(Eckert, 1947; Johnson, 1947, 1948; Johnson and Stadel, 1946; Milne,

Transcription

1 THE INFLUENCE OF ANTIBIOTICS AND SULFA DRUGS ON BACILLUS LARVAE, CAUSE OF AMERICAN FOULBROOD OF THE HONEYBEE, IN VITRO AND IN VIVO' H. KATZNELSON2 Division of Bacteriology and Dairy Research, Department of Agriculture, Ottawa, Canada Received for publication January 5, 1950 American foulbrood is a disease of the larvae of the honeybee caused by a sporeforming organism, Bacillus larvae White (1920), and is considered the most serious of all bee diseases. The unusual resistance of the spores to heat and disinfectants (White, 1920; Sturtevant, 1926; Burnside, 1940) has resulted in the development of a variety of methods for control based on their effectiveness in destroying these spores. The burning of diseased and contaminated material has been univerally accepted as the most efficacious of these methods, although it is a costly procedure. For this reason the recent announcement of the use of sulfa drugs in controlling American foulbrood (Haseman and Childers, 1944) was of great interest to beekeepers. These drugs not only prevented the development of the disease but also resulted in strengthening colony morale to the extent that the bees cleaned out all evidence of the disease (dead larvae, scale) from the hive. However, when feeding of the drugs was discontinued, the disease recurred (Eckert, 1947; Johnson, 1947, 1948; Johnson and Stadel, 1946; Milne, 1947; Reinhardt, 1947). This raised many problems concerning the effect of sulfa drugs on the vegetative cells and spores of Bacillus larvae. Attempts to use antibiotics such as penicillin to control American foulbrood in the apiary have not been on the whole successful (Johnson, 1947; Katznelson and Gooderham, 1949). Experiments with antibiotics and sulfa drugs were therefore initiated to determine their bacteriostatic effect on Bacillus larvae, in vitro and in the apiary, and to obtain some information as to the mode of action of the drugs in the larva. It has already been demonstrated by Katznelson and Gooderham (1949) that para-aminobenzoic acid reverses the effect of sulfathiazole in the apiary. No attempt will be made to review the literature on the influence of sulfa drugs and antibiotics on bacteria as this has already been done exhaustively (Henry, 1943; Northey, 1948; Waksman, 1947). EXPERIMENTAL RESULTS In vitro studies. The sulfonamides and antibiotics tested are listed in table 1. In the first series of experiments only the first eight antibiotics were available; subsequently the last five were obtained and tested. However, at this time the Bacillus larvae cultures used at first (Wi and 10) had died out and were replaced with strain III and 4ST as indicated. In addition to B. larvae two strains each 1 Contribution No. 300 (Journal Series) from the Division of Bacteriology and Dairy Research, Science Service, Department of Agriculture, Ottawa, Canada. 2Bacteriologist. 471

2 472 H. KATZNELSON [VOL. 59 of Bacillus alvei and Bacillus para-alvei, organisms associated with foulbrood diseases of bees, were included for comparison. Concentrated stock solutions of the substances used were prepared aseptically and added to the fluid media used in the tests. For B. larvae the medium employed contained inorganic salts (Lochhead, 1942); KNO3, 0.25 g; yeast extract, 2 g; peptone, 5 g; and glucose 2 g per liter. For the remaining organisms the medium used contained inorganic salts; yeast extract, 4 g; and glucose, 5 g per liter. The media were adjusted to ph 7.0 before sterilization and were dispensed aseptically in 1-ml amounts into sterile 100-by-13-mm test tubes. One ml of concentrated solution of drug or antibiotic was added to 1 ml of medium; 1 ml of this dilution was transferred to 1 ml of medium; and this was repeated until the desired dilution was obtained. The inoculum was prepared as follows: B. larvae was grown for 48 hours on Holst and Sturtevant's (1940) stock medium, after which the cells were suspended in 8 drops of the test medium and one 3-mm loopful was used per tube; B. alvei and B. para-alvei were grown for 24 hours in nutrient broth and one 1-mm loopful was used per tube. Readings were made visually at 24, 48, and 96 hours. Suitable controls were also included. Table 1 gives a summary of the results obtained; only dilutions showing complete inhibition are presented. It is apparent that the sulfa drugs are the least inhibitory of the substances tested, to all six of the cultures-a phenomenon that is undoubtedly due to the presence of drug antagonists in the yeast extract and peptone used in the test media. However, sulfathiazole appeared, on the whole, to be more potent than sulfanilamide or sulfapyridine. Polymyxin, an antibiotic active chiefly against gramnegative bacteria (Stansly and Schlosser, 1947), had little effect on the bacilli except in high concentrations. The most potent antibiotic (especially against B. larvae) was aureomycin; penicillin was next, followed by chloromycetin, streptomycin, clavacin, gliotoxin, subtilin, and tyrothricin. Marked differences in the response of the three species to certain of these substances were observed. B. larvae is more sensitive to bacitracin, streptothricin, subtilin, and aureomycin than are B. alvei and B. para-alvei, which are, however, more sensitive to gliotoxin. B. para-alvei is much less sensitive to aureomycin than B. alvei. Considerable strain variation in response to the antibiotics is also evident. Since it is well recognized that sulfa drug antagonists in a medium interfere with the action of these drugs in vitro (Henry, 1943; Northey, 1948), an attempt was made to determine the bacteriostatic effect of sulfathiazole (sodium) in a synthetic medium lacking these inhibitory substances (p-aminobenzoic acid, methionine, purine bases, etc.). The recently developed synthetic medium for B. para-alvei (Katznelson and Lochhead, 1947) was therefore used and it was found that growth could be inhibited by a dilution of 1:40,000 of the drug as compared with 1:1,000 in the test medium containing yeast extract. Since considerable variation of sensitivity to certain antibiotics was observed with two strains of a species, it was considered desirable to determine the limits of sensitivity among a larger number of strains. Accordingly 14 strains of B. larvae were tested in dilutions of aureomycin ranging from 1:1,000,000 to 1:300,- 000,000. The results tabulated in table 2 show considerable variation among

3 1950] INFLUENCE OF ANTIBIOTICS AND SULFA DRUGS 473 strains. Cultures NW4, WB, and 23 showed partial inhibition even at 1:300,000,- 000 of aureomycin; 50 per cent of the strains showed definite inhibition at a TABLE 1 Bacteriostatic effect of sulfa drugs and antibiotics on organisms associated with foulbrood diseases of bee8* BACIUS LAVAE BACILUS LVE3I BACILLUS PARA-ALWI ACTIVE An Strain Strain Strain WI Sulfanilamide... <1 <1 <1 <1 <1 <1 Sulfapyridine... <1 <1 <1 <1 <1 <1 Sulfathiazole 1... < Clavacin Penicillin... 5,120 5,120 10,240 2, ,240 10,240 Citrinin Aspergillic acid Gliotoxin Streptomycin Streptothricin Tyrothricin III 4ST Bacitracin... 1, Polymyxin... 1I Subtilin Aureomycin ,000 50,000 16,000 8, Chloromycetin * Reciprocals of dilutions in thousands causing complete inhibition of growth. TABLE 2 Bacteriostatic effect of aureomycin on different strains of Bacillus larvae DILTION In 4sT WB WC NW ? + 4?? ? ? b Check * Reciprocals of dilutions in millions. t -, no growth;?, faint growth; :41, distinct growth but definitely poorer than in check; +, growth as in check. dilution of 1:200,000,000. Strain NW4 was the most sensitive of the 14, giving very little growth at a dilution of 1:150,000,000. The bacteriostatic and bactericidal effects of sodium sulfathiazole on 40 strains of B. larvae (isolated from different samples of infected comb) were also studied.

4 A47A H. KATZNELSON [VOL. 59 However, since the significance of the results was questionable owing to the presence of drug antagonists in the test medium, they will be mentioned only briefly. All strains were completely inhibited by 1:100 of the drug; three were completely inhibited and the remainder partly by 1:200, 26 partially and one completely by 1:400, 5 partially by 1:800, and one by 1:1600 and 1:3,200. The strongest solution tested (1:100) was bactericidal. Attempts to adapt these strains to the drug in a semisolid agar, yeast, carrot extract medium were also made, starting with 0.1 per cent and with 0.05 per cent increments until 1 per cent was reached. Thirty-six of the strains finally grew, though rather poorly, in the presence of 1 per cent of the drug. Since the amount of drug antagonists in the medium used was constant, it is considered that some adaptation to the drug occurred. However, after two subcultures in the absence of the drug, the strains lost their ability to develop in media containing 1 per cent of the drug. Loss of resistance to drugs as a result of subculturing in their absence has been observed with other organisms (Northey, 1948). Microscopic examination of the cells growing in the presence of the drug showed elongated filamentous forms as compared with the shorter, thicker cells in normal cultures (Lochhead, 1928; Waksman, 1947). Spores of Bacillus larvae in both water and honey solutions containing 1 per cent sodium sulfathiazole or 0.01 per cent clavacin remained viable for at least 15 months. As will be shown later, spores in water containing 15 and 30 per cent of the drug for 7 months still retained their virulence. Apiary studies. In earlier studies Katznelson and Gooderham (1949) corroborated the findings of Johnson (1947, 1948) and Eckert (1947, 1948) that sodium sulfathiazole prevented infection of colonies of bees with American foulbrood and that the presence of the drug in a colony apparently increased its morale so that infected material (dead larvae, dried larval scale, etc.) was rapidly removed and no visible trace of the disease left in the hive. They also showed that p-amiinobenzoic acid reversed the effect of the drug, resulting in the infection of the colony. The experiments to be described represent an extension of this work to include various antibiotics, another sulfa drug, and other drug antagonists such as methionine, guanine, xanthine, and folic acid (Henry, 1943; Hotchkiss, 1948; Northey, 1948; Woods, 1947). In the spring of 1949 healthy colonies were fed these different substances in a 1: 1 sugar-water syrup from a perforated honey pail placed directly over the comb containing larvae in all stages of development. Spores of B. larvae derived from American foulbrood scale were fed in the same syrup at the rate of about 2 billion per gallon. One gallon of medicated, inoculated syrup was fed to each colony under treatment. The amount of each substance used is given in table 3. It is quite clear that not one of the antibiotics was effective in preventing infection, although procaine penicillin retarded it initially. Aureomycin, which was bacteriostatic in vitro at a dilution of 1:50,000,000, was ineffective in vivo at a dilution of about 1: 1,000,000 (treatment 5). On the other hand, sulfadiazine and sulfathiazole were both effective, no infection occurring in the drug-treated

5 1950] INFLUENCE OF ANTIBIOTICS AND SULFA DRUGS 475 TABLE 3 Influence of antibiotic8 and 8ulfa drugs on American foulbrood in the apiary TREATMENT (A'R GALON SYRU) EXANATION OF COLONES 17 days 32 days 1. Procaine penicillin ("duracillin") Very light infection Extensive infection 300,000 units 2. Clavacin 0.05 grams Extensive infection Extensive infection 3. Bacitracin 0.05 grams Extensive infection Extensive infection 4. Subtilin 0.05 grams Extensive infection Extensive infection 5. Aureomycin grams Extensive infection Extensive infection 6. Aureomycin grams Extensive infection Extensive infection 7. Sulfadiazine 0.5 grams No infection No infection 8. Na-sulfathiazole 0.5 grams No infection No infection 9. Na-sulfathiazole + PABA 0.5 grams Light infection Moderate infection each 10. Na-sulfathiazole + guanine, xanthine, No infection Light infection methionine, 0.5 grams each 11. Na-sulfathiazole 0.5 grams + folic acid No infection No infection 0.1 gram 12. Na-sulfathiazole 0.5 grams + folic acid No infection Light infection 0.1 gram + guanine, xanthine, methionine 0.5 grams each 13. Na-sulfathiazole 0.5 grams + methi- No infection No infection onine 1.0 gram 14. Control-inoculum only Extensive infection Extensive infection TABLE 4 Influence of different treatment8 on the virulence of American foulbrood spores for honeybee larvae SPORE TREATMNT EXAMNATION OF COLONIS 17 days 32 days 1. 1% sodium sulfathiazole in water, 15 Extensive infection Extensive infection months 2. 1% sodium sulfathiazole in honey, 15 Extensive infection Extensive infection months 3. 15% sodium sulfathiazole in water, 7 Extensive infection Extensive infection months 4. 30% sodium sulfathiazole in water, 7 Extensive infection Extensive infection months 5. In water only, 15 months Extensive infection Extensive infection 6. In moist soil, 7 months Very light infection Extensive infection 7. Amosol-treated Very light infection Extensive infection colonies throughout the suimmer. p-aminobenzoic acid reversed the sulfa effect and resulted in infection. Of particular interest was the reversal of the sulfa effect in colonies fed, in addition, methionine plus the purines, guanine and

6 476 H. KATZNELI3ON xanthine. However, neither methionine nor folic acid alone interfered with the drug's action. In addition to the experiments above, virulence tests were conducted with spores receiving different treatments. The spore suspensions were each added at the rate of 500,000,000 to 1,000,000,000 per gallon of syrup. Those spores that had been suspended in sodium sulfathiazole solutions were first washed three times in water and, prior to feeding them, 0.5 g of p-aminobenzoic acid was added to each gallon of syrup to overcome the effect of any sulfa drug that might not have been removed by washing. Spores were also treated with the disinfectant "amosol" by suspending 2 billion3 in 150 ppm for 30 minutes at 180 F, washing by centrifugation, and adding to syrup. Again, one colony received 1 gallon of inoculated syrup. The results in table 4 indicate clearly that the spores survived immersion in solutions of sodium sulfathiazole in amounts up to 30 per cent. The light initial infection by spores from soil or from "amosol" solutions may be due to the loss of virulence of the spores as a result of treatment or to interference with germination and subsequent vegetative development in the larvae by substances in the soil or by residual "amosol." However, the disease developed and later was spread by the bees so that the colonies became extensively infected. DISCUSSION [VOL. 59 The use of antibiotics for controlling American foulbrood would seem to be promising from the results obtained in vitro, but even aureomycin, the most potent of the antibiotics tested, failed as a prophylactic agent. This may be due to its instability or to its being inactivated in the syrup or in the larvae. Penicillin, one of the most effective substances tested in vitro, retarded though did not eliminate infection, as has also been reported by others (Johnson, 1947; Katznelson and Gooderham, 1948). Haseman (1948) has recently reported some success with both penicillin and streptomycin but did not recommend their use owing to the difficulty in applying them as compared with sulfa drugs. However, regardless of their possible efficacy in preventing the growth of B. larvae in the digestive tract of the larva, these antibiotics are not on the whole sporicidal agents (except penicillin, Waksman, 1947) and consequently will have no effect on spores in honey, in pollen stores, in wax, in scale, or on any of the hive parts. Their use may result rather in masking the disease and in its dissemination. The same criticism applies to the sulfa drugs. Both sulfadiazine and sodium sulfathiazole effectively controlled the disease, as has also been reported by Johnson (1948), although Haseman and Childers (1944) did not obtain satisfactory results with sulfadiazine. Since the former drug is, on the whole, less toxic than the latter (Northey, 1948), its use is preferable, as it would allay the fears of many apiarists that toxic amounts of the drug may be stored in the surplus (market) honey. These fears are without serious foundation owing to the extremely small amount (at most a few parts 8 Since this number of spores is considerably in excess of that on which recommendations for "amosol" are based, the results should not be construed as being due necessarily to the ineffectiveness of this material.

7 1950] INFLUENCE OF ANTIBIOTICS AND SULFA DRUGS 477 per million) that may find its way into the honey when the drug is fed to infected colonies (Eckert, 1948). Since this is done chiefly in the spring or fall, when there is little if any honey flow, the chances for such contamination are slight. The reversal of the sodium sulfathiazole effect by p-aminobenzoic acid (PABA) and by the purines, guanine and xanthine (in the presence of methionine), throws some light on the possible mode of action of the drugs in the intestinal tract of the larva. Holst (1946) has pointed out that the spores of B. larvae germinate shortly after they are taken into the digestive tract with the larval food. It is in this vegetative stage that the organism is susceptible to any agent consumed along with the food and retaining its antibacterial property. The sulfa drugs are evidently such agents and inhibit further development of the vegetative cells of B. larvae. The reversal of this effect by guanine and xanthine (treatments 10 and 12, table 3) suggests that the drug interferes with the utilization of these substances, for they are essential for the growth of B. larvae (Katznelson and Lochhead, 1948). PABA also reversed the inhibition by sulfa drugs, although it has not been found to be essential for B. larvae when added to a synthetic medium (Katznelson and Lochhead, 1948). However, if, as Fildes (1940), Woods (1940), and Woods and Fildes (1940) contend, PABA is an "essential metabolite," the organism must synthesize its own requirement, as has been reported for other bacteria (Northey, 1948). The sulfa drugs may then interfere with its utilization competitively, an effect that is reversed by more PABA. Since guanine and xanthine also antagonize inhibition by sulfa compounds, it is possible that it is their further utilization (such as in the synthesis of nucleic acids), in which PABA may play a part directly or indirectly (Woods, 1947), with which sulfa drugs interfere. PABA may also be involved in the synthesis of other amino acids (Lampen et al., 1949) required by B. larvae, a function that may be inhibited by sulfa drugs; this inhibition may be competitively reversed by additional PABA. Further work is required to clarify these points. The effective use of sulfa drugs as chemotherapeutic agents has frequently been seriously hindered by the appearance of resistant or drug-fast organisms (Henry, 1943; Northey, 1948). It is conceivable that a similar phenomenon may arise in American-foulbrood-infected larvae receiving sulfa drugs since it has been mentioned earlier that strains of B. larvae could adapt themselves to a tenfold increase in the amount of the drug. However, this possibility may not be serious owing to the short period of contact of the vegetative cells in the larvae with the drug and to the rapidity with which the adapted organisms may lose their "resistance" on further growth in the absence of the drug. To many beekeepers, the announcement of the preventive properties, often perverted to "curative properties," of sulfa drugs for American foulbrood was the answer to the problem. Before long, however, with the repeated observation of disease recurrence after sulfa drug treatment, it was realized that considerable caution was required in the use of these drugs, that they did not cure an infected colony since they did not kill the spores of B. larvae, that indiscriminate and careless use and undue reliance on their effectiveness might result in masking the

8 478 H. KATZNELSON [VOL. 59 disease and in disseminating it widely, and that their application at the wrong part of the season was not only ineffective but also resulted often in the contamination of market honey with small but detectable traces of the drugs. For these reasons the industry has been warned to use these drugs sparingly and only under supervision by provincial or state inspectors or apiculturists until more is known of the conditions under which the drugs are most effective. ACKNOWLEDGMENT The author is greatly indebted to Drs. H. B. Woodruff, G. W. Rake, E. A. Doisy, J. M. Hardy, J. Ehrlich, R. G. Shepherd, and A. J. Salle for supplying the antibiotics, to the technical staff of the Bee Division, Experimental Farms Service, for the manipulation of the bee colonies, and to Mrs. C. M. Derraugh for technical assistance. 1SUMMARY The bacteriostatic effect of 13 antibiotics and 3 sulfonamide drugs on strains of Bacillus larvae, Bacillus alvei, and Bacillus para-alvei was determined. Aureomycin was the most potent (especially against B. larvae), penicillin was next, followed by chloromycetin, streptomycin, clavacin, gliotoxin, subtilin, and tyrothricin. The sulfa drugs were the least inhibitory owing undoubtedly to the presence of drug antagonists in the test media. Marked species and strain differences in response to certain of these substances were observed. Certain strains of B. larvae showed partial inhibition by aureomycin at a dilution of 1:300,000,000. Considerable variation among 40 strains of B. larvae in response to sodium sulfathiazole was also noted. In the medium tested, a 1 per cent solution was bactericidal. Thirty-six of the strains were adapted to grow in this solution but lost this ability after two transfers in the absence of the drug. Spores of B. larvae in both water and honey containing 1 per cent sodium sulfathiazole remained viable for at least 15 months. Procaine penicillin, clavacin, bacitracin, subtilin, and aureomycin were ineffective in preventing the development of American foulbrood in honeybee larvae in the apiary. Both sulfadiazine and sodium sulfathiazole were completely effective in this regard. The sulfa drug inhibition was reversed by p-aminobenzoic acid and the purines, guanine and xanthine, but not by folic acid or methionine. Spores of B. larvae immersed for 15 months in 1 per cent sodium sulfathiazole solution or in 15 and 30 per cent for 7 months retained their virulence, as did spores kept in moist soil for 7 months and spores treated with the disinfectant "amosol." The mode of action of the sulfa drugs and their use in the prevention of American foulbrood in the apiary are discussed. REFERENCES BUM8IDE, C. E. 33, The thermal resistance of Bacillus larvae. J. Econ. Entomol.,

9 1950] INFLUENCE OF ANTIBIOTICS AND SULFA DRUGS 479 ECKERT, J. E Use of sulfa drugs in treatment of American foulbrood disease of honeybees. J. Econ. Entomol., 40, ECKERT, J. E The use of sodium sulfathiazole in the treatment of American foulbrood disease of honeybees. J. Econ. Entomol., 41, FILDES, P A rational approach to research in chemotherapy. Lancet, 1, HASEMAN, L Further studies with sulfathiazole for control of foulbrood. J. Econ. Entomol., 41, 120. HASEMAN, L., AND CHILDERS, L. F Controlling American foulbrood with sulfa drugs. Univ. Missouri Agr. Expt. Sta., Bull HENRY, R. J The mode of action of sulfonamides. Bact. Revs., 7, HOLST, E. C Newer knowledge of American foulbrood. Gleanings in Bee Culture, March, HOLST, E. C., AND STURTEVANT, A. P Relation of proteolytic enzymes to phase of life cycle of Bacillus larvae, and two new culture media for this organism. J. Bact., 40, HOTORKISS, R. D The mode of action of chemotherapeutic agents. Ann. Rev. Microbiol., 2, JOHNSON, J. P Sulfathiazole for American foulbrood disease of honeybees. Second report. J. Econ. Entomol., 40, JOHNSON, J. P Sulfa drugs for American foulbrood of honeybees. Third report. J. Econ. Entomol., 41, JOHNSON, J. P., AND STADEL, R Sulfathiazole as a medication for American foulbrood disease of honeybees. J. Econ. Entomol., 39, KATZNELSON, H., AND GOODERHAM, C. B Sulfathiazole in relation to American foulbrood. Sci. Agr., 29, KATZNELSON, H., AND LOCHHEAD, A. G Nutritional requirements of Bacillus alvei and Bacillus para-alvei. J. Bact., 63, KATZNELSON, H., AND LOCHHEAD, A. G Nutritional requirements of Bacillus larvae. J. Bact., 55, LAMPEN, J. O., JONES, M. J., AND ROEPKE, R. R Mutant strains of Escherichia coli unable to synthesize p-aminobenzoic acid. J. Biol. Chem., 180, LOCHHEAD, A. G Cultural studies of Bacillus larvae White. Sci. Agr., 9, LOCHHEAD, A. G Growth factor requirements of Bacillus larvae White. J. Bact., 44, MILNE, P. S Sulphonamide treatment of American foulbrood. Agriculture, 54, NORTHEY, E. H The sulfonamides and allied compounds. Reinhold Publishing Corp., New York. REINHARDT, J. F The sulfathiazole cure of American foulbrood; an explanatory theory. J. Econ. Entomol., 40, STANSLY, P. G., AND SCHLOSSER, M. E Studies on polymyxin: isolation and identification of Bacillus polymyxa and differentiation of polymyxin from certain known antibiotics. J. Bact., 54, STURTEVANT, A. P The sterilization of American foulbrood combs. U. S. Dept. Agr., Circ WAKSMAN, S. A Microbial antagonisms and antibiotic substances. 2d ed. The Commonwealth Fund, New York. WHITE, G. F American foulbrood. U.S. Dept. Agr., Bull WOODS, D. D The relation of p-aminobenzoic acid to the mechanism of the action of sulphanilamide. Brit. J. Exptl. Path., 21, WOODS, D. D Bacterial metabolism. Ann. Rev. Microbiol., 1, WOODS, D. D., AND FILDES, P The antisulphonamide activity (in vitro) of p-aminobenzoic acid and related compounds. Chemistry & Industry, 59,