Plasmid Eviction to Restore Susceptibility in Carbapenem-Resistant Enterobacteriaceae

 描述（由申请方提供）：碳青霉烯类耐药肠杆菌科是一类新兴的多重耐药细菌病原体，这些病原体要么无法有效治疗，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”，从而恢复碳青霉烯对这些菌株的敏感性。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敏感性。因此，寻求通过质粒驱逐疗法对抗碳青霉烯耐药性的原理证据。为了实现这一目标，在R21阶段，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。筛选策略基于能够定量评估质粒损失的技术。具体地，新型转座子将用于将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，从而提供标准化质粒数量的容易测量的读数。然后使用中等大小的高通量筛选抗质粒剂来鉴定质粒维持的有效抑制剂。将测试抑制剂恢复碳青霉烯类药物敏感性的能力，即，抗微生物活性在R33阶段，将进行更大规模的高通量筛选，以鉴定适合进一步开发的其他强效抗质粒药物。然后将使用迭代的结构-活性关系研究来鉴定具有增强的药理学潜力的类似物。最后，将在鼠感染模型中表征选择的抗质粒剂的活性以建立体内功效。综上所述，这些实验应该为这种连续的抗菌治疗及其作为一种新的人类治疗方法的潜在用途建立理论基础。

 DESCRIPTION (provided by applicant): Carbapenem-resistant Enterobacteriaceae 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 restoring carbapenem susceptibility to these strains. Considered more broadly, this strategy might also be employed to restore susceptibility to many other antimicrobials as well. Therefore, proof of principle is sought for combating carbapenem resistance through plasmid eviction therapy. To accomplish this goal, in the R21 phase, a screening strategy will be developed, validated, and implemented to identify small molecule inhibitors of plasmid maintenance. The screening strategy is based on technology that will allow quantitative assessment of plasmid loss. Specifically, novel transposons will be used to integrate reporter genes into the carbapenemase resistance plasmid and bacterial chromosome of a screening strain, thereby providing an easily measured readout of normalized plasmid number. A mid-size high throughput screen for anti-plasmid agents will then be used to identify potent inhibitors of plasmid maintenance. The inhibitors will be tested for their ability to restore carbapenem susceptibility, i.e., adjunctive antimicrobial activity. In the R33 phase, a larger high throughput screen will be performed to identify additional potent anti-plasmid agents appropriate for further development. Iterative structure-activity relationship studies will then be used to identify analogues with enhanced pharmacological potential. Finally, the activity of select anti-plasmid agents will be characterize in murine infection models to establish in vivo efficacy. Taken together, these experiments should establish the theoretical basis for this adjunctive antimicrobial therapy and its potential use as a new human therapeutic.