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: 6204152
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6204152
• 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.

高稳定性超广谱头孢菌素的引入 在20世纪80年代初， 出现几种头孢菌素耐药的超广谱抗生素 克雷伯菌临床分离株中鉴定的β-内酰胺酶 肺炎。 这些酶的同源性研究表明， 细菌通过改变（氨基酸）来适应抗生素 取代）现有的质粒介导的β-内酰胺酶， 扩大他们的活动范围。 随后，编码 改变的β-内酰胺酶在革兰阴性菌中传播， 临床分离株。 这导致β-内酰胺类抗生素 临床革兰氏阴性菌耐药性。 在这一提议中， 将进行广泛的调查， 表征新的β-内酰胺酶（衍生自氨基酸 在克雷伯氏菌临床分离株中鉴定的 肺炎。 头孢菌素耐药肺炎克雷伯菌将是 通过Kirby-Bauer纸片扩散敏感性试验鉴定， 用头孢菌素（头孢他啶、头孢噻肟和 头孢曲松）。 表现出超广谱活性的分离株为 与敏感的E.大肠杆菌感染者 可传播的β-内酰胺抗性基因 筛选是否存在TEM或SHV型β-内酰胺酶 PCR和Southern blot检测基因 杂交 通过PCR获得的产物被测序以鉴定 新的突变 克隆携带突变的克隆， 重新排序以确认原始结果。数据来源于此 该提案将有助于（1）早期发现突变体 TEM和SHV型β-内酰胺酶用于适当治疗，（2） 最大限度地使用目前可用的抗生素，（3） 建立新的治疗形式;（4）阐明趋势 以及TEM的耐药和传播机制， 医院内爆发的SHV型β-内酰胺酶 这 项目将涉及学生在各个方面。 学生将获得 DNA基础知识的宝贵知识和训练 操作，PCR技术，临床样本分析，DNA 测序、数据收集和手稿准备。 培训 从这项研究中获得的将为学生准备博士研究， 细胞、分子和微生物学