Evaluation of a 10 µg cefoxitin disc for the detection of methicillin resistance in Staphylococcus aureus by BSAC methodology

Sir, The detection of methicillin resistance in Staphylococcus aureus has always presented problems for diagnostic laboratories because test conditions have a marked effect on the expression of resistance. Detection of the mecA gene or PBP2a are considered the reference methods, but are impractical for routine use and phenotypic methods have therefore been used. A recent publication has shown the value of using cefoxitin as an indicator of resistance. The aim of this study was to evaluate a 10 mg cefoxitin disc by the BSAC standardized disc methodology. Two hundred consecutive clinical isolates (repeat isolates from the same patient were excluded) of S. aureus, were disc tested in each of the five centres. Briefly, the method used was Iso-Sensitest agar (ISA; Oxoid, Basingstoke, UK) poured to a depth of 4 mm (–0.5 mm), an inoculum equivalent to semi-confluent growth, a 10 mg cefoxitin disc and incubation at 35 C (–1 C) for 18–20 h. Obvious zones of inhibition were measured using a ruler or callipers, but zones were examined carefully in good light to detect colonies within the zone of inhibition, in which case the organism was considered to be resistant. Multiplex PCR for the amplification of mecA and nuc was carried out using primers and conditions as previously described. Template DNA was prepared by boiling bacterial colonies in water. Figure 1 shows the zone diameter distribution for the organisms tested, all of which were nuc-positive. Of the isolates studied, 328 were mecA-positive and 224 (68%) of these gave no zone of inhibition. The zones of inhibition for the remaining mecA-positive isolates (104, 32%) ranged from 7–19 mm. Zones of inhibition for the methicillin-susceptible population ranged from 21–34 mm. A susceptible zone diameter breakpoint of ‡22 mm was chosen to avoid interpreting mecA-positive isolates at the tail end of the resistant zone distribution as falsely susceptible. Using this criterion, all mecA-positive isolates were correctly categorized as resistant, but two (0.2%) mecA-negative organisms were incorrectly categorized as resistant. Resistant control strain NCTC 12493 and susceptible control NCTC 6571 gave zones of 6 mm (no zone), and a range of 24–30 mm, respectively. The BSAC currently recommends using Columbia agar supplemented with 2% salt and incubation at 30 C for testing methicillin against both S. aureus and coagulase-negative staphylococci (CoNS). Although a reliable method of testing, this has the disadvantages that the medium and incubation temperatures differ from those used for testing other anti-staphylococcal agents, that strains sensitive to salt appear falsely susceptible and that some hyperproducers of b-lactamase appear falsely resistant and require latex or PCR tests for confirmation of resistance. The method described above using cefoxitin as the indicator antibiotic circumvents many of these disadvantages in particular that hyper-producers of b-lactamase appear susceptible. One important feature of the method is that the incubation temperature must not exceed 36 C. Experiments have shown that there is unacceptable merging of the resistant and susceptible populations when tests are incubated at 37 C (data not shown). Although presently not recommended for