Probing Staphylococcus aureus diversity through a novel fluorescent amplified fragment length polymorphism assay
Abstract number: P1701
Desai D., Ellington M.J., Arnold C., Desai M.
Objectives: FAFLP is a robust, high-resolution, PCR-based methodology used for the analysis of genetic diversity within bacterial genomes, without prior knowledge of genome sequence. It combines the principle of restriction fragment length polymorphism with selective PCR amplification of subsets of fragments generated by restriction endonucleases. Here we sought to evaluate and establish a novel fluorescent amplified fragment length polymorphism (FAFLP) assay to assess the genetic diversity within Staphylococcus aureus including major methicillin resistant clones.
Methods: A total of 53 S. aureus isolates, from 11 major multilocus sequence types, including 15 isolates of the Epidemic MRSA-15 clone were used for comparative analysis using FAFLP. Genomic DNA was restricted using the endonuclease combination Csp6I and BglII. A combination of four one-base selective fluorescently-labelled BglII primers (selective base +A, T, G & C) with a non-selective Csp6I primer (+0) were evaluated for the development of a multiplex assay. FAFLP products were analysed using the ABI 3730 sequencer and GeneMapper software (v.4.0) for data analysis. Clusters from the generated profiles were compared with staphylococcal cassette chromosome mec (SCCmec) and multilocus sequence typing (MLST) data.
Results: FAFLP generated 240 to 290 amplified fragments ranging in size from 60600bp: these fragments constituted an FAFLP profile. Thirty-five unique profiles were exhibited amongst the 53 isolates. The multiplex assay differentiated EMRSA-15 (ST22) from EMRSA-1, 3 and 16 (ST36) isolates as well as isolates that were ST30 and ST5. Genetic heterogeneity within the EMRSA-15's was also revealed. Comparison of FAFLP data with SCCmec data showed that FAFLP data differentiated isolates harbouring type I and II as well as IV; it also discriminated between isolates harbouring different sub-types of SCCmecIV.
Conclusions: The established multiplex FAFLP assay increased the discriminatory power by approximately four-fold compared to a singleplex, as the number of data points spanning the genome increased. This approach provides a cost-effective and high-throughput method applicable to outbreak analysis through to global epidemiological studies and has the potential for use in hospital infection control. The identification of differential fragments between different lineages of MRSA will be incorporated into a PCR-based diagnostic assay.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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