施肥至农田土壤的抗生素抗性基因的污染特征和传播扩散机制研究

The abuse of antibiotics in medical and breeding industry, and the emissions without criterion of pharmaceutical industry, have greatly promoted antibiotics and ARGs pollution in environment. Meanwhile, It is the widespread use of fertilization with poultry/animal feces and sludge, that cause a potential risk of rapid proliferation and spread of ARGs in soil. Therefore, the methods of RTQ-PCR and metagenomics high-throughput sequencing are used in this study, and field-investigation combining soil-microcosm simulation experiment are objective to grasp pollution situation and dynamic changes of ARGs in soil, also to explore the molecular mechanism of ARGs proliferation and spread in soil bacteria. The specific sequences of plasmids are sequenced and analyzed with bioinformatics methods, which includes those genes encoding a key structure protein assembling the cell conjugation, a start replication protein and flank linked genes. The extracellular ARGs and intracellular ARGs are then analyzed on the basis of DNA extraction, and population polymorphism comparison of soil bacteria are also conducted. Finally, the mechanism of ARGs proliferation and spread can be deduced on the basis of these results. This study will provide us the first-hand data of antibiotics and ARGs pollution in soil, and provide us the basis for setting the standards. The mechanism of ARGs proliferation and spread in soil bacteria will also provide us the way to control and manage the antibiotics and ARGs pollution in soil.

抗生素在医疗和养殖业的滥用及在制药业的无标准排放，严重污染了环境，并造成了ARGs泛滥。针对普遍采用畜禽粪便和污泥施肥农田土壤这一现状，以及这一现状下抗生素抗性基因在土壤中增殖和扩散所造成的潜在风险，本研究拟采用分子生物学研究手段RTQ-PCR和最新的Metagenomics高通量测序等技术，野外采样和土壤微宇宙室内模拟相结合，研究土壤中ARGs的消长规律，以及增殖和扩散的分子机制。通过对携带ARGs的质粒特异序列分析，包括细胞接合装置结构蛋白编码基因、复制相关基因和连锁基因等，结合抗性基因存在形态分析，以及土壤细菌种群多态性比较分析，揭示抗生素抗性基因经施肥至土壤并增殖和传播扩散的分子机制。项目研究成果将有助于了解我国土壤环境抗生素和抗性基因污染现状，为标准制定提供基础参考数据；并基于土壤介质中抗性基因增殖和扩散机制的揭示，为实际控制和治理土壤环境抗性基因提供参考。

抗生素在医疗和养殖业的滥用以及在制药业的无标准排放，严重污染了环境，并造成环境抗生素抗性基因泛滥。针对普遍采用畜禽粪便和污泥施肥农田土壤这一现状，以及这一现状下抗生素抗性基因在土壤中增殖和扩散所造成的潜在风险，本研究基于前期建立的方法，进一步优化了抗生素和抗性基因提取和检测的方法学，并分析研究了土壤中抗生素污染水平和分布特征、土壤中抗生素定量表征、土壤中抗性基因分布及消长规律、建立以RTQ-PCR技术研究土壤中抗性基因的方法、Real time-PCR方法定量表征土壤多种类抗性基因、土壤典型抗性基因的来源研究、畜禽粪便施肥土壤中的典型抗性基因的来源研究、泥施肥土壤中的典型抗性基因的来源研究、土壤中抗性基因的增殖-传播机制及影响因素研究、土壤细菌获得抗性基因和传递抗性基因的机制及影响因素研究、模拟实验研究抗性基因在土壤土著细菌间的水平转移规律、施肥土壤中抗生素抗性基因的消长规律、纳米银粒胁迫土壤细菌群落和土壤抗性基因的消长规律、新型有机溶剂-离子液体胁迫抗性基因消长规律、重金属胁迫土壤抗性基因增殖传播扩散机制。通过研究，揭示土壤中抗生素抗性基因的来源、阐明土壤环境抗生素抗性基因的污染特征、建立土壤抗生素和抗性基因分析方法、揭示土壤中抗生素抗性基因增殖和传播扩散的机制、揭示纳米材料和新型有机溶剂离子液体及重金属等污染物胁迫土壤抗生素抗性基因的消长规律。本研究成果有助于了解我国土壤环境抗生素和抗性基因污染现状，对污染现状敲响警钟，为污染排放管理政策制定提供基础参考数据，并基于土壤介质中抗性基因增殖和扩散机制的揭示，为实际控制和治理土壤环境提供科学参考。