a Institute of Microbiology, University of Ancona, Via Ranieri, Monte d'Ago, 60131 Ancona; b Hoechst Marion Roussel, Medical Scientific Department, 20020 Lainate, Italy
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Abstract |
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Introduction |
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In Italy, the rate of erythromycin resistance increased considerably in the 1990s in both Streptococcus pyogenes and Streptococcus pneumoniae isolates, with incidences of >40%2 and >30%,3 respectively. In the present study, the activities of telithromycin and HMR 3004 were evaluated in vitro against erythromycin-susceptible and erythromycin- resistant Italian isolates of these two species. Erythromycin-resistant test strains were preliminarily characterized according to their susceptibilities to antibiotics of the macrolide, lincosamide and streptogramin (MLS) group, and assigned to the constitutive resistance (cMLS) phenotype, the inducible resistance (iMLS) phenotype or the more recently described M phenotype.4,5 Inducibly resistant S. pyogenes strains were further subdivided into the three distinct subtypes iMLS-A, -B and -C; the characteristics of these subtypes and genotypic correlations with erythromycin resistance genes have been delineated recently.6
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Materials and methods |
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A total of 311 strains of S. pyogenes (100 erythromycinsusceptible and 211 erythromycin-resistant) and 157 of S. pneumoniae (100 erythromycin-susceptible and 57 erythromycin-resistant), all isolated from clinical specimens between mid-1996 and mid-1999, were obtained from various Italian laboratories. Strain identification was confirmed in our laboratory and erythromycin susceptibility (MIC 0.25 mg/L) or resistance (MIC
1 mg/L) quantified by a broth microdilution method. Using the same method, pneumococci were categorized as penicillin-susceptible (MIC
0.06 mg/L), -intermediate (MIC 0.121 mg/L) or -resistant (MIC
2 mg/L). Erythromycin-resistant strains of S. pyogenes were assigned to the cMLS, iMLS or M phenotype, and iMLS strains were further subdivided into the three subtypes iMLS-A, iMLS-B and iMLS-C on the basis of a triple-disc test (erythromycin plus clindamycin and josamycin).6 The resistance phenotype of erythromycin-resistant strains of S. pneumoniae was identified using a double-disc test (erythromycin plus clindamycin).4,6 Attempts at distinguishing between inducibly and constitutively resistant pneumococci were also made on the basis of rokitamycin MICs as described by Agouridas et al.7 (taking
1 and >4 mg/L as the breakpoints for inducibly and constitutively resistant strains, respectively). Strains were maintained in glycerol at 70°C and subcultured twice on blood agar before susceptibility testing.
Antibiotics
Telithromycin and HMR 3004 were provided by Hoechst Marion Roussel (Lainate, Italy). Erythromycin, clindamycin and benzylpenicillin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Josamycin and rokitamycin were obtained from ICN Biomedicals (Costa Mesa, CA, USA) and Formenti (Milan, Italy), respectively.
Susceptibility tests
MICs were determined by a standard microdilution procedure using MuellerHinton II broth (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with 3% lysed horse blood. The inoculum was 5 x 105 cfu/mL. The antibiotics were tested at final concentrations (prepared from two-fold dilutions) ranging from 0.015 to 128 mg/L. The MIC was defined as the lowest concentration that yielded no visible growth. In the absence of established interpretive standards, the MIC breakpoints proposed unofficially for telithromycin (susceptible, 1 mg/L; intermediate, 2 mg/L; resistant,
4 mg/L)8 were considered tentatively for both ketolides. S. pneumoniae ATCC 49619 was used as a quality control.
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Results |
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Among the S. pyogenes isolates (Table I), the erythromycin-susceptible strains, erythromycin-resistant strains with the M phenotype and inducibly resistant strains of the iMLS-B and -C subtypes were uniformly susceptible to telithromycin and HMR 3004. The most potent ketolide activity was recorded against iMLS-C isolates (MICs of both telithromycin and HMR 3004 were invariably
0.015 mg/L), followed by iMLS-B and erythromycin-susceptible isolates (MIC50s
0.015 mg/L; MIC90s 0.06 mg/L) and M isolates (MIC50 and MIC90, 0.12 and 0.25 mg/L, respectively, for telithromycin, and 0.06 and 0.12 mg/L for HMR 3004). Susceptibility to ketolides of the cMLS and iMLS-A phenotypes usually ranged from the upper limits of susceptibility (MIC 0.51 mg/L) to low-level resistance (MIC, 48 mg/L), with MIC50s of 1 mg/L for HMR 3004 and 24 mg/L for telithromycin; however, one highly ketolide-susceptible cMLS isolate (telithromycin MIC 0.03 mg/L; HMR 3004 MIC
0.015 mg/L) was encountered.
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Discussion |
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The uniform activity of ketolides against pneumococci, including erythromycin- and/or penicillin-resistant isolates, suggests that ketolides may offer a valuable alternative for the treatment of pneumococcal infections. Once pharmacological and toxicological investigations of telithromycinthe ketolide currently considered for clinical useare completed, clinical trials should be performed to test this hypothesis. Against erythromycin-resistant strains of S. pyogenes, ketolide antibiotics appear to be highly active when resistance is mediated by an efflux system5 (M isolates) and even more so when it is mediated by the methylase encoded by the recently described ermTR gene15 (iMLS-B and -C isolates);6 in contrast, their activity is lower when resistance is mediated by the conventional ermAM (ermB)-encoded methylase (cMLS and iMLS-A isolates).6
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Notes |
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References |
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Received 20 January 2000; returned 29 March 2000; revised 6 April 2000; accepted 7 June 2000