisawa, K., Konishi, M., Saito, K., Numata, K., Ishikawa, H., Miyaki, T., Tomita, K., and Kawaguchi, H. (1973). J. Antibiot. 26, 351. 151. Sugawara, S., Inaba, S., Madate, M., Saeki, H., Ohashi, Y., Shimada, Y., and Oki, E. (1973). lap. Antibiot. Res. Ass. 187th Meet. Paper No. 2. 151a. Woo, P. W. K. (1973). U.S. Patent 3,743,634. 152. Umezawa, S., Jikihara, T., Tsuchiya, T., and Umezawa, H. ( 1972). J. Antibiot. 25, 322. 153. Hanessian, S., Butterworth, R. F., and Nakagawa, T. (1973). Carbohyd. Res. 26, 261. 154. Umezawa, S., Ikeda, D., Tsuchiya, T., and Umezawa, H. (1973). J. Antibiot. 26, 304. 155. Endo, T., and Perlman, D. (1972). J. Antibiot. 25, 681. 156. Stark, W. M., Hoehn, M. M., and Knox, N. G. (1968). Antimicrob. Ag. Chemother. p. 314. 157. Stark, W. M., Knox, N. G., and Wilgus, R. M. (1970). FoZia Microbiol. (Prague) 16, 205. 158. O’Connor, S., and Lam, L. K. T. (1973). Amer. Chem. SOC., Diu. Med. Chem., Abst. Symp. Advan. Aminogly. Chem. 159. Wick, W. E., and Welles, J. S. (1968). Antimicrob. Ag. Chemother. p. 341. 160. Mann, R. L., and Bromer, W. W. (1958). J. Amer. Chem. SOC. 80,2714. 160a. McGuire, J. M., and Mann, R. L. (1962). U.S. Patent 3,018,220. 161. Neuss, N., Koch, K. F., Molloy, B. B., Day, W., Huckstep, L. L., Dorman, D. E., and Roberts, J. D. ( 1970). Helv. Chim. Acta 53, 2314. 162. Shoji, J., and Nakagawa, Y. (1970). J . Antibiot. 23, 569. 163. Kondo, S., Sezaki, M., Koike, M., Shimura, M., Akita, E., Satoh, K., and Hara, T. (1965). J. Antibiot., Ser. A 18, 38. 164. Inouye, S., Shomura, T., Watanabe, H., Totsugawa, K., and Niida, T. (1973). J. Antibiot. 26, 374. 165. Umezawa, H. (1967). “Index of Antibiotics from Actinomycetes,” p. 250. Univ. Park Press, State College, Pennsylvania. 166. Lemieux, R. U. (personal communication). 167. Kugelman, M., Mallams, A. K., and Vernay, H. F. (1973). J. Antibiot. 26, 394. 168. Daniels, P. (personal communication). 169. Shomuri, T., Ezaki, N., Tsuruoka, T., Niwa, T., Akita, E., and Niida, T. (1970). J. Antibiot. 23, 155. 169a. Meiji Seika Kaisha, Ltd. (1973). British Patent 1,303,974. 170. Akita, E., Tsuruoka, T., Ezaki, N., and Niida, T. (1970). J. Antibiot. 23, 173. 171. Haskell, T. H., Rodebaugh, R., Plessas, N., Watson, D., and Westland, R. D. 1973). Carbohyd. Res. 28, 263. 172. Heifetz, C. L., Fisher, M. W., Chodubski, J. A., and DeCarlo, M. 0. (1972). Antimicrob. Ag. Chemother. 2, 89. 173. Tsukiura, H., Saito, K., Kobaru, S., Konishi, M., and Kawaguchi, H. (1973). J. Antibiot. 26, 386. 174. Woo, P. W. K., Dion, H. W., Coffey, G. L., Fusari, S. A., and Senos, G. D. (1970). US. Patent 3,541,078.

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

305

175. Howells, J. D., Anderson, L. E., Coffey, G. L., Senos, G. D., Underhill, M. A., Vogler, D. L., and Ehrlich, J. (1972). Antimicrob. Ag. Chemother. 2, 79. 176. Dion, H. W., Woo, P. W. K., Willmer, N. E., Kern, D. L., Onaga, J., and Fusari, S. A. (1972). Antimicrob. Ag. Chemother. 2, 84. 177. Woo, P. W. K., Dion, H. W., and Bartz, Q. R. (1971). Tetrahedron Lett. p. 2625. 178. Ikeda, D., Tsuchiya, T., Umezawa, S., and Umezawa, H. (1972). J . Antibiot. 25, 741. 179. Kawaguchi, H. (unpublished data). 180. Price, K. E. (unpublished data). 181. Kawaguchi, H., Naito, T., Nakagawa, S., and Fujisawa, K. (1972). J . Antibiot. 25, 695. 182. DeFuria, M. D., and Claridge, C. A. (1973). Abstr. 73rd Annu. Meet. ASM, Miami Beach, Florida p. 13. 183. Kojima, M., and Satoh, A. (1973). J. Antibiot. 26, 784. 184. Kojima, M., Inouye, S., and Niida, T. (1973). J . Antibiot. 26, 246. 184a. Onioto, S., Inouye, S., Kojima, M., and Niida, T. (1973). 1. Antibiot. 26, 717. 185. Umezawa, S., Tsuchiya, T., Ikeda, D., and Umezawa, H. (1972). J . Antibiot. 25, 613. 185a. Ikeda, D., Tsuchi-, T., Umezawa, S., and Umezawa, H. (1973). J . Antibiot. 26, 799. 186. Ikeda, D., Tsuchiya, T., Umezawa, S., Umezawa, H., and Hamada, M. (1973). J . Antibiot. 26, 307. 187. Haskell, T. H. (personal communication). 188. Culbertson, T. P., Watson, D. R., and Haskell, T. H. (1973). J. Antibiot. 26, 790. 189. Umezawa, S., Watanabe, I., Tsuchiya, T., Umezawa, H., and Hamada, M. 1972). J. Antibiot. 25, 617. 190. Horii, S., Hitomi, H., and Miyake, A. (1966). US. Patent 3,277,078. 191. Mori, T., Kyotani, Y., Watanabe, I., and Oda, T. (1972). J. Antibiot. 25, 317. 192. Mori, T., Ichiyangi, T., Kondo, H., Tokunaga, K., Oda, T., and Munakata, K. (1971). J. Antibiot. 24, 339. 193. Kobayashi, F., Nagoya, T., Yoshimura, Y., Kaneko, K., and Ogata, S. ( 1972). J. Antibiot. 25, 128. 194. Oda, T., Mori, T., Ito, H., and Kunieda, T. (1971). J. Antibiot. 24, 333. 195. Oda, T., Mori, T., Kyotani, Y., and Nakayama, M. ( 1971). J. Antibiot. 24, 511. 196. Kobayashi, F., Nagoya, T., Yoshimura, Y., Kaneko, K., and Ogata, S. (1972). J. Antibiot. 25, 128. 197. Yamaguchi, M., Kobayashi, F., and Mitsuhashi, S. (1972). Antimicrob. Ag. Chemother. 1 , 139. 198. Mori, T., Kyotani, Y., Watanabe, I., and Oda, T. (1972). J. Antibiot. 25, 149. 199. Shier, W. T., Rinehart, K. L., Jr., and Gottlieb, D. (1969). Biochemistry 63, 198. 200. Shier, W. T., Rinehart, K. L., Jr., and Gottlieb, D. (1972). US. Patent 3,669,838. 201. Davies, J. ( 1970). Biochim. Biophys. Acta 222, 674.

306

KENNETH E. PRICE, JOHN C. GODFREY, AND HIROSHI KAWAGUCHI

202. Watanabe, I., Tsuchiya, T., Umezawa, S . , and Umezawa, H. ( 1973). J. Antibiot. 26, 310. 203. Umezawa, S., Tsutiya, T., Watanabe, I., and Umezawa, H. (1973). Jap. Antibiot. Res. Ass., 187th Meet. Paper No. 5. 203a. Watanabe, I., Tsuchiya, T., Uniezawa, S . , and Umezawa, H. ( 1973). J. Antibiot. 26, 802. 204. Yamamoto, H., Kondo, S . , Maeda, K., and Umezawa, H. (1972). J . Antibiot. 25, 487. 205. Yamamoto, H., Kondo, S., Maeda, K., and Umezawa, H. (1972). J. Antibiot. 25, 485. 206. Shier, W. T., and Rinehart, K. L., Jr. ( 1973). J. Antibiot. 26, 547. 207. Higgens, C. E., and Kastner, R. E. (1968). Antimicrob. Ag. Chemother. p. 324. 207a. Koch, K. F., Davis, F. A., and Rhoades, J. A. (1973). J. Antibiot. 26, 745. 208. Takagi, Y., Miyake, T., Tsuchiya, T., Umezawa, S., and Umezawa, H. ( 1973). J. Antibiot. 26, 403. 209. Kawaguchi, H., Naito, T., Nakagawa, S., and Fujisawa, K. (1972). J. Antibiot. 25, 695. 210. Naito, T., Nakagawa, S., Abe, Y., Toda, S . , Fujisawa, K., Miyaka, T., Koshiyama, H., Ohkuma, H., and Kawaguchi, H. (1973). J. Antibiot. 26, 297. 211. Kondo, S., Iinuma, K., Hamada, M., Maeda, K., and Umezawa, H. ( 1974 ). J Antibiot. 27, 90. 212. Lemieux, R. U. (personal communication). 213. Kondo, S., Iinuma, K., Yamamoto, H., Ikeda, Y., Maeda, K., and Umezawa, H. ( 1973). J. Antibiot. 26, 705. 214. Umezawa, H., Nishimura, Y., Tsuchiya, T., and Umezawa, S. ( 1972). J. Antibiot. 25, 743. 215. Godfrey, J. (unpublished data). 216. Umezawa, S., Tsuchiya, T., Muto, R., Nishimura, Y., and Umezawa, H. (1971). J. Antibiot. 24, 274. 217. Umezawa, H., Umezawa, S., Tsuchiya, T., and Okazaki, Y. (1971). J. Antibiot. 24, 485. 218. Umezawa, H., Tsuchiya, T., Moto, R., and Umezawa, S. (1972). BUZZ. Chem. SOC. lap. 45, 2842. 219. Inouye, S. (1968). Chem. Pharm. Bull. 16,573. 220. Inouye, S. (1965). Symp. Chem. Natur. Prod. Qth,1965. 221. Kobayashi, T., Tsuchiya, T., Tatsuta, K., and Umezawa, S. (1970). J. Antibiot. 23, 225. 222. Endo, T., and Perlman, D. (1972). J. Antibiot. 25, 751. 223. Lemieux, R. U.,Nagabhushan, T. L., Clemetson, K. S., and Tucker, L. C. N. (1973). Can. J. Chem. 51, 53. 224. Fujii, A., Maeda, K., and Umezawa, H. (1968). J. Antibiot. 21, 340. 225. Daniels, P. J. L., Kugelman, M., Mallams, A. K., Tkach, R. W., Vernay, H. F. Weinstein, J,, and Yehaskel, A. (1971). Chem. Commun. p. 1629. 225a. Weinstein, M. J., Luedemann, G. M., Oden, E. M., and Wagman, G. H. (1966). Antimicrob. Ag. Chemother. p. 816. 226. Schering Corp., Ltd. ( 1970). Belgian Patent 768,796. 227. Weinstein, M. J,, Marquez, J. A., Testa, R. T., Wagman, G. H., Oden, E. M., and Waitz, J. A. (1970). J. Antibiot. 23, 551. 227a. Weinstein, M. J,, Wagman, G. H., Marquez, J. A., Testa, R. T., and Waitz, J. A. (1973). Abstr. 13th ICAAC, Washington, D.C. No. 136.

MODIFICATION OF AMINOGLYCOSIDE ANTIBIOTICS

307

228. Wagman, G. H., Testa, R. T., Marquez, J. A., Waitz, J. A., and Weinstein, M. J. (1973). Abstr. 13th ICAAC, Washington D.C. No. 138. 229. Wagman, G. H., Testa, R. T., and Marquez, J. A. (1970). J. Antibiot. 23, 555. 230. Waitz, J. A., Moss, E. L., Jr., Oden, E. M., and Weinstein, M. J. (1970). J. Antibiot. 23, 559. 231. Daniels, P. J. L., and Yehaskel, A. S. (1973). Abstr. 13th ICAAC, Washington, D.C. No. 135. 232. Testa, R. T., and Tilley, B. (1973). Abstr. 13th ICAAC, Washington, D.C. No. 46. 233. Marquez, J. A,, Murawski, A., and Wagman, G. H. (1973). Abbr. 13th ICAAC, Washington, D.C. No. 47. 234. Daniels, P. J. L., Yehaskel, A. S., and Morton, J. (1973). Abstr. 13th ICAAC, Washington, D.C. No. 137. 235. Merck & Co., Inc. (1971). Netherlands Patent 2,234,804. 236. Daniels, P. J. L., Mallams, A. K., and Wright, J. F. ( 1973). J. Chem. SOC., Chem. Commun. p. 675. 237. Mallams, A. K., Vernay, H. F., Crowe, D. F., Detre, G., Tanabe, M., and Yasuda, D. M. (1973). J. Antibiot. 26, 782. 238. Cooper, D. J., Weinstein, J., and Waitz, J. A. (1971). J . Med. Chem. 14, 1118. 239. Weinstein, M. J., Wagman, G. H., and Taber, R. I. (1966). Antimicrob. Ag. Chemother. p. 227.

This Page Intentionally Left Blank

Recent Developments of Antibiotic Research and Classification of Antibiotics According to Chemical Structure

JANOS B ~ R D Y Research Institute for Pihamceutical Chemistry, Budapest, Hungary

I. Introduction ...................................... 11. Antibiotic Research in the Past Decade ............... A. Shift in the Nature of Antibiotic Research ......... B. Diverse Applications; Increase of Nonclinical Use C. ‘Specialization and Concentration of Research. Revision of Screening Methods .................. D. New Antibiotic Sources ........................ E. Multiphasic Study of Microorganisms that Produce Known Antibiotics ..................... F. Extensive Investigation of Known Antibiotics 111. Systematization of Antibiotics ....................... A. General Problems of Systematization . . . . . . . . . . . . . B. Problems of Classification Examined from Different Points of View ....................... IV. Classification of Antibiotics According to Chemical Structure .............................. A. Basic Principles of Chemical Classification ......... B. Proposed System for the Chemical Classification of Antibiotics V. Conclusions ...................................... A. Some Nomenclatural Problems of Antibiotics B. Epilog and Summary . . . . . . . .. : . ............... References .......................................

..

......

..................... ......

309 310 310 317 321 326 332 335 338 336 337 345 345

359 397 397 401 402

I. Introduction The history of antibiotic research may be divided into three periods. The first period, up to the early 1940’s, is characterized by slow development and accumulation of basic observations as well as by some fundamental discoveries. The therapeutic utilization of penicillin and the beginning of systematic screening of actinomycetes constitute a milestone in the “Golden Era” of antibiotic research. The second period is characterized by fundamental discoveries made as a result of frenzied research during the next 15-20 years. In the 1960’s, however, the situation changed: with basic therapeutic demands fulfilled, the period of rapid dynamic successes and spectacular, easily attainable results came to an end. 309

310

J ~ N O SB ~ D Y

The main characteristics of the latest period, beginning about 10-12 years ago, are the following: 1. Declining development rate in research on microbial antibiotics; emergence of semisynthetic antibiotics 2. Search for new fields of application; increasing importance of nontherapeutic antibiotic applications 3. Specialization and concentration of research; revision of classical screening methods 4. Intensified search for new antibiotic sources 5. Extensive many-sided study of microorganisms producing known antibiotics 6. Extensive investigation of the biochemistry, pharmacology, and chemistry of known antibiotics 7. Emergence of classification and nomenclatural problems The main characteristics of the past decade are demonstrated herein, when possible by means of statistical data. After a short summary of problems of antibiotic classification, a classification system according to chemical structure is proposed. The conclusions drawn are based on the utilization of a punch-card system, reported upon in our earlier papers (Bkrdy, 1961; B6rdy and Magyar, 1968). The statistical evaluations analyze papers published through December 31,1972. II.

Antibiotic Research in the Past Decade

A. SHIFT IN

THE

NATUREOF ANTIBIOTICRESEARCH

1. Decline of Research on Microbial Antibiotics Several authors stressed unanimously ( Conover, 1971; Kurylowicz, 1965) that the intensity of research in the field of new microbial antibiotics declined after 1959-1960. Most antibacterial antibiotics of the greatest importance in chemotherapy had been discovered by 1960 (Table I). Looking at the discovery and introduction of nonantibacterial agents however, the decline is far less pronounced (Fig. l a ) . The causes of the diminishing rate of development may be the following: 1. The most urgent clinical demands were already satisfied, consequently the vacuum in the field of chemotherapy that speeded up research in the 1950s’no longer exists. 2. Because of the higher standards for medical acceptance of new drugs, it is more difficult to get regulatory approval for a new antibiotic today. Very often new agents not possessing significant advantages over

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

PRACTICAL

Year of publication

TABLE I APPLICATION O F MICROBIAL ANTIBIOTICS Antibiotics

Antibacterial

Other therapeutic Agricultural

Pyocyanina Penicillin G Tyrothricin Gramicidin Streptomycin Chloramphenicol* Chlortetracycline Oxytetracycline Neom ycin Penicillin VC Polymyxin B Colistin Erythromycin Oleandomycin Leucomycin Spiramycin Viomycin Cycloserineb Penicillin Oc Tetracycline1 Cephalosporin Ce Gramicidin J (S) Xanthocillind Carbomycinc Albomycina Novobiocin Vancom ycin Kanamycin Demethylchlortetracycline Paromomycin (USA) Aminosidin (Italy) Pristiqamycin (France) Ristocetin Rifamycin Be Polymyxin Md Fusidic acid Lincom ycin Capreom ycin Gentam ycin (Lysostaphin)

Griseofulvin

Bacitracin

Fumagillin

Nisin Antim ycin Cycloheximide

Nystatin Trichomycin Candicidind Lucensomycin Actinomycin C Actinomycin D Sarcomycin Carzinophyllin Azaserinea Puromycin“ Aureothricina

Grisin (SU) Virginiam ycin

Amphotericin Pentam ycind Pimaricin Variotin Hamycin (India) Mitomycin C Chromomycin Mithramycin Thiazolidomycin’ Heliomycind Pyrrolnitrinb Porfiromycin Bleomycin Daunomycin Olivomy cin Spectinomycin Azalomycind Siccanind (Asparaginase) Adriamycin

Mikamycin Hygromycin B Tylosin Atterrimina Blasticidin S Cellocidinb Stendomycin“ Thiostrepton

~~

Through 1945

1946-1950

1951-1955

1956- 1960

1961-1965

1966-1970

1971-

Josamycin Enduracidin Kanendom ycinc Ribostamycin Tobramycin Mydecamycin

~~

No longer available as a commercial product. Produced mainly or exclusively by chemical synthesis. Produced by “biosynthetic method.” Produced in small amount, mainly for topical purposes. * Historically important, not used as an antibacterial drug. f Produced also by “synthetic method.” a

311

Siomycin Moenom ycin Destom ycin Kasugam ycin Polyoxin Phytobacteriomycin

Monensin Validam ycin

Thiopeptin

312

z

l0i

I -1945 L6-50 51-55 56-60 61-65 66-70

71-

period

FIG. 1. Development of microbial antibiotics with practical application in five-year , Antibacterial; 0,other therapeutic; H , agricultural antibiotics. ( a ) periods. . Year of discovery. ( b ) Year of introduction.

existing drugs are not even introduced in therapy, or their marketing is stopped altogether ( e.g., carbomycin, ristocetin) . 3. Development and commercialization require too much time, sometimes even 5-6 years (e.g., tobramycin, spectinomycin). It should be stressed that it seems to be more difficult to introduce a new antibiotic in some countries (United States, United Kingdom), because of their strict requirements, than in others. Very often the performance of an individual antibiotic can be evaluated only after 10-12 years. For instance, gentamicin, discovered in 1963, has only recently been recognized as an antibiotic of major importance. 4. To gain widespread use of new products at acceptable prices, unless they exhibit significant advantages, requires a big material investment because of the relative cheapness of comparable antibiotics already on the market. A more realistic picture of the rate of research on useful antibiotics emerges if one considers not the date of discovery, but that of wider

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

313

clinical utilization (Fig. l b ) . Griseofulvin, for instance, was described in 1939; its practical utilization, however, was established only after 1958. Actinomycins have been known since 1940; they were applied only in the past decade. Colistin was described in 1950, but only after 1962 did it gain wider popularity. No forecast can be made as to which of the antibiotics (or their derivatives) that became known only a couple of years or so ago may become a pharmaceutical drug (e.g., phosphonomycin, bicyclomycin, maridomycin, megalomicin, butirosin, lividomycin, sisomicin, negamycin, cephamycins, tuberactinomycin, polyfungin, levorin, mycoheptin, methylpartricin, distamycin, mycophenolic acid, neocarzinostatin, coumermycin, nigericin, diumycin, axenomycin, aabomycin) , A completely different picture of the rate of development emerges if the number of yearly publications or the number of new antibiotics described is taken into consideration (Fig. 2 ) . The rate has remained virtually unchanged, except for a short break between 1957 and 1963, though in the special case of actinomycete antibiotics it slowed down somewhat around 1960.

19L5

1950

1955 1960 Year

1965

1970

FIG. 2. Number of new antibiotics published year by year, according to original publication.

314

J ~ N O SB ~ D Y

The true situation is better characterized by the following statistics. About 5%of the antibiotics described up to 1960 have become established. This ratio diminished to 2.6%of those described between 1961 and 1965, and to 1%of those described between 1966 and 1971. These data indicate unequivocally the declining efficiency of antibiotic research. What is behind this decline? Lack of enthusiasm in this field of science? or exhaustion of the natural reservoir of antibiotic-producing microorganisms? Either is most improbable. The decline of. effectiveness in this field is a natural phenomenon. Most of the antibiotics exhibiting high in vitro activity and produced in sufficient quantity by microorganisms that grow easily under usual conditions have surely been isolated already. The rediscoder of substances already described becomes more and more I Y frequent. New antibiotics recognized recently belong to well known antibiotic groups and usually do not exhibit superior microbiological properties; consequently quantitative development does not necessarily mean qualitative improvement as well, Despite this declining trend, however, the total growth of antibiotic armamentarium shows a steady increase. This is due mainly to the success of new semisynthetic antibiotics.

2. Success of Semisynthetic Antibiotics The “Golden Era” of antibiotic research has yielded examples of the production of antibiotics of chemically or biochemically modified structure, e.g., dihydrostreptomycin and some penicillin and tetracycline derivatives. But the decisive change was made after 1959 with the introduction of semisynthetic penicillin, then later of cephalosporin and rifamycin, and recently of modified aminoglycoside antibiotics (Table 11; Fig. 3 ) . About 60 different antibiotic derivatives have gained general acceptance in medicine; these include true semisynthetic antibiotics ( e.g., 6-aminopenicillanic acid and 7-aminocephalosporanic acid derivatives, clinda-

.,

Z

19L6-50 51-55 56-60 61-65 66-70 Period

71-

FIG.3. Development of antibiotic derivatives used in medicine in five-year periods. Semisynthetic antibiotics; 17, simple derivatives.

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

TABLE I1 ANTIBIOTIC DERIVATIVES USED Year of publication

Semisynthetic 8-lactam antibiotics

Through 1950 1951-1955 1 9 5 61960

Pheneticillin Propicillin Methicillin

1961- 1965

Ampicillin Oxacillin Cloxacillin Nafcillin Ancillin Phenbenicillin Quinacillin Cephalothin Cephaloridin Carbenicillin Hetacillin Dicloxacillin Rixapen Penamec ycline Cephaeolin Cephalexin Cephaloglicin Amoxycillin Flucloxacillin Sulbenicillin Pivampicillin Cephradine Cephapirin Ccphacetrile Tricarcillin? Indanylcarbenicillin ? Cephamedin?

1966-1970

1971-

IN

Other semisynthetic antibiotics Dihydrostreptomycin (Tetracycline) Thiamphenicol Dihydrodeoxystreptomycin Rollitetracycline

Lymecycline Methac ycline Rifamycin SV Rifamide

315

CHEMOTHERAPY Simple antibiotic derivatives Procaine penicillin Beneathine penicillin Chlorprocaine penicillin Chloramphenicol palmitate Colistin methanesulfonate Sulfomyxin Erythromycin estolate Erythromycin stearate Erythromycin succinate Chloramphenicol succinate Hydrobamine penicillin Triacet yloleandom ycin Erythromycin lactobionate Erythromycin glucoheptonate Chloramphenicol cinnamate

Minocycline Doxycycline Morfocycline Rifampicin Clindamycin Rifaein

Acetylspiramycin

3,4-Dideoxykanamycin B Clindamycin palmitate? BB-K-g? = amikacin

Josamycin propionate, Maridomycin propionate?

mycin, rif ampicin, doxycycline ) and other simple antibiotic derivatives formed by esterification or salt formation ( e.g., triacetyloleandomycin, procaine penicillin, erythromycin esters, chloramphenicol esters ) . Conse-

316

J ~ N O SB ~ D Y

quently the importance of antibiotic derivatives and their advantages are by no means of equal value. Superiority may consist in the following characteristics: 1. Higher activity at unchanged or lower toxicity (e.g., cephalothin, cephaloridine, clindamycin, rifamycin derivatives) 2. Broadening of the range of antimicrobial activity (e.g., ampicillin, carbenicillin ) and/ or microbial activity against resistant organisms ( e.g., 3,4-dideoxykanamycin,minocycline ) 3. Improved pharmacokinetic characteristics ( e.g., doxycycline, rifampicin, cephalexin ) 4. Lower toxicity and side effects (e.g., dihydrostreptomycin, clindamycin ) . 5. Enhanced solubility (e.g., rollitetracycline, chloramphenicol esters, N-acylnolyene derivatives) 6. Improved stability ( e.g., tetracycline, penicillin V ) 7. Other advantages: elimination of bitter taste, local irritation, etc. ( e.g., chloramphenicol derivatives, macrolide esters ) . The amount of research carried out in the field of semisynthetic antibiotics is demonstrated in Table 111. It is apparent that only a small part of all derivatives synthesized ( 0.1-0.8%) was introduced in practice. Modification of the chemical structure of antibiotics generally leads, as in the mutagenic treatment of antibiotic-producing strains, to diminished, or eventually lost, antimicrobial activity; consequently, drugs of superior quality may be expected only in negligible proportion. Research in the field of new penicillin and cephalosporin derivatives continues unabated and may result in the synthesis of further new pharTABLE 111 TOTAL NUMBERO F ANTIBIOTIC I ~ R I V A T I VPREPARED ES COMPARED WITH NUMBER USED IN MEDICINE Antibiotic type Penicillin Cephalosporin Tetracycline Rifam ycin Kanam ycin Chloramphenicol Lincom ycin Streptomycin Kasugam ycin Coumermycin

Approximate number of derivatives prepared

Clinically used

20,000

23 8 6

4,000 2,500 1,000 500

500 450

300 300 230

4

2 1 1 2 -

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

317

maceuticals of improved properties. Of the derivatives of rifamycin and tetracycline, rifampicin and doxycycline appear to represent the peak of achievement. Recently, there has been interest in the modification of aminoglycoside antibiotics by chemical ( 3‘,4‘-dideoxykanamycins, BBK-8) (Yagisawa, 1973; Kawaguchi et al., 1972) as well as by biosynthetic means by utilizing different mutants ( hybrimycins, kanendomycin ) ( Shier et al., 1969; Meiji Seika Kaisha Ltd., 1968). At present the prospect of achieving results in the field of semisynthetic antibiotics seems more promising than in that of natural ones. Further elucidation of structure-activity relationships would eventually enable the synthesis of antibiotic analogs of a structure designed in advance. Synthetic modifications, however, are unable to change the fundamental quality of microbial activity. It now seems most improbable, for instance, that antifungal or antitumor penicillin derivatives could ever be synthesized, as was formerly expected by certain scientists. Thus research in the field of microbial antibiotics promises the discovery of fundamentally new types of agents exhibiting new types of microbial activity as well as new modes of action. In the past decade new natural antibiotics and antibiotic derivatives were introduced in almost equal numbers. It is most probable that new active drugs will be found in both categories. The establishment of semisynthetic antibiotics doubtless played a decisive role in the development of chemotherapy during the past 10-12 years. Except for the first 23 years, however, when they exerted a discouraging effect on research bearing on microbial antibiotics, they did not alter the rate of discovery in this field (see Fig. 2 ) . In the followig sections, only the aspects of research in the field of microbial antibiotics, obtained by means of fermentation, are discussed.

B. DIVERSEAPPLICATIONS; INCREASE OF NONCLINICAL USE 1. Agricultural Antibiotics The success of antibiotics in chemotherapy was soon followed by the discovery of their role in food preservation, in animal nutrition, and as plant-protecting agents. Until the 1960’s, antibiotics established in human chemotherapy were utilized for these purposes (e.g., penicillin, tetracyclines, streptomycin). Serious problems that arose from this practice include the development of resistant microorganisms. At present, the United Kingdom limits legally the agricultural utilization of therapeutic antibiotics (Swann, 1969), and in Western Europe and Japan similar limitations are prevalent. As a result, today the series of antibiotics used therapeutically differs from “feed-grade” and “pesticide” groups. The therapeutic utilization of bacitracin and tylosin, used earlier in human

318

JLNOS

B ~ D Y

therapy as well, was stopped altogether. Some “feed-grade’’ antibiotics ( for which quality requirements are less stringent) including bacitracin, tylosin, flavomycin ( bambermycin ) as well as the anthelminthics hygromycin and destomycin, are manufactured yearly on a 100-1000 ton scale, virginiamycin, mikamycin, thiostrepton, and other antibiotics are produced on a smaller scale. A series of antibiotics active against various plant pathogens are manufactured on a large scale, mainly in Japan (blasticidin S, cellocidin, kasugamycin, polyoxin, validamycin ) and also in other countries ( grisin, phytobacteriomycin, cycloheximide, antimycin ) . Outstanding results were achieved with blasticidin S-which is active against Piricularia oryzae, a rice parasite, and is manufactured in Japan on a scale of at least 10,000 tons yearly (Perlman, 1968)-as well as with cellocidin, produced synthetically. It is mostly due to these achievements that Japan is no longer compelled to import rice, and can even export a considerable amount of it today. Owing to several strict measures in the preserving industry, the role of therapuetic antibiotics is in a steady decline. In this field it is nisin, applied for the time being only in England on a large scale, which has a promising future. The enhanced importance of nontherapeutic antibiotics may be deduced also from manufacturing data. The distribution of world production of antibiotics at the beginning of the 1970’s is summarized in Table IV. 2. New Horizons of Chemotherapy Beyond the use of antibiotics and other metabolite of microorganisms in antibacterial and antifungal chemotherapy, the search for their utilizaTABLE I V ANNUALWORLDPRODUCTION (EARLY1970’s) OF ANTIBIOTICS VARIOUSAPPLICATIONS

OF

Therapeutic antibiotics Antibacterial antibiotics, Antifungal antibiotics, Antiprotozoal antibiotics] Other (antitumor) antibiotics]

10,000-12,000 tons

30-40 tons

5-10 tons

Agricultural antibiotics Plant-protecting antibiotics,b “Feed additive” antibiotics,c Antibiotics as preservatives,

20,000-25,000 tons

5-6,000 tons About 10 tons

kilogram amounts

* Together with semisynthetic antibiotics.

Together with synthetic cellocidin. Together with therapeutic feed-additive antibiotics and anthelminthic antibiotics.

RESEARCH AND CLASSIFlCATION OF ANTIBIOTICS

319

tion in new fields of chemotherapy and diverse other terrains is constantly broadening. Some of these areas have long been in the center of interest; other areas came to the forefront only recently. a. Antitumor screening programs, started relatively early, at the beginning of the 1950’s, brought many partial results. The rate of research may best be characterized by the fact that up to now the antitumor (cytostatic and cytotoxic) activity of more than 600 new antibiotics has been described. Starting in 1955, within the CCNSC program 120,000 culture broths were screened in 10 years, as a result 30 new antitumor antibiotics were isolated for preclinical trials ( Schepartz, 1965). As yet only mithramycin has been introduced in therapy, and eventually streptonigrin and antibiotic NSC-A-649 (olivomycin) may be judged to be promising therapeutic agents. Antitumor screening programs in the USSR (Larionov, 1970; Gause, 1972) and Japan (Umezawa, 1972a) were executed on a similar scale ( e.g., sarcomycin, carzinophilin, mitomycin C, chromomycin, bleomycin, olivomycin, bruneomycin, sybiromycin) . Although at present 12 antitumor antibiotics are used in cancer therapy-similarly to synthetic antitumor agents-not one has become an overwhelming success. Even though these substances are utilized advantageously in some special fieIds (e.g., actinomycin D in Wilms’s tumor, bleomycin in skin cancer, mithramycin in testicular tumor ) , the chemotherapy of cancer may succeed only if its origin and nature are elucidated in detail. Even statistical data demonstrate the abating interest in research on new antitumor antibiotics since 1960 ( Fig. 4). On the other hand, the reinvestigation of previously described and discarded substances may increase the number of antitumor antibiotics (e.g., mycophenolic acid). b. The antiviral and antiphage screening program has not as yet resulted in any new drug active against true viruses, which may be applied 100 %

0 %

80 %

20 %

t

60 %

-19L5 6 - 5 0

51-55 56-60 61-65 66-70

10 %

71-

FIG. 4. Percentage distribution of antibiotics according to type of activity. H, Antimycoplasma activity; m, anthelminthic activity; m, anti-Xunthomonas activity.

320

JdNOS BdRDY

in therapy (though the introduction of distamycin may be expected soon, and there are some results with rifampicin) although the efforts invested in antiviral research are comparable to those invested in antitumor antibiotic research. Up to now more than 250 antiviral antibiotics have been described. According to statistical data (Fig. 4), the volume of research did not change appreciably in the past 10-15 years, but most likely research efforts will increase. c. The number of newly described antimycoplasma agents again shows a rapid growth and looks promising. The search for new antibiotics exhibiting antispirochetal activity, however, has brought no results since the introduction of spectinomycin. d. Among antiprotozoal antibiotics, coccidiostatic monensin, used now in veterinary practice, as well as the discovery of some new antiprotozoal agents not as yet utilized, constitute a new aspect of development. e. The search for anthelminthic agents also shows a steady increase: hygromycin and destomycin are well established drugs, and the recently discovered axenomycin (Della Bruna, 1973) may mark a new step forward in this area. 3. Other Physiologically Active Substances of Microbial Origin

Although they cannot be placed strictly among antibiotics, nontherapeutic microbial metabolites have recently considerably increased in number. Primarily, intense Japanese efforts in the area of enzyme inhibitors deserve to be mentioned. Many protease inhibitors (e.g., leupeptin, antipain, chimostatin, pepstatin) were isolated from different Streptomyces species. Some of them may be applied even in ulcer therapy (Umezawa, 1972b). Another line of interest seems to be the search for tyrosine and dopamin-p-hydroxylase inhibitors among various microbial metabolites. These substances ( e.g., fusaric acid, oudenon, oosponol, dopastin, aquayamycin ) exhibit considerable hypotensive activity, which may be exploited therapeuticially ( Umezawa, 1 9 7 2 ~ )Other . metabolites of Streptomycetales ( pimprinin ) inhibit monoamineoxidase ( Takeuchi et al., 1973). Presently, the number of enzyme inhibitors isolated from microorganisms is about 50. The discovery of p-lactamase ( penicillinase) inhibitors of Streptomyces origin looks highly promising (Hata et al., 1972; Umezawa et al., 1973). These inhibitors render penicillin-resistant organisms sensitive against penicillin G. In the past couple of years, the search for insecticides (e.g., destruxin, aspochracin, piericidin, versimide, tetranactin ) and growth regulators of plants ( e.g., gibberellin, helminthosporal, fragin) of microbial origin (Tamura, 1972) was also enhanced. That microorganisms may produce a series of agents of manifold physiological activity (Perlman and Peruzzotti, 1970; Floss, 1972) is proved

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

321

by the isolation of microbial metabolites, diverse, biologically active agents, such as alkaloids ( e.g., nigrifactin, physostigmine, muscarine, slaframine) , microbiologically inactive growth promoters ( e.g., zeranol) , cholesterol-lowering agents ( e.g., lentinacin ) , hallucinogens ( e.g., psylocibin ) , phytotoxins, mycotoxins, chelate-forming drugs ( e.g., desferrioxamine B ) . 4. Antibiotics as Research Tools

Antibiotics played an important role in the development of basic sciences, primarily in biochemistry and microbiology and to a minor extent also in organic chemistry and in pharmacology, and had a decisive influence on molecular biology. It was the antibiotics that enabled for the first time a deeper insight into the mechanism of formation and metabolism of such important biopolymers as proteins and nucleic acids. Antibiotics provide some of the best opportunities to understand drug-receptor interactions at molecular levels. The role of antibiotics in the preparation of culture media suitable for the selective isolation of different microorganisms has long been known. As specific inhibitors the antibiotics are increasingly utilized in biochemical research. Primarily different cytotoxic agents ( e.g., cytochalasins, actinomycins) , specific inhibitors of protein-, RNA-, and DNA-synthesis ( e.g., chloramphenicol, guromycin ), electron-transport inhibitors ( e.g., oligomycin, antimycin ) , substances affecting cation transport through the cell membrane (e.g., valinomycin, dianemycin, nonactin, alamethicin ) may gain growing importance in research executed at molecular biological levels.

C. SPECIALIZATION AND CONCENTRATION OF RESEARCH, REVISION OF SCREENING METHODS

1. Research in Various Countries. Specialization and Concentration In the past, individual research laboratories generally concentrated their efforts to the screening of microbial agents exhibiting specific types of activity and to the screening of well defined groups of microorganisms. Earlier successes as well as health conditions and specific problems of a particular country may define the profile of research there. In Japan many laboratories specialized in research on plant-protecting agents ( anti-Piricularia and anti-Xanthomonas substances ) and on antitumor antibiotics. In the United States, the following special research lines may be observed: antimetabolites ( Hoffmann-La Roche, Upjohn), coccidiostatic polyether antibiotics ( Eli Lilly ), antibiotics of Micromonospora origin ( Schering ) , semisynthetic cephalosporins ( Eli Lilly ), penicillins ( Bristol) .

322

J ~ N O SB ~ R D Y

In England, research is primarily on semisynthetic p-lactam antibiotics, insecticides, and other fungal metabolites ( Beecham). In Switzerland and Canada, the search for antibiotics of fungal origin is favored. In the USSR, research is virtually limited to antibiotics originating from Actinomycetales. In the Research Institute for Antibiotics in Leningrad, antifungal polyene antibiotics constitute the main line of research, and in the Academic Research Institute for New Antibiotics in Moscow, antitumor, mainly anthracycline-type antibiotics are stressed. In India, anthelminthic, antiprotozoal, and antifungal polyene antibiotics are investigated; in Czechoslovakia, antiprotozoals of fungal origin; and in Brasil, primarily agents of plant origin. Naturally big firms of large research capacity do not limit their field of interest to a single area. Upjohn, Eli Lilly, Institute of Microbial Chemistry (Tokyo), Takeda, etc. execute many-sided research projects. The steady rise of research expenditure led necessarily to the concentration of research within an individual country. The discovery of a new antibiotic, which is expensive in itself, is followed by its commercialization, which also requires steadily rising investment costs. This can be borne only by firms of large economic potential. According to statistics in the United States, only the following firms continue their original antibiotic screening program with unabated intensity: Upjohn, Eli Lilly, Lederle, Schering, Bristol Laboratories, Hoffmann-La Roche, and Squibb Institute for Medical Research. Pfizer has almost abandoned it, and Parke-Davis, Abbott Laboratories, and Merck Sharp & Dohme have considerably lowered the rate of their new antibiotic research efforts. In the past decade, the withdrawal of the university faculties, which formerly had an important part in this field also became apparent. In Japan too, research is gaining ground in the big firms (primarily Meiji, Takeda, and Fujisawa) . The concentration of research is not extensive here. About 20 laboratories execute systematic screening programs, among them many financially subsidized university institutes and special research laboratories. In the USSR wide, but highly specialized, antibiotic research is performed in two industrial institutes and one academic institute. Italian, French, West German, English, and Swiss antibiotic research is also concentrated in 2 or 3 industrial research laboratories. This concentration can be observed also in the distribution of antibiotics discovered according to individual countries : research in the United States and Japan are outstanding both in amount of scientific work and in results obtained. In England research is continued primarily on semisynthetic antimicrobials and on metabolites of fungal origin. Soviet research has overtaken that in England in the number of new antibiotics found. Declining tendencies are prevalent in research on new antibiotics of microbial origin in Switzerland, France, and Belgium, too, and in

323

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

Sweden it has entirely stopped. In the past 5-10 years, however, an enhanced rate of research has been observed in Japan, Italy, India, Canada, and West Germany. Table V demonstrates the total number of antibiotics of actinomycete, fungal, and bacterial origin described by scientists of various countries. Enhanced concentration may be deduced also from the fact that, until 1965, 55%of newly discovered antibiotics was described by United States and Japanese scientists and the share of the first four countries (United States, Japan, USSR, United Kingdom) amounted to 73%.After 1965 this ratio shifted to 62 and 77%,respectively. Concentration of research is especially acute in the highly expensive fields of antitumor and antiviral research: 71 and 78% (formerly 65 and 71%) of new antitumor and antiviral antibiotics, respectively, described after 1965 were discovered by US and Japanese scientists. TABLE V NUMBERO F ANTIBIOTICS DESCRIBED IN DIFFERENT COUNTRIES ~

~

~~~~

~~

~

Sources Country Japan USA USSR UK West Germany Switzerland Italy India France Canada East Germany Brazil Hungary Czechoslovakia Poland Holland Sweden Denmark Belgium Other 18 countries Used in practice

Actinomycetales 784 566 2 18 38 86 56 78 63 55 6 26 26 21 6 11 8 -

Fungi

Today used in Bacteria

Total

153 165 35 169 12 50 18 19 14 38 6 1 2 14 2 5 8 5 4

90 104 29 48 13 2 3 15 19

12 2074

52 -

24 -

772

376

88 3222

76

8

12

96

7 7

7 1 1 2 8 2 6 1

I

1027 835 282 255 111 108 99 97 88 51 33 27 24 22 21 15 14 13 12

Human therapy

Agriculture 9

20 35 3 4 2 5 1 2 1

-

1

1

75

21

7 2 I 1

324

J ~ N O SB ~ R D V

2. New Screening Methods General screening methods have not changed fundamentally in the past 30 years. Many millions of different microorganisms-primarily actinomycete species originating from soil samples-were screened all over the world by means of the classical methods of Waksman and Dubos. Parallel screening programs frequently led simultaneously to the independent discovery of the same antibiotic (e-g., carbomycin, novobiocin, cycloserine) ( Neelamegham et al., 1970). The rediscovery of known substances has become more frequent. The introduction of rapid identification methods may improve this situation, but would not fully resolve it. A t the beginning of the 1950’s the screening of 10,000 microorganisms resulted in one single useful agent (Woodruff and MacDaniel, 1958); later the screening of 400,000 microorganisms over a 10-year period led to a total of three utilizable drugs (Nelson, 1961). At present, although no exact information is available, probably many hundred thousands of microorganisms must be looked over to find one valuable substance. Apparently even a high degree of automatization and mechanization would increase the number of screened strains infinitely. New means and techniques have to be found to solve the problems of chemotherapy. By the end of the 195O’s, antibiotics of microbial origin-and by the 1960’s semisynthetic antibiotics-appeared to have solved the most urgent probIems of chemotherapy. Primary infections due to resistant, gram-positive strains could successfully be treated with semisynthetic antibiotics (lincomycin and fusidic acid) ; gram-negative infections could be treated with gentamicin, cephalosporin derivatives, ampicillin, carbenicillin, and recently with tobramycin. In antitubercular therapy, capreomycin and especially rifampicin brought a further decisive improvement. Even a new, potent antigonorrheal agent, spectinomycin, was introduced in chemotherapy. In spite of these results, several urgent therapeutic problems are yet to be solved. a. The degree of hospital infections is still very high: $lo%. These constitute most frequently urinary infections, pneumonia, bacteremia, and infections following surgery. The infections are mainly due to gramnegative bacteria and some streptococci. Mortality rate due to bacterial ( Streptococcus D ) endocarditis and pneumococcal meningitis is even higher ( 2 0 4 0 % ) . b. Growing resistance of gram-negative bacteria ( R factor) requires a search for further agents exhibiting different modes of action compared to those already in use. c. Infections caused by microorganisms that were generally nonpatho-

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

325

genic up to now (Escherichia coli, Klebsiella pneumoniae) are increasingly frequent. d. Systemic mycoses of the internal organs have no effective remedy as yet. Amphotericin B did not fulfill the hopes it raised, and resistance rapidly emerges upon flucytosine treatment. e. Combined treatments as well as treatments with unsuitable drugs or too high doses often resulted in enhanced bacterial resistance. f. Gonorrhea again poses problems due to declining effectiveness of agents used up to now in some countries. g. Penicillin allergy (&lo%),oto- and nephrotoxicity of aminoglycosides, leukopenia-causing effect of chloramphenicol, and damaging side effects of other antibiotics, all call for further research. h. Unresolved questions of antitumor, antiviral, and antiprotozoal chemotherapy also require further new agents. i. Special new, nonclinical antibiotics for use as feed additives, plantprotecting agents, and preserving materials are urgently needed too. All these problems cannot be expected to be solved solely by the use of semisynthetic antibiotics or synthetic chemotherapeutics. New types of antibiotics, exhibiting new profiles of activity and mechanism of action, may be found mainly by further systematic screening of microorganisms, performed according to fundamentally new principles. Throughout the world efforts have been made to introduce new techniques in the isolation of new antibiotic-producing strains, in their storage, and in their culturing conditions. New and sensitive detection methods, in vivo and genetic techniques found their way in the research laboratories. Noteworthy is the recent discovery of two nontoxic antibiotics: bicyclomycin ( Miyoshi et al., 1972), active against gram-negative microorganisms and nearly devoid of in vitro activity, but in vivo a potent antibiotic comparable to ampicillin; and the well known broadspectrum phosphonomycin. New, specific screening methods led also to the simultaneous, but independent, discovery of new cephem antibiotics, produced by Streptomyces, by Merck Sharp & Dohme and Eli Lilly scientists (Stapley et al., 1972; Nagarajan et al., 1971). The newly developed methods enable ever more frequently the dropping, at an early stage of research of agents that are highly potent in vitro, but toxic. This is also reflected perhaps in the statistics: up to 1960, 4050% of antibiotics found were highly toxic (LDS0< 15 mg/kg, iv); after 1960 this number diminished to 15-20%, and the nontoxic agents (LD,, > 500 mg/kg, iv) rose from %lo% to 20-25%. Further new approaches may be found in the production of antibiotics in extraordinary fermentation conditions. Although no new types of useful antibiotics were found among metabolites of thermophilic and psychrophilic microorganisms, their further investigation is promising. It is

324.3

J ~ N O SB ~ R D Y

an interesting result that Malbranchea pulchella var. sulfurea ( a thermophilic ascomycete) produced penicillin G at 43-45C0 ( Aragozzini et al., 1970) and Streptomyces griseus subsp. psychrophilus at 5-7Co produced a new peptide antibiotic, cryomycin (Yoshida et al., 1972). Change in fermentation times and classical media, fermentations executed with the exclusion of light (spirillomycin) (Domnas, 1968) may surely result in the discovery of further new, eventually useful antibiotics. The culturing of some Corynebacterium and Nocardia strains on n-paraffins led, for instance, most surprisingly to new chloramphenicol analogs, corynecynes ( Suzuki et al., 1972). “Chemical screening” based on various chemical reactions ( Umezawa et al., 1970a) also resulted in some new types of substances (dienomycins, arglecin) . The screening of antimycoplasma agents and the search for agents active against various plant pathogens, e.g., Xanthomonas oryzae and Piricularia oryzae, in the past decade has gained in intensity. The recently published screening method for antifungal agents on the basis of action mechanism ( Arai and Mikami, 1973) is of interest. Special antiphage screening recently resulted in a new substance, requinomycin, which could inhibit R-factor transfer and has no effect on cells ( Hori and Takemoto, 1972). Many new screening methods, both in vitro and in vivo, were introduced in the search for antitumor substances. Most probably the performance of the usual screening programs too may further decline. This would necessitate even more the extensive investigation of slowly growing organisms that are difficult to isolate and require special media. As to the future, I would like to quote Waksman’s forecast: “Any new methods for the isolation of antibiotics producing organisms will tend to yield new types of antibiotics” (Waksman, 1969). D. NEWANTIBIOTIC SOURCES

1. Antibiotics from Microorganisms The main sources of microbial antibiotics are the following groups of microorganisms: ( a ) Actinomycetales, ( b ) bacteria, and ( c ) microscopic fungi. Up to the end of the 1940’s, fungi, and to a lesser extent bacteria, furnished the greatest number of antibiotics discovered. Between 1955 and 1962, however, about 80% of antibiotics found originated from Actinomycetales species. In the past 10 years, again the ratio of antibiotics isolated from Actinomycetales diminished decisively and the ratio of new antibiotics, especially those found in fungi, shows a tendency to increase (Fig. 5 ) .

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

327

FIG. 5. Percentage distribution of antibiotics according to producer type of microorganisms.

a. Actinomycetales. Great screening programs started after 1945, which utilized simple methods for the isolation of a large number of strains from soil samples, resulted in the production of hundreds of antibiotics of Actinomycetales origin (Fig. 2 ) . The initial great successes justified this growing interest. After a peak of achievement in 1957, a slow decline followed, at least in the absolute numbers of agents discovered, and only after 1964 could a development, though considerably slower than earlier, be registered. The importance of non-Streptomyces Actinomycetales is an interesting and much studied problem in antibiotic research. According to various investigations ( Lechevalier and Lechevalier, 1967; Rangaswami et al., 1967) the ratio of Streptomycetales species in Actinomycetales isolated from soil samples amounts to about 90-95%. Consequently about 92.5% of antibiotics of Actinomycetales origin, described up to now, were isolated from Streptomyces species (Table VI). The ratio of antibiotics of non-Streptomyces origin, however, amounted to 4-5% up to 1965 and has since risen to 10%.Gentamicin, rifamycin, and ristocetin take their origin from Micromonospora and Nocardia species. Some problem is posed by the uncertain identification of the microorganisms themselves. Rifamycin-producing Nocardia mediterranei was for a long time supposed to be a Streptomyces species (Thiemann et al., 1969). It is the Actinomycetales which furnished the greatest part of antibiotics in commercial use; altogether 72 of them are utilized even today for various purposes. Despite their declining ratio, Actinomycetales still promise to remain the richest sources of useful antibiotics in the future too. In the past 10 years, all new antibiotics introduced in practice, altogether 25, were furnished exclusively by Actinomycetales, except for fusidic acid and such agents of negligible importance, for instance, pyrrolnitrin and siccanin.

J ~ N O SBBRDY

328

TABLE VI NUMBER OF ANTIBIOTICS PRODUCED BY I ~ F F E R E N SCHIZOMYCETES T Pseudomanadales Pseudomonaceae Psrudomonas Acrobacter Spirillaceae Vibrio Eu bacteriales Aaotobacteriaceae Rhizobiaceae Achromobacteriaceae Enterobacteriaceae Brucellaceae Micrococcaceae Lactobacillaceae Propionibacteriaceae Brevibacteriaceae Corynebacteriaceae Bacillaceae Bacillus Clostridium Actinomycetales Mycobacteriaceae Ac tinoplanaceae Actinoplanes Spirillospora Streptosporangitm Microcellobosporia Streptomycetaceae Streptomyces (Actinomyces Krass.) Streptoverticil lium Chainia K itasatoa Micromonosporaceae Micromonospora Thermoactinomycetaceae Thermoactinom yces Microbispora l’hermomonospora Nocardiaceae Nocard ia (Proactinomyces Krass.) M icropolyspora Thcrmomonospora , Myxobacteriales (Cytophaga, ~ f y x o c o c c u sctc.) Mycoplasmatales (Mycoplasma) Total:

87 84 82 2 3 3

274 1 2 9 36 3 16 28 2 1 5 171

167 4 2078 4 18

1950 1922 19 8 1 41 41 17 9 4 4

48 45 2 1 9

2 2450

329

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

0%

100

50 ' l a

50 '10

100 'la

'la

g 4 % z y L , $ l Z IZ Ll ? - '

0%

m ,

~

~

~

=

K P

P

p

!

FIG.6. Percentage distribution of antibiotics according to type of bacteria.

b. Eubacteriales and Pseudomonadeles. Among other Schizomycetales, Eubacteriales and to a smaller extent members of the order Pseudomonadeles may be of importance as potential sources of new antibiotics. Within the Pseudomonadales, exclusively Pseudomonas species, and within Eubacteriales only Bacillus species, deserve attention. Table VI and Fig. 6 demonstrate the ratio of antibiotics of non-Bacillus origin, primarily those from Pseudomonas, whose ratio has lately shown considerable growth. Up to now about 10 agents of bacterial origin have been introduced: colistin, polymyxins B and M, gramicidin, gramicidin S , tyrothricin, bacitracin, and atterimin from Bacillus species; pyocyanin and pyrrolnitrin from Pseudomonas species; and finally nisin from other Eubacteriales. Except for pyrrolnitrin, all were discovered before 1950. Nor were bacteriolytic and cancerolytic lysostaphin and asparaginase produced by bacilli, but by Eubacteriales. c. Fungi. Primarily Penicillium and Aspergillus, belonging to the Aspergillaceae family and some fungi imperfecti species are of greater practical value with respect to antibiotic pr0duction.l Many antibiotics have been isolated from Basidiomycetes, but none is worth mentioning. Recently, coriolin derivatives may have attained some functions as antitumor agents. Antibiotics isolated from fungi are listed in Table VII. Figure 7 shows that in the past years primarily the ratio of antibiotics from different Monililales and those from other fungi has grown considerably, and the ratio of antibiotics from Aspergillaceae diminished decisively. Several antibacterial agents were isolated recently from Dermatophytons, and some yeasts too. From antibiotics of fungal origin up to now 10 have 1 Recent taxonomic systems list the Aspergillaceae family, because of their sexual behavior, similarly to asporogenic yeasts, among Ascomycetes ( Lechevalier and Pramer, 1971).

330 TABLE VII NUMBEROF ANTIBIOTICSPRODUCED B Y DIFFERENT FUNQI Myxothallophytes (Myxomycotinia) (Fuligo, Physarum, Lycologa sp.) Eumycotinia (Mycophyta), “true fungi” Phycomycetes (Phytophtora, Mucor, Rhizopus sp.) Ascomycetes Protoascomycetes (Ascosporogen yeasts) Euascomycetes Plectascales Aspergillaceae Penicillium Aspergillus Other orders of Euascomycetes Basidiom ycetes Fungi imperfecti Moniliales Moniliaceae Cephalosporium Trichoderma Oospora A crostalagmus Cylindrocladium Ilemetaciae Hclminthosporium (Ophiobolus) Alternaria Phoma Tuberculariae Fusarium Myrothecium Stilbaceae (Zsaria, Mclarrhgzium) Mclanconiales Mycelia sterilia Dermatophyta Asporogen yeasts Unidentified fungi iinperfecti

4 768 14 299

8 291 248 242 123 115 43 140 315 269 102 20 13 10 7 9 69 23 10 8 79 46 26

10 1 5 9 19 12

been commercialized: penicillin G, penicillin V, penicillin 0, cephalosporin, griseofulvin, fumagillin, variotin, fusidic acid, siccanin, and xanthocillin. Unfortunately, as yet no new antibiotic of fungal origin, comparable in importance to p-lactam antibiotics, has been isolated; only griseofulvin and fusidic acid are worth mentioning specifically. It is interesting to note that the very antibiotics of fungal origin applied most frequently in therapy-penicillins, cephalosporin C, and fusidic acid, were equally found among metabolites of different fungal species (see Section 111,BJ). Consequently the conclusion may be drawn that a wide variety of fungi are able to produce the same individual antibiotic types. The occurrence of cephamycins, helvolic acid, as well as that

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

a

331

1

50 %

100 Y o

FIG.7. Percentage distribution of antibiotics according to type of fungi,

of penicillin among metabolites of Streptomycetales, emphasize this fact even more. That fungi and bacteria as eventual sources of further new and useful antibiotics do not reach the importance of Streptomycetes can be concluded from actual results. However, the systematic screening of a large number of bacterial types, e.g., Eubacteriales, Myxobacteriales, Mycoplasmatales, as well as about 50,000 fungal species neglected up to now, and further screening of algae and lichens may lead eventually to the discovery of further new types of antibiotics. The finding of new ways to replace the accustomed old ones may produce methodological and technical problems, specially in the study of new types of microorganisms that are difficult to isolate or to cultivate, or both; these efforts, however, may sooner or later furnish appreciable results. For the time being it is an open question whether microorganisms that have not as yet been found to produce antibiotics are able to synthesize antagonistic substances under suitable conditions, or whether antibiotic-production capacity is characteristic of certain microorganisms. The question of whether antibiotic production in the world of microorganisms is a relatively rare property, as we see it today, may be decided by future studies. 2. Antibiotics from Higher Forms of Life Table VIII lists the number of antibiotics and antitumor agents found up to now in higher forms of life. Especially recently, there is enhanced interest in antibacterial substances that may be isolated from marine organisms, including marine microorganisms, from algae, seaweeds, marine animals, primarily from sponges. In the past couple of years, 18 antibiotics were isolated from Porifera species; unfortunately they were of no practical use, although they were essentially new types of substances containing halogens, mainly bromine.

332

JANOS BBRDY TABLE VIII ANTIBIOTICS (ANTITUMOR AGENTS)FROM

OTHER

Thallophytes different from fungi Algae Lichens Higher plants Antibiotic substances from the animal kingdom Protozoa Insects Molluscs Sponges (Porifera sp.) Worms Vertebrates

SOURCES 732 23 56 653

122 8 24 3 18 3 66

Antibacterial agents of plant origin have long been well known ( phytoncides ) ; however, they never gained practical introduction except for the antitumor Vinca alkaloids, On the other hand, there is great development in the field of cytotoxic substances of plant origin, primarily in research on different terpene derivatives. Kupchan (1971) alone isolated nearly 100 such, sometimes new types of compounds, for instance ansa-macro-lactam maytansine ( Kupchan et aZ., 1972). Some of them exhibit marked antitumor .activity in uiuo; their practical value, however, will be decided upon later. The world of plants may in any case be a rich source of new natural substances of possible antibacterial activity. Of some 500,000 plant species, about 10%were investigated from a phytochemical point of view, and only a negligible portion for antibacterial activity. A number of antibiotics have been isolated from various insects, vermin, and tissues of higher animal organs, but for the time being they are not recognized as useful antibiotic agents. The nearly inexhaustible living world may doubtless offer new possibilities for the discovery of new agents exhibiting biological activity. The periodical reinvestigation of microorganisms by means of new, more sophisticated methods and the inclusion of new types of organisms in overall screening undoubtedly would further enrich our chemotherapeutic drug arsenal. THATPRODUCE E. MULTIPHASICSTUDYOF MICROORGANISMS KNOWN ANTIBIOTICS

I. Biosynthesis, Directed Fermentations The economic feasibility of antibiotic production requires the raising of microoganisrn productivity to a maximum. In the case of strains that

RESEARCH A N D CLASSIFICATION OF ANTIBIOTICS

333

produce several active components, the shifting of the component ratio in the required direction, the repression of minor component production ( neamine, neomycin C, mannosidostreptomycin, etc. ) and/or just the promotion of these minor component yields (e.g., cephalosporin C, tobramycin ) may be equally desirable. Sometimes simple strain selection and strict physical control of fermentation conditions suffice to obtain good economic results. In the past decade, however, metabolically controlled “directed fermentations” ( Perlman, 1973) gained ground in an increasing number. The utilization of different ( 1 ) precursors (phenylacetic acid), ( 2 ) inhibitors of metabolism (sulfonamides, methionine), ( 3 ) enzyme stimulators, i.e., inducers (barbiturates), ( 4 ) specific culture media, and (5) different genetic methods (starting with blocked mutants up to recombination and transduction methods) may be encountered with enhanced frequency. As regards practical results it is sufficient to mention the example of rifamycin B, demethylchlortetracycline, and kanendomycin. The influencing of fermentation in the desired direction requires a detailed knowledge of biosynthesis. Research in directed fermentations, based on modified biosynthetic pathways, has led beyond improved production yields and economic advantages to the discovery of a number of new substances, e.g., new lincomycins, rifamycins, actinomycins, tyrocidines (Argoudelis et al., 1973; Lancini and White, 1973; Katz, 1971). The proportion of antibiotics obtained for the first time by these means has grown from an initial 1-2% in earlier years to nearly 5%after 1965. Some of them are generally listed among antibiotics modified by biological methods-for instance, penicillin V-though they do not much differ, in regard to their production, from “normal” antibiotics obtained by simple fermentation. They may be produced in the course of usual precursor-free fermentations, though in trace quantities. Even 6-aminopenicillanic acid, the classical example of antibiotic modification by microbiological means, is produced in the course of every “normal” penicillin fermentation. 2. Minor Components Antibiotic-producing microorganisms produce at the rarest occasions one single antibiotic. In nearly 50% of cases, strains produce a variety of substances of different nature and biological activity (Porter et al., 1964). The simultaneous production of antibacterial antibiotics (mainly water-soluble basic substances ) and antifungal polyene ones is very frequent. It depends generally on fermentation conditions which of the antibiotics can be isolated in sufficient quantity. Some antibiotic complexes, mostly those of the macrolide, aminoglycoside, polyene, peptide, and glycosidic family, contain besides one or two major components,

334

J ~ N O SB ~ R D Y

a whole series of various accompanying minor components, which differ only in the nature of one or two substituents, and an amino acid or carbohydrate moiety. Many antibiotics seem to consist of one single component only until they are investigated by more refined, sophisticated methods. Recently, with the rapid development of separation techniques, these minor components, formed usually only in small proportions at normal fermentations, have been isolated more and more frequently. Some substances, supposed to be uniform, proved to consist sometimes of several components, even 8-10 ( e.g., leucomycin, nystatin, phleomycin) . In our own studies, for instance, a Micromonospora strain produced no fewer than 48 aminoglycoside antibiotics of related properties; we succeeded in separating 26 of them. Bleomycin-producing Streptomyces verticillatus forms 13 sorts of bleomycins, and by means of precursor addition about 100 different bleomycins could be isolated ( Umezawa, 1971). The importance of minor components often overshadows that of the main one. Originally cephalosporin C was such a minor component of yield the Cephdosporium strain Brotzu. Kanendomycin, produced in 14% along with kanamycin A has in many respects properties more advantageous than those of kanamycin itself; its 3,4-dideoxy derivative may yet prove to be one of the most promising semisynthetic aminoglycoside antibiotics. The yield of kanendomycin could be raised with the help of precursor addition and utilization of new mutants, perhaps to 30-40%, i.e., to an industrially realizable proportion (Meiji Seika Kaisha Ltd., 1968). The investigation of other biologically inactive metabolites or agents with an activity not hitherto recognized, which were formed simultaneously with antibiotics, is gaining in intensity. These include odor substances ( geosmin, 2-methylborneol), different coumarin derivatives, and simply organic acids. The metabolites isolated consist also of intermediaries of antibiotic biosynthesis, or are products of independent biosynthetic pathways. The role of antibiotics in the ecology of microorganisms producing them is a much discussed question. Today it is generally accepted that antibiotics are not products of some unusual biosynthetic pathway, but are secondary metabolites or normal metabolism and thus do not play a decisive part in the ecology of microorganisms. Antibiotics form only a part of the secondary metabolites of microorganisms. Their discovery in such large number is due to their easily detectable biological activity, which is lacking in other secondary metabolites or has not yet been detected. The conclusion drawn by Dhar and Khan (1971) is worth mentioning. They consider at least some of the antibiotics to be products of the detoxification procedure for agents toxic to the producing micro-

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

335

organisms themselves (heterocyclic substances, specific carbohydrates, etc. ) .

F. EXTENSIVE INVESTIGATION OF KNOWNANTIBIOTICS

I. Biological Characteristics In the past few years the detailed pharmacokinetic investigation of known antibiotics was undertaken with enhanced intensity, as well as the elucidation of their exact mode of action and possibilities of widening clinical application fields, together with the determination of eventual side effects utilizable in other areas. The ever increasing number of papers published underline this intensified activity. Many entirely new, nonchemotherapeutic types of activities were detected in a number of antibiotics (Perlman and Peruzzotti, 1970). The anti-inflammatory effect of many antibiotics was recognized ( griseofulvin, fusafungin, SL-21429) as well as the cardiotonic and serum-lipid repressive effect of polyenes and the lowering of the calcium level by mithramycin. The spasmolytic activity of colisan and patulin, the tranquilizing effect of monorden, and the manifold physiological activity range of a number of microbial metabolites ( parasympathomimetic-cholinergic, ion-chelating, estrogenic, anabolic, immunosuppressive, immunodepressive, cholesterol level-lowering, vasodilator, antiallergenic, salivation-inducing, serotonin-antagonistic effects ) proves unequivocally that antibiotics and other microbial metabolites may exhibit, beyond their microbiological activity, a large variety of physiological effects. If suitable screening methods are developed, microorganisms may furnish a series of agents utilizable in various therapeutic fields. Recently, streptozotocin has been applied clinically ( Editorial, 1970) as a diabetogen, and fusaric acid was employed as a useful drug in alcohol detoxication cures (Ogota, 1971). The investigation of the mode of action and the nature of damaging side effects of antibiotics may have results, beyond developments in pharmacology, in chemotherapy and in molecular biology, in the planning of new, potent, antibiotic derivatives free from side effects. Today the structure of semisynthetic penicillin derivatives, effective against resistant microorganisms or exhibiting a broad antibacterial spectrum, may generally be designed in advance. After the recent discovery of the site of attack of enzymes produced by resistant, aminoglycosideinactivating bacteria (Mitsuhashi et al., 1973), there are sufficient points of departure at our disposal to permit us to synthesize new, more potent aminoglycoside derivatives. Experience gained up to now prove that the presence of some structural moieties is essential for certain effects. In many cases, however, the therapeutic effect of antibiotics seems to be due not solely to a

336

J ~ N O SB ~ R D Y

single chemical structural element, but much more to the entire molecule and the conformation of the active groups of the molecule itself. Sometimes slight changes, even only conformational ones, resulted in the complete loss of biological activity. It is as yet not possible to predict the entire range of biological activity of a planned molecule. There is, however, much experience which permits us to influence the activity of a given biologically active molecule in the desired direction with more or less success. 2. Chemical Structures The elucidation of the chemical structure of antibiotics enriched science by uncovering highly interesting features. The rapid development of physical and chemical analytical methods ( e.g., mass spectrometry, infrared ( I R ) and nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, amino acid analyzer, optical rotatory dispersion ( ORD ) spectroscopy, gas chromatography) greatly improved the detailed investigation of the chemical structure of antibiotics. Recently, the formula of antibiotics of relatively simple structure is generally published at once, and more complex structures of already known antibiotics are revealed. The full structure of more than 50%of antibiotics published after 1965 is known. This proportion amounts, even together with the large number of the latest structure elucidations, only to 35% of antibiotics described between 1955 and 1965. Today the total synthesis of 8%of all known antibiotics has been published (see Table X ) . In the 1950’s the elucidation of the relatively simple structure of neomycin, carbomycin, and tetracyclines took several years, but today the structure determination of many antibiotic types has become a routine matter. This growing knowledge has created a good basis for the suitable classification of antibiotics, discussed in detail below. 111.

Systematization of Antibiotics

A. GENERALPROBLEMS OF SYSTEMATIZATION The systematization of the huge body of knowledge acquired in the investigation of the biological, chemical, and pharmacological properties of antibiotics has become an urgent necessity. The basic principles of a classification of antibiotics were discussed in detail at a Round Table Discussion (1970) held at the VIth International Congress of Chemotherapy. The opinions of the participants reflected the obvious urgency of the problem. Numerous fundamental features of the necessity, purpose, and means of realization of a classification of antibiotics came into the limelight.

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

337

The systematization of antibiotics according to both practical and theoretical points of view is highly important. The physicians request a concise classification of the most important clinical antibiotics according to their effectiveness. Theoretical pharmacologists, biochemists, and clinical scientists need an exhaustive systematization according to mechanism of action. Microbiologists prefer a classification according to the origin of antibiotics, biochemists seek one based on biosynthetic pathways. Chemists naturally want to classify antibiotics according to chemical structure. Any kind of classification implies difficulties due to a variety of reasons. a. A system of practical value not only should include agents already existing but should be flexible enough to accommodate future developments as well. b. A satisfactory classification needs to be unambiguously and logicafly arranged, but at the same time must be adaptable to specific requirement. It is extremely difficult to reach a compromise between the viewpoints of the experts of various Faculties. c. Our present knowledge about the characteristics of antibiotics is manifold. Antibiotics of both practical and theoretical importance have been extensively studied; their structure, biosynthesis, and mode of action have been elucidated. Most of the other antibiotics were characterized unsatisfactorily. d. Because of lack of sufficient data, some of the principles listed above proved adequate for an exact, extensive classification at present (for instance, Streptomyces taxonomy, molecular-biological basis of mechanism of action ) . The extent and profoundness of a classification may be highly variable. A review of therapeutic antibiotics is amply sufficient from the point of view of the physician, and partly sufficient from that of the clinical scientists. There is no difficulty in systematizing antibiotics of a known mode of action or biosynthetic pathway, since they amount to no more than 200-300 substances. But to classify satisfactorily the 4000 antibiotics, of manifold characteristics, known today, constitutes a hard task. OF CLASSIFICATION EXAMINED FROM B. PROBLEMS DIFFERENT POINTSOF Vmw

1. Classification According to Origin The primary classification of antibiotics may be performed according to their origin, i.e., by means of a systematization based on the nature of the producer microorganisms. Questions relating to this classification method have been reviewed by Okami (1970) and Kurylowicz (1970).

338

J ~ N O SB ~ R D Y

One of the advantages of this method is that all antibiotics can be placed in several large groups, but beyond their listing according to individual taxonomic families, there is no possibility for further rational subdivision. A systematization according to origin alone seems to be insufficient for the following reasons: a. Antibiotic-producing ability, as it is accepted generally, is not a stable intrinsic feature, innate to a given microorganism. b. Various species of some genus produce a large variety of substances of diverse chemical structure and activity. Today it is impossible to subdivide the more than 2000 antibiotics of Streptomyces origin on this basis. Even within the Streptomyces genus itself there is no final taxonomy yet. No correlation could be observed between properties of antibiotics produced by Streptomyces strains belonging to the same series. Neither is the phylogenetic interrelationship of Streptomycetales satisfactorily known. Consequently, Streptomyces antibiotics ought to be classified on a purely chemical basis, as proposed by Kurylowicz (1966) and Korzybski et al. (1967). The situation is similar with antibiotics of the Aspergillaceae and Bacillaceae too. c. Identical antibiotics are often produced by entirely different organisms, sometimes even by those belonging to quite different families, orders, classes, and divisions, as is summarized in Table IX. There exist even more examples of the production of structurally related antibiotics by different microorganism types. For instance, ribostamycin ( Shomura et al., 1970) and butirosin (Woo et al., 1971), which have identical structural skeletons, are formed by a Streptomyces species and a Bacillus species, respectively, Cephalosporin C and penicillins are exclusively synthesized by fungi; in the past years, however, five cephalosporin C skeleton-containing antibiotics were isolated from cultures of Streptomyces strains, Iodinin is produced by Pseudomonales, Eubacteriales, and Actinomycetales, (Prauser and Eckardt, 1967) ; its methoxy derivative, myxin, is produced by a Sorangium species belonging to the order Myxobacteriales (Peterson et al., 1966). d. A given species may produce many different antibiotics. Thus, for instance, Bacillus subtilis forms 66, and Bacillus brevis 23, different antibiotic substances, though mainly of polypeptide character. Species belonging to Streptomyces hygroscopicus or Streptomyces griseus series form a set of manifold antibacterial agents, as do Pseudomonas aeruginosa strains, which produce about 40 various sorts of antibiotics, mainly of N-heterocyclic skeleton. e. A given antibiotic may very often be produced by many different species of the same genus, Thus oxytetracycline is formed by at least 19 kinds of Streptomyces species ( Kurylowicz, 1971).

TABLE IX SOMEANTIBIOTICSPRODUCED BY DIFFERENT GROUPSOF ORGANISMS" Main groups of organisms Schizomycetes

Antibiotics Bovinocidin (fl-nitropropionic acid) Nebularin Helvolinic acid Penicillin N Questiomycin A, B Hadacidin Pyocyanin (cyanomycin) Cycloserine (0-Carbamyh-serine) Iodinin Phenazine-1-carboxylic acid 1,6Dihydroxyphenazine Prodigiosin Toxoflavin (xanthotricin) Anticapsin &N-Hydroxy-barginine Citrinin Penicillin G Fusidic acid Phthiocol Lecanoric acid

StreptoOther myces Actino- Bacillus sp. mycetdes sp.

X X X X X X X X X X X X X X X

Fungi

Other Eubacterides

Pseudommas sp.

Asper- Basidiogillaceae mycetes

Fungi imperfecti

Other fungi

X

Higher plants

E R ti

X

R

X X X X

X

X

x

X X

X

X X

X

X X X X

X X

EE

x

ii

b

ij 2

X

8

X

m"

5 s

X

x

* 3

X X X

X X

X

E

X X X

X X

Antibiotics produced by Streptomyces species and other Actinomycetdes: aburamycin, azomycin, bottromycin, carbomycin, chelocardin, chloramphenicol, fervenulin, formycin B, fradicin, fungichromin, gentamicin, histidomycin, hydromycin B, mycoheptin, neomycin, polymycin, streptocardin, siomycin, taitomycin, undecylprodigiosin.

8 CD

$2 0 TABLE X PERCENTAGE DISTRIBUTION OF VARIOGSTYPESOF ANTIBIOTICS FROM DIFFERENTGROUPSOF MICROORGANISMS A N D EXTENT O F THEIR CH.4R.4CTERIZATION Main microorganism groups Antibiotic types

Carbohydrates Macrolides Quinones Peptides N-containing heterocycles 0-containing heterocycles Alicycles Aromatic compounds Aliphatic compounds Degree of knowledge Total numbers of substances Number of substances with known character Percentage Number of substances with known structure Percentage Number of synthesized substances Percentage

2 10 94 18 2 5 1 -

17 10 68 66 8 1 6 -

-

3 4

2 6

111 85 82 11 10

376 279 77 88 24 42 11

-

5 2 1 6 167 126 73 35 21 17 10

87 68 80 42 48 18 21

7 6

7 11 18 2 6 38 14 4

269 237 89

158 60 30 11

11 8 4 8 25 6 28

6 3 20 25 4 8 16 9 9

4 4 12 21 3 11 22 14 9

20 23 15 25 8 2,4 2,4 2 2.2

15 17 13 27 8 4 7 5 4

140 106 75 84 60 18 13

121 76 66 64 53 14 12

772 600 38 445 58 106 13

2074 1803 87 722 35 125 6

3222 2682 83 1255 39 273 8

2 3 12 30 2 20 7 20 4

10 -

242 181 74 139 59 44 18

2

-

7 3 20 g27 ,o 26 m 10 5 %

F

653 472 72 380 58 78 12

Key to groups: 1, Bacillus sp.; 2, Pseudomonadales; 3, other bacteria; 4, all Eubacteriales*; 5, Moniliales; 6, Aspergiilaceae; 7, Basidiomycetales; 8, other fungi; 9, all fungi; 10, Actinomycetales; 11, total microbial antibiotics; 12, antibiotics from higher plants. Including Mycobacteria, Myxobacteria, and Mycoplasma sp.

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

341

The production of some structural types seems to be more or less characteristic for a given type of producing microorganisms. Table X summarizes the distribution of the main structural types of antibiotics according to different sources. Bacillus species synthesize almost exclusively polypeptide derivatives; Pseudomonas species, mainly N-heterocyclic phenazine derivatives. Some fungi produce alicyclic aromatic or simple O-heterocyclic substances with outstanding frequency. Macrolide antibiotics, tetracyclines, anthracyclines, and aminoglycoside antibiotics again are exclusively characteristic for Actinomycetales species. On the other hand, bacteria are unable to produce quinone, macrolide, and alicyclic substances. Antibiotics of higher plant origin are mainly heterocyclic and alicyclic substances.

2. Classification According t o Biosynthesis The classification according to biosynthesis ( Abraham and Newton, 1959) has a significant theoretical and biochemical background ( Gottlieb and Shaw, 1967; Snell, 1966). However, the relatively low number of agents investigated (the biosynthesis of about 200 antibiotics is known) limits the possibilities of systematization. Further difficulties are encountered because very often similar, general biosynthetic pathways metabolize manifold chemical structures with different actions ( macrolides, polyenes, tetracyclines, terpenes, polyines, etc. ) . Mixed biosynthetic pathways are highly frequent when one part of the molecule is built up in a different metabolic way from the other. The classification according to biosynthesis is closely connected with chemical systematization. Similar chemical structures are generally formed by identical or similar biosynthetic pathways. Systematization according to biosynthesis is based on the frequently uncertain pathway of metabolism; chemical classification, on the other hand, is founded on the final result of the exact chemical structure. In isolated cases the relationship between a classification based on biosynthesis and origin is obvious; identical microorganism groups may generally form similar types of chemical structure by identical metabolic pathways. 3. Spectrum of Actioity

The classification according to the spectrum of antibiotic activity is time-honored in usage and is of fundamental value in practice. The spectrum of activity is well known in the case of almost every important antibiotic, In the course of the usual determination of an antibacterial spectrum, antibiotics active against the following microorganisms are differentiated: gram-positive and gram-negative bacteria, Mycobacteria, fungi, yeasts, protozoa, and such specific microorganisms as rickettsiae, mycoplasma, and spirochetes. Some microbial metabolites have either

342

J ~ N O SB ~ R D Y

antitumor, antiviral, insecticidal, etc., activity alone or, more frequently, combined with antibacterial activity. As can be seen in Table XI, these activities exist in various combinations Even among antibiotics active against bacteria and fungi, there exist broad-spectrum antibiotics, i.e., agents equally effective against nearly all gram-positive and gram-negative bacteria, fungi, and yeasts investigated, but there are also some that exhibit specific activity against only one or two distinct organisms ( e.g., sarcidin, validamycin, saramycetin ) . With respect to unambiguous classification of therapeutic antibiotics, problems are posed by the fact that paromomycin, for instance, is generally ranged among antiprotozoal substances, although its entire spectrum of activity, not to mention its chemical structure, is almost identical with that of other aminoglycosides (mentioned as antibacterial anibiotics); e.g., with neomycin, kanamycin, except that it exhibits a much higher antiprotozoal activity than either of them. It is difficult to obtain precise data about the true frequency of individuaI types of activity among microbial metabolites because the determination of the activity spectrum is very uneven. Frequently, only isolated activity data are published, measured on one or two distinct test organisms, or solely the potency conforming to the screening program is determined at all. Consequently the correct classification of most antibiotics is impossible. The simple system ranging the useful antibiotics in groups designated “narrow spectrum” or “broad spectrum” agents, antifungal, antitumor, etc., antibiotics, is unsuitable for any systematization of scientific value. There is another interesting question-“the summarized activity” of antibiotic substances in accordance with various points of view. A characteristic picture can be gained if the activity of all antibiotics described up to now against most frequently used test organisms, virus, TABLE XI PERCENTAQE O F ANTIBIOTICS ACTIVEAGAINST VARIOUSMICROORGANISMS Activity type

Percentage

Gram-positive bacteria (including Mycobacteria) Gram-positive and gram-negative bacteria Gram-negative bacteria Gram-positive bacteria, fungi, and yeasts Gram-positive, gram-negative bacteria and fungia Fungi and yeasts Tumor Other (virus, protozoa, etc.)

32

a

Including yeasts.

25 1

10 13 16 2

1

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

343

and tumor cells, etc., is considered with respect to its frequency (Table XII). It seems that the frequency of an individual antibiotic activity varies according to its origin. Thus an “overall spectrum of activity” is obtained which is sufficiently characteristic for antibiotic-producing microorganisms. The activity of agents of Streptomyces origin is generally more manifold. Antibiotics of fungal origin, especially in those derived from Moniliales, activities against saprophytic and phytopathogenic fungi, and cytotoxic effects are prevalent; their potency against Mycobacteria, Proteus and Mycoplasma, however, is rather low. Antibiotics of Basidiomycetales are generally potent against gram-positive bacteria but are inactive against yeasts. Antibiotics of Bacillus origin exhibit high activity against gram-positive bacteria, Mycobacteria and algae, and their activity against anaerobic clostridia is rather distinct. The above data suggest that there is an unambiguous difference between antibiotics of various origins, i.e., in the intrinsic frequency of antagonistic properties of different organisms producing them, even in cases where screening is performed according to the entire spectrum by means of utilizing equally sensitive detection methods.

4. Classification According to Mechanism of Action Classification based on mechanism of action represents a profound fundamental meaning for theoretical scientists. There are many examples for the systematization of antibiotics of known mechanism of action (Gottlieb and Shaw, 1967; Evans, 1965; Miura, 1969; Gause, 1970). Recently interest has grown rapidly for this field of molecular biology. In spite of the accumulated mass of data there are still many aspects awaiting clarification, From all the known antibiotics, the mode of action of about 200 has been described, at least partially. The mechanism of action only of antibiotics in commercial use can be regarded as exhaustively elucidated. Many problems still await clarification. a. In experiments investigating mechanism of action, it is often diffcult to identify primary and secondary processes. b. It is difficult to construct viable cell models in a cell-free system. Results obtained in vitro do not necessarily reflect in viuo mechanisms. Experiments are usually performed in a cell-free system that is not completely identical with living cell. c. In addition, some antibiotics exhibit different types of mechanisms of action; for instance, gramicidin S affects membrane function as well as oxidative phosphorylation. d. Further problems arise from the fact that substances of completely diverse chemical structure often exhibit a similar or identical mode of action. For instance, aminoglycosides and macrolides both inhibit protein synthesis, and the site of their attack is primarily 30 S ribosomes.

TABLE XI1 PERCENTAGE DISTRIBUTION OF ACTIVITYOF ANTIBIOTICSAGAINST DIFFERENT MICROORGANISMS

(ACCORDING TO ORIGIN) Main microorganism groups" Test organisms or type of activity Stap hylowccus aureus Bacillus subtilis Clostridium sp. Escherichia coli Pseudomonas aeruginosa Proteus sp. Brucella sp. Mycobacterium sp. Xanthmonas sp. Candida sp. Saccharomyces sp. Dermatophytons Phytopathogen fungi Saprophyta fungi Protozoon Rickettsiae Mycoplasma (PPLO) sp. Spirochetae Algae Anthelminthic activity Insecticide activity Antiviral activity Antitumor (cytotoxic) activity Phytotoxic effect

1

2

3

4

47 40 12 40 19 12 8 22 0.6 18 13 18 20 18 9 2 -

57 60 3 25 7 14 7 25 7 26 12 25 22 26 5

38 23 3 20 8 8 1 15

44 39 7 28 13 11 5 20 2 15 9 15 17 16 7 0.8 1.5 1.3 0.3 0.6 6 10 3

1

4 3 3

-

2 1 1 6 10 7

1

3 4 3 6 5 3 1 2 5 10 17 1

5

37 34 2 15 6 8 2 15 5 27 15 25 33 34 10

-

0.8 0.8 1.3 1.3 8 38 7

6 44 37 9 22 8 8 1 17 2.5 16 10 14 16 16 6 6 4 13 17 4

7 64 45 2 30 14

8

9

10

40 36 1 21 2 3 2 10 3 17 10 11 28 30 5 3 -

41 34 4 19 7 6 2 16 3.6 16 10 16 21 22 7 0.4 2 0.1 0.5 2 8.5 23 5

65 63 4 33 15 18 7 31 7.5 23 19 16 18 20

4 4 26 5 8 7 12 14 18 4 0.6 -

-

3 19 1

6 11 5

-

11

1.4 3.5 2 0.2 1.8

0.7 8 23 1.2

11

57 53 5 31 12 15 6 24 6 21 16 16 19 20 10 1

2.5 2 0.3 1.3 1 8 21 2

12 39 23 2 18 5 5 4 20

2.

6

"

7 12 4

%

5 i-

0.2 0.3 0.5

1.2 4.5 22 1

4 Key to groups: 1, Bacillus sp.; 2, Pseudomonadales; 3, other bacteria; 4, all Eubacterialesb; 5, Moniliales; 6, Aspergillacetie;7, Basidiomycetales; 8, other fungi; 9, all fungi; 10, Actinomycetales; 11, total microbial antibiotics; 12, antibiotics from higher plants. b Including Mycobacteria, Myxobacteria, and Mycoplasma sp.

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

345

These systematizations ought to be revised according to the increasing the changing aspects of science. In the distant future, however, a systematization according to mode of action may be useful.

5. Classification According to Physicochmical Properties Antibiotics may be classified solely according to their physical or chemical properties. Antibiotics may be differentiated on the basis of their water solubility or solubility in organic solvents, of whether they have acidic, basic, or amphoteric character, of indicator properties, or of those containing N or other elements. Attempts were made to classify antibiotics on the basis of their elementary analysis and functional groups, too. Ranging antibiotics according to such physicochemical characteristics as UV, IR, NMR spectra, paper-chromatographic or thin-layer chromatographic data is of utmost importance from a practical point of view, since it facilitates the screening and rapid identification of new antibiotics. These classifications, however, though of practical value, do not constitute a theoretical improvement. It ought to be stated, however, that because of lack in suitable experimental data, some of the above-mentioned methods may not be ripe yet for use in a final, extensive systematization. We must stress the fact that none of the present classification systems can satisfy both theoretical and practical requirements, serving simultaneously the needs of chemists, pharmacologists, microbiologists, biochemists, and physicians. The elaboration, within a reasonable time, of such an overall satisfactory system, meeting all the above requirements, is inconceivable. On the other hand, in my opinion there is a special field where the time is ripe for drawing up a meaningful, uniform classification system of practical and theoretical importance. This is the area, to be discussed exhaustively in Section IV, of the classification of antibiotics according to chemical structure. IV.

Classification of Antibiotics According to Chemical Structure

A. BASICPRINCIPLESOF CHEMICAL CLASSIFICATION 1. Interrelationship of Different Points of View Chemical structure is the unique property which unambiguously distinguishes one antibiotic from another. It is this chemical structure that determines all its physical, chemical, microbiological, pharmacological, and finally clinical properties. The interrelationship between structure and physicochemical characteristics is obvious, but not less so between

346

J ~ N O SB ~ R D Y

structure and biosynthetic pathway ( Section III,B,2). There may be found connections between structure and origin too (Section III,B,l and Table IX) . Relationship between structure and various biological activities are quite obvious. Antibiotics of similar structural skeleton mostly exhibit similar overall microbiological activity. For instance, macrolides are active against gram-positive bacteria, gram-negative cocci, and Mycoplasma species, are moderately toxic, and have similar side effects. Polyene macrolides exhibit a broad antifungal spectrum and are generally inactive against bacteria. Tetracyclines have a broad antibacterial spectrum; anthracycline glycosides are active only against gram-positive bacteria but generally have cytotoxic and antitumor properties. Specific biological activity frequently is inherent in some smaller structural moieties, -CO-C=X ( X=CH2, N, groups) connected with cytotoxic antitumor effects (e.g., sarcomycin, azaserine, terpene lactones) . In the case of water-soluble, basic, aminoglycoside or polypeptide antibiotics, the increasing number of amino groups, i.e., enhanced basicity, shifts the antibacterial spectrum in the direction of gram-negative bacteria. Slight changes in structure are followed often by dramatic changes in overall potency. A methodical and extensive systematization according to chemical structure may further the comprehension of several deeper interrelationships in the field of both molecular biology and chemotherapy. 2. New Types of Chemical Structure The early systematizations according to structure ( Waksman and Lechevalier, 1962; Shemyakin and Khohlov, 1957; Umezawa, 1964) had to be rudimentary because of the lack of sufficient knowledge about chemical structures of antibiotics. Presently more than 4000 antibiotic substances derived from natural sources are known. Even those of microbial origin amount to more than 3200 (Tables V, VI, VII). Omitting close homologs and nearly identical analogs, their number still is above 2500. Owing to the tremendous development of structure elucidation methods, today the exact structure of 1255 microbial, and altogether more than 1700 natural, antibiotics is known. Additionally about 800 structures have been elucidated to a sufficient extent ( degradation products, empirical formulas, partial structures ) for meaningful classification. The known chemical and physical characteristics of about 1000 antibiotics suffice for a tentative classification too. The number of known antibiotics grows from year to year. But the discovery of important, entirely new antibiotic families comparable to macrolides, tetracyclines, polyenes, or aminoglycosides is highly improb-

347

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

able. There is a real possibility, however, of the recognition of several new and useful antibiotic groups. In the past ten years the new groups of moenomycin ( Slusarchyk, 1971), ascochlorin ( Minato et al., 1972), cytochalasin ( Binder and Tamrn, 1973), and bleomycin type antibiotics, as well as daunomycin and nogalamycin type anthracyclines, gentamicin type aminoglycosides, albocycline and maridomycin type marcolides, enduracidin and grisellimycin type peptides, and pyrrolnitrin, polyoxin, or formycin type heterocyclic antibiotic groups were recognized. In the past 2-3 years, further completely new types of antibiotics have been described. Beyond kasugamycin, phosphonomycin, showdomycin and negamycin (compound I ) (Kondo et al., 1971) mentioned by Conover ( 1971) in his exhaustive review, I list below a series (11-XXII) of antibiotics representing completely new types of agents: negamycin ( I ) ( Kondo et al., 1971 ) bicyclomycin ( I1 ) ( Kamiya et al., 1972 )

YHz

(R)

Go

$€*I

OH I FHS H,NCH,CHCH,CHCHICONHNCH,COOH

E C H .

HN

(R) (1)

(11)

ikarugamycin (111) (Kazuyoshi et al., 1972; Ito and Hirata, 1972)

fi (In)

0

validamycin (IV) (Horii and Kameda, 1972) HOH,C,

HO

\

OH

axenomycins ( V ) (Arcamone et al., 1973a,b)

OH

OH

OH

OH

OH

OH

OH

OH

OH

CH,

0

\

H,C CO

0

(V)

antibiotic K-16 (VI) ( Batelaan et al., 1972)

0 COOH C-CH, II -(!!-COOQ

@O

0

I

H

NH-CO-C-

I

0NHS '

N H s -H H z I c ~ o

CH,OH

H

(VI)

mocimycin (VII ) ( Vos and Verwiel, 1973) antibiotic X-5108 (VII) (Maehr et al., 1974)

H3C

R I

OH

OH

0

HO

OH

1. R = C H , , Antibiotic X-5108 (Goldinodox) 2. R = H, Mocimycin (Delvomycin) (VII)

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

349

maytansine (VIII ) (Kupchan et al., 1972)

R=H (VIII)

myriocin (thermazymocidin) ( IX) (Bagli et al., 1973) aphidicolin ( X ) (Brundet et al., 1972)

vermiculin ( X I ) ( Sedmera et al., 1973) lipoxamycin (XII) ( Whaley, 1971 )

J=Fc0-cH3

0

H,C,

0

0 0 H O O 1 I 1 I I I1 ,CH( CH,),C( CH,), CCH2CH2N- CCHNH,CH,OH

HSC

(XI)

SF-666 A and B antibiotics (XIII) ( I t o et al., 1971)

OH

HO (XIII)

(XI)

350

J ~ N O SB ~ R D Y

1233 A antibiotic (XIV) ( Aldridge et aZ., 1971 )

HOCH,CH-CH

I 1 oc-0

*

Me Me Me I I I CH, * CH, . CH, . CH, * CH . CH, . C=CH C=CH. CO,H 3

(rn) N6-phosphono)methionhe-S-sulfoxyminylalanylalanine ( XV ) et al., 1973) phosphotricinylalanylalanine ( XVI ) ( Bayer et nl., 1972)

( Pruess

L-(

0 0 0 I1 11 I1 H,NCHCNHCHCNHCHCOH

0 0 0 I1 II 11 H,NCHCNHCHCNHCHCOH I I I CH, CH, CH,

0

11 HO -P-NI

OH

I

I

I

7%

S =O I CH,

kinamycins (XVII) (Omura et al., 1971)

CH,

0% 0% 0

0%

Component Rl A COCH,

R2

%

COCH,

COCH,

H

R4

B

H

COCH,

H

H

C

COCH,

H

COCH,

COCH,

D

COCH,

H

COCH,

H

(xvn) pseudomonic acid (XVIII) (Fuller et al., 1971; Beecham Group Ltd., 1971 ) OH

CH * CO,(CH,),CO,H

II

2H. CH,C. CH,

A-25822 antibiotics (XIX) (Lilly & Co., 1972)

R , , R, = H, CH, & = OH, AcO

(XIXI

oleficin (XX) ( HorvAth et ol., 1973) 0

COOR, R, = H; % = p-o-Digitoxoside; n = 5

-0-0 - Digitoxoside

(XX)

a-lipomycin XXI) (Schabacher and Zeeck, 1973) 0

OR" CHS

CHS R'O,C

dienomycins (XXII) (Umezawa et al., 1970b)

H

H

H

A R = OCCH(CH,), B: R = OCCH, C R=H

(xxn)

OR

352

JANOS

B~FIDY

New types of structures were discovered also in substances known for a long time, but for which structure elucidation was completed only in the past few years, as, for instance, in the case of compounds

(XXII1)-( XXXII) : bleomycins (XXIII) (Takita et al., 1972)

( R Terminal amine) (XXIII)

albofungin (XXIV) (Gurevich et al., 1972)

Me

OH Me0 (XXN)

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

bundlines (T-2636 antibiotics) (XXV) (Harada et al., 1971) oligomycin B ( XXVI) (von Glehn et al., 1972)

I'

3'

Me

2'

NHCOC:

% '

Me

0

(xxv) T-2636A T-2636C T-2636D T-2636F

RL -COMe -H -COMe -H

%

=O =O

-H, -OH -H, -OH

primycin (XXVII) ( Aberhart et aZ., 1970)

oxazinomycin (XXVIII) (Hashimoto et al., 1972)

(XXVI)

353

354

JINOS B~RDY polyoxin A (XXIX) (Isono and Suzuki, 1968) HOOC CH,C

HCOH

I

H°CH

HO

I

OH

CH,OCONH, (XXIX)

ascochlorin (XXX) (Nawata et al., 1969)

(XXX) thiostrepton (XXXI) (Anderson et al., 1970)

zQ

Pyruvic acid

eucine

0

I Theostreptine

Nitrogen

0 Oxygen

0

Sulfur

355

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

chlorothricin ( XXXII ) ( Muntwyler and Keller-Schierlein, 1972 ) COOH

CO

x

=

c1

(XXXII)

Among new types of antiobiotics, individual agents, built up by the surprising combination of earlier known and widely distributed moieties, may be found. These include, besides bleomycins (XXIII) already mentioned (which contains amino acids, sugars, p-lactam), compounds (XXXII1)-( XXXVI) : cephamycins (XXXIII) (Stapley et al., 1972) (cephem nucleus, aromatic acids ) 0

HOOC-CHI

CH,-

CH,-

CH,-

11

C-N

H OCH3

NH,

p>

0

A

COOH

R

Cephamycin -C=C I

O O S 0 , H

OCH,

B

- CI = ! O O H

OCH, C

CH,-0-C-R

-NH,

(XXXIII)

0 I1

356

JANOS

B~RDY

thermorubin ( XXXIV) (Moppett et al., 1972) (anthracene, xanthone)

Me0

O/H..*

H

OR"

R"O RO

H

H

R = R' = H; R" = Me

(XXXrV)

prumycin (XXXV) (Omura et ol., 1972) (diaminosugar, amino acid)

r-:z2

0 1

LXP"

I CH,NHCOCH CH, I

NH2 (XXXV)

593 A substance (XXXVI) ( Arison and Beck, 1973) (diketopiperazine, chlorpiperidine) and myomycin ( French et al., 1973 ) (guanidinosugar, inositol, p-lysine)

(XXXVI)

There are many antibiotics the structure of which is still completely unknown, not similar to any of the known antibiotics, which may belong to some new types of substances. Their proportion has grown considerably in the past few years. Up to 1965 they made up 10%of antibiotics described; after 1965, 16%. In spite of this large number of examples, most antibiotics discovered recently are new members of well known antibiotic types. Particularly, many new macrolides, polyenes, polyethers, and oligopeptides containing unusual amino acid residues have been described recently.

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

357

On the basis of the above-mentioned findings, however, our present knowledge seems to be sufficient for drawing up a useful and fairly flexible classification system based on the chemical structures of antibiotics. 3. Criteria of Chemical Classification

The classification of antibiotics based on their chemical structure raises a number of special problems. Contrary to other groups of natural substances, e.g., alkaloids, steroids, enzymes, terpenes, etc., representing a fairly narrow range of structural skeletons, the antibiotics have structures expressing the features of the entire range of organic chemistry. Especially conspicuous is the presence of some specific moieties, skeletons, and configurations not found, or very rarely found, in other substances of natural origin, which cannot be synthesized by simple routine methods. Consequently the generally utilized inflexible system of organic chemistry is unsuitable for the classification of antibiotics unless it is altered and adapted to our purposes. Since the antibiotics are originally metabolites of living organisms and thus are biosynthesized from simple organic substances (sugars, amino acids, purine or pyrimidine bases, fatty acids, active acetyl and propinonyl groups), it is obvious that these fundamental moieties must constitute the first approach for the basic principle of classification. However, this approach alone is insufficient for the construction of an uniform, unambiguous system. Differentiation between groups of substances generally should not be based on a single criterion. Some criteria are more important than others. Consequently, the system proposed by Sensi (1970; Sensi and Coronelli, 1971), which classifies antibiotics of Streptomyces origin according to the occurrence of six basic moieties in different combinations, includes several contradictions. It does not recognize any difference in the importance of individual residues. Thus agents of most diverse structural skeletons and of entirely different biological properties are placed in the same group. The outlined system built up by Yonehara (1970) is logical, though in my opinion the glutarimide and glycoside antibiotic families break up the unity of the system to a certain degree. The classification of Waksman and Lechevalier ( 1962), Shemyakin and Khoklov (1957), as well as that of Umezawa (1964, 1967), is criticized by Kurylowicz (1970) in a constructive way. In my opinion, a logical system, free of contradictions, has to take into account, beyond chemical principles, the most specific characteristics of the classified objects-in our case the antimicrobial activity of the respective antibiotics. From the chemical point of view this is represented by the very moiety of the molecule primarily responsible for antimicrobial activity. The particular importance of this molecular fragment ( p r i m

358

JANOS

B~RDY

cipal constituent) has to be an outstanding feature of our system. In the case of small molecules of simple structure, generally the entire molecule is responsible for effectiveness. In more complex compounds, the principal constituent responsible for activity needs to be selected out of a number of other moieties, and the systematization executed accordingly. There exist some structural moieties that occur almost exclusively in antibiotics and play an outstanding role in microbiological activity; these include macrocyclic lactone and p-lactam rings, some quinonelike systems, such as the tetracyclines, anthracyclines, 2-deoxystreptamine. These constituents have to be considered with specific emphasis. One of the most important prerequisites of a chemical classification is that agents of similar structure and physicochemical characteristics as well as microbiological activity should be placed, if possible, in identical groups. At the same time it is essential that any individual antibiotic should be listed in only one single group. There exist a number of antimicrobial agents, especially those consisting of a more complicated molecule, which have several, sometimes three or four principal constituents of different types. In these cases, to avoid any duality, the principal structural feature responsible for microbiological activity has to be selected, if possible, and this may constitute the basic principle of classification. It is easy to make a choice; for instance, in the case of penicillins and cephalosporins, where the p-lactam ring, which contains the penicillin and the cephalosporin nucleus, can unambiguously be considered as the part of the molecule exhibiting microbiological activity. There are situations, however, where one must compromise in the selection of the basic structural element. Some antibiotics of similar structure but devoid of less important structural moieties might still exhibit some degree of microbiological activity. In this case it seems obvious that classification should be perfomed according to the simplest molecular residue still exhibiting microbiological activity, and/or according to the most characteristic common feature of several active agents. For instance, the molecules of amicetin and septacidin, devoid of their amino acid, aromatic, and aliphatic residues, show a remaining pyrimidine and adenine glycoside nucleus, which closely resembles that of other highly potent agents. Consequently, amicetin and septacidin may both be placed in the group of heterocyclic glycosides. In the case of antibiotics containing characteristic structural elements of approximately equal importance ( e.g., streptothricins, lincomycin-type antibiotics), it is very difficult to make a proper decision. The p-lysine moiety of the streptothricins, as well as the aromatic nucleus in celesticetin, seems to be nonessential, whereas no unambiguous choice can

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

359

be made between the remaining amino sugar moiety and heterocyclic amino acid residue of both compounds. Because of generally very similar physical, chemical, and microbiological characteristics, their listing in the group of carbohydrate antibiotics would seem justified. There is great similarity between their physicochemical properties and activity, especially in the case of streptothricins, and those of other water-soluble, basic aminoglycoside (carbohydrate) antibiotics. Based on identical principles, the moenomycins may be assigned as carbohydrate antibiotics too. In constructing an unambiguous logical system, there must sometimes be exceptions. Antibiotics of different structural features, but having a close biogenetic relationship and general overall characteristics as well-such as alicyclic cycloheximides, aromatic actiphenol, and aliphatic protomycin-should be listed in the same group, but in a different subgroup. It seems to be of fundamental importance to find the specific structural moiety generally common to the antibiotics investigated and which is no general constituent of other natural organic substances. Furthermore in addition to purely chemical characteristics, aspects based on effectiveness, chemical and physical similarity, as well as biogenetics, have to be considered. In spite of these basic principles, the listing of some antibiotic groups, primarily the chromomycin-type agents, aromatic glucosides, and tetracyclines, seems to be somewhat arbitrary. B. PROPOSED SYSTEMFOR THE CHEMICAL CLASSIFICATION OF ANTIBIOTICS'

1. Primary Classification On the basis of the above-mentioned principles, the antibiotics may be arranged primarily into families according to the following criteria. a. Among the basic constituents in the construction of antibiotics, it is doubtless the carbohydrates that occur most frequently and are at the same time related to the greatest variety of other moieties. Almost all antibiotic families have several members containing sugars, generally in glycosidic linkage. Sugar-containing antibiotics where the carbohydrate moiety is not a predominant part of the molecule, although important with respect to antimicrobial activity, cannot be placed, according to the rules of primary classification, in the family of carbohydrate antibiotics. Consequently in different sugar-containing macrolides, anthraA previous compilation ( BBrdy, 1972 ), prepared in collaboration with the International Center of Information on Antibiotics, contains this section of the present paper.

360

JANOS

B~RDY

cyclines, purine-pyrimidine glycosides, aromatic glycosides, the sugar residue cannot constitute the basis of systematization; such antibiotics should be classified according to their aglycons. The establishment of a separate “family of glycosidic antibiotics,” although it seems logical, would only disrupt the unity of the system. The family of “carbohydrate antibiotics” consists of antimicrobial agents containing sugar as the sole basic constituent, as well as those where the sugar moiety constitutes the main skeleton of the molecule. b. The grounds for the existence of two important, highly specific antibiotic families, the macrocyclic lactones and quinone-indicator antibiotic families, represent a specific problem. On the basis of rigid systematization principles, they might be placed in the group of aliphatic or aromatic antibiotics. These two specific families consist of a large number of highly important antibiotics, besides many special naturally occurring substances, where the microbial activity is connected with the entire molecule, or at least with the main part of it, and consequently constitute a principal constituent in themselves. Beyond their frequent occurrence and importance, their complete new biological properties, different from those of normal aliphatic or aromatic antibiotics, justify my proposal to list them not as a subgroup of aliphatic or aromatic antibiotics, but as a separate family of antibiotics. Independently from all other basic constituents present, every antibiotic having a larger than 8-membered lactone or polylactone ring may be placed in the family of macrocyclic antibiotics. The family of quinone antibiotics consists, besides normal quinones, of other agents having pseudoquinone moieties or polycondensed ring systems with mobile electrons and being very often indicators (except anthocyanides ) as, for instance, tetracyclines-and of partially saturated or epoxide quinone derivatives. The basis for the primary classification of the rest of antibiotics is formed unambiguously by the next principal constituents: amino acids, heterocycles, alicycles, aromatic ring systems, and aliphatic carbon chains. c. The family of peptide antibiotics consists of antimicrobial agents constructed solely from one single amino acid or from amino acids and of those agents having some supplementary substituents of a different character. These other residues are most frequently fatty acids in the side chain ( polymyxins, enduracidin, glumamycin), simple aliphatic acids ( depsipeptides ) , heterocyclic chromophores ( e .g., actinomycins, echinomycins, virginiamycins) , specific heterocycles in the peptide skeleton of the molecule (thiostrepton, bleomycin), and aromatic rings (ilamycin). In this family, the occurrence of amino acids having an unusual structure or configuration, not to be found in nature, is very frequent. Some individual members of this family, for instance, the penicillins,

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

361

may be assignable to the heterocycles as well, but biosynthetically they can be definitely derived from amino acids. The family of peptide antibiotics may be divided further into two subgroups-one containing antibiotics constructed solely of residues coupled by peptide bonds, and the other of peptolides built up by additional lactone linkages. d. The heterocyclic antibiotics are best divided into families of nitrogen- and oxygen-containing heterocyclic ring systems. As yet, no antibiotic has been discovered where a sulfur-containing skeleton is a solely basic constituent. Similar structural moieties, however, may be deduced from amino acids (e.g., thiostrepton, penicillin). All antibiotics having a heterocyclic ring or polycondensed heterocyclic ring systems-but which did not figure among the formerly listed sugar (except glycosides), macrocyclic lactone, quinone, or amino acid-containing principal constituents, as well as the glutarimides-should be placed in this family. Antibiotics having a heterocyclic skeleton condensed with aromatic or alicyclic rings or a long aliphatic chain belong to this family. In the family of oxygen-containing heterocyclic antibiotics are listed all small cyclic lactone derivatives which may be deduced from aromatic or aliphatic acids, as well as agents of polyether character (nigericin). Alkaloids of antimicrobial activity may be also classed as a subgroup of nitrogen-containing heterocyclic substances. e. Antibiotics of an alicyclic skeleton having an aromatic ring (siccanin) or aliphatic chain (steroid antibiotics) might be placed in the family of alicyclic antibiotics. The terpene lactones having a small lactone ring (4-6 members) may be listed here as a separate subgroup (and not in the heterocyclic system containing oxygen). f. The family of aromatic antibiotics consists of nonquinonoid agents containing an aromatic system as well as an aliphatic side chain. g. As a consequence of the basic rules of classification, the family of aliphatic antibiotics contains agents of a relatively simple character, saturated or unsaturated aliphatic compounds having no other characteristic principal constituents. In summary, the principal rules of the classification of antibiotics according to the chemical structure are the following: I. Assignment to antibiotic families is performed according to the following nine principal constituents: ( a ) sugar; ( b ) macrocyclic lactone ring (rings containing more than eight members); ( c ) quinone (or quinonelike ) skeleton; ( d ) amino acid; ( e ) nitrogen-containing heterocyclic systems; ( f ) oxygen-containing heterocyclic systems; ( g ) alicyclic skeleton; ( h ) aromatic skeleton; ( i ) aliphatic chain. 11. In the case of simple molecules of a sole basic moiety, the molecular skeleton is the basis of classification. 111. In antibiotics of more complicated structure, having a variety

362

J ~ N O SB ~ D Y

of constituents, the principal constituent responsible for biological activity should be considered as the basis of classification. IV. When the characteristic structural moiety cannot be established unambiguously, the main constituents of the molecule have to’be considered in a sequence defined in rule I. V. In particular cases, biogenetic aspects, as well as similarities in physicochemical characteristics and activity, have to be taken into account.

2. Secondary Classification Within the above-established families, the antibiotics are placed into subgroups according to the characteristics of the individual antibiotics. The main rules are the following: 1. The so-called “pure antibiotics” consisting of one single constituent, form a separate subgroup. 2. Antibiotics containing several characteristic constituents are subdivided according to the quality of the so-called “secondary constituents.” 3. A further basis for classification is the size of the molecule (i.e., oligopeptides, polypeptides ) . 4. The variants of similar or identical structural skeletons represent an important feature of classification (e.g., macrolide ring systems of different sizes, open-chain or cyclic peptides, saturated or unsaturated side chain), 5. Antibiotic types of practical importance having frequently occurring structural features (e.g., moenomycin-type agents, streptothricins, anthracyclines, prodigiosin-type substances ) should be regarded as separate types, if possible, even if respective structural features would warrant a different grouping ( glutarimide antibiotics). 6. In certain cases, the specific type of linkage, or quality of chromophore in the molecule, i.e., in the polyenic antibiotics, may be the basis of a finer systematization. 7. Similar antibiotics originating from diverse microorganisms often form an individual subgroup (differentiation according to origin of resistomycin-herqueiquinone type; nidulin-type agents and depsidones originating from lichens; alkaloids; etc.) . 8. The secondary classification may be done according to common biological activity, as, for instance, in the case of anticarcinogenic proteins. Their further subdivision based on physicochemical properties appeared to be highly advantageous. The great advantage of the system is that not only antibiotics of well established or almost elucidated structure can be classified, but also that those sufficiently characterized by physical, chemical, and biological data or by degradation products may be classed in the individual

RESEARCH AND CLASSIFICATION O F ANTIBIOTICS

363

groups with more or less exactitude. Consequently, we encountered no difficulty in assigning macrolides, polyenes, oligo- and polypeptides, quinones, purine-pyrimidine glycosides, etc., of unknown structure. There is even a possibility of performing the tentative secondary subdivision of an entirely unknown agent according to its most probably basic skeleton. There exist some well established types of antibiotics, for instance in the family of quinones (luteomycin and xanthomycins), where we have only vague ideas as to their structure (they are most probably heterocyclic ring systems containing naphthoquinones, with some sugar content). Since all groups contain many well characterized antibiotics, they should be considered as separate subgroups within the quinone antibiotics. A very similar situation exists in the case of polypeptide antibiotics of completely unknown structure, which cannot be placed anywhere. From about 3000 chemicallv fairly well defined antibiotics, about 2500 may be classified with sufficieit accuracy. Antibiotics still to be discovered as well as antibiotic groups of unknown structure, which are placed at present in miscellaneous antibiotic groups, may be listed, after elucidation of their structure, in appropriate existing classes or in new classes constructed within the framework of the system. At first glance, the proposed system may seem too detailed for widespread practical purposes, but methodologically and from the point of view of a deeper comprehension of more profound connections, the maximally detailed listing of the existing data seems to be of utmost importance.

3. Methods of Classification. Code System To facilitate classification and to promote an overall orientation, the individual groups received code numbers. On the basis of these code numbers, orientation among the antibiotic groups would be easy with the help of various punch-card systems or computers. In our institute the identification of new antibiotics has long been performed by means of an edge-punch-card system (Bkrdy, 1961; B6rdy and Magyar, 1968). Our system registers, in addition to the various physical, chemical, and biological data, the code numbers of the most characteristic structural features of all the known (more than 4000, excluding synonyms) natural antibiotics. The recently developed code system, based on decimal principles, assigns a code number, containing generally four elements, to any individual antibiotic division. The first element of this code number shows to which of the nine families the antibiotic belongs. The second figure

364 KEY TO

THE

TABLE XI11 MAIN ANTIBIOTIC FAMILIES

~~~~~~

Primary code numbers 1 1.1 1.2 1.3 1.4 2 2.1 2.2 2.3 2.4 3 3.1 3.2 3.3 3.4 4 4.1 4.2 4.3 4.4 4.5 5 5.1 5.2 5.3 6 6.1 6.2 6.3 6.4 6.5

7 7.1 7.2 7.3 8 8.1 8.2 8.3 8.4 9 9.1 9.2 9.3 0

CARBOHYDRATE ANTIBIOTICS Pure saccharides Aminoglycoside antibiotics Other (N- and C-) glycosides Various sugar derivatives MACROCYCLIC LACTONE (LACTAM) ANTIBIOTICS Macrolide antibiotics Polyene antibiotics Other macrocyclic lactone antibiotics Macrolactam antibiotics QUINONE AND SIMILAR ANTIBIOTICS Linearly condensed polycyclic compounds Naphthoquinone derivatives Benzoquinone derivatives Various quinonelike compounds AMINO ACID, PEPTIDE ANTIBIOTICS Amino acid derivatives Homopeptides Heteromer peptides Peptolides High molecular weight peptides NITROGEN-CONTAINING HETEROCYCLIC ANTIBIOTICS Noncondensed (single) heterocycles Condensed (fused) heterocycles Alkaloids with antibiotic (antitumor) activity OXYGEN-CONTAINING HETEROCYCLIC ANTIBIOTICS Furan derivatives Pyran derivatives Benzo[r]pyran derivatives Small lactones Polyether antibiotics ALICYCLIC ANTIBIOTICS Cycloalkane derivatives Small terpenes Oligoterpene antibiotics AROMATIC ANTIBIOTICS Benzene compounds Condensed aromatic compounds Nonbenzoid aromatic compounds Various derivatives of aromatic compounds ALIPHATIC ANTIBIOTICS Alkane derivatives Aliphatic carboxylic acid derivatives Aliphatic compounds with S or P content MISCELLANEOUS ANTIBIOTICS (with unknown skeleton) (May be distributed according to physical, chemical, and microbiological properties)

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

365

indicates the subfamily. Table XI11 demonstrates the key to numbering and primary classification of antibiotics according to main classes. Further subdivision of the main classes: ( i ) the third figure indicates the group (e.g., 2.1.1 is the group of small macrolides); (ii) the fourth figure gives the type (e.g., 2.1.1.3 as the type of 14-membered small macrolides with sugar); (iii) the fifth figure represents the subtype (e.g., 2.1.1.3.1is the subtype of picromycin-type antibiotics). The less well-known agents receive 2- to 3-membered code numbers, characterizing the subfamily or group where the antibiotic can be placed with absolute certainty. For instance, the code number of the poorly characterized tetraenes is 2.2.2. In certain fields (for instance, macrolides or anthracyclines ) in which we know a large number of exact structures, a further more detailed subdivision is possible. A given code system enables the subdivision of every class into ten further subclasses. In a general antibiotic list, it is possible to number the substances in each subdivision by means of serial numbering, as used for natural terpenes (Devon and Scott, 1972), or in alphabetical order. By utilization of further code numbers, leading to newer subdivisions, the system may gain in size and depth, and this would enable us to refine it to a desired degree. Table XIV registers the exact subdivision of the individual families in subfamilies, groups, types, and eventually subtypes, together with their respective code numbers, as well as almost all known representatives of the individual types. This table includes not only antibiotics of microbial origin, but also those isolated from higher orders of organisms as well as agents exhibiting solely antitumor, antiviral, anthelminthic, and insecticidal activity.

PROPOSED CODESYSTEMFOR Code numbers

TABLE XIV CHEMICAL CLASSIFICATION OF ANTIBIOTICS

THE

Divisions

Representatives

1.1

1. CARBOHYDRATE ANTIBIOTICS Pure Saccharides

1.1.1 1.1.1.1 1.1.1.2 1.1.1.2.1 1 . 1 , l .2 . 2

MONO-A N D DISACCHARIDES Simple sugars Aminosaccharides Monoamino sugars Aminodisaccharides

1.1.1.3

Various simple sugar derivatives

SF-666 A,-B, isomaltose-like antibiotic

Nojirimycin, 3-amino-3-deoxy-~-glucose Mannosyl-1-D-glucoseaminide, trehalosamine (nocardin) Prumycin, streptoeotocin

366

jhos B~RDY

TABLE XIV (Continued) Code numbers

Divisions

1.1.2 1.1.2.1

POLYSACCHARIDES Glycan-type polysaccharides

1.1.2.2 1.1.2.3

Polysaccharide-protein complexes Lipopolysacc harides

1.1.2.4

Other polysaccharides

1.2 1.2.1 1.2.1.1

1.2.1.2 1.2.1.3

1.2.1.4

1.2.2

Representatives

Aeroglycan, isolichenin, lentinan, lichenin, laminarin, mannan, nigeran, pustulan, schysophyllan, CPS (Crepidotus polysaccharide), heteroglycans, G-2, GE2,-3, EP-7, LC-33, P-2, PC-3, etc. Propionin A,-B,-C,-D, statolon, vivomycin, K-4, 173-t Acetoxan, gangliosides, prodigiosan, ungulic acid, CWP (cell wall protein) Hemicellulose, pectin, sarganan, soedomycin, staphylococcin A, talaron, KCG, etc.

Aminoglycoside antibiotics

STREPTAMINE DERIVATIVES Streptomycin type (streptidine derivatives)

Bluensomycin type Spectinomycin type (N,Ndimethylstreptamine derivatives) Hybrimycin type (streptamine and epistreptamine paeudoaaccharidea) 2-DEOXYSTREPTAMINE

Streptomycin, mannosido-, hydroxymannosidohydroxy-, dihydro-, N-demethylstreptomycin, aygomycin B, G.B-229, 1943 Bluensomycin (glebomycin), €3-255 H Spectinomycin (actinospectacin, M-141)

Hybrimycin AI,-A2,-BI,-B2,-C1,-C2

DERIVATIVES

1.2.2.1 1.2.2.1.1

1.2.2.1.2 1.2.2.1.3 1.2.2.2

4,5-Disubstituted deoxystreptamine derivatives Neomycin type (pseudotetrasacc harides)

Ribostamycin type (pseudotrisaccharides) Neamine type (pseudodisaccharides) 4,6-Disubstituted deoxystreptamine derivatives

Neomycin B,-C,-LP I,-LP 11, phosphamidoneomycin, paromomycin 1,-I1 (catenulin, aminosidin, monomycin, hydroxymycin, SF-767 P, lividomycin C, etc.), monomycin 3 (N-acetyl paromomycin) , lividomycin A (mannosylparomomycin) ,-B (mannosyldeoxyparomomycin) ,-D (2230 C, deoxyparomomycin), SF-767 A, L, K Ribostamycin (SF-733), butirosin A,-B,-Ei,-Ez Neamine (neomycin A), paromamine

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

367

TABLE XIV (Continued) Code numbers

Divisions

1.2.2.2.1

Kanamycin type

1.2.2.2.2

Gentamicin type

1.2.2.3

Hygromycin B type

1.2.3

OTHER AMINOCYCLITOL

1.2.3.1 1.2.3.2 1.2.3.3 1.2

1.3 1.3.1

(Cyclohexanol) DERIVATIVES Validamycin type

Kasugamycin type Myomycin type lesser known aminoglycosidelike antibiotics

Representatives Kanamycin A,-B (kanendomycin),-C, NK-1001, NK-1003, NK-1012-1, NK1012-2, NK-1012-3, NK-1013-1, NK1013-2, nebramycin 1,-2,-3,-4,-5,-5’,-6 (tobramycin) Gentamicin A,-B (Sch.14342),-B,,-CI, -Cla,-C2,-X (X2), sisomicin, verdamicin, G-418 Hygromycin B (marcomycin, A-396 11), destomycin A,-B, A-396 I (SS-56 D), 88-56 C

Validamycin A,-B,-C,-D,-E,-F, validoxylamine A,-B Kasugam y cin Myomycin A,-B,-C Amycin, anthelmycin, eurimycin, eurymycin, glucomycin, miusidin, roseomycin, senomycin, solemycin (ferromycin), A-4696, Ac3/203, H 702, H-1250, F-1001, MA-1267, SOB-7, T-82, 15 A,-B,-C,-D,-E,-F, 460 A,-B,-C

Other (N- and C - ) glycosider

N-GLYCOSIDES (STREPTOTHRICIN GROUP)

1.3.1.1

Streptothricin type (with 0-lysin)

1.3.1.2

“Pseudostreptothricin” type (without B-lysin)

1.3.1.3

Other streptothricin types (dextrarotatory)

Actinorubin, akimycin, evericin, fuscomycin, grasseriomycin, grisamin, lavendulin, luridin, musashimycin, mycothricins, neonocardin, neothricin, nourseothricins, novomycin, phagomycin, phytobacteriomycins, pleocidins, polymyeins, racemom ycins, roseocitrins, roseothricins, streptin, streptolins, streptothricin A,-B,-C, -D,-E,-F,-X, virothricins, virusin, yazumicins, A-6, A-20, A-249, A-8265, B-637, F-20, H-56, H-146, IN-183-T, 0-24, P-9, 43-127, 136, 539, 587/13, 700, 24010 B-I, etc. LL-AC-541 (BY-81), E-749 C, 1483 A, citromycin, LL-AB-664 (BD-12), LLBL-136, SF-701, sclerothricin Bagacidin (A-7907), boseimycin 111, fucothricin, geomycin, lemacidin, S-15-1, 156

368

JANOS

B~RDY

TABLE XIV (Continued) Code numbers 1.3.2

Divisions

Representatives

C-GLYCOSIDES (OLIGOSACCHARIDES WITH AROMATIC CHROMOPHORES)

1.3.2.1

Ristocetin type

1.3.2.2

Vancomycin type

1.3.2.3

Chromomycin type

1.4 1.4.1 1.4.1.1

Actinoidin A,-B,-C, ristocetin A,-B (ristomycin 111,-IV), ristomycin 1,-I1 Avoparcin (LL-AV-290), K-288, LL-AM374, vancomycins Chromomycin AX-As,-B,C (aburamycin A,-B,-D, M-5-18903, SK-229), aburamycin C, olivomycin (demethylchromomycin) A,-B,-C,-D (NSC-A-649 A,-B, 232), variamycin (6604-9A), mithramycin (aureolic acid, LA-7017, 7193, 11296), mithramycin B,-C, gelbecidin, chrysomycin, RP-9768, RP16978, 144-3

Various sugar derivatives

SUGARESTERS, AMIDES Everninomycin type (evernic acid esters)

1.4.1.2

Lincomycin type (lincoseamine derivatives)

1.4.2 1.4.2.1

OTHERSUGAR DERIVATIVES (sugar lipids) Moenomycin type

1.4.2.2

Glycolipids

1.4.2.3

Other sugar derivatives

Everninomycin (R-451) A,-B,-C,-D,-E, avilamycin A, curamycin, cineromyciii A, exfoliatin Lincomycin A,-B,-C,-D,-K,-S, U-24166, 1-demethylthiolincomycin, lincomycin sulfoxide and other lincomycin derivatives, celesticetin A,-B,-C,-D, desalicetin, N-demethyl-, 7-O-demethyl-, N-demethyl-7-0-demethylcelesticetin Moenomycins (bambermycin, prasinomycin) A,B1,B2,C,-D,-E,-F,-G,-H, diumycins (A,A’,B,B’), macarbomycins, RP-8036, ItP-11837, RP-19402 “Glycolipid,” pyolipic acid, rhamnolipids (R-1, R-a), trehalose lipid, tuliposide A,-B, ustillagic acid A,-B Labilom ycin

2. MACROCYCLIC LACTONE (LACTAM) ANTIBIOTICS

2.1

Macrolide antibiotics

2.1.1

SMALLMACROLIDES 12-Membered ring (methymycin type)

2.1.1.1

Methymycin, neomethymycin, NA-181, PA-133 A, YC-17 (deoxymethymycin)

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

369

TABLE XIV (Continued) Code numbers 2.1.1.2 2.1.1.3 2.1.1.3.1

Divisions

Representatives

14-Membered ring without sugar (albocycline type) 14-Membered ring with sugar(s) Picromycin type

Albocycline, cineromycin B, ingramycin

2.1.2

Griseomycin (lomycin), narbomycin, PA133 B, picromycin (albomycetin, amaromycin, argomycin, proactinom ycin A, etc.), proactinomycin B Erythromycin type Erythromycin A,-B,-C,-E, flavucidin, oleandomycin,-B, proactinomycin C Megalomicin type Megalomicin A,-B,-C,,-CZ Lankamycin type Bandamycin, kujimycin A, lankam ycin (kujimycin B), T-2636 B MEDIUM( ~ G M E M B E R E D )

2.1.2.1

With UV maxima a t 232 nm

2.1.1.3.2 2.1.1.3.3 2.1.1.3.4

MACROLIDES

(-C=C-C=C-

I

I

I

(4I -OH

I

2.1.2.1.1

chromophore) Leucomycin type

2.1.2.1.2

Spiramycin type

2.1.2.2

With UV maxima a t 240 nm 0 0

II

/\

(-c-c-c=c-c-

I

I

2.1.2.2.1

chromophore) Angolamycin type

2.1.2.2.2 2.1.2.2.3

Carbomycin (A) type Cirramycin type

2.1.2.3

Coleimycin (K-231), espinomycin AI-As, -B, josamycin (leucomycin Aa),-S, leucomycin Al-A9,-Bl-B4,-U,-V,mydecamycin (medemycin, SF-837), llP-6237, SF-837 Az, tertiomycin B, turimyein (JA-6599), YL-704 A1,-Azr-BiJ-B2,-Cz, 446 Foromacidin D, spiramycin I,-II(accty1spiramycin),-111, (foromacidin A,-B,-C)

With UV maxima a t 278283 nm chromophore)

Angolamycin (It-491 A), shincromycin A,-B, PA-148 Carbomycin A, sekazin, S-4C-33 Acumycin (B-38941), cirramycin Al-As, -B, juvenimycin A,-Ad, rosamicin

370

JLNOS B ~ R D Y TABLE XIV (Continued) ~

Code numbers

Divisions

2.1.2.3.1

Carbomycin B type

2.1.2.3.2

Tylosin type

2.1.2.5 2.1.2.5. I

Without chromophore (maridomycin type) Neutral medium macrolides Neutramycin type

2.1.2.5.2 2.1.3

Aldgamycin E type OTHER (17-18-MEMBERED)

2.1.2.4

Representatives Abbot-29119, carbomycin B, niddamycin (F-3463), CC-10232, M-188, PA-108, SF-837-A3,-AdJYL-704-Wi,-Wz,-W3 Tylosin,-B (desmycosin),-C, juvenimycin B, lactenocin, macrocin, relomycin Maridomycin (B-5050)T,-II,-III(YL-704

cl),-Iv,-v,-vI

Bandamycin B, chalcomycin (aldgamycin I), mikonomycin), megacidin, neutram ycin Aldgamycin C,-E

MACROLIDE-TYPE ANTIBIOTICS

2.1.3.1

Bundlin type

2.1.3.2 2.2 2.2.1

Borrelidin type

2.2.1.1

Trienin type

2.2.1.2 2.2.2 2.2.2.1

Other trienes

2.2.2.2

Ritnocidin type

2.2.2.3

Nystatin type

2.2.2

OTHERLESS

Bundlin A (T-2636 C, lankacidin),-B (T-2636 A), T-2636 D,-E,-F Borrelidin

Polyene antibiotics

TRIENES (PROBABLY M ACROLIDES)

Mycotrienin, trienin, MM-8, 141-18, triene from Chainiu minutisclerotina Proticin, resistaphyllin, robigocidin

TETR.4ENES

Pimaricin type

KNOWN

TETRAENES

2.2.3 2.2.3.1

PENTAENES Aldopentaenes (methylpentaenes) (neutral pentaenes without sugar)

Arenomycin B (P-42 E), etruscomycin (lucensomycin), pimaricin (tennecetin), tetramycin, terin A,-B, PA-166 Akitamycin, albotetraen, rimocidin (PA-86), toyamamycin, LL-BH-890 a,+, RP-9971 Nystatin A1,-Az,-A~(polyfungin A,-Az), polyfungin B, amphotericin A, niycoheptin B, plumbomycin A,-B Antimycoin A, candistine, chromin, flavoviridomycin, guamycin, endomycin A (helixin A), ornamycin, protocidin, sistomycosin, tetraenin, tetramedin, tetramycoin A,-B, unamycin A, xanthalicin A, ACz-43.i, MYC-4, PA-85, RP-7071, RP-17732, 11-1 Aurenin, cabacidin, chainin, norchainin, homochainin, filipin I,-11,-111,-IV (durhamycin), lagosin (fungichromin, moldcidin B, pentamycin), fungichro-

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

371

TABLE X I V (Continued) Code numbers

Divisions

2.2.3.2

Eurocidin type (amphoteric pentaenes with sugar)

2.2.3.3 2.2.3.4 2.2.4 2.2.4.1

Capacidin type Gangtokomycin type HEXAENES Probable macrocyclic hexaenes

2.2.4.2

Fradicidin type (unknown structures) HEPTAENES Aromatic heptaenes Candicidin type

2.2.5 2.2.5.1 2.2.5.1.1

2.2.5.1.2

Aureofungin type

2.2.5.1.3 2.2.5.2

Perimycin type Nonaromatic heptaenes

2.2.5

OTHERLESSER

KNOWN

HEPTAEN ES

2.2.6 2.2.6.1 2.2.7 2.2.7.1

OCTAENES Ochramycin type OXOPOLYENES Flavofungin type (oxopentaenes)

Representatives matin, pentaneicin, neopentaene, rubrochlorin, xanthalicin B, JA-4495, HA-106, HA-135, HA-145, HA-176, T-2636-M, 661 Aliomycin, chantalmycin, distamycin C, eurocidin A,-B, fumanomycin, misionin, moldcidin A, onomycin I, pentafungin, pentaene G-8, quinquamycin, compound No. 58, A-228, PA-153, LL-BL-2 17-01,-&JA-1015, X-1019, 17-41 B, 1579, 2814 P Capacidin, lienomycin Gangtokomycin, genimycin, 1454/69 Cryptocidin, hexamycin, hexin, endomycin B (helixin B), flavacid, mediocidin, sapromycetin (B-34), LIA-0677 B Eurotin, fradicin, kotomycin, mycellin, mycellin IMO, A-207, A-1404

Antifungin 4915, ascosin, ayfactin (AYF, AE-56, aureofacin), candicidin (G-252, PA-150), eurotin A, gerobriecin, hamycin,-x, hepcin, heptafungin A, levorin Ao,A~-A~,-A~,-B, takamycin, trichomycin A,-B, AF-1231, DJ-400 Bl,-B,, G-83, X-63, X-68, 616, 0704 (levorin A2), 2339, 2789 Aureofungin, candimycins, cryptomycins, HA-496 Perimycin (NC-1968) Amphotericin B, candidin, candidoin, candidinin, monicamycin, mycoheptin (2814 H), nursimycin, partricin, tbilimycin, LL-BK 217-7 Azacolutin, champamycin A,-B, grubilin, heptamycin, neoheptaene, A-113, A-264, A-583, LL-BL 617, 528, 757, 1645-PI, 1900 C,, etc. Ochramycin Flavofungin A,-B (mycoticin A,-B), flavomycoin, roseofungin, bruneofungin, 2381

372

J ~ N O SB ~ D Y

TABLE XIV (Continued) Code numbers 2.2.7.2 2.2.8 2.2.8.1

Representatives

Divisions Dermostatin type (0x0hexaene) MIXED POLYENES Tetraesin type (tetraene hexaene)

Dermostatin (viridofulvin)

+

2.3

Other macrocyclic lactone antibiotics

2.3.1

LARGE(MACROLIDE-LIKE)

2.3.1.1

Oligomycin type

2.3.1.2 2.3.1.3 2.3.1.4 2.3.1.5

Venturicidin type Axenomycin type Primycin type Other probable macrolactones

2.3.2 2.3.2.1

SIMPLELACTONES Eight-membered lactones

2.3.2.2 2.3.3 2.3.3.1

Nine-membered lactones DILACTONES Antimycin type

2.3.3.2 2.3.3.3 2.3.4 2.3.4.1

Boromycin type Pyrenophorin type POLYLACTONES Cyclopolylactones (nonactin type)

2.3.4.2

Fluorin type

2.3.5

MACROLACTONES CON-

Tetraesin (tetrahexin)

MACROLACTONES

Botrycidin, cryptomycins, hondamycin (albimycin), oligomycin A,-B,-C, olimycin, phycomycin, rutamycin (oligomycin D), 1642-P2, 5339 C, B-41 Aabomycin, venturicidin A,-B,-X Axenomycin A,-B,-D Primycin Azalomycin B, blasticidin A, camphomycin, eliaophyllin, folimycin, humidin, ikutamycin, kokubomycin, neohumidin, sapromycetin A, scopamycin A,-B, suitamycin, PA-128 7-Hydroxy-4-oxo-2-enoic acid lactone (pyrenophorin monomer) Vermiculin Antimycin Ao-As (blastmycin, phyllomycin, virtosin, vulgarin, K M 57-h), 1-2743 C, neoantimycin (trilactone) Boromycin (ivomycin) Pyrenophorin, pyrenophorol (inactive) Desideus, dinactin, laidlomycin, lustericin, monactin, nonactin, peliomycin, perlimycin (Sq-15859), tetranactin (S-3466 C), trinactin, werramycin, macrotetrolides B,-C,-D,-G, SF-1195, SP-351 A,-B,-C,-D Bichoromycin, duamycin, fluorin, leucocidin, longisporin, A-9828, 291 1/2

D E N S E D WITH OTHER

2.3.5.1

RINQ(S) Chlorothricin type

2.3.5.2

Cytochalasin type

Bromothricin, chlorothricin, dechlorothricin Cytochalasin A (j-dehydrophomin),-B (phomin),-E,-F

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

373

TABLE XIV (Continued) Code numbers

Divisions

2.3.5.3

Brefeldin type

2.3.5.4

Zygosporin type (nonmacrolides, biogenetically and structurally closely related to cytochalasins)

2.4 2.4.1 2.4.1.1

ANSAMYCINS Rifamycin type

2.4.1.2 2.4.1.3 2.4.2

Tolypomycin type Geldanamycin type MAYTANSINE TYPE

Representatives Brefeldin A (ascotoxin, cyanein, decumbin),-B, monorden (radicicol) Zygosporin A (cytochalasin D),-13,-C, -D,-E,-F,-G, chaetoglobosin A,-B proxiphomin, protophomin

Macroloctam antibiotics

Rifamycin (nancymycin, C-521) A,-B, -C,-D,-E,-L,-0,-S,-SV,-Y, 27-demethyl-, 27-deacetylrifamycins, streptovaricin A,-B,-C,-D,-E,-F,-G Halomicin B, tolypomycin R,-Y Geldanam ycin Maytansine, maytanprine, maytanbutine, colubrinol

3. QUINONE AND SIMILAR ANTIBIOTICS

3.1

linearly condensed polycyclic compounds

3.1.1

TETRACY CLIN E-TY PE

3.1.1.1

Tetracyclines and tetracycline-like compounds Tetracyclines

SUBBTANCES

3.1.1.1.1

3.1.1.1.2

Acetamido tetracyclines

Bromtetracycline, chlortetracycline, demethyltetracyclines, tetracycline, fluorotetracycline, oxytetracycline, other various tetracycline derivatives 2-Acetyl-2-decarboxamidotetracycline, -oxytetracycline, -chlortetracycline, chelocardin Aranciamycin, chromocyclomycin, pillaromycin A,-Bl,-Bz,-C Quinocycline A,-B, isoquinocycline A,-B, nocardorubin (rufinosporin)

3.1.1.2

Pillaromycin type

3.1.1.3

Quinocycline type

3.1.2

ANTHRACYCLINES (Anthraquinones, linearly condensed with alicycles) Anthracycline glycosides Rhodomycin type p-Rhodomycin I,-11,-111,-IV,-V (S-583), p-rhodomycin- (Roal,Rods),- (Roa2,Rod),- (deo,Fuc ,Roa, Rod), p-isorhodomyoin I,-II, az-rhodomycin, 7-rhodomycin I,-11,-111,-IV, mycetin B,,-Ba, -E, retamycins, violamycins, B-5794, etc.

3.1.2.1 3.1.2.1.1

374

J ~ N O SB

~

Y

TABLE XIV (Continued) Code numbers

Divisions

3.1.2.1.2

Cinerubin type

3.1.2.1.3

Aklavin type

3.1.2.1.4

Uaunomycin type

3.1.2.1.5

Nogalamycin type

3.1 .2.2

Anthracyclinones

3.1.2

OTHER

LESS KNOWN

ANTHRACYCLINE-LIKE

ANTIBIOTICS

3.1.3

A N T H RAQUI N O N E DERIVA-

3.1.3.1

Simple anthraquinono antibiotics

3.1.3.2

Dianthraquinone derivatives Benx[a]anthraquinones

Representatives Cinerubin A,-B (tavromycetins), galirubin A, pyrromycin, rutilanthin Aklavin, galirubin B, requinomycin, LL-AH-272-cu1-p Carminomycin 1,-2,-3, daunomycin (rubidomycin A, rubomycin C), duborimycin (dihydrodaunomycin, ltP-20798), adriamycin, mitochromin D, rubidomycin B,-C, rubomycin A,-B1,-B2, B-107 (daunosaminyldaunomycin) Beromycin A,-B,-C, citotetrin A,-B,-C, -D,-E, nogalam ycin, ruticulomycin A,-B, A-195 Ciclacidin (7-pyrromycinon), galirubinon L) (ryemycin At, 7-deoxyaklavinon), various rhodomycinones, ryemycin A, lateriomycin B, various pyrromycinones, vaccinocidin, violacin, violarin, M-770 Cyclamycin A,-B, cirolerosus, lateriomycin A,-F, miniatomycin, mycetins, ryemycin C, trypanomycin A2, violarin B, 135/I, 719, 1683, etc.

TIVES

3.1.3.3

3.1.3.4

3.1.3.5

3.1.3.6 3.2 3.2.1

Anthraquinones condensed with oxygen containing heterocycles Pluramycin type

Phenanthrenequinones Naphthoquinone derivatives

SIMPLE

NAPHTHOQUINONE

DERIVATIVES

Asperginon, cassic acid, chrysophanic acid, endochrocin, minomycin, purpurin, tectoquinone, verdoskyrin Luteoskyrin, rugulosin, skyrin Aquayamycin, ochromycinone, rabelomycin, tetrangomycin and probable ayamycins (A-435) Averufin, metabolite 111, phenocyclinon, versicolorin A,-B,-C Griseophagin A,-B,-C, hedamycin, indomycin A,-B,-C, iyomycin B1,-B2,-B3, -B4, kidamycin F (iyomycin F), neopluramycin, pluramycin A,-B, plurallin-chromophore, rubiflavin, tumim ycin, 44 1S-I,-II Piloquinone, 14-hydroxypiloquinone

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

375

TABLE X I V (Continued) Code numbers

Divisions

3.2.1.1 3.2.1.2

o-Naphthoquinones p-Naphthoquinones

3.2.2

NAPHTHOQUINONI: I S CON-

Representatives Biflorin, mycochryson Alkannin (A-2), chymaphillin, droseron, javanicin, norjavanicin, fusarubin, novarubin, juglon, juglomycin A,-B, lapachol, lawson, mollisin, naphthomycin, phthiocol, plumbagin, plumbagol, shikonin, solaniol, thermophyllin (lenzitin) , 5,8-dihydroxy-2,7-dimethoxy-lJ4-naphthoquinone, 2,3-dimethoxy-1,4-naphthoquinone, 2-methyl5-hydroxy-1,4-naphthoquinone, &methyl-l,4-naphthoquinone, 2-rnethoxy-lJ4-naphthoquinone

DENSED WITH OTHER RI N 0(S)

3.2.2.1

3.2.2.1.1 3.2.2.1.2 3.2.2.2

3.2.2.2.1 3.2.2.2.2

Naphthoquinones condensed with alicyclic rings Cervicarcin type Julimycin type Naphthoquinones condensed with oxygen containing heterocycles Actinorhodin type Rubromycin type

3.2.2.2.3

Granaticin type

3.2.2.2.4

Various derivatives

3.2.2.3

3.2.3

Naphthoquinones condensed with N-containing heterocycles OTHERPROBABLY NAPHTHO-

3.2.3.1

Luteomycin type

Altersolanol, bostrycin (rhodosporin), cervicarcin, narangomycin, R-12 Julimycins and julichromes (19 different substances)

Actinorhodin, 199, 206 Griseorhodin A,-B,-C,-K,-L, prunacetin AE, rubromycin-a(col1inomycin) ,-p (tuorom ycin) ,-7, 3 1-MA, 2844-31, 2929, etc. Granaticin A,-B, kalamycin, TA/30, WR-145 Bikaverin (lycopersin), norbikaverin, cryptosporin, erythrostominon, 4-deoxyerythrostomaninon, frenolicin, deoxyfrenolicin, herbarin, dehydroherbarin, methylherbarin, lambertellin, marticin, isomarticin, xyloidon Bostricoidin, kinamycin A,-B,-C,-D

Q U INONE DERIVATIVES

Luteomycin (289, H-2053, K-349-3), mezzanomycin, variomycin, vinacetin, Sax-10, N-109, Y-107

376

J ~ N O SB ~ D Y

TABLE XIV (Continued) ~

Code numbers

Divisions

3.2.3.2

Xanthomycin type

3.3 3.3.1 3.3.1.1

BENZOQUINONE ANTIBIOTICS

3.3.1.2 3.3.1.3 3.3.2

Representatives Gancidin A,-C, kuranomycin, lemonomycin, lysotoxin, miromycin, rubradirin, xanthomycin, 0-5

Benzoquinone derivatives

Simple p-benzoquinone derivatives

Benzoquinones with other constituents Biquinones BENZOQUINONEB CON-

Auranthiogliocladin, fumigatin, gonylleptidins, coprinin, primin, rapanon, shanorellin, spinulosin, stemphone, thymochynon, methoxybenzoquinone, 2,5- and 2,6-dimethoxybenzoquinone, 4-methoxytoluquinone, p-toluquinone Gliorosein, helicobasidin, polyporic acid, pleurotin, rubrogliocladin Phenicin, oosporein

DENSED WITH OTHER RINGS

3.3.2.1

Mitomycin type

3.3.2.2

Streptonigrin type

3.3.2.3

Benzoquinones condensed with 0-containing heterocycles

3.4 3.4.1 3.4.1.1

Simple quinone methides

3.4.1.2

Other quinone methides

3.4.1.3 3.4.1.3.1

Polycondensed quinone methides ltesistomycin type

3.4.1.3.2

Herqueinone type

Mitomycin A,-B,-C,-R,-Y, mitiriomycin, porfiromycin, G-253 A,-Bl,-Bz,-C Streptonigrin, streptonigrin A,-F,-P (rufochromomycin, bruneomycin, valacidin), streptocardin Cyperaquinone, dihydrocyperaquinone. etc.

Various quinonelike compounds SEMlQUlNONEs

3.4.2

OTHER QUINONELIKE

3.4.2.1

Epoxydon type

3.4.3.2

Bromine containing benzoquinone derivatives

Ascochytin, citrinin, fuscin, dihydrofuscin, pulvilloric acid, sclerotiorines, taxodon, taxodion Celastrol, maitenin, pristimerin, pterigospermin, uliginosin A,-B Itamycin, resistomycin (croceomycin, heliomycin, X-340), reaistoflavin, 11-98 Atroventin, herqueichrysin, herqueinone, deoxyherqueinone

GUBSTANCES

Phyllosinol (epoxidon), terreic acid, terremutin, panepoxydon, panepoxydion, neopanepoxydon, isopanepoxydon, phyllostine, sphaeropsidin, KD-16 Ul Aeroplysinin-l,Z, l,l-dimethyl-4-acetamido-4-hydroxy-2,5-cyclohexadiene,

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

377

TABLE XIV (Continued) Code numbers

3.1 or 3 . 2 or 3 . 3 or 3.4

Divisions

Other lesser known quinoneindicator antibiotics

Representatives

2,6-dibromo-4-acetamido-4-hydroxy2,5-cyclohexadien- 1-one Albidin, antivirubin, aurovertin, cremeomycin, crothiomycin, denamycin, ericamycin, kirromycin, kundrymycin, mekemycin, microcins, nocardianin, nigrin, phytorubin, ractinomycin A,-B, rhodocidin, rhodomycetin, rubranigrin, streptorubin A,-B, steffisburgensimycin, umbrinomycin A,-B, A-10598, A,,,, AB-64, AH-14, B-21085, F-6, 0-2, X-1285, 54

4. AMINO ACID, PEPTIDE ANTIBIOTICS

4.1 4.1.1 4.1.1.1

4.1.1.2 4.1.1.2.1

4.1.1.2.2

Amino acid derivatives

SIMPLEAMINO ACIDS Diazo-, nitroso amino acid derivatives Amino acids Aliphatic amino acids

4.1.1.2.3

Amino acids with ether bond Alicyclic amino acids

4.1.1.2.4

Heterocyclic amino acids

4.1.1.3 4.1.2

Amino acid analogs ANTIBIOTICS DERIVED BIOGENETICALLY FROM AMINO ACIDS

4.1.2.1

@-Lactam antibiotics

Azaserine, diazooxonorleucine (DON), duazomycin A,-B,-C (alazopeptin, azotomycin), alanosin, hadacidin, primocarcin Armentomycin, ~-4-azaleucine,fumarylalanine, N-nitroglycine, 2-amino-4methyl-5-hexenoic acid (dehydroleucine), 2-amino-4-methoxy-lrans-3-butenoic acid, 2-amino-4-pentynoic acid, r,-lhreo-a-amino-p,r-dihydroxybutyric acid, 0-carbamyl-D-serine, N - (1-iminoethyl)ornithine, HON (6-hydroxy-roxonorvaline) , N-&-hydroxy-~-arginine, O-[~-norvalyl-5]isourea, ~-2-amino-4(2-aminoethoxy)-3-butenoic acid Canavanine, 4-oxalysine1 rhizobitoxin, dihydro-rhizobitoxin Anticapsin, cyclohexenyl-Z-glycine, L-2,5dihydrophenylalanine (FN-1636), MM-27 Azetidine-2-carbonic acid, azirinomycin, cycloserine, threomycin (furanomycin), U-42126 Aspergillomarasmin, lycomarasmin

378

Jh0.9 B h D Y

TABLE XIV (Continued) Code numbers 4.1.2.1.1 4.1.2.1.2

Representatives

Divisions Penicillin type Cephalosporin type

4.1.2.2

Pyrrothin type

4.1.2.3

Actithiazic acid type

4.1.3

DIKETOPIPERAZINE

4.1.3.1

Diketopiperasine antibiotics

4.1.3.2

Diketodithiopiperazine antibiotics Gliotoxin type

6-Aminopenicillanic acid, penicillins Cephalosporin C, N, deacetoxycephalosporin C, emercillopsin, cephamycin A, B, C, A-16886-11-II, A-16884 Auroethricin, holomycin, holothin, isobutyropyrrothin, thioaurin, thiolutin, thiomycin, VD 844, VD-846, HA-9, WS-1921 Actithiazic acid, lydimycin, a-methylbiotin, a-methyldethiobiotin

DERIVATIVES

4.1.3.2.1 4.1.3.2.2

Chaetocin type

Albonursin, mycellianamide, rhodotorulic acid, DCS [2,5-bis(aminooxymethyl)3,&diketopiperazine], 593 A, 3,&dioxohexahydropyrrolo [ 1,2,a]pyrazine Aranotin, acetylaranotin (LL-S-88 01)) apoaranotin, gliotoxin, gliotoxin acetate, dehydrogliotoxin, virocytin Chaetocin, 11,ll’-dihydroxychaetocin, chaetomin, melinacidin I,-11,-111,-IV, oryzachlorin, verticillin A,-B,-C Sporidesmin A,-B,-C,-D,-E,-F,-G Hyalodendrin and its trithia derivative Aspergillic acid, neoaspergillic acid, neohydroxyaspergillic acid, hydroxyaspergillic acid, mutaaspergillic acid, pulcherrimin, pulcherrimic acid, AO-1,2,3, etc.

4.1.3.2.3 4.1.3.2.4 4.1.3.3

Sporidesmin type Hyalodendrin type Aspergillic acid type

4.2 4.2.1

OLIGOPEPTIDES (INCLUDING

Homopaptidar DIPEPTIDES)

4.2.1.1 4.2.1.1.1

4 .2 . 1 . 1 . 2 4.2.1.2

Aminopyrrolecarboxylic acid peptides Netropsin type

Noformycin type Linear oligopeptides

Anthelvencin A,-B, azomultin, congocydin, distamycin, griseococcin, -D, kikumycin A,-B, netropsin, violacetin, A-4993 A,-B, S-685, 583, 12782 Noformycin, myxoviromycin (amidinomycin) Actinonin, karginyl-D-allothreonyl-Lphenylalanine, bacilysin (tetain), linatine, negamycin, leucylnegamycin,

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

379

TABLE XIV (Continued) Code numbers

4.2.1.3 4.2.2 4.2.2.1 4.2.2.2 4.2.2.3

Divisions

Cyclic oligopeptide-like antibiotics LINEARHOMOPEPTIDES Gramicidin A type

4.2.2.4

Edein type Probably linear other sulfur (1anthionine)-containing peptides Bacillomycin type

4.2.3 4.2.3.1

CYCLIC HOMOPEPTIDES Tyrocidine type

4.2.3.2 4.2.3.3

Bacitracin type Viomycin type

4.2.3.4

Ilamycin type

4.2.3.5

Various other cyclopeptides

4.3 4.3.1

CYCLOPEPTIDES WITH

4.3.1.1

Glumamycin type

4.3.1.2

Polymyxin type

4.3.1.3

Others

4.3.2

PEPTIDES WITH

Representatives L-(N6-phosphono)methionine-S-sulfoxyminylalanylalanine, phosphothricinylalanylalanine (SF-1293), stravidin (MSD-235) Sz,-S3, A-19009, K-16 Bicyclomycin (5879), ikarugamycin, griseoviridin Graniicidin A,-B,-C,-D (leucyl-, valyl-, isoleucyl-gramicidins), colisan, esein Edein-Al,-Az,-B,,-B2,-D Cinnamycin, duramycin

Bacillomycin A,-B,-C,-R, bacillocin, fungocin, mycosubtilin, toximycin, antib. AL Bresein, gramicidin S, tyrocidine A,-B, -C,-D,-E, B-456 Bacitracins Capreomycin 1,-11,tuberactinomycin A,-N,-0, viomycin (tuberactinomycin B), delysylviomycin, XK-33-F-2 Ilamycin A,-B1,-B2,-C1,-C2,rufomycin A,-B Alamethicin, alboverticillin, aspercolorin, aspochracin, cerein B2, epidermidines, fungisporin, malformin A,-B, mycobacillin, subsporin, triculamin, trichotoxin A, aspercolorin, I&

Heteromer peptides FATTY ACIDS

SULFUR

C ON TAI NI NG HETERO-

CYCLES

Amphomycin (aspartocin), crystallomycin, glumamycin, laspartomycin, parvulin, tsushimycin, eaomycin Circulin A,-B, jolipeptin, polymyxin A (M),-B,-C,-D,-E (colistin, colimycin), -P, polypeptin, thianosin, 4205-A, -B,-C, E-49 ~r,@,y,G Antiamoebin, iturin A,-B,-C, peptidolipid antibiotic, stilbellin, S-520

380

JdNOs B l h Y

TABLE XIV (Continued) Code numbers

Divisions

4.3.2.1 4.3.2.1.1

Thiazolyl peptides Thiostrepton type

4.3.2.1.2

Althiomycin type

4.3.2.1.3 4.3.2.2

Saramycetin type Bottromycin type

4.3.2.3

Other peptides probably with sulfur-containing heterocycles

4 .3 . 3

CHELATE-FORMING PEP-

4.3.3.1

Sideromycins (peptides with N-heterocycles) Albomycin-grisein type

Representatives

Micrococcin, sulfactin, siomycin (mutabillicin, sporangiomycin), thiostrepton, thiopeptin-B, A-59-A, 1456, 2337 Althiomycin, garlandosus, matamycin, RP-10206 Saramycetin (X-5079-C) Bottromycin Al,-A*,-B1,-B2,-C2,amethobottromycin, methobottromycin Actinothiocin, arsimycin, berninamycin, micropolysporin A,-B, peptiomycin A,-B, phalamycin, sulfomycin I,-II, -111, stysadin, sulfactin, thermothiocin, theiomycetin

TIDES

4.3.3.1.1 4.3.3.1.2

Ferrimycin type

4.3.3.1.3 4.3.3.1.4

Succinimycin type Pseudosiderom ycins

4.3.3.1.5

Sideramines

4 .3 . 3 . 2

Bleomycin type (glycopeptides)

4.3.3.3

Other chelate-forming antibiotics OTHER LESS KNOWN PEP-

4.3.4

TIDE ANTIBIOTICS

4.4 4.4.1 4.4.1.1

Albomycins (A,-As), griseins (1-8) (Ro5-2667, Ro-7-7730, 7731), alveomycin, A-1787, LA-5352, LA-5937, 1965, 3510 Ferrimycins (Al-Az,-B, pilosomycin), grisonomy cin Succinimycin (A-22765), danomycin Gluconimycin, ferramidochloromycin, ASK-753 Ferrichrysin, ferrichrocin, ferroverdin, nocardamin Bleomycin A1,-A,-B1,-Ba, about 80 biosynthetic bleomycins, phleomycins (C-I), eorbamycin, zorbonamycin B,-C, YA-56 X,-Y Actinomycellin, iaquirin 111, matchamycin, viridomycin, proviridomycin Almarcetin, botrocidin, chymostatin, histidomycin A,-B, hodydamycin, jolipeptin, gatavaline, leucinostatin, leucopeptin, lilacin, myroridin, practomycin, radicicolin, rhieomycin, yemenimycin, BA-17039 A, I.C.1-13595, NRC-101, RP-19042, 1415

Peptolider (Cyclic peptides with -CO-0linkage)

CHROMOPEPTOLIDES Actinomycin type

Actinomycin 1,-11,-111,-IV,-V,-V1,-VII and various actinomycin complexes

(A,-B,-C,-G,-H,-K,-M,-P,-S,-U,-X,-Z)

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

381

TABLE XI V (Continued) Code numbers

Divisions

Representatives

4.4.1.2

Echinomycin type

4.4.1.3

Various other chromopeptides

4.4.2

PEPTOLIDES WITH

and about 30 biosynthetic actinomycins (auranthines, actinolevalin, actinoleucine, pipecolic acid compounds, etc.), actinomycin monolactone, MT-10 Echinomycin (quinomycin A, levomycin, X-948, etc.), quinomycin B,-C,-D,-El triostin A,-B,-C, and about 10 biosynthetic quinomycin and triostin, guamycin, biquinaaomycin, quinazomycin, X-1008 Etabetacin, multhiomycin, taitomycin A,-B,-C,-D, R P 9671

FATTY

ACIDS

4.4.2.1

Enduracidin type

4.4.2.2 4.4.2.3

Stendomycin type Esperin type

4.4.3

PEPTOLIDES WITH N-CON-

4.4.3.1 4.4.3.1.1

4.4.3.1.2 4.4.3.1.3 4.4.3.2

Enduracidin A,-B,-C,-D,-SA,-SB, janiemycin Stendomycin A,-B (A-116 SA,-SO) Esperin, isariin I,-11,-111, surfactin (analysin, subtilysin, streptolysin 0 ) , viscosin

TAINING HETEROCYCLES 3-Hydroxypyridinecarboxylic acid-containing antibiotics Staphylomycin (virginiamycin) S,-SI,-S~, Virginiamycin type -S3, vernamycin Ba-Ba,-C (doricin), (mikamycin B, pristinamycin-I, synergistin-PA-1 14, ostreogrycin B), ostreogrycin Ba, lathumycin, patricin Etamycin (viridogrisein, F-1370 A, 6613, Etamycin type K-179) Pyridomycin (eryzomycin) Pyridomycin type Monamycin A,-B,-C,-D,-E,-F,-G,-H,-I, Monamycin type bromomonamycin 1,-2,-3

4.4.4

PEPTOLIDES WITHOUT

4.4.4.1

Telomycin type

4.4.4.2

Grisellimycin type

4.4.5 4.4.5.1

UEPSIPEPTIDES Valinomycin-enniatin type

OTHER CONSTITUENTS

Telomycin, neotelomycin OP,-P, LLAO-341 A,-B, C-159 Cycloheptamycin, destruxin A,-B, grisellimycin A,-B (RP-l1072),-C,-D Avenacein, enniatin A,-B,-C (baccatin, lateritin I), fructigenin, fusafungin, lateritin 11, sambucin, valinomycin (aminomycin), yakusimycin C, beauvericin

382

J ~ N O SB ~ R D Y

TABLE X I V (Continued) Code numbers

Representatives

4.4.5.2

Serratamolide type

4.4.5.3

Ostreogrycin A type (depsipeptide-like antibiotics)

4.5 4.5.1 4.5.1.1

4.5.1.2 4.5.1.3

High molecular weight peptider

POLYPEPTIDES Acidic polypeptides (unknown structures) Basic polypeptides (unknown structures) Amphoteric polypeptides (unknown structures)

4.5.2 4.5.2. I 4.5.2.1.1

PROTEINS Anticancer proteins Acidic proteins

4.5.2.1.2

Basic proteins

4.5.2.1.3

Amphoteric proteins

4.5.2.2

Serratamolide A,-B, sporidesmolide I-IV, Is,-I, from Pseudomonas rollandii Ostreogrycin A,-G (mikamycin A, pristinamycin 11, staphylomycin M, streptogrammin A, PA-114 A, vernamycin A), mesenterin

Other proteins

Brevin, globicin, komamycin A,-B, phytoactin, phytostreptin, polcillin, rhizobacidin, suzukacillin A,-B, N-44-A-21, x-73 Alvein, brevolin, laterosporin, mareesin, ussamycin, XK-19-2, 6431-36 Bacillin, chalcidin, comirin, cryomycin A,-B, fungistatin (XG), fluvomycin (efsiomycin, riomycin, vivicil), lactolin, lichenoformines, nisin, pumilin, saihochin A,-B, subtilin, syringomycin, subtenolin, trichomatycin, AB-22, B-344 A,-B,-C, E-91, Nx-1, 17-41 A, 175, 362, 2725 Neocarzinostatin A,-B,-C, actinoxanthin, bioactin (2135), cephalomycin, iyomycin A, lymphomycins, macroinomycin, melanomycin, mitomalcin, raromycin, A-280 Actinin (mycetin), actinocarsin, carcinomycin (ganmycin), enomycin, flammulin, phenomycin, peptimycin, stellin, triprotamine, thymostatin, A-216, M-741, M-6672, TPP C-2 Abrin, calvacin, carzinostatin A, carzinocidin, kunomycin, marinamycin, mitogillin, mutamycin, neocid, pacibillin (PCC-45), pepticarcin, regulin, restrictocin, ricin, a-sarcin, yesivin, BU-306, “M-substance,” sanitamyein, 10484 Amodin A, bactericidin, caseicidin, cuticullin A,-B, cyclopin, diplococcin, helicidin, lactobrevin, leukin, magnopeptin, malucidin, melittin, mycomycetin, phagocytin, purothionin, streptavidin

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

383

TABLE XIV (Continued) Code numbers

Divisions

Representatives

L (MSD-235-L), “thymus factor,” KA-107, MM-4550, MC-696 SY-2 4.5.3 4.5.3.1

PROTEIDS Chromoproteids

4.5.3.2

Glucoproteids

4.5.3.3

Nucleoproteids (viral particles)

4.5.3.4

Enzymes and enzymelike factors

4.5.3.5

Bacteriocines

4.5.3.6

Interferon-like substances

Celikomycin, largomycin I,-11,-111,micromonosporin, plurallin, poricin, prunacetin A, pyoverdin, roseolic acid, spirillomycin, TA-2590 Actinogan, “boivin substance,” calvatin, coliformin A,-B, deutomycin, mercenene, mycobactocidin, streptogan, AVS, St.-142, 81, etc. Helenin, hortensin, xerosin (APM), EAP, LB-51 (antibioticum bulgaricum), 19514/1, 6-MFA Actinolysin, actinomycetin, ascaricidin (ascarinase), asparaginases (glutaminases), bacteriolysin, cellolidin (protoplast dissolving factor), canavalin, lysozymes, “lytic enzyme from B. cereus,” lysostaphnin, mucinase, notatin (glucose oxidase, penatin, corylophyllin, microcid, etc.), polysaccharidase, streptozyme, subtilin-11, ALE, LA-7, S-35, Z-1 AA, etc. Colicins, clostocins, enterocin, hemolysins, megacins, pesticin, phagicin, pyocines, staphylococcines, thermocin, vibriocin and various other bacteriocines, boticin, pneumocin, thuricin, protescin, pyocin B-39 Interferon, inhibitor E, antivirin, etc.

5. NITROGEN-CONTAINING HETEROCYCLIC ANTIBIOTICS

5.1

Noncondensed (single) heterocycles

5.1.1 5.1.1.1

FIVE-MEMBERED RINGS Pyrrolnitrin type

5.1.1.2

Prodigiosin (tripyrrol) type

5.1.1.3

Pyrrolidenon antibiotics

Pyrrolnitrin, isopyrrolnitrin, 2-chlor-, 4’-flour-, 3‘-dechlorpyrrolnitrin, bromonitrin A,-B,-C, pentabrompseudilin, rugosin H, luteolin, pyoluteorin, aerugonic acid Prodigiosin, norprodigiosin, nonyl-, undecyl-, metacycloprodigiosin, pelletrin, vitamycin A, 1435/4

384

J ~ N O SB ~ R D Y

TABLE XIV (Continued) Code numbers

Divisions

5 . 1 . 1 . 3 . I. 5.1.1.3.2 5.1.1.3.3 5.1.1.4

Tenuazonic acid type Streptolydigin type Oleficin type Pyrrolidine antibiotics

5.1.1.5

Five-membered ring with 2 heteroatoms

5.1.2 5.1.2.1

Simple pyridine derivatives

5.1.2.2 5.1.2.3

Mocimycin type Piperidine derivatives

5.1.2.4

Heterocycles condensed with alicyclic ring Six-membered rings with 2 heteroatoms

5.1.2.5

Representatives Tenuazonic acid, magnesidin Streptolydigin, tirandamycin Oleficin, lypomycin a,+, erythroskyrin Anisomycin, ahygroscopin A, desdanin (pyracrymycin A, cyclamidomycin), desdamethine, ethesdamin, jatropham, pyracrymycin B, verrucarin E Azomycin, 3-nonylpyrazole, uroconic acid

SIX-MEMBERED RINGS

5.1.3

PYRIMIDINE A N D SIMILAR

5.1.3.1 5.1.3.1.1

Cytosin N-glycosides Amicetin type

5.1.3.1.2

Blasticidin S type

5.1.3.1.3

Gougerotin type

Caerulomycin, fusaric acid, dehydrofusaric acid, fusariolic acid, a-picolinic acid, piericidin A,-B Mocimycin (MYC-8003), X-5108 Ilienomycin A,-B,-C, dihydropiperine, flavipucine (glutamycine), nigrifactin, oryzocydin, solenopsin A,-B,-C Abikoviromycin (lathumcidin), dehydroabikoviromycin, virocidin Bacimethrin, emimycin, sparsomycin

GLYCOSIDES

5.1.3.1.4 5.1.3.1.5 5.1.3.2 5.1.3.2.1

Azacytosine glycosides Ezomycin type Uracil N-glycosides Polyoxin type

5.1.3.2.2

Mycospocidin type

5.1.3.3

Various (5-6 membered rings) C-glycosides

5.2 5.2.1

HETEROCYCLES CONDENSED

5.2.1.1

Indole derivatives

Amicetin A (allomycin, sacromycin), amicetin B (plicacetin), (R-285), amicetin C, bamicetin, grisamine Blasticidin S (cytovirin), cytomycin, cellostatin, pentopyronines, U-1 Aspiculamycin, gougerotin (asteromycin), hikiaimycin 5-Azacitidin Ezomycin Al,-BI,-C Polyoxin A,-B,-C,-D,-El-F,-GI-HI-I,-J,-K -L,-MI-N,-0, harimycin, piomycin, 5-fluoropolyoxin L,-M Mycospocidin, streptovirudines, tunicamycin, 323/58, 9408, 24010 Minimycin (oxazinomycin), pyrazomycin A,-B, showdomycin

Condensed (fused) heterocycles WITH AROMATIC RING(S)

Indolmycin, indole-3-acetonytril, -indolylacetic acid, bufotenin, echinulin,

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

385

TABLE XIV (Continued) Code numbers

5.2.1.2

Divisions

5.2.1.3

Quinoline-quinoxazoline derivatives Phenazine derivatives

5.2.1.4

Phenoxazine derivatives

5.2.1.5

Albofungin type

5.2.1.6

Various condensed heterocycles

5.2.2

CONDENSED HETERO-

5.2.2.1

Anthramycin (benzdiazepine) type

5.2.2.2

Purine, azapurine derivatives Fervenulin (pyrimidotriazine) type

Representatives pimprinin, teleocidin A,-B, tryptophol, serotonin, violacein, NP-522, 83, “bromoindole antibiotics” Pyo compounds (Ia,-Ib,-Ic,-II,-III,-IV), substances B-A, BB, B-C, MSD-819 Pyocyanine (cyanomycin), hemipyocyanine, chlororaphin, griseolutein A,-B, lomondomycin, oxychlororaphnin, piricularin, ruticin, tubermycin A,-B, 1,6dihydroxyphenazine, l,&dimethoxyphenazine, 6methoxy-1-phenazinol, 1-methoxyphenasine, 1,Bphenazine dicarboxylate, F-10 B, Px, 7413, iodinin, myxin, crysalloiodinin, l-phenazinol10-oxide, caulerpin Cinnabarin, questiomycin A, 2-acetamidophenoxazone, 2-amino-1-carboxy-3H-p henoxazin-3-on Albofungin A,-B (chlor-albofungin), kanchanomycin, BA-181314, P-42 Deoxynybomycin, febrifungin, lycoricidinol, narciclasine, nybomycin, tryptanthrin, 2(3)-benzoxazolinone, 6-methoxy-2 (3)-benzoxazolinone

CYCLIC RINGS

5.2.2.3

5.2.3

P U R I N E AND PURlNELIKE

5.2.3.1 5.2.3.1.1 5.2.3.1.2

Purine type N-glycosides Purine glycosides Simple adenine glycosides

Anthramycin (refuin, A-6413, M-259), cyclopenin, cyclopenol, dextrochrysin, sybiromycin, tomaymycin Lentinacin, hormycin, thioguanine, pathocidin (8-azaguanine), vasocidin Fervenulin (planomycin), 2-methylfervenulon (MSD-92), reumycin, toxoflavin (xanthotricin), 10204-B 11, iso-xanthopterin, pseudoiodinin

QLYCOSIDES

Nebularin (9-8-D-ribofuranosylpurine) Angustmycin A (angstomycin, decoyinin), angustmycin C (psicofuranine), Ara A (9-8-D-arabinofuranosyladenine), cordycepin, nucleocidin (T-3018), 3’-amino-3’-deoxyadenosine, 3-acetamido-3-deoxyadenosine,A-9145

386

J ~ N O SB ~ R D Y

TABLE XIV (Continued) ~-

Code numbers 5.2.3.1.3 5.2.3.1.4

5.2.3.1.5 5.2.3.2

5.2.3 5.3

Divisions Complicated adenine glycosides Deazaadenine glycosides

Representatives Puromycin, septacidin

Monillin, sangivamycin (B-14437), toyokamycin (ahygroscopin B, naritheracin, vengicid, unamycin B, 1037), tubercidin (sparsomycin A), B-15645 Adenine glycoside analogs Aristerom y cin C-glycosides, formycin type Formycin A, formycin B (laurusin), coform ycin, kirigamycin A,-& oyam ycin, raurycin Ablastmycin, bulgerin, canarius, sarcidin, OTHER LESS KNOWN RP-6798, SF-98, E-352 GLYCOSIDES Achyranthin, aconitine, acromycin, anaAlkaloids with antibiotic basine, berbamine, berberine (cheldo[antitumor) activity xanthine), camptotecin, carpaine, casimiroin, cassilysine, cassipurine, cepharanthine, chackranin, chacsin, canthin-6-one, cheleritrin, chelidonine, homochelidonine, chinin, cissamparaine, citriodorol, colochicin, coptisine, coronaridin, coryanthin, crosemperin, cryptopleurine, cyclobuxine-11, “dauricin-like,” demethyltolyphorinine (compound C), deacetylvinealeucoblastine, ellipticin, emetine, fargarinone, febrifungin, isofebrifungin (dichroin), gerrardin, glaucine, gindarinin, gindricinc, girinimbin, gramin, harman, norharman, harringtonine, deoxyharringtonine, ibogatine, jatrorrhizin, koputin, leurosin, lutenurin, mahanimbin, matrine, oxymatrine, marmalin, obomegine, ockrobamine (ochopannine), olivacin, oxyacanthin, oxoaporphine, palmitine, peltatine, perivine, physostigmine, piptanthin, plakellin, dibrom-plakellin, protopin, pteleatine, rugulovasin-A,-B, sanguinarin, shikonin, spartein, sphaeroplysin, thalicflavin, thalidasine, thalrugosamine, thilophoridine, trilobin, tylocrebin, umbellatin, valerin, vinblastine, vincristine, vinalin, 2-X

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

387

TABLE XTV (Continued) Code numbers

Divisions

Representatives

6. OXYGEN-CONTAINING HETEROCYCLIC ANTIBIOTICS

6.1 6.1.1 6.1.1.1 6.1.1.2 6.1.2 6.1.2.1

Furan derivatives

SIMPLEFURANS Monofurans (tetrahydrofurans) Ilifuran (lignan) type FURANS CONDENSED Aflatoxin type

Altenin, botryodiploidin, carlina oxide, ipomearon, oudenon, sphydrofuran Asarinin, eudesmin, pinoresinol, sesamin Aflatoxins, aspertoxin, parasiticol, sterigmatocystin, 5-, Gmethoxysterigmatocystin Genipic acid, genipinic acid

6.1.2.2 6.1.3 6.1.3.1 6.1.3.2 6.2 6.2.1 6.2.1.1 6.2.1.2

Other condensed furans BENZOFURAN DERIVATIVES Dibenzofuran derivatives Usnic acid type

6.2.2 6.2.2.1

a - P ~ ~ Asperline type

6.2.2.2

Other simple a-pyranones

6.2.2.3

a-Pyranones condensed with alicyclic ring a-Pyranones with aliphatic- Alternaric acid, bisnoryangonin, goniothalamin, hispidin, kawain, dihydroaromatic side chain kawain, luteoreticulin, 74-7 Y-PYRANONES Aureothin, distacin, luteothin, mycoAureothin type lutein, neoaureothin, F-10-C, 74-a, 2814 K Citreoviridin Citreoviridin type Funicone, kojic acid, LL-Z-1220, A-1, Other simple y-pyranones R-2 (kojic acid dimer)

6.2.2.4

6.2.3 6.2.3.1

6.2.3.2 6.2.3.3

Didymic acid, strepsilin Usnic acid, isousnic acid, C-2167

Pyran derivatives

SIMPLEPYRAN DERIVATIVES Single pyrans Condensed pyrans ~

6.3

Benr-y-pyran (fiavone) derivatives

6.3.1

SIMPLEFLAVONE:

k

Centrolobin, funginon, oleuropein Aucubin, plumericin, valtratum, isovaltratum ~ ~ ~ ~ ~ Asperlin, deacetoxydeoxyasperlin, phomalactone, 3-(1,2-epoxypropyl)-5,6dihydro-5-hydroxy-6-methylpyran2-on, LL-Z-1276 Dehydracetic acid, dhalwengine, mucidone, parasorbic acid, sorbic acid, LL-P 880 01 Fulvoplumierin, irridomyrmercin

DERIVA-

TIVES

6.3.1.1

Flavone type

Eupafolin, eupatorin, eupatilin, flavone, hesperetin, luteone, nobiletin, pinocembrin, tageritin

388

JANOS

B ~ D Y

TABLE XIV (Continued) Code numbers

Divisions

6.3.1.2

Flavonol type

6.3.1.3 6.3.1.4

Flavanone type Anthocyanides

6.3.2 6.3.2.1

Simple isoflavones

Representatives Avicularin, centaureidin, cbloroflavonin, datiscetin, eupalitin, eupatin, eupatorein, galangine, guaijaverin, morin, leucocyanidol, quercetin, rhamnetin, urensin Naringenin, toxifolin Amylocyanin, coelicolorin, cyanidin, litmocidin, litmomycin, mycorhodin, rubidin, A-25, XK-46, 1016-31, 1321

IBOFLAVONES

6.3.2.2

Pterocarpan (phytoalexin) derivatives

6.3.2.3 6.3.3 6.3.3.1 6.3.4

Rotenone type NEOFLAVONES Dalbergion type OTHER CONDENSED

6.3.4.1

Dibenz-(71-pyranone derivatives

6.3.4.2

Morellin type

6.3.4.3

7-Pyranones condensed with other ring

Biochanin (A-I), pratol (formomenthin, A-2) Maackianin (demethylpterocarpin), pterocarpin, homopterocarpin, medicarpin, kieviton, phaseolin, hydroxyphaseolin, phaseolinisoflavone, phaseolidin, pisatin, saphojaponicin, trifolirrhizin, tuberosin Rotenones, deguelin, etc. Dalbergion, methoxydalbergion, etc.

y-PYRANONES

0.4 6.4.1 6.4.1.1

Mangostin, pinselin, secalonic acids (chrysergic acid, enthotein), 1,6-dihydroxyxanthon Guttiferines, guttiferinic acids, morellin, morellin M, isomorellin (gambogic acid) Fomannosin, frequentic acid (cytromycetin) radicinin (stemphylon), LL-D253 y

Small lactoner

SIMPLELACTONES (4-5 membered lactones) Four-membered &lactones

6.4.1.2

Five-membered simple 7-lactones

6.4.1.3

Lichesteric acid type

4-Acetamido-4-hydroxy-2-butenicacid lactone, 1233 A Acetomycin, anemonin, protoanemonin, narithresin, tulipalin (a-methylen-rbutyrolactone), 2-acetamido-2,5-dihydrod-oxofuran, 2( 13-carboxy-14,15diacetoxyhexadecenyl)-2-penten-4olide, 4-hydroxy-3-methoxy-2-buten4-olide Lichesteric acid, nephromopsic acid, nephrosterinic acid, picacic acid, protolichesteric acid

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

389

TABLE XIV (Continued) Code numbers 6.4.1.4

6.4.1..5 6.4.1.6 6.4.2

Divisions

Representatives

Aspertetronin, penicillic acid, tetrenolin, PA-147, 2-carboxymethyl-3-N-hexylmaleic anhydride Simple dilactone derivatives Avenaciolide, canadensolide, dihydrocanadensolide, ethisolide Calycin, pinastric acid, pulvinic acid Vulpic acid type lactone, vulpic acid, xerocomic acid CONDENSED SMALL

Tetramic acids

LACTONES

6.4.2.1

6.4.2.2

6.4.2.3

6.4.2.4

6.4.2.5

6.5 6.5.1 6.5.1.1

6.5.1.2

Burseran, monascin, patulin (clavacin), peltatines, pentanelolactone (PA-132), podophyllotoxins, etc. Mycophenolic acid, -methyl ester, mycoCoumaron derivatives chromenic acid, nidulol, 5-methoxycoumaron Coumarin (benzo[a]pyrone) Esculetin, callophyllic acid, daphnetin, dicoumarol, herniarin, aurapten, galderivatives banic acid, kotanin, mammein, marmin, marmesin, ostruhin, pilosellin (umbelliferone), prangolarin, scopoletin, toddalolactone, mesuol, mesuon, surangin A,-B Coumarins condensed with Ayapin, bergapten, byakangelicin, imperatorin, luvangetin, peucedanin, other ring pimpinellin, psoralen, psoral aldehyde, sesellin, xanthiletin, xanthotoxjn Isocoumarin (3,4-benzo[a]- Actinobolin, canescin A,-B (malignin), cladosporin, diaporthin, duclauxin, pyrone) derivatives mellen, 5-methylmellein, Gmethoxymellein, monocerin, ochratoxin A, -B,-C, oosponol, reticulol, 4-acetyl6,8-dihydroxy-5-methyl-isocoumarin, 4-(2-hydroxyacetyl)-8-hydroxyisocoumarin, 8-hydroxy-6-methoxy-3-rnethylisocoumarin, 5-chlor-, 7-chlor-, 5,7dic hlor-3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin

Small lactones condensed with hetero- or alicycles

Polyether antibiotics

FURAN-PYRAN POLYETHERS Nigericin type

Unsaturated polyether antibiotics

Grisorixin, monensin A,-B,-C,-D, nigericin (asalomycin M, helixin C, polyetherin, K-178,X-464), deoxynigericin (K-358), X-206, A-204 A,-B, A-28695 A,-B Dianemycin,'A-130 A

390

JANOS

B~RDY

TABLE XIV (Continued) Code numbers 6.5.1.3 6.5.1.4

Divisions Aromatic polyethcr antibiotics Other polyether-like antibiotics

Representatives X-537 A Camphomycin, imoticidin, ramnacin, rotaventin, salinomycin

7. ALlCYCLlC ANTIBIOTICS

7. I 7.1.1

CYCLOPENTANE DEKIVA-

7.1.1.1

Simple cyclopcntanes

7.1.1.2

Cyclopentenonc derivatives

7.1.1.3

7.1.3

Complicated cyclopentane derivatives CYCLOHEXANE DERIVATIVES Fumagillin, ovalicin, graphinone, crotFumagillin type epoxide Cyclohexenone derivatives Frequentin, pallitanthin, humulone, lupulone (iso- and adhumulones and lupulones), humulinone Cyclohcxene-cyclohexaKetomycin (cyclohexenyl-1-glyoxalic acid), cyclohexenyl-1-hydroxyacetic diene derivatives acid, abscisin, dormin, oryzoxymycin CYCLOHEXIMIDE-CLUTARI-

7.1.3.1

Actidione type

7.1.3.2

Actiphcnol type (aromatic glutarimides) Streptimidone type (aliphatic glutarimides) Other less known glutarimides

Cycloalkane derivatives TIVES

7.1.2 7.1.2.1 7.1.2.2 7.1.2.3

Caldariomycin, sarkomycin, vertimycin, xanthocidin, “substance 111” from Periconia macrospinosa Cryptosporiopsin, methylenomycin A,-B, oudcnon, pentanenomycin 1,-11,terrein, “substances I and 11” from Sporomia a f i n i s (2-t-allyl-3-chlor-lhydroxy-4-0x0-2-cyclopentene carboxylic acid derivatives) Pactamycin (cranomycin)

M I D E ANTIBIOTICS

7.1.3.3 7.1.3.4 7.2 7.2.1

Small terpenes

SIMPLETERPENES

Cycloheximide (Actidione, naramycin A, A-67), dehydrocycloheximide, d-isocycloheximide, inactone, levoristatin F, fermicidin, naramycin B, niromycin A,-B, streptovitacin A,-B,-C,,-C,, -D,-El E-73 (acetocycloheximide) Actiphenol (C-73), C-73-X Protomycin, atreptimidon Inomycin, moldcidin A (J-4-A), “niger factor,” streptocin, viractin

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

391

TABLE XIV (Continued) Code numbers

Divisions

7.2.1.1

Monoterpenes

7.2.1.2

Sesquiterpencs

7.2.1.3

Diterpenes

7.2.1 , 4

Sesterterpenes

7.2.1.5

Terpene glycosides

7.2.2 7.2.2.1

Sesquiterpene lactones

7.2.2.2

Diterpene lactones

7.2.2.3

Various complicated terpene lactone derivatives

Representatives Bornyl acetate, cannabidol, cannabidiolic acid, chamovic acid (dihydrothujic acid), dormin (cineol) a-d-Bisabolol, helminthosporal, helminthosporol, capsidiol, hinociic acid, lubimin, rishitin, 12-carboxyeudesma3,11(13)-diene, selina-4( 14),7(11)dien-9-01, LL-Z-1271-& arenamycin C,-D,-E, chinoic acid, coriolin A,-B,-C, illudin M, illudin S (lunamycin, lampterol), hirsutic acid A,-N, aonarol, isozonarol Aphidicolin, cyathine A3,-Bar-C1, allocyathines, eupacunin, isoroseolic acid, jatrophon, liatrin, oregonensin, pachydictyol A, phytuberin, pleuromutillin (drosophyllin B), siccanin, stemodin, triptolide, tripdiolide Ophiobolin A (cochliobolin A), ophiobolin B (zizanin B, cochliobolin B, ophiobolosin A), ophiobolin C (aieanin A), ophiobolin D (cephalonic acid), ophiobolin F, cochliobolin C, ophiobolosin B, variabilin Virescenoside A,-B,-C,-D,-E,-F,-G, anagallosides

TERPENE LACTONES Alantolactone, angustibalin, arctiopicrin, arteglasin, canin, costunolide, crepin, cnicin, damsin, elephantin, elephantopin, encelin, eremathine, eriolangin, eriolanin, eupachlorin, euparotin, eupaserrin, deacetyleupaserrin, eupatolide, fomannosin, gaillerdin, ludovicin, helenalin, marasmic acid, molephanthin, parthenolide, psilostachin, paucin, ridentin, tulipinolide, vernolepin, vernodalin, vernomygdin, xanthatin, xanthinin, LL-Z-l271a,y Enmein, enmein 3-acetate, eunicin, provincialin, rosololactone (rosein 11), stegenacin, stegenanin, LL-S-491-P,r Bruceanthin, fusariocines, fusicoccines, glaucarubin, holacanthon, hyperforin, rubratoxin A,-B, scytalidin (scytalidic acid), taxol

392

JANOS

B~RDY

TABLE XI\.’ (Continued) Code numbers 7.3 7.3.1

Divisions

Representatives

Oligoterpene antibiotics

ANTIBIOTICSWITH

STEROID

SKELETON

7.3.1.1 7.3.1.1 , 1

7.3.1.1.2

Steroids Fusidic acid type

Polyporcnic acid type

7.3.1.2

Steroid alkaloids

7.3.1.3

Cardcnolides, bufadienolidcs, withanolides, sterol glycosidcs

7.3.1.4

Steroid analogs--viridin type

7.3.1.5 7.3.2 7.3.2.1

Asasteroids TRITERPENES Simple triterpcnes

7.3.2.2

Triterpene glycosides

7.3.2.3

Saponins

7.3.3 7.3.3.1 7.3.3.1.1 7.3.3.1.2

TEKPENOIDS Scirpene type Trichothecin type Trichodermin type

Cephalosporin P1,LP2,-P3,-P4,isoccphalosporin P1methyl ester, monodcacetylcephalosporin PI, fusidic acid (ramnacyn), dehydrofusidic acids, helvolic acid, 7-deacctoxyhelvolic acid, hclvolinic acid, viridominic acid A,-B,-C, EPF Campestrin, cucurbitacines (colocynthin, elatericines), eburicoic acid, echinodol, datiscosid, datiscacin, obliquol, paecylornycerol, polyporenic acid A (ungulinic acid), -C (gypsogenin) Conessin, demissin, solanocapsin, solanine (chaconine), tomatin (lycopersicin) Abocanosin, amplexoside A, bersaldigenines, calotropin, gitoxigcnin, hellebrigenine acetatcs, oleandrigenin, rhodexin B, withafterincs, withanolides Viridin, viridiol, wortmannin (SL-2052), 11-deacetoxy-wortmannin (5-3 1’J6), herquenin A-25822 A,-B,-D,-H,-L,-M,-N Glycirrhetinic acids, oleanic acid, psidiolic acid, squalene, seorin Escin, asiaticoside, oxyasiaticoside, avenacin, caulosides, gymnemic acid, muscunin, stichoposides Acerotin, anagallosid, calagualine, convallamaroside, cyclamin, entagenic acid, eupteleosid A,-B, forsythin, hederin, hirudigenin, holothurin A,-B, holotoxin, “myrsene saponin,” parillin, “saponin P,” “spinasaponin,” ASP (asterosaponin)

Trichothecin, crotociri, cephalotecin Trichodermin, trichodermol (roridin C), anguidin (diacetoxyscirpenol), deacetylanguidin, calonectrin, nivalenol, acetylnivalenol (fusarenone), diacetyl-

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

393

TABLE XIV (Continued) Code numbers

Divisions

7.3.3.2

Scirpene lactones

7.3.3.3

Other scirpenelike toxins

Representatives nivalenol, triacetylnivalenol, neosolaniol, T-2 toxin, HT-2 toxin, A-2 (diacetyl-verrucarol), MM-4462 Dendrodochin, roridin A,-B,-D,-E,-H, verrucarin A (muconomycin A), verrucarin B,-C,-D,-F,-G,-J,-K (glutinosin, myrothecines, 379-Y), 2-dehydroverrucarin A, 602 A Ncmatolin, necrocitin

8. AROMATIC ANTIBIOTICS

8.1 8.1.1

Benzene compounds

MONOCYCLIC BENZENIG DERIVATIVES WITHOUT OTHER CONSTITUENTS

8.1.1.1

Simple phenols

8.1.1.2

Phthalyl aldehyde derivatives

8.1.1.3

Simple carboxylic acids

8.1.1.4

Other benzene derivatives

8.1.2

MONOCYCLIC BENZENE

Amudol, catechol, chromocyatin, drosophyllin A, inethyldrosophyllin A, gentisyl alcohol, chlorgentisyl alcohol, gentisin acetate, miconidin, questiomycin B (o-aminophenol), versicolin (2,3,6-trihydroxytoluene),2,5-dimethoxy-3,6-dimethylhydroquinone, 2,4diacetylphloroglucine, 4-methoxyresorcylaldehyde, PH-A Cyclopaldic acid, flavipin, fomecin A,-B, gladiolic acid, dihydrogladiolic acid, quadrilineatin Caffeic acid, gallic acid, gallic acid methyl and ethyl esters, gentisic acid, 6-methylsalicylic acid, 4-hydroxybenzoic acid, sparassol, ustic acid Peyocactin (hordenine), phenethyl alcohol, p-phenylethyl isothiocyanate, p-hydroxybenzyl isothiocanate, benzyl-2-hydroxyethyl trisulfide

DERIVATIVES WITH ALIPHATIC SIDE C H A I N

8.1.2.1 8.1.2.1.1 8.1.2.1.2

Benzenes with peptide linkages Chloramphenicol type Others

Chloramphenicol, bromamphenicol, dechlorchloramphenicol, corynecin A,-B,-C, tuberin, N-acetyltyramine Spinamycin, viridicatin, viridicatol

394

JINOS B~RDY TABLE XIV (Continued)

Code numbers

Divisions

Representatives

8.1.2.2

Benzenes with terpene side chain-ascochlorin type

8.1.2.3

8.1.3

Benzenes with aliphatic side chain (saturated) Benzenes with polyene side chain Benzenes with polyine side chain ISOLATED POLYCYCLIC

Ascochlorin (LL-Z-1271-7), ascofuranon, colletochlorin, cylindrocladin A,-B, cylindrochlorin A,-B,-C, chloronectrin, grifolins, ilicicolin A,-B,-C,-E,-F, -G,-H, LL-Z-1272 a,P,A,(, ICI-232573 Panosialines

8.1.3.1

Xanthocillin type

8.1.3.2

Stilbene derivatives

8.1.3.3

Diphenyl derivatives

8.1.3.4

Other isolated polycyclic compounds

8.1.2.4 8.1.2.5

Aspergin, anacardic acids, demetric acid, fumigachlorin, pyriculol, 31-1, 31-2 Agropyren, capillarin, capillen, capillin, phenylhepta-1,3,5-triyne,siccayne

B E N Z E N E DERIVATIVES

8.2 8.2.1 8.2.1.1

Griseofulvin type

8.2.1.2 8.2.2 8.2.2.1 8.2.2.2

Geodin type NAPHTHALENE DERIVATIVES Carzinophyllin type Others

8.2.3

ANTHRACENE-PHENAN-

8.2.3.1 8.2.3.2 8.3 8.3.1 8.3.1.1

Anthracene derivatives Phenanthrene derivatives

8.3.1.2 8.3.2 8.3.2.1

Others AZULENE Lactaroviolin type

Xanthocillin X,-Y,,-Y,,-Z, methoxyxanthocillin X dimethylether, xanthocillin X mono- and dimethyl ethers, emerin, endrin, puberulin Chlorophorin, obtusastyren, pinosylvin, pinosylvin monomethyl ether, tetrahy droxystilbene Alternariol, altenuene, altenuisol, ellagic acid Curcumin, nordihydroguiaretic acid, phloretin

Condensed aromatic compounds SPIRO COMPOUNDS

Annualene, annujane, griseofulvin, dechlorogriseofulvin, bromogriseofulvin, hydroxygriseofulvin Geodin, erdin, estin, nordin, trypacidin Asperflavin, carzinophyllin A Gossipol, scytalone, 3,4-dehydro-3,4,8trihydroxy-l[2H]naphthalenone

THRENE DERIVATIVES

Barbaloin, thermorubin A, BT-3-3 Aristolochic acids, hirchinol, orchinol

Nonbenzoid aromatic compounds

TROPOLONES Puberulic acid type

Puberulic acid, puberulonic acid, stipi, tatic acid, thujaplicin ' ~ , @ , ythujic acid Nootkatin, sepedonin, GF-1 Lactaroviolin

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

395

TABLE XIV (Continued) ~

Code numbers

~~

Divisions

8.4

Various derivatives of aromatic compounds

8.4.1 8.4.1.1

AROMATICETHERS

8.4.1.2 8.4.2

Aromatic-aliphatic ethers

8.4.2.1

Hygromycin A type (benzene glycosides)

8.4.2.2

Chartreusin type (naphthalene glycosides) Coumarin glycosides Novobiocin type

Diphenyl ether derivatives

Representatives

LL-Ir-125a, bromodiphenyl ethers (5 compounds) from Dysidea herbacea Zinninol

AROM.4TIC GLYCOSIDESGLYCOSIDIC ANTIBIOTICS

8.4.2.3 8.4.2.3.1 8.4.2.3.2 8.4.3 8.4.3.1 8.4.3.1.1

8.4.3.1.2

Coumermycin type ESTERS AROMATIC Depsidones Nidulin type

Depsidones from lichens

8.4.3.2

Depsides

8.4.3.3

Phenol esters

8.4.3.4

Other aromatic esters

Hygromycin A (homomycin), hygromycin C,-D,-E,-F, sporaviridin, totomycin, 11-906, 1703-18 B Chartreusin A (X-465 A, G-72, 68-36, (2-261 A, etc.), coerulomycin, G-261 B Novobiocin, methoxynovobiocin, chlorobiocin (RP-18631), antiprotozin, 5-800 Coumermycin A1,-A2, sugordomycin D-la,-lb,-lc,-ld, D-2, D-3, D-4 Nidulin, nornidulin (ustin I), dechlornornidulin (ustin 11), estin, terrecin, yasimin (trisdechlornornidulin), haiderin, rubinin, skirin Collatolic acid, diploicin, fumarcetraric acid, lobaric acid, pannarin, physodic acid, psoromic acid, protocetraric acid, salazinic acid Atronarin, anziacic acid, barbatic acid, boninoic acid, caperidin, diffractic acid, divaricatic acid, everinic acid, gyrophoric acid, lecanoric acid, obtusatic acid, olivetoric acid, perlatolic acid, picrolichenic acid, ramanilovic acid, sekiacic acid, spherophorin, thammnolic acid, usminic acid Selovicin, -B, tannic acids, gallotannin, etc. Benzylgentisinate, chlorogenic acid, echinacoside, phlorizin, trichocarpin

9. ALIPHATIC ANTIBIOTICS

9.1

Alkane derivatives (excluding carboxylic acids)

9.1.1

SIMPLEALKANE TIVES

DERIVA-

396

J ~ N O SB ~ R D Y

TABLE XIV (Continued) Code numbers

Divisions

Representatives

9.1.1.1

Hydrocarbons, alcohols, 0x0 derivatives

9.1.1.2 9.1.1.3 9 . 1 . 1. 4 9.1.2 9.1.2.1

Aliphatic amines Elaiomycin type (azoxy derivatives) Lipoxarnycin type POLYINES, ALLENES Polyine alcohols (alkanes)

9 . 1. 2 , 2

Polyine acids

9.1.2.3 9.2

Polyenes

9.2.1

SMALLCARBOXYLIC

Disparolone, methylglyoxal (BPC), 2,sdimethyl-1-pentene, 1,8-pentadecadiene, 1,2,4-trihydroxyheptadeca-16ene from “avocado pear,” 4-hydroxy-2ketobutyraldehyde (retine), 2-hexenal, decanoylacetaldehyde Spermine, spermidine Elaiomycin, LL-BH-872 C U , ~ ,ascomycin, cacaomycetin, hygroscopin A,-B Lipoxamycin Hexa-1,3,5-triyne, biformine, biforminic acid, cortinellin, (+) and (-)-marasin, quadrifidin, mucidin, safynol, trideca-5,7,9,1 l-tetrayne-1,2,3,4-tetraol, cis-9,17, octadecadiene-12,14-diyne1,16-diol Agrocybin, clitocybin, dermadine, diatretin 1,2,3, drosophyllin C,-D,-E,-F, mycomycin, nemotin, nemotinic acid, nudic acid A, odyssin, odyssic acid, passicol, quadrifidin B, wyerone, methyl-8-hydroxy-5,6-octadienoate, R ( -)-16-hydroxy-cis-9,17-octadecadien-l2,14-diynoic acid Limocrocin, lusomycin, jawaharan

Aliphatic carboxylic acid derivatives ACID

DERIVATIVES

9.2.1.1

Enteromycin type (acrylic acid derivatives)

9.2.1.2

Acetylene-dicarboxylic acid derivatives

9.2.1.3

Simple carboxylic acid derivatives FATTY ACID DERIVATIVES Simple saturated fatty acids

9.2.2 9.2.2.1

Acrylic acid, t-3-methylthioacrylic acid, acrylamidine, cis-p-acrylamidine (U-20904), enteromycin, enteromycin carboxamide (thermicetin), melrosporus, seligocidin, versimide (MM4086), zedalan, RP-7080 Cellocidin (lenamycin, aquamycin), 1418 A-1, acetylene-dicarboxylate methyl ester Bovinocidin (p-nitropropionic acid), pmeth ylthiopropionic acid, brevimycin A Diphtherinic acid, palmitic acid, rangiformic acid, roccellic acid, globismycin, 10-methyldodecanoic acid, 12-methyltridecanoic acid

RESEARCH A N D CLASSIFICATION OF ANTIBIOTICS

397

TABLE XIV (Continued) Code numbers

Divisions

Representatives

9.2.2.2

Unsaturated fatty acids

9.2.2.3

0x0-oxy fatty acids

9.2.2.4

Fatty acid esters (glycerides) Fatty acid amides

9.2.2.5

Bacilipin A,-B, encalines, isostatin, chaulmoogric acid, hydnocarpic acid, linoleic, linolenic acids, ricinolic acid, 10-undecynoic acid, octadecenoic acid, octadecadienoic acid, caperatic, ungulinic acids, bongkrekik acid, 34, 99, 1085, 534 Alternaric acid, myrmycacin, phomic acid, 9-hydroxy-2-decenoic acid, 9-oxo2-decenoic acid, 10-hydroxy-2-decenoic acid, 1233 B Monoolein, monolaurin, 534, 737, coixenolid, pseudomonic acid A,-B Caulerpicin, cerulenin, dopastin, eulicin, fragin, helicocerin, siolipin, solapalmitin, solapalmitenin, variotin (paecilocin) Myriocin (thermoeymocidin)

9.2.2.0 9.3

Long-chain amino acids

9.3.1

SULFUR-CONTAINING SIMPLE

9.3.1.1 9.3.1.2 9.3.1.3

Thioformine derivatives Sulfoxides Isothiocyanates

Aliphatic compounds with S and

P contents ALIPHATIC COMPOUNDS

9.3.1.3 9.3.2

Fluopsin C,-F, YC-73, 4601 Allicin, alliin Cheirolin, erysolin, allylisothiocyanate, raphanin (sativin), sulforaphen, senfolomycin, proceomycin Ju n ip a1 Others PHOSPHORUS-CONTAINING Phosphonomycin SIMPLE ALIPHATIC COM-

POUNDS

V.

Conclusions

PROBLEMS OF ANTIBIOTICS A. SOME NOMENCLATURAL The problems of antibiotic nomenclature, similar to those of antibiotic classification, came into the foreground in the past few years. Several papers have discussed the question (Round Table on Nomenclature of Antibiotics, 1966; Delcambe, 1970; Kowazyk-Gindifer, 1971) ; the satisfactory regulation of nomenclature seems to be more urgent even than that of classification. The problems under consideration are highly complex, and wide-ranging discussions revealed several contrasting opinions. The most urgent and obvious problems appear to be the following:

398

JINOS B ~

D Y

1. In the literature there are several different substances under identical names, e.g., aminomycin, antimycin, albomycin, vertimycin, mycoin, mycerin, staphylomycin, colimycin, flavomycin, resistomycin. 2. The first names (nonproprietary ) given by discoverers are generally not identical with later adopted commercial names, i.e., tradenames. 3. Designations used in patents constitute a problem in itself. These frequently only numbers or letter-number combinations, are generally not identical with names applied in later publications ( nonproprietary names), and even less so with commercial-medical ones utilized after eventual commercialization. Examples are summarized in Table XV. 4. There is nearly complete lack of uniform denomination for antibiotics described by different scientists and found later to be identical. Usually the name used by the author making the first publication is retained ( desdanin, netropsin, neomycin, etc. ) . In other cases the designation given by the scientific team which recognized the practical value of the respective agent for the first time, or investigated it extensively, is used ( e.g., paromomycin, not catenulin; verrucarin, not glutinosin; novobiocin, not griseoflavin); or a new general name is created (bambermycin, virginiamycin) . Unfortunately, very often several names for one agent are in general use in various publications : paromomycin-aminosidin-monomycin; leucomycin-josamycin; mithramycin-aureolic acid; pentamycin-lagosin-fungichromin; trichomycin-candicidin-hamycin-levorin; decumbin-cyaneinbrefeldin A; streptonigrin-bruneomycin; variotin-paecilocin; daunomycin (rubidomycin, daunorubicin)-rubomycin C; nigericin-polyetherin A; actinomycin C-auranthin. Some of the duplications are justified because of a slightly different component ratio. At present there are nearly a thousand superfluous antibiotic names in current use. TABLE XV DIFFERENTNAMESFOR THE SAMEANTIBIOTICS Patent designation

Nonproprietary name

Tenebrim ycin

Nebram ycin

Moenomycin Quintomycin (antibiotic 2230) SF-733 antibiotic Rickam ycin RP-7293 M-141, U-18409 2-Aminodeox ykanamycin Marcomycin

Bambermycin Lividom ycin

Commercial name Tobramycin (nebramycin factor 6) Flavom ycin ?

Ribostamycin Vistam ycin 6640 antibiotic (sisomicin) Sisomicin ? Pristinam ycin Pyostatin (Pyoclastin) Actinospecta cin Spectinomycin (Kanamycin B) kanendomycin Bekanamyein Hygromycin B Hygromix

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

399

5. Some antibiotic names are very similar in sound and in spelling; this may pose problems in oral or written communications: monomycin-moenomycin; mycoticin-mycithricin; halomycin-holomycin; patricin-partricin; prodigiosin-prodigiosan. Proposals made by the Committee on Antibiotic Nomenclature at the Fifth Interscience Conference of Antimicrobial Agents and Chemotherapy in 1965 for the denomination of newly described antibiotics ought to be taken into consideration. Unfortunately, in practice, even after a suitable wide-ranging propagation of the above recommendations, there still appear illogical, complicated or fantastic denominations-e,g., tuber-, actinomycin, steffisburgensimycin, bikaverin. Some progress has been made, however, in the designation of antibiotics of Streptomyces origin, designated “-mycin” and those of Micromonospora origin ending with “-micin.” Agents of uncertain and/or completely unknown character are more and more often identified at first by letters or numbers; they receive a final name only after their properties have been investigated more exhaustively and their “new antibiotic” character unequivocally proved. Substances of simple chemical structure usually are characterized by their chemical name ( ~-4-azaleucine,cyclohexenylglycine) . Within one antibiotic family, there are promising openings for a uniform nomenclature at international levels (scirpene antibiotics, polymyxines) and recently also for virginiamycins (Croy and De Neys, 1972), cytochalasins, (Binder et al., 1973), rhodomycins, etc. Nevertheless the construction and general introduction of a uniform, simple, intelligible, expressive nomenclature constitutes a nearly unsurmountable problem: 1. The fundamental right to denominate an antibiotic cannot be refused to the scientist who discovered it, although the choice of a suitable name may be justly expected from him. Unfortunately, uncontrolled naming is unavoidable. 2. It is impossible to change names now widely used, though less expressive but euphonious ( e.g., nystatin, lincomycin) . 3. Chemical designations, because of their complexity and length, are completely unsuitable for use in solving problems of nomenclature. 4. The number of “free” denominations that meet recommendations concerning chemical structure and chemical or biological properties (USAN proposal points l., 3., 4b) is rather limited. 5. Lack of satisfactory characterization of an antibiotic and other considerations often result in designations based on the geographical site of isolation of the producing strain. The choice in these names is unlimited, and the names may sound rather pleasing, but they are not usually characteristic of any property of the antibiotic or the producing organism.

400

JANOS

B~RDY

6. Denominations based on taxonomic or physiological characteristics of the producing strain are more expressive, but do not reveal any information to an outsider. The natural requirement, that the name of an antibiotic should unequivocally reveal the substantial properties of an agent as regards either structure type, activity, properties, or origin, cannot be satisfied even by the most consistent designations referring directly to structure and characteristics. Physicians require a rapid and simple guide as to structure, activity, clinical indications, and side effects of antibiotics, rather than an unattainable uniform denomination of antibiotic drugs. In the case of antibiotics of medical value, one of the solutions might be the worldwide acceptance of Simon’s proposals (1970). Although of great practical value, it is unsuitable for resolution of the problems of nomenclature of all known antibiotics. What is to be done in such a chaotic situation? Simultaneous development of the classification and uniform nomenclature of enzymes, another important group of natural substances, constitutes an interesting analogy (IUPAC and IUB, 1972). Their uniform and internationally accepted nomenclature became established on the basis of classificaton according to reactions catalyzed by them, with the use of respective code numbers for classification. Presently there is no uniform point of view covering the effect of antibiotics; systematization on the basis of mode of action might serve this purpose, but only in the distant future. The chemical classification described above, primarily the code numbers applied therein, seems to be suitable, as are code numbers utilized in enzyme nomenclature, to supplement the original distinct names of antibiotics, and thus promote their rapid recognition and identification. Utilization of similar code numbers has been proposed at the Round Table Discussion about Classification of Antibiotics, held during the Sixth International Congress of Chemotherapy ( BBrdy, 1970). A well constructed antibiotic list, periodically compiled and distributed by an international organization and containing the names, code numbers, and eventual supplemental serial numbers, chemical structure, and important properties, would enable the rapid definition of the respective antibiotic. In any chemical classification or in a systematization according to other aspects, for instance according to activity or origin, code numbers may be formed that supplement the name of the respective antibiotic and promote rapid orientation and unequivocal communication. In my opinion sooner or later this will become unavoidable. The final elaboration of such an extensive classification and nomen-

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

401

clature can be performed only at the international level-for instance, within the range of the International Center of Information on Antibiotics.

B. EPILOGAND SUMMARY The first part of the present paper, on the basis of literature data and supported by the elucidation of statistical interrelationships, gives an exhaustive picture about the main trends in present antibiotic research. This kind of review in naturally somewhat subjective, primarily because of the chemical nature of investigative methods. Some valuable information is not available, and many specific problems are not mentioned at all because of limited space. It is obvious that antibiotic research entered a new phase 10-12 years ago. It is impossible, however, to draw a sharp line, since the aspects of the new era will become apparent probably only in forthcoming years. The past decade should be regarded as a transition period. In my opinion, in the future well defined and organized research, planned in advance and based on structure-activity relationships and exact knowledge of microbial metabolism, will be performed. According to other opinions, the “era of microbiologists” will be followed by the “era of chemists.” In fact, this new era should be characterized by the close and successful team work of microbiologists, biochemists, and chemists. To find the right approach to antibiotic-systematization problems, it is essential that we review our present pool of knowledge from the most diverse points of view. In some fields only rudimentary or narrow practical classification is possible; in yet others, present data are not sufficient for systematization. The proposed system is certainly not the sole possibility for classification according to chemical structure. It may be possible to make a computerized exact system on the basis of molecular skeleton and functional group-biological activity relationships. As mentioned before, the assignment of some individual antibiotic groups is somewhat arbitrary, but this was unavoidable in order to avoid ambiguity and to ensure homogeneity, for instance in nomenclature. Even so it is expedient to revise the system periodically with respect to newly recognized structures and to rearrange eventual new groups, types, and subtypes. Certainly this system may be improved and refined. The proposed system seems to be the most extensive classification as yet of antibiotics according to chemical structure, though classification in the field of antibiotics may never have such an important role as in other scientific fields, such as plant or microbiological taxonomy, crystallography, or astronomy. Beyond the importance of classification as an end in itself, it would appear to be a very good aid in orientation

402

J~NOS B ~ D Y

among the continuously growing number of antibiotics and identification of new substances, and it may promote the development of a uniform numerical antibiotic nomenclature. Extensive analysis of origin, mode of action, and various physical, chemical, microbiological, pharmacological and clinical characteristics of individual series of antibiotics, in connection with structure, structural elements, molecular conformations, etc., may enhance comprehension of some practical and theoretical relationships. Statistical investigations, employing a simple punch-card system or computers, executed on the basis of time of discovery of an antibiotic, nationality of the scientist publishing it as well as his firm or affiliation, production data, and economic analysis, or from other view points, may draw attention to outstanding trends in general research and to historical, organizational, and economic trends. ACKNOWLEDGMENTS I wish to thank Professor I. Horvith and Dr. J. Gyimesi who read and corrected the manuscript. The author is indebted to Dr. L. Delcambe for the valuable discussions about the chemical classification of antibiotics. I am grateful to many colleagues in different parts of the world who have been most helpful by sending me reprints of their papers.

REFERENCES Aberhart, J., Fehr, T., Jain, R. C., de Mayo, P., Motl, O., Baczynskyj, L., Gracey, D. E. F., MacLean, D. B., and Sziligyi, I. (1970). J . Amer. Chem. SOC. 92, 5816-5817. Abraham, E. P., and Newton, G. G. F. (1959). PTOC. lnt. Congr. Biochem., 4th, 1958 Vol. 5, pp. 42-63. Aldridge, D. C., Giles, D., and Turner, W. B. ( 1971). J . Chem. SOC., C pp. 3888-389 1. Anderson, B., Crowfoot-Hodgkin, D., and Viswamitra, M. A. ( 1970). Nature (London) 225, 233-235. Aragozzini, F., Toppino, P., and Rindone, B. ( 1970). Ann. Microbiol. 20, 43-55. Arai, T., and Mikami, Y. (1973). Abstr., Int. Congr. Chemother., 8th, 1972 A-423. Arcamone, F., Barbieri, W., Franceschi, G., Penco, S., and Vigevani, A. (1973a). J. Amer. Chem. SOC.95, 2008-2009. Arcamone, F., Franceschi, G., Gioia, B., Penco, S., and Vigevani, A. (1973b). J . Arner. Chern. SOC. 95, 2009-2011. Argoudelis, A. D., Johnson, L. E., and Pyke, T. R. ( 1973). I . Antibiot. 26, 429436. Arison, B. H., and Bleck, J. L. (1973). Tetrahedron 29, 2743-2748. Bagli, J. F., Kluepfel, D., and St. Jacques, M. (1973). J . Org. Chem. 38, 1253-'1260. Batelaan, J. G., Barnick, J. W. F. K., van der Baan, J. L., and Bickelhaupt, F. (1972). Tetrahedron Lett. pp. 31073110. Bayer, E., Gugel, K. H., Hagle, K., Hagenamier, H., Jessipow, S., Konig, W. A., and Zahner, H. (1972). Helv. Chim. Acta 55, 224-239. Beecham Group Ltd. ( 1971 ) . German Patent 2,227,739. Bkrdy, J. (1961). Ph.D. Thesis, Debrecen (in Hungarian).

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

403

BBrdy, J. ( 1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chemother., 6th, 1969 Vol. 11, pp. 1006-1009. Bbrdy, J. 1972). Inform. Bull., Int. Cent. Inform. Antibiot. 10, 1-65. BBrdy, J., and Magyar, K. (1968). Process Biochem. 3, 45-50. Binder, M., and Tamm, C. (1973). Angew. Chem. 85,369-381. Binder, M., Tamm, C., Turner, W. B., and Minato, H. (1973). J. Chem. Soc., Perkin Trans. 1, 1146-1147. Brundret, K. M., Dalziel, W., Hesp, B., Jarvis, J. A. J., and Neidle, S. ( 1972). Chem. Commun. pp. 1027-1028. Conover, L. H. (1971). Aduan. Chem. Ser. 108, 33-80. Croy, P., and De Neys, R. (1972). 1. Antibiot. 25, 371-372. Delcambe, L. ( 1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chemother, 6th, 1969 Vol. 11, pp. 1001-1005. Della Bruna, C., Ricciardi, M. L., and Sanfilippo, A. (1973). Antimicrob. Ag. Chemother. 3, 708-710. Devon, T. K., and Scott, A. I. (1972). “Handbook of Naturally Occurring Compounds,’’ Vol. 2. Academic Press, New York. Dhar, M. M., and Khan, A. W. (1971). Nature (London) 233, 182-184. Domnas, A. (1968). J . Elisha Mitchell Sci. Soc. 84, 16-23. Editorial. ( 1970). J. Amer. Med. Ass. 214, 907. Evans, H. M. (1965). “The Chemistry of Antibiotics Used in Medicine.” Pergamon, Oxford. Floss, H. G. (1972). Proc. Int. Ferment. Symp., 4th, 1972 pp. 407412. French, J. C., Bartz, Q. R., and Dion, H. W. (1973). J. Antibiot. 26, 272-283. Fuller, A. T., Mellows, C., Woolford, M., Banks, C. T., Barrow, K. D., and Chain, E. B. (1971). Nature (London) 234,416417. Gause, G. F. (1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chemother., 6th, 1969 Vol. II., pp. 972-973. Cause, C. F. ( 1972). Aduan. Antimicrob. Antineoplastic Claemother. Proc. Int. Congr. Chemother., 7th, 1971. Vol. II., pp. 75-80. Gottlieb, D., and Shaw, P. D., (1967). “Antibiotics,” Vols. I and 11. Springer-Verlag, Berlin and New York. Curevich, A. I., Karapetyan, M. G., Kolosov, M. N., Omelchenko, V. N., Onopirenko, V. V., Petrenko, G. I., and Popravko, S. A. (1972). Tetrahedron Lett. pp. 1751-1754. Harada, S., Kishi, T., and Mizuno, K. ( 1971). J . Antibiot. 24, 13-22. Hashimoto, T., Takahashi, S., Nagasawa, H., Takita, T., Maeda, K., and Umezawa, H. (1972). 1. Antibiot. 25, 330-355. Hata,, T., Omura, S . , Iwai, Y., Ohno, H., Takeshima, H., and Yamaguchi, N. ( 1972). J. Antibiot. 25, 473474. Hori, M., and Takemoto, K. ( 1972). J . Antibiot. 25, 393-399. Horii, S., and Kameda, Y. (1972). Chem. Commun. pp. 747-748. HorvPth, Gy., Gyimesi, J., and Mdhesfalvi-Vajna, Zs. ( 1973). Tetrahedron Lett. pp. 3643-3658. Isono, K., and Suzuki, S. (1968). Tetrahedron Lett. pp. 1133-1137. Ito, S., and Hirata, Y. ( 1972). Tetrahedron Lett. pp. 1185-1188. Ito, T., Ezaki, N., Tsuruoka, T., and Niida, T. (1971). Carbohyd. Res. 17, 375-382. IUPAC and IUB ( 1972). “Enzyme Nomenclature Recommendations,’’ p. 443. Elsevier, Amsterdam. Kamiya, T., Maeno, S., Hashimoto, T., and Mine, Y. (1972). J. Antibiot. 25,576-581. Katz, E. (1971). Pure Appl. Chem. 28, 551-570.

404

J ~ N O SB ~ R D Y

Kawaguchi, H., Naito, T., Nakagawa, S., and Fujisawa, K. (1972). J. Antibiot. 25, 695-708. Kazuyoshi, J., Kuroda, Y., Ajisaka, M., and ‘Sakai, H. (1972). J . Antibiot. 25, 271-280. Kondo, S., Shibahara, S., Takahashi, S., Maeda, K., Umezawa, H., and Ohno, M. (1971). J. Amer. Chem. SOC. 93, 6305-6306. Korzybski, T., Kowszyk-Gindifer, Z., and Kurylowicz, W. ( 1967). “Antibiotics. Origin, Nature and Properties.” Pergamon, Oxford. Kowszyk-Gindifer, Z. ( 1971 ). Postepy Mikrobiol. 10, 219-247. Kupchan, S. M. (1971). Aduan. Chem. Ser. 108, 1-13. Kupchan, S. M., Komada, Y., Court, W. A., Thomas, G. J., Smith, R. M., Karim, A., Gilmore, C. J., Haltiwanger, R. C., and Bryan, R. F. (1972). J. Amer. Chem. SOC. 95, 1354-1356. Kurylowicz, W. (1965). Postepy H i g . Med. Dosw. 19, 469491. Kurylowicz, W. ( 1966). Rev. Immunol. 30, 253-270. Kurylowicz, W. ( 1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chemother., 6th, 1969. Vol. 11, pp. 976-981. Kurylowicz, W. ( 1971 ), Antibiotiki ( Moscow ) 16, 395-400. Lancini, G., and White, R. J. ( 1973). Process Biochem. 8, 14-16. Larionov, L. F. ( 1970). Cancer Chemother. Rep. 54, 71-77. Lechevalier, H. A., and Lechevalier, M. P. (1967). Annu. Reu. Microbtol. 21, 71-100. Lechevalier, H. A., and Pramer, D. ( 1971). “The Microbes,” p. 537. Lippincott, Philadelphia, Pennsylvania. Lilly, Eli & Co. ( 1972). Belgian Patent 795,918. Maehr, H., Williams, T. H., Leach, M., and Stempel, A. (1974). Helu. Chim. Acts 57, 212-213. Meiji Seika Kaisha Ltd. (1968). British Patent 1,244,042. Minato, H., Katayama, T., Hayakawa, S., and Katagiri, K. (1972). J . Antibiot. 25, 315-316. Mitsuhashi, S., Kawabe, H., Iyobe, S., and Inoue, M. (1973). Abstr. Int. Congr. Chemother., 8th, 1972, A-39. Miura, Y. ( 1969). “Antibiotics and Replication.” Published and distributed by Calbiochem. Miyoshi, T., Miyairi, N., Aoki, H., Kohsaka, M., Sakai, H., and Imanaka, H. (1972). J. Antibiot. 25, 569-575. Moppett, C. E., Dix, D. T., Johnson, F., and Coronelli, C. ( 1972). J. Amer. Chem. SOC. 94, 3269-3272. Muntwyler, R., and Keller-Schierlein, W. ( 1972). Helu. Chim. Acta 55, 2071-2094. Nagarajan, R., Boeck, L. D., Gorman, M., Hamill, R. L., Higgens, C. E., Hoehn, M. M., Stark, W. M., and Whitney, J. G. (1971). J. A m r . Chem. SOC. 93, 2308-2310. Nawata, Y., Ando, K., Tamura, G., Arima, K., and Iitaka, Y. (1969). J. Antibiot. 22, 511-512. Neelameghan, A., Buche, V. V., and Gupta, B. S. S. (1970). Curr. Sci. 39, 511-512. Nelson, T. C. (1961). Nut. Acad. Sci.-Nut. Res. Counc. Publ. 891. Ogata, M. ( 1971). Igaku N o Ayumi 77, 534-537. Okami, Y. ( 1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congv. Chemother., 6th, 1969. Vol. 11, pp. 974-975. Omura, S., Nakagawa, A., Yamada, H., Hata, T., Furusaki, A,, and Watanabe. T. (1971). Chem. Pharm. Bull. 19,2428-2430.

RESEARCH AND CLASSIFICATION OF ANTIBIOTICS

405

Omura, S., Tishler, M., Katagiri, M., and Hata, T. (1972). Chem. Commun. pp. 633-634. Perlman, D. ( 1968). Process Biochem. 3, 54-58. Perlman, D. ( 1973). Process Biochem. 8, 18-20. Perlman, D., and Peruzzotti, G. P. (1970). Advan. Appl. Microbiol. 12, 277-294. Peterson, E. A., Gillespie, D. C., and Cook, F. D. (1966). Can. J. Microbiol. 12, 221-226. Porter, J. N., Wilhelm, J. J., Kirby, J. P., and Hansmann, W. K. ( 1964). Antimicrob. Ag. Chemother. pp. 100-103. Prauser, H., and Eckardt, K. ( 1967). 2. AZZg. MikrobioZ. 7,409-410. Pruess, D. L., Scanell, J. P., Ax, E. A,, Kellett, M., Weiss, F., Demny, T. C., and Stempel, A. ( 1973). J . Antibiot. 26, 261-266. Rangaswami, G., Oblisami, G., and Swaminatham, R. ( 1967). Antagonistic Actinomycetes in the Soils of South India.” Phoenix Press, Visveswarapuram, Bangalore, India. Round Table Discussion.. ( 1970). Prog. Antimicrob. Anticancer Chemother., Proc. lnt. Congr., Chemother., 6th 1969. Vol. 11, pp. 967-1009. Round Table on Nomenclature of Antibiotics. ( 1966). Antimicrob. Ag. Chemother. pp. 1102-1103. Schabacher, K., and Zeeck, A. (1973). Tetrahedron Lett. pp. 2691-2694. Schepartz, S. A. (1965). Antimicrob. Ag. Chemother. pp. 500-504. Sedmera, P., Vokoun, J,, Podojil, M., Vanek, Z.,Fuska, J., Nemes, P., and Kuhr, I. ( 1973 ). Tetrahedron Lett. pp. 1347-1348. Sensi, P. ( 1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chemo. ther. 6th, 1969. Vol. 11, pp. 967-968. Sensi, P., and Coronelli, C. (1971). Inform. Bull., Int. Cent. Inform. Antibiot. 9, 2-18. Shemaykin, M. M., and Khokhlov, A. S. (1957). “Chemistry of Antibiotic Substances.” Goskhimizdat., Moscow ( in Russian). Shier, W. T., Rinehart, K. L., Jr., and Gottlieb, D. (1969). Proc. Nat. Acad. Sci. US. 63, 198-203. Shomura, T., Ezaki, N., Tsuruoka, T., Niwa, T., Akita, E., and Miida, T. (1970). 3. Antibiot., Ser. A 23, 155-161. Simon, H. J. (1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chemother., 6th, 1969 Vol. 11, pp. 963-985. Slusarchyk, W. A. (1971). Biotechnol. Bioeng. 13, 399-407. Snell, J. F., ed. (1966). “Biosynthesis of Antibiotics,” Vol. 1. Academic Press, New York. Stapley, E. O., Jackson, M., Hernandez, S., Zimmerman, S. B., Currie, S. A., Mochales, S., Mata, J. M., Woodruff, H. B., and Hendlin, D. (1972). Antimicrob. Ag. Chernother. 2, 122-131. Suzuki, T., Honda, H., and Katsumata, R. (1972). Agr. Biol. Chem. 36, 2223-2228. Swann, M. (1969). “The Use of Antibiotics in Animal Husbandry and Veterinary Medicine.” Report of the Joint Committee, London. Takeuchi, T., Ogawa, K., Iinuma, H., Suda, H., Ukita, K., Nagatsu, T., Kato, M., Umezawa, H., and Tanabe, 0. (1973). J. Antibiot. 26, 162-167. Takita, T., Muruoka, Y., Yoshioka, T., Fujii, A., Maeda, K., and Umezawa, H. (1972). 3. Antibiot. 25, 755-756. Tamura, S. (1972). Proc. Int. Ferment. Symp., 4 t h 1972 pp. 413-418. Thiemann, J. T., Zucco, G., and Pelizza, G. (1969). Comb. Nou. Arch. Microbiol. 67, 147-155.

406

J ~ N O SB ~ D Y

Umezawa, H. (1964). “Recent Advances in Chemistry and Biochemistry of Antibiotics.” Microb. Res. Found., Tokyo. Umezawa, H., ed. (1967). “Index of Antibiotics from Actinomycetes,” Penn. State Univ. Press, University Park, Pennsylvania. Umezawa, H. ( 1971). Pure Appl. Chem. 28, 665-680. Umezawa, H. ( 1972a). Aduan. Antimicrob. Antineoplust. Chemother. Proc. lnt. Congr. Chemother, 7th, 1971. Vol. 11, pp. 81-82. Umezawa, H. (197213). Enzyme Inhibitors of Microbial Origin.” Univ. of Tokyo Press, Tokyo. Umezawa, H. ( 1 9 7 2 ~ )Proc. . Int. Ferment. Symp., 4th, 1972. pp. 19-29 and 401-405. Umezawa, H., Mitsuhashi, S., Hamada, M., Iyobe, S., Takahashi, S., Utahara, R., Osato, Y., Yamazaki, S., Ogawara, H., and Maeda, K. (1973). J. Antibiot. 26, 51-54. Umezawa, S., Tsuchiya, T., Tatsuoka, K., Horiuchi, Y., and Usui, T. (1970a). J. Antibiot. 23, 20-27. Umezawa, S., Tatsuta, K., Horiuchi, Y., Tsuchiya, T., and Umezawa, H. ( 1970b). J . Antibiot. 23, 28-34. von Glehn, M., Norrestam, R., Kierkegaard, P., Maron, L., and Ernster, L. (1972). FEBS Lett. 20, 267-269. Vos, C., and Verwiel, P. E. J. (1973). Tetrahedron Lett. pp. 51736176. Waksman, S. A. (1966). Antimicrob. Ag. Chemother. pp. 9-19. Waksman, S. A. (1969). Adoun. Appl. Microbiol. 11, 1-16. Waksman, S. A., and Lechevalier, H. A. (1962). “The Actinomycetes,” Vol. 111. Williams & Wilkins, Baltimore, Maryland. Whaley, H. A. ( 1971). J . Amer. Chem. SOC. 93,3767-3769. Woo, P. W. K., Dion, H. W., and Bartz, Q. R. (1971). Tetruhedron Lett. pp. 2625-2628. Woodruff, H. B., and MacDaniel, L. E. (1958). Symp. Soc. Gen. Microbiol. 8, 94. Yagisawa, Y. ( 1973). Japanese Medical Gazette 1973, March 20. Yonehara, H. ( 1970). Prog. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chernothef., 6th, 1969 Vol. 11, pp. 969-971. Yoshida, N., Tani, Y., and Ogata, K. ( 1972). J. Antibiot. 25, 653-659.

SUBJECT INDEX

A

Actinomudura Lechevalier et Lechevalier, 174 Actinomycete taxonomy cardinal classification, 134-135 differential characters acid-fast staining, 136-137 bacteriocin typing, 141 cell wall composition, 141-143 chemical composition, 137-138 DNA analyses, 162-172 lipid composition, 143-144 metabolism regulation, 145-153 phage typing, 138-140 protein dynamic state, 153-162 serological analyses, 144-145 early classification schemes, 133-134 numerical taxonomy, 135 Aflatoxins, 58-59 Albofungin, 352 Aldrin, metabolism, 5-6 Aleukia, 57 Algal toxins, 59-60 Alicyclic antibiotics cycloalkane derivatives, 390 oligoterpene antibiotics, 392-393 small terpenes, 390-391 Aliphatic antibiotics alkane derivatives, 395396 aliphatic carboxylic acid derivatives, 396-397 aliphatic compounds with S and P contents, 397 Amino acid and peptide antibodies amino acid derived, 377-378 heteromer peptides, 379-380 high molecular weight peptides, 382-383 homopeptides, 379 peptolides, 380-382 Ammonia emission, 13-16 Antibiotics, see also specific substances 1233% structure, 350 A-25822, structure, 351 anti-tumor, p. 319, 332 classification, based on bioactivity, 341-343 biosynthesis, 341

chemical structure, 345 mechanism of action, 343-345 origin, 337-341; 344 physiochemical properties, 345 by directed fermentation, 332-333 families, 364 K-16, structure, 348 naturally occurring, currently in clinical and agricultural use, 311 nonclinical uses, 317 number of derivatives prepared, 316 occurrence in microorganisms, 326331 screening laboratories, 321 screening methods, 324 semisynthetic, in clinical and agricultural use, 315 SF-666A and B, structure, 349 X-5108, structure, 348 Aphidicolin, structure, 349 Apramycin (nebramycin factor 2 ) , 224-227 Aromatic antibiotics, benzene compounds, 393-394 condensed aromatic compounds, 394 nonbenzoid aromatic compounds, 394 various derivatives of aromatic compounds, 395 Arsenic compounds, microbial formation, 46-47 Ascochlorin, structure, 354 B

Benzoquinones, 376 Bicyclomycin, structures, 347 Bleomycins, structures, 352 Bundlins, structures, 353 Butirosins, 228 -24 1 C

Carbohydrate antibiotics, 365 aminoglycosides, 366-367 C-glycosides, 365 N-glycosides, 367-368 407

408

SUBJECT INDEX

pure saccharides, 365-366 sugar esters and amides, 368 Carbon dioxide, microbial formation, 31-33 Carbon monoxide, microbial formation, 29-31 Carcinogens, microbial formation, 53 Cephamycins, structures, 355 Chlorothricin, structure, 355 2-Chloro-6- ( trichloromethyl )pyridine dehalogenation, 12 Citrinin, natural occurrence, 61 D

DDT, microbial metabolism, 5 Deoxystreptamine antibiotics, see also specific antibiotics antibacterial spectrum, 205-207 enzymatic inactivation, 217-219 Dichlorbenil, metabolism of, 11 2,4-Dichlorophenol formation of, 8-10 metabolism of, 8 2,4-dichlorophenoxyacetic acid ( 2,4 D ), metabolism, 6-7 Dienomycins, structure, 351 Diphenamide, metabolism 13 Diuron, metabolism, I1 E

Ergotism, 5 6 5 7 Ethylene, microbial formation, 28-29 G

Gentamicins, 274-282 chemistry, 204 production, 203 H

Heptachlor, metabolism, 5 Hydrogen sulfide, microbial formation, 37-40 Hydroxylamine, formation, 23-24 Hygromycin B, 226-229

I

Ikarugamycin, structure, 347 K

Kanamycins, 252-274 chemistry, 203 production, 201-203 Kinamycins, structure, 350 1

0-Lipomycine, structure, 351 Lipoxamycin, structure, 349 M

Macrocyclic lactone ( Lactam) antibiotics, 368 macrolactam, 373 macrolactones, 373 other macrocyclic lactones, 372-373 polyene antifungals, 370-372 Maytansine, structure, 349 Mercury compounds, microbial formation, 43-46 Mine waters, microbial formation of acids, 34-37 Mocimycin, structure, 348 Mycobacterium Lehniann et Neumann, 172-1 73 Mycobacterium species M . abscessus 183 M . auium, 183 M . flavescens, 183 M . fortuitum, 183 M . gastra, 183 M . gordonae, 183 M . intracellulare, 183 M . kansasii, 183 M . leprae, 183 M. lepremurium, 183 M. nonchromogenicum, 183 M . paratuberculosis, 183 M . scrofulaceum, 183 M . smegmatis, 183 M . terras, 183 M . trivale, 183 M . tuberculosis, 183, 184 M . ulcerans, 183 M . uaccae, 183 M . renopi, 183

409

SUBJECT INDEX

Myriocin ( Thermazymocidin ), structure, 349 N

Naphthoquinones, 374-375 Neamine, 219-224 Neomycins, 241-251 production, 199-200 relationships to paramomycins, 200-201 Nitrate formation, 16-20 Nitrite formation, 20-23 Nitrogen-containing heterocyclic antibiotics alkaloids with antibiotic (antitumor) activity, 386 condensed ( fused ) heterocycles, 384-386 noncondensed ( single ) heterocycles, 383-384 Nitrogen oxides, microbial formation, 25-28 Nitrosamines, microbial formation, 50-53 Nocardia species, N . asterckes, 177 N . brasiliensis, 177 N . caviae, 178 N . treuisan, 173-74 0

Odors, microbial production, 62 Oerskouia Prauser, Lechevalier et Lechevalier, 175-176 Oleficin, structure, 351 Oligomycin B, structure, 353 Organic acids, microbial formation in soil, 33-34 Organic sulfur compounds, volatile compounds formed by microbial systems, 41-43 Oxazinomycin, structure, 353 Oxygen, microbial formation, 49-50 Oxygen-containing heterocyclic antibiotics benz-ypyran( flavone ) derivatives, 387-388 furan derivatives, 387 polyether antibiotics, 389-390 pyran derivatives, 387 small lactones, 388-389

P

Patdin, natural occurrence, 61 Pentachlorobenzyl alcohol, metabolism, 11-12 Pentachloronitrobenzene, metabolism, 12 Pesticide biodegradability, 119 persistence in soil, 116-118 trade names and chemical designation, 123-124 transformation chlorobenzilate, 79 chlorones, 79 cornetabolisrn, 78 conjugate formation, 80 DDT, 79 3,5-dichlprocatechol, 79 p,p’-dichlorodiphenyl-methane,79 mechanisms, 77 3-nitrophenol, 79 nutrient source, 78 2,4,5-T, 79 2,3,6-TBA, 79 transformation, mechanisms aromatic ring oxidation, 90-94 p-Oxidation, 94-95 dealkylation, 84 N-dealkylation, 85-87 0-dealkylation, 87-89 dehalogenation, 103 with hydrolytic cleavage, 105-106 with reductive cleavage, 105-106 epoxidation, 95-96 ether cleavage, 89-90 hydrolysis, 99-103 hydroxylation, 81-84 methylation, 109-114 reductions, 97-98 sulfoxidation, 97 L - ( Nj-phosphono )methionine-Ssulfoxyminylalanylalanine,structure, 350 Phosphorothionates dimethoate, 3 disulfoton, 4 metabolites from, 3 phorate, 3 Phosphotricinylalanylalanine, structure, 350

410

SUBJECT INDEX

Polyoxin A, structure, 354 Primycin, structure, 353 Proactinomyces Jensen, 174-175 Proactinomyces species P. corallinus, 179-180 P. gbberdus, 180-181 P. opacus, 180 Propanil, metabolism of, 10 Prumycin, structure, 356 Pseudomonic acid, structure, 350

SF-666A and B, structure, 349 Streptomycin, antibacterial properties, 194-195 bacterial resistance, 196 mechanisms of inactivation, 197-198 structures resistant to enzyme inactivation, 198 Sulfur dioxide, microbial formation,

40-41 T

Q

Quinones, 373-377, see also specific quinones linearly condensed polycyclic compounds, 373-374 R

Ribostamycin, 22 8-241

Tellurium compounds, microbial formation, 4 7 4 9 Thermorubin, structure, 356 Thiostrepton, structure, 354 Thiram, metabolism, 12 V

Validamycin, structure, 347 Vermiculin, structure, 349

S

Selenium compounds, microbial formation, 4 7 4 9

Zytron, metabolism, 8

CONTENTS OF PREVIOUS VOLUMES Volume 1

Protected Fermentation Milo5 HeroM and Jan Ne&d.sek

A Commentary on Microbiological Assaying F . Kavanagh

The Mechanism of Penicillin Biosynthesis Arnold L. Demain

Application of Membrane Filters Richard Ehrlich

Preservatlon of Foods and Drugs by Ionizing Radiations W . Dexter Belhmy

Microbial Control Brewery Gerhard J . Hass

The State of Antibiotics in Plant Disease Control David Pramer

Newer Development in Vinegar Manufactures Rudolph J. Allgeier and Frank M . Hildebrandt

Microbial Synthesis of Cobamides D. Perlman Factors Affecting the Antimicrobial Activity of Phenols E. 0. Bennett Germfree Animal Techniques and Their Applications Arthur W . Phillips and James E. Smith Insect Microbiology S. R. Dutky The Production of Amino Acids by Fermentation Processes Shukuo Kinoshita Continuous Industrial Fermentations Philip Gerhardt and M . C. Bartlett The Large-Scale Growth of Higher Fungi Radclifle F. Robinson and R. S. Davidson AUTHOR INDEX-SUBJECT

in

the

The Microbiological Transformation of Steroids T. H . Stoudt Biological Transformation of Solar Energy William I. Oswald and Clarence G. Golueke SYMPOSIUM ON ENGINEERING ADVANCES IN FERMENTATION PRACTICE Rheological Properties of Fermentation Broths Fred H. Deindoerfer and John M . West Fluid Mixing in Fermentation Processes J. Y. Oldshue Scale-up of Submerged Fermentations W . H . Bartholemew Air Sterilization Arthur E. Humphrey

INDEX

Volume 2

Newer Aspects of Waste Treatment Nandor Porges Aerosol Samplers Harold W . Batchelor

Methods

Sterilization of Media for Biochemical Processes Lloyd L. Kempe Fermentation Kinetics and Model Processes Fred H . Deindoerfer 41 1

412

CONTENTS OF PREVIOUS VOLUMES

Continuous Fermentation W . D. Maron

Volume 4

Control Applications in Fermentation George 1. Fuld AUTHOR INDEX-SUBJECT

INDEX

Volume 3

Preservation of Bacteria by Lyophilization Robert J. Heckly

Sphaerotilus, Its Nature and Economic Significance Norman C . Dondero Large-Scale Use of Animal Cell Cultures Donald J. Merchant and C. Richard Eidum Protection Against Infection in the Microbiological Laboratory: Devices and Procedures Mark A. Chatigny Oxidation of Aromatic Compounds by Bacteria Martin H. Rogoff Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M. Schabel, Jr., and Robert F. Pittillo The Classification of Actinomycetes in Relation to Their Antibiotic Activity Eli0 Baldacci The Metabolism of Cardiac Lactones by Microorganisms Elwood Titus Intermediary Metabolism and Antibiotic Synthesis J. D. Bu’Lock Methods for the Determination of Organic Acids A. C . Hulme AUTHOR INDEX-SUB JECT INDEX

Induced Mutagenesis in the Selection of Microorganisms S . I . Alikhanian The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F. 3. Babel Applied Microbiology in Animal Nutrition Harlow H . Hall Biological Aspects of Continuous Cultivation of Microorganisms T. Holme Maintenance and Loss in Tissue Culture of Specific Cell Characteristics Charles C. Morris Submerged Growth of Plant Cells L. G. Nickell AUTHOR INDEX-SUB JECT INDEX

Volume 5

Correlations between Microbiological Morphology and the Chemistry of Biocides Adrien Albert Generation of Electricity by Microbial Action I. B. Davis Microorganisms and the Molecular Biology of Cancer G. F. Gause Rapid Microbiological with Radioisotopes Gilbert V. Levin

Determinations

The Present Status of the 2,3-Butylene Glycol Fermentation Sterling K. Long and Roger Patrick Aeration in the Laboratory W . R. Lockhart and R. W . Squires

CONTENTS OF PREVIOUS VOLUMES

Stability and Degeneration of Microbial Cultures on Repeated Transfer Fritz Reusser Microbiology of Paint Films Richard T . Ross The Actinomycetes an,d Their Antibiotics Selman A. Waksman Fuse1 Oil A. Dinsmoor Webb and John L. Zngraham AUTHOR INDEX-SUB JECT INDEX

413

Volume 7

Microbial Carotenogenesis Alex Ciegler Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility M . Alexander Cold Sterilization Techniques John B. Opfell and Curtis E . Miller Microbial Production of Metal-Organic Compounds and Complexes D. Perlman

Volume 6

Global Impacts of Applied Microbiology: An Appraisal Carl-Goran Hedbn and Mortimer P . Starr Microbial Processes for Preparation of Radioactive Compounds D. Perlman, Aris P. Bayan, and Nancy A. Giuffre Secondary Factors in Fermentation Processes P. Margalith Nonmedical Uses of Antibiotics Herbert S. Goldberg Microbial Aspects of Water Pollution Control K. Wuhmann Microbial Formation and Degradation of Minerals Melvin P. Silverman and Henry L. Ehrlich Enzymes and Their Applications Irwin W . Sizer

A Discussion of the Training of Applied Microbiologists B. W . Koft and Wayne W . Umbreit AUTHOR INDEX-SUE JECT INDEX

Development of Coding Schemes for Microbial Taxonomy S. T. Cowan Effects of Microbes on Germfree Animals Thomas D. Lackey Uses and Products of Yeasts and Yeastlike Fungi Walter J . Nickerson and Robert G. Brown Microbial Amylases Walter W . Windish and Nagesh S. Mhatre The Microbiology of Freeze-Dried Foods Gerald J . Silverman and Samuel A. Goldblith Low-Temperature Microbiology Judith Farrell and A. H. Rose AUTHOR INDEX-SUB JECT INDEX

Volume 8

Industrial Fermentations an.d Their Relations to Regulatory Mechanisms Arnold L. Demain Genetics in Applied Microbiology S . G. Bradley

414

CONTENTS OF PREVIOUS VOLUMES

Microbial Ecology and Applied Microbiology Thomas D. Brock

Cellulose and Cellulolysis Brigitta Norkram

The Ecological Approach to the Study of Activated Sludge Wesley 0. Pipes

Microbiological Aspects of the Formation and Degradation of Cellulosic Fibers L. Jurdek, 1. Ross Colvin, and D. R. Whitaker

Control of Bacteria in Nondomestic Water Supplies Cecil W . Chambers and Norman A. Clarke

The Biotransformation of Lignin to Humus-Facts and Postulates R. T. Oglesby, R. F. Christman, and C. H . Driuer

The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods Stephen Alan Kollins Oral Microbiology Heiner Hoflrnan

Malo-lactic Fermentation Ralph E . Kunkee AUTHOR INDEX-SUB JECT INDEX

Media and Methods for Isolation and Enumeration of the Enterococci Paul A. Hartman, George W . Reinbold, and Deui S . Saraswat Crystal-Forming Bacteria Pathogens Martin H. Rogof?

Bulking of Activated Sludge Wesley 0. Pipes

as

Insect

Mycotoxins in Feeds and Foods Emanuel Borker, Nino F. Insalata, Colette P. Levi, and john S . Witzeman AUTHOR INDEX-SUB JECT INDEX

Volume 9

The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. Russell, June Jenkins, and I. H . Harrison Antiserum Production in Experimental Animals Richard M . Hyde Microbial Models of Tumor Metabolism G. F. Gause

Volume 10

Detection of Life in Soil on Earth and Other Planets. Introductory Remarks Robert L. Starkey For What Shall We Search? Allan H . Brown Relevance of Soil Microbiology to Search for Life on Other Planets G. Stotzky Experiments and Instrumentation Extraterrestrial Life Detection Gilbert V. Levin

for

Halophilic Bacteria D. J . Kushner Applied Significance of Polyvalent Bacteriophages S. G. Bradley Proteins and Enzymes as Taxonomic Tools Edward D. Garber and John W . Rippon

CONTENTS OF PREVIOUS VOLUMES

Mycotoxins A l a Ciegler and Eivind B. Lillehoi

Ergot Alkaloid Fermentations William J. Kelleher

Transformation of Organic Compounds by Fungal Spores Claude Vkzina, S. N. Sehgal, and Kartar Singh

The Microbiology of the Hen's Egg R. G. Board

Microbial Interactions in Continuous Culture Henry R. Bungay, I l l and Mary Lou Bungay

415

Training for the Biochemical Industries 1. L. Hepner AUTHOR INDEX-SUB JECT INDEX

Chemical Sterilizers ( Chemosterilizers ) Paul M . Borick Antibiotics in the Control of Plant Pathogens M . 3. Thirumalachar AUTHOR INDEX-SUB J ECT INDEX

CUMULATIVEAUTHOR INDEX-CUMULATIVE TITLEINDEX

Volume 1 2

History of the Development of a School of Biochemistry in the Faculty of Technology, University of ManChester Thomas Kennedy Walker Fermentation Processes Employed Vitamin C Synthesis Milo& Kulhdnek

in

Flavor and Microorganisms P. Margalith and Y. Schwartz Volume 1 1

Successes and Failures in the Search fox Antibiotics Selmun A. Waksmun Structure-Activity Relationships of 'Semisynthetic Penicillins K. E . Price Resistance to Antimicrobial Agents J. S . Kiser, G. 0. Gale, and G. A. Kemp

Micromonospora Taxonomy George Luedemann Dental Caries and Periodontal Disease Considered as Infectious Diseases William Gold The Recovery and Purification of Biochemicals Victor H . Edwards

Mechanisms of Thermal Injury in Nonsporulating Bacteria M . C. Allwood and A. D. Russell Collection of Microbial Cells Daniel 1. C. Wang and Anthony Sinskey

I.

Fermentor Design R. Steel and T . L. Miller The Occurrence, Chemistry, and Toxicology of the Microbial PeptideLactones A. Taylor Microbial Metabolites as Potentially Useful Pharmacologically Active Agents D. Perlman and G. P. Peruzzotti AUTHOR INDEX-SUB JECT INDEX

416

CONTENTS OF PREVIOUS VOLUMES

Volume 13

Chemotaxonomic Relationships Among the Basidiomycetes Robert G. Benedict Proton Magnetic Resonance Spectroscopy -An Aid in Identification and Chemotaxonomy of Yeasts P. A. J . Gorin and j . F. T. Spencer Large-Scale Cultivation of Mammalian Cells R. C . Telling and P. J. Radlett Large-Scale Bacteriophage K . Sargeant

Production

Microorganisms as Potential Sources of Food Jnanendra K . Bhattacharjee Structure Activity Relationships Among Semisynthetic Cephalosporins M . L. Sassiver and Arthur Lewis Structure-Activity Relationships in the Tetracycline Series Robert K. Blackwood and Arthur R. English Microbial Production of Phenazines 1. M . Ingram and A. C . Blackwood The Gibberellin Fermentation E. G. jeflerys Metabolism of Acylanilide Herbicides Richard Bartha and David Prarner Therapeutic Dentrifices J. K. Peterson Some Contributions of the US. Department of Agriculture to the Fermentation Industry George E . Ward Microbiological Patents in International Litigation John V . Whittenburg

Industrial Applications of Continuous Culture: Pharmaceutical Products and Other Products and Processes R. C. Righelato and R. Elsworth Mathematical Models for Fermentation Processes A. G. Fredrickson, R. D. Megee, I l l , and H. M. Tsuchiya AUTHOR INDEX-SUB JECT INDEX

Volume 1 4

Development of the Fermentation Industries in Great Britain John J. H. Hastings Chemical Composition as a Criterion in the Classification of Actinomycetes H . A. Lechevalier, Mary P. Lechevalier, and Nancy N . Gerber Prevalence and Distribution of Antibiotic-Producing Actinomycetes John N . Porter Biochemical Activities of Nocardia R. L. Raymond and V . W . Jamison Microbial Transformations of Antibiotics Oldrich K . Sebek and D. Perlman

In Vivo Evaluation of Antibacterial Chemotherapeutic Substances A. Kathrine Miller Modification of Lincomycin Barney J . Magerlein Fermentation Equipment G. L. Solomons The Extracellular Accumulation of Metabolic Products by Hydrocarbon-Degrading Microorganisms Bernard J. Abbott and William E. Gledhill AUTHOR INDEX-SUB JECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 15

Medical Applications Enzymes Irwin W . Sizer

of

Microbial

Immobilized Enzymes K. L. Smiley and G . W . strandberg Microbial Rennets Joseph L. Sardinas Volatile Aroma Components of Wines and Other Fermented Beverages A. Dinsmoor W e b b and Carlos J. Muller Correlative Microbiological Assays Ladisluv 1. Hahka Insect Tissue Culture W . F. Hink Metabolites from Animal and Plant Cell Culture Irving S . Johnson and George B. Boder Structure-Activity Relationships in Coumermycins John C . Godfrey and Kenneth E. Price Chloramphenicol Vedpal S. Malik Microbial Utilization of Methanol Charles L. Cooney and David W . Levine Modeling of Growth Processes with Two Liquid Phases: A Review of Drop Phenomena, Mixing, and Growth P . S . Shuh, L. T . Fan, I. C . Kao, and L. E . Erickson Microbiology and Fermentations in the Prairie Regional Laboratory of the National Research Council of Canada 1946-1971 R . H . Haskins

417

Intestinal Microbial Flora of the Pig R. Kenworthy Antimycin A, a Piscicidal Antibiotic Robert E . Lennon and Claude Vbzinu Ochratoxins Kenneth L. Applegate and John R. Chipley Cultivation of Animal Cells in ChemicaIly Defined Media, A Review Kiyoshi Higuchi Genetic and Phenetic Classification of Bacteria R. R. Colwell Mutation and the Production of Secondary Metabolites Arnold L. D e m i n Structure-Activity Relationships in the Actinomycins Johannes Meienhofer and Eric Atherton Development of Applied Microbiology at the University of Wisconsin William B. Sarles AUTHOR INDEX-SUB JECT INDEX Volume 17

Education and Training in Applied Microbiology Wayne W. Umbreit Antimetabolites from Microorganisms David L. Pruess and James P. Scannell Lipid Composition as a Guide to the Classification of Bacteria Norman Shaw

AUTHORINDEX-SUB JECT INDEX Volume 16

Public Health Significance of Feeding Low Levels of Antibiotics to Animals Thomas H . Jukes

Fungal Sterols and the Mode of Action of the Polyene Antibiotics J . M . T . Hamilton-Miller Methods of Numerical Taxonomy for Various Genera of Yeasts I . Campbell

418

CONTENTS OF PREVIOUS VOLUMES

Microbiology and Biochemistry of Soy Sauce Fermentation F . M . Yong and €3. J. B . Wood

Linear Alkylbenzene Sulfonate: Biodegradation and Aquatic Interactions William. E . Gledhill

Contemporary Thoughts on Aspects of Applied Microbiology P. S. S . Dawson and K . L. Phillips

The Story of the American Type Culture Collection-Its History and Development (1899-1973) William A. Clark and Dofothy H . Geary

Some Thoughts on the Microbiological Aspects of Brewing and Other Industries Utilizing Yeast G . G. Stewart

Microbial Penicillin Acylases E . J. Vandamme and J. P . Voets SUBJECT INDEX

A 4 6 5 C 6

D ?

E F G H

8 9 O 1

1 2 J 3