GLYCOPEPTIDE RESISTANCE LOCI IN STAPHYLOCOCCUS AUREUS

描述(改编自申请人的摘要)：糖肽(GP) 万古霉素和替考拉宁是治疗的首选药物 由耐甲氧西林葡萄球菌引起的感染 对其他常用药物具有多重抗药性。GP耐药性一直是 在致病性较低的凝固酶阴性葡萄球菌中被识别 多年来。引起极大关注的是最近出现的中间体 金黄色葡萄球菌耐甲氧西林菌株对gp的耐药性 在日本和美国，对VM治疗有反应的患者。虽然 葡萄球菌对gp耐药的许多生化相关因素 目前，耐药机制仍不清楚。此外，技术 临床实验室中用于鉴定耐药菌株的方法已经证明 不充分和不可靠。因此，随机扫描人类基因组的方法 Gp耐药决定因素的临床分离株可能被证明 是识别抗性机制的有效手段。我们建议 执行几种互补的随机基因组扫描技术，转座子 (TN)诱变、抗GP质粒库的筛选 临床分离株及mRNA差异显示。糖蛋白耐药菌株TN的诱变作用 分离株可能导致对GP耐药性的必要因素的鉴定。 筛选质粒文库将确定足够的抗药性和 MRNA差异显示将导致识别出 在GP敏感株和耐药株之间存在差异表达。推定 通过这些技术确定的决定因素将用于互补 分析和插入失活实验以确定它们在 抵抗。我们已经从一株对VM敏感的Tn551突变体中分离出 实验室衍生的耐Vm菌株，523K。我们建议确定该基因的位置 在523K中Tn551插入的侧翼负责降低电阻 表型。耐gp的临床分离株也将直接用于 TN的诱变研究、文库筛选和mRNA差异显示。这些 策略可以揭示导致认同的抗拒机制 抗菌治疗和耐药菌株检测的新靶点。

DESCRIPTION (Adapted from the applicant's abstract): The glycopeptides (GP) vancomycin (Vm) and teicoplanin (Tco) are the agents of choice in treating infections caused by methicillin-resistant staphylococci which are often multiply resistant to other commonly used agents. GP resistance has been recognized among the less-pathogenic coagulase-negative staphylococcal species for many years. Of great concern has been the recent emergence of intermediate GP resistance in methicillin-resistant S. aureus isolates obtained from patients who did respond to Vm therapy in Japan and the United States. Although many biochemical correlates of GP resistance among staphylococci have been identified, the mechanism of resistance is still unclear. Moreover, techniques for identifying resistant isolates in the clinical laboratory have proved insufficient and unreliable. Therefore, random approaches to scan the genome of GP-resistant clinical isolates for determinants of GP resistance may prove to be an effective means to identify the resistance mechanism. We propose to perform several complementary random genomic scanning techniques, transposon (Tn) mutagenesis, screening of plasmid DNA libraries produced from GP-resistant clinical isolates and mRNA differential display. Tn mutagenesis of GP-resistant isolates may lead to identification of factors essential for GP resistance. Screening plasmid libraries will identify loci sufficient for resistance and mRNA differential display will lead to the identification of genes that are differentially expressed between GP susceptible and resistant strains. Putative determinants identified by these techniques will be used in complementation analyses and insertional inactivation experiments to establish their role in resistance. We have already isolated a Vm-susceptible Tn551 mutant from a laboratory-derived Vm-resistant strain, 523k. We propose to identify the locus flanking the Tn551 insertion in 523k responsible for decreasing the resistance phenotype. The GP-resistant clinical isolates will also be used directly in the Tn mutagenesis studies, library screening and mRNA differential display. These strategies could reveal the mechanism of resistance leading to identification of novel targets for antimicrobial therapy and detection of resistant isolates.