Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus
Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus
复制标题
实时 PCR 检测与金黄色葡萄球菌 grlA 突变相关的氟喹诺酮耐药性
DOI：
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发表时间：
2003
影响因子：
9.4
通讯作者：
M. Bergeron
中科院分区：
文献类型：
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作者：
P. Lapierre;A. Huletsky;Véronique Fortin;F. Picard;P. H. Roy;M. Ouellette;M. Bergeron
摘要金黄色葡萄球菌临床分离株对氟喹诺酮类药物的耐药性已成为临床问题。因此，一种快速鉴定金黄色葡萄球菌及其对氟喹诺酮类药物敏感性的方法可以为临床医生在医疗保健环境中正确使用这些抗菌剂提供有用的工具。在这项研究中，我们建立了一种快速实时定量PCR方法来检测金黄色葡萄球菌以及编码DNA拓扑异构酶IV的grlA基因的Ser-80和Glu-84密码子突变，这些突变与氟喹诺酮类药物敏感性降低有关。该方法的检测下限为每次反应10个基因组拷贝。26例Non-S患者的DNA检测结果均为阴性。检测了金黄色葡萄球菌的细菌种类。对85株对氟喹诺酮类药物耐药的金黄色葡萄球菌进行了聚合酶链式反应检测。与常规培养方法相比，该聚合酶链式反应检测方法正确识别了100%的金黄色葡萄球菌分离株。环丙沙星、左氧氟沙星、加替沙星MIC值与聚合酶链式反应结果的符合率为98.8%。鉴定金黄色葡萄球菌和测定其对氟喹诺酮类药物敏感性所需的总时间约为45min，包括DNA提取。这种新的快速聚合酶链式反应方法是检测金黄色葡萄球菌及其对氟喹诺酮类药物敏感性的有效方法。
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.