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 plusTaqI 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.

Genetic stability of strains preserved on LENTICULE discs and by freeze-drying: a comparison using fluorescent amplified fragment length polymorphism analysis

“…and Salmonella spp. and endonucleases EcoRI and MseI for Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes as described previously (Desai et al, 1998;Arnold et al, 1999;Grady et al, 1999;Desai et al, 2001;Lawson et al, 2004). FAFLP reactions were carried out in duplicates for each strain from the same DNA preparation.…”

“…The reverse primer had one or two selective bases at the 3 0 end ( Table 2). For each of the bacterial genera in this study, previously standardized endonucleases and selective primers were used for the FAFLP reactions (Desai et al, 1998;Arnold et al, 1999;Grady et al, 1999;Desai et al, 2001;Lawson et al, 2004). FAFLP products were separated on an ABI 377 automated DNA sequencer (Applied Biosystems, Warrington, UK), as described previously (Desai et al, 2001).…”

Fluorescent amplified-fragment length polymorphism analysis of the MRSA epidemic

“…We have previously demonstrated the use of FAFLP analysis to identify and to subtype isolates of phage type EMRSA-15 [8]. In this study, we have demonstrated how FAFLP analysis can be used to identify the full range, and to characterise the putative genetic interrelationships of, EMRSA phage types.…”

“…Endonucleases EcoRI and MseI were used to digest approximately 500 ng genomic DNA from each isolate, and restriction fragments were ligated to double-stranded adaptors as previously described [8]. PCR was then performed in a ¢nal volume of 25 Wl using 5 pmol of MseI adaptor-speci¢c primer (MseI+C) and 2 pmol of the EcoRI adaptor-speci¢c primer (EcoRI+0) labelled with 5-carboxy£uorescein.…”

“…Reproducible DNA ¢ngerprinting of EMRSA-15 isolates was accomplished by £uorescent ampli¢ed-fragment length polymorphism (FAFLP) [8], a PCR-based technique which ampli¢es and labels a selected subset of small DNA restriction fragments distributed throughout the genome. These are separated and sized on an automated DNA sequencer, yielding a reproducible clone-speci¢c DNA pro¢le.…”