水禽源大肠杆菌中ISCR2元件协同floR基因水平传播机制研究

The insertion sequence common region (ISCR) element is a novel gene-capturing system and it may be involved in the development of drug resistance and accelerate the spread of drug resistance. However, this component responsible for the mobility of resistance gene floR from Escherichia coli isolated from waterfowl is still unclear. We have previously found that ISCR2 and floR were simultaneously located in the plasmid of Riemerella anatipestifer, and then it is speculated that ISCR2 may be involved in the dissemination of floR in the bacteria isolated from waterfowl. Therefore, on the basis of the strains preserved previously in our laboratory, E. coli isolated from waterfowl and the environmental media (soil, pond water, sediment, vegetables and so on) were collected and strains carrying both ISCR2 and floR were selected. By primer walking method, digital PCR, molecular biology, whole genome sequencing and bioinformatics analysis, the diversity of genetic contexts of ISCR2 with floR, the incompatibility group, characteristics of genetic evolution, stability of plasmids and factors influencing horizontal transfer of plasmids were studied in order to explore the mechanism of horizontal transmission of ISCR2 with floR. The expected research results can provide a new theoretical basis for understanding the molecular mechanism of horizontal transmission of floR, and provide scientific theoretical guidance for reducing and controlling the spread of the floR derived from waterfowl and the risk of drug resistance transmission.

插入序列共同区（ISCR）元件是一种新的基因捕获系统，并认为其可能参与耐药性的形成，加速耐药性的传播，然而这类元件与水禽源大肠杆菌floR耐药基因转移的关系仍不清楚。申请者前期发现，在鸭疫里氏杆菌中同时携带ISCR2和floR的质粒，推测ISCR2可能参与水禽源细菌floR基因的扩散。因此，本项目拟在实验室保存菌株的基础上，继续从水禽及环境介质（土壤、池塘水、底泥、蔬菜等）分离大肠杆菌并筛选同时含有ISCR2和floR菌株，通过引物步移法、数字PCR技术、分子生物学技术、全基因组学和生物信息学分析研究ISCR2携带 floR的基因环境多样性和可水平传播质粒的类型、遗传进化特征、稳定性和影响水平转移因素等，探讨ISCR2协同floR水平传播的机制。预期研究结果可为揭示floR水平传播的分子机制提供新的理论依据，为降低和控制水禽源floR基因的扩散和耐药性传播风险提供科学理论指导。

本项目对广东省部分地区鸭源大肠杆菌进行流行病学调查，了解鸭源大肠杆菌的耐药现状、ISCR2、floR及其他耐药基因情况流行情况，并研究ISCR2与floR共同传播的耐药机制。研究结果表明： 11个鸭场共分离出大肠杆菌654株，615株分离自麦康凯琼脂培养基，39株分离自含黏菌素LB琼脂培养基。分离菌对阿莫西林的耐药率最高，为83.0%，其次是氟苯尼考78.4%，氨苄西林77.4%，复方新诺明71.1%，多西环素69.0%。615株大肠杆菌中，mcr-1基因的阳性率为10.6%，floR基因的阳性率为74.5%，ISCR2的阳性率为11.1%，rmtB的阳性率为0.3%。从普通琼脂培养基和黏菌素耐药培养基中得到同时含有ISCR2和floR的菌株共67株。对16株不同来源的菌株的基因组和基因环境研究，发现ISCR2-hdR-floR是主要的遗传骨架，且具有不同的方向， 还包括APH(3”)-lb-APH(6)-lb-tnpA-tetR-tetA-ΔISCR2-hdfR-floR；IS110-hp-sul2-glmM-ISCR2-hdfR-floR等基因环境，并存在于不同来源的菌株之中，这表明其对酰胺醇类、氨基糖苷类、磺胺类、四环素类以及其它耐药基因的传播十分重要。mcr-1阳性菌株中，共有23株菌携带floR，11株菌携带ISCR2，8株菌中ISCR2元件存在于floR基因周围，主要以ISCR2-HP-floR-hdfR-HP-tetA-tetR和floR-hdfR-ISCR2为结构转移耐药性，其余floR阳性菌株则携带IS91家族移动元件。 23株floR基因的菌株可分为13种MLST型，最主要的是ST2144，其余的有三株ST93、ST1011，两株ST602、ST48和ST1638、ST8377、ST349、ST224、ST457、ST1414、ST542、ST410。含有ISCR2和floR的菌株分为7种MLST型，其中2株ST1011，其余的有ST410、ST542、ST349、ST2144、ST8733、ST48各占一种，说明携带ISCR2的菌株在不同ST型的菌株中广泛存在。水禽养殖场可能在通过环境以及食物链传播耐药基因方面发挥了重要的作用，因此监测水禽养殖场中耐药基因以及其他移动元件的传播趋势，寻找不同环境下耐药基因的传播驱动因素十分重要。