Loss of mecA gene in Staphylococcus epidermidis after prolonged therapy with vancomycin.

ibility and erythromycin resistance phenotypes. DNA from test samples was prepared by both Qiagen extraction and rapid boiling. As the efficiencies of the two methods were comparable, the rapid boiling method was used throughout. Primers were as published for erm(A), erm(B), erm(C), mef(A/E) and erm(TR). Donor and acceptor probe-pairs were designed for erm(A), erm(TR), erm(B), erm(C) and mef(A/E) respectively: ERMADP1, CTGCAACGAGCTTTGGGTTTACTA and ERMAAP1, AATGGTGGAGATGGATATAAAAATGC; DF-ERMA1, GTCAAGCGAAATATAGCTACCTT and DR-ERMA1, TGTAGAGAGGGGATTTGCTA; ERMA/MD1, CGTGTCACTTTAATTCACCAAGATand ERMA/ MA1, TCTACAGTTTCAATTCCCTAACAAA; ERM/CD1, GTATGGTTCCAAGAGAATAT and ERM/CA1, TCATCCTAAACCTAAAGTGAA; MEFA/ED1, TATCCGTAGCATTGGAACAGCT and MEFA/EA1, TTCATACCCCAGCACTCAATGCGGT. All donor probes were 30 end-labelled with fluorescein. Acceptor probes specific for erm(A), erm(B), erm(C) and erm(TR) were 50 end-labelled with LC Red 640; for mef (A/E), LC Red 705. Each acceptor probe had a 30-phosphate blocking group. Each 20 mL PCR mixture consisted of: 2 mL of FastStart DNA Master Hybridisation Probes (Roche Applied Sciences), 1.6 mL of 25 mM MgCl2, 50 pmol of primers, 10 pmol of each probe pair, 8.4 mL of (PCR standard) water and 5 mL of bacterial DNA preparation. Two negative controls (PCR standard water and an erythromycin-sensitive GBS) were included in each run. Cycling conditions were: 95 C for 10 min, temperature transition rate (TTR) 20 C/s; 60 cycles 95 C for 0 s, TTR 20 C/s, annealing at 60 C for 10 s, TTR 20 C/s, and 72 C for 15 s, TTR 2 C/s; followed by a melting curve of: 99 C for 15s, TTR 20 C/s, 40 C for 15s, TTR 0.2 C/s to 95 C with continuous fluorescent acquisition. Fluorescence was measured on channel F2 for probes labelled with LC Red 640 and channel F3 for the LC Red 705-labelled probe. Clinical isolates were tested four times using primers and probes specific for either mef(A/E), erm(A), erm(TR) or erm(B) genes. The erm(C) gene was not sought as its presence has only been reported in staphylococci. Figure 1 shows the characteristic melting peaks for the positive controls; similar peaks were produced by the clinical isolates. erm(TR) and erm(B) were detected as the only erythromycin resistance genes in 41 (67%) and five (8%), respectively, of clinical strains with theMLSB phenotype. All strains with theM-phenotype contained mef(A/E) only, while among those with the MLSB phenotype, seven (11%) possessed double mechanisms of resistance: erm(TR) + erm(B), erm(TR) + mef(A/E) or erm(B) + mef(A/E). Two (3%) clinical strains expressing MLSB phenotype were negative for erythromycin resistance genes. No isolate carried the erm(A) gene. This assay, with the rapid boiling method for DNA extraction, provides results within 1 h of sample preparation. It is sensitive and specific, successfully identifying resistance genes in 97% of the erythromycin-resistant clinical strains of group B, C and G streptococci. With the increasing availability of Light-Cyclers, the assay can be used as a rapid tool for local, national and international surveillance of erythromycin resistance in streptococci, thereby guiding therapy. In agreement with a previous study, we found erm(TR) to be the most frequent resistance gene. However, for definitive erythromycin resistance genotyping, isolates should be tested for the most common mef and erm genes, as more than one resistance gene may be present. Many of the existing PCR assays are multiplex, reducing the number of reactions required. The characteristic melting peaks detected in the different channels are being exploited in developing a multiplex assay, which will reduce further the turn-around time and cost.