Rapid detection of methicillin-resistant staphylococci using polymerase chain reaction

AbstractObjective: A comparison was made of polymerase chain reaction (PCR), Southern blot, and routine susceptibility testing for determination of methicillin resistance in clinical isolates of staphylococci.Methods: A PCR-based test and Southern hybridization were used to detect the mecA gene in staphylococci. A broth double-dilution method was used to determine the minimum inhibitory concentration (MIC) for oxacillin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then fluorography were used to test the affinities of penicillin-binding proteins of Staphylococcus aureus ATCC 25923 for oxacillin.Results: The presence or absence of a meth icillin-resistant gene (mecA) in 228 clinical isolates of staphylococci was examined by PCR and Southern blot analyses. The results were compared in relation to those of the MIC assay to oxacillin. A total of 57 of 58 oxacillin-resistant S. aureus strains were mecA-positive, whereas 3 of 126 oxacillin-susceptible strains were mecA-positive. For 21 oxacillin-resistant coagulase-negative staphylococci, 100% of the strains were mecA-positive. but 9 of 23 oxacillin-susceptible coagulase-negative staphylococci were mecA-positive. The PCR test identified methicillin-resistant staphylococci in less than 3 hours, using as few as 300 cells or 3 pg crude extract DNA as the PCR template. The results of the PCR test correlated well with those of DNA hybridization. Dot blot hybridization could detect as little as 4 ng DNA. Five penicillin-binding proteins were identified in S. aureus.Conclusions: Identification of methicillin-resistant staphylococci by PCR (confirmed by DNA hybridization) offers a specific, sensitive, and rapid alternative to traditional susceptibility testing and serves as a guide for the rational treatment of infections caused by staphylococci