Development of a real-time PCR assay on the Roche Light-Cycler for the detection of erm and mef erythromycin resistance genes in β-haemolytic streptococci

Abstract

Sir, Although erythromycin resistance in streptococci is predominantly due to target site modification by rRNA methylases (encoded by erm genes) or drug efflux (encoded by mef genes), novel resistance mechanisms continue to emerge. Furthermore, susceptibility to macrolides, lincosamides, streptogramins and ketolides varies depending on the distribution of different erythromycin resistance mechanisms. Therefore, phenotypic and genotypic erythromycin resistance surveillance data are required as an adjunct in determining therapy for streptococcal infections. In the UK, erythromycin resistance in Lancefield group B (GBS), C (GCS) and G (GGS) streptococci is increasing, but data on the molecular basis of resistance are limited. PCR with agarose gel analysis or microwell-format probe hybridization can be used to detect erm and mef genes; however, both require additional handling steps and gel analysis lacks the specificity of sequencing or hybridization analyses. Real-time PCR has the dual benefits of rapid amplification and specific detection, occurring simultaneously in the same tube, reducing the risks of contamination. The aim of this study was to develop a real-time PCR assay to detect both erm and mef genes and characterize erythromycin resistance genes in GBS, GCS and GGS isolated from clinical samples. This approach has not been used before in detecting erm and/or mef genes in streptococci. Positive control strains containing erm and mef genes used were: 02C1064 [Streptococcus pyogenes, mef(A/E)]; 02C1110 [S. pyogenes, erm(A) subclass erm(TR)]; RN1389 [Staphylococcus aureus, erm(A)]; JH2-2 [Enterococcus faecalis, erm(B)] and RN4220 [S. aureus, erm(C)]. Sixty-one erythromycinresistant streptococci (14 GBS, four GCS and 43 GGS) collected from patients with community-acquired infections at two different hospitals during two different 6 month periods were also tested.