禽源大肠杆菌IncI1型质粒流行特征及多重耐药区重组机制研究

超广谱β-内酰胺酶和16S rRNA甲基化酶是肠杆菌科细菌对β-内酰胺类和氨基糖苷类抗生素高度耐药的主要原因。blaCTX-M-65、rmtB已在动物源大肠杆菌中广泛传播。目前携带blaTEM-1、blaCTX-M-65，rmtB，fosA3的质粒多重耐药区已出现，但blaCTX-M-65和rmtB重组到一个质粒的分子机制尚不清楚。本项目拟以禽源大肠杆菌为研究对象，采用质粒分析，PCR定位、分子克隆法研究携带blaCTX-M-65和rmtB多重耐药区的侧翼序列，以新一代测序技术法对临床分离多重耐药质粒和试验诱导重组质粒进行全序列测序与比较；利用不同PCR产物构建重组质粒研究IS1294的功能，以期阐明携带blaCTX-M-65和rmtB 的IncI1型质粒流行特征及多重耐药区的重组机制，对进一步揭示blaCTX-M-65、rmtB快速传播规律有重要意义，为防控多重耐药菌提供理论依据。

Increasing resistance to β-lactams and aminoglycoside antibiotics is becoming a serious clinical problem in bacteria. High-level resistance to these antibiotics is mainly due to the extented-spectrum β-lactamase (ESBLs) and 16S rRNA methylase produced by Enterobacteriaceae. In China, blaCTX-M-65 and rmtB genes are widely spreading in Escherichia coli isolated from animal. It is worth noting that the conjugative plasmids with the multidrug-resistance regions (MRR) harbouring blaTEM-1, blaCTX-M-65, fosA3 and rmtB were recently found in Escherichia coli isolates from animal, however, the molecular mechanism which they are recombined into the same plasmid is still not clear. Plasmids from the multi-drug resistant E. coli isolates recovered from poultry were characterized by conjugation experiments, incompatibility group, restriction enzyme digestion and Southern hybridization analysis. The genetic environment of MRR harboring blaCTX-M-65 and/or rmtB was determined by PCR mapping and cloning method. A recombinant plasmid carrying blaCTX-M-65 and rmtB genes was induced by conjugation or transformation experiments. The complete sequence of the wild and recombinant plasmids were determined using a next-generation sequencing (NGS) approach. The sequence was compared using bioinformatic tools with available resistance region sequences. The role of insert sequence IS1294 in MRR was characterized by using different length of PCR products and constructing of recombination plasmids. So, the purpose of this project is to determine the epidemiological characteristics of the IncI1 plasmids in avian Escherichia coli isolates and explore the molecular mechanism which blaCTX-M-65 and rmtB are colocalized on the same conjugative plasmid. This project is also to provided theoretical basis for prevention and control disseminations of multidrug resistant Avian E. coli.