title: High-level resistance to moxifloxacin and gatifloxacin associated with a novel mutation in gyrB in toxin-A-negative, toxin-B-positive Clostridium difficile.
abstract: OBJECTIVE
  To determine the mechanism of high-level resistance to fluoroquinolone antimicrobials in toxin-A-negative, toxin-B-positive (A- B+) Clostridium difficile isolates.
  
  
  METHODS
  Following culture 16-23S PCR ribotyping was used to determine genomic relationships between A- B+ C. difficile isolates. Antimicrobial susceptibilities were determined using Etests in the presence and absence of the efflux pump inhibitors reserpine (20 microg/mL), L-phenylalanine-L-arginine-beta-naphthylamide (PAbetaN; 20 microg/mL) and verapamil (100 microg/mL). Genomic regions including the quinolone-resistance-determining-region (QRDR) of gyrA and gyrB were amplified and characterized.
  
  
  RESULTS
  PCR ribotyping profiles identified one major cluster of A- B+ C. difficile, universally resistant to the fluoroquinolones tested (ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin and gatifloxacin; MICs > 32 mg/L). All isolates with high-level resistance had a transversion mutation (A-->T) resulting in the amino acid substitution Asp-426-->Val in gyrB. Non-clonal isolates were susceptible to moxifloxacin and gatifloxacin (MICs 0.3 and 0.4 mg/L, respectively) with reduced susceptibility to levofloxacin (MIC 3 mg/L) consistent with the wild-type genotype. The MICs for resistant isolates were not significantly affected by the addition of any of the efflux pump inhibitors. No amino acid substitutions were identified in the QRDR of gyrA.
  
  
  CONCLUSIONS
  High-level resistance to fluoroquinolones in A- B+ C. difficile is associated with a novel transversion mutation in gyrB. The emergence of universal resistance in different C. difficile strain types may be a factor promoting outbreaks in hospitals.
authors.affiliation: Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland. denise.drudy@ucd.ie
authors.forename: Denise
authors.initials: D
authors.lastname: Drudy
authors.forename: Teresa
authors.initials: T
authors.lastname: Quinn
authors.forename: Rebecca
authors.initials: R
authors.lastname: O'Mahony
authors.forename: Lorraine
authors.initials: L
authors.lastname: Kyne
authors.forename: Peadar
authors.initials: P
authors.lastname: O'Gaora
authors.forename: Séamus
authors.initials: S
authors.lastname: Fanning
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001372:Aza Compounds / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis; D016360:Clostridioides difficile / Q000187:drug effects* / Q000235:genetics; D027081:DNA Gyrase / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004151:Dipeptides / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004768:Enterotoxins / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D024841:Fluoroquinolones / Q000494:pharmacology*; D000077734:Gatifloxacin; D008826:Microbial Sensitivity Tests / Q000379:methods; D008969:Molecular Sequence Data; D000077266:Moxifloxacin; D009154:Mutation; D016133:Polymerase Chain Reaction; D011804:Quinolines / Q000494:pharmacology*; D012110:Reserpine / Q000494:pharmacology; D021521:Ribotyping; D017422:Sequence Analysis, DNA; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001372:Aza Compounds; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C488583:Clostridium difficile lethal toxin B; D004269:DNA, Bacterial; D004151:Dipeptides; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D024841:Fluoroquinolones; D011804:Quinolines; C419365:phenylalanine arginine beta-naphthylamide; C041348:tcdA protein, Clostridium difficile; D012110:Reserpine; D014700:Verapamil; D027081:DNA Gyrase; D000077734:Gatifloxacin; D000077266:Moxifloxacin