Combination on Ampicillin-Susceptible and -Resistant Haemophilus influenzae Type B

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1979, p. 415-419 0066-4804/79/03-0415/05$02.00/0 Vol. 15, No. 3 Effect of Ampicillin and Chloramphenicol Alone and in Combination on Ampicillin-Susceptible and -Resistant Haemophilus influenzae Type B F. SESSIONS COLE,t* ROBERT S. DAUM,: LYNN TELLER, DONALD A. GOLDMANN, AND ARNOLD L. SMITH Division of Infectious Diseases, Department of Medicine, The Children's Hospital Medical Center, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 Received for publication 27 December 1978 To evaluate ampicillin (Amp) and chloramphenicol (Cm) alone and in combination against Haemophilus influenzae type b, we examined the viability of 5 log10 colony-forming units (CFU) of early-log-phase organisms per ml after 4 and 8 h.of incubation with the drug(s). Nine Amp-susceptible (Amp8) and five Ampresistant (Ampr) systemic isolates were examined. Antibiotic concentrations included: the minimum inhibitory concentration (MIC) of Amp, 50% of the MIC of Amp, 25% of the MIC of Amp, the MIC of Cm, 50% of the MIC of Cm, 25% of the MIC of Cm, and nine combinations of these concentrations. Both Amp and Cm at their MIC significantly reduced bacterial titers of Amps H. influenzae type b after 8 h of incubation (1.36 and 1.47 logio CFU/ml, respectively; both p < 0.01); only Cm at its MIC significantly reduced the number of viable organisms after 4 h (0.91 logio CFU/ml; P < 0.001). With Ampr organisms, significant reductions in mean bacterial titers occurred after 4 and 8 h of incubation in the presence of Amp at its MIC (1.66 and 2.06 log10 CFU/ml, respectively; both P < 0.02); smaller but significant reductions were noted after 4 and 8 with Cm at its MIC (0.56 and 0.87 log2o CFU/ml, respectively; both P < 0.025). Antagonism with Amps or Ampr strains was not seen. We conclude that combinations of Amp and Cm have indifferent effects on Amps and Ampr H. influenzae type b. Recognition of ampicillin-resistant (Ampr) Haemophilus influenzae led to the recommendation that initial treatment of children with bacterial meningitis of unknown etiology include a penicillin (penicillin G or Amp) and chloramphenicol (Cm) (8). Previous studies suggested that this combination was less active than penicillin alone against susceptible bacteria both in vitro and in animal models (4, 6, 9, 17-19, 31). However, recent data failed to demonstrate antagonism of this combination against H. influenzae using a checkerboard technique (1, 12); clinical evaluations have been inconclusive (2, 3, 14, 15, 22, 26, 32). We therefore examined the effects of Amp and Cm alone and in combination on H. influenzae type b (Hib), using a killingcurve technique. (These data were presented at The American Federation for Clinical Research, Eastern Section Meeting, Boston, Mass., October 1978.) t Present address: Department of Biology, Georgetown University, Washington, DC 20057. t Present address: Department of Pediatrics, Tulane University School of Medicine, New Orleans, LA 70112. Present address: Department of Pediatrics, University of Washington School of Medicine, Seattle WA 98105. MATERIALS AND METHODS Antibiotics. Amp sodium was kindly supplied by Bristol Laboratories, Syracuse, N.Y.; Cm was obtained from Sigma Chemical Co., St. Louis, Mo. Bacterial strains. The nine strains of Amp-susceptible (Amp8) Hib and five strains of Ampr Hib used in this study were among those isolated from blood or cerebrospinal fluid at this institution in the previous 4 years. Hib were identified according to standard criteria (10). Amp resistance was ascribed to strains possessing,8-lactamase activity and having a minimum inhibitory concentration (MIC) by agar dilution of -2 jlg/ml (5, 23, 28). All strains were frozen in skim milk at -70 C until testing. Culture media. All experiments used brain heart infusion broth or agar (Baltimore Biological Laboratory, Cockeysville, Md.) supplemented with,f-nicotinamide adenine dinucleotide (Sigma Chemical Co.) and horse erythrocyte lysate (Massachusetts Biological Laboratories) as previously described (10). Broth and agar media were refrigerated at 4 C and used within 7 days of preparation. Determination of MIC. The MICs of nine Amp" and five Ampr strains of Hib were determined by agar dilution (11) with an initial inoculum of 5 logi0 colonyforming units (CFU)/ml. Mean values are shown in Table 1. 415

416 COLE ET AL. Killing kinetics. Early-log-phase Hib from a broth culture were inoculated into supplemented brain heart infusion broth to yield a final concentration of 5 log,o CFU/ml. Antibiotics were added at selected concentrations (Table 2) and incubated with shaking at 370C. Before the addition of antibiotics and after 4, 8, and 24 h, aliquots were removed, diluted in chilled 6 mm phosphate-buffered saline (ph 7.4), and plated on solid media for enumeration. The significance of differences in bacterial densities was assessed by the two-tailed Student t test (7). No viable Amp' organisms could be detected in the presence of Amp or Cm at their MIC after 24 h of incubation. No viable organisms from three Ampr strains could be detected in the presence of Cm at its MIC after 24 h of incubation. The data derived from 24 h of incubation were therefore not included in the analyses. The effect of each antibiotic combination was assessed by comparison of mean bacterial densities and by evaluation of the effect of each antibiotic combination on each strain. The following definitions were used to describe antibiotic combination effects: (i) synergy-a reduction of viable organisms 2 log,o CFU/nl or greater than that occurring with either antibiotic alone; (ii) addition-a reduction in bacterial density at least 1 log,o CFU/ml greater than that occurring with either antibiotic alone; (iii) indifference-any combination whose effect was within 1 logio CFU/ml of that produced by either antibiotic alone; (iv) single antagonism-bacterial density with the combination equal to or greater than that of the less effective antibiotic, when the bacterial titer of the less effective antibiotic was 1 log,o CFU/ml greater than the more effective antibiotic; (v) mutual antagonismbacterial titers at least 2 log,o CFU/ml greater than those observed at either antibiotic concentration alone. Assessment of error. The error of enumeration was assessed by four replicate experiments with a single Amp' strain after 4 and 8 h of incubation. For all samples, the standard error of the mean expressed as a percentage was 32 (range, 8 to 66%). RESULTS Amp' strains. With Amp at its MIC, no change in the mean number of viable organism TABLE 1. Mean MICs ofamp and Cm for Amps and Amp' Hib MIC (Ag/ml) of: Strain Amp (range) Cm (range) Amp' (n = 9) Ampr (n = 5) 0.33 (0.20-0.63) 235 (50-800) 0.79 (0.30-1.5) 0.73 (0.60-1.25) ANTIMICROB. AGENTS CHEMOTHER. was observed after 4 h of incubation (Table 3). Cm at its MIC, however, reduced the mean bacterial density 0.91 logio CFU/ml (P < 0.001). Addition of Cm to the MIC of Amp also significantly reduced the number of viable organisms (P < 0.02). Amp, however, when added to the MIC of Cm, produced no significant differences in the number of viable organisms. Of the nine combinations of Amp and Cm tested, seven had indifferent effects, and two were additive when examined after 4 h of incubation. No synergy or antagonism was noted. In contrast to the effect seen after 4 h of incubation, Amp at its MIC produced a mean reduction of 1.36 logio CFU/ml after 8 h of incubation (P < 0.01) (Table 3). Cm at its MIC produced a mean decrease of 1.47 logio CFU/ml (P < 0.001). Addition of Cm did not further reduce bacterial densities produced by Amp at its MIC. Similarly, Amp did not alter reductions in mean bacterial titers produced by Cm at its MIC. Of the nine combinations of Amp and Cm tested, six had indifferent effects, one had an additive effect, and two had synergistic effects. No antagonism was noted. The effect of the nine combinations of antibiotics, tested individually against nine Amp8 strains after 4 and 8 h of incubation, is shown in Table 4. The majority of combinations had indifferent effects; no mutual antagonism was observed. Ampr strains. Amp at its MIC, one-half its MIC, and one-fourth its MIC reduced mean TABLE 3. Mean bacterial densities of nine Amp' Hib in the presence ofamp and Cm after 4 and 8 h of incubation Mean bacterial density (log,o CFU/ml) Amp MIC Amp Amp 0 MIC/2 MIC/A Amp 4 h Cm MIC 4.18 3.67 4.04 4.32 Cm MIC/2 4.71 4.18 4.61 5.04 Cm MIC/4 4.67 4.67 5.48 5.97 0 Cm 5.34 4.94 5.90 6.45 8 h Cm MIC 3.63 3.28 4.58 3.76 Cm MIC/2 3.80 4.51 5.00 6.49 Cm MIC/4 4.36 5.04 5.95 6.91 0 Cm 3.94 5.41 6.48 8.15 TABLE 2. Concentrations ofamp and Cm used in killing kinetic studies' Amp MIC Amp MIC + Cm MIC Amp MIC + Cm MIC/2 Amp MIC + Cm MIC/4 Amp MIC/2 Amp MIC/2 + Cm MIC Amp MIC/2 + Cm MIC/2 Amp MIC/2 + Cm MIC/4 Amp MIC/4 Amp MIC/4 + Cm MIC Amp MIC/4 + Cm MIC/2 Amp MIC/4 + Cm MIC/4 Control Cm MIC Cm Mic/2 Cm MIC/4 a In each killing curve, the test organism's MICs for Amp and Cm were used.

VOL. 15, 1979 Amp AND Cm VERSUS H. INFLUENZAE 417 bacterial densities by more than 1 log1o CFU/ml after 4 hours of incubation (all P < 0.02) (Table 5). Cm at its MIC produced a smaller, but significant, decrease in mean bacterial density (P < 0.025). However, in contrast to the effects observed with Amp8 Hib, bacterial density increased when Amp at its MIC was combined with Cm. Reductions in bacterial densities by Cm at its MIC were not altered by the addition of Amp. Of the nine combinations of Amp and Cm tested, seven had indifferent effects, and two had singly antagonistic effects. No synergy or mutual antagonism was noted. Amp at its MIC, one-half its MIC, and onefourth its MIC reduced mean bacterial titers by more than 1.5 logio CFU/ml after 8 h of incubation (all P < 0.01) (Table 5). Cm at its MIC reduced mean bacterial density by 0.87 logio CFU/ml (P < 0.01). When Cm was combined with Amp at its MIC, increasing bacterial density was again noted. Of the nine combinations of Amp and Cm tested, eight had indifferent effects, and one was singly antagonistic. Neither synergy nor mutual antagonism was noted. The effects of the nine combinations of antibiotics against five strains of Ampr Hib after 4 and 8 h of incubation are shown in Table 6; again, neither synergy nor mutual antagonism was observed. TABLE 4. Assessment of effects ofamp and Cm in, combination against nine strains ofamp' Hib8 % of combinations exhibiting: Length of in- Mutual cubation (h) Indiffer- Single an- SynergY Addition autag eneagomntago ence tagonism nism 4 9 23 63 5 0 8 6 12 73 9 0 8Total number of combinations (nine strains of Amp' Hib tested against nine combinations of antibiotics) is 81. The definitions are given in the text. TABLE 5. Mean bacterial densities of five Ampr Hib in the presence ofamp and Cm after 4 and 8 h of incubation Mean bacterial density (log,o CFU/ml) AmpMC Amp Amp Am Amp MIC MIC/2 MIC/4 OAmp 4 h Cm MIC 4.65 4.58 4.64 4.72 Cm MIC/2 4.40 4.49 5.00 5.95 Cm MIC/4 3.80 4.58 4.63 6.28 0 Cm 3.68 3.81 4.38 6.72 8 h Cm MIC 4.23 3.87 4.56 4.41 Cm MIC/2 3.96 5.00 5.38 6.53 Cm MIC/4 3.59 4.69 5.30 7.23 0 Cm 3.28 3.59 3.80 8.18 TABLE 6. Assessment of effects ofamp and Cm in combination against five strains ofampr Hib' % of combinations exhibiting: Length of in- Mutual cubation (h) Synergy Addition Ince ence tagomam tani antago- nism 4 0 13 58 29 0 8 0 13 60 27 0 atotal number of combinations (five strains of Ampr Hib tested against nine combinations of antibiotics) is 45. The definitions are given in the text. DISCUSSION Antibiotic susceptibility testing of H. influenzae presents a number of technical difficulties (10, 13, 16, 20, 21, 25, 27, 29). Its fastidious nature, requiring hemin and f8-nicotinamide adenine dinucleotide, as well as the effects of media composition, incubation period, and inoculum size, all represent variables which need to be controlled to evaluate antibiotic susceptibility (16, 27). By using killing curves standardized for initial inoculum, antibiotic concentrations, and media characteristics, the effect of Amp, Cm, and combinations of these antibiotics on H. influenzae can be examined in a controlled fashion Ȧmp is classified as a bactericidal agent: it reduces the number of viable organisms in an inoculum. Cm is defined as a bacteriostatic drug: the number of viable organisms remains unchanged in its presence (24). This classification was derived from experiments in which the number of viable organisms was evaluated after 18 or 24 h of incubation with the antibiotic. However, recent work suggests that Cm is bactericidal for Hib. Turk, evaluating 25 strains after a 6-h incubation, noted that Cm at concentrations of 1 to 20,ug/ml produced a reduction in the number of viable Hib at two inocula, 7 and 4 log10 CFU/ml (30). After 24 h of incubation, similar results were observed. MICs of the strains were not reported. Rahal and Simberkoff found that the minimum bactericidal concentration of Cm against nine of eleven strains of H. influenzae was 0.78 to 1.56,ug/ml (J. J. Rahal and M. S. Simberkoff, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 17th, New York, N.Y., Abstr. no. 5, 1977). We also observed a bactericidal effect of Cm. Several authors questioned the possible antagonistic effect of Amp and Cm when used against Hib (1, 12). According to the initial observations of Jawetz and early in vitro and animal studies of similar antibiotic combinations (6, 18, 19, 31), antagonistic effects of these drugs were predicted. Using an inoculum of 4 logio

418 COLE ET AL. CFU/ml and a checkerboard technique, Ahronheim found no antagonism between Amp and Cm after 12 h of incubation (1). Using a similar technique, Feldman studied 13 Amp8 and 8 Ampr strains (12). He also found no evidence of antagonism with Amp and Cm; synergy with these drugs against the majority of Amp' strains (at two inocula, 4 and 7 logio CFU/ml) was described. Additive effects were observed with the remainder of the isolates. These results are consistent with one of the observations reported here: antagonism does not occur when Amp and Cm are used against Amp' and Ampr Hib. However, indifferent (not additive or synergistic) effects are observed with killing curves. Interference between drugs occurs more frequently with Ampr than with Amp8 strains. The clinical significance of antibiotic synergy and antagonism has recently been reviewed (24). The lack of in vitro antagonism with Amp and Cm against Hib suggests no in vivo antagonism. However, data available concerning the effect of penicillin and Cm on Streptococcus pneumoniae, viridans streptococci, group A streptococci, and Klebsiellapneumoniae indicate antagonism (4, 18, 31). To ensure non-antagonistic combination chemotherapy for bacterial meningitis or sepsis of unknown etiology, the effect of Amp and Cm on other common pathogens should be examined. ACKNOWLEDGMENTS We thank Alan L. Schwartz for his thoughtful review of this manuscript and Vassiliki Syriopoulou, A. Lynn Harding, Patricia L. Cole, and Walter Siedlecki for their encouragement of and contributions to this work. R.S.D. is a Fellow of the Research Council of Canada. LITERATURE CITED 1. Ahronheim, G. A. 1975. Hemophiius influenzae type B: lack of in vitro antagonism between penicillins and chloramphenicol. Pediatr. Res. 9:337. 2. Barrett, F. F., W. A. Eardley, M. D. Yow, and H. A. Leverett. 1966. Ampicillin in the treatment of acute suppurative meningitis. J. Pediatr. 69:343-353. 3. Barrett, F. F., L H. Taber, C. R. Morris, H. B. Stephenson, D. J. Clark, and M. D. Yow. 1972. A 12 year review of the antibiotic management of Hemophilus influenzae meningitis. J. Pediatr. 81:370-377. 4. Carrizosa, J., W. D. Kobusa, and D. Kaye. 1975. Antagonism between chloramphenicol and penicillin in streptococcal endocarditis in rabbits. J. Lab. Clin. Med. 85:307-317. 5. Center for Disease Control. 1975. Ampicillin resistant Haemophilus influenzae. Morbid. Mortal. Weekly Rep. 24:205-206. 6. Chang, T.-W., and L. Weinstein. 1966. Inhibitory effects of other antibiotics on bacterial morphologic changes induced by penicillin G. Nature (London) 211:763-765. 7. Colton, T. 1974. Statistics in medicine, pp. 129-131. Little, Brown and Co., Boston. 8. Committee on Infectious Diseases. 1974. Ampicillinresistant strains of Haemophilus influenzae type b. Pediatrics 55:145-147. ANTIMICROB. AGENTS CHEMOTHER. 9. Dowling, H.F. 1957. Mixtures of antibiotics. J. Am. Med. Assoc. 164:44-48. 10. Emerson, B. A., A. L Smith, A. L Harding, and D. H. Smith. 1975. Haemophilus influenzae type B susceptibility to 17 antibiotics. J. Pediatr. 86:617-620. 11. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing: report of an intemational collaborative study. Acta Pathol. MicrobioL Scand. Sect. B 217(Suppl.):9-87. 12. Feldman, W. E. 1978. Effect of ampicillin and chloramphenicol against Haemophilus influenzae. Pediatrics 61:406-409. 13. Finland, M., C. Garner, C. Wilcox, and L D. Sabath. 1976. Susceptibility of pneumococci and Haemophilus influenzae to antibacterial agents. Antimicrob. Agents Chemother. 9:274-287. 14. Finland, M., C. V. Pryles, and W. F. Jones. 1958. Antibiotic combinations: antibacterial action of plasma of human subjects after ingestion of penicillin V or chloramphenicol or both. N. Engl. J. Med. 258:817-824. 15. Fleming, P. C., J. D. M. Murray, M. W. Fujiwara, J. S. Paichard, and G. A. McNaughton. 1966. Ampicillin in the treatment of bacterial meningitis. Antimicrob. Agents Chemother. 6:47-52. 16. Gray, B. M., C. A. Hubbell, and H. C. Dillon. 1977. Manner and meaning of susceptibility testing of ampicillin-resistant Haemophilus influenzae. Antimicrob. Agents Chemother. 11:1021-1026. 17. Jawetz, E., and J. B. Gunnison. 1952. Experimental basis of combined antibiotic action. J. Am. Med. Assoc. 150:693-695. 18. Jawetz, E., J. B. Gunnison, R. S. Speck, and V. R. Coleman. 1951. Studies on antibiotic synergism and antagonism. The interference of chloramphenicol with the action of penicillin. Arch. Intern. Med. 87:349-359. 19. McBryde, V. E., H. F. Dowling, and M. Mellody. 1965. Comparison of tube and plate methods for testing combinations of antibiotics against Haemophilus influenzae. Antimicrob. Agents Chemother. 5:267-272. 20. McGowan, J. E., P. M. Terry, and A. J. Nahmias. 1976. Susceptibility of Haemophilus influenzae isolates from blood and cerebrospinal fluid to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Antimicrob. Agents Chemother. 9:137-139. 21. McLinn, S. W., J. D. Nelson, and K. C. Haltalin. 1970. Antimicrobial susceptibility of Hemophilus influenzae. Pediatrics 45:827-838. 22. Mathies, A. W., J. M. Leedom, L D. Thrupp, D. Ivler, B. Portnoy, and P. F. Wehrle. 1965. Experience with ampicillin in bacterial meningitis. Antimicrob. Agents Chemother. 5:610-617. 23. Medeiros, A. A., and R. F. O'Brien. 1975. Ampicillin resistant Haemophilus influenzae type b possessing a TEM-type /i-lactamase but little permeability barrier to ampicillin. Lancet i:716-719. 24. Rahal, J. J. 1978. Antibiotic combinations: the clinical relevance of synergy and antagonism. Medicine 57: 179-195. 25. Sell, S. H. W. 1967. In vitro sensitivity studies of Hemophilus influenzae-typable and non-typable strains. Pediatrics 39:214-219. 26. Shackelford, P. G., J. E. Bobinsk, R. D. Feigin, and J. D. Cherry. 1972. Therapy of Haenwphilus influenzae meningitis reconsidered. N. Engl. J. Med. 287: 634-638. 27. Thornsberry, C., C. N. Baker, L. A. Kirven, and J. M. Swenson. 1976. Susceptibility of ampicillin-resistant Haemophilus influenzae to seven penicillins. Antimicrob. Agents Chemother. 9:70-73. 28. Thornsberry, C., and L. A. Kirven. 1974. Ampicillin resistance in Haemophilus influenzae as determined by a rapid test for beta-lactamase production. Antimicrob. Agents Chemother. 6:653-654.

VOL. 15, 1979 Amp AND Cm VERSUS H. INFLUENZAE 419 29. Thornsberry, C., and L. A. Kirven. 1974. Antimicrobial susceptibility of Haemophilus influenzae. Antimicrob. Agents Chemother. 6:620-624. 30. Turk, D. C. 1977. A comparison of chloramphenicol and ampicillin as bactericidal agents for Haemophilus influenzae type B. J. Med. Microbiol. 10: 127-131. 31. Wallace, J. F., R. H. Smith, M. Garcia, and R. G. Petersdorf. 1967. Studies on the pathogenesis of meningitis. VI. Antagonism between penicillin and chloramphenicol in experimental pneumococcal meningitis. J. Lab. Clin. Med. 70:408-418. 32. Wehrle, P. F., A. W. Mathies, J. M. Leedom, and D. Ivler. 1967. Bacterial meningitis. Ann. N.Y. Acad. Sci. 145:488-498.