Figure 1 Pentaplex PCR assay profile with reference strains. M, 100-bp marker; lane 1, negative control; lane 2, Staphylococcal positive control; lane 3, ATCC 33591 (16S rRNA, femA-S. aureus, mecA); lane 4, ATCC 33592 (16S

PARP inhibitor trial rRNA, femA-S. aureus, mecA); lane 5, ATCC 43300 (16S rRNA, femA-S. aureus, mecA); lane 6, ATCC 25923 (16S rRNA, femA-S. aureus, lukS); lane 7, ATCC 49775 (16S rRNA, femA-S. aureus, lukS); lane 8, ATCC 51153 (16S rRNA, femA-S. aureus); lane 9, CoNS methicillin-resistant clinical isolate (16S rRNA, mecA); lane 10, ATCC 14990 (16S rRNA); lane 11, ATCC 29970 (16S rRNA); lane 12, ATCC 13518 (16S rRNA); M, 100-bp marker Table 1 Bacterial species and strains used in this study and results of pentaplex PCR. No. Reference strains 16S rRNAa femA mecAb lukS Internal control 1. S. aureus (ATCC 33591) + + + – + 2. S. aureus (ATCC 33592) PS-341 research buy + + + – + 3. S. aureus (ATCC 43300) + + + – + 4. S. aureus (ATCC 25923)d + + – + + 5. S. aureus (ATCC 49775) + + – + + 6. S. aureus (ATCC 51153)e + + – - + 7. S. epidermidis (ATCC 14990) + – - – + 8. Staphylococcus haemolyticus (ATCC 29970) + – - – + 9. Staphylococcus saprophyticus (ATCC 13518)d + – - – + 10. CoNS methicillin-resistante + – + – + 11. Streptococcus spp. Group A (ATCC 19615)e – - – - + 12. Streptococcus spp. Group B (ATCC 12401)e – - – - + 13. Streptococcus spp. Group

Ge – - – - + 14.Streptococcus spp. Group Fe – - – - + 15. Bacillus subtilis (ATCC 6633)e – - – - + 16.Listeria monocytogenes (ATCC 7644)e – - – - + 17. Enterococcus faecium LMG 16192c – - – - + 18. Enterococcus faecalis (ATCC 29212)e – - – - + 19. Corynebacterium sppe – - – - + 20. Escherichia coli (EHEC)e – - – - + 21. E. coli (EPEC)e – - – - + 22.E. coli (ETEC)e – - – - + 23. Klebsiella pneumoniae (ATCC 10031)e – - – - + 24. Shigella sonnei (ATCC 25931)e – - – - + 25. Shigella flexneri (ATCC 12022)e – - – - + 26.

Shigella boydii (ATCC 9207)e – - – - + 27.Proteus mirabilis (ATCC 29245)e – - – - + 28. Salmonella typhi e – - – - + 29. Pseudomonas aeruginosa (ATCC 27853)e – - – - + 30.Yersinia enterocolitica (ATCC 23715)e – - – - + 31. Vibrio cholerae (O1 classical)e – - – - + 32. Citrobacter freundii (ATCC 8090)e – - – - + 33.Gardnerella sppe – - – - + 34.Candida albicans (ATCC 10231)e Ribonucleotide reductase – - – - + a Staphylococcus genus b methicillin-resistant genotype c Reference strains from Belgian Co-ordinated Collections of Micro-organisms (BCCM), Ghent, Belgium d Obtained from Institute for Medical Research, Malaysia e Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia. Upon completion of the standardization of the methicillin-resistant pentaplex PCR assay with reference strains, the assay was validated with 230 clinical isolates. Among these, all had 16S rRNA, 82 contained mecA, 178 had femA and none had lukS genes by pentaplex PCR.