In Vitro Activity of Coumermycin A1

Transcription

1 APPLIED MICROBIOLOGY, Nov. 1969, p Vol. 1, No. 5 Copyright 1969 American Society for Microbiology Printed in U.S.A In Vitro Activity of Coumermycin A1 JOSEPH FEDORKO, SOL KATZ, AND HEDI ALLNOCH Bacteriology Research Laboratory, Veterans Administration Hospital, Washington, D.C. 422 Received for publication 29 July 1969 The in vitro activity of coumermycin Al was compared with that of novobiocin, ampicillin, and minocycline. Coumermycin was found to be the most active antibiotic of the four against Staphylococcus aureus. It was about times more active than novobiocin or minocycline against the strains tested. Coumermycin also showed good activity against group A streptococci and pneumococci, moderate activity against Escherichia coli, indole-positive Proteus species, and Pseudomonas aeruginosa, and poor activity against Klebsiella-Enterobacter and enterococci. Against P. mirabilis, however, coumermycin activity was almost equal to that of ampicillin. The new antibiotic was further found to be greatly reduced in activity in the presence of plasma, but its minimal inhibitory concentration was not greatly affected by inoculum size. Coumermycin was found to be bacteriostatic in its action, and resistance to it developed slowly. Also, cross-resistance was present with novobiocin but absent with ampicillin or minocycline. Just prior to the report in 1965 by Berger et al. (1) on the discovery and subsequent synthesis of a new antibiotic originating from a then unidentified species of Streptomyces, Kawaguchi et al. (6, 7) in Japan published reports on an identical agent named coumermycin. It was isolated from a species of Streptomyces named S. rishiriensis. From all indications, the antibiotic is basically antistaphylococcal with moderate activity against other gram-positive bacteria and with possible usefulness against selected species of gram-negative bacilli () and diplococci (4). Coumermycin is structurally related to novobiocin. It contains the same association of sugar and coumarin moieties (9) İn this report, the in vitro activity of coumermycin Al is compared with that of novobiocin, that of the broad-spectrum penicillin ampicillin, and that of minocycline, a member of the tetracycline family of antibiotics with exceptional antistaphylococcal activity. MATERIALS AND METHODS Coumermycin is only very slightly soluble in water and is unstable at above ph 9. It was dissolved in dimethylsulfoxide (DMSO) and diluted to a DMSO concentration of 1:4 in a ph 6. phosphate buffer. Further dilutions were made in DMSO-phosphate buffer (1:4). All media except Mueller-Hinton medium (Difco) contained DMSO at 2% to prevent possible precipitation of the drug. Except for Staphylococcus aureus 9P, the organisms used in this study were recent isolates from clinical specimens. Tables 1 and 2 list the organisms studied. The minimal inhibitory concentrations (MIC) of the antibiotics were determined in Trypticase Soy Broth (BBL) by the usual twofold serial dilution test. The inocula consisted of 0.5 ml of a - dilution of a -hr culture for the staphylococci and gramnegative rods and 0.5 ml of a -2 dilution of a -hr culture with 1.5% horse blood for the streptococci and pneumococci. Final volume was 1.0 ml. The last tube in a series to show no visible growth after 1 hr at 7 C was considered the MIC. The effect of inoculum size on MIC was tested in the same way. A culture of S. aureus 9P was diluted to provide inocula containing approximately to organisms per 0.5 ml. N-methylglucamine has been said to aid absorption of coumermycin after oral administration (South African Patent 65/5644). The effect of this drug on the MIC of coumermycin was assessed by incorporating it into the coumermycin standard solution at a N- methylglucamine to coumermycin ratio of 4:1 (w/w). The effect of human plasma on antibiotic activity was ascertained by determining the sensitivity of S. aureus 9P in broth alone and in broth containing % plasma. The effect of blood on coumermycin activity was further determined by performing disc sensitivity tests in the presence and in the absence of blood. Swabs moistened in overnight broth cultures were used to inoculate Mueller-Hinton medium with and without 5% horse blood. Disc potencies included 2,, 0, 60, and 0,g. Resistance of S. aureus 9P to coumermycin was induced by growing the organism on Trypticase Soy Agar containing increasingly larger concentrations of coumermycin. The growth from the highest concentration of drug that allowed it was suspended in broth and swabbed on agar plates containing the same and higher concentrations of the drug. After subcultures, when the organism was able to grow well on 69

2 70 FEDORKO, KATZ, AND ALLNOCH APPL. MICROBIOL. agar containing jig of coumermycin per ml, it was checked for cross-resistance with the other drugs employed in the study. The antibacterial action of coumermycin was further evaluated by growing S. aureus 9P in the presence of the MIC of coumermycin in ml of Trypticase Soy Broth. The average inoculum was 1.4 X 6 organisms/ml. At intervals of 1,, 6,, and hr, samples were removed, appropriately diluted, and plated for viable counts by the pour plate method. Staphylococcus aureus Escherichia coli Klebsiella-Enterobacter Proteus mirabilis Other Proteus species Pseudomonas aeruginosa TABLE 1. RESULTS Coumermycin showed its greatest activity against S. aureus (Table 1). Of 0 strains tested, almost % were susceptible to 0.004,ug/ml or less. The majority of these very sensitive strains were susceptible to about 0.2 Isg of novobiocin per ml. Therefore, in most cases, coumermycin is about times more active than novobiocin Sensitivity studies Organism No. of MIC (yg/ml) strains or > or < or > Per cent inhibited -inocydine Couem~y- Novobiocin Ampicillin Minocycline cin S S

3 VOL. 1, 1969 TABLE 2. Organism ACTIVITY OF COUMERMYCIN A71 Sensitivity of gram-positive cocci other than Staphylococcus aureus Avg MIC of No. of strains Coumermycin Novobiocin Ampicillin Minocycline Streptococcus pyogenes Diplococcus pneumoniae Enterococci TABLE. Effect of inoculum size on minimal inhibitory concentrations (MIC) of' coumermycin and novobiocin for S. aureus 9P Inoculum (organisms/ml) Coumermycin MIC (pg/ml) Novobiocin > , against S. aureus. A high percentage of the strains tested were resistant to ampicillin. However, 9% of the staphylococci were inhibited by at least.1 gg of minocycline per ml. This is even higher than previously reported (2). Against Escherichia coli, Klebsiella-Enterobacter, and indole-positive Proteus species, coumermycin was much more active than novobiocin, but it showed no advantage over ampicillin or especially minocycline. Against P. mirabilis, however, coumermycin approached ampicillin in activity: 71% of the strains were susceptible to.12 j&g or less of coumermycin per ml, and 94% were susceptible to the same concentration of ampicillin. Coumermycin was more active against Pseudomonas aeruginosa than the other antibiotics tested. However, even the lowest inhibitory concentrations were quite high. The MIC of coumermycin against S. aureus strains and 5 P. mirabilis strains was determined with N-methylglucamine incorporated in the standard. The inhibitory concentrations in the presence of N-methylglucamine were not significantly different from those in its absence. The average MIC values of coumermycin and the other antibiotics against gram-positive bacteria other than S. aureus are shown in Table 2. Pneumococci and group A streptococci were sensitive to the new antibiotic, but it was only occasionally more active than novobiocin against the strains tested. Coumermycin was about half as active as minocycline against group A strepto- TABLE 4. Effect of /co plasma on antibiotic minimal inhibitory concentration (MIC) for S. aureus 9P Antibiotic Broth MIC (pg/ml) Plasma increase Coumermycin , 562 Novobiocin Ampicillin Minocycline cocci and about one-tenth as active against pneumococci. Ampicillin, which showed the greatest activity against these organisms, was about 0 times more active than coumermycin. Enterococci were uniformly resistant to all of the antibiotics except ampicillin, which showed its usual moderate activity. The activity of coumermycin in broth was not greatly influenced by inoculum size (Table ). Most laboratories use between 4 and 6 organisms/ml in their tube sensitivity studies. Within this inoculum range, the MIC of coumermycin, at least for staphylococci, remained unchanged. However, with very high or very low numbers of organisms there was a considerable change in the MIC. The MIC of novobiocin decreased progressively with a decrease in inoculum size. Plasma greatly affected the MIC of coumermycin and novobiocin for S. aureus (Table 4). Plasma at a concentration of % in the medium caused many-fold increases in the MIC of coumermycin and novobiocin. In contrast, the MIC of ampicillin was increased only twofold. However, even in % plasma, coumermycin was still times as active as novobiocin. Further evidence of the depressing effect of blood constituents on coumermycin activity is given in Table 5. Zones of inhibition were greatly reduced in the presence of blood. Whereas a 19-mm zone was produced by a 2-,ug disc in the absence of blood, it took about 0 times more coumermycin to produce a comparable zone in the presence of blood. When S. aureus 9P was repeatedly subcultured on agar containing increasing concentra-

4 72 FEDORKO, KATZ, AND ALLNOCH APPL. MICROBIOL. tions of coumermycin, it showed an increase of over 0,000-fold in its ability to tolerate the drug (Table 6). Cross-resistance with novobiocin was demonstrated in that it took 0 times more novobiocin to inhibit the coumermycin-resistant strain. Cross-resistance with ampicillin and minocycline was not present. The resistance thus induced was permanent, and the resulting strain was only slightly altered; it grew a little less vigorously and coagulated plasma a little less intensely than the parent strain. The MIC of coumermycin for this organism had not changed even after 12 biweekly transfers. Other characteristics, including phage type, were identical with those of the parent. Figure 1 shows coumermycin to be bacteriostatic in its action. After an initial decrease, the live count began to rise but never at the rate of the control. The initial decrease was probably the result of a prolongation of the lag period during which older cells were dying off at a rate not overcome by multiplication. Subculture of one 5-mm loop from all clear tubes from 0 staphylococcal sensitivity tests revealed less than confluent growth (2+) from the MIC tube and a few colonies from each of the other subcultures through the tube containing 0.5 jsg/ml. Even at 12 times the MIC, a few cells remained viable after 1 hr. Subculture of tube contents from the novobiocin sensitivity studies on the same group of staphylococci gave identical results, i.e., 2 + growth at the MIC tube and a few colonies from each of the remaining clear tubes. TABLE 5. Influence of 5% horse blood in agar on coumermycin activity against S. aureus ( sensitive strains) Coumermycin concn (jg/disc) Without blood Avg zone size (mm) With blood TABLE 6. Development of resistance of S. aureus 9P when grown in the presence of coumermycin Antibiotic Initial Final Fold M MIC increase Coumermycin , 0 Novobiocin Ampicillin Minocycline FIG. 1. T T VE 'I 0 u R s CONTROL COUIAERMYC IN Growth of S. aureus 9P in the presence of lig of coumermycin Al per ml in ml of Trypticase Soy Broth at 7 C. DISCUSSION The outstanding feature of coumermycin, of course, is its activity against S. aureus. We encountered no resistant strains, and the MIC for these organisms ranged from 0.2 to Atg/ml. However, only 2 strains among 0 tested were inhibited by jtg/ml, and only 1 strain was inhibited by jug/ml. The most frequent MIC was 0.004,g/ml. As a whole, our results vary somewhat from the early in vitro studies of Kawaguchi et al. (7), who found that 17% of their 1 clinical isolates were inhibited by Ag/ml, and an occasional strain was even inhibited by jsg/ml. For the majority of strains, however, the MIC was between 0.01 and jsg/ml, and could very well have been reported as or 0.00 jug/ml if the tests had been performed by a serial twofold technique. It appears, however, that the clinical S. aureus strains tested in the study mentioned were more sensitive to coumermycin than the 0 strains tested by us. This could be the result of differences in technique. Results similar to ours were subsequently reported by Kawaguchi et al. (), when a twofold dilution agar plate method was used. Hoeprich (5), in a study on antistaphylococcal agents, found coumermycin to be one of the most active of the agents tested. About 51 % of his S. aureus strains were susceptible to Aug/ml (',umole). Our strains were more sensitive in that 9 % were inhibited by '5mole or less. Another striking thing about coumermycin is its greatly reduced activity in the presence of plasma. This does not necessarily imply inadequacy in vivo, however. In vivo influences go beyond protein binding and are not fully understood. The limited number of reports on chemotherapeutic action in animals (, 4) and our own clinical studies to be reported subsequently indicate that coumermycin is active in vivo and can be used successfully even in the treatment of infections due to organisms other than S. aureus.

5 VOL. 1, 1969 ACTIVITY OF COUMERMYCIN Al 7 Keil et al. (9) mentioned the poor absorption of coumermycin Al when the drug is administered orally as one of the reasons for the search for a replacement, i.e., the development of a coumermycin of higher solubility and greater oral absorption. N-methylglucamine, shown here to have no influence on drug activity, is useful in enhancing the absorption of coumermycin Al (H. L. Newmark, unpublished data). Preliminary studies indicate that an oral dose per human of 0 mg of coumermycin Al with N-methylglucamine produces a level at hr comparable to or better than that produced by a 0-mg dose without the additive. In our studies, nine patients showed a range of 1.2 to 5. Ag/ml of blood, with an average of.9,4g/ml, hr after ingestion of 0 mg of coumermycin Al with N-methylglucamine (assays performed by J. Berger, Hoffmann-La Roche, Inc., Nutley, N.J.). Presumptive evidence indicates that these levels are higher than those obtainable without N-methylglucamine. The studies reported here demonstrate that coumermycin Al has promise as an agent for the treatment of infections due to staphylococci, group A streptococci, and pneumococci among the gram-positive organisms, and perhaps some usefulness against the gram-negative enteric organisms, especially P. mirabilis. Extensive clinical studies are being conducted to confirm these assertions. LITERATURE CITED 1. Berger, J., A. J. Schocher, A. D. Batcho, B. Pecherer, 0. Keller, J. Maricq. A. E. Karr, B. P. Vaterlaus, A. Furlenmeier, and H. Spiegelberg Production, isolation and synthesis of the coumermycins (sugordomycins), a new streptomycete antibiotic complex. Antimicrob. Agents Chemother , p Fedorko, J., S. Katz, and H. Allnoch In vitro activity of minocycline, a new tetracycline. Amer. J. Med. Sci. 5: 2-.. Grunberg, E., and M. Bennett Chemotherapeutic properties of coumermycin Al. Antimicrob. Agents Chemother , p Grunberg, E., R. Cleeland, and E. Titsworth Further observations on chemotherapeutic activity of coumermycin Al. I. Activity against Neisseria meningitidis type A and meningopneumonitis. Antimicrob. Agents Chemother , p Hoeprich, P. D Susceptibility of staphylococci to new antimicrobial agents. Antimicrob. Agents Chemother , p Kawaguchi., H. T. Naito, and H. Tsukiura Studies on coumermycin, a new antibiotic. II. Structure of coumermycin Al. J. Antibiot. (Tokyo) Ser. A 1: Kawaguchi, H., H. Tsukiura, and M. Okanishi Studies on coumermycin, a new antibiotic. I. Production, isolation and characterization of coumermycie Ai. J. Antibiot. (Tokyo) Ser. A 1:1-.. Kawaguchi, H., H. Ueda, and S. Ishiyama Laboratory and clinical studies on coumermycin Al, a new acid antibiotic. 5th Int. Congr. Chemother., vol. 1, pt. 2, p Keil, J. G., L. R. Hooper, M. J. Cron, 0. B. Fardig. D. E. Nettleton, F. A. O'Herron, E. A. Ragan, M. A. Rousche, H. Schmitz, R. H. Schreiber, and J. C. Godfrey Semisynthetic coumermycins. I. Preparation of -acylamido- 4-hydroxy-- methyl (5 methyl -2-pyrrolylcarbonyl) noviosyloxy coumarins. J. Antibiot. (Tokyo) 21: