Title: In vitro activity of telithromycin (HMR 3647) against Greek Streptococcus pyogenes and Streptococcus pneumoniae clinical isolates with different macrolide susceptibilities
Abstract: The susceptibilities to macrolides and telithromycin of 161 Streptococcus pyogenes and 145 Streptococcus pyogenes strains, consecutively isolated from five Greek hospitals, were determined by Etest. Moreover, mechanisms of resistance to macrolides were phenotypically and genetically determined by double disk induction test and PCR, respectively. Of the S. pneumoniae and S. pyogenes isolates, 42.8% and 30.8%, respectively, were found to be resistant to erythromycin. Of the erythromycin-resistant S. pneumoniae and S. pyogenes isolates, 57.5% and 59.5%, respectively, displayed the M phenotype and harbored the mefA/E gene. Telithromycin was found to be more active than both erythromycin and clarithromycin against both species, with considerably lower MIC50 and MIC90 values. The susceptibilities to macrolides and telithromycin of 161 Streptococcus pyogenes and 145 Streptococcus pyogenes strains, consecutively isolated from five Greek hospitals, were determined by Etest. Moreover, mechanisms of resistance to macrolides were phenotypically and genetically determined by double disk induction test and PCR, respectively. Of the S. pneumoniae and S. pyogenes isolates, 42.8% and 30.8%, respectively, were found to be resistant to erythromycin. Of the erythromycin-resistant S. pneumoniae and S. pyogenes isolates, 57.5% and 59.5%, respectively, displayed the M phenotype and harbored the mefA/E gene. Telithromycin was found to be more active than both erythromycin and clarithromycin against both species, with considerably lower MIC50 and MIC90 values. An increase in the rate of isolation of erythromycin A-resistant pneumococci and other streptococci has been observed in the past few years in many parts of the world. Data from the Greek Electronic Network for the Surveillance of Antibiotic Resistance revealed an increase in macrolide resistance from approximately 5% for Streptococcus pyogenes and 12% for S. pneumoniae in 1995, to approximately 20% and 30%, respectively, in 2000 [1The WHONET Network Macrolide resistance in Greece, 1996–99.Arch Hellenic Med. 2000; 17: 113Google Scholar]. In the study reported here, the in vitro activity of telithromycin, a new ketolide drug, against clinical strains of S. pyogenes and S. pneumoniae, recently isolated in various Greek hospitals, was compared to the activity of erythromycin A, clarithromycin, and clindamycin, and related to the macrolide resistance phenotypes. In total, 306 clinical isolates, 161 S. pyogenes and 145 S. pneumoniae, consecutively isolated in three adult and two pediatric hospitals of the Athens–Piraeus area during the period October 1999 to February 2000, were tested. Identification to species level was performed with standard methodology. Susceptibilities to erythromycin A, clindamycin, clarithromycin and telithromycin were determined by Etest (AB Biodisk, Solna, Sweden), using Mueller–Hinton agar (Oxoid Ltd, Basingstoke, UK) supplemented with 5% blood, and incubated in an ambient atmosphere, according to the manufacturer's recommendations. The susceptibility breakpoints as defined by the National Committee for Clinical Laboratory Standards (NCCLS) were used [2National Committee for Clinical Laboratory Standards Performance standards for antimicrobial susceptibility testing, Eleventh Informational Supplement. M100-S11. NCCLS, Wayne, PA2001Google Scholar]. For telithromycin, the breakpoints defined by The European Medicines Evaluation Agency (EMEA) (A. Bryskier, personal communication) were used (Table 1).Table 1Susceptibilities of clinical isolates of S. pneumoniae and S. pyogenes to the antimicrobials used in this studyAntibioticBreakpoints (mg/L)%R%IMIC50MIC90Geometrical MeanMIC RangeS. pneumoniae (n = 145)ErythromycinS ≤ 0.25 R ≥ 142.83.40.252561.2530.023–256ClarithromycinS ≤ 0.25 R ≥ 149.03.41320.8960.0064–256ClindamycinS ≤ 0.25 R ≥ 113.816.60.1930.3530.019–256TelithromycinS ≤ 0.5 R ≥ 23.48.30.0940.750.1140.008–6S. pyogenes (n = 161)ErythromycinS ≤ 0.25 R ≥ 130.83.10.19320.6220.016–256ClarithromycinS ≤ 0.25 R ≥ 128.83.10.094240.3370.0047–256ClindamycinS ≤ 0.25 R ≥ 11.23.80.1250.250.1360.0094–0.75TelithromycinS ≤ 0.5 R ≥ 23.111.20.06410.0810.008–2 Open table in a new tab Erythromycin-resistant isolates (MIC > 0.5 mg/L) were divided into constitutive resistant (CR) and inducible resistant (IR) MLSB and M phenotypes by the double disk test, using erythromycin and clindamycin disks as described previously [3Seppala H Nissinen A Yu Q Huovinen P Three different phenotypes of erythromycin-resistant Streptococcus pyogenes in Finland.J Antimicrob Chemother. 1993; 28: 885-891Crossref Scopus (276) Google Scholar]. Erythromycin resistance genes (ermB, ermTR, and mefA/E) were detected by PCR as previously described [4Kataja J Huovinen P Skurnik M Sepala H Erythromycin resistance genes in group A streptococci in Finland.Antimicrob Agents Chemother. 1999; 43: 48-52PubMed Google Scholar, 5Luna VA Coates P Eady EA Cove J Nguyen TTH Roberts MC A variety of Gram-positive bacteria carry mobile mef genes.J Antimicrob Chemother. 1999; 44: 19-25Crossref PubMed Scopus (133) Google Scholar, 6Roberts MC Chung WO Roe D et al.Erythromycin resistant Neiseria gonorrheae and oral commensal Neisseria spp carry known rRNA methylase genes.Antimicrob Agents Chemother. 1999; 43: 1367-1372PubMed Google Scholar]. Data were analyzed using the WHONET 5 software (WHO Collaborating Center for Surveillance of Antibiotic Resistance, Boston, USA). Table 1 shows the antibacterial activities of telithromycin and the other antibiotics against the 145 S. pneumoniae and the 161 S. pyogenes isolates. High rates of resistance to both macrolides studied were observed in both species, with 42.8% of S. pneumoniae isolates and 30.8% of S. pyogenes isolates being resistant to erythromycin (Table 1). Similar results were observed for clarithromycin. In total, 59.5% of the erythromycin-resistant S. pyogenes isolates and 57.5% of the S. pneumoniae isolates tested displayed the M phenotype (Table 2). Within the MLSB phenotype, all S. pyogenes isolates displayed the inducible type of resistance (IR), whereas 16 of the 17 S. pneumoniae isolates displayed the constitutive type (CR). Genetic analysis showed that the majority of the M phenotype isolates (23 of 25 S. pyogenes isolates and 22 of 23 S. pneumoniae isolates) carried mefA/E. The ermTR gene was found in the majority (16/17) of S. pyogenes IR isolates, while all 16 of the S. pneumoniae CR, constitutive type of MLSB resistance; IR, inducible type of MLSB resistance; M, M phenotype (efflux mechanism). CR isolates carried ermB (data not shown in the table).Table 2Phenotypes of resistance to erythromycin and clindamycin of the erythromycin-resistant (MIC > 0.5 mg/L) isolatesS. pneumoniaeS. pyogenesPhenotypeNo.%No.%CR1640.000IR12.51740.5M2357.52559.5All tested40100.042100.0CR constitutive type of MLSB resistance;IR, inducible type of MLSB resistance;M, M phenotype(efflux mechanism). Open table in a new tab CR constitutive type of MLSB resistance;IR, inducible type of MLSB resistance;M, M phenotype(efflux mechanism). In S. pyogenes, the M phenotype (mefA/E) is prevalent in Spain [7Alos JI, Aracil B, Oteo J, Torres C, Gomez-Garcés JL, the Spanish Group for the Study of Infection in the Primary Health Care Setting High prevalence of erythromycin-resistant, clindamycin/miocamycin-susceptible (M phenotype) Streptococcus pyogenes: results of a Spanish multicentre study in 1998.J Antimicrob Chemother. 2000; 45: 605-609Crossref PubMed Scopus (55) Google Scholar], Finland [4Kataja J Huovinen P Skurnik M Sepala H Erythromycin resistance genes in group A streptococci in Finland.Antimicrob Agents Chemother. 1999; 43: 48-52PubMed Google Scholar], and Belgium [8Descheemaeker P Chapelle S Lammens C et al.Macrolide resistance and erythromycin resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates.J Antimicrob Chemother. 2000; 45: 167-173Crossref PubMed Scopus (119) Google Scholar], whereas in France [9Arpin C Canron M-H Noury P Quentin C Emergence of mefA and mefE genes in beta-haemolytic streptococci and pneumococci in France.J Antimicrob Chemother. 1999; 44: 133-134Crossref PubMed Scopus (36) Google Scholar], Germany [10Arvand M Hoeck M Hahn H Wagner J Antimicrobial resistance in Streptococcus pyogenes isolates in Berlin.J Antimicrob Chemother. 2000; 46: 621-624Crossref PubMed Scopus (38) Google Scholar] and Portugal [11Melo-Cristino J Fernandes ML Streptococcus pyogenes isolated in Portugal: macrolide resistance phenotypes and correlation with T types. Portuguese Surveillance Group for the Study of Respiratory Pathogens.Microb Drug Resist. 1999; 5: 219-225Crossref PubMed Scopus (37) Google Scholar] the majority of macrolide resistance seems to be due to the MLSB phenotype. Other countries such as Italy [12Savoia D Avanzini C Bosio K et al.Macrolide resistance in group A streptococci.J Antimicrob Chemother. 2000; 45: 41-47Crossref PubMed Scopus (33) Google Scholar, 13Ripa S Zampaloni C Vitali LA et al.SmaI macrorestriction analysis of Italian isolates of erythromycin-resistant Streptococcus pyogenes and correlations with macrolide-resistance phenotypes.Microb Drug Resist. 2001; 7: 65-71Crossref PubMed Scopus (34) Google Scholar] present a mixture of mechanisms. The predominance of the M phenotype in S. pyogenes in Greece has also been shown by other investigators [14Tzelepi E Kouppari G Mavroidi A Zaphiropoulou A Tzouvelekis LS Erythromycin resistance amongst group A beta-haemolytic streptococci isolated in a paediatric hospital in Athens, Greece.J Antimicrob Chemother. 1999; 43: 745-746Crossref PubMed Scopus (21) Google Scholar], whereas, to our knowledge, this is the first report of the predominance of the M phenotype and of mefA/E in S. pneumoniae in Greece. It should be noted that the results of the PCR assay were compatible with the phenotype characterizations of macrolide resistance. Interestingly, the efflux mechanism is the predominant form of erythromycin resistance in S. pneumoniae strains in the USA and Canada, whereas in Europe the majority of erythromycin-resistant pneumococci carry an erm methylase [15Tait-Kamradt A Davies T Appelbaum PC et al.Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America.Antimicrob Agents Chemother. 2000; 44: 3395-3401Crossref PubMed Scopus (232) Google Scholar]. Telithromycin was found to be more active than both erythromycin A and clarithromycin against both species (Table 1). More specifically, for S. pneumoniae, the MIC50 and MIC90 of telithromycin were found to be 0.094 mg/L and 0.75 mg/L, respectively. The corresponding values for erythromycin were 0.250 mg/L and 256 mg/L, and those for clarithromycin were 1 mg/L and 32 mg/L. Similarly, for S. pyogenes, the MIC50 and MIC90 of telithromycin were found to be 0.064 mg/L and 1.00 mg/L, respectively, whereas the respective values for erythromycin were 0.190 mg/L and 32 mg/L, and those for clarithromycin were 0.094 mg/L and 24 mg/L. Neither Giovanetti et al. [16Giovanetti E Otanari MP Marchetti F Varaldo PE In vitro activity of ketolides telithromycin and HMR 3004 against Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility.J Antimicrob Chemother. 2000; 46: 905-908Crossref PubMed Scopus (41) Google Scholar] nor Jalava et al. [17Jalava J Kataja J Seppala H Huovinen P In vitro activities of the novel ketolide telithromycin (HMR 3647) against erythromycin resistant Streptococcus species.Antimicrob Agents Chemother. 2001; 45: 789-793Crossref PubMed Scopus (77) Google Scholar] reported resistance to telithromycin in S. pneumoniae (MIC > 1 mg/L), whereas all S. pyogenes isolates found by these authors to display resistance to telithromycin (MIC > 1 mg/L) were of the MLSB resistant phenotype. Bemer-Melchior et al. [18Bemer-Melchior P Juvin ME Tassin S Bryskier A Schito GC Drugeon HB In vitro activity of the new cetolide telithromycin compared with those of macrolides against Streptococcus pyogenes: influences of resistance mechanisms and methodological factors.Antmicrob Agents Chemother. 2000; 44: 2999-3002Crossref PubMed Scopus (54) Google Scholar] reported that telithromycin could overcome resistance due to the efflux mechanism. Moreover, the activity of ketolides against inducible strains can be attributed to their weak inducing ability [19Bonnefoy A Girard AM Agouridas C Chantot JF Ketolides lack inducibility properties of MLS (B) resistance phenotype.J Antimicrob Chemother. 1997; 40: 85-90Crossref PubMed Scopus (167) Google Scholar], whereas the differences in susceptibility between the two streptococcal species could be due to ribosomal structural differences in the two bacterial species [17Jalava J Kataja J Seppala H Huovinen P In vitro activities of the novel ketolide telithromycin (HMR 3647) against erythromycin resistant Streptococcus species.Antimicrob Agents Chemother. 2001; 45: 789-793Crossref PubMed Scopus (77) Google Scholar]. Three of our S. pneumoniae isolates and five S. pyogenes isolates were found to display an MIC of telithromycin greater than 1 mg/L. Of these, two S. pneumoniae isolates were found to display the CR phenotype (MIC 2 and 4 mg/L, respectively) and one the M phenotype (MIC 1.5 mg/L), whereas all five S. pyogenes isolates were of the M phenotype, with MIC values of 1.5 mg/L (three strains) and 2 mg/L (two strains). In conclusion, telithromycin was found to be more active in vitro than erythromycin and clarithromycin against clinical isolates of S. pneumoniae and S. pyogenes. It remained active even against erythromycin-resistant S. pyogenes and S. pneumoniae of both phenotypes (MLS and M), with an MIC up to 4 mg/L in only a few exceptions. This activity makes ketolides suitable for further investigation as effective alternatives for the treatment of streptococcal infections. This work was supported by a grant from Aventis Pharma (Romainville France). We are grateful to Dr A. Bryskier for his help, and also to Dr G. Cornaglia, P. Huovinen and J. Jalava for providing control strains with known resistance to macrolide genes.