Rapid Detection of Genomic Mutations in gyrA and parC Genes of Escherichia coli by Multiplex Allele Specific Polymerase Chain Reaction.

BACKGROUND: Fluoroquinolone (FR) resistant Escherichia coli infection has become a global problem. The FR resistance usually occurs mainly due to specific point of mutations within the quinolone resistance-determining regions (QRDRs) at the gyrA codon of Ser83 and Asp87 and the parC codon of Ser80 and Glu84. Here, we appraised type and frequency of the QRDR mutations in FR-resistant E. coli isolates, and developed multiplex allele specific PCR (MAS-PCR) for the detection of "hot spot" mutations. METHODS: A total of 111 ciprofloxacin-resistant E. coli from Ramathibodi Hospital in Bangkok, Thailand, were performed Minimum Inhibitory Concentration (MIC) by Etest® and investigated for gyrA and parC genes' mutations by MAS-PCR. Sensitivity and specificity of MAS-PCR were compared to the sequencing method's. RESULTS: Ninety-nine of 111 (89.19%) E. coli isolates had mutation at least one point in the QRDRs. Six usual amino acid substitutes were reported, including Ser83-Lue, Asp87-Asn, Asp87-Tyr, Ser80-Ile, Glu84-Gly, and Glu84-Val. MAS-PCR detected codons 83 and 87 in gyrA and codons 80 and 84 in parC mutations, yielding 96.97%, 100%, 100%, and 93.33% sensitivity, respectively, and 100%, 100%, 100%, and 98.48% specificity, respectively. CONCLUSION: MAS-PCR may be used for rapid detection of FR resistance in routine laboratory as well as in epidemiology study.