Molecular mechanisms of decreased susceptibility to fluoroquinolones in avian Salmonella serovars and their mutants selected during the determination of mutant prevention concentrations

Abstract

Objectives: Salmonella enterica isolates of six serovars and mutants obtained during determination of mutant prevention concentrations (MPCs) were investigated for mechanisms of decreased susceptibility to fluoroquinolones. Methods: The quinolone resistance determining regions(QRDRs) of gyrA, gyrB, parC and parE genes were sequenced. MIC values were determined in the presence/absence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or Phe-Arg-β-naphthylamide (PAbN). PCR assays for the quinolone resistance genes qnrA, qnrB, qnrS or aac(6′)-Ib-cr were applied. The MPC values of ciprofloxacin (MPCCIP) were determined for all isolates and selected mutants were investigated for their quinolone resistance mechanisms. Results: In contrast to 11 nalidixic acid-susceptible isolates, 24 nalidixic acid-resistant isolates exhibited single mutations in gyrA (Asp-87 → Tyr, Gly, Asn or Ser-83 → Phe, Tyr) or parC (Thr-57 → Ser). While CCCP had no influence on the MICs, PAβN decreased the MICCIP values by 1-3 dilution steps and MICNAL values by up to 6 dilution steps. Of the resistance genes investigated, only qnrS was present, in a single Salmonella Infantis isolate. The MPCCIP values were 4-64-fold higher than the MICs and ranged between 1-16 and 0.12-1 mg/L, respectively, for isolates resistant or susceptible to nalidixic acid. Only mutants obtained from formerly nalidixic acid-susceptible isolates developed single mutations in gyrA or gyrB. Conclusions: In field isolates and mutants, target site mutations and efflux seem to be important mechanisms for decreased fluoroquinolone susceptibility. Mutants derived during MPC determination from field isolates already harbouring single-step mutations in gyrA did not exhibit further mutations in any target genes. © The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.