Detection of ciprofloxacin resistance mutations inCampylobacter 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 ≥ 16 μg/ml] was closely associated with one type of single‐point mutation at codon 86 (Thr‐lle). Two strains with MICs of 8 and 1 μg/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‐lle), and type VI to mutation at codon 86 (Thr‐lle) 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. © 1996 Wiley‐Liss, Inc.

gyrA Polymorphism in Campylobacter jejuni : Detection of gyrA Mutations in 162 C. jejuni Isolates by Single-Strand Conformation Polymorphism and DNA Sequencing

“…A point mutation in the quinolone resistance-determining region (QRDR) of the gyrA gene at codon 86 (ACA to ATA), substituting isoleucine for threonine, is the most common cause of class-wide fluoroquinolone resistance among C. jejuni isolates (17,24,27). There have also been anecdotal reports of mutations leading to additional amino acid changes, as well as silent nucleotide mutations (1,24,27).…”

Acquisition of Quinolone Resistance and Point Mutation of the gyrA Gene in Campylobacter jejuni Isolated from Broilers and in vitro-Induced Resistant Strains

“…Mutations in gyrA have been found in a variety of bacteria including Staphylococcus aureus [14], Escherichia coli [6], Salmonella spp. [17] and C. jejuni [3]. After C. jejuni gyrA was cloned and sequenced [25], a number of rapid methods for the detection of single nucleotide mutations of the gene without sequencing, including nonradioisotopic single-strand conformation polymorphism [3] and a mismatch amplification mutation PCR assay for the detection of the point mutation within the Thr-86 codon of C. jejuni gyrA [27], were established.…”

“…[17] and C. jejuni [3]. After C. jejuni gyrA was cloned and sequenced [25], a number of rapid methods for the detection of single nucleotide mutations of the gene without sequencing, including nonradioisotopic single-strand conformation polymorphism [3] and a mismatch amplification mutation PCR assay for the detection of the point mutation within the Thr-86 codon of C. jejuni gyrA [27], were established. In this study, a rapid and simple assay system was developed for the detection of mutations within the Thr-86, Asp-90 and Ala-70 codons of C. jejuni gyrA.…”

Molecular Detection of Antimicrobial Resistance

“…Meanwhile, several investigators have applied SSCP analysis to the detection of mutations, mainly in gyrA, in several bacterial species (47,203,230,325,331,350). In the most recent study, Takenouchi et al (332) studied gyrA point mutations in 335 clinical P. aeruginosa isolates by nRI-SSCP analysis and direct sequencing.…”