STAPHYLOCOCCAL METHICILLIN RESISTANCE LOCUS

金黄色葡萄球菌甲氧西林耐药基因座

• 批准号: 6137189
• 负责人: Gordon Lee Archer
• 金额: $ 35.89万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6137189
• 关键词: Staphylococcus; bacterial genetics; gene expression; gene induction /repression; gene mutation; genetic regulation; human subject; methicillin; multidrug resistance; nucleic acid sequence; phenotype; plasmids; polymerase chain reaction; regulatory gene; reporter genes; restriction fragment length polymorphism; transposon /insertion element

Staphylococci causing hospital-acquired infections are increasingly resistant to multiple antimicrobial agents. The trait that defines the multidrug-resistant phenotype is resistance to beta-lactam or methicillin resistance (MR). The chromosomal gene responsible for MR, mecA, encodes a penicillin binding protein with low beta-lactam affinity. MecA is found only in resistant isolates, is common to all resistant staphylococcal species and is accompanied by >30 kb of DNA (mec DNA) that is also unique to resistant isolates. Contained within the mec DNA, just 5' to mecA, are two genes, mecRI and mecI, that regulate the transcription of mecA. This proposal will investigate the regulation of mecA by mecRI and mecI; the contribution of regulation to phenotype; and the mutations, insertions, deletions and rearrangements of mec-associated DNA that alter regulatory genes in clinical isolates. In addition, the organization and extent of the entire mec locus will be determined in three staphylococcal isolates. The regulatory loci will be cloned by PCR from wild isolates of defined phenotype and introduced into a MR S. aureus strain with deleted regulatory genes. The contribution of intact and altered regulatory genes to phenotypic resistance and mecA expression will be investigated; induction will be studied by analyzing mRNA transcripts and the PBP2A gene product; the specific role of mutations in determining phenotype will be assessed by making mecR1-mecI fusions; and the failure of reporter gene induction will be used to locate additional inactivated chromosomal regulatory loci. The total extent and composition of mec DNA will also be determined among MR S. aureus and coagulase-negative staphylococci by cloning large, overlapping DNA fragments and DNA sequencing and PCR amplification, generating probes that can be used to compare mec restriction fragment length polymorphisms among a large number of clinical MR isolates. These studies will help to define the role of specific mec-associated DNA in the expression of resistance; identify other regulatory circuits involved in signal transduction; further assess the clonality of MR staphylococci and identify specific DNA sequences that can be used for assessing the molecular epidemiology of hospital-acquired staphylococci.

引起医院获得性感染的葡萄球菌越来越多， 对多种抗菌剂有抗性。 定义 多重耐药表型是对β-内酰胺或甲氧西林耐药 电阻（MR）。 负责MR的染色体基因mecA编码一个 具有低β-内酰胺亲和力的青霉素结合蛋白。 发现MecA 仅在耐药分离株中，常见于所有耐药葡萄球菌 种，并伴随着>30 kb的DNA（mec DNA），这也是独特的 对耐药的分离物。 在mec DNA中，mecA的5'端， 调节mecA转录的两个基因mecRI和mecI。 这 建议将调查mecRI和mecI对mecA的监管; 调节对表型的贡献;以及突变，插入， mec相关DNA的缺失和重排，改变了调节 临床分离株中的基因。 此外，组织和范围 在三种葡萄球菌分离株中测定整个MEC基因座。 调控基因座将通过PCR从定义的野生型分离株中克隆。 表型并引入到MR S中。缺失调节基因的金黄色菌株 基因. 完整的和改变的调控基因的贡献， 将研究表型抗性和mecA表达;诱导 将通过分析mRNA转录物和PBP 2A基因产物进行研究; 突变在确定表型中的具体作用将通过以下方法进行评估： 制造mecR 1-mecI融合体;报告基因诱导的失败将 用于定位另外的失活的染色体调节基因座。 的 还将确定MRS中mec DNA的总范围和组成。 金黄色葡萄球菌和凝固酶阴性葡萄球菌通过克隆大，重叠 DNA片段和DNA测序以及PCR扩增，生成探针 其可用于比较MEC限制性片段长度多态性 在大量的临床MR分离株中。 这些研究将有助于 确定特定mec相关DNA在表达中的作用 电阻;识别信号中涉及的其他调节电路 转导;进一步评估MR葡萄球菌的克隆性并鉴定 可以用于评估分子的特定DNA序列 医院获得性葡萄球菌的流行病学