Aventis Pharma, Infectious Diseases Group, Microbiology, 102, Route de Noisy, 93235 Romainville Cedex, France
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Abstract |
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Introduction |
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Ketolides are a new class of semi-synthetic 14- membered-ring macrolides, active against respiratory tract pathogens and also enterococci.3,4 Introduction of a fluorine moiety in position 2 of the macrolactone skeleton improves the overall antibacterial spectrum, as reported previously for novel 2-fluoro-ketolides,5 such as HMR 3562. Here, we focused on the in vitro and in vivo antibacterial activity of HMR 3562 against enterococci, including vancomycin-resistant strains.
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Materials and methods |
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HMR 3562, erythromycin A, clarithromycin and azithromycin were prepared by Aventis (Romainville, France). Pristinamycin was provided by Aventis (Vitry, France). Teicoplanin and vancomycin were purchased from Sigma (St Louis, MO, USA).
Bacterial strains
Ninety-five strains were tested: 51 E. faecalis, 40 E. faecium, two Enterococcus gallinarum, one E. durans and one Enterococcus casseliflavus. Thirty-six isolates were vancomycin resistant. All the strains tested were clinical isolates from various European and US hospitals, except the reference strain E. faecalis ATCC 29212 used for quality control.
MIC determination
MICs were measured by a two-fold agar dilution method in MuellerHinton (MH) agar medium (pH 7.4; Diagnostic Pasteur, France).6 A standard inoculum of 104 cfu/spot was used throughout the study. All plates were incubated at 37°C for 24 h. The MIC was defined as the lowest concentration at which no visible growth could be detected on agar plates. According to NCCLS breakpoints, MICs for susceptible strains were 0.5,
4 and
8 mg/L for erythromycin, vancomycin and teicoplanin, respectively.
Systemic infection model
The present studies were approved by the Internal Animal Ethics Committee. Male C3H and C57Bl/6 Charles River mice were used to study the antibacterial activity of compounds in a peritonitis model against vancomycin-susceptible and -resistant strains, respectively. Each dosing group was composed of 10 animals weighing 2022 g. Mice were infected intraperitoneally with 0.5 mL of an overnight culture suspended in physiological buffer containing 5% pig mucin (Sigma) to a final cell density corresponding to 10100 times the minimal lethal dose, i.e. approximately 109 cfu. Bovine haemoglobin (2%) (Sigma) was added when testing vancomycin-resistant strains. Under these conditions, untreated animals died by 4872 h. Suspensions of compounds (0.5 mL) were administered in carboxymethyl cellulose (Sigma) by the oral route, except for vancomycin, immediately and 4 h post-infection. Vancomycin was administered subcutaneously in saline buffer. Mice were observed for 810 days following the inoculation, and the 50% protective dose (PD50) expressed as the unit dose that protected 50% of the animals from death was calculated by the probit method of Litchfield & Wilcoxon.7
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Results |
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Table I indicates the in vitro activity of HMR 3562 against 95 enterococci. The MICs of erythromycin, vancomycin and teicoplanin for E. faecalis ATCC 29212 were 1.2, 2.5 and 0.08 mg/L, respectively, which are within NCCLS quality control breakpoint limits.
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PD50 determinations
Table II gives the PD50s of HMR 3562, clarithromycin, pristinamycin and vancomycin obtained in five different lethal infections induced by one E. faecalis and four E. faecium isolates in a murine peritonitis model. The activity of HMR 3562 did not depend on the resistance phenotype of the strains tested and was the highest, with PD50 ranging from 3.4 to 21.8 mg/kg. As expected, vancomycin was completely inactive against vancomycin-resistant isolates.
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Discussion |
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As shown previously,8,9 HMR 3562 displayed potent activity against E. faecalis and E. faecium, irrespective of their erythromycin A susceptibility status. The MIC90 of HMR 3562 for erythromycin A-resistant enterococci was 0.6 mg/L, while all of the macrolides tested remained inactive. As vancomycin resistance is commonly associated with macrolide resistance in enterococci, it is worth noting that HMR 3562 remained active against vancomycinresistant strains. However, as with telithromycin and macrolides,4 HMR 3562 demonstrated bacteriostatic activity against both erythromycin A-susceptible and erythromicin A-resistant strains (data not shown).
There is little published information about therapy of enterococci in murine models, particularly with ketolides.3,10 In experimental peritonitis, we confirmed the in vitro activity of HMR 3562, with consistent efficacy against all of the five strains tested, irrespective of their macrolide resistance phenotype. The PD50 range was 3.421.8 mg/kg, while MICs ranged from 0.01 to 0.15 mg/L. On the other hand, despite its favourable MICs, pristinamycin did not show any therapeutic efficacy at the doses tested, probably due to disadvantageous kinetics.
In conclusion, HMR 3562 shows promise as an antimicrobial agent for use against multi-resistant enterococci.
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Acknowledgments |
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Notes |
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References |
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2 . Moellering, R. C. (1991). The Garrod Lecture. The enterococcus: a classic example of the impact of antimicrobial resistance on therapeutic options. Journal of Antimicrobial Chemotherapy 28, 112.[ISI][Medline]
3 . Agouridas, C., Bonnefoy, A. & Chantot, J. F. (1997). Antibacterial activity of RU 64004 (HMR 3004), a novel ketolide derivative active against respiratory pathogens. Antimicrobial Agents and Chemotherapy 41, 214958.[Abstract]
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Malathum, K., Coque, T. M., Singh, K. V. & Murray, B.E. (1999). In vitro activities of two ketolides, HMR 3647 and HMR 3004 against gram-positive bacteria. Antimicrobial Agents and Chemotherapy 43, 9306.
5 . Bonnefoy, A., Denis, A., Bretin, F., Fromentin, C. & Agouridas, C. (1999). In vitro antibacterial activity of novel 2-fluoro-ketolides. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 2153, p. 351. American Society for Microbiology, Washington, DC.
6 . Barry, A. (1991). Procedures and theoretical considerations for testing antimicrobial agents in agar media. In Antibiotics in Laboratory Medicine, 3rd edn, (Lorian, V., Ed.), pp. 116. Williams & Wilkins, Baltimore, MD.
7 . Litchfield, J. T. & Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. Journal of Pharmacology and Experimental Therapy 96, 99113.[ISI]
8 . Bonnefoy, A., Denis, A., Bretin, F., Fromentin, C. & Agouridas, C. (1999). In vitro antibacterial activity of two ketolides, HMR 3562 and HMR 3787, against respiratory pathogens. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 2155, p. 351. American Society for Microbiology, Washington, DC.
9 . Felmingham, D., Robbins, M. J., Mathias, I. & Bryskier, A. (1999). In vitro activity of two ketolides, HMR 3562 and HMR 3787 against clinical bacterial isolates. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 2154, p. 351. American Society for Microbiology, Washington, DC.
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Singh, K. V., Zscheck, K. K. & Murray, B. E. (2000). Efficacy of telithromycin (HMR 3647) against enterococci in a mouse peritonitis model. Antimicrobial Agents and Chemotherapy 44, 34347.
Received 7 December 2000; returned 26 February 2001; revised 19 March 2001; accepted 17 April 2001