title: Phenotypic and genetic characterization of macrolide resistance in Francisella tularensis subsp. holarctica biovar I.
abstract: OBJECTIVE
  Francisella tularensis subsp. holarctica strains are classified as biovars I and II, which are susceptible and naturally resistant to the macrolide erythromycin, respectively. The present study was aimed at both selecting biovar I strains with increased levels of erythromycin resistance and characterizing the underlying genetic mechanisms.
  
  
  METHODS
  Serial cultures in the presence of increasingly high erythromycin concentrations were performed to select independent high- and intermediate-level erythromycin-resistant mutants from each of three different biovar I strains. The mutants were characterized for cross-resistance to several antibiotics, presence of mutations in the genes encoding the 23S rRNA and the L4 and L22 ribosomal proteins, and overexpression of efflux pumps.
  
  
  RESULTS
  Mutants displayed cross-resistance to all macrolide compounds tested but not to other classes of antibiotics. We found mutations in domain V of the 23S rRNA gene (G2057A, A2058G, A2058T and C2611T) and in the gene encoding L22, leading to either the G91D substitution or the M82K83R84 deletion. Analysis of mutants with intermediate resistance levels obtained over the course of the selection process revealed both a positive correlation between the number of mutated ribosomal operons and the resistance level, and an additional resistance mechanism in the early steps of selection.
  
  
  CONCLUSIONS
  We showed that high-level resistance to macrolides can be easily obtained in vitro in F. tularensis subsp. holarctica biovar I strains, thereby suggesting that in vivo selection for resistance may explain reported failures of antibiotic treatment. Ketolides were the most effective macrolides tested, which may limit the risk of selection for resistance.
authors.affiliation: CNRS UMR 5163 and Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier Grenoble 1, BP 170, F-38042 Grenoble Cedex 9, France.
authors.forename: Brieuc
authors.initials: B
authors.lastname: Gestin
authors.forename: Eric
authors.initials: E
authors.lastname: Valade
authors.forename: François
authors.initials: F
authors.lastname: Thibault
authors.forename: Dominique
authors.initials: D
authors.lastname: Schneider
authors.forename: Max
authors.initials: M
authors.lastname: Maurin
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D001693:Biological Transport, Active; D024881:Drug Resistance, Bacterial; D005604:Francisella tularensis / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D018942:Macrolides / Q000494:pharmacology*; D009154:Mutation; D012329:RNA, Bacterial / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D012641:Selection, Genetic; D012692:Serial Passage
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D018942:Macrolides; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012269:Ribosomal Proteins