猪粪中三种兽药残留与土壤微生物的互作效应研究

There are interaction effects between veterinary drug residue and environmental microorgnaism, including the effcets of veterinary drug residue on community structure, quantity and function of microorgnaism, the production and dissemination of veterinary drug-resistance genes after the addaption of microorgnaism to veterinary drug, and the microbial degradation effects on veterinary drug. This project would study the interaction effects between soil microorgnaism and doxycycline, tylosin and ciprofloxacin which were commonly used in swine industry. Through excretion experiment and field investigation, environmental substance samlples such as swine feces, soil, water and mud samples were collected and detected to obtain the concentration of veterinary drugs prototype and their main metebolite, and the concentration of microorgnaism degrading veterinary drug and veterinary drug-resistance genes. Two methods including using manure collect from swine administered with veterinary drug and directly adding of veterinary drug to soil would be used to study the effects of veterinary drug residue on community structure, quantity and function of soil microorgnaism, to confirm the fuction of veterinary drugs, their main metebolite and swine manure carrying veterinary drugs in the effects. These matrix include the swine feces containing veterinary drugs, the blank swine manure plused veterinary drugs, and the veterinary drugs were used to study the degrading behavior of doxycycline, tylosin and ciprofloxacin in soil under different environmental conditions, to analyze the variation between veterinary drugs residue and soil microorgnaism degrading veterinary drug and veterinary drug-resistance genes, and to confirm the adaption of soil microorgnaism to veterinary drugs. The project could clarify the interaction effects between veterinary drugs residue and soil microorgnaism, procide a more accurate method to study the effect of veterinary drugs residue on environmental microorgnaism, and to accuratly evaluate the ecotoxicologyl effects of veterinary drugs after they entered environment.

兽药残留与环境微生物间存在互作效应，一方面兽药残留可能影响环境微生物的群落结构、数量和功能；另一方面环境微生物可以通过适应产生和传播耐药基因，还可通过微生物降解作用减弱或消除兽药的影响。因此本项目将以养猪生产中常用的强力霉素、泰乐菌素和环丙沙星为研究对象，通过排泄试验和现场调研采样检测猪粪、土壤等环境介质中三种兽药及其主要代谢产物的含量，并检测样品中可降解兽药微生物含量和兽药耐药基因水平。采用经体内代谢法和直接加入法研究兽药残留对土壤微生物群落结构、数量和功能的影响，确定兽药原形、主要代谢产物、兽药的载体-猪粪在其中的作用；研究不同环境条件下土壤微生物对兽药残留的降解效应，分析土壤中可降解兽药微生物和兽药耐药基因的变化规律，确认土壤微生物对兽药残留的适应。本项目可阐明兽药与土壤微生物的互作效应，为研究兽药残留对环境微生物的影响提供更为准确地方法，更为准确地评价兽药的生态毒理学效应。

兽用抗生素可能影响环境微生物的数量、结构和功能；环境微生物可通过适应产生和传播耐药基因，而且还可主动降解兽用抗生素。因此，明确兽用抗生素与环境微生物间的互作效应对于兽用抗生素生态毒理学评价具有重要意义。本项目首先采集了广东省18家不同规模养猪场猪粪、污水和土壤样品，检测了环丙沙星和泰乐菌素浓度及相应耐药基因含量；然后分别以泰乐菌素A（TYLA）、环丙沙星（CIP）和强力霉素（DOX）为研究对象，研究兽用抗生素与土壤微生物的互作效应。结果表明，所采样品中环丙沙星检出率最高，其中猪粪中环丙沙星浓度范围为14.01mg/kg~316.75mg/kg；各样品中也可检出兽用抗生素耐药基因，其中猪场原水中四环素类耐药基因拷贝数的对数值高达12.39/L。添加TYLA未显著影响土壤细菌总数，但可降低土壤微生物群落结构多样性；TYLA的加入可增加土壤中大环内酯类耐药基因ermB和ermF的数量，而且微生物对土壤中TYLA的降解起重要作用，如由于采用空白土壤+TYLA组细菌总数和无丙二酸柠檬酸杆菌数量显著低于其它两种试验组，进而导致该组土壤中TYLA降解最慢。添加CIP可显著降低土壤细菌总拷贝数，改变土壤微生物的群落结构，而且这种影响与CIP的添加方式有关，如经体内代谢含CIP猪粪+空白土壤组土壤细菌总数最高，空白猪粪+CIP+空白土壤组次之，CIP+空白土壤组最低；土壤微生物对CIP在土壤中的降解有促进作用，而且除aac(6’)-Ib-cr外，CIP的加入不会促进耐药基因gryA、parC在土壤中的扩散。DOX可抑制土壤细菌、放线菌总数，而且还可增加产碱杆菌属（Alcaligenes）相对丰度，降低拟杆菌目（Bacteroidales）相对丰度；DOX的加入浓度高时，对耐药基因tetA、tetM、tetW、tetX的扩散起抑制作用；微生物对土壤中DOX的降解起主要作用，表现为非灭菌处理组强力霉素的降解半衰期显著快于相应灭菌处理组。本项目还从长期施用粪肥的菜地土壤分离纯化出9株强力霉素耐药菌，其中以埃希氏杆菌降解性能最强，降解率为92.52±0.33%。综上所述，TYLA、CIP和DOX可显著影响土壤微生物的数量和结构，微生物也可通过耐药性产生适应，并启动兽用抗生素的微生物降解。本项目阐明了兽用抗生素与土壤微生物间存在互作效应，能更准确地评价兽用抗生素的生态毒理学效应。