Direct detection of mecA, blaSHV , blaCTX-M , blaTEM and blaOXA genes from positive blood culture bottles by multiplex-touchdown PCR assay.

ABSTRACT

Methicillin-resistant staphylococci (MRS) and ESBL(Extended-Spectrum ß-Lactamase)-producing bacteria are the most important resistant pathogens in sepsis. In this study, a new multiplex-touchdown PCR method (MT-PCR) was developed to detect rapidly and simultaneously the presence of mecA, blaSHV , blaCTX-M , blaTEM and blaOXA genes from positive blood culture bottles. The technique showed a sensitivity of 103  CFU ml-1 for mecA detection and of 102  CFU ml-1 for other genes, and 100% specificity in the detection of all genes. All genes were detected in the spiked blood culture bottles artificially contaminated with reference strains. Three methicillin-resistant S. aureus (MRSA), two methicillin-resistant S. epidermidis (MRSE) and 32 ESBL-producing bacteria, were isolated from the clinical blood culture specimens in 48 h by standard microbiological procedures. The corresponding genes were detected directly in the three MRSA, two MRSE and 29 ESBL-producing bacteria from the clinical blood culture specimens in 4 h by MT-PCR assay. None of the blaSHV , blaCTX-M , blaTEM and blaOXA genes were detected in three other bottles with ESBL-producing bacteria because of other ESBL genotypes in the pathogens. Likewise, all bottles proven negative by culture remained negative by PCR. The proposed method was rapid, sensitive and specific, and was able to directly detect the genes of MRS and ESBL-producing bacteria from the blood culture bottles. SIGNIFICANCE AND IMPACT OF THE STUDY Many studies on the development of PCR for the detection of resistance genes have already been published, including multiplex PCR methods. However, cross-amplification reactions can be a major concern in multiplex PCR methods. In this study, we developed a highly sensitive and specific multiplex-touchdown PCR assay for simultaneous detection of mecA, blaSHV , blaCTX-M , blaTEM and blaOXA genes from positive blood culture bottles, cross-amplification was absent and false-positive results were not obtained.