Abstract
Recent reports have noted a discernible increase in the number of community- associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes the Panton-Valentine Leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis (PFGE) type USA300, multi-locus sequence type 8 (ST8), and carries the methicillin resistance cassette SCCmec type IV. At present, molecular characterization of MRS A such as USA300 can be time consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCC mec element in 44 USA300 MRSA isolates and identified a signature ‘AT repeat’ sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3' end of the Jl-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of >6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had >6 AT repeats but were PVL negative). Using a locked nucleic acid (LNA) primer specific for >6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting the method has promise as a means of rapidly identifying these problem isolates.