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：
8891557
负责人：
JAMES E KIRBY
金额：
$ 21.75万
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/8891557
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 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 public health relevance resistance gene screening small molecule 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（由应用程序提供）：产生生物的克雷伯氏菌肺炎（KPC）是一类新兴的多药耐药细菌病原体，它们可以有效地无法治疗，或者只能用有毒抗菌药物治疗。它们对碳青霉烯的抵抗力尤其有问题，因为这些药物通常是对耐药病原体的最后防御。因此，CDC现在将这种抗碳青霉烯抗杆菌科（CRE）分类为顶级抗生素耐药性威胁水平。迫切需要新的反感染策略。碳青霉酶基因（以及对许多其他抗菌剂的耐药性）都在大的低拷贝数质粒上携带。该提案的一个基本假设是，应该有可能将这些质粒靶向“驱逐”，从而使菌株易感性。更广泛地认为，该策略也可以用于恢复对许多其他抗菌剂的抵抗。因此，在一个特定的目的中，寻求原理证明来通过质粒驱逐疗法对抗碳青霉酶的耐药性。为了实现这一目标，将制定，验证和实施筛查策略，以识别质粒维持的小分子抑制剂并恢复对碳青霉烯的易感性。（1）筛选策略基于将允许对质粒损失进行定量评估的技术。具体而言，新型的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酶抗性质粒和细菌染色体中，从而允许对质粒数的归一化测量。（2）然后将验证并执行抗质粒剂的高吞吐量屏幕。（3）将测试质粒维持的有效抑制剂，以恢复碳青霉烯敏感性，即辅助抗菌活性。（4）主要研究者有大量的CRE隔离株，将其基因组对其进行测序并注释，作为与Broad Institute CRE基因组项目合作的一部分。初步序列分析提出了共享质粒维持机制的潜力，这些机制可能代表广泛作用的抗质粒剂的靶标。彼此之间，还将测试具有辅助抗菌活性的化合物针对这种CRE收集的活性，以确定广泛作用疗法的潜力。原则证明的验证将为未来的大规模筛查工作提供依据和动力。