Genotyping of epidemic methicillin-resistant Staphylococcus aureus phage type 15 isolates by fluorescent amplified-fragment length polymorphism analysis

Abstract

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for its ability to identify and subtype isolates of an epidemic methicillin-resistant phage type of Staphylococcus aureus, EMRSA-15. These isolates were also characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) of the coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP, DNA was double digested with restriction enzymes ApaI plus TaqI or EcoRI plus MseI. Site-specific adaptors were ligated to one or the other set of restriction fragments, and PCR amplification was carried out with adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software generated FAFLP profiles for all the isolates. The presence or absence of fragments was scored, similarity coefficients were calculated, and UPGMA (unweighted pair group method using arithmatic averages) cluster analysis was performed. Either enzyme-primer combination readily differentiated EMRSA-15 from other methicillin-resistant S. aureus (MRSA) isolates and also revealed heterogeneity within the phage type. The discriminatory power of FAFLP was high. By combining both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates. PFGE could discriminate well between isolates but was not as reproducible as FAFLP. All S. aureus and MRSA isolates in this study were typeable by FAFLP, which was easy to perform, robust, and reproducible, with evident potential to subtype MRSA for purposes of hospital infection control.