A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii

ABSTRACT

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.