大黄鱼中抗生素耐药基因从养殖到销售过程中的分布特征及播散机制

Antibiotic resistance has become a major public health concern. As for the fact of the spread of antibiotic resistance in aquaculture environment and aquaculture products, this project will target at Pseudosciaena crocea, one of the major mariculture species in our country, systematically investigate the distribution characteristics and dissemination mechanism of antibiotic resistance genes (ARGs) in Pseudosciaena crocea during the process from farm to market, on both microbiota level and strain level. The bacterial community structure, the variety and abundance of ARGs and mobile genetic elements (MGEs) in fish and water samples at different stage will be measured by metagenomics combined with quantitative real-time PCR, and their relationship on microbiota level will be analyzed using biostatistics; Then at the strain level, microbial drug resistance measurement and molecular biology techniques such as PCR detection, Southern blot, conjugation test, phylogeny analysis and sequencing of multidrug resistant plasmids, will be used to study the horizontal gene transfer of representative ARGs mediated by MGEs, so as to illustrate the impact of different stages, during the process from farm to market, on the distribution of ARGs in Pseudosciaena crocea, the critical control points of ARGs pollution, and the dissemination mechanism of representative ARGs. Results from this project will provide a scientific basis for early warning and effective control of foodborne antibiotic resistance pollution resulting from ARGs in aquaculture products, also contribute to ensure the safety of food and public health.

抗生素耐药性已成为人类健康巨大威胁。针对抗生素耐药性在水产养殖环境及水产中泛滥现状，本项目拟以我国海水养殖量最大的鱼类之一（大黄鱼）为考察对象，从菌群和菌株水平系统研究大黄鱼中抗生素耐药基因（ARGs）从养殖到销售过程中的分布特征和播散机制。通过宏基因组学结合实时荧光定量PCR考察不同环节大黄鱼及养殖水样中菌群结构及ARGs、可移动基因元件（MGEs）的种类和丰度，通过生物统计法分析ARGs与菌群结构、MGEs在菌群水平的相关性，通过在菌株水平考察耐药表型结合PCR检测、Southern杂交、接合转移、同源性分析、多重耐药质粒测序等分子生物学方法研究典型ARGs由MGEs介导的水平转移，以揭示从养殖到销售过程的环节差异对大黄鱼中ARGs分布的影响、ARGs污染控制关键环节及典型ARGs的播散机制。为水产中ARGs引起的食源耐药性污染的预警和有效控制提供科学依据，保障食品安全和公共卫生安全。

抗生素耐药性已成为人类健康巨大威胁。本项目旨在研究大黄鱼中抗生素耐药基因（ARGs）从养殖到销售过程中的分布特征和播散机制。高通量测序测定各阶段鱼样的菌群结构，结合PCoA分析发现各阶段菌群结构存在差异。平板涂布法测定了复合磺胺、四环素、环丙沙星、氯霉素、头孢噻肟和链霉素的耐药菌数量，表明检测的养殖和市售鱼及养殖海水样都含耐药菌，分别为10~10^9CFU/g、100~10^5CFU/g和10~10^3CFU/g。高通量测序表明养殖阶段鱼样的三类耐药菌的优势菌是革兰氏阴性菌，而市售阶段样品中革兰氏阳性菌占比升高。定量PCR结果表明所有样品都检出intI1、aac(6')-Ib、aadA1、blaTEM、sul1和sul2。ARGs平均丰度为5.3x10^-4~1.59x10^-1/16S rRNA，sul1在所有样品中丰度高。市售样品ARGs丰度总量低于养殖阶段。intI1平均丰度为6.97x10^-2/16S rRNA，和sul1、tetE强正相关。网络分析表明养殖阶段ARGs持留菌多为Proteobacteria，市售阶段则多为Firmicutes。一些ARGs和其持留菌贯穿于整个供应链。抗生素平板初筛发现，95.42%的菌株为多重耐药菌。药敏实验和MIC测定表明大部分菌株对10种以上抗生素耐受且有较高MIC值。利用PCR在883株多重耐药菌中检出了23种耐药基因，检出最多为sul1（42.92%）。利用16S rDNA测序鉴定耐药基因携带菌株得9个菌属，即柠檬酸杆菌、变形杆菌、假单胞菌、摩根氏菌、希瓦氏菌、不动杆菌、气单胞菌、芽孢杆菌和产碱杆菌。质粒检出率为16.35%且大多编码耐药基因。PCR检测发现39.75%的菌有intI1，8.38%的菌有intI2。在I类整合子中检出11种基因盒排列：arr-3+aac(6')-Ib、arr-3+dfrA27、dfrA12+DUF100+aadA2、aadA1、aadA4、aadA5、blaCARB-2、catB3、dfrA1、dfrA5、aacA34。养殖和市售鱼样分离菌株分别检测到4种、8种耐药基因盒。同源性分析表明耐药基因盒可在不同宿主、菌属中传播。结果表明市售阶段ARGs潜在污染源不同于养殖阶段，需要被重视。研究结果为水产中ARGs引起的食源耐药性污染的预警和有效控制提供科学依据，保障食品安全和公共卫生安全。