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Journal of Clinical Microbiology, July 2003, p. 3246-3251, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3246-3251.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus

Pascal Lapierre,1,2 Ann Huletsky,1,2 Véronique Fortin,1 François J. Picard,1 Paul H. Roy,1,3 Marc Ouellette,1,2 and Michel G. Bergeron1,2*

Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL),1 Division de Microbiologie, Faculté de Médecine,2 Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Sainte-Foy, Québec, Canada3

Received 29 January 2003/ Returned for modification 17 March 2003/ Accepted 28 April 2003

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.

* Corresponding author. Mailing address: Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL), 2705 boul. Laurier, Sainte-Foy, Québec, G1V 4G2, Canada. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: michel.g.bergeron{at}crchul.ulaval.ca.

Journal of Clinical Microbiology, July 2003, p. 3246-3251, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3246-3251.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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