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: 6107310
• 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: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107310
• 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.

高稳定超广谱头孢菌素的介绍 在20世纪80年代初，S很快就被 几种头孢菌素耐药超广谱菌株的出现 临床分离克雷伯菌中的β-内酰胺酶 肺炎。对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 替换)现有的质粒介导的β-内酰胺酶 扩大他们的活动范围。随后，编码的质粒 革兰氏阴性菌中传播的β-内酰胺酶变异 临床分离株。这导致了β-内酰胺类抗生素的增加 临床革兰氏阴性菌的耐药性分析。在这份提案中， 将进行广泛的调查，以确定和 新型β-内酰胺酶的特性(来源于氨基酸) 在临床分离的克雷伯氏菌中发现的 肺炎。对头孢菌素耐药的肺炎克雷伯菌 经Kirby-Bauer纸片扩散敏感性试验鉴定 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株是 与敏感的大肠杆菌受体结合以识别 可传播的β-内酰胺类耐药基因转接物为 TEM型或SHV型β-内酰胺酶的筛查 聚合酶链式反应和Southern杂交检测基因 混血。通过聚合酶链式反应获得的产物进行测序以鉴定 新的突变。含有突变的克隆被克隆并 重新测序以确认最初的发现。从本网站获得的数据 建议将有助于(1)突变的早期检测 适当治疗的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 以及耐药机制和传播机制。 医院暴发流行的SHV型β-内酰胺酶这 项目将涉及到学生的方方面面。学生将受益 无价的DNA基础知识和培训 操作、聚合酶链式反应技术、临床样本分析、DNA 测序、数据收集和手稿准备。培训 这项研究将使学生为博士学习做好准备 细胞、分子和微生物学。