These three PVL negative clones harbor few additional resistance and virulence genes which paradoxically may account for their success. Methods Isolates The isolates studied are representative of the 83 CA-MRSA unique PFGE strains identified in WA from 1989 to 2010 (Figure 3). They include five strains isolated from indigenous inhabitants living

in remote WA rural communities in 1989 (WA5 WBG7583 [20]) and 1995 (WA1 WBG 8287, WA2 WB8366, WA3 WBG8378, and WA4 WBG8404 [42]); and 78 strains identified from 24,368 CA-MRSA referred to ACCESS Typing and Research between July 2003 and June 2010. Figure 3 Dendrogram of the 83 pulsed-field gel electrophoresis patterns of CA-MRSA isolated in PF-01367338 Western NCT-501 Australia. nuc and mecA S. aureus species and methicillin resistance was confirmed by the detection of nuc (thermostable extracellular buy FRAX597 nuclease) and mecA

(methicillin resistance) genes by PCR [43]. Susceptibility testing An antibiogram was performed by disk diffusion on Mueller-Hinton agar according to the Clinical and Laboratory Standards Institute (CLSI) recommendations [44]. A panel of eight antimicrobial drugs was tested: erythromycin (15 μg), tetracycline (30 μg), trimethoprim (5 μg), ciprofloxacin (5 μg), gentamicin (10 μg), rifampin (5 μg), fusidic acid (10 μg), and mupirocin (5 μg). CLSI interpretive criteria [45] were used for all drugs except fusidic acid [46] and mupirocin [47]. PVL PCR for the detection of PVL determinants was performed as previously described [48]. PFGE Electrophoresis of chromosomal DNA was performed as previously described [49], using a contour-clamped homogeneous electric field (CHEF) DR III system (Bio-Rad Laboratories Pty Ltd). Chromosomal patterns were examined visually, scanned tuclazepam with a Quantity One device (Bio-Rad Laboratories Pty Ltd), and digitally analyzed using FPQuest (Bio-Rad Laboratories

Pty Ltd). S. aureus strain NCTC 8325 was used as a reference strain. MLST and spa typing Chromosomal DNA for MLST and spa typing was prepared using a DNeasy tissue kit (Qiagen Pty Ltd). MLST was performed as previously described [50]. The sequences were submitted to http://​www.​mlst.​net/​ where an allelic profile was generated and an ST assigned. Clonal complex (CC) was determined using the eBURST V3 algorithm at the same website. Clones that diverged at no more than one of the seven MLST loci were considered to belong to the same CC. Double locus variants (dlvs) were included if the linking single locus variant (slv) was present in the MLST database. spa typing, a DNA sequenced-based analysis of the protein A gene variable region was performed as previously described [51] using the nomenclature as described on the Ridom website (http://​spa.​ridom.​de/​). SCCmec typing The strategy used for SCCmec typing was as previously described [32].