鸡源沙门氏菌中质粒介导广谱头孢菌素类、多粘菌素类耐药基因（blaCTX-M、mcr-1）共传播机理研究

Salmonella is a significant zoonotic pathogen. Broad-spectrum cephalosporins are very important antimicrobial agents for the treatment of bacterial infection in humans and animals. blaCTX-M encoded extend-spectrum β-lactamases are the predominant mechanism for Salmonella resistant to broad-spectrum cephalosporins. Polymyxin is the last storage of antimicrobials for the treatment of G- bacteria. Liu YY et al. (2016) found the plasmid-mediated polymyxin resistance gene mcr-1 for the first time. Rhouma M (2016) reported co-resistance to both broad-spectrum cephalosporins and polymyxin, which brought about worldwide attention. A plasmid co-harboring blaCTX-M and mcr-1 mediating resistance to broad-spectrum cephalosporins and polymyxin was found by this applicant in chicken-origin Salmonella in 2016. This research will figure out the prevalence and genetic structure of the plasmids carrying blaCTX-M and mcr-1 in Salmonella. hereditary stability and fitness cost will be determined by competition experiment. The critical regulatory genes and proteins in the fitness compensatory mechanism will be sought through transcriptome sequencing, CRISPR-Cas and high expression vector construction. This study will illuminate the co-transmission mechanism of blaCTX-M and mcr-1 and evaluate the transmission risk, which has important scientific significance on the guidance of antimicrobials use and the control of the transmission of broad-spectrum cephalosporins and polymyxin resistance.

沙门氏菌是重要的人畜共患病原菌。广谱头孢菌素类是治疗人和动物细菌感染的重要抗生素，blaCTX-M编码超广谱β-内酰胺酶是沙门氏菌对广谱头孢菌素类耐药主要机制。多粘菌素类是人感染G-细菌的最后储存药物，Liu YY等2016首次发现质粒介导多粘菌素耐药基因mcr-1。Rhouma M 2016报道广谱头孢菌素和多粘菌素共耐药，引起世界关注。申请人2016发现鸡源沙门氏菌中携带blaCTX-M、mcr-1基因质粒，介导广谱头孢菌素类和多粘菌素类共耐药。拟采用全基因组测序、转录组测序、CRISPR-Cas、高表达载体等研究携带blaCTX-M、mcr-1基因质粒的流行和遗传结构特征、分析遗传稳定性和适应性代价、寻找关键调控基因和蛋白、探明适应性代偿机制。阐明鸡源沙门氏菌中质粒介导blaCTX-M、mcr-1基因共传播机理和风险，对指导科学用药、控制广谱头孢菌素类、多粘菌素类耐药有重要科学意义。

在该项目资助下，课题组完成了以下研究工作：1、开展了鸡源沙门菌广谱头孢菌素和多粘菌素耐药性流行情况调查，探明我国鸡源沙门菌中介导blaCTX-M 和mcr-1共传播质粒分布及遗传进化特征，发现了4株携blaCTX-M和mcr-1共传播质粒的沙门菌，带并阐明遗传结构特征，通过接合转移实验证明blaCTX-M和mcr-1共传播质粒可以在沙门菌的转移传播。2、阐明了blaCTX-M和mcr-1共传播质粒的水平接合转移频率、遗传稳定性及对沙门菌造成的适应性代价。3、全基因组测序结合转录组测序分析发现了耐药质粒适应代偿调控基因cse1，完成了cse1基因的定点敲除，获得cse1基因缺失株，探究了基因缺失后对沙门菌适应性代价的影响，确定了cse1基因在沙门菌blaCTX-M和mcr-1共传播质粒的适应性代偿调节过程中发挥一定的作用，为解决耐药质粒传播扩散问题提供新思路。4、项目执行期间，发表SCI论文5篇，全面完成项目要求的内容。质粒是介导多重耐药传播的重要水平转移元件，而鸡源沙门菌中blaCTX-M 和mcr-1共传播质粒的流行及其亚型分布情况未见报道，研究阐明鸡源沙门氏菌中blaCTX-M 和mcr-1共传播质粒的遗传进化特征、适应性代价及适应性代偿机制，对多重耐药鸡源沙门菌防治具有重要基础理论意义。