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PCR-restriction fragment length polymorphism assay for detection of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli.

ABSTRACT: A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.

SUBMITTER: Alonso R

PROVIDER: S-EPMC529227 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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PCR-restriction fragment length polymorphism assay for detection of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli.

Alonso Rodrigo R Mateo Estibaliz E Girbau Cecilia C Churruca Estibaliz E Martinez Irati I Fernández-Astorga Aurora A

Antimicrobial agents and chemotherapy 20041201 12

A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation. ...[more]

PMID: 15561873

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Fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli from poultry and human samples assessed by PCR-restriction fragment length polymorphism assay.

Project description:The objective of this study was to determine fluoroquinolone resistance in Campylobacter spp from poultry and human isolates. Forty-one Campylobacter jejuni isolates (30 of poultry origin and 11 of human origin) and 11 Campylobacter coli isolates (10 of human origin and 1 of poultry origin) were examined for ciprofloxacin, norfloxacin, and nalidixic acid resistance using the minimal inhibitory concentration (MIC) method. Thereafter, the isolates were analyzed by PCR-Restriction Fragment Length Polymorphism (RFLP) assay for detection of Thr-86 mutation. Finally, DNA sequencing was performed for confirmation of gyrA gene mutation. A complete correlation was observed between MICs, PCR-RFLP assay, and sequencing. The results revealed high quinolone resistance rates for C. jejuni (100%) and C. coli (100%) isolates obtained from poultry and moderate resistance for C. jejuni (9.1%) and C. coli (40%) samples of human origin. A mutation in codon 86 of the gyrA gene with a Thr-to-Ile substitution is reported to be the main cause of high resistance to quinolones. This mutation can be analyzed by PCR-RFLP assay, which has been proven to be a simple and fast method for the detection of fluoroquinolone resistance in Campylobacter spp.

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