Detection of epidemic USA300 community-associated methicillin-resistant Staphylococcus aureus strains by use of a single allele-specific PCR assay targeting a novel polymorphism of Staphylococcus aureus pbp3

Abstract

In recent years, the dramatic increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections has become a significant health care challenge. Early detection of CA-MRSA is important because of its increased virulence associated with the arginine catabolic mobile element (ACME), Panton-Valentine leukocidin (PVL), and other toxins that may contribute to disease severity. In particular, the USA300 epidemic clone has emerged and now represents the cause of as much as 98% of CA-MRSA skin and soft tissue infections in the United States. Current diagnostic assays used to identify CA-MRSA strains are based on complex multiplex PCRs targeting the staphylococcal cassette chromosome mec (SCCmec) DNA junction, a multitude of genes, and noncoding DNA fragments or on a number of lengthy sequence-typing methods. Here, two nucleotide polymorphisms, G88A and G2047A, that were found to be in strict linkage disequilibrium in the S. aureus penicillin-binding protein 3 (pbp3) gene were also found to be highly associated with the USA300 clone of CA-MRSA. Clinical isolates that contained this pbp3 allele were also positive for the presence of SCCmec type IV, the ACME, and the PVL toxin gene and matched the t008 or t121 molecular spa types, which are associated specifically with the USA300 CA-MRSA clone. A single allele-specific PCR targeting the G88A polymorphism was developed and was found to be 100% sensitive and specific for the detection of USA300 CA-MRSA and 91.5% sensitive and 100% specific for the detection of all CA-MRSA isolates in this study.

Fig 1

Allele-specific PCRs to detect G88A and G88 pbp3 alleles in S. aureus. Allele-specific PCR assays that detect the USA300 G88A (A) and non-USA300 G88 (B) pbp3 sequences were performed, and gel electrophoresis on a 1% agarose gel was used to detect amplification for the following bacterial strains: lanes 1 and 14, Affymetrix 1-kb Plus ladder; lane 2, HA-MRSA, SCCmecI, BAA-38; lane 3, HA-MRSA, SCCmecII, 0158p; lane 4, HA-MRSA, SCCmecIII, BAA-39; lane 5, CA-MRSA, SCCmecIV, BAA-1556; lane 6, Streptococcus agalactiae A909; lane 7, Streptococcus agalactiae NEM316; lane 8, Streptococcus agalactiae O90R; lane 9, methicillin-susceptible S. aureus ATCC 29213; lane 10, Streptococcus pyogenes ATCC 19615; lane 11, Escherichia coli ATCC 11303; lane 12, Staphylococcus epidermidis ATCC 12228; lane 13, no-template control.