Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR

Abstract

Objectives:Emergence of methicillin resistant Staphylococcus aureus (MRSA) is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information.Methods:We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors.Results:We found 38.33% isolates as methicillin resistant. α -toxin, the major cytotoxic factor, was detected in 40% whereas β-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible.Conclusions:These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures.

Circadian protection against bacterial skin infection by epidermal CXCL14-mediated innate immunity

“…For the binding assay for RAW264.7 cells, cells in 24-well plates were treated with S. aureus DNA in the presence or absence of 300 nM CXCL14 at 4 °C for 1 h. The treated cells were lysed to isolate genomic DNA. The copy ratio of S. aureus DNA to mouse genome DNA was determined by qPCR using primer sets for S. aureus Nuc [used for copy number validation ( 53 )] and mouse rRNA ( SI Appendix , Table S1 ).…”