Abstract
The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.
Highlights
Staphylococcus aureus is considered as an important cause of a wide variety of diseases in humans such as: food poisoning, pneumonia, wound and nosocomial infections [1,2]
The altered penicillin-binding protein (PBP2a) is associated with methicillin resistance. This protein has a reduced affinity for blactam antibiotics [5,6], and is encoded by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec) [5]
CA-Methicillin-resistant S. aureus (MRSA) strains are more likely to encode the Panton– Valentine leukocidin (PVL) toxin, which is a pore-forming toxin considered as a virulence factor [7,8]
Summary
Staphylococcus aureus is considered as an important cause of a wide variety of diseases in humans such as: food poisoning, pneumonia, wound and nosocomial infections [1,2]. Methicillin-resistant S. aureus (MRSA) is an increasing cause of health care-associated (HA-MRSA) [1], community-associated (CA-MRSA) [2], and livestock-associated (LA-MRSA) infections worldwide [4]. The altered penicillin-binding protein (PBP2a) is associated with methicillin resistance. This protein has a reduced affinity for blactam antibiotics [5,6], and is encoded by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec) [5]. CA-MRSA strains are more likely to encode the Panton– Valentine leukocidin (PVL) toxin, which is a pore-forming toxin considered as a virulence factor [7,8]. The PVL toxin has been related to life-threatening CA-MRSA infections and deaths, primarily severe skin infections and tissue necrosis [9]
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