title: Real-time PCR assay for detection of fluoroquinolone resistance associated with grlA mutations in Staphylococcus aureus.
abstract: Resistance to fluoroquinolones among clinical isolates of Staphylococcus aureus has become a clinical problem. Therefore, a rapid method to identify S. aureus and its susceptibility to fluoroquinolones could provide clinicians with a useful tool for the appropriate use of these antimicrobial agents in the health care settings. In this study, we developed a rapid real-time PCR assay for the detection of S. aureus and mutations at codons Ser-80 and Glu-84 of the grlA gene encoding the DNA topoisomerase IV, which are associated with decreased susceptibility to fluoroquinolones. The detection limit of the assay was 10 genome copies per reaction. The PCR assay was negative with DNA from all 26 non-S. aureus bacterial species tested. A total of 85 S. aureus isolates with various levels of fluoroquinolone resistance was tested with the PCR assay. The PCR assay correctly identified 100% of the S. aureus isolates tested compared to conventional culture methods. The correlation between the MICs of ciprofloxacin, levofloxacin, and gatifloxacin and the PCR results was 98.8%. The total time required for the identification of S. aureus and determination of its susceptibility to fluoroquinolones was about 45 min, including DNA extraction. This new rapid PCR assay represents a powerful method for the detection of S. aureus and its susceptibility to fluoroquinolones.
authors.affiliation: Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL), Université Laval, Sainte-Foy, Québec, Canada.
authors.forename: Pascal
authors.initials: P
authors.lastname: Lapierre
authors.forename: Ann
authors.initials: A
authors.lastname: Huletsky
authors.forename: Véronique
authors.initials: V
authors.lastname: Fortin
authors.forename: François J
authors.initials: FJ
authors.lastname: Picard
authors.forename: Paul H
authors.initials: PH
authors.lastname: Roy
authors.forename: Marc
authors.initials: M
authors.lastname: Ouellette
authors.forename: Michel G
authors.initials: MG
authors.lastname: Bergeron
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D024841:Fluoroquinolones; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
chemical_list: D000890:Anti-Infective Agents; D024841:Fluoroquinolones; D027101:DNA Topoisomerase IV