Nonmedical uses of Antibiotics

Nonmedical Uses of Antibiotics HERBERT S. GOLDBERG Department of Microbiology, School of Medicine, Uniuersity of Missouri, Columbia, Missouri

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Economic Aspects ...................................... 11. Antibiotics in Animal Nutrition . . , . , . . . . . . . A. Current Status . . . . . . . .. . . . . . . .. .. . . . B. Antibiotic Levels . . . . . .. .. . . .. . . . . . . . . . . . . .. . . . . . . .. C. Mode of Action of Antibiotic Growth Stimulation . . . . . . . . 111. Antibiotics in Plant Disease Control . . . . . . . . . . . . . . . . . . . . . . . Antibiotics of Importance . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Antibiotics in Food Preservation . . . . . . . . . . . . . . . . . . . . . . . . . . A. Canning . . . . . .. .. .. . . . . . , . . . . B. Dairy Products . . . . . . . . . . . . . . . . C. Fresh Meats and Poultry . .. . . . . . . . . . . . . . . . . .. D. Fruits and Vegetables . . . . . . . . . . . ........... E. Fish and Sea Food . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . V. Antibiotics as Adjuncts in Microbiological Techniques and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Tissue Culture . . . . . .. . . . . .. .. . . ... B. Microbial Classification . . . . . . . . . . ... VI. Public Health Aspects of Nonmedical Uses of Antibiotics . . . . . A. Tetracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Streptomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Penicillin . . . . D. Nisin-Subtilin-Bacitracin . .. .. . . . . . . . . . . .. . . .. .. . . . E. Tylosin-Oleandomycin .. . .. . . .. . . . . . . F. Research .................. References . ..................

91 92 93 93 94

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98 98 100 100 101 102 103 104 106 106 108 109 110 112 112 113 113 114 114

1. Introduction The impact of antibiotics on the treatment of infectious disease and indeed on the practice of medicine is well appreciated by most individuals. However, the multiple uses of antibiotics in areas outside of human and veterinary medicine, although quite extensive, are not so well known. It is the purpose of this review to present the current status of nonmedical uses of antibiotics, revealing its past, present, and potential contribution to food and agriculture. In addition, the microbiologist in particular has found ways in which antibiotics have helped solve problems in the research laboratory, and these will also be discussed. 91

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ECONOMIC ASPECTS In order to assess the relationship between antibiotics produced for nonmedical and medical (including veterinary) uses, a brief look at some figures is in order (Table I). The three major nonmedical uses of antibiotics are in animal feed supplements, crop protection, and food preservation. The data indicate that in 1961 more than half of the total United States antibiotic production was for nonmedical use. Over a 10-year span, 1951-1961, antibiotic usage, nonmedically, increased to the point where almost two million pounds were allocated for food preservation and feed and crop usage. A $45,000,000 annual industry has become a wellestablished part of the economy. TABLE I ANTIBIOTIC PRODUCTION FOR NONMEDICAL USES ( UNITEDSTATES)1951-1961

Year 1951 1952 1953 1954 1956 1960 1961 1961

Antibiotic use Feed supplement Feed supplement Feed supplement Feed supplement Feed-food-crops Feed-food-crops Feed-food-crops All uses-medical and nonmedical

Pounds

Value in millions of dollars

236,000 258,000 434,000 479,000 779,000 1,200,000 1,800,000 3,311,000

17.0 17.0 19.0 25.0 28.2 39.4 45.4 114.6

The specific antibiotics of major concern in nonmedical usage include tetracyclines, streptomycin, penicillin, and bacitracin along with several others of lesser importance. Consequently, the antibiotics of medical importance are also the antibiotics of nonmedical importance. This fact complicates the role of regulatory agencies as they are presented with the problem of potential public health hazards, such as toxicity, hypersensitivity, and emergence of microbial resistance. The entire aspect of nonmedical usage of antibiotics presents itself in a complex form. It seems appropriate at this time to first examine the current status of the efficacy of antibiotics used nonmedically and then to analyze the public health aspects of such usage.

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II. Antibiotics in Animal Nutrition A. CURRENT STATUS It is generally held today that certain antibiotics are capable of stimulating the growth rate of a variety of livestock and fur-bearing animals. This was the first broad nonmedical use of antibiotics and was given impetus by the investigation of Moore et al. in 1946. Excellent reviews on the use of antibiotics in animal nutrition are available by Jukes (1955), Ferrando and Jacquet (1958), and Luckey ( 1959). These reviews emphasize the fact that antibiotics stimulate appetite, increase food efficiency, reduce requirement for vitamins, increase survival, and, most significant of all, increase the growth rate. The evidence is clear that antibiotics are effective only in the early growing period and in particular in situations in which animals are undergoing stress. Animals that are weak or runts, in poor environmental conditions, or on inadequate diets do much better on antibiotics than do normal animals, reared under good management and fed a complete diet. The animal species generally accepted as requiring antibiotics for maximum production efficiency include poultry, swine, calves, lambs, and fur-bearing animals. Some would also include beef cattle. Since the effectiveness of antibiotics is limited to the early growing period the age periods as shown in the following tabulation have been recommended for feeding (WHO Tech. Rept., 1963). Animal Poultry Swine Calves Beef cattle Lambs Fur-bearinc animals

Ace 8-10 weeks 4-6 weeks 3 months 18 months 2 months 2-3 months

The most frequently used antibiotics for the purpose of animal growth are penicillin, chlortetracycline, and oxytetracycline, primarily, and bacitracin, erythromycin, oleandomycin, spiramycin, streptomycin, and tylosin. All of these have shown growth-stimulatory activity in the animals mentioned in the above tabulation and are used for this purpose commercially in the United States and abroad.

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Even with the above facts well established there exists, however, current controversy over two aspects of antibiotics in animal feeds. One, a practical problem, concerns levels of antibiotic necessary to the ration and the other, a fundamental problem, concerns the mechanism of action of antibiotic growth stimulation.

B. ANTIBIOTIC LEVELS In the early 1950s the antibiotic level, used in feeds to bring about growth stimulation, varied from 5-20 p.p.m. of the “total ration.” The “total ration” being defined as “the total daily feed intake on a dry-matter basis.” Since then the tendency has been to increase the level of antibiotics in commercial animal feeds. Levels as high as several hundred p.p.m. and higher are currently used. Table I1 indicates the maximum permissible levels in representative TABLE I1 NATIONALREGULATIONS FOR ANTIBIOTIC FEEDSUPPLEMENTS Countrya Austria Belgium

Antibiotics OTC-CTC-penicillin OTC-CTC-penicillinbaci tracin Denmark OTC-CTC-penicillin Fin 1and OTC-CTC France OTC-CTC-penicillinbacitracinb Germany OTC-CTC-penicillin Great Britain OTC-CTC-penicillin Holland Not specified Norway OTC-CTC-penicillin Sweden OTC-CTC-penicillin Switzerland OTC-CTC-penicillinbacitracin United States OTC-CTC-penicillinbacitracinb a b

Animals Pigs-poultry Calves-pigs-poultry

Maximum level (P.P.m. ) 60

Young growing animals Pigs-poultry-fur bearers Pigs-poultry Pigs-poultry-calves Growing pigs-poultry Pigs-poultry-calves Pigs-poultry-calves Pigs-poultry-calves-mink All except dairy cattle Pigs-poultry-calves

50

25 50 200

200 100 100 50 20 50

2000

Ireland, Greece, Israel, G y , and Portugal have no restrictions. Also several other antibiotics.

countries as of 1961. It can be readily seen that the concentration of antibiotics necessary for the growth effect has been universally exceeded. The reasons for this are not too clear and have never been satisfactorily explained on a nutrition basis. In general, levels of antibiotic in feeds have been divided into “low-level feeding,”

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“prophylactic feeding,” and “therapeutic feeding.” Unfortunately, these levels are not clearly defined nor are they well regulated, particularly in the United States. “Low-level feeding is best defined, perhaps, as the minimum level which achieves the growth effect. In most instances 20 p.p.m. should be the maxium necessary for this result. However, the United States accepts low-level feeding as that which occurs at up to 50 p.p.m. Christensen (1956) and Carlson (1957) have judged 50 p.p.m. as being in excess of that needed for the growth effect. It is at this level that the term “prophylactic feeding should apply. The question arises, however, is “prophylactic feeding” needed routinely to prevent infection or should it be used only when needed to stop a suspected disease in a herd or flock? In the United States the “prophylactic level” is assumed to be 100-400 p.p.m. and is used all too often in a routine feeding program. Still higher levels of antibiotic are often used, when necessary, to treat disease. This is properly carried out under veterinary control. However, up to 2000 p.p.m. can be so used, and there is much evidence to indicate that this “therapeutic level” feeding is not always used as intended by statute. The basic concern over the amount of antibiotic in feed is caused by potential public health hazards which may occur when the antibiotic develops a tissue level and is then presented to the consumer in meat. Those levels which result in tissue residues and become implicated in a threat to the public health are those which exceed the level necessary for the simple growth effect. Very few publications of investigations carried out to determine tissue residues in animals fed dietary antibiotics are available. This is unquestionably a neglected area from the standpoint of evaluating any public health aspects of this use of antibiotics. It is undoubtedly true that animals are coming to slaughter with antibiotic residues in their tissues, particularly those which have been on prophylactic or therapeutic dosage. Some data have been obtained in this area and are described. Broquist and Kohler (1954) point out that, following nutritional feeding of chlortetracycline to farm animals in amounts of 10-20 gm.per ton of feed, the antibiotic was not present in the serum or tissues of the animals. When the antibiotic was fed to chickens or pigs for an extended time at levels above 50 gm. per ton, the antibiotic could be detected in the serum. At levels of 1000 gm. per ton, the antibiotic could be recovered from tissue in the order of 1 part

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per 10,000 parts of tissue, These amounts of chlortetracycline resulting from massive chlortetracycline dosage rapidly disappeared from the tissues when the antibiotic was removed from the diet 1 or 2 days prior to slaughter, or when the meat was cooked. [This latter statement is controversial, Goldberg ( 1959).] Thus far, antibiotics used as feed supplements have not led to untoward effects in man, Residues of bacteriologically active antibiotics are not encountered in the flesh of animals that have received recommended levels of antibiotics ( u p to 20 p.p.m.) in feeds throughout their life span. Furthermore, detectable levels of bacteriologically active antibiotic residues in the flesh of animals fed antibiotic supplements disappear rapidly when antibiotic feeding is discontinued for a few days prior to slaughter. There then would seem to be little public health danger from the use of antibioticsupplemented feed, if feeding were discontinued after growth stimulation occurred and well before the time of slaughter. T. H. Jukes (1955) has summed up the public health hazards of antibiotics used in animal nutrition very clearly as follows. “The prolonged feeding of comparatively high levels of the common antibiotics to animals has not raised problems in public health as regards the commption of animal products. Broquist and Kohler (1954) found no detectable amounts of chlortetracycline in the liver and muscle of chicks receiving chlortetracycline, 200 mg/ kg of diet. Barely detectable amounts were present in the blood serum. At unusually high dietary levels, traces of the antibiotics were found in the liver and muscle. Chicken breast muscle containing 3 mg/g of chlortetracycline was found to be free from the antibiotic after boiling for 15 minutes or after roasting for 30 minutes at 230°C. No toxic reactions are imaginable from the traces of chlortetracycline which might be present in the meat of animals fed high levels of the antibiotic. Indeed, these traces of antibiotic were only of the order of 50 mg/lb or less of muscle tissue even when the chicken received 2000 gm of chlortetracycline per ton of diet and the antibiotic was destroyed by ordinary cooking procedures.” Analyzing the residue of antibiotics from use in feedstuffs has not yet been done on a large scale. Although there are data to show that high-level feeding is required to achieve tissue levels, it is not known how many animals came to slaughter following high-level (prophylactic or therapeutic) feeding.

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C. MODEOF ACTION OF ANTENOTICGROWTH STIMULATION It appears to be widely accepted that the effect of antibiotics upon growth is initiated by modification of the enteric flora. Nevertheless, there is a formidable array of evidence that much beyond the antimicrobial effect is involved. In a limited series of experiments with germfree chicks, Luckey (1956) showed that under some conditions (1-15 p.p.m.) the growth of these chicks was stimulated with antibiotics. However, this work has not been repeated with the same antibiotic levels, and some question exists as to the magnitude of this response. Using somewhat higher levels (25-50 p.p.m.1, Forbes et al. (1959) and Gordon et al. (1957) got no growth response in germfree chicks. There is no dispute, however, over the fact that antibiotic feeding results in a thinning of the intestinal wall (H. G. Jukes et al., 1956; Coats et al., 1955) and alteration of bowel motility (Leaders et al., 1956; Clegg, 1962). This is theorized by some to result in better ab,sorption of nutrients. Several workers have also shown that inactivated antibiotics (without antimicrobial activity) continue to bring about a growth response. This includes heat-, enzyme-, and metal-inactivated penicillin (W. L. Williams et al., 1953; T. K.Jukes, 1955; Taylor and Gordon, 1955). Further evidence for a mode of action beyond an effect on enteric flora is presented by the fact that some antifungal antibiotics produce a growth promotion effect. These include nystatin and griseofulvin (Coats, 1962). It seems unlikely that the antifungal activity of these agents would play a significant role toward influencing the enteric flora of most animals. Evidence substantiating the influence of the enteric flora on growth is best provided by the recent paper of Lev (1962). His work demonstrates the effect of CEostridium welchii toxin on growth rate of chicks. The author indicates clearly that when this organism is inhibited in the gut, growth proceeds at a higher rate. It is suggested that this and other microbial toxins are deleterious to growth and that antibiotics in the feed help control bacteria producing these harmful agents. Luckey (1959) has summarized several modes of action for antibiotic growth stimulation. They are described below because they appear to best represent the available documented opinion.

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SOMEPROPOSED MODESOF ACTION OF ANTIBIOTIC GROWTHSTIMULATION (modified from Luckey, 1959)

A. Indirect Action 1. Via intestinal microflora a. Increased numbers of “good” microorganisms, such as vitamin synthesizers b. Decrease numbers of “bad” microorganisms, such as vitamin users, toxin producers, and pathogens or potential pathogens 2. External (or intestinal) mileux reaction a. Detoxication b. Chelation activator c. Reduce p H of intestinal mileux B. Direct Action 1. Cells a. Permeability of cell wall b. Biological stabilizer against stress c. Activate anabolic regulator d. Mitotic stimulant 2. Tissues a. Intestinal wall length, weight, and thickness made more efficient b. Increased absorption c. Increased apparent utilization of metabolites d. Decreased energy expenditure

It can be concluded that the mode of antibiotic action as a growth stimulant is multifaceted and that alteration of enteric flora is not a complete explanation.

Ill. Antibiotics in Plant Disease Control ANTIBIOTICSOF IMPORTANCE In a recent review Goodman (1959) listed 25 bacterial diseases of plants that are amenable to antibiotics for treatment or prevention. In addition almost 50 fungal diseases of plants are described that respond similarly to antibiotics. In all of these cases there are only 3 antibiotics which play any significant role. These antibiotics are streptomycin, griseofulvin (Brian et aE., 1946), and cycloheximide (Whiffen et al., 1946). The latter two are antifungal in their activity while streptomycin is able to inhibit many bacterial and some fungal phytopathogens. Although streptomycin is well established as a medical antibiotic, griseofulvin and cycloheximide are less well known. The structures below indicate that these are

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99

compounds of low molecular weight ( 250450) with cycloheximide a weak acid and griseofulvin a neutral compound.

0 Cy cloheximide

Griseof ulvin

Cycloheximide is the more prominent of these antifungal agents in phytopathology. However, now that griseofulvin is produced by large-scale fermentation as an oral antibiotic for treatment of human fungal disease (Barnett, 1960; D. I. Williams, 1960), it is possible that an extension of its use in agriculture may occur. Streptomycin is primarily effective against bacterial diseases caused by Xanthomonas, Pseudomonm, and Erwinnia spp. and Phytophthora and Peronospora fungi (Zaumeyer, 1956). Cycloheximide is most active against Cocomyces (cherry leaf spot) and turf diseases (Hamilton et al., 1956). Griseofulvin was most recently reported to be active against powdery mildews, melon cankers, and certain Fusarium species (Rhodes, 1962). The fundamental advantage of antibiotics over previous methods of plant disease control has been the fact that antibiotics are absorbed by the plant and are effective within the plant. That is to say the antibiotics are systemic in their action. Prior to the advent of antibiotics, plant disease control was based upon external protectants. Such protectants were applied to surfaces, where they remained until diluted, inactivated, or degraded. They were “preventive” in their action. There is evidence to show that antibiotics are both protective and “therapeutic.” Not only can the antibiotics prevent plant disease, they can also eradicate existing disease by virtue of their ability to act systemically and be translocated. Of course, results vary with the nature of the antibiotic and plant tested. Such influences as phytotoxicity (Goodman, 1962), antibiotic concentration, method of application, temperature, and humidity play an important role in the outcome of this action. Since applications may be made by spraying, dusting, dipping, and soil routes the stability and persistence of the antibiotic will vary as will its

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usefulness in disease control. Another contributing factor is, of course, economic. The current use of antibiotics in plant disease control is undoubtedly limited by this factor. Detailed analysis of stability, persistence, and movement of antibiotics in plants have been made by Pramer (1959), Goodman (1959, 1962), Goodman and Dowler (1960), and Goodman and Goldberg (1960). This use of antibiotics also has a public health aspect since many plants so treated ultimately are used for food. The problem of antibiotic residues from plant disease control is discussed in the next section on antibiotics in food preservation.

IV. Antibiotics in Food Preservation A. CANNING The antibiotic most evaluated as an adjunct to mild heat for the canning preservation of foods has been subtilin. Originally, several optimistic reports (Anderson and Michener, 1950) and ( Burroughs and Wheaton, 1951) indicated this polypeptide antibiotic could preserve foods in cans with the addition of mild heat. It soon became apparent, however, that species of Clostridiurn botulinum, the organism of botulism, an often fatal type of food poisoning, were not destroyed by this procedure (Cameron and Bohrer, 1951). Subsequently, although much work on antibiotics in canned foods was continued, the fear of botulism negated acceptance of any antibiotic agent as the sole source of sterilization of canned products. More recently, however, two additional antibiotics, nisin ( Mattick and Hirsch, 1957), a polypeptide, and tylosin, a macrolide (Greenberg and Silliker, 1962a,b, c ) , have reawakened interest in this subject. These have been tested with some success for ability to reduce thermophilic spoilage bacteria in all types of canned foods including fruits, vegetables, meats, fish, soups, and dairy products. Under this new approach the search for an antibiotic capable of destroying botulinum spores is abandoned and the antibiotics in canned foods are used as a supplement to traditional botulinum destroying methods of processing. In this manner, saprophytic spoilage organisms may be controlled as well as the agent of botulism. In order for agents to be successful against saprophytic spores in canned foods, they must be present in the product throughout its shelf life. Thus, an antibiotic residue is necessarily to be encountered in this procedure. From the available data, it would appear

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that such residues would probably fall in the range of 1-20 p.p.m. (Goldberg, 1959).The actual residue depends, of course, upon the nature of the product, the pH, moisture content, bacterial flora, and on the degree of thermal application. Although many additional antibiotics such as tetracyclines, streptomycin, chloromycetin, and penicillin have been tested for their ability to contribute to preservation of canned foods, they have, in general, been eliminated from canning because of objectionable chemical or physical properties. Consequently, subtilin, nisin, and tylosin remain the antibiotics of choice for inhibition of spoilage bacteria, particularly of the thermophilic variety. B.

DAmY

PRODUCTS

In technologically advanced areas there is little reason to consider the possible use of antibiotics to keep milk fresh. However, fresh milk is unavailable to much of the world's population, partly because of the impossibility of distributing it without adequate pasteurization, refrigeration, and transportation facilities. Consequently, some study has been given to the possible use of antibiotics in this regard. Early observations indicated that penicillin, streptomycin, and other narrow-spectrum antibiotics, although capable of interfering with lactic acid production (souring), were of little value against putrefaction. Various combinations were somewhat more effective, but, as observed with other foods, the broad-spectrum antibiotics proved to be of the greatest value. Several reports indicate that chlortetracycline (CTC) or oxytetracycline (OTC) can substitute to a considerable extent for refrigeration. If 1 p.p.m. of a tetracycline antibiotic is added to raw milk directly after milking, the onset of spoilage is delayed for about one day at 37°C. If the milk is pasteurized, these antibiotics will preserve it without refrigeration from 2, days to several weeks, depending on storage conditions and the level of antibiotics used (Wrenshall, 1959). Residue data for antibiotics in milk preservation indicate some interesting phenomena. Low-level antibiotic residues are remarkably stable in milk. In addition, there is ample evidence that pasteurization temperatures have little or no effect and some constituents of milk offer considerable protective effect. Residue levels for increasing the shelf life of fresh milk can be anticipated at 1-2 p.p.m., and this level is ample for preservative

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purposes. Studies have been carried out to show that OTC looses only 2.5% of its activity during pasteurization when inoculated at 0.5-1.0 p.p.m. in fresh milk; streptomycin loss and OTC loss are about the same in similar conditions. Cheese products, both processed and natural, may often be spoiled by certain clostridial species. Here the antibiotic nisin has seemed to be most effective and also at a rather low level, less than 5 p.p.m. However, no exact level for nisin can be established since it is found naturally in cheeses. It should be noted here that a recent committee of experts has indicated that hydrogen peroxide is a more practical milk preservative than antibiotics (WHO/FAO Technical Rept., 1960). C. FRESH MEATSAND POULTRY Rather remarkable preservation powers have been reflected in the use of tetracycline antibiotics applied to red meats and poultry. Three methods of introducing the antibiotic have been used with success. The first consisted of dipping steaks and chops in antibiotic solution (Tarr et aZ,, 1952); this retarded bacterial spoilage quite effectively. As many as 20 antibiotics were used but oxytetracycline and chlortetracycline proved best. In a second method, the entire carcass was infused with antibiotic just after slaughter (Weiser et d.,1953). Here again chlortetracycline was able to keep beef successfully at temperatures as high as 80°F. for several days. A third successful method utilized ante-mrtem injection of oxytetracycline for preservation of meat of cattle, lamb, and other species (McMahan et al., 1956). In all cases of tetracycline preservation of meat, it is quite apparent that some low-level residue will reach the consumer. Although residues of OTC are somewhat higher than CTC, there is actually little difference. The initial level in the raw meat ranges from 1 4 p.p.m. Following cooking, a decrease in residue occurs but there is still detectable residue no matter if the meat is cooked rare, medium, or well done. This residue is almost always less than 1.0 p.p.m. (Goldberg, 1959). This point when applied to poultry is somewhat altered. It has been shown that the tetracyclines are the antibiotics of choice for poultry and dipping in antibiotic solution is the preferred method. However, by limiting the dip to 55 p.p.m. concentration, the residue level on the bird is less than 7 p.p.m., and all of this is destroyed

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by cooking (Hines, 1956). The subject of antibiotics in poultry preservation has recently been reviewed ( Ayres, 1962).

D. FRUITS AND VEGETABLES Antibiotic treatment of fruits and vegetables has taken two directions. The first concerns antibiotics in plant disease control. In this instance, streptomycin is clearly the antibiotic of choice (Goodman, 1959). It is applied to apple, pear, peach, and other trees during the growing season to prevent bacterial disease. Among vegetable crops it is applied to beans, tomatoes, cabbage, etc., for the same purpose. In treating the fruit trees, no residue is found in the fruit because the disease treatment is applied before flowering and fruiting. Vegetables, however, are sprayed until time of harvest, and they may show streptomycin residue at detectable levels within the vegetable tissues. The extremely perishable nature of fresh vegetables, especially the green leafy varieties, is one of the most adverse factors encountered in their distribution. Substantial losses due to spoilage are accepted as normal and are a major factor in determining the price consumers must pay for such products. The need for methods of delaying vegetable spoilage has been accentuated by recent trends in distribution involving the prepackaging of these commodities in ready-to-use form. The most prevalent cause of spoilage is bacterial soft rot. Modern methods of handling raw agricultural commodities after harvest materially reduce development of bacterial spoilage. Such methods as hydrocooling and refrigeration, however, only inhibit microbial decay by the influence of temperatures adverse to growth of microorganisms. When perishable produce is removed from these

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spinach by a l-minute dip in a 500 p.p.m. solution of streptomycin sulfate. Koch and Carroll (1957) reported reduction in decay of spinach with oxytetracycline, streptomycin, polymyxin, and neomycin dip treatments. Cox (1955) and associates also reported complete control of radish pit after 5 days' storage at temperatures of 5"35"C., using 5-minute dip treatments of 50 p.p.m. oxytetracycline solutions. Streptomycin at the same concentration was not as effective and did not give control at 35°C. Carroll and associates (1957) studied decay in a salad mix and each of its components, using dip treatments of antibiotics. Vegetables which deteriorate rapidly appeared to establish the rate of decay for the other components in a salad mix. Degree of spoilage could be judged easily by measuring the volume of liquid exudate. The effects of antibiotics and refrigeration were additive. Oxytetracycline was by far the most effective antibiotic tested. Goodman and associates (1958) confirmed the effects of antibiotics in prolonging the refrigerated shelf life of spinach. They also studied the active antibiotic residues present in various vegetables after treatments with oxytetracycline and streptomycin. Residues of streptomycin were found to be persistent, and there was no assurance that they would be destroyed by cooking. While the information available is still somewhat fragmentary, the future appears to hold considerable promise for the use of antibiotics on vegetable products. Certainly, antibiotics appear capable of contributing to the solution of serious spoilage problems in this area. Realization of these potential values must, however, await clarification of the public health significance of antibiotic residues. Summing up we find that the antibiotics of importance in fruit and vegetable preservation are oxytetracycline, chlclrtetracycline, and streptomycin. The residues are < 5.0 p.p.m. for the tetracyclines and 2-40 p.p.m. for streptomycin.

E. FISHAND SEAFOOD In this area most work has been done by Canadians, Japanese, and English workers concerned with preservation of one of the most perishable of foods (Tarr et al., 1952; Tomiyama et al., 1955; Ingram et al., 1956). As is true with certain other foods, stability of antibiotics is often enhanced, particularly in conjunction with fish skin. Ample studies have shown that only tetracycline antibiotics

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have the capability of controlling spoilage in this product. Application of these antibiotics in the form of ice or dipping fillets in antibiotic solution are both highly effective in increasing storage ability. Although much antibiotic residue is decreased in cooling, it is still necessary to consider the tetracycline residues in concentration of < 1.0-5.0 p.p.m. in fish treated for preservation by antibiotics, Table I11 sums up the current status of antibiotics permitted for food preservation in various countries. It can be quite easily seen TABLE I11 FOODPRESERVATION BY ANTIBIOTICS IN DIFFERENT COUNTRIES

Country

Antibiotics permitted

Tolerance permitted (p.p.m. 1

Used for

Argentina

Chlortetracycline Oxytetracycline

5-10

Meat, poultry, fish

Canada

Chlortetracycline Oxytetracycline

7 5

Poultry Fish preservation in ice Fresh fillets in dipping tanks

10

Great Britain

Chlortetracyclinea 5 Oxytetracyclinea Nisina No limit Nystatin

On the skin but not in the flesh 5

Japan

Chlortetracycline Oxytetracycline

Norway

Chlortetracycline Oxytetracycline

250

United States

Chlortetracycline Oxytetracycline

5

Chlortetracycline Oxytetracycline

7

Chlortetracycline

5

USSR

'Raw fish Cheese and certain canned goods Bananas

Fish preservation in ice; fish for fish pasta; salmon for cannine Slaughterhouse offals for minks in the warm weather Deriod Fish preservation in ice; preservation of shrimps and scallops Poultry preservation in slush-ice tanks Codfish preservation in ice and for transuort

a The use of these antibiotics for the purposes and in the amount stated is a proposal only, made by the Antibiotic Panel of the Ministry of Health.

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that the experimental results above have not been readily accepted by regulatory agencies of governments due to unanswered public health questions.

V. Antibiotics as Adjuncts in Microbiological Techniques and Procedures

A. TISSUE CULTURE There are generally two broad areas in which the microbiologist has extensively utilized antibiotics in the laboratory. The first is in the selective isolation of specific microorganisms and the second is the characterization of microorganisms for purposes of classification via “antibiograms.” In the area of selective isolation Goldberg (1959) and Cruickshank (1960) have reviewed the subject in detail with primary emphasis on selecting bacteria and fungi of animal or plant pathogenic origin. More recent advances have been in the area of tissue culture growth of virus with attempts to prevent fungal and yeast contamination. Penicillin and streptomycin or chloromycetin alone appear to control satisfactorily the bacterial contamination in tissue cultures. The most recent of the agents to be used in tissue culture to control fungal and yeast contamination is amphotericin B (Gold et al., 1956). It was investigated by Perlman et al. (1961) for effect on several tissue culture cell lines with a variety of media. The results indicated that amphotericin B is not toxic in tissue culture at a recommended concentration of 2.0pg./ml. It has also been shown that normal growth of viruses is not interfered with by amphotericin B. Although this antibiotic is slowly inactivated at 37°C. it does not loose potency in the refrigerator (Table I V ) . As large-scale tissue cultures are produced for growing virus vaccines the need for yeast and fungal control becomes more important. Undoubtedly, amphotericin B will find more widespread use in the future. Nystatin (Hazen and Brown, 1951) continues to be used in tissue culture as an antifungal agent (Table V). It is a polyene antibiotic, as is amphotericin B, and has many similar uses. However, this antibiotic has the disadvantage of being unstable at 37°C. and must be added to tissue culture media to maintain an antifungal level; it thus may be ultimately replaced by amphotericin B.

STABILITYOF AMPHOTERICINB

TABLE IV VARIOUSTISSUE CULTUREMEDIAAT 37°C.

IN

Medium without cells; amphotericin B concentrations (pg./ml.) at: Mediuma

0 day

2 days

4 days

A

5 50 5 50

3.5 50 1.2 43 3.9 44 3.4 43

2.7 29 0.6 37 2.8 37 1.9 39

B C

5 50

D

5

50 a

b

I

7 days Mediuma 1.7 29 0.5 27 2.0 33 1.0 34

A

B C D

Medium with cells;b amphotericin B concentrations ( pg./ml.) at: 0 day 2 days 4 days 7 days 5 50 5 50 5 50 5 50

3.6 46 1.3 41 4.4 50 3.4 42

3.3 16 0.9 33 3.7 39 2.9 40

Composition of media: A-Waymouth‘s MB 752/1 supplemented with 10% (v./v.) calf serum and 0.03% methylcellulose. B-Waymouth‘s MB 752/1 supplemented with 0.5% methylcellulose. CZiegler’s modification of Eagle’s medium containing 10% ( v./v. ) calf serum. D-Eagle’s medium containing 10% (v./v.) calf serum. Cell line: Earle’s L cells NCTC 929 (mouse fibroblast).

2.9 13 0.9 28 3.3 35 2.1 38

r

C m

N

IA

5

H

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HERBERT S. GOLDBERG

TABLE V ANTIFUNGAL SPECTRUM

OF

NYSTATIN Minimum inhibitory

concentration (units/ml. ) a

Organism Aspergillus fumigatus Bhtomyces dermatidis (yeast phase) Candida albicam Candida guillermondi Candida krusei Cryptococcus neofomnans Geotrichum luctb H i s t o p h a capsulatum (yeast phase) PetaicilEium spp. Rhodoturulu muciluginosa Saccharomyces cereuisiae Saccharomyces pastorianus Trichophyton mentagrophytes a

6.25 1.56 3.13 3.13 6.25 1.56 6.25 1.56 13.0 1.56 3.13 3.13 6.25

As determined by a twofold dilution test.

Nystatin’s most recent use has been in submerged culture of plant cells (Bryne and Koch, 1962) and with marine fish-cell tissue culture (Clem et al., 1961). In summarizing this use of antibiotics the data above together with the aforementioned recent reviews of Goldberg (1959) and Cruickshank (1960) indicate that viral specimens, chick embryo cultures, and tissue cultures require penicillin and streptomycin or chloromycetin for bacteria-free conditions and amphotericin B or nystatin for yeast- and fungi-free conditions. B. MICROBIAL CLASSIFICATION Antibiotics have been used in the past as an epidemiologic tool much as phage typing or serology. It is only recently, however, within the last 10 years, that formal classification has been attempted by antibiotic sensitivity patterns or “antibiograms.” The majority of the work published so far has concerned the Pseudomom-Achromobacter group. Shewan et al. ( 1954, 1960) classified Pseudomonas by penicillin sensitivity and divided Achromobacter, Alcaligenes, and Spirillum from Flauobacterium, Pseudomonas, Vibrio, and Aeromonas by penicillin response. The following genera have also been taxonomically evaluated by antibiotic patterns: Coynebacterium (Seaman and Woodbine, 1962), Listeria

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(Jones and Woodbine, 1961), Erysipelothrix ( Wix and Woodbine, 1955), Microbacterium (Turbitt et al., 1959, 1960). Renoux (1960) used erythromycin at 15pg./ml. to differentiate Brucella spp., and Lutz et al. (1958) showed that the Proteus species can be separated by novobiocin. Goldberg and Barnes (1962) have used antibiograms as aids in identifying gram-negative anaerobic rods. They were able to separate Bacteroides from Spherophorus and Fusobacterium genera by the use of penicillin, neomycin, and polymyxin B. Bacteroides were inhibited by penicillin and not by the other two. The Spherophorus and Fusobacterium were inhibited by penicillin and polymyxin B but not by neomycin. Seaman and Woodbine (1962) analyzed a genus which contained both plant and animal pathogens. They concluded that the Corynebacterium from a single habitat could be usefully separated by antibiotics. However, results were unsatisfactory when, for example, the plant and animal pathogens were tested as a single group. One weakness of this method of classification is similar to that inherent in all taxonomic schemes, i.e., lack of consistent, standardized methods with large numbers of strains. It is anticipated that this situation will improve with reawakened interest in the problems of microbial classification (Ainsworth and Sneath, 1962).

VI. Public Health Aspects of Nonmedical Uses of Antibiotics

Untoward effects of antibiotics have been described in numerous reports in the literature of clinical medicine. For the most part, these effects have appeared following long-term therapeutic dosage or have been due to individual idiosyncrasy and drug reaction. The reactions have been emergence of antibiotic-resistant bacteria, hypersensitivity, toxicity, and superinfection or overgrowth of indigenous flora resulting in a pathogenic process, i.e., candidiasis. The question at hand is whether or not low levels of these antibiotics are capable of elucidating the same effects. In most instances the dose of antibiotic ingested from food would be 100-500 times less than a therapeutic daily dose of the same antibiotic. This can be illustrated with chlortetracycline. The level in foods preserved with CTC is 1-10 p.p.m., usually at the lower range. If an individual consumed 1-2 kg. of food per day, the total intake would be 5-10

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mg. This is about 100300 times less than the therapeutic daily dose of CTC. In order to consider the low levels of these antibiotics each can be reviewed in turn with regard to resistance, superinfection, hypersensitivity, and toxicity rate at low levels. A. TETRACYCLINES The tetracyclines of importance in foods and feeds are chlortetracycline and oxytetracycline. The maximum level to be encountered would seem to be less than 10 p.p.m. according to the preceding data. The foods involved are all of the fresh variety and include meats, poultry and fish, fruits, and vegetables. Many reports have been made on the effect of these antibiotics on intestinal bacteria of laboratory animals. Unfortunately, the data are conflicting, particularly with regard to coliform population and resistance (Schwachman et al., 1951; Goldberg et al., 1958, 1959). However, there have appeared more satisfactory studies with humans on long-term OTC or CTC, and these data are worthy of comment. Schwachman and associates (1951, 1952; Schwachman and Kulczycki, 1958) in several reports have used chlortetracycline or oxytetracycline in treating chronic cystic fibrosis over a period of 8 to 9 years. These antibiotics were given in daily doses up to 25 mg./kg. body weight. Although development of resistance by Staphylococcus and Proteus species occurred and an increase in yeasts was found, the authors continued treatment and were satisfied with results, since only clinically beneficial responses were noted. Sprunt and McVay (1953) used CTC at a daily level of 500mg. for 19 months in geriatric patients as a routine prophylactic. Toxic effects were minimal and did not require cessation of antibiotic. A reduction in respiratory and urinary infections was observed, although no studies on enteric microorganisms were carried out. Other studies support the efficacy of therapeutic daily doses of tetracycline antibiotics as prophylactic agents over long periods. However, bacteriologic analyses were not reported ( Stowens, 1951 ). Studies of long-term, low-level tetracyclines in humans are confined for the most part to attempts to correct growth failure in malnourished children. Joliffe et al. (1956) fed 20 mg. CTC daily for 7 months to undernourished children. When compared with

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controls, these children showed greater weight gains. No untoward effects were reported, although no bacteriology was done. Similarly, Scrimshaw is reported by Carey (1958) to have fed 50mg. CTC daily to children for up to 30 months. No toxic effects were noted. Physical examination and hematological study supported this claim, although no bacteriology was done. Bacteriologic analysis was carried out by Loughlin et al. (1958), a study which evaluated OTC as a growth-promoting agent in 243 young children. Daily 10 and 50 mg. doses were administered for 12 months to several groups of these children. Cultures of Staphylococcus aureus and enterococci were isolated from rectal swabs and checked for OTC resistance prior to and periodically after the study began. A skin test was also employed to determine allergic sensitization to OTC. The results showed complete absence of toxic effects, gastrointestinal upsets, increased resistance, moniliasis, and positive skin tests. More recently a few limited studies have been carried out to investigate specifically the effect of CTC or OTC levels from food preservation on human intestinal flora. Kuwahara et al. (1958) administered CTC to five healthy adults in a daily dose of 0.0-20 mg. for 20-107 days and observed change in flora and skin reaction for sensitivity to CTC. No change was observed in cases given daily doses of 10 and 20 mg., except a tendency toward constipation and a concurrent increase in colon bacilli and enterococci in stool during administration. In all the cases, resistant strains emerged during drug administration, especially among strains of colon bacilli which were found to show resistance to 100 pg./ml. However, these strains disappeared after stopping the administration of CTC. Over a 4-month period, after the end of the administration, skin tests were carried out using CTC solution; the results were negative in all cases. Knothe (1957, 1958) found that coliform resistance developed when more than 25 mg. CTC/kg. of food were present. However, the resistant flora disappeared quickly on cessation of antibiotic in the food. Finally, Goldberg ef al. (1961), working with 46 persons, fed 10 mg. OTC/kg. of food and found that resistant coliforms existed prior to antibiotic administration, that OTC-induced resistance was transitory, that no OTC blood levels appeared, and that hypersensitivity to OTC failed to occur. These experiments were carried out over 14 months, and all individuals acted as their own controls.

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On the basis of available information, it seems questionable that any hazard exists from ingestion of OTC or CTC at levels in foods that are below 10 p.p.m. in antibiotic residue. With the knowledge that hypersensitivity to these antibiotics is extremely rare, this too presents a doubtful hazard. One relatively unknown area, however, concerns breakdown products of the tetracyclines such as isochlortetracycline. This heat degradation product appears to be harmless; further work is needed in this area.

B.

STREPTOMYCIN

Streptomycin has many characteristics subject to objection when used therapeutically. A t the lower levels it is found useful in food preservation, although many of these criticisms still remain. In almost all studies on intestinal flora in animals using high or low levels, this antibiotic brings about rapid resistance. Streptomycin has been shown to be extremely stable in milk, in tissues of vegetables, and in other plant foods. Furthermore, the toxicity of this antibiotic for the eighth cranial nerve is well documented. Consequently, it would seem that streptomycin can be a public health hazard in foods. Streptomycin in plant disease control is not a hazard so long as field workers do not react allergically when applying streptomycin to plants. Studies on development of hypersensitivity by field workers have been carried out, however, and appear to be of no significant hazard (Goldberg, 1962). Streptomycin in animal feedstuffs also does not appear to be hazardous since oral streptomycin is not absorbed into the tissue of animals and is excreted in feces and urine. C. PENICILLIN This antibiotic has little role in food preservation. Its limited antibacterial spectrum and the prominent ability to stimulate allergic responses in a large number of individuals precludes its use in this manner. On the other hand, this antibiotic plays an important role in animal dietary supplements. Levels are fed that are quite low and little, if any, penicillin gets into animal tissues. The main public health concern is environmental. Several investigators have reported resistant staphylococci in human attendants working in areas where penicillin supplements were used. In addition, the

NONMEDICAL. USES OF ANTIBIOTICS

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animals appeared to be carriers of penicillin-resistant organisms at a higher percentage than expected (Smith and Crabb, 1957).

D. NISIN-SV~TILIN-BACITRACIN These three antibiotics are all polypeptide in nature and can be considered as a group although they have individual characteristics which contribute to their role in foods and foodstuffs. As a group, they are not absorbed from the gut into tissues and thus their influence on other than intestinal flora is limited. They are of little consequence in hypersensitivity and nontoxic at low levels by the oral route. Nisin and subtilin have a useful role in canned food preservation as a supplement to heating. They can prevent spoilage successfully, and in acid foods, where botulism is not a threat, they can supplement reduced heating processes. Since they are narrow spectrum antibiotics, they do not alter the intestinal flora and, in fact, are acted upon by digestive enzymes of the body. Of the two, nisin has the advantage of occurring in nature in many dairy products and thus is consumed by many, daily, as part of their diet. As long as these antibiotics are used as advocated in canned foods, at levels below 20 p.p.m., and accompanied by botulinum-destroying processing, they appear to lack any hazard to public health. Bacitracin has found a useful role in the zinc form as an animal dietary supplement. Public health does not seem endangered by such use.

E. TYLOSIN~LEANDOMYCIN The macrolide antibiotic, tylosin, has recently been advocated as a supplement to canned food preservation. The claim that it is not used medically is not quite valid since it is closely related to erythromycin and oleandomycin and shares some cross resistance with these medical antibiotics. The spectrum of tylosin is somewhat broader than nisin and subtilin and should be further investigated along with other canning antibiotics. In general, published reports on tylosin and tylosin residues in foods are promising (Denny et al., 1961; Greenberg and Silliker, 1962a, b, c ) . This antibiotic is also used in animal feeds, particularly for swine. It has also been advocated for prophylactic and therapeutic use in pleuropneumonialike organism (PPLO) diseases of poultry. At the present time,

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HERBERT S. GOLDBERG

tylosin and oleandomycin show a minor role in nonmedical usage of antibiotics.

F. RESEARCHNEEDS Because of the paucity of noncommercial laboratories working in the area of nonmedical use of antibiotics, there are many unanswered questions. The comments in the preceding pages are based upon the author’s own investigations and the published literature. It wouId appear, however, at this time that considerable studies are needed to further results, protect the public health, and expand the worlds food supply. The necessary public health studies fall into three categories.

1. Laboratory Studies Most needed here are acceptable standardized methods for the assay of antibiotics in all types of foods, Obviously, intelligent evaluation of residues cannot be made until such tests are available and standardized. The techniques of the F.D.A., available on request, go a long way in this direction.

2. Animal Studies These must be directed to acute and chronic oral toxicity studies with the antibiotic compounds and their breakdown products. These studies should be oriented toward finding safe levels. If it is clear that levels are going to be present, what are the safe ranges toxicologically? 3. Human Studies

Studies such as those completed by Kuwahara et al. (1958), Knothe (1957, 1958), and Goldberg (1962; Goldberg et aZ., 1961) should be expanded and extended by direct studies on human volunteers. One can take the laboratory and animal data and achieve meaningful results. However, studies on resistance, toxicity, and hypersensitivity should be encouraged in human volunteers whenever possible.

REFERENCES Ainsworth, G . C., and Sneath, P. H. A. (1962). “Microbial Classification.” Cambridge Univ. Press, London and New York. Anderson, A. A., and Michener, H. D. (1950). Food Technol. 4, 188.

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Ayres, J. C. ( 1962). “Antibiotics in Agriculture” (Woodbine, ed.), pp. 244266. Buttenvorths, London. Barnett, S. M. (1960). Wisconsin Med. J. 59, 421. Bonde, R. (1953). Am. Potato J . 30, 143. Brian, P. W., Curtis, P. J., and Hemming, H. G. (1946). Trans. Brit. Mycol. SOC. 29, 173. Brody, H. D., and Francis, F. J. (1956). Pre-Pack-Age 10, 29. Broquist, H. P., and Kohler, A. R. (1954). Antibiotics Ann. 2953-54, p. 409. Burroughs, J. D., and Wheaton, I. E. (1951). Canner 112, 50. Byrne, A. F., and Koch, R. B. (1962). Science 135, 215. Cameron, E. J., and Bohner, C. W. ( 1951). Food Technol. 5, 340. Carey, B. W. (1958). Intern Congr. Biochem. 5th Congr., p. 208. Carlson, C. W. (1957). Feed Age 7, 42. Carroll, V. J., Benedict, R. A., and Wrenshall, C. L. (1957). Food Technol. 11, 490. Christensen, J. J. (1956). First Intern. Conf. Antibiotics in Agr., Proc. Natl. Acad. Sci. 397, 13. Clegg, F. G. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 361-369. Buttenvorths, London. Clem, L. W. et al. (1961). Proc. Soc. Exptl. B i d . Med. 108, 762. Coats, M. E. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 203-208. Butterworths, London. Coats, M. E., Davies, M. K., and Kar, S. K. (1955). Brit. J. Nutrition 9, 110. Cox, R. S. (1955). Plant Diseuse Reptr. 39, 421. Cruickshank, R. (1960). Brit. Med. Bull. 16, 79. Denny, C. B. et al. (1961). Food Technol. 15, 338. Fernando, R., and Jacquet, J., Jr. (1958). Reu. Hyg. Med. SOC. 6, 77. Forbes, J. J., Parks, J. T., and Lev, M. (1959). Ann. N . Y. Acad. Sci. 78, 321. Gold, W. et al. (1956). Antibiotics Ann. 1955-56, p. 579. Goldberg, H. S., ed. ( 1959). “Antibiotics, Their Chemistry and Non-medical Uses.’’ Van Nostrand, Princeton, New Jersey. Goldberg, H. S. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 389-400. Butterworths, London. Coldberg, H. S., and Barnes, E. M. (1962). Abstr. Intersci. Conf. Antimicrobial Agents and Chemotherapy, Am. SOC.Microbiol. p. 46. Goldberg, H. S., Read, B. E., and Goodman, R. N. (1958). Antibiotics Ann. 1957-58, p. 144. Goldberg, H. S., Goodman, R. N., and Lanning, B. (1959). Antibiotics Ann. 1958-59, p. 930. Goldberg, H. S . et al. (1961). Antimicrobial Agents and Chemother. 1961, pp. 80-88. Goodman, R. N. (1959). In “Antibiotic, Their Chemistry and Non-medical Uses” ( H . S. Goldberg, ed.), Van Nostrand, Princeton, New Jersey. Goodman, R. N. ( 1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 165-180. Butterworths, London.

116

HERBERT S. GOLDBERG

Goodman, R. N., and Dowler, W. N. (1960). Intern. Congr. Plant Protect. 2, 1559. Goodman, R. N., and Goldberg, H. S. (1960). Phytopathology 50, 851. Goodman, R. N., Johnston, M. R., and Coldberg, H. S. (1958). Antibiotics Ann. 1957-58,p. 236. Gordon, H. A., Wagner, M., and Wostmann, B. (1957). Antibiotics Ann. 195657 p. 218. Greenberg, R. A., and Silliker, J. H. (1962a). J. Food Sci. 27, 64. Greenberg, R. A,, and Silliker, J. H. (1962b). Food Sci. 27, 60. Greenberg, R. A., and Silliker, J. H. (1962~).Bacteriol. Proc. p. 1963. Hamilton, J. M. et al. (1956). Science 123, 1175. Hazen, E. L., and Brown, R. (1951). Proc. Soc. Exptl. Biol. Med. 76, 93. Hines, L. R. (1956). Proc. Natl. Acad. Sci. U.S. 397, 227. Ingram, M., Barnes, E. M., and Shewan, J. M. (1956). Food Sci. Abstr. 28. Joliffe, N. et al. (1956). Antibiotics Ann. 1955-56, p. 19. Jones, S. M., and Woodbine, M. (1961). Vet. Res. Ann. 7, 39. Jukes, H. G., Hill, D. C., and Branian, H. D. (1956). Poultry Sci. 35, 716. Jukes, T. H. (1955). “Antibiotics in Nutrition,” M. D. Encyclopedia, New York. Knothe, H. (1957). Deut. Med. Wochschr. 82, 1685. Knothe, H. (1958). Arzth. Wochschr. 13, 179. Koch, G., and Carroll, V. J. (1957). Antibiotics Ann. 195657, p. 1010. Kuwahara, S. et al. (1958). Japan. J . Microbiol. 2, 225. Leaders, F., Pittinger, C. B., and Long, J. P. (1960). Antibiot. Chemotherapy 10,503. Loughlin, E. H.et al. (1958). Antibiotics Ann. 1957-58, p. 95. Luckey, T. D. (1956). Proc. Natl. Acad. Sci. U . S. Luckey, T. D. (1959). In “Antibiotics, Their Chemistry and Non-medical Uses” (H. S. Goldberg, ed.), Van Nostrand, Princeton, New Jersey. Lutz, A. et al. (1958). Ann. Inst. Pasteur. 94, 44. McMahan, et al. ( 1956). Antibiotics Ann. 1955-56, p. 727. Mattick, A. T. R., and Hirsch, A. (1944). Nature 154, 551. Moore, P. R., Evenson, A., Luckey, T. D., McCoy, E., Elvehem, C. A., and Hart, E. B. (1946). J. Biol. Chem. 165, 437-441. Perlman, D.et al. (1961). Proc. Soc. Exptl. Biol. Med. 106, 880. Pramer, D. (1959). Adoan. Appl. Microbiol. 1, 75. Renoux, G. (1960). Arch. Inst. Pasteur. Tunis 37, 23. Rhodes, A. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 101-121.Buttenvorths, London. Schwachman, H., and Kulczycki, L. L. (1958). Am. J. Diseases Children 96, 6. Schwachman, H., Foley, G. E., and Cook, C. D. (1951). J. Pediat. 38, 91. Schwachman, H. et al. (1952). J. Am. A . 149, 1101. Seaman, A., and Woodbine, M. (1962). In “Antibiotics in Agriculture” ( Woodbine, ed. ), pp. 333-346.Buttenvorths, London. Shewan, J. M., Hodgkiss, W., and Liston, J. (1954). Nature 173, 208. Shewan, J. M., Hobbs, G., and Hodgkiss, W. (1960). J . Appl. Bacteriol. 23, 379.

NONMEDICAL USES OF ANTIBIOTICS

117

Smith, H. W., and Crabb, W. E. (1957). Vet. Res. 69, 24. Smith, L. W. (1953). Phytopathology 42, 475. Sprunt, D. H., and McVay, L. V. (1953). Antibiotics Ann. 1953-54, p. 273. Stowens, D. (1951). Pediatrics 8, 60. Tarr, H. L. A. et al. (1952). Food Technol. 6, 363. Taylor, J. H., and Gordon, W. S. (1955). Nature 176, 312. Tomiyama, T., Kuroki, S., and Nomura, M. (1955). Bull. Japan. SOC. Sci. Fisheries 21, 958. Turbitt, P. A,, Seaman, A., and Woodbine, M. (1959). J . Appl. Bacteriol. 22. Turbitt, P. A., Seaman, A,, and Woodbine, M. (1960). Dairy Sci. Abstr. 22, 543. Weiser, H. H. et al. (1953). Food Technol. 7, 495. Whiffin, A. J., Bohonos, N., and Emerson, R. L. (1946). Phytopathology 44, 509. Williams, D. I. (1960). Practitioner 184, 383. Williams, W. L. et al. (1953). Federation PTOC. 12, 290. Wix, P., and Woodbine, M. (1955). Brit. Vet. J. 111, 432. World Health Org. Tech. Rept. (1960). Milk Hyg. No. 197. World Health Org. Tech. Rept. (1963). Public Health Aspects of the Use of Antibiotics in Food and Feedstuffs No. 260. Wrenshall, L. ( 1959). In “Antibiotics, Their Chemistry and Non-medical Uses” ( H . S. Goldberg, ed.), Van Nostrand, Princeton, New Jersey. Zaumeyer, W. J. (1956). Proc. Intern. Conf. Antibiotics, 1st Conf. pp. 171187.