Evaluation of different methods to detect oxacillin resistance in Staphylococcus aureus and their clinical laboratory utility

Abstract

Methicillin-resistant Staphylococcus aureus strains (MRSA) are major human pathogens responsible for nosocomial and serious community-acquired infections worldwide. Methicillin resistance in S. aureus is primarily mediated by a modified penicillin-binding protein (PBP2a) encoded by the mecA gene, which is not present in susceptible strains [1–3]. Several additional gene products, such as those encoded by fem (factor essential for methicillin resistance) genes and auxiliary genes, can also contribute to MRSA resistance [1, 2, 4]. Rapid and accurate identification of MRSA is necessary for therapeutic and epidemiological purposes. Currently, several methods are available for detecting oxacillin resistance including phenotypic methods, such as disk diffusion, E-test and broth dilution tests, and genotypic methods that detect the mecA gene [1, 5, 6]. PCR amplification of the mecA gene has been used successfully and is presently considered the gold standard for detecting methicillin resistance in S. aureus [1]. Detection of oxacillin resistance using phenotypic methods is problematic because of variation in the degree of mecA gene expression in heterogeneous populations of MRSA; thus, routine oxacillin disk diffusion tests often fail to detect heterogeneous MRSA populations. The Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) has consequently recommended use of the cefoxitin disk diffusion test rather than the oxacillin disk diffusion test [7]. In the study presented here, the performance and utility of several different methods for detecting MRSA was evaluated. PCR for detecting the mecA gene was used as the gold standard assay against which we compared oxacillin and cefoxitin disk diffusion tests and the GenoType MRSA molecular genetic assay (Hain Lifescience, Nehren, Germany) for detecting MRSA. The objective was to determine whether these disk diffusion tests and GenoType MRSA could detect all MRSA as accurately as mecA PCR and to evaluate their suitability as routine methods for detecting oxacillin resistance in S. aureus in clinical microbiology laboratories. GenoType MRSA is a new DNA-based strip assay kit designed to detect oxacillin resistance in S. aureus. Its performance is based on amplification of a 71 bp fragment of the mecA gene and an 85 bp sequence highly specific for S. aureus and subsequent hybridization of the denatured amplicons to their complementary sequences coated on DNA strips. A total of 224 isolates of S. aureus collected from patients of Suleyman Demirel University Hospital were included in this study. The isolates were collected from different clinical specimens and at different times. All strains were identified using biochemical procedures. Reference strains included ATCC33591 (MRSA) and ATCC29213 (MSSA). Disk diffusion tests were performed for each of the 224 isolates using the following method recommended by the National Committee for Clinical Laboratory Standards [7]: 1 μg oxacillin disks (Oxoid, Basingstoke, UK) were incubated at 35°C and at 30°C on Mueller-Hinton agar (Difco, Detroit, MI, USA) with and without 2% NaCl, and 30 μg cefoxitin disks (Oxoid) were incubated at 35°C on Mueller-Hinton agar. After incubation, inhibition zone sizes for oxacillin and cefoxitin were noted and compared. For cefoxitin, isolates showing a zone of inhibition of ≤19 mm were considered Eur J Clin Microbiol Infect Dis (2006) 25:410–412 DOI 10.1007/s10096-006-0153-8