Detecting Carbapenem and Quinolone Resistance in Klebsiella pneumoniae using PCR/

描述（由申请人提供）：在革兰氏阴性菌中，对碳青霉烯类和喹诺酮类耐药的肺炎克雷伯菌引起的医院感染正迅速成为一个全球性问题。新出现的亚胺培南耐药主要由KPC β-内酰胺酶介导;喹诺酮类耐药是由于gyrA和parC的喹诺酮类耐药决定区（QRDR）突变以及获得质粒介导的喹诺酮类耐药基因qnrA、-B、-S、修饰酶（aac（6 '）-Ib-cr）和外排泵所致。对碳青霉烯类和环丙沙星耐药的克雷伯菌的早期检测与分析。肺炎将极大地帮助临床医生治疗严重感染的患者。为此，建立了一种快速、灵敏、高通量的检测、鉴定、基因分型和确定克雷伯氏菌抗生素耐药性的方法。计划使用PCR结合电喷雾电离质谱法（PCR/ESI-MS）检测肺炎链球菌。本研究的具体目的是：1）比较K. pneumoniae pneumoniae的脉冲场凝胶电泳和rep-PCR，以建立一种快速和可重复的方法，可以应用于任何设施; 2）确定的序列的KPC型β-内酰胺酶和喹诺酮类药物耐药决定簇的工作收集43 K。肺炎分离株;（3）将1和2中的知识结合起来，同时检测PCR/ESI-MS检测blaKPC基因和喹诺酮类耐药决定簇的能力。通过选择具有最小变异性的基因（blaKPC、gyrA等），该方法有望提高准确性并减少检测碳青霉烯和喹诺酮耐药性所需的时间。希望这一努力将作为一种模式，用于开发其他“从实验室到床边”的方法，以发现其他肠杆菌的耐药表型，并形成实施真实的时间感染控制和诊断范例的基础。公共卫生相关性：本申请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关于适当施用抗生素以治疗感染的挽救生命的医疗决定。我们建议开发一种快速、灵敏、经济有效的诊断方法，用于检测、鉴定、基因分型和确定碳青霉烯和喹诺酮类耐药克雷伯菌的抗生素耐药性。肺炎。我们的目标是，这种基于新技术的方法将形成实施真实的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.