title: New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.
abstract: Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.
authors.affiliation: Center for Antimicrobial Resistance, Calgary Health Region-Calgary Laboratory Services-University of Calgary, Alberta, Canada T2L 2K8. kunyan.zhang@cls.ab.ca
authors.forename: Kunyan
authors.initials: K
authors.lastname: Zhang
authors.forename: Jennifer
authors.initials: J
authors.lastname: Sparling
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Chow
authors.forename: Sameer
authors.initials: S
authors.lastname: Elsayed
authors.forename: Zafar
authors.initials: Z
authors.lastname: Hussain
authors.forename: Deirdre L
authors.initials: DL
authors.lastname: Church
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Gregson
authors.forename: Thomas
authors.initials: T
authors.lastname: Louie
authors.forename: John M
authors.initials: JM
authors.lastname: Conly
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D003030:Coagulase / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006801:Humans; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance; D008826:Microbial Sensitivity Tests; D016712:Mupirocin / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013210:Staphylococcus / Q000145:classification* / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013211:Staphylococcus aureus / Q000145:classification* / Q000187:drug effects / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003030:Coagulase; D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D016712:Mupirocin; D008712:Methicillin