水源产ESBL大肠杆菌耐药质粒在小鼠肠道内水平转移规律及其分子机理研究

Accumulating evidence indicates that extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli (E. coli) has become a frequently detected antibiotic resistance bacterium in water environments. Our previous studies showed that ESBL-producing E. coli in water environments can retain and multiply in mouse intestinal tract through drinking water route; more importantly, mouse intestinal flora can become recipient bacteria of the antibiotic resistant plasmid from water-borne ESBL-producing E. coli. Based on our previous research, we speculate that water-derived ESBL-producing E. coli can horizontally transfer antibiotic-resistant plasmid to animal or human intestinal flora, therefore triggering extensive dissemination of antibiotic-resistant gene. To confirm this hypothesis, we will develop a mouse model of antibiotic resistance plasmid horizontal transfer through antibiotic resistance gene insertion and dual fluorescence labeling techniques, and further make clear distribution characterization of water-derived ESBL-producing E. coli in mouse intestinal tract, and elucidate the abundance and species of transconjugants in mouse digestive tract. Based on the experiment above, both plasmid and intestinal niche will be analyzed to explore horizontal transfer mechanism of antibiotic resistance plasmid of water-derived ESBL-producing E. coli in mouse intestinal tract via drinking water route. This proposal will clarify the molecular mechanism of water-derived antibiotic resistance gene horizontal transfer in human or animal intestinal tract from a new perspective, and provide new targets and theoretical basis for the prevention and control of antibiotic resistance genes diffusion.

产超广谱β-内酰胺酶(ESBL)大肠杆菌已成为一类广泛污染水源的耐药菌。我们前期工作已证实，水源产ESBL大肠杆菌会通过饮水途径在小鼠肠道内滞留且大量繁殖，并与肠道固有菌群之间发生了耐药质粒的水平转移。因此我们推测：当人和动物通过饮水摄入水源产ESBL大肠杆菌后，这些耐药菌株会与肠道固有菌群之间发生耐药质粒的水平转移，是造成ESBL耐药基因大面积散播的重要途径。为证实这一假说，我将通过抗性基因插入和双荧光标记技术构建水源产ESBL大肠杆菌耐药质粒水平转移的小鼠模型，探讨水源产ESBL大肠杆菌在小鼠肠道内的分布规律，明确肠道内接合子的种属和丰度，然后从质粒本身和肠道微环境等多个角度探讨饮用含有水源产ESBL大肠杆菌的水后，耐药质粒在小鼠肠道内水平转移规律及其分子机理。本课题将从新的视角阐明耐药基因水平转移的机制，为耐药性扩散的防控提供新的靶点和理论依据。

项目背景：水体环境中的耐药基因，尤其是耐药质粒，通过饮水途径进入动物和人的肠道会造成耐药基因扩散，给公共安全造成危害。但到目前为止，水体环境中耐药质粒在肠道内散播规律及其水平转移机制还有待于进一步阐述。.研究内容：本研究首先用双荧光标记技术构建水源产ESBL大肠杆菌耐药质粒(K12CAT-TD-TOMATO :W121-P-EGFP) 在小鼠肠道内水平转移模型，然后采用流式细胞分选和16S rRNA高通量测序技术分析耐药菌和耐药基因在粪便和肠道内的分布规律、菌群结构的改变和接合子种属特点，最后利用分子生物学技术探讨水源产ESBL耐药质粒在小鼠肠道内水平转移的分子机理。.重要结果：灌服水源产ESBL大肠杆菌后，小鼠肠道微生物菌群多样性显著降低；耐药质粒在肠道菌群间发生了水平转移，且盲肠是耐药质粒转移的主要场所；变形菌门是接合子的主要优势菌门，肠道大肠杆菌作为接合子在属水平丰度最高。进一步的机制研究发现，灌服水源产ESBL大肠杆菌后造成的小鼠肠道炎症，肠道菌群活性氧含量和细胞膜通透性增加，以及质粒接合转移相关基因的上调表达是引起耐药质粒在肠道内水平转移的重要驱动力。.科学意义：本研究不仅展示了水源产ESBL大肠杆菌耐药质粒通过饮水途径在小鼠肠道内进行耐药质粒水平转移的规律，而且其具体机制也得到了进一步阐述。这不仅进一步拓展了人们对耐药基因散播途径的认知，而且为水源耐药机制的散播防控也提供了相关的科学数据。