Received for publication August 23, RESULTS AND DISCUSSION substances. Commercial neomycin which is commonly

A CORRELATION BETWEEN THE STRUCTURE OF AND ITS ACTION ON BACTERIA OLDRICH K. SEBEK Research Laboratories, The Upjohn Company, Kalamazoo, Michigan Received for publication August 23, 1957 Streptomyces fradiae is known to synthesize a tance at 660 m,u (Coleman Junior spectrophotometer). mixture of antibiotics of which two, neomycin B (Regna and Murphy, 1950) and neomycin C (Dutcher et al., 1951), are isomeric antibacterial RESULTS AND DISCUSSION substances. Commercial neomycin which is commonly used in therapeutic treatments is preman and Lechevalier, 1949), chemical work re- Following the discovery of neomycin (Waksdominantly neomycin B. Its growth inhibitory vealed that this antibiotic contained two closely effect, both alone and in combinations with related antibacterial substances, neomycin B various other compounds on different microorganisms, has been extensively investigated. On (Dutcher et al., 1951). In spite of such close (Regna and Murphy, 1950) and neomycin C the other hand, the antibacterial effect of neomycin C has been studied very little. In an atcept of biological specificity suggested that bac- chemical similarity of these two agents, the contempt to obtain more information on the functional relationship of these two antibiotics, their inhibited only by one but not the other neomycin. teria might exist in nature whose growth would be effect on bacterial growth was investigated and If this supposition were indeed found to be correct, such a property could logically be associated preliminary results surmmarized (Sebek, 1955a). The data which are reported here in some detail with those parts of the respective neomycin which demonstrate the differences in the growth-inhibitory characteristics of these agents. In the In an attempt to find organisms which would are structurally different. case of at least two bacterial species, it was possible to identify a chemically defined part of the bacterial species to neomycin B and C were possess such a specificity, the responses of 24 neomycin B molecule as the site of a specific determined. For comparison, neamine-a substance chemically related to the neomycins-was growth inhibitory effect. also included (table 1). MATERIALS AND METHODS The results of this survey indicated that: Preparations of neomycin B, C, and neamine (a) With the exception of Alcaligenes faecalis, used were those reported by Ford et al. (1955). whose growth was not affected even at the 5,000 In the growth-inhibitory experiments, each antibiotic was dissolved in 0.1 M phosphate buffer the three antibiotics to the same extent. These,ug per ml level, no organism was susceptible to (ph 7.9). The solutions were then serially diluted observations corroborated the previously published data of Hamre et at. (1952); (b) In most in 1-ml volumes so that each dilution in the series was twice the preceding one. To these volumes, cases, neomycin B possessed a higher antibacterial 4-ml portions of brain heart infusion broth activity than either neomycin C or neamine; and (Difco), which had been inoculated with the organism under investigation, were added. The (referred to as a coccobacterium by Sebek, 1955a) (c) Two bacteria, namely Corynebacterium sp. tubes were then incubated at 37 C for 2 days. and Sarcina lutea, exhibited unusually different After that period, a record was made of the responses t. the antibiotics. While the growth of highest concentration of each antibiotic at which these two organisms was inhibited by approximately 250,ig of neomycin C or neamine per ml, the organisms were still able to grow (the maximum tolerated concentration, MTC). In order to only 0.4-1.6,ug of neomycin B per ml was needed relate bacterial growth to concentration of the to bring about the same effect. Table 1 also shows antibiotic, the amount of the 2-day bacterial similarly specific but less pronounced responses of mass was measured in terms of the light transmit- Micrococcus lysodeikticus to these agents. 199

200 SEBEK [VOL. 75 TABLE 1 Responses of bacteria to neomycin B, neomycin C, and neamine A two-day incubation at 37 C in 5 ml of brain heart infusion broth (Difco) Organism Maximum Tolerated Concentration (pug/ml) Neomycin B Neomycin C Neamine Serratia marcescens, ATCC 60... 0.8 12.5 1.5 Micrococcus pyogenes var. albus, ATCC 4840... 0.8 1.5 6 Bacillus brevis*... 0.8 6 6 Corynebacterium diphtheroides, ATCC 6931... 1.5 6 6 Neisseria catarrhalis, OSU 581t... 1.5 6 6 Aerobacter aerogenes, ATCC 211... 1.5 3 12.5 Klebsiella pneumoniae, ATCC 10031... 3 6 12.5 Escherichia coli, ATCC 26... 3 12.5 12.5 Micrococcus pyogenes var. aureus, ATCC 6538P... 3 12.5 25 Shigella dysenteriae, ATCC 9665... 6 25 25 Salmonella paratyphi, Upjohn 109... 6 25 25 Proteus vulgaris, ATCC 6380... 6 50 25 Salmonella pullorum, MSDH: 75... 6 25 50 Shigella paradysenteriae var. sonnei, Upjohn 133... 12.5 25 25 Salmonella schottmuelleri, ATCC 9149...... 12.5 50 > 100 Salmonella hirschfeldii, MSDHt SF 22375......12.5 100 >100 Pseudomonas aeruginosa, ATCC 10145... 12.5 >100 > 100 Streptococcus pyogenes, C 203... 25 25 12.5 Streptococcus viridans, 25-101... 25 25 12.5 Diplococcus pneumoniae, Felton I MSDHt... 25 50 50 Alcaligenes faecalis, ATCC 212... >100 >100 > 100 Micrococcus lysodeikticus, ATCC 4698... 1.5 50 >100 Corynebacterium sp.li... 1 250 250 Sarcina lutea, ATCC 9341... 1 250 250 * From Dr. T. M. Rivers, Rockefeller Inst., New York, N. Y. t From Dept. of Bacteriology, Ohio State Univ., Columbus, Ohio. t From Dr. Wm. F. Ferguson, Michigan State Dept. of Health, Lansing, Mich. From Dr. J. B. Gunnison, Univ. of Calif., San Francisco, Calif. From Dr. H. R. Prentice, Prentice Clinical Lab., Kalamazoo, Mich. From Dr. B. L. Hutchings, Lederle Laboratories, American Cyanamid Co., Pearl River, N. Y. (Hutchings et al., 1948). Because of such striking responses, this phe. nomenon was investigated in more detail with the corynebacterium and the sarcina. Since the responses of both bacteria to neomycin B and C were practically identical, only the findings obtained with the corynebacterium are here described. The response of this organism to the individual antibiotics is given in figure 1. These data show that 1.5,ug of neomycin B per ml completely suppressed the growth and that 0.2,ug per ml allowed the bacterium to grow with no measurable inhibition. In the case of neomycin C or neamine, however, not even 100,ug of antibiotic per mlwhich was the highest concentration tested-was sufficient to suppress the growth completely. Neomycin B was then combined in varying ratios and increasing concentrations with either neomycin C or neamine, or simultaneously with both of these antibiotics. When the bacterium was incubated in the presence of such mixtures, results were obtained which are summarized graphically in figures 2-4. Figure 2 illustrates the response of the organism to the mixtures of neomycin B with neomycin C, figure 3 to the mixtures of neomycin B with neamine, and figure 4 to

19581 CORRELATION BETWEEN STRUCTURE AND ACTION OF 201 il/ / ~ ~~~~~.Z yl x/~~ f ~ ~~~~~~~~~~~~ NEAMINE NEOMYCIN C 0.2 0.4 0.8 L5 30 6 12 mcg ANTIBIOTIC/ml Figure 1. Growth response of Corynebacteriuim sp. to neomvcin B, C, and neamine (a 2-day growth at 37 C). 100-90- z s 70~ a: 70-100- 90 w 80 m 70 60 50', 80-60- 5-50- x x ' <T. / + NEOMYCIN C X:.N C -- ---y- NEOMYCIN C 0.2 0.4 0.8 15 3 6 12 mcq ANTIBIOTIC /ml Figure 2. Growth response of Corynebacterium sp. to the mixtures of neomycin B and C. The amounts of the antibiotics are expressed in terms of neomycin B only. At each neomycin B level tested, neomycin C was present in ratios varying between 1:0.25 and 1:2 (a 2-day growth at 37 C). several combinations of all three antibiotics. These results show that the inhibitory range of the antibiotic mixtures closely coincided with the inhibitory range of neomycin B, and that neomycin C and neamine had very little growth inhibitory effect at the concentrations used. The relationship between the inhibitory properties and chemical structures of these antibiotics was then further investigated by the crossresistance technique. The basis for this technique lies in the assumption that related antibiotics exert their growth inhibitory action through the same or closely similar groupings in their respective molecules. By this method, presumptive evidence for identity or for a close similarity of unknown antibiotics may also be obtained (Szybalski and Bryson, 1952). J 50 100 In line with this reasoning, the corynebacterium was made resistant separately to neomycin C, neamine, and neomycin B by a series of transfers in brain heart infusion broth to which increasing amounts of the respective antibiotic were added. At each transfer, the mutant with the most highly developed resistance to one antibiotic was tested for the simultaneous development of resistance to the other two (table 2). The results showed that 100-90' w 80 2 704 z 1- ro60 50' X-I... I A A- -- - - - - 0.2 0.4 0.8 1.5 3 mcg ANTIBIOTIC/ml + NEAMINE NEAMINE 6 12 Figure S. Growth response of Corynebacterium sp. to the mixtures of neomycin B and to neamine. The amounts of the antibiotics are eypressed in terms of neomycin B only. At each neomycin B level tested, neamine was present in ratios varying between 1:0.25 and 1:2 (a 2-day growth at 37 C). 00 + NEOMYCIN C,., 80 [/;' + NEAMINE 60- ~ NEAMINE / 60 A NEAMINE NEOMYCIN C 0.2 04 3 6 Q8 I5 12 mcg ANTIBIOTIC/ml Figure 4. Growth response of Corynebacterium sp. to the mixtures of neomycin B, C, and neamine. The amounts of the antibiotics are expressed in terms of neomycin B only. At each neomycin B level tested, neomycin C and neamine were present in the following ratios: 1:1:1, 1:0.25:0.25, 1:0.25:1, and 1:1:0.25 (a 2-day growth at 37 C).

202 SEBEK [VOL. 75 TABLE 2 Resporse to neomycin C, neamine and neomycin B of Corynebacterium sp. and its resistant mutants The organism was grown in the presence of increasing amounts of the specified antibiotic at 37 C. At each transfer the mutants with the most highly developed resistance were tested for the degree of resistance which simultaneously developed to the remaining two antibiotics. Maximum Tolerated Concentration (jig/ml) Developed Resistance Antibiotic Tested Transfer Parent- First Second Third Sixth Neomycin C Neomycin C 125 250 1000 3000 50,000 Neamine 250 1000 1500 3000 25,000 Neomycin B 1 3 1.5 6 12 Neamine Neamine 250 500 1000 3000 50,000 Neomycin C 125 250 500 500 12,000 Neomycin B 1 1.5 3 6 12 Neomycin B Neomycin B 1 3 6 25 50 Neomycin C 125 250 250 250 250 Neamine 2E0 250 500 500 500 in 6 successive transfers of the bacterium in the presence of neomycin C or neamine, the organism increased its resistance to these antibiotics 100 to 400 times. In both cases this increase in resistance proceeded at similar but not identical rates. This indicated that these two antibiotics did not inhibit the microorganism by an identical mechanism. At the same time, however, the resistance of these mutants to neomycin B increased comparatively little (approximately 12 times). When, on the other hand, the resistance of this organism to neomycin B increased 50 times in 6 successive transfers, the resistance to neomycin C and neamine increased only twice in each instance. Once the organisms acquired a considerably high level of resistance, they retained it rather tenaciously. Thus, the corynebacterium made resistant to 20,000 ug of neomycin C per ml, retained this high resistance after 21 daily transfers in antibiotic-free medium. However, when the organism made resistant to lower levels of neomycin C (1000 to 2000,ug per ml), was subcultured in antibiotic-free medium, it reverted almost completely to its original sensitivity. None of these antibiotics was either destroyed or inactivated, for even after a 5-day incubation with the organism in shaken flasks, their titers remained unchanged when measured by the plate assay method and identified by paper chromatographic means (Sebek, 1955b). Although the organism thrived in the presence of 50,000 jig neomycin C per ml, no destruction of the antibiotic was detected. The specific inhibitory effect of neomycin B on the corynebacterium and sarcina, and the failure of these organisms to develop reciprocal crossresistance to neomycin B and C, indicated that the two antibiotics affected the growth of these bacteria through very different mechanisms. As a corollary, these data also provided biological evidence that neomycin B and C were not identical chemical entities. It is known that these two substances are isomeric and that they have, according to the more recent data, a formula of C23H48N6013 (Ford et al., 1955) or C23H46N6013 (Rinehart et al., 1957a). Upon methanolysis (or hydrolysis with 6 N HCl) both give the same non-reducing base, neamine, C12H26N406 (Leach and Teeters, 1951) which is identical with neomycin A (Peck et al., 1949; Leach and Teeters, 1952). In addition to neamine, the methanolysis of neomycin B yields neobiosamine B (isolated and identified as methyl neobiosaminide B), and neomycin C gives neobiosamine C (also isolated as its methyl glycoside, methyl neobiosaminide C). Neobiosamine B and C were shown to be isomeric diaminohexosido-pentoses (C1uH22N208, Rinehart et al., 1957a). The pentose moiety in both neobiosamines has been isolated and identified as D-ribose (Rinehart et al., 1957b). From this chemical evidence, the composition

1958] CORRELATION BETWEEN STRUCTURE AND ACTION OF 203 Neomycin B Neobiosamine B Diaminohexose B Neamine (Neomycin A) D-Ribose Neomycin C Neobiosamine C Diaminohexose C Figure 6. Degradation products of neomycin B and neomycin C. of the neomycin molecule may be written in the following fashion: C12H25N40.-O-C5H60(OH)-O-C6H70(OH)2(NHD2 Neamine D-Ribose Diaminohexose (B or C) It may also be seen that the difference between the neomycin B and C molecules lies only in their respective diaminohexose moieties (figure 5). Since neomycin B, but not neomycin C, was highly inhibitory for the growth of the corynebacterium and the sarcina, it is concluded that it is the diaminohexose portion of neomycin B with its particular configuration or linkages to other parts of the molecule, that is instrumental in the specific inhibition of growth of these two bacteria. Although the inhibition of these two organisms by neomycin B was very pronounced, it was not an isolated phenomenon. The inhibitory levels of this agent for most of the bacteria listed in table 1 were invariably lower than those of neomycin C. This indicates that neomycin B affected these organisms also through its diaminohexose portion.'1 2 Other inhibitory mechanisms could have simultaneously been at play. Observations of this nature may be found in the literature. Thus, Welsch et al. (1954) observed marked differences in the growth inhibitory activity of three closely related antibiotics, chlor- 1 The whole neomycin B molecule is essential for its high growth-inhibitory effect since neither methyl neobiosaminide B alone nor in combination with neamine arrested the growth of these organisms even at 100 ug per ml. 2 Amino sugars have been found in several other antibiotics (e. g., angolamycin, carbomycin, erythromycin, foromacidine, griseomycin, miamycin, narbomycin, picromycin, puromycin, spiramycin, streptomycin). When more chemical data become available, the use of suitable organisms may reveal that these moieties play a specific role in the action of these antimicrobial agents. tetracycline, oxytetracycline and tetracycline, on 338 strains of staphylococci and noted later (Reedy et al., 1955) a similar differential effect on nine additional bacterial genera. Maxwell and Nickel (1954) as well as Hahn et al. (1954) found that D(-)threo chloramphenicol was highly inhibitory for the growth of Escherichia coli and Bacillus subtilis, respectively, while its L( +)- erythro stereoisomer was only slightly bacteriostatic for these bacteria. The latter group of investigators (Hahn et al., 1956) postulated that, in addition to other considerations, the structural features of the chloramphenicol molecule have a specific influence on its antibacterial action. On the other hand, erythromycin and erythromycin B, which are closely related, showed no significant differences in their antibacterial spectra (Grundy et al., 1955). Since, however, the development of resistance to these agents proceeded at different rates, organisms may be found-as evidenced by our observationswhich would be sensitive to only one of the erythromycins. Such exacting organisms might also be useful in detecting specific components in mixtures of closely related antibiotics. ACKNOWLEDGMENTS Grateful acknowledgment is made to Mrs. E. E. Nelson forher able assistance, and to Dr. K. L. Rinehart, Jr., University of Illinois, who provided us with the data on the neomycin chemistry prior to their publication. SUMMARY Growth-inhibitory effect of neomycin B, neomycin C and neamine on twenty-four bacterial species was investigated. Differences in such an effect were found in most of the organisms tested. They were particularly striking in two bacteria, Corynebacterium sp. and Sarcina lutea. The data reported here on the corynebacterium showed that its growth was inhibited by approximately 250,ug of neamine or neomycin C per ml but by only 0.4 to 1.6,ug of neomycin B per ml. The inhibitory concentrations of neomycin B remained practically unchanged even when neomycin C and neamine were added in varying ratios and in different combinations. The organism readily acquired resistance and cross-resistance to neamine and neomycin C to a high degree but only slightly to neomycin B. The data indicate that the diaminohexose portion of neo-

204 SEBEK [VOL. 75 mycin B is responsible for the specific growthinhibitory effect of this antibiotic on these two bacteria., REFERENCES DUTCHER, J. D., HOSANSKY, N., DONIN, M. N., AND WINTERSTEINER, 0. 1951 Neomycins B and C and some of their degradation products. J. Am. Chem. Soc., 73, 1384-1385. FORD, J. H., BERGY, M. E., BROOKS, A. A., GARRErT, E. R., ALBERTI, J., DYER, J. R., AND CARTER, H. E. 1955 Further characterization of neomycin B and neomycin C. J. Am. Chem. Soc., 77, 5311-5314. GRUNDY, W. E., ALFORD, E. F., RICKHER, C. J., BOWER, R. R., RDZOK, E. J., AND SYLVESTER, J. C. 1955 A microbiological comparison of erythromycin and erythromycin B. Antibiotics & Chemotherapy, 5, 212-217. HAHN, F. E., WISSERMAN, C. L., JR., AND Hopps, H. 1954 Mode of action of chloramphenicol. II. Inhibition of bacterial D-polypeptide formation by an L-stereoisomer of chloramphenicol. J. Bacteriol., 67, 674-679. HAHN, F. E., HAYES, J. E., WISSEMAN, C. L., JR., Hopps, H. E., AND SMADEL, J. E. 1956 Mode of action of chloramphenicol. VI. Relation between structure and activity in the chloramphenicol series. Antibiotics & Chemotherapy, 6, 531-543. HAMRE, D. M., PANSY, F. E., LAPEDES, D. N., PERLMAN, D., BAYAN, A. P., AND DoNoVICm, R. 1952 Some characteristics of the neomycins. Antibiotics & Chemotherapy, 2, 135-141. HUTCHINGS, B. L., STOKSTAD, E. L. R., BOHONOS, N., SLOANE, N. H., AND SUBBAROW, Y. 1948 The isolation of the fermentation Lactobacillus casei factor. J. Am. Chem. Soc., 70, 1-3. LEACH, B. E. AND TEETERS, C. M. 1951 Neamine, an antibacterial degradation product of neomycin. J. Am. Chem. Soc., 73, 2794-2797. LEACH, B. E. AND TEETERS, C. M. 1952 The identity of neamine and neomycin A. J. Am. Chem. Soc., 74, 3187. MAXWELL, R. E. AND NICKEL, V. S. 1954 The antibacterial activity of the isomers of chloramphenicol. Antibiotics & Chemotherapy, 4, 289-295. PECK, R. L., HOFFHINE, C. E., JR., GALE, P., AND FOLKERS, K. 1949 Streptomyces antibiotics. XXIII. Isolation of neomycin A. J. Am. Chem. Soc., 71, 2590-2591. REEDY, R. J., RANDALL, W. A., AND WELCH, H. 1955 Variations in the antimicrobial activity of the tetracyclines. II. Antibiotics & Chemotherapy, 5, 115-123. REGNA, P. AND MURPHY, F. X. 1950 The isolation of neomycin B. J. Am. Chem. Soc., 72, 1045-1046. RINEHART, K. L., JR., WOO, P. W. K., ARGOUDELIS, A. D., AND GIESBRECHT, A. M. 1957a Chemistry of the neomycins. I. A partial structure of neobiosamines B and C. J. Am. Chem. Soc., 79, 4567-4568. RINEHART, K. L., JR., WOO, P. W. K., AND ARGOUDELIS, A. D. 1957b Chemistry of the neomycins. II. The pentose moiety. J. Am. Chem. Soc., 79, 4568. SEBEK, 0. K. 1955a The differential action of neomycins on bacteria. Bacteriol. Proc., 1955, 78. SEBEK, 0. K. 1955b The synthesis of neomycin- C14 by Streptomyces fradiae. Arch. Biochem. Biophys., 57, 71-79. SZYBALSKI, W. AND BRYSON, V. 1952 Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of Escherichia coli to fifteen antibiotics. J. Bacteriol., 64, 489-499. WAKSMAN, S. A. AND LECHEVALIER, H. A. 1949 Neomycin, a new antibiotic active against streptomycin-resistant bacteria, including tuberculosis organisms. Science, 109, 305-307. WELCH, H., RANDALL, W. A., REEDY, R. J., AND OSWALD, E. J. 1954 Variations in antimicrobial activity of the tetracyclines. Antibiotics & Chemotherapy, 4, 741-745.