Selection and characterization of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin

Significant levels of fluoroquinolone resistance were obtained in Campylobacter jejuni isolates after an unique step of selection using enrofloxacin. An Asp90-to-Asn and a Thr86-to-Ile change in the gyrase subunit GyrA were found associated with a low (MIC less than or equal to 8 mug/ml) or a high (MIC greater than or equal to 16 mug/ml) level of, resistance to ciprofloxacin, respectively. An association of both mutations conferred a higher level of resistance (MIC greater than or equal to 128 mug/ml). Further steps of selection increased the MICs of fluoroquinolones but did hot result in a multiple antibiotic resistance phenotype. The Thr86-to-Ile change was found to confer different levels of resistance, pointing out other mechanisms of resistance. However, sequencing revealed no mutation in gyrB, and several attempts did not enable any amplification of the parC gene coding for topoisomerase IV, suggesting an absence of this secondary target in C. jejuni. In addition, no difference in the major outer membrane protein expression was found among the isolates. Furthermore, the use of the recently identified efflux pump inhibitor Phe-Arg-beta-naphthylamide did not result in a significant decrease of fluoroquinolone MICs or change in the frequency of isolation of enrofloxacin-resistant mutants, and thus appears ineffective against fluoroquinolone-resistant C. jejuni isolates. Results obtained during ciprofloxacin accumulation studies confirmed that efflux probably plays a minor role in fluoroquinolone resistance of C. jejuni.