Development of multiplex polymerase chain reaction assay for rapid detection of <i>Staphylococcus aureus</i> and selected antibiotic resistance genes in bovine mastitic milk samples

Abstract

To improve the diagnosis of Staphylococcus aureus mastitis and its potential antibiotic resistance in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for simultaneous species identification and detection of penicillin, erythromycin, and tetracycline resistance genes. The assay was first verified using 85 clinical isolates of S. aureus, and its effectiveness was evaluated by testing 99 mastitic milk samples. The multiplex PCR assay had high detection sensitivity in samples from both groups (100% agreement with single-gene PCR), with detection limits of 10(0)-10(3) colony-forming units (cfu)/ml. The detection limits of the current assay for nuc, blaZ, erm B, erm C, tet K, and tet M genes were 10(1), 10(1), 10(2), 10(0), 10(2), and 10(3) cfu/ml, respectively. There was a good correlation between genotypic analysis by PCR and phenotypic determination using S. aureus species identification and susceptibility testing methods. High diagnostic sensitivities and specificities were shown for the target resistance genes (blaZ gene, 97.2% and 92.9%; ERM resistance genes, 100% and 95.6%; TET resistance genes, 100% and 96%). Results suggested that the current PCR assay could be used as an alternative diagnostic method in the routine diagnosis for rapid, sensitive, and specific detection of S. aureus and its associated antibiotic resistance genes in mastitic milk samples.