The first line of choice for the treatment of infections caused by Methicillin-Resistant Staphylococcus aureus is the antibiotic vancomycin. MRSA strains which do not respond to treatment with vancomycin can be characterized as being either resistant (VRSA) or intermediate (VISA). VISA strains present cell wall thickening as a result of a string of mutations in possibly different sets of genes, leading to vancomycin resistance. Furthermore, some strains present a heterogeneous mode of resistance (hVISA) in which sub-populations within a colony present a minimal inhibitory concentration (MIC) of vancomycin higher than that of the median and modal MIC of the entire population. These sub-populations with higher than median MICs of vancomycin may present mutations in genes which would not be detected by sequencing of DNA extracted from a cultured mass of cells because the majority wild-type polymorph would dominate the population. To circumvent this predicament, we propose to use a single-cell approach in order to identify which of the mutated genes attributed to the VISA phenotype are already found in all hVISA populations and which are present in only higher MIC subpopulations. Genomic DNA extracted from single hVISA cells isolated by laser microdissection will be amplified by MDA using phi29 DNA polymerase and sequenced using the SOLiD platform. The same will be carried out for VISA cells and vancomycin susceptible S. aureus (VSSA) cells. Alignment of all genome scaffolds obtained will permit the identification of coding sequence polymorphisms within hVISA populations and between VISA, hVISA and VSSA.
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