Detection of ciprofloxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single-strand conformation polymorphism and direct DNA sequencing

Abstract

A total of 27 strains of Campylobacter jejuni (24 clinical strains and three laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis with silver stain. Direct DNA sequencing of the polymerase chain reaction (PCR)-amplified DNA fragments confirmed the results obtained by non-RI SSCP analysis and revealed that in clinical strains high-level quinolone resistance [minimal inhibitory concentration (MIC) to ciprofloxacin > or = 16 micrograms/ml] was closely associated with one type of single-point mutation at codon 86 (Thr-Ile). Two strains with MICs of 8 and 1 microgram/ml showed point mutations at codons 86 and 70, respectively. Furthermore, transitions at codon 119 of the gyrA QRDR were identified in 17 strains. Six types of bands were separated in a single electrophoretic step with silver stain within 2 hours after PCR amplification of the gyrA QRDR as follows: type I associated to mutation at codon 70 (Ala-Thr), type II to mutation at codon 90 (Asp-Asn), type III to variant with transition at 119, type IV to wild-type, type V to mutation at codon 86 (Thr-Ile), and type VI to mutation at codon 86 (Thr-Ile) and transition at codon 119. Using four DNA extracts from Cambylobacter coli organisms as templates for amplification of the gyrA QRDR, no PCR products were obtained. Non-RI SSCP was proved to be a simple, rapid, and useful screening method for detecting gyrA mutations associated with ciprofloxacin resistance in C. jejuni.