title: Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals.
abstract: Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.
authors.affiliation: UCD Centre for Food Safety & Centre for Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Maria
authors.initials: M
authors.lastname: Karczmarczyk
authors.forename: Marta
authors.initials: M
authors.lastname: Martins
authors.forename: Teresa
authors.initials: T
authors.lastname: Quinn
authors.forename: Nola
authors.initials: N
authors.lastname: Leonard
authors.forename: Séamus
authors.initials: S
authors.lastname: Fanning
mesh_terms: D000822:Animal Husbandry; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary*; D029968:Escherichia coli Proteins / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology*; D020869:Gene Expression Profiling; D007494:Ireland; D026901:Membrane Transport Proteins / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009268:Nalidixic Acid / Q000494:pharmacology; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; D026901:Membrane Transport Proteins; D009268:Nalidixic Acid; D002939:Ciprofloxacin; D027081:DNA Gyrase