title: Evolution toward high-level fluoroquinolone resistance in Francisella species.
abstract: OBJECTIVE
  Francisella tularensis, a CDC class A potential bioterrorism agent, is a Gram-negative bacterium responsible for tularaemia. Understanding the mechanisms of resistance to antibiotics used as first-line treatment is of major security relevance.
  
  
  METHODS
  We propagated the three parental reference strains Francisella tularensis subsp. holarctica live vaccine strain, Francisella novicida and Francisella philomiragia with increasing concentrations of ciprofloxacin, a fluoroquinolone used as curative and prophylactic treatment for tularaemia. This evolution procedure provided us with high-level ciprofloxacin-resistant mutants and all evolutionary intermediates towards high-level resistance. We determined the resistance levels to other fluoroquinolones (levofloxacin and moxifloxacin) and other antibiotic families (aminoglycosides, tetracyclines and macrolides) and characterized the genetic changes in the fluoroquinolone target genes encoding DNA gyrase and topoisomerase IV.
  
  
  RESULTS
  All high-level resistant mutants shared cross-resistance to the tested fluoroquinolones, while some also revealed striking levels of cross-resistance to other clinically relevant antibiotic classes. High-level resistant mutants carried one to three mutations, including some not previously reported. We mapped all mutations onto known topoisomerase three-dimensional structures. Along the pathways towards high-level resistance, we identified complex evolutionary trajectories including polymorphic states and additional resistance mechanisms likely to be associated with efflux processes.
  
  
  CONCLUSIONS
  Our data demonstrated the efficiency and speed of in vitro production of mutants highly resistant to fluoroquinolones in Francisella species. They emphasize the urgent need to identify all antibiotic resistance mechanisms in these species, develop molecular tools for their detection and design new therapeutic alternatives for tularaemia.
authors: Centre Hospitalier Universitaire Grenoble, CS10217, 38043 Grenoble cedex 9, France.
authors: Vivien
authors: V
authors: Sutera
authors: Maxime
authors: M
authors: Levert
authors: Wim Pascal
authors: WP
authors: Burmeister
authors: Dominique
authors: D
authors: Schneider
authors: Max
authors: M
authors: Maurin
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics; D004252:DNA Mutational Analysis; D027101:DNA Topoisomerase IV / Q000235:genetics; D024881:Drug Resistance, Bacterial; D024841:Fluoroquinolones / Q000494:pharmacology*; D005603:Francisella / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D006801:Humans; D008826:Microbial Sensitivity Tests; D012641:Selection, Genetic; D012692:Serial Passage
chemical_list: D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase
keywords: DNA gyrase; antibiotic resistance; ciprofloxacin; topoisomerase IV; tularaemia; type II topoisomerase