title: Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila.
abstract: OBJECTIVE
  Fluoroquinolone resistance has been poorly studied in Legionella pneumophila, an intracellular pathogen responsible for legionellosis. Our goal was to further characterize molecular mechanisms involved in fluoroquinolone resistance in this species.
  
  
  METHODS
  Eight independent lineages were founded from a common fluoroquinolone-susceptible L. pneumophila ancestor and propagated by serial passages in moxifloxacin-containing culture medium. We identified the substituted mutations that affected the DNA topoisomerase II-encoding genes, determined the order of substitution of the mutations leading to the stepwise MIC increases of moxifloxacin over evolutionary time and demonstrated their direct involvement in the resistance process.
  
  
  RESULTS
  Adaptation occurred through parallel stepwise increases in the moxifloxacin MICs up to 512-fold the MIC for the parental strain. Mutations affected the topoisomerase II-encoding genes gyrA, parC and gyrB, reflecting a high degree of genetic parallelism across the independent lineages. During evolution, the T83I change in GyrA occurred first, followed by G78D or S80R in ParC and D87N in GyrA, or S464Y or D426N in GyrB. By constructing isogenic strains, we showed that the progressive increase in resistance was linked to a precise order of mutation substitution, but also to the co-existence of several subpopulations of bacteria bearing different mutations.
  
  
  CONCLUSIONS
  Specific mutational trajectories were identified, strongly suggesting that intermolecular epistatic interactions between DNA topoisomerases underlie the mechanism of fluoroquinolone resistance in L. pneumophila. Our results suggest that L. pneumophila has strong potential to become resistant to fluoroquinolone compounds and warrant further investigation of resistance in clinical and environmental strains of this pathogen.
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier Grenoble 1, Institut Jean Roget, Campus Santé, Domaine de la Merci, BP 170, F-38042 Grenoble cedex 9, France.
authors.forename: Iyad
authors.initials: I
authors.lastname: Almahmoud
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kay
authors.forename: Dominique
authors.initials: D
authors.lastname: Schneider
authors.forename: Max
authors.initials: M
authors.lastname: Maurin
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001372:Aza Compounds / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D027081:DNA Gyrase / Q000235:genetics; D004252:DNA Mutational Analysis; D027101:DNA Topoisomerase IV / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D024881:Drug Resistance, Bacterial; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D016952:Legionella pneumophila / Q000187:drug effects* / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D000077266:Moxifloxacin; D020125:Mutation, Missense; D011804:Quinolines / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001372:Aza Compounds; D001426:Bacterial Proteins; D024841:Fluoroquinolones; D011804:Quinolines; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D000077266:Moxifloxacin