Rapid detection of methicillin-resistant staphylococci using polymerase chain reaction

Abstract

Objective: 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.