Antibiotic pressure and selection of TCA cycle mutants in Staphylococcus epidermi

描述(由申请人提供)：Kohanski等人最近的一份报告。证明了多种杀菌抗生素刺激羟基自由基的产生，从而加速了大肠杆菌中细菌细胞的死亡。尽管不同类别的抗生素有独特的作用机制，但他们的数据表明，它们通过涉及羟基自由基的共同途径来介导杀戮。通过一种未知的机制，杀菌抗生素通过TCA循环瞬间增加NAD+的还原，增加ETC中超氧化物的产生。超氧化物破坏铁-硫团簇；因此，释放的亚铁离子可用于Fenton反应，产生羟基自由基。验证了这些研究，TCA循环酶乌头酸酶(AcnB)和异柠檬酸脱氢酶(IcdA)的等位基因替换基因敲除消除了杀菌抗生素的杀菌效果。此外，杀菌抗生素不能诱导TCA循环突变体中的羟基自由基的形成。本申请中提供的数据表明，26%的临床分离的表皮葡萄球菌是TCA周期缺陷的。此外，苯唑西林对表皮葡萄球菌1457乌头酸酶突变体(1457 ACNA：：TETM)的时间杀灭研究表明，与野生型相比，杀伤率降低了2.6log10。因此，TCA循环活性的丧失代表了细菌对抗生素压力普遍存在的环境的生理适应。这一应用的中心假设是，抗生素压力选择了表皮葡萄球菌种群中的TCA周期突变体。这一假说将首先通过确定杀菌抗生素是否诱导表皮葡萄球菌中的羟基自由基形成来检验。其次，我们将确定临床相关的表皮葡萄球菌在TCA循环功能方面存在缺陷的比例。最后，我们将使用豚鼠组织笼模型来确定TCA周期突变是否对抗生素治疗更加顽固。这项提议将产生关于细菌在抗生素压力下生长的生理适应的重要新信息。作为这些研究的结果，可能会提出预防和治疗细菌感染的新方法。公共卫生相关性：本申请中提出的实验旨在确定某些抗生素的使用是否选择了由于常见代谢途径突变而产生抗药性的细菌。

DESCRIPTION (provided by applicant): A recent report by Kohanski et al. demonstrated that multiple classes of bactericidal antibiotics stimulate the production of hydroxyl radicals thus accelerating bacterial cell death in Escherichia coli. Although different classes of antibiotics have unique mechanisms of action, their data suggest that they mediate killing through a common pathway involving hydroxyl radicals. By an unknown mechanism, bactericidal antibiotics transiently increase the reduction of NAD+ via the TCA cycle, increasing superoxide generation in the ETC. Superoxide damages iron-sulfur clusters; the released ferrous ion is thus available for the Fenton reaction generating hydroxyl radicals. Validating these studies, allelic replacement gene knockouts of the TCA cycle enzymes aconitase (acnB) and isocitrate dehydrogenase (icdA) abrogated the killing effect of bactericidal antibiotics. In addition, bactericidal antibiotics did not induce hydroxyl radical formation in the TCA cycle mutants. Data presented in this application demonstrate that 26% of clinical Staphylococcus epidermidis isolates are TCA cycle defective. In addition, oxacillin time-kill studies against a S. epidermidis 1457 aconitase mutant (1457 acnA::tetM) revealed that killing was decreased 2.6 log10 compared to wild type. Therefore, the loss of TCA cycle activity represents a physiological adaptation of the bacterium to an environment where antibiotic pressure is prevalent. The central hypothesis of this application is that antibiotic pressure selects for TCA cycle mutants within the S. epidermidis population. This hypothesis will be tested by first determining if bactericidal antibiotics induce hydroxyl radical formation in S. epidermidis. Secondly, we will determine the percentage of clinically relevant S. epidermidis isolates that are defective in TCA cycle function. Lastly, we will determine if TCA cycle mutants are more recalcitrant to antibiotic therapy using a guinea pig tissue cage model. This proposal will yield significant new information regarding the physiological adaptation of bacteria allowing growth under antibiotic pressure. Novel means to prevent and treat bacterial infections may be suggested as a result of these studies. PUBLIC HEALTH RELEVANCE: Experiments proposed in this application are designed to determine whether the use of certain antibiotics selects for bacteria that are resistant because of mutations in a common metabolic pathway.