Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli.
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.
Cambau
Emmanuelle
E
Université Paris12, Creteil, France. emmanuelle.cambau@sls.aphp.fr
Matrat
Stephanie
S
Pan
Xiao-Su
XS
Roth Dit Bettoni
Romain
R
Corbel
Céline
C
Aubry
Alexandra
A
Lascols
Christine
C
Driot
Jean-Yves
JY
Fisher
L Mark
LM
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
D000900:Anti-Bacterial Agents; D001381:Azepines; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D059005:Topoisomerase II Inhibitors; C522124:besifloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase