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VALIDATION OF A HIGH THROUGHPUT SCREEN FOR KPC PLASMID EVICTION

KPC 质粒驱逐的高通量筛选的验证

基本信息

批准号：
8990439
负责人：
JAMES E KIRBY
金额：
$ 26.1万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8990439
关键词：
Anti-Infective Agents Antibiotic Resistance Antibiotics Bacterial Chromosomes Biological Assay Carbapenems Centers for Disease Control and Prevention (U.S.)Chromosomes Clinical Microbiology Collaborations Collection DNA Development Drug resistance Escherichia coli Frequencies Funding Future Genome Goals Health Infection Institutes Klebsiella Klebsiella pneumonia bacterium Label Lead Learning Maintenance Measures Multi-Drug Resistance Organism Output Performance Plasmids Predisposition Principal Investigator Reporter Reporter Genes Resistance Sequence Analysis Signal Transduction Specificity Staging Technology Testing Therapeutic Validation Work antimicrobial base carbapenem resistance carbapenem-resistant Enterobacteriaceae carbapenemase clinical efficacy combat drug resistant bacteria genome sequencing high throughput screening inhibitor/antagonist meetings novel pathogen resistance gene screening small molecule inhibitor technology development

项目摘要

DESCRIPTION (provided by applicant): Klebsiella pneumonia carbapenemase (KPC)-producing organisms are an emerging class of multi-drug resistant bacterial pathogens that are either effectively untreatable or only treatable with toxic antimicrobials. Their resistance to carbapenems is especially problematic, as these agents are often the last line of defense against drug-resistant pathogens. Therefore, the CDC now categorizes such carbapenem-resistant Enterobacteriaceae (CRE) in their top antibiotic resistance threat level. New anti-infective strategies are urgently needed. Carbapenemase genes (and resistance to many other antimicrobials) are carried on large, low copy number plasmids. An underlying hypothesis of this proposal is that it should be possible to target these plasmids for "eviction", thereby rendering strains carbapenem susceptible. Considered more broadly, this strategy might also be employed to restore resistance to many other antimicrobials as well. Therefore, in one specific aim, proof of principle is sought for combating carbapenemase resistance through plasmid eviction therapy. To accomplish this goal, a screening strategy will be developed, validated, and implemented to identify small molecule inhibitors of plasmid maintenance and restore susceptibility to carbapenems. (1) The screening strategy is based on technology that will allow quantitative assessment of plasmid loss. Specifically, novel transposons will be used to integrate luminescent and fluorescent reporter genes into the carbapenemase resistance plasmid and bacterial chromosome of a screening strain, thereby allowing a normalized measure of plasmid number. (2) A high throughput screen for anti-plasmid agents will then be validated and performed. (3) Potent inhibitors of plasmid maintenance will be tested for their ability to restore carbapenem susceptibility, i.e., adjunctive antimicrobial activity. (4) The principal investigator has a large collection of CRE isolates that will have their genomes sequenced and annotated as part of a collaboration with the Broad Institute CRE genome project. Preliminary sequence analysis suggests potential for shared plasmid maintenance mechanisms that could represent targets for broadly acting anti-plasmid agents. Accordingly, compounds with adjunctive antimicrobial activity will also be tested for activity against this CRE collection to establish the potential for broadly acting therapeutics. Validation of proof of principle would provide rationale and impetus for future large scale screening efforts for lead compound identification and development.

描述（由申请方提供）：产肺炎克雷伯菌碳青霉烯酶（KPC）微生物是一类新兴的多重耐药细菌病原体，其无法有效治疗或仅可使用毒性抗菌剂治疗。它们对碳青霉烯类的耐药性尤其成问题，因为这些药物通常是抵抗耐药病原体的最后一道防线。因此，CDC现在将这些碳青霉烯类耐药肠杆菌科（CRE）归类为最高抗生素耐药威胁级别。迫切需要新的抗感染策略。碳青霉烯酶基因（以及对许多其他抗菌剂的耐药性）携带在大的低拷贝数质粒上。这一提议的一个基本假设是，应该可以靶向这些质粒进行“驱逐”，从而使菌株对碳青霉烯敏感。更广泛地考虑，这种策略也可以用于恢复对许多其他抗菌剂的耐药性。因此，在一个特定目的中，寻求通过质粒驱逐疗法对抗碳青霉烯酶耐药性的原理证明。为了实现这一目标，将开发、验证和实施筛选策略，以鉴定质粒维持的小分子抑制剂并恢复对碳青霉烯类的敏感性。(1)筛选策略基于可定量评估质粒丢失的技术。具体而言，新型转座子将用于将发光和荧光报告基因整合到筛选菌株的碳青霉烯酶抗性质粒和细菌染色体中，从而允许质粒数量的标准化测量。(2)然后将验证并进行抗质粒试剂的高通量筛选。(3)将检测质粒维持的有效抑制剂恢复碳青霉烯敏感性的能力，即，抗微生物活性(4)主要研究者收集了大量的CRE分离株，这些分离株将进行基因组测序和注释，作为与布罗德研究所CRE基因组项目合作的一部分。初步的序列分析表明，共享的质粒维护机制，可以代表广泛作用的抗质粒剂的目标的潜力。因此，还将测试具有连续抗微生物活性的化合物对该CRE集合的活性，以确定广泛作用的治疗剂的潜力。验证的原则证明将提供理论基础和动力，为未来的大规模筛选工作的铅化合物的识别和发展。