Detecting Carbapenem and Quinolone Resistance in Klebsiella pneumoniae using PCR/

描述(申请人提供)：在革兰氏阴性细菌中，对碳青霉烯类和喹诺酮类药物耐药的肺炎克雷伯菌医院感染正迅速成为一个全球性问题。对亚胺培南的耐药主要由KPCβ-内酰胺酶介导；对喹诺酮类药物的耐药是由gyrA和parc基因的喹诺酮类耐药决定区突变以及获得的质粒介导的喹诺酮类耐药基因qnrA、-B、-S、修饰酶(aac(6‘)-Ib-cr)和外排泵引起的。对碳青霉烯类和环丙沙星耐药肺炎克雷伯菌的早期检测和分析将极大地帮助临床医生治疗严重感染患者。为此，我们计划建立一种快速、灵敏、高通量的检测、鉴定、分型和确定肺炎克雷伯菌耐药谱的方法--聚合酶链式反应/电喷雾电离质谱仪(PCR/ESI-MS)。这项建议的具体目的是：1)比较电喷雾电离质谱仪(PCR/ESI-MS)和脉冲场凝胶电泳法和REP-PCR法检测肺炎克雷伯菌中七种“看家”基因的能力，以建立一种快速、重复性好的方法，适用于任何机构；2)测定43株肺炎克雷伯菌的KPC型β-内酰胺酶和喹诺酮类耐药决定簇的序列；3)整合从1和2中获得的知识，同时检查PCR/ESI-MS在检测blaKPC基因和喹诺酮耐药决定簇中的能力。通过选择变异最小的基因(blaKPC、gyrA等)这种方法有望提高检测碳青霉烯类和喹诺酮类耐药所需的准确性和时间。希望这项工作将成为开发其他“床到床”方法以发现其他肠道杆菌耐药表型的典范，并为实施实时感染控制和诊断范例奠定基础。公共卫生相关性：这项应用解决了肺炎克雷伯菌快速基因分型和抗生素耐药性分析的主要未满足需求。及时和正确的识别可以直接影响到关于正确使用抗生素治疗感染的挽救生命的医疗决策。我们建议开发一种快速、敏感和经济有效的诊断方法，用于检测、鉴定、分型和确定对碳青霉烯类和喹诺酮类耐药肺炎克雷伯菌的耐药性。我们的目标是，这种基于新技术的方法将成为实施实时感染控制和诊断范例的基础。

DESCRIPTION (provided by applicant): Among Gram-negative bacteria, nosocomial infections by Klebsiella pneumoniae that are resistant to carbapenems and quinolones are rapidly becoming a global problem. The emerging resistance to imipenem is primarily mediated by KPC beta-lactamases; resistance to quinolones occurs as a result of mutations in the quinolone resistance determining region (QRDR) of gyrA and parC as well as the acquisition of plasmid mediated quinolone resistance genes qnrA, -B, -S, modifying enzymes (aac(6')-Ib-cr) and efflux pumps. The early detection and analysis of carbapenem- and ciprofloxacin- resistant K. pneumoniae will enormously assist clinicians treating patients with serious infections. To that end, a rapid, sensitive, and high-throughput assay to detect, identify, genotype, and determine the antibiotic resistance profile of K. pneumoniae using PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is planned. The specific aims of this proposal are to: 1) compare electrospray ionization mass spectrometry (PCR/ESI-MS) of seven "housekeeping" genes in K. pneumoniae to pulsed field gel electrophopresis and rep-PCR to develop a fast and reproducible method that can applied in any facility; 2) determine the sequence of KPC-type beta-lactamases and quinolone resistance determinants in a working collection of 43 K. pneumoniae isolates; 3) Integrate the knowledge acquired from 1 and 2 to check simultaneously the ability of PCR/ESI-MS in detection of blaKPC genes and quinolone resistance determinants. By choosing genes that have minimal variability (blaKPC, gyrA, etc.) this approach promises to increase accuracy and to reduce the time needed to detect carbapenem and quinolone resistance. It is hoped that this effort will serve as a model for the development of other "bench to bedside" methods for discovering resistant phenotypes in other enteric bacilli and form the basis for the implementation of real time infection control and diagnostic paradigms. PUBLIC HEALTH RELEVANCE: This application addresses a major unmet need for rapid genotyping and antibiotic resistance profiling of Klebsiella pneumoniae. The timely and correct identification can directly influence lifesaving medical decisions regarding the proper administration of antibiotics to treat infection. We propose to develop a rapid, sensitive, and cost-effective diagnostic method that will be used to detect, identify, genotype, and determine the antibiotic resistance profile of carbapenem- and quinolone-resistant K. pneumoniae. It is our goal that this method, based upon a novel technology, will form the basis for the implementation of real time infection control and diagnostic paradigms.