title: In vitro selection of resistance to pradofloxacin and ciprofloxacin in canine uropathogenic Escherichia coli isolates.
abstract: This study explored and compared the mechanisms and selective concentration of resistance between a 3rd (pradofloxacin) and 2nd (ciprofloxacin) generation fluoroquinolone. Pradofloxacin- and ciprofloxacin-resistant mutants were selected by stepwise exposure of Escherichia coli (E. coli) to escalating concentrations of pradofloxacin and ciprofloxacin. The sequence of the quinolone resistance determining region (QRDR) and the transcriptional regulator soxS were analyzed, and efflux pump AcrAB-TolC activity was measured by quantitative real-time reverse transcription-PCR (qRT-PCR). First-step mutants reduced the fluoroquinolone sensitivity and one mutant bore a single substitution in gyrA. Four of six second-step mutants expressed ciprofloxacin resistance, and displayed additional mutations in gyrA and/or parC, while these mutants retained susceptibility to pradofloxacin. All the third-step mutants were fluoroquinolone resistant, and each expressed multidrug resistance (MDR) phenotypes. Further, they displayed resistance to all antibacterials tested except cefotaxime, ceftazidime and meropenem. The number of mutations in QRDR of gyrA and parC correlated with fluoroquinolone MICs. Mutations in parC were not common in pradofloxacin-associated mutants. Moreover, one second- and one third-step ciprofloxacin-associated mutants bore both mutations at position 12 (Ala12Ser) and 78 (Met78Leu) in the soxS gene, yet no mutations in the soxS gene were detected in the pradofloxacin-selected mutants. Altogether, these results demonstrated that resistance emerged relatively more rapidly in 2nd compared to 3rd generation fluoroquinolones. Point mutations in gyrA were a key mechanism of resistance to pradofloxacin, and overexpression of efflux pump gene acrB played a potential role in the emergence of MDR phenotypes identified in this study.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, PR China.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA.
authors.forename: Caterina
authors.initials: C
authors.lastname: Lazzaroni
authors.affiliation: Department of Medical Microbiology and Immunology, College of Medicine, Assiut University, Assiut, Egypt.
authors.forename: Sherine A
authors.initials: SA
authors.lastname: Aly
authors.affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA.
authors.forename: Kamoltip
authors.initials: K
authors.lastname: Thungrat
authors.affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA. Electronic address: boothdm@auburn.edu.
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Boothe
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics; D004283:Dog Diseases / Q000188:drug therapy / Q000382:microbiology*; D004285:Dogs; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D004927:Escherichia coli Infections / Q000188:drug therapy / Q000382:microbiology / Q000662:veterinary*; D029968:Escherichia coli Proteins / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000662:veterinary; D009154:Mutation; D014552:Urinary Tract Infections / Q000188:drug therapy / Q000382:microbiology / Q000662:veterinary*; D056689:Uropathogenic Escherichia coli / Q000187:drug effects* / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; D002939:Ciprofloxacin; C507250:pradofloxacin; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase
keywords: Ciprofloxacin; Escherichia coli; Pradofloxacin; Resistance mechanism; Stepwise mutation