GLYCOPEPTIDE RESISTANCE LOCI IN STAPHYLOCOCCUS AUREUS

描述（改编自申请人摘要）：糖肽类（GP） 万古霉素（Vm）和替考拉宁（Tco）是治疗 耐甲氧西林葡萄球菌引起的感染， 对其他常用药剂具有多重抗性。GP抗性已经 在致病性较低的凝固酶阴性葡萄球菌中被识别 多年来最近出现的中间体引起了极大的关注。 耐甲氧西林链球菌对GP耐药。金黄色葡萄球菌分离株， 在日本和美国对Vm治疗有反应的患者。虽然 葡萄球菌中GP耐药性的许多生化相关性已经被 鉴定，耐药机制仍不清楚。此外，技术 用于在临床实验室中鉴定耐药分离株， 不足和不可靠。因此，随机方法来扫描基因组， GP耐药决定因素的GP耐药临床分离株可能证明， 是识别耐药机制的有效手段。我们建议 进行几种互补随机基因组扫描技术，转座子 (Tn)诱变，筛选从GP抗性菌株产生的质粒DNA文库， 临床分离株和mRNA差异显示。抗GP突变株的Tn突变 分离物可能导致鉴定GP抗性所必需的因子。 筛选质粒文库将鉴定足以产生抗性的基因座， mRNA差异显示将导致识别基因， GP敏感株和抗性株之间的差异表达。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 阻力我们已经从一个受Vm影响的突变体中分离出了一个Tn551突变体。 实验室来源抗Vm菌株，523k。我们建议找出 在523k中负责降低抗性的Tn551插入的侧翼 表型。GP耐药临床分离株也将直接用于 诱变研究、文库筛选和mRNA差异显示。这些 策略可以揭示导致识别的抗性机制， 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.