鸡源沙门氏菌中多重耐药基因岛SGI1新亚型及与毒力、生物被膜的相关性阐明

Salmonella enterica is a zoonotic pathogen that threatens to the health breeding of livestock and poultry, as well as the safety of animal products. Layer-origin S. enterica can cause illness of layer and pose a threat to chicken and egg products. In the previous study, a lot of S. enterica strains were isolated from layer farms in twenty-five provinces, China. Antimicrobial susceptibility test showed seventy percent of strains displayed multidrug resistance (MDR) profiles. Salmonella genomic island 1 (SGI1) is a chromosomal mobilizable element mediating MDR. SGI1 carrying S. enterica appears to have higher virulence and biofilm formation ability. We characterized three novel SGI1 variants (SGI1-W, SGI1-X and SGI1-Y) in 2014. In the present study we will detect the SGI1 in layer-origin S. enterica isolates and clarify the novel variants and genetic characteristics by using Tail-PCR and whole-genome scan. Then the horizontal transfer ability, hereditary stability and fitness cost will be figured out by using conjugation, competition and continuous passage experiment. The pathogenicity test and scanning electron microscope will be used to confirm the virulence and biofilm formation of transconjugants. It will be verified by gene knock-out, cloning and expression that whether SGI1 is correlated with virulence and biofilm formation. This study will clarify the genetic characteristic of SGI1 and its relation to virulence and biofilm in layer-origin S. enterica isolates, which has crucial theoretical significance in the prevention and control of MDR layer-origin S. enterica.

沙门氏菌是一类重要的人畜共患病原菌，严重危害畜禽健康养殖和动物产品安全。鸡源沙门氏菌引起鸡发病和影响蛋品、鸡肉安全。申请人前期积累了来自我国25个省的鸡源沙门氏菌分离株，药敏结果显示菌株多重耐药率高达70%。沙门氏菌基因岛SGI1是介导多重耐药的重要水平转移元件，携带SGI1的沙门氏菌具有更强的毒力和生物被膜形成能力。2014年申请人发现了三个SGI1新亚型（SGI1-W、SGI1-X、SGI1-Y）。拟进一步调查SGI1的流行特征，采用Tail-PCR、全基因组扫描分析SGI1的遗传进化特征和新亚型；采用接合转移、竞争试验等阐明SGI1的水平转移能力、适应性代价及遗传稳定性；通过致病性试验、电镜扫描、基因敲除、克隆表达等阐明SGI1与沙门氏菌毒力、生物被膜的相关性。研究将阐明鸡源沙门氏菌中SGI1的遗传进化特征及与毒力、生物被膜相关性，对多重耐药鸡源沙门氏菌防治具有重要基础理论意义。

在该项目资助下，课题组完成了以下研究工作：1、鸡源沙门氏菌中多重耐药基因岛SGI1新亚型鉴定及遗传进化特征阐明：开展了沙门氏菌多重耐药基因岛SGI1的流行情况调查，探明其亚型分布及遗传进化特征，发现SGI1-XJ9S、PGI2、SGI1-PmBC1123、SGI1-PmSC1111、AGI1-D等新亚型并阐明遗传结构特征。2、鸡源沙门氏菌中SGI1的水平转移能力、适应性代价及遗传稳定性研究：采用接合转移实验证实SGI1及其新亚型具有水平接合转移能力；连续传代培养及适应性代价研究表明，SGI1新亚型对沙门氏菌引起的适应性代价低，具有较高的传播扩散风险。3、SGI1与沙门氏菌毒力、生物被膜形成的相关性阐明：采用小鸡口服菌液的方式比较SGI1阳性沙门氏菌、接合子及受体菌的毒力差异，证明SGI与沙门氏菌毒力相关；分别采用试管法和微孔法测定肠炎沙门氏菌获得SGI1基因岛新亚型前后生物被膜形成能力，表明肠炎沙门氏菌获得SGI1后的生物被膜形成能力显著增强。项目执行期间，发表SCI论文6篇，中文核心期刊1篇，申请专利3项，全面完成项目要求的内容。SGI1是介导多重耐药的重要水平转移元件，研究阐明了鸡源沙门氏菌中SGI1的遗传进化特征、适应性代价及与毒力、生物被膜的相关性，对多重耐药鸡源沙门氏菌防治具有重要基础理论意义。