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

Abstract

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. 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-beta-naphthylamide (PA beta N). PCR assays for the quinolone resistance genes qnrA, qnrB, qnrS or aac(6')-Ib-cr were applied. The MPC values of ciprofloxacin (MPC(CIP)) were determined for all isolates and selected mutants were investigated for their quinolone resistance mechanisms. 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, PA beta N decreased the MIC(CIP) values by 1-3 dilution steps and MIC(NAL) values by up to 6 dilution steps. Of the resistance genes investigated, only qnrS was present, in a single Salmonella Infantis isolate. The MPC(CIP) 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. 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.