Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant pathogens for which new treatments are desperately needed. Carbapenemases and other types of antibiotic resistance genes are carried almost exclusively on large, low-copy-number plasmids (pCRE). Accordingly, small molecules that efficiently evict pCRE plasmids should restore much-needed treatment options. We therefore designed a high-throughput screen to identify such compounds. A synthetic plasmid was constructed containing the plasmid replication machinery from a representative Escherichia coli CRE isolate as well as a fluorescent reporter gene to easily monitor plasmid maintenance. The synthetic plasmid was then introduced into an E. coli K12 tolC host. We used this screening strain to test a library of over 12,000 known bioactive agents for molecules that selectively reduce plasmid levels relative to effects on bacterial growth. From 366 screen hits we further validated the antiplasmid activity of kasugamycin, an aminoglycoside; CGS 15943, a nucleoside analog; and Ro 90-7501, a bibenzimidazole. All three compounds exhibited significant antiplasmid activity including up to complete suppression of plasmid replication and/or plasmid eviction in multiple orthogonal readouts and potentiated activity of the carbapenem, meropenem, against a strain carrying the large, pCRE plasmid from which we constructed the synthetic screening plasmid. Additionally, we found kasugamycin and CGS 15943 blocked plasmid replication, respectively, by inhibiting expression or function of the plasmid replication initiation protein, RepE. In summary, we validated our approach to identify compounds that alter plasmid maintenance, confer resensitization to antimicrobials, and have specific mechanisms of action.

耐碳青霉烯肠杆菌科 (CRE) 是耐多药病原体，迫切需要新的治疗方法。碳青霉烯酶和其他类型的抗生素抗性基因几乎完全由大的低拷贝数质粒 (pCRE) 携带。因此，有效驱逐 pCRE 质粒的小分子应该恢复急需的治疗选择。因此，我们设计了一个高通量筛选来识别此类化合物。构建了一个合成质粒，其中包含来自代表性大肠杆菌CRE 分离株的质粒复制机制以及荧光报告基因，可轻松监测质粒维护。然后将合成质粒导入大肠杆菌K12 tolC主持人。我们使用这种筛选菌株测试了一个包含 12,000 多种已知生物活性剂的库，这些分子可以选择性地降低质粒水平（相对于对细菌生长的影响）。通过 366 次筛选，我们进一步验证了春日霉素（一种氨基糖苷类）的抗质粒活性；CGS 15943，一种核苷类似物；和 Ro 90-7501，一种联苯并咪唑。所有三种化合物都表现出显着的抗质粒活性，包括在多个正交读数中完全抑制质粒复制和/或质粒驱逐，以及碳青霉烯美罗培南对携带大 pCRE 质粒的菌株的增强活性，我们从中构建了合成筛选质粒. 此外，我们发现春雷霉素和 CGS 15943 分别阻止了质粒复制，通过抑制质粒复制起始蛋白 RepE 的表达或功能。总之，我们验证了我们的方法来识别改变质粒维持的化合物，赋予对抗菌剂的再敏感性，并具有特定的作用机制。