Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay

Abstract

Fluoroquinolones are one class of antimicrobial agents commonly used to treat severe Campylobacter jejuni infection. C. jejuni strains resistant to high levels of the fluoroquinolone ciprofloxacin (MIC ≥16 μg/ml) have been predominantly characterized with a C→T transition in codon 86 of gyrA. The gyrA gene encodes one subunit of DNA gyrase, which is a primary target for fluoroquinolone antibiotics. This study establishes a rapid PCR-based TaqMan method for identifying ciprofloxacin-resistant C. jejuni strains that carry the C→T transition in codon 86 of gyrA. The assay uses real-time detection, eliminating the need for gel electrophoresis. Optimization of the assay parameters using purified Campylobacter DNA resulted in the ability to detect femto-gram levels of DNA. The method should be useful for monitoring the development of ciprofloxacin resistance in C. jejuni. Compiled nucleotide sequence data on the quinolone resistance-determining region of gyrA in Campylobacter indicate that sequence comparison of this region is a useful method for tentative identification of Campylobacter isolates at the species level.