抗生素胁迫下大肠埃希菌和质粒的适应性进化导致耐药水平增高的机制研究

Antibiotic resistance is a serious threat to human health and new ways to combat it are urgently needed. The worldwide increase in antibiotic resistance has motivated numerous studies aimed at understanding the phenotypic and genotypic evolution of antibiotic resistance. A key aspect of bacterial survival is the ability to evolve while migrating across spatially varying environmental challenges. In our preliminary study, We found that under the treatment of antibiotic, the copy number of plasmid, plasmid stability with and without antibiotic, plasmid transfer frequency and minimal inhibitory concentration of E. coli SM10λπ (pUCP24T) with continuously subculture had been enhanced significantly. However, little is known about the mechanisms. We speculated that antibiotic selection drove plasmid-host compensator revolution and adaptive evolution, resulting in increased resistance ability, with a potential molecular mechanism relating to genes mutations and differential expression in host and plasmid. In this project, to illuminate the mechanisms of antibiotic resistance strengthened by adaptive evolution of E. coli and plasmid under antibiotic stress, we will: ①obverse the biology changes of E. coli harboring resistance plasmids after continuously subculture under the treatment of antibiotic, the biology changes including minimal inhibitory concentration, the fitness cost imposed by resistance plasmids on E. coli hosts, the copy number of the resistance plasmids, plasmid stability with and without antibiotic, and plasmid transfer frequency; ②explore the adaptive adaptation to this fitness cost by chromosomal and/or plasmid mutations using whole-genome sequencing; ③identify differential expression genes and small non-coding RNA (sRNA) by RNA sequencing; ④confirm the functions of those candidate differential expression genes and sRNA regulating the adaptive evolution by over-expression and knockout technology. We hope to provide rationale strategies to blocking-up horizontal gene transfer, ameliorate antibiotic resistance mediated by plasmids, provide the drug target for novel antibiotics discovery studies and enrich the theory of antibiotic resistance.

在抗细菌感染中，细菌因适应性进化导致耐药已显著占据上风。前期发现，抗生素胁迫下，携带耐药质粒的大肠埃希菌连续多次传代后对抗生素的MIC、质粒拷贝数、质粒稳定性和质粒转移能力均显著增高，机制尚不清楚。我们假设，在选择压力胁迫下，细菌与质粒相互适应性进化并导致其耐药能力增强，潜在的分子机制可能涉及细菌和质粒的基因突变及差异表达。本课题拟研究：①抗生素胁迫下，携带耐药质粒的大肠埃希菌连续多次传代后的生物学变化；②高通量测序检测多次传代后细菌全基因组和质粒的基因突变和突变导致的结果；③转录组测序检测多次传代后细菌和质粒的差异表达基因和sRNA；④筛选调节适应性变化的差异表达基因和sRNA，过表达和基因敲除这些基因，明确其对适应性进化的调控作用，阐明抗生素作用下细菌和质粒适应性进化和导致耐药能力增强的分子机制，为阻断耐药基因通过质粒水平转移、为抗菌药物研发提供新靶点和丰富细菌耐药发生的理论提供依据。

细菌耐药对全球公共卫生健康造成巨大威胁，研究耐药进化机制对遏制细菌耐药具有重要意义。本课题研究了抗生素压力下，宿主菌和质粒发生耐药性进化的机制。采用接合试验和药敏实验研究了抗生素胁迫下，携带耐药质粒的宿主菌多次传代后发生的耐药性进化情况，采用生长曲线、配对竞争、质粒稳定性实验等方法检测耐药性进化前后宿主菌的适应代价变化，并进一步采用全基因组测序、RNA-seq、real-time PCR、基因敲除与过表达、报告系统构建等一系列细胞和分子生物学手段，研究了质粒与宿主菌耐药性进化后的基因突变位点、功能和差异表达sRNA对细菌适应性和耐药性的影响。研究发现持续抗生素压力下，大肠埃希菌和不同类型质粒发生了不同的耐药性进化，并从基因组和转录组层面分别揭示了细菌耐药性进化机制。在E. coli SM10λπ (pUCP24T)进化模型中发现：1) 30 μg/mL庆大霉素持续压力下，E. coli SM10λπ (pUCP24T) 发生了接合频率增高的耐药性进化；2) E. coli SM10λπ (pUCP24T)发生耐药性进化时伴随着适应性代价；3) 通过转录组测序结合生物信息学分析筛选出庆大霉素作用下发生表达变化且可能参与接合调控的非编码小RNA（sRNA）IsrC和GcvB；4)过表达sRNA IsrC和GcvB接合反应受到抑制，提示sRNA IsrC和GcvB调控大肠埃希菌的接合反应。在E. coli C600 (pNDM-5)进化模型中发现：1) 4 μg/mL美罗培南压力下E. coli C600 (pNDM-5)发生耐药性进化，耐药性增强且接合频率增高；2) E. coli C600 (pNDM-5) 发生耐药性进化未伴随适应性代价；3) pNDM-5质粒的复制蛋白repA D140Y (GAT→TAT)突变导致质粒拷贝数增高，从而介导了细菌耐药性进化。在E. coli C600 (pGZ49260)进化模型中发现：2 μg/mL多粘菌素B压力下E. coli C600 (pGZ49260)的适应性和耐药性未发生明显变化。研究结果阐明了抗生素压力下宿主菌与质粒发生耐药性进化的新机制，拓展了sRNA的生物学功能，具有较大的科学意义。