CHARACTERIZATION OF CEFTAZIDIME RESISTANCE IN KLEBSIELLA PNEUMONIAE

肺炎克雷伯菌对头孢他啶耐药的特征

• 批准号: 6338812
• 负责人: James Edward Raynor
• 金额: $ 3.95万
• 依托单位: FAYETTEVILLE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6338812
• 关键词: Klebsiella pneumoniae; antibacterial agents; beta lactamase; cephalosporins; drug resistance; gene expression; gene mutation; genetic strain; human tissue; molecular cloning; nosocomial infections; nucleic acid hybridization; nucleic acid sequence; plasmids; polymerase chain reaction; pulsed field gel electrophoresis; serology /serodiagnosis; southern blotting

The introduction of highly stable Extended-Spectrum cephalosporins at the beginning of the 1980's was quickly followed by the emergence of several cephalosporin resistant Extended-Spectrum beta-lactamases identified among clinical isolates of Klebsiella pneumoniae. Homology studies of these enzymes suggested that bacterial have adapted to the antibiotics by altering (amino acid substitutions) existing plasmid-mediated beta-lactamases such to expand their spectrum of activity. Subsequently, plasmids encoding altered beta-lactamases were disseminated among gram-negative clinical isolates. This resulted in increased beta-lactam antibiotic resistance among clinical gram-negative bacteria. In this proposal, an extensive investigation will be conducted to identify and characterize novel beta-lactamases (derrived from amino acid substitutions) identified in clinical isolates of Klebsiella pneumoniae. Cephalosporin resistant Klebsiella pneumonia will be identified by Kirby-Bauer disk diffusion susceptibility testing using disk impregnated with cephalosporins (ceftazidime, cefotaxime, and ceftriaxone). Isolates demonstrating Extended-Spectrum activity are conjugated with susceptible E. Coli recipients to identify transmissible beta-lactam resistance genes Transconjugates are screened for the presence of TEM or SHV-type beta-lactamase genes by polymerase chain reaction and Southern blot hybridizations. Products obtained by PCR are sequenced to identify novel mutations. Clones harboring mutations are cloned and resequenced to confirm original findings. Data obtained from this proposal will be instrumental in (1) the early detection of mutant TEM and SHV-type beta-lactamases for proper treatment, (2) maximizing the use of antibiotics currently available, (3) establishing new forms of treatment, and (4) elucidating the trends and mechanisms of drug resistance and transmission of TEM and SHV-type beta-lactamases during nosocomial outbreaks. This project will involve students in every aspect. Students will gain invaluable knowledge and training in the fundamentals of DNA manipulations, PCR technology, analysis of clinical samples, DNA sequencing, data collection, and manuscript preparation. Training obtained from this study will prepare students for doctoral studies in cell, molecular, and microbiology.

推出高度稳定的广谱头孢菌素 20 世纪 80 年代初，紧随其后的是 几种头孢菌素耐药的广谱药物的出现 在克雷伯氏菌临床分离株中鉴定出 β-内酰胺酶 肺炎杆菌。 这些酶的同源性研究表明 细菌通过改变（氨基酸 取代）现有的质粒介导的β-内酰胺酶，例如 扩大他们的活动范围。 随后，质粒编码 改变的β-内酰胺酶在革兰氏阴性菌中传播 临床分离株。 这导致β-内酰胺抗生素增加 临床革兰氏阴性菌的耐药性。 在这个提案中， 将进行广泛的调查，以确定并 表征新型 β-内酰胺酶（源自氨基酸 克雷伯氏菌临床分离株中鉴定出的替换） 肺炎杆菌。 肺炎克雷伯菌对头孢菌素耐药 通过 Kirby-Bauer 纸片扩散敏感性测试确定 用头孢菌素（头孢他啶、头孢噻肟和 头孢曲松）。 表现出广谱活性的分离物是 与易感大肠杆菌受体结合以识别 传染性β-内酰胺抗性基因转缀物是 筛选是否存在 TEM 或 SHV 型 β-内酰胺酶 通过聚合酶链反应和 Southern blot 检测基因 杂交。 通过 PCR 获得的产物进行测序以鉴定 新的突变。 克隆携带突变的克隆并 重新测序以证实最初的发现。由此获得的数据 该提案将有助于（1）突变体的早期检测 用于正确治疗的 TEM 和 SHV 型 β-内酰胺酶，(2) 最大限度地使用现有抗生素，(3) 建立新的治疗形式，以及（4）阐明趋势 以及 TEM 和透射电镜的耐药性和传播机制 医院内暴发期间的 SHV 型 β-内酰胺酶。 这 项目将让学生参与各个方面。 学生将收获 DNA 基础知识和培训非常宝贵 操作、PCR 技术、临床样本分析、DNA 测序、数据收集和手稿准备。 训练 从这项研究中获得的知识将为学生攻读博士学位做好准备 细胞学、分子学和微生物学。