title: Analysis of the mechanisms of fluoroquinolone resistance in urinary tract pathogens.
abstract: OBJECTIVE
  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 spectrophotometrically 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.
authors.affiliation: Department of Microbiology, School of Microbiology and Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa.
authors.forename: Hafizah Y
authors.initials: HY
authors.lastname: Chenia
authors.forename: Balakrishna
authors.initials: B
authors.lastname: Pillay
authors.forename: Dorsamy
authors.initials: D
authors.lastname: Pillay
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D002939:Ciprofloxacin / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D027081:DNA Gyrase / Q000235:genetics; D027101:DNA Topoisomerase IV / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects*; D004927:Escherichia coli Infections / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D008070:Lipopolysaccharides / Q000032:analysis; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014552:Urinary Tract Infections / Q000382:microbiology*
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; D008070:Lipopolysaccharides; C485384:Qnr protein, E coli; D002939:Ciprofloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase