VALIDATION OF A HIGH THROUGHPUT SCREEN FOR KPC PLASMID EVICTION

 描述(申请人提供)：肺炎克雷伯菌碳青霉烯酶(KPC)产生菌是一种新兴的多重耐药细菌病原体，要么实际上无法治疗，要么只能用有毒的抗菌剂治疗。他们对碳青霉烯类抗生素的耐药性尤其成问题，因为这些药物往往是对抗耐药病原体的最后一道防线。因此，美国疾病控制与预防中心现在将这种耐碳青霉烯类肠杆菌科(CRE)归类为最高的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药的耐药性)是在大的、低拷贝数的质粒上携带的。这一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”，从而使碳青霉烯类菌株变得敏感。从更广泛的角度来看，这一策略也可能被用来恢复对许多其他抗菌素的耐药性。因此，在一个特定的目标中，寻求通过质粒驱逐疗法对抗碳青霉烯酶耐药的原则证据。为了实现这一目标，将开发、验证和实施一种筛选策略，以识别质粒维持的小分子抑制剂并恢复对碳青霉烯类抗生素的敏感性。(1)筛选策略基于的技术将允许对质粒丢失进行定量评估。具体地说，新的转座子将被用于将发光和荧光报告基因整合到碳青霉烯酶抗性质粒和筛选菌株的细菌染色体中，从而实现对质粒数量的标准化测量。(2)随后将验证并进行高通量的抗质粒药物筛选。(3)将测试有效的质粒维持抑制剂恢复碳青霉烯类药物敏感性的能力，即辅助抗菌活性。(4)首席研究员收集了大量Cre分离株，作为与布罗德研究所Cre基因组项目合作的一部分，这些分离物将对其基因组进行测序和注释。初步的序列分析表明，可能存在共享的质粒维护机制，这些机制可能代表广泛作用的抗质粒制剂的靶标。因此，还将测试具有辅助抗菌活性的化合物对这种Cre集合的活性，以确定广泛作用疗法的可能性。原则证明的确认将为今后鉴定和开发铅化合物的大规模筛选工作提供理由和动力。

 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.