title: Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli.
abstract: OBJECTIVE
  Besifloxacin is a new fluoroquinolone in development for ocular use. We investigated its mode of action and resistance in two major ocular pathogens, Streptococcus pneumoniae and Staphylococcus aureus, and in the reference species Escherichia coli.
  
  
  METHODS
  Primary and secondary targets of besifloxacin were evaluated by: (i) mutant selection experiments; (ii) MIC testing of defined topoisomerase mutants; and (iii) inhibition and cleavable complex assays with purified S. pneumoniae and E. coli DNA gyrase and topoisomerase IV enzymes.
  
  
  RESULTS
  Enzyme assays showed similar besifloxacin activity against S. pneumoniae gyrase and topoisomerase IV, with IC(50) and CC(25) of 2.5 and 1 microM, respectively. In contrast to ciprofloxacin and moxifloxacin, besifloxacin was equally potent against both S. pneumoniae and E. coli gyrases. DNA gyrase was the primary target in all three species, with substitutions observed at positions 81, 83 and 87 in GyrA and 426 and 466 in GyrB (E. coli numbering). Topoisomerase IV was the secondary target. Notably, resistant mutants were not recovered at 4-fold besifloxacin MICs for S. aureus and S. pneumoniae, and S. aureus topoisomerase mutants were only obtained after serial passage in liquid medium. Besifloxacin MICs were similarly affected by parC or gyrA mutations in S. aureus and S. pneumoniae and remained below 1 mg/L in gyrA-parC double mutants.
  
  
  CONCLUSIONS
  Although mutant selection experiments indicated that gyrase is a primary target, further biochemical and genetic studies showed that besifloxacin has potent, relatively balanced activity against both essential DNA gyrase and topoisomerase IV targets in S. aureus and S. pneumoniae.
authors.affiliation: Université Paris12, Creteil, France. emmanuelle.cambau@sls.aphp.fr
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Cambau
authors.forename: Stephanie
authors.initials: S
authors.lastname: Matrat
authors.forename: Xiao-Su
authors.initials: XS
authors.lastname: Pan
authors.forename: Romain
authors.initials: R
authors.lastname: Roth Dit Bettoni
authors.forename: Céline
authors.initials: C
authors.lastname: Corbel
authors.forename: Alexandra
authors.initials: A
authors.lastname: Aubry
authors.forename: Christine
authors.initials: C
authors.lastname: Lascols
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Driot
authors.forename: L Mark
authors.initials: LM
authors.lastname: Fisher
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001381:Azepines / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics / Q000302:isolation & purification; D004252:DNA Mutational Analysis; D027101:DNA Topoisomerase IV / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects*; D024841:Fluoroquinolones / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D013211:Staphylococcus aureus / Q000187:drug effects*; D013296:Streptococcus pneumoniae / Q000187:drug effects*; D059005:Topoisomerase II Inhibitors
chemical_list: D000900:Anti-Bacterial Agents; D001381:Azepines; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D059005:Topoisomerase II Inhibitors; C522124:besifloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase