Abstract

Inhibitory Activities of Quinolones against DNA Gyrase and Topoisomerase IV of Enterococcus faecalis Yoshikuni Onodera * , Jun Okuda , Mayumi Tanaka and Kenichi Sato New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan ABSTRACT We have cloned the DNA gyrase and topoisomerase IV genes of Enterococcus faecalis to examine the actions of quinolones against E. faecalis genetically and enzymatically. We first generated levofloxacin-resistant mutants of E. faecalis by stepwise selection with increasing drug concentrations and analyzed the quinolone resistance-determining regions of gyrA and parC from the resistant mutants. Isogenic mutants with low-level resistance contained a mutation in gyrA , whereas those with higher levels of resistance had mutations in both gyrA and parC . These results suggested that gyrA is the primary target for levofloxacin in E. faecalis . We then purified the recombinant DNA gyrase and topoisomerase IV enzymes of E. faecalis and measured the in vitro inhibitory activities of quinolones against these enzymes. The 50% inhibitory concentrations (IC 50 s) of levofloxacin, ciprofloxacin, sparfloxacin, tosufloxacin, and gatifloxacin for DNA gyrase were found to be higher than those for topoisomerase IV. In conflict with the genetic data, these results indicated that topoisomerase IV would be the primary target for quinolones in E. faecalis . Among the quinolones tested, the IC 50 of sitafloxacin (DU-6859a), which shows the greatest potency against enterococci, for DNA gyrase was almost equal to that for topoisomerase IV; its IC 50 s were the lowest among those of all the quinolones tested. These results indicated that other factors can modulate the effect of target affinity to determine the bacterial killing pathway, but the highest inhibitory actions against both enzymes correlated with good antienterococcal activities.