The coagulase BIX 1294 mouse plasma test (Remel, Lenexa, KS, USA) was performed on organisms that exhibited typical staphylococcal colony morphology, to allow for discrimination of S. aureus from CoNS. Susceptibility testing for methicillin resistance and other antibiotic resistance phenotypes was carried out by the Kirby-Bauer methods [44]. MIC of methicillin was determined by E-test kits (AB Biodisk, Solna, Sweden). The results were categorized according to CLSI standards. Reference strains used as

controls were S. aureus (ATCC 33591), S. aureus (ATCC 25923), and S. epidermidis (ATCC 12228) (Table 1). GDC0449 primer design for pentaplex PCR assay The 16S rRNA of Staphylococcus genus, and gene sequences for femA, mecA and lukS of S. aureus were obtained from GenBank [45], for DNA sequence alignment and primer design. The ClustalW program in Vector NTI version 9.0 software (Invitrogen,

Carlsbad, CA, USA) was used to align the DNA sequences. The conserved and non-conserved regions of the DNA sequence alignments were visualized using GeneDoc software [46]. Based on the conserved regions of the alignment, specific primer pairs were designed to amplify the Staphylococcus genus. Specific primers of S. aureus species were designed based on the non-conserved regions of femA gene sequences. Methicillin-resistance specific primers were CX 5461 designed based on the conserved regions of mecA DNA sequences. For the PVL-encoding gene, specific primers were designed based on lukS gene. The five primer pairs (Research Biolabs, KL, Malaysia) were designed in such a way that the PCR products ranged from 151 to 759 bp. The specificity of the designed primers was checked using BLAST, which is available at the GenBank website [47]. The

primer sequences for the five genes and expected PCR product sizes are shown in Table 2. A primer pair based on hemM gene was designed (759 bp) and was used as an internal control (Table 2). Table 2 Sequences of primers Protein kinase N1 used for the pentaplex PCR. Gene Primer Name 5′———————————3′ Gen Bank accession number Product size Internal IC-F AGCAGCGTCCATTGTGAGA AF227752 759 bp control hem M IC-R ATTCTCAGATATGTGTGG     16S rRNA 16S rRNA-F GCAAGCGTTATCCGGATTT D83356 597 bp   16S rRNA-R CTTAATGATGGCAACTAAGC     fem A femA-F CGATCCATATTTACCATATCA CP000255 450 bp   femA-R ATCACGCTCTTCGTTTAGTT     mec A mecA-F ACGAGTAGATGCTCAATATAA NC_003923M 293 bp   mecA-R CTTAGTTCTTTAGCGATTGC     luk S lukS-F CAGGAGGTAATGGTTCATTT AB186917 151 bp   lukS-R ATGTCCAGACATTTTACCTAA     Pentaplex PCR assay DNA-contamination is a major problem encountered in the routine use of the PCR; we followed all contamination prevention measures in the PCR daily work to avoid pre and post-PCR contamination [48]. The monoplex PCR for each gene and the pentaplex PCR assay were standardized using genomic DNA extracted from reference Staphylococcus spp. A mixture of DNAs from two reference strains, namely S.