Comparison of Mismatch Amplification Mutation Assay PCR and PCR-Restriction Fragment Length Polymorphism for Detection of Major Mutations in gyrA and parC of Escherichia coli Associated with Fluoroquinolone Resistance.

Abstract

Fluoroquinolones are the drug of choice for most of the infections caused by Escherichia coli, and their indiscriminate use has resulted in increased selective pressure for antibiotic resistance. At present, sequencing is the only reliable and direct technique to detect mutations in the quinolone resistance determining region (QRDR). In this study, a rapid and reliable mismatch amplification mutation assay (MAMA) PCR to detect mutations in the QRDR was evaluated and compared to PCR-restriction fragment length polymorphism (PCR-RFLP). One hundred one clinical isolates of E. coli were subjected to MAMA-PCR and PCR-RFLP to detect QRDR mutations. Overall, 92 (91.08%) resistant isolates harbored a point mutation of S83L in gyrA. Double mutations in gyrA were also detected in 45 (44.55%) isolates. Similarly, 41 (40.59%) isolates possessed a point mutation at parC 80, and 25 (24.75%) isolates possessed a point mutation at parC 84. Additionally, MAMA-PCR-the first of its kind-was also standardized to detect mutations in regions gyrB 447 and parE 416, although no mutations were detected in these regions. The rapid and sensitive MAMA-PCR method evaluated in this study would be helpful in exploring the underlying mechanism of fluoroquinolone resistance to enhance control strategies.