典型高浓度PM污染下环境介质和人体排泄物中抗生素残留的测定及耐药基因分布特征研究

Air is an environmental factor to depend for survival of humans and animals. Misuse of antibiotics in animal feeding would be caused residual antibiotic, resistant bacteria and antibiotic resistance genes existing in the environment, and diffusing spread with PM particles as carrier, air as media (microbial aerosol), and would be resulted in polluting food source environment, such as atmosphere, soil, and finally enters the body through the food chain. That could be made to strengthen antibiotic resistance of bacteria in the human body. Junlebao’ cattle farm is a bigger farm at Shijiazhuang of Hebei province, which PM is very serious pollution. Low concentrations, varieties and difference of property of residual antibiotic and antibiotic resistance genes at PM particles, soil and in human excreta at up and down the wind of the farm, brings to difficult analysis of testing. The project will be established HPLC-Ag(III)-luminol chemiluminescence detection new technique based on the previous research work, and coupled with HPLC/MS/MS method validation to resolve the above analysis questions in these samples at the same time. Metagenomic sequencing technique will be used to identify gut microbiota in the body. Antibiotic resistance genes, resistance gene identification and distribution characteristics will be analyzed by real time quantitative PCR technique. Through the establishment and evaluation on the new analysis method, safety of food source media of Hebei and Jing-Jin-Ji region will be given.

空气是人和动物赖以生存的环境因素，动物饲养中滥用抗生素造成环境中的抗生素残留、病原菌及耐药基因以PM颗粒物为载体、空气为媒介扩散传播，污染大气、土壤等食品源环境，并最终通过食物链进入人体，使人体内细菌的抗生素耐药性增强。针对河北PM污染重、君乐宝奶牛养殖场上下风向大气颗粒物载体、土壤介质及周边人群排泄物中抗生素残留及耐药基因浓度低、种类多、性质差异大给分析检测带来的难题，本项目拟在前期工作基础上建立高效液相色谱HPLC-Ag(III)-鲁米诺化学发光高灵敏检测新技术，并协同HPLC-MS/MS方法验证，实现养殖场上下风向空气颗粒物、土壤、人体尿液中多种类抗生素残留的同时测定；通过宏基因组测序技术鉴定人体肠道菌群的变化；应用实时定量PCR技术实现对上述分析物中耐药基因的种类鉴定及分布特征分析。通过上述抗生素残留新方法的建立及耐药基因污染分布分析等为河北及京津冀地区食品源介质安全评估提供支持。

项目背景：兽用抗生素在动物源食品生产中的过度使用及滥用，致使环境介质中的抗生素残留、病原菌及耐药基因以PM颗粒物、土壤、水等介质扩散传播；河北PM污染重，对境内规模化奶牛养殖场大气颗粒物、土壤、水质及周围人群排泄物中抗生素残留及耐药基因的调查研究对建立养殖领域细菌耐药监测网络有重要意义。主要研究内容：①建立环境中抗生素残留的高效提取技术、结合液相色谱、毛细管电泳高灵敏检测技术，协同HPLC-MS/MS方法验证，实现养殖场周围空气颗粒物、土壤、水体、人体尿液中多种抗生素残留的同时测定；②应用实时定量PCR技术实现对上述分析物中耐药基因的种类鉴定、丰度及分布特征分析。③通过第三代测序技术，分析周围环境介质中的微生物多样性。重要成果：① 制备了聚吡咯涂布尼龙-6纳米纤维膜（PPy-PA6-NFsM）、聚吡咯修饰Fe3O4磁性纳米颗粒（PPy/Fe3O4 MNPs）、三聚氰酸-咪唑树状大分子磁性功能纳米粒子（Fe3O4@SiO2-NH2-G2）、磁性纳米孔有机高分子材料（Fe3O4@SiO2-NH2-COP）等磁性功能性纳米材料吸附剂，高效吸附水、土壤、颗粒物中的抗生素残留；②结合毛细管电泳-二极管阵列检测技术、高效液相色谱分离分析技术，分别建立了奶牛养殖场周围土壤、水、空气颗粒物及人体尿液中的抗生素残留检测新方法，对β-内酰胺类、磺胺类等抗生素的检测灵敏度达到ng mL-1；③提取鉴定了养殖场周围空气颗粒物、土壤及水体中的耐药基因，给出了各类耐药基因的丰度及分布特征；结果显示，耐药基因的丰度为：颗粒物﹥土壤﹥水体。④提取并定性定量了市区河水、土壤中的抗生素残留及耐药基因的种类和丰度。通过上述抗生素残留检测新方法的建立及耐药基因的分布分析，为抗生素高暴露区域环境介质的安全评估及建立养殖领域细菌耐药监测网络提供数据支持。