Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates

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 Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, 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 SCCmec 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 J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of > or =6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had > or =6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for > or =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 that the method has promise as a means of rapidly identifying USA300 isolates.

FIG. 1.

Diagram showing the location of the multiplex PCR primers with reference to S. aureus strain COL (GenBank accession number CP000046). The black bar is the SCCmec resistance element. DR is the 3′ direct repeat region flanking the SCCmec element. Gray indicates the 3.3-kb chromosomal sequence 3′ to SCCmec, including SACOL0058 and the AT repeat sequence. PCR amplification using primers lnaAT (nucleotides 69490 to 69511) and ATreg-2 (nucleotides 70831 to 70855) indicates the presence of ≥6 AT repeats. SACOL0058 is detected using ATreg-1 (nucleotides 69954 to 69977) and ATreg-2. Amplification of the PVL genes using primers designed by Lina et al. (21) occurs at a separate location within the chromosome. The lower black box demonstrates hybridization of the lnaAT primer with the AT repeat sequence as it would occur in a USA300 MRSA strain.

FIG. 2.

(A) LNA primer identification of USA300 MRSA strains. Lane 1, 1-kb DNA ladder (Invitrogen, Carlsbad, CA); lanes 2 and 3, USA300:ST8 strains CRG-1130 and CRG-1128, containing eight and six AT repeats, respectively; lane 4, strain CRG-930 (USA500:ST250) containing five AT repeats; and lane 5, water control. (B) Multiplex PCR assay differentiates USA300 strains from other MRSA strains. Lane 1, 1-kb DNA ladder (Invitrogen); lanes 2 and 3, USA300:ST8 strains CRG-1130 and CRG-1128, containing eight and six AT repeats, respectively, as well as SACOL0058 and PVL; lane 4, strain CRG-1112 (mecA negative) containing five AT repeats; lane 5, strain CRG-1231 (USA500:ST8) containing six AT repeats and SACOL0058; lane 6, strain MW2 (CC1:ST1) (SACOL0058 negative); and lane 7, water control.