mecA Locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone

Abstract

An emerging public health phenomenon is the increasing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of mecA in combination with a single nucleotide polymorphism (SNP) defined by the S. aureus multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of mecA were determined. The CA-MRSA isolates were found to contain a truncated mecA downstream region consisting of mecA-HVR-IS431 mec-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different mecA downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the S. aureus MLST database, and the arcC 272G polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated mecA downstream region in combination with the interrogation of arcC position 272 provided an unambiguous identification of WSPP isolates.

FIG. 1.

Binding sites for primers used to determine mecA downstream arrangements. The scale is in kilobases.

FIG. 2.

Downstream mecA arrangement of S. aureus ATCC 49476 (arrangement J). The scale is shown in kilobases.

FIG. 3.

Downstream mecA arrangements of hospital-acquired isolates. The scale is shown in kilobases.

FIG. 4.

(A) Positions of the three primer pairs used to discriminate the downstream mecA arrangements associated with community-acquired isolates. The scale is shown in kilobases. (B) Amplification products from the primer set specific for arrangement I. Lanes 1 to 3, isolate 1; lanes 4 to 6, isolate 7; lanes 7 to 9, isolate 13; lanes 10 to 12, isolate 31. The isolates shown in lanes 1 to 9 were community acquired and show the amplification of the three fragments at the expected sizes. Lanes 10 to 12 show results for a hospital-acquired isolate that does not have arrangement I: a truncated dcs is present (arrangement B). MW, molecular weight marker (Roche X).