title: Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay.
abstract: In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.
authors.affiliation: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Negar Shafiei
authors.initials: NS
authors.lastname: Sabet
authors.forename: Geetha
authors.initials: G
authors.lastname: Subramaniam
authors.forename: Parasakthi
authors.initials: P
authors.lastname: Navaratnam
authors.forename: Shamala Devi
authors.initials: SD
authors.lastname: Sekaran
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008712:Methicillin / Q000494:pharmacology*; D016106:Methicillin Resistance / Q000235:genetics; D008826:Microbial Sensitivity Tests; D046915:Penicillin-Binding Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000617:Aminoglycosides; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C096686:FemA protein, Bacteria; D005456:Fluorescent Dyes; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus; D008712:Methicillin