Cambau Emmanuelle, Matrat Stephanie, Pan Xiao-Su, Roth Dit Bettoni Romain, Corbel Céline, Aubry Alexandra, Lascols Christine, Driot Jean-Yves, Fisher L Mark
Université Paris12, Creteil, France.
J Antimicrob Chemother. 2009 Mar;63(3):443-50. doi: 10.1093/jac/dkn528. Epub 2009 Jan 15.
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.
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.
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.
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.
贝西沙星是一种正在研发的新型用于眼部的氟喹诺酮类药物。我们研究了其对两种主要眼部病原体肺炎链球菌和金黄色葡萄球菌以及参考菌株大肠杆菌的作用方式和耐药性。
通过以下方法评估贝西沙星的主要和次要靶点：（i）突变体选择实验；（ii）对确定的拓扑异构酶突变体进行最低抑菌浓度（MIC）测试；（iii）用纯化的肺炎链球菌和大肠杆菌DNA旋转酶及拓扑异构酶IV酶进行抑制和可裂解复合物测定。
酶活性测定表明，贝西沙星对肺炎链球菌旋转酶和拓扑异构酶IV具有相似的活性，IC（50）和CC（25）分别为2.5和1微摩尔。与环丙沙星和莫西沙星不同，贝西沙星对肺炎链球菌和大肠杆菌旋转酶的活性相当。DNA旋转酶是所有三种菌株的主要靶点，在GyrA的81、83和87位以及GyrB的426和466位（大肠杆菌编号）观察到替代。拓扑异构酶IV是次要靶点。值得注意的是，在金黄色葡萄球菌和肺炎链球菌的4倍贝西沙星MIC浓度下未获得耐药突变体，并且金黄色葡萄球菌拓扑异构酶突变体仅在液体培养基中连续传代后才获得。金黄色葡萄球菌和肺炎链球菌中parC或gyrA突变对贝西沙星MIC的影响相似，在gyrA-parC双突变体中仍低于1毫克/升。
尽管突变体选择实验表明旋转酶是主要靶点，但进一步的生化和遗传学研究表明，贝西沙星对金黄色葡萄球菌和肺炎链球菌中必需的DNA旋转酶和拓扑异构酶IV靶点均具有强大且相对平衡的活性。
