VALIDATION OF A HIGH THROUGHPUT SCREEN FOR KPC PLASMID EVICTION

KPC 质粒驱逐的高通量筛选的验证

• 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
• 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

 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（由申请人提供）：产生肺炎克雷伯菌碳青霉烯酶（KPC）的生物体是一类新兴的多重耐药细菌病原体，其要么无法有效治疗，要么只能用有毒抗菌剂治疗。它们对碳青霉烯类药物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，疾病预防控制中心现在将此类碳青霉烯类耐药肠杆菌科细菌 (CRE) 归类为最高抗生素耐药性威胁级别。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药物的耐药性）携带在大的、低拷贝数的质粒上。该提议的一个基本假设是，应该有可能针对这些质粒进行“驱逐”，从而使碳青霉烯菌株变得敏感。从更广泛的角度考虑，这种策略也可用于恢复对许多其他抗菌药物的耐药性。因此，在一个具体目标中，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药物的敏感性。 (1) 筛选策略基于能够定量评估质粒损失的技术。具体来说，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，从而实现质粒数量的标准化测量。 (2) 然后将验证并进行抗质粒剂的高通量筛选。 (3) 将测试质粒维持的有效抑制剂恢复碳青霉烯敏感性的能力，即辅助抗菌活性。 (4) 主要研究者拥有大量 CRE 分离株，作为与 Broad Institute CRE 基因组项目合作的一部分，将对其基因组进行测序和注释。初步序列分析表明共享质粒维持机制的潜力，可以代表广泛作用的抗质粒剂的目标。因此，具有辅助抗菌活性的化合物也将针对该 CRE 集合进行活性测试，以确定广泛作用的治疗的潜力。原理证明的验证将为未来先导化合物鉴定和开发的大规模筛选工作提供理论依据和动力。