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

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.

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。

超广谱β-内酰胺酶（ESBLs）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。本项目研究了禽源大肠杆菌携带IncI1质粒的流行特征，重点解析了5个IncI1和1个IncZ的质粒全序列，已获得NCBI序列号KY748188（pEC006），KY748189（pEC007），KY748190 （pEC008），KT282968（pEC012），MG545909（pEC013），MG545910（pEC014）。pEC012质粒全长为139，622 bp，携带有blaTEM-1，blaCTX-M-65，rmtB，fosA3，floR，aac(3)-IV，oqxAB等多个耐药基因，由IS26构成的多个复合型转座子组成一个复杂的多重耐药区（MRR），特别是IS26-fosA3-orf1-orf2-∆orf3-IS26、IS26-fip-∆ISEcp1-blaCTX-M-65-IS903D-iroN-IS26、IS26-tnpR-blaTEM-1-rmtB-IS26的重组方式，进一步解释了携带blaCTX-M-65，rmtB，fosA3多重耐药区的重组机制。pEC012还携带floR和oqxAB的IS26复合型转座子，也构成了MRR的一部分，这些结构表明由IS26构成的复合型转座子在多重耐药区的形成中发挥了重要作用。另外，在pEC012首次发现了一个新的插入序列元件ISEc57（https://www-is.biotoul.fr//）。3个IncI1 ST136质粒pEC006～pEC008全序列测定表明，pEC006质粒的接合转移区traI基因由于被移动元件IS10L插入截断而失去接合转移能力，pEC008质粒含有一个复杂的多重耐药区：耐药基因blaTEM-1、rmtB、aacC2、tetA、folR、strAB和1型整合子 （intI1-dfrA12-orfF-aadA2-qacE∆1-sul1）经Tn2、Tn1721、Tn1722、Tn5393、ISCfr1、IS5057、ISCR1、ISCR、IS26等多个转座子和插入序列重组成一个复杂的嵌合体。该项目进一步解析了携带blaCTX-M和rmtB的MRR重组机制，进一步引起公众的注意和形成共识：合理使用抗生素，预防和控制耐药细菌的传播，促进人类、动物的健康发展。