Abstract
The microdilution MICs of HMR 3647, erythromycin A, azithromycin, clarithromycin, roxithromycin, and pristinamycin against 50/90% of 249 Haemophilus influenzae and 50 Moraxella catarrhalis isolates were 2/4, 0.06/0.125; 8/16, 0.25/0.25; 2/4, 0.06/0.125; 16/16, 0.25/0.25; 32/>32, 1/2; and 2/4, 0.5/0.5 μg/ml. Azithromycin was bactericidal against all 10 H. influenzae and 3 of 5 M. catarrhalis isolates and HMR 3647, erythromycin A, clarithromycin, roxithromycin, and pristinamycin were bacteriostatic, against all 15 strains after 24 h at the MIC.
Haemophilus influenzae and Moraxella catarrhalis remain important causes of respiratory tract infections (2, 10). The in vitro susceptibility of H. influenzae to macrolides is variable, with azithromycin showing the lowest MICs, followed by clarithromycin, erythromycin A, and roxithromycin. Pristinamycin and other streptogramins are also active in vitro against H. influenzae (5, 7, 8, 11, 12, 18). M. catarrhalis has been reported to be more susceptible to all of the above compounds than H. influenzae (2).
HMR 3647 is a new ketolide with expanded activity against many multiresistant (especially erythromycin A-resistant) gram-positive organisms (4, 7, 20); excellent activity against M. catarrhalis and activity against H. influenzae equivalent to that of azithromycin have recently been documented (1, 3, 4, 6, 7, 20). The problem of macrolide activity against H. influenzae is complicated by the difficulty of in vitro susceptibility testing (21). Haemophilus test medium (HTM), the medium recommended by the National Committee for Clinical Laboratory Standards (NCCLS), has a shelf life of only a few weeks and is therefore difficult to obtain commercially. Viability counts should also be performed on each suspension used for susceptibility testing (10, 21).
The current study attempted to shed light on the above by using freshly prepared HTM and Mueller-Hinton broth with added Fildes extract (MHF) to test the activity of HMR 3647, erythromycin A, azithromycin, clarithromycin, roxithromycin, and pristinamycin against 249 H. influenzae and 50 M. catarrhalis isolates by microdilution. The activity of each drug against 10 H. influenzae and 5 M. catarrhalis isolates was also tested by time-kill assay.
The organisms used were all recent clinical isolates. β-Lactamase testing was by the nitrocefin disk method (Cefinase; BBL Microbiology Systems, Cockeysville, Md.). Powders were obtained from the respective manufacturers. Microdilutions were performed on 249 H. influenzae and 50 M. catarrhalis strains by the NCCLS microdilution method (9). Inocula were prepared from chocolate agar plates (BBL) incubated for 24 h by the direct colony suspension method. Final organism suspensions in trays yielded colony counts of 3 × 105 to 8 × 105 CFU/ml. Inoculum checks were performed in every case; preparation of strains with inocula which did not conform to the standard was repeated.
Frozen microdilution trays (MicroMedia Systems Inc., Cleveland, Ohio) each contained all five antimicrobials prepared in freshly prepared HTM and MHF. HTM was prepared by adding 0.5% yeast extract, 15-μg/ml hematin, and 15-μg/ml NAD to cation-supplemented Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.), and MHF was prepared by adding 1% yeast extract and 5% Fildes enrichment (Difco) to cation-supplemented Mueller-Hinton broth (Difco). Wells were inoculated with 100-μl suspensions and incubated in air at 35°C for 20 to 24 h (9). Standard quality control strains (9) were included in each run.
Time-kill experiments were carried out with HTM as previously described (13, 14). Dilutions required to obtain the correct inoculum were determined by prior viability studies with each strain. Only tubes containing an initial inoculum between 5 × 105 and 5 × 106 CFU/ml were acceptable. Viability counts of antibiotic-containing suspensions were performed at 0, 3, 6, 12, and 24 h, respectively (13, 14), on chocolate agar plates incubated for up to 48 h in CO2. Colony counts were performed in duplicate, and means were taken.
Time-kill assays were analyzed by determining the number of strains which showed viable count decreases of 1, 2, and 3 log10 CFU/ml compared to the counts at 0 h. Drugs were considered bacteriostatic if they yielded a decrease in the count of <3 log10 CFU/ml compared to that at 0 h. With the sensitivity threshold (250 CFU/ml) and inocula used, bactericidal activity (99.9% killing and a decrease in the count of >3 log10 CFU/ml) could be determined when present. Bacterial carryover was minimized by dilution (13, 14).
Of 249 H. influenzae strains, 118 (47.4%) were β-lactamase positive. All 50 M. catarrhalis strains were β-lactamase producers. MICs did not differ for β-lactamase-positive and -negative H. influenzae strains (data not shown). Microdilution MICs are presented in Table 1. HMR 3647, azithromycin, and pristinamycin had the lowest MICs against H. influenzae, followed by erythromycin A, clarithromycin, and roxithromycin. All compounds were active against M. catarrhalis, with HMR 3647 and azithromycin having the lowest MICs. Although MICs were sometimes higher in MHF than in HTM, the values did not differ significantly (Table 1).
TABLE 1.
MICs for H. influenzae and M. catarrhalis strains in HTM and MHF
| Drug, organism, and medium | MIC (μg/ml)
|
||
|---|---|---|---|
| Range | For 50% of isolates | For 90% of isolates | |
| HMR 3647 | |||
| H. influenzae | |||
| HTM | 1.0–8.0 | 2.0 | 4.0 |
| MHF | 1.0–>8.0 | 4.0 | 8.0 |
| M. catarrhalis | |||
| HTM | 0.06–0.25 | 0.06 | 0.125 |
| MHF | 0.06–0.25 | 0.125 | 0.125 |
| Erythromycin A | |||
| H. influenzae | |||
| HTM | 2.0–>16.0 | 8.0 | 16.0 |
| MHF | 2.0–>16.0 | 8.0 | 16.0 |
| M. catarrhalis | |||
| HTM | 0.125–0.5 | 0.25 | 0.25 |
| MHF | 0.125–0.5 | 0.25 | 0.5 |
| Azithromycin | |||
| H. influenzae | |||
| HTM | 0.5–8.0 | 2.0 | 4.0 |
| MHF | 1.0–16.0 | 4.0 | 4.0 |
| M. catarrhalis | |||
| HTM | 0.06–0.125 | 0.06 | 0.125 |
| MHF | 0.06–0.125 | 0.125 | 0.125 |
| Clarithromycin | |||
| H. influenzae | |||
| HTM | 4.0–>32.0 | 16.0 | 16.0 |
| MHF | 4.0–>32.0 | 16.0 | 32.0 |
| M. catarrhalis | |||
| HTM | 0.25 | 0.25 | 0.25 |
| MHF | 0.25 | 0.25 | 0.25 |
| Roxithromycin | |||
| H. influenzae | |||
| HTM | 8.0–>32.0 | 32.0 | >32.0 |
| MHF | 8.0–>32.0 | >32.0 | >32.0 |
| M. catarrhalis | |||
| HTM | 0.25–4.0 | 1.0 | 2.0 |
| MHF | 0.5–4.0 | 1.0 | 2.0 |
| Pristinamycin | |||
| H. influenzae | |||
| HTM | 1.0–8.0 | 2.0 | 4.0 |
| MHF | 1.0–16.0 | 2.0 | 4.0 |
| M. catarrhalis | |||
| HTM | 0.5–1.0 | 0.5 | 0.5 |
| MHF | 0.5–1.0 | 0.5 | 0.5 |
Azithromycin had the best kill kinetics against H. influenzae, with 99.9% killing of all strains after 24 h at the MIC. Pristinamycin was bactericidal against all strains after 24 h at twice the MIC. HMR 3647, erythromycin A, and clarithromycin had similar kill kinetics, with 99.9% killing of seven or eight strains at twice the MIC after 24 h and 90% killing of all strains after 24 h at two to four times the MIC. The kill kinetics of roxithromycin were slower. After 24 h, HMR 3647, erythromycin A, and clarithromycin had bacteriostatic activity at the MIC for all of the strains tested. All compounds were bactericidal at 24 h for three or four of the five M. catarrhalis strains at the MIC, and all were bacteriostatic at the MIC after 24 h.
Our results indicate that against both H. influenzae and M. catarrhalis, HMR 3647 and azithromycin had the greatest activity by MIC determination and azithromycin had the greatest activity by time-kill test followed by erythromycin A, clarithromycin, and roxithromycin. Pristinamycin gave MICs similar to those of HMR 3647 but showed more rapid kinetics of H. influenzae killing. Our HMR 3647 MICs were similar to those obtained by Felmingham et al. (4) and Wise and Andrews (20) but higher than those obtained by Agouridas and colleagues for HMR 3004, an older ketolide (1). However, ketolides consistently yield lower MICs than other macrolides, lincosamides, and streptogramins against these species (1, 3, 4, 6, 7, 20). Medium-related problems are probably responsible for published MIC differences.
Although HTM is recommended by the NCCLS as the method of choice for Haemophilus testing (9), the medium cannot be made reliably commercially and must be used within 2 to 3 weeks of in-house preparation for optimal growth. MICs in MHF did not differ significantly from those in HTM. Because most strains of Haemophilus and Moraxella have no specific macrolide-lincosamide-streptogramin mechanism such as erm or mef (16, 17), pharmacokinetic and pharmacodynamic factors (time above the MIC, area under the concentration-time curve/MIC, and concentration in specific body fluids) must also be taken into therapeutic consideration (10, 11, 19). There is an urgent need for a method of Haemophilus susceptibility testing which can readily be adapted for use in the clinical laboratory.
Despite the above technical problems, HMR 3647 and azithromycin had the lowest MICs of all of compounds tested. HMR 3647 was also bacteriostatic against all of the strains tested at the MIC after 24 h, with kill kinetics similar to those of erythromycin A and clarithromycin. HMR 3647 is very active against macrolide-susceptible and -resistant pneumococci (15), as well as other organisms responsible for community-acquired pneumonia (4, 7, 20). If results of pharmacokinetic, pharmacodynamic, and animal studies support its in vitro activity, clinical testing of HMR 3647 in respiratory infections is indicated.
Acknowledgments
This study was supported by a grant from Hoechst-Marion Roussel, Division of Clinical Anti-infectives, Paris, France.
REFERENCES
- 1.Agouridas C, Bonnefoy A, Chantot J F. Antibacterial activity of RU 64004 (HMR 3004), a novel ketolide derivative active against respiratory pathogens. Antimicrob Agents Chemother. 1997;41:2149–2158. doi: 10.1128/aac.41.10.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Berk, S. L., J. H. Kalbfleisch, and The Alexander Project Collaborative Group. 1996. Antibiotic susceptibility patterns of community-acquired respiratory isolates of Moraxella catarrhalis in Western Europe and in the USA. J. Antimicrob. Chemother. 38(Suppl. A):85–96. [DOI] [PubMed]
- 3.Bryskier A, Agouridas C, Chantot J F. Ketolides: new semisynthetic 14-membered ring macrolides. In: Zinner S H, Young L S, Acar J F, Neu H C, editors. Expanding indications for the new macrolides, azalides and streptogramins. New York, N.Y: Marcel Dekker, Inc.; 1996. pp. 39–50. [Google Scholar]
- 4.Felmingham D, Robbins M J, Leakey A, Cooke R, Dencer C, Salman H, Ridgway G L, Grüneberg R N, Bryskier A. The comparative in vitro activity of HMR 3647, a ketolide antimicrobial, against clinical bacterial isolates, abstr. F-116. Washington, D.C: American Society for Microbiology; 1997. p. 166. . Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. [Google Scholar]
- 5.Goldstein, F. W., M. E. Emiran, A. Coutrot, and J. F. Acar. 1990. Bacteriostatic and bactericidal activity of azithromycin against Haemophilus influenzae. J. Antimicrob. Chemother. 25(Suppl. A):25–28. [DOI] [PubMed]
- 6.Jamjian C, Biedenbach D J, Jones R N. In vitro evaluation of a novel ketolide antimicrobial agent, RU 64004. Antimicrob Agents Chemother. 1997;41:454–459. doi: 10.1128/aac.41.2.454. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Jones R N, Biedenbach D J. Antimicrobial activity of RU-66647, a new ketolide. Diagn Microbiol Infect Dis. 1997;27:7–12. doi: 10.1016/s0732-8893(96)00181-2. [DOI] [PubMed] [Google Scholar]
- 8.Maskell, J. P., A. M. Sefton, and J. D. Williams. 1990. Comparative in-vitro activity of azithromycin and erythromycin against gram-positive cocci, Haemophilus influenzae and anaerobes. J. Antimicrob. Chemother. 25(Suppl. A):19–24. [DOI] [PubMed]
- 9.National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. NCCLS publication no. M7-A4. Villanova, Pa: National Committee for Clinical Laboratory Standards; 1997. [Google Scholar]
- 10.Needham C A. Haemophilus influenzae: antibiotic susceptibility. Clin Microbiol Rev. 1988;1:218–227. doi: 10.1128/cmr.1.2.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Neu, H. C. 1991. The development of macrolides: clarithromycin in perspective. J. Antimicrob. Chemother. 27(Suppl. A):1–9. [DOI] [PubMed]
- 12.Olsson-Liljequist, B., and B. M. Hoffman. 1991. In-vitro activity of clarithromycin combined with its 14-hydroxy metabolite against Haemophilus influenzae. J. Antimicrob. Chemother. 27(Suppl. A):11–17. [DOI] [PubMed]
- 13.Pankuch G A, Jacobs M R, Appelbaum P C. Study of comparative antipneumococcal activities of penicillin G, RP 59500, erythromycin, sparfloxacin, ciprofloxacin and vancomycin by using time-kill methodology. Antimicrob Agents Chemother. 1994;38:2065–2072. doi: 10.1128/aac.38.9.2065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Pankuch G A, Lichtenberger C, Jacobs M R, Appelbaum P C. Antipneumococcal activities of RP 59500 (quinupristin-dalfopristin), penicillin G, erythromycin, and sparfloxacin determined by MIC and rapid time-kill methodologies. Antimicrob Agents Chemother. 1996;40:1653–1656. doi: 10.1128/aac.40.7.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Pankuch G A, Visalli M A, Jacobs M R, Appelbaum P C. Susceptibilities of penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, compared with susceptibilities to 17 other agents. Antimicrob Agents Chemother. 1998;42:624–630. doi: 10.1128/aac.42.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother. 1996;40:2562–2566. doi: 10.1128/aac.40.11.2562. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Tait-Kamradt A, Clancy J, Cronan M, Dib-Hajj F, Wondrack L, Yuan W, Sutcliffe J. mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrob Agents Chemother. 1997;41:2251–2255. doi: 10.1128/aac.41.10.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Vallée, E., A. Azoulay-Dupuis, R. Swanson, E. Bergogne-Bérézin, and J. J. Pocidalo. 1991. Individual and combined activities of clarithromycin and its 14-hydroxy metabolite in a murine model of Haemophilus influenzae infection. J. Antimicrob. Chemother. 27(Suppl. A):31–41. [DOI] [PubMed]
- 19.Vazifeh D, Abdelghaffar H, Labro M T. Cellular accumulation of the new ketolide RU 64004 by human neutrophils: comparison with that of azithromycin and roxithromycin. Antimicrob Agents Chemother. 1997;41:2099–2107. doi: 10.1128/aac.41.10.2099. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Wise R, Andrews J M. The in vitro activity of the new ketolide HMR 3647 against a wide range of clinical isolates, abstr. F-114. Washington, D.C: American Society for Microbiology; 1997. p. 165. . Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. [Google Scholar]
- 21.Yeo S F, Akalin E, Arikan S, Auckenthaler R, Bergan T, Dornbusch K, Howard A J, Hryniewicz W, Jones R N, Koupari G, Legakis N J, McLaughlin J, Ozkuyumcu C, Percival A, Phillips I, Reeves D, Spencer R, Warren R E, Williams J D. Susceptibility testing of Haemophilus influenzae—an international collaborative study in quality assessment. J Antimicrob Chemother. 1996;38:363–386. doi: 10.1093/jac/38.3.363. [DOI] [PubMed] [Google Scholar]