JAC Advance Access originally published online on March 16, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):886-892; doi:10.1093/jac/dkm072
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Molecular mechanisms of decreased susceptibility to fluoroquinolones in avian Salmonella serovars and their mutants selected during the determination of mutant prevention concentrations
1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany 2 Bayer HealthCare AG, Animal Health Division, Clinical Research & Development Antiinfectives, 40789 Monheim, Germany 3 Unité Infectiologie Animale et Santé Publique, Institut National de la Recherche Agronomique, 37380 Nouzilly, France
Received 7 December 2006; returned 26 January 2007; revised 12 February 2007; accepted 14 February 2007
* Corresponding author. Tel: +49-5034-871-242; Fax: +49-5034-871-246; E-mail: corinna.kehrenberg{at}fal.de
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-ß-naphthylamide (PAßN). 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, PAßN 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.
Keywords: ciprofloxacin , nalidixic acid , QRDR , efflux pump systems , qnr genes , fluoroquinolone modifying enzyme aac(6')-Ib-cr
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