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

disc testing was used to determine antibiotic susceptibility 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) 5 and erm(TR). 4 Donor and acceptor probe-pairs were designed for erm(A), erm(TR), erm(B), erm(C) and mef(A/E) respectively: ERMADP1, CTGCAACGA-AACAGCT and MEFA/EA1, TTCATACCCCAGCACTCAATG-CGGT. All donor probes were 3 0 end-labelled with fluorescein. Acceptor probes specific for erm(A), erm(B), erm(C) and erm(TR) were 5 0 end-labelled with LC Red 640; for mef (A/E), LC Red 705. Each acceptor probe had a 3 0-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 MgCl 2 , 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 eryth-romycin-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 the MLS B phenotype. All strains with the M-phenotype contained mef(A/E) only, while among those with the MLS B 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 MLS B phenotype were negative for erythromycin resistance genes. No isolate carried the …