Objectives: To characterize the mechanisms of fluoroquinolone resistance in urinary tract pathogens exhibiting a multiple antibiotic resistance phenotype as well as a high-level resistance to fluoroquinolones. Methods: Nineteen Escherichia coli urinary tract infection pathogens exhibiting high-level resistance to fluoroquinolones were characterized in this study. Alterations in outer membrane proteins (OMPs) and lipopolysaccharide (LPS) were analysed by PAGE. Changes to the quinolone resistance-determining regions (QRDRs) of GyrA and ParC were determined by PCR and DNA sequencing. The presence of the qnrA gene was determined by PCR amplification. Ciprofloxacin uptake was determined spectrophoto-metrically using the quinolone accumulation assay. Results: OMP analysis showed decreased expression, the absence of certain proteins or the presence of proteins with altered molecular weights when compared with wild-type strains. Most isolates possessed a smooth LPS phenotype. Isolates had double mutations in GyrA codons 83 and 87, in addition to a ParC alteration at Ser-80/Glu-84. Isolates accumulated varying levels of ciprofloxacin, and upon the addition of carbonyl cyanide m-chlorophenylhydrazone, increased accumulation was observed in all instances. E. coli isolates with a rough LPS phenotype appeared to accumulate higher levels of ciprofloxacin compared with those with a smooth LPS phenotype expressing OmpF normally, or even those not possessing OmpF. All E. coli isolates tested demonstrated active efflux of ciprofloxacin. Plasmid-mediated quinolone resistance (presence of the qnrA gene) was observed in 36.8% of isolates. Conclusions: A combination of target gene alterations, altered OM permeability, presence of the qnrA gene and active efflux appear to act together to produce a high-level, multiresistance phenotype.
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