1 The BSAC Standardized Method Development Centre, City Hospital NHS Trust, Birmingham, UK; 2 Addenbrookes Hospital, Cambridge, UK; 3 Freeman Hospital, Newcastle, UK; 4 St Thomas's Hospital, London, UK; 5 Glasgow Royal Infirmary, Glasgow, Scotland, UK
Keywords: S. aureus , staphylococci , mecA , MRSA , methicillin
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,1 but are impractical for routine use and phenotypic methods have therefore been used.2 A recent publication has shown the value of using cefoxitin as an indicator of resistance.3 The aim of this study was to evaluate a 10 µg 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 µg cefoxitin disc and incubation at 35°C (±1°C) for 1820 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.4 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 719 mm. Zones of inhibition for the methicillin-susceptible population ranged from 2134 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 2430 mm, respectively.
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References
1. Chambers HF. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microb Rev 1997; 10: 78191.[Abstract]
2. Brown DFJ. Detection of methicillin/oxacillin resistance in staphylococci. J Antimicrob Chemother 2001; 48 Suppl S1: 6570.
3.
Skov R, Smyth R, Larsen AR et al. Evaluation of cefoxitin 5 and 10 µg discs for the detection of methicillin resistance in staphylococci. J Antimicrob Chemother 2005; 55: 15761.
4.
Louie L, Matsumura SO, Choi E et al. Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus. J Clin Microb 2000; 38: 21703.
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