title: Chromosomal and plasmid-mediated fluoroquinolone resistance mechanisms among broad-spectrum-cephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA.
abstract: OBJECTIVE
  To determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and investigate mutations in gyrase and topoisomerase genes that may contribute to increased fluoroquinolone resistance in canine and feline Escherichia coli isolates in the USA that displayed reduced susceptibility to extended-spectrum cephalosporins. This study was undertaken because previous epidemiological studies identified a potential correlation between extended-spectrum cephalosporins and fluoroquinolone resistance.
  
  
  METHODS
  Isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime were screened by PCR for the presence of PMQR determinants and gyrase and topoisomerase genes were sequenced. Isolates were further characterized by conjugation and phylogenetic analyses.
  
  
  RESULTS
  PMQR determinants aac(6')-Ib-cr, qnrS and qepA were identified in 30, 23 and 5 isolates, respectively. Multiple mutations were identified in the quinolone resistance-determining region, including the novel substitutions of Glu-84 → Ala and Leu-88 → Gln in ParC and Arg-432 → Ser and Glu-460 → Val in ParE. The isolate that exhibited the highest level of enrofloxacin resistance (MIC > 256 mg/L) had a double mutation in gyrA (Ser-83 → Leu and Asp-87 → Asn) and a triple mutation in parC (Ser-80 → Ile, Glu-84 → Gly and a novel mutation, Leu-88 → Gln). The presence of PMQR genes increased the ciprofloxacin MIC values 4-fold to 8-fold in transconjugants relative to the recipient strain. Approximately 39% of the isolates belonged to phylogenetic group D and 30% to group B2, which typically contain an increased number of virulence determinants compared with other groups.
  
  
  CONCLUSIONS
  Novel mutations in topoisomerase genes and PMQR determinants aac(6')-Ib-cr, qnrS and qepA genes were detected among extended-spectrum β-lactamase-producing E. coli in the USA.
authors.affiliation: Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR, USA.
authors.forename: Bashar W
authors.initials: BW
authors.lastname: Shaheen
authors.forename: Rajesh
authors.initials: R
authors.lastname: Nayak
authors.forename: Steven L
authors.initials: SL
authors.lastname: Foley
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Boothe
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002415:Cats; D002511:Cephalosporins / Q000494:pharmacology; D002876:Chromosomes, Bacterial; D003227:Conjugation, Genetic; D026942:DNA Topoisomerases / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004285:Dogs; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary*; D024841:Fluoroquinolones / Q000494:pharmacology*; D005798:Genes, Bacterial; D057805:Pets / Q000382:microbiology*; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014481:United States
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D026942:DNA Topoisomerases