Synergism of penicillin or ampicillin combined with sissomicin or netilmicin against enterococci
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1 Journal of Antimicrobial Chemotherapy (78) 4, Synergism of penicillin or ampicillin combined with sissomicin or netilmicin against enterococci Chatrchal Watanakunakoni and Cheryl Glotzbecker Infectious Disease Division, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, U.S.A. The in vitro activity of penicillin, ampicillin, sissomicin and netilmicin against 35 strains of enterococci were studied. Tests for synergism of penicillin or ampicillin combined with sissomicin or netilmicin against all strains of enterococci were performed using the killing-curve method. Synergism was demonstrated against the majority of strains. Synergism was demonstrated slightly more often with penicillin than ampicillin in combination with sissomicin or netilmicin. There were slightly more strains killed synergistically by sissomicin than netilmicin in combination with penicillin or ampicillin. Introduction Sissomicin and netilmicin are two recently developed aminoglycosides with molecular structures closely resembling gentamicin Q., a component of gentamicin complex (Cooper, Jaret & Reimann, 7; Wright, 76). Preliminary studies indicate that netilmicin is less toxic than gentamicin in experimental animals (Miller, Arcieri, Weinstein & Waitz, 76; Luft, Yum & Kleit, 76). As with gentamicin, neither sissomicin nor netilmicin alone is very effective against enterococci (Watanakunakom & Glotzbecker 78). Penicillin-gentamicin as well as ampicillin-gentamicin combination have been shown to be synergistic against enterococci (Watanakunakom, 7; Harwick, Kalmanson & Guze, 73). Since sissomicin and netilmicin will probably be used in a similar fashion as gentamicin, we studied the effects of sissomicin or netilmicin combined with penicillin or ampicillin against enterococci. Materials and methods Enterococci Thirty-five strains of enterococci were studied, 3 of which were isolated from blood cultures of patients with bacteremia or endocarditis. All strains gave a positive reaction on bile-aesculin agar and grew in Brain Heart Infusion broth supplemented with 6-5% (w/v) of NaCl (Facklam, 72) /78/ /0 78 The British Society for Antimicrobial Chemotherapy
2 540 C. Watanakmmkorn and C Glotzbecker Antibiotic susceptibility test The minimal inhibitory concentration (MIQ of sissomicin, netilmicin, penicillin and ampicillin was determined simultaneously by the WHO-ICS agar dilution method (Ericsson & Sherris, 7). Antibiotics in twofold decreasing concentrations from 64 to 0-25 ug/ml were incorporated into Mueller Hinton agar. The inoculum was 0002 ml of a 0"' dilution of an overnight culture (approximately 0* to 0 4 organisms), delivered by a Steers replicator (Steers, Foltz & Graves, 5). The MIC was the lowest concentration of antibiotic which totally inhibited the growth of enterococci or allowed only growth of one colony after 8 h of incubation at 37 C. Test for synergism The killing-curve method was utilized. The enterococci were grown overnight and diluted with Mueller Hinton broth to give between 0 s and 0 7 colony-forming units (CFU) per ml and incubated with different antibiotics in a waterbath at 37 C. The final antibiotic concentrations were as follows: (A) penicillin 20 ug/ml, ampicillin 20 ug/ml; (B) sissomicin 4 ug/ml, netihnicin 4 ug/ml ;(C) combination of each antibiotic in (A) with each antibiotic in (B). A culture with no antibiotic was set up as a control. At 6, 24 and 48 h of incubation, viable CFU were enumerated by serial 0-fold dilutions and subcultures on Mueller Hinton agar. Colonies were counted after incubation at 37 C for 48 h. Results The MIC of the 4 antibiotics for the 35 strains of enterococci is listed in Table I. Ampicillin was slightly more active than penicillin. The enterococci were relatively resistant to sissomicin and netihnicin. Antibiotic Ampicillin Penicillin Sissomicin Netilmicin Table L Antimicrobial susceptibility of 35 strains of enterococci No. of strains with MIC (ng/ml) of: >64 0 The effect of antibiotic combinations in comparison with single antibiotics against the 35 strains of enterococci is shown in Table EL An antibiotic combination was considered synergistic when it killed more enterococci (by a factor of at least x logj 0 ) than the most effective antibiotic alone at designated time intervals. In general, synergism became more obvious as the period of incubation increased. There were slightly more strains killed synergistically by sissomicin than netilmicin in combination with penicillin or ampicillin. Synergism was demonstrated slightly more often with penicillin than ampicillin in combination with sissomicin or netihnicin. For all strains tested, none of the culture systems became sterile at the end of 6, 24 or 48 h. Data were then analysed according to the number of viable CFU at 6, 24 or 48 h of
3 Penicillin aminoglycosjde synergtsm 54 Table. EL Effects of antibiotic combinations in comparison with single antibiotics against 35 strains of enterococci Antibiotic Combinations No. of strains showing synergism* at: 6h 24 h 48h 6, 24, or 48 h Penicillin + sissomicin Penicillin + netilmicin Ampicillin + sissomicin Ampicillin + netilmicin 3 7 * Decrease in viable colony counts 0 >fold as compared with the most effective antibiotic alone at designated time intervals. Table HL Effects of antibiotic combinations against 35 strains of enterococci Antibiotic combinations Penicillin 4- sissomicin Penicillin + netilmicin Ampicillin + sissomicin Ampicillin + netilmicin No. of strains with viable CFU < x lo'/ml (< X lovml) at: 6h 5(0) (0) 3() (0) 24 h 6(5) (0) 2(3) (0) 48 h 25(5) 5(0) 8(5) (0) e ^ - Figure. Synergism of antibiotic combinations against a strain of enterococci. PEN = penicillin 20 jig/ml, AMP = ampicillin 20 jig/ml, SIS => sissomicin 4 iig/ml, NET = netilmicin 4 pg/ml.
4 542 C. Watanaktmakorn and C. Glotzbecker incubation. Table IQ shows the number of strains with viable CFU<x lc/ml and < X KP/ml in different antibiotic concentrations at different time intervals. The results were in accordance with the trend shown by analysis of synergism. Figure is an example of synergism demonstrated by all antibiotic combinations against a strain of enterococci. Discussion The combination of penicillin-streptomycin has been used to treat enterococcal endocarditis because of the relative resistance of enterococci to penicillin and the synergistic action of this antibiotic combination against enterococci. However, it has been shown repeatedly that penicillin-streptomycin has no synergistic effect against a large number of enterococcal strains (Standiford, demaine & Kirby, 70; Watanakunakora, 7; Moellering, Wennersten & Weinberg, 7; Ruhen & Darrell, 73; Gutschik, Jepsen & Mortensen, 77). However, penicillin-gentamicin and ampicillin-gentamicin combinations have been shown to be synergistic against almost all enterococci tested (Watanakunakorn, 7; Moellering, Wennersten & Weinberg, 7; Ruhen & Darrell, 73; Harwick, Kalmanson & Guze, 73; Gutschik, Jepsen & Mortensen, 77). The results of the present study indicate that penicillin or ampicillin combined with sissomicin or netilmicin are synergistic against the majority of enterococci tested. Similar results have been reported with peniciuin-sissomicm (Calderwood, Wennersten, Moellering, Kunz & Krogstad, 77; Sanders, 77) and penicillin-netilmicin combinations (Sanders, 77). Sissomicin and netilmicin are newer aminoglycosides which may be used in combination with penicillin or ampicillin in the treatment of serious enterococcal infections. Because so many aminoglycosides are now available, it is advisable to do in vitro testing for synergism between penicillin and different aminoglycosides against the infecting strain of enterococci. Selection of the best aminoglycoside then can be made on the basis of the degree of in vitro synergism and the toxic potential of a given aminoglycoside. If confirmed in human studies, netilmicin may have an advantage of being less toxic. Acknowledgements This study was supported by a grant from the Schering Corp. Bloomfield, N.J., and the Morton Hamburger Memorial Fund. We thank Dexter Balterman for his assistance in this study. References Calderwood, S. A., Wennersten, C, Moellering, R. C, Jr., Kunz, L. J. & Krogstad, D. J. Resistance to six aminoglycosidic aminocyclitol antibiotics among enterococci: prevalence, evolution, and relationship to synergism with penicillin. Antimicrobial Agents and Chemotherapy 2: 40-5 (77). Cooper, D. J., Jaret, R. S. & Reimann, H. Structure of sisomicin, a novel unsaturated aminoglycoside antibiotic from Micromonospora inyoensis. Journal of the Chemical Society Chemical Communication 7: (7). Ericsson, H. M. & Sherris, J. C. Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathologica et Microbiobgica Scandinavica, Section B, Supplement 27: -0 (7). Facklam, R. R. Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Applied Microbiology 23:3- (72).
5 Penicillin aminoglycoside synergisin 543 Gutschik, E., Jcpsen, O. B. & Mortensen, I. EflFect of combinations of penicillin and aminoglycosides on Streptococcus faecalis: a comparative study of seven aminoglycoside antibiotics. Journal of Infectious Diseases 35: (77). Harwick, H. J., Kalmanson, G. M. & Guze, L. B. In vitro activity of ampicillin or vancomycin combined with gentamicin or streptomycin against enterococci. Antimicrobial Agents and Chemotherapy 4: (73). Luft, F. C, Yum, M. N. & Kleit, S. A. Comparative nephrotoxicity of netilmicin and gentamicin in rats. Antimicrobial Agents and Chemotherapy 0: 845- (76). Moellering, R. G, Jr., Wennersten, C. & Weinberg, A. N. Synergy of penicillin and gentamicin against enterococci. Journal of Infectious Diseases 24 (Suppl.): S207-S2O (7). Miller, G. H., Arcieri, G., Weinstein, M. J. & Waitz, J. A. Biological activity of netilmicin, a broad-spectrum semisynthetic aminoglycoside antibiotic. Antimicrobial Agents and Chemotherapy 0: (76). Ruhen, R. W. & Darrell, J. H. Antibiotic synergism against group D streptococci in the treatment of endocarditis. Medical Journal of Australia 2: 4-6 (73). Sanders, C. C. Synergy of penkillin-netilmicin combinations against enterococci including strains highly resistant to streptomycin or kanamycin. Antimicrobial Agents and Chemotherapy 2: (77). Standiford, H. D., demaine, J. B. & Kirby, W. M. M. Antibiotic synergism of enterococci. Archives of Internal Medicine 26: 255- (70). Steers, E., Foltz, E. L., Graves, B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiotics and Chemotherapy : 307- (5). Watanakunakorn, C. Penicillin combined with gentamicin or streptomycin: synergism against enterococci. Journal of Infectious Diseases 24: 58-6 (7). Watanakunakorn, C. & Glotzbecker, C. In vitro activity of sisomicin and netilmicin alone and in combination with nafcillin, oxacillin and methicillin against enterococci. Chemotherapy 24: 236- (78). Wright, J. J. Synthesis of -ethylsisomicin: a broad spectrum semi-synthetic aminoglycoside antibiotic. Journal of the Chemical Society Chemical Communications 76: (76). (Manuscript accepted 0 April 78)
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