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 ≤ 8 μ g/ml) or a high (MIC ≥ 16 μ g/ml) level of resistance to ciprofloxacin, respectively. An association of both mutations conferred a higher level of resistance (MIC ≥ 128 μ g/ml). Further steps of selection increased the MICs of fluoroquinolones but did not 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- β -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 .