title: In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid.
abstract: OBJECTIVE
  To investigate quinolone resistance mechanisms in an Escherichia coli clinical isolate (Ar2) resistant to ofloxacin but susceptible to nalidixic acid selected after 10 days of ofloxacin therapy in a patient with prostatitis.
  
  
  METHODS
  Molecular typing (ERIC-PCR and RAPD), antibiotic susceptibility and gyrA, gyrB, parC and parE QRDR sequences were compared for E. coli Ar2 and a wild-type E. coli (Ar1) isolated 2 months earlier in the same patient. Ofloxacin-resistant mutants were selected in vitro in order to reproduce the mutations observed and the original phenotype.
  
  
  RESULTS
  The two strains were similar with regard to antibiotic susceptibility except quinolones and for ERIC-PCR and RAPD patterns, suggesting a clonal relationship and acquisition of quinolone resistance by chromosomal mutation. Quinolone MICs were 3, 0.12, 0.05 and 0.02 mg/L of nalidixic acid, ofloxacin, levofloxacin and ciprofloxacin, respectively, for E. coli Ar1 and 6, 32, 8 and 1 mg/L, respectively, for E. coli Ar2. The strain Ar2 harboured two substitutions, Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC. Introduction into E. coli Ar2 of the wild-type gyrA fully complemented fluoroquinolone resistance. Although the strain was not a hypermutator, ofloxacin first-step resistant mutants with gyrA mutations were easily obtained from E. coli Ar1 and 25% of them were at codon 81. In vitro stepwise combination of Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC reproduced the original phenotype in E. coli KL16.
  
  
  CONCLUSIONS
  A double topoisomerase mutant was selected in vivo by 10 days ofloxacin. The mutations were originally combined for a result of ofloxacin resistance but nalidixic acid susceptibility.
authors.affiliation: Service de Bactériologie-Virologie-Hygiène, AP-HP CHU, Henri Mondor Université Paris XII, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil Cedex, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Cattoir
authors.forename: Philippe
authors.initials: P
authors.lastname: Lesprit
authors.forename: Christine
authors.initials: C
authors.lastname: Lascols
authors.forename: Erik
authors.initials: E
authors.lastname: Denamur
authors.forename: Patrick
authors.initials: P
authors.lastname: Legrand
authors.forename: Claude-James
authors.initials: CJ
authors.lastname: Soussy
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Cambau
mesh_terms: D000368:Aged; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D016470:Bacteremia / Q000097:blood / Q000382:microbiology; D026942:DNA Topoisomerases / Q000235:genetics*; D004351:Drug Resistance; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000097:blood / Q000382:microbiology; D006801:Humans; D064704:Levofloxacin; D008297:Male; D009268:Nalidixic Acid / Q000494:pharmacology*; D015242:Ofloxacin / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D009268:Nalidixic Acid; D064704:Levofloxacin; D015242:Ofloxacin; D026942:DNA Topoisomerases