a Area Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006 Logroño, Spain; b Laboratorio Microbiología, Hospital San Millán, Logroño, Spain
Sir,
Two main mechanisms of resistance to macrolidelincosamide-streptogramin (MLS) antibiotics have been described in group B (GBS), group C (GCS) and group G (GGS) ß-haemolytic streptococci. Target site modification by rRNA methylases, encoded by erm genes, confers the MLSB resistance phenotype [erythromycin-resistant clindamycin resistant (ERYR-CLIR)]. The mechanism of active efflux confers a dissociate M resistance phenotype [erythromycin-resistantclindamycin-susceptible (ERYR-CLIS)]. The objective of the present study was to determine the incidence of MLS resistance in vitro among 136 GBS, eight GCS and 28 GGS consecutive clinical isolates recovered from the Hospital San Millán (Logroño, Spain) between 1997 and 2000, and to characterize the resistance mechanisms involved. Susceptibility testing was performed for ERY, CLY, spiramycin (SPY) and virginiamycin (VGY) by the disc diffusion method. MICs were also determined for ERY, penicillin and cefotaxime using the E-test method. The MLS resistance mechanisms were determined by PCR amplification of erm genes [erm(A), erm(B), erm(C)1 and erm(TR)2]. The efflux pump mechanism was also analysed by PCR amplification of the msr(A)3 and mef(A/E) genes.4 According to the new nomenclature for macrolide and MLSB resistance determinants,5 we consider that positive PCR amplifications obtained using either erm(A) and/or erm(TR) primers correspond to the presence of the erm(A) gene. Similarly, and according to Roberts et al.,5 we have designated as mef(A) genes all the expected-size PCR products obtained with mef(A/E) primers. The presence of the mre(A) gene was also determined in all GBS isolates by PCR.
Twenty of 136 (14.7%) GBS isolates were ERYR, and ERY MIC50 and MIC90 for these isolates were 0.094 and >256 mg/L, respectively. Penicillin and cefotaxime MIC90s were 0.064 mg/L and all isolates remained susceptible to these antibiotics. All ERYR GBS isolates with MIC in the range 4>256 mg/L (n = 19), showed the MLSB resistance phenotype (ERYR-SPIR-CLIR), and one additional ERYR GBS isolate (MIC 2 mg/L) showed the M phenotype (ERYR-CLIS). The expression of the MLSB resistance mechanism was either inducible (six isolates) or constitutive (13 isolates) (Table). PCR analyses were performed for all ERYR (n = 20) and for 11 of 106 ERYS GBS isolates, in order to determine the resistance mechanisms. The mre(A) gene was detected by PCR and sequencing in all GBS, either with ERYR or ERYS phenotypes, suggesting that it could be ubiquitous in this streptococcal species, as has been reported previously.6 Other macrolide resistance PCR analyses were negative in all 11 ERYS isolates studied (ERY MICs in the range 0.0320.38 mg/L). The mef(A) gene was detected by PCR in the only isolate with M resistance phenotype (MIC 2 mg/L), and negative results were obtained for the other macrolide resistance genes. The erm(B) gene was detected, either alone (11 isolates with MIC range 8>256 mg/L) or associated with erm(A) (one isolate with MIC > 256 mg/L) in 12 of 13 ERYR isolates with the constitutive MLSB resistance phenotype. The remaining constitutive MLSB isolate (MIC > 256 mg/L), showed only the erm(A) gene by PCR analysis. The presence of the erm(A) gene was demonstrated, either alone (five isolates with MIC range of 4>256 mg/L) or associated with the erm(B) gene (one isolate with MIC > 256 mg/L) in all six ERYR isolates with the inducible MLSB resistance phenotype. Neither the msr(A), nor the mef(A) gene was detected in any of these MLSB-resistant isolates. Transfer of the mef(A) gene was demonstrated by filter mating conjugation from the GBS isolate S385 (M resistance phenotype) to Enterococcus faecalis strain JH2-2. Transconjugants showed an ERY MIC of >32 mg/L and the mef(A) gene was shown by PCR analysis.
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Previous studies carried out in our group showed that the M phenotype is prevalent among ERYR Streptococcus pyogenes, and curiously, this phenotype is rarely found among other streptococcal species, such as GBS, GCS and GGS, in our region. This fact should be considered when deciding empirical therapy.
Acknowledgments
This work was supported in part by a grant from the Ministerio de Salud y Consumo of Spain (FIS 00/0545). Aránzazu Portillo has an FPI fellowship from the Ministerio de Educación y Ciencia of Spain.
Notes
J Antimicrob Chemother 2001; 47: 115116
* Corresponding author. Tel: +34-941-299750; Fax: +34-941-299721; E-mail: carmen.torres{at}daa.unirioja.es
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