Ince Dilek, Zhang Xiamei, Silver L Christine, Hooper David C
Division of Infectious Disease and Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114-2696, USA.
Antimicrob Agents Chemother. 2002 Nov;46(11):3370-80. doi: 10.1128/AAC.46.11.3370-3380.2002.
We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 x 10(-12) to 4.0 x 10(-11)) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 micro g/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5' terminus of grlB and the 3' terminus of gyrA.
我们通过遗传学和生化研究确定了新型去氟喹诺酮类药物加替沙星（BMS - 284756，T - 3811ME）在金黄色葡萄球菌中的靶酶相互作用。我们发现加替沙星对野生型金黄色葡萄球菌的活性比环丙沙星高4至8倍。单一的拓扑异构酶IV或gyrase突变仅使加替沙星的MIC增加2至4倍，而两个位点的突变组合则导致显著增加（128倍）。NorA外排泵的过表达对加替沙星耐药性的影响最小。加替沙星浓度为MIC的两倍时，耐药突变体（<7.4×10⁻¹²至4.0×10⁻¹¹）的选择比环丙沙星少5至6个对数单位。在单步突变体中选择了拓扑异构酶IV或gyrase或两者的喹诺酮耐药决定区（QRDR）内部或外部的突变，表明拓扑异构酶IV和gyrase存在双重靶向作用。遗传学实验表明，其中三个新突变与耐药性有关。对来自金黄色葡萄球菌的纯化拓扑异构酶IV和gyrase的研究还表明，加替沙星对拓扑异构酶IV和gyrase具有相似的活性（50%抑制浓度分别为1.25至2.5和1.25μg/ml），虽然其对拓扑异构酶IV的活性比环丙沙星高2倍，但其对gyrase的活性高10倍。本研究提供了首个遗传学和生化数据，支持喹诺酮类药物对金黄色葡萄球菌中的拓扑异构酶IV和gyrase进行双重靶向作用，同时也为将QRDR扩展至包括grlB的5'末端和gyrA的3'末端提供了遗传学证据。
