Evaluation of a new 3-h hybridization method for detecting the mecA gene in Staphylococcus aureus and comparison with existing genotypic and phenotypic susceptibilty testing methods

Abstract

A new 3-h hybridization assay for detection of the staphylococcal mecA gene and the Staphylococcus aureus nuclease gene was evaluated by comparing the assay with existing genotypic and phenotypic methods. A total of 275 S. aureus strains were tested, including 257 epidemiologically unrelated strains (135 mecA-positive and 122 mecA-negative; collection I), and 18 strains with known borderline resistance to methicillin (collection II). Complete agreement was obtained for both collections when comparing the new assay with genotypic methods. We further evaluated a range of phenotypic susceptibility methods recommended in Europe and/or USA using the presence of the mecA gene as the defining standard. For collection I a high degree of agreement was found for both Etests (256 strains) and the oxacillin screen plate test (255 strains); the degree of agreement was lower for agar dilution methicillin (250 strains) and oxacillin 1 microg discs (239 strains). For the borderline strains a high degree of agreement was only obtained by the oxacillin screen plate test (17 of 18 strains). The other tests were less accurate, in the following order: agar dilution methicillin, Etest methicillin, Etest oxacillin and oxacillin discs with disagreement for four, five, nine and 13 strains, respectively. In conclusion, the new hybridization assay is a rapid and exact method for detecting the mecA gene and the S. aureus nuclease gene. This study confirms that phenotypic tests for methicillin resistance in S. aureus strains creates both false-susceptible and false-resistant results, especially for borderline resistant strains.

Epidemiology of European Community-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 80 Type IV Strains Isolated in Denmark from 1993 to 2004

“…The presence of mecA and nuc genes in all MRSA isolates was confirmed using an Evigene MRSA detection kit following the manufacturer's instructions (SSI, Copenhagen, Denmark) (39). Susceptibility tests for penicillin, cefoxitin, streptomycin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, fusidic acid, rifampin, and ciprofloxacin were performed using Neosensitabs (Rosco, Taastrup, Denmark) on Danish blood agar (SSI, Copenhagen, Denmark), using a semiconfluent (10 6 CFU/ml) inoculum and overnight incubation in ambient air at 35 to 36°C.…”

Methicillin resistance in <i>Staphylococcus aureus</i>: a review of the molecular epidemiology, clinical significance and laboratory detection methods

“…In the clinical laboratory, mecA gene can be detected using the three hours DNA hybridization probe. 17 This is highly sensitive and specific and correlates with phenotypic susceptibility testing and other genotyping methods. However, the polymerase chain reaction (PCR) is now the most frequently used genotypic …”

“…15,16 Molecular detection of mecA gene using DNA probe or its products PBP 2' by immunoblot assay and PCR based methods are now considered the 'gold' standard for MRSA confirmation. 17 Epidemiological typing methods to characterize MRSA strains involved in outbreaks have also moved from the use of bacteriophage typing, protein antigen electrophoresis profile and plasmid fingerprinting to pulse field gel electrophoresis (PFGE) of SmaI-digest of genomic DNA, ribotyping, multilocus sequence typing (MLST), PCR amplification of target genes such as staphylococcus protein A (spa) and coagulase (coa) genes, and more recently sequencing of these DNA. 18 From published literatures available, MRSA reporting in Nigeria and other parts of Africa appear very few.…”

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) in Blood with the EVIGENE MRSA Detection Kit

“…Nevertheless, there is a need for rapid diagnosis of MRSA bacteremia so that appropriate therapeutic decisions can be made. This study evaluated a commercial gene probe hybridization assay (EVIGENE MRSA Detection Kit; Statens Serum Institut, Copenhagen, Denmark) that targets staphylococcus-specific 16S rRNA, mecA, and nuc gene sequences, thereby differentiating coagulase-negative staphylococci and S. aureus and determining methicillin resistance (14).…”