抗性基因（ARGs）的环境污染已经成为备受国际瞩目的公共安全问题。城镇污水厂作为城市-城郊关键带的重要组成部分，其出水的排放过程是人类活动富集的ARGs向自然环境扩散的重要环节。近些年，城镇污水厂出水排放引起的自然水体抗性基因组富集现象已经引起学界重视，但目前的研究对出水受纳水体中ARGs的富集机理尚不明确，主要表现为:1)出水中不同营养组份对不同ARGs宿主菌群的选择性富集作用不明确；2)ARGs在受纳水体微生物群系中的水平基因转移扩散规律也缺乏深入研究。针对以上研究不足，本项目拟利用基于三代单分子纳米孔测序的宏基因组学方法，系统调研出水水质等环境因子对受纳水体中ARGs环境行为的影响。同时借助纳米孔测序得到的DNA序列甲基化信息，鉴定多重耐药菌携带的可移动质粒并追踪其原始宿主，解析水体中由可移动性质粒介导的ARGs水平基因转移规律。

The widespread of antibiotic-resistant pathogens and the related increase of human morbidity and mortality has aroused global concern on the dissemination of antibiotic resistance genes (ARGs) in the environment. Domestic wastewater treatment plants (WWTPs) as the intersect between the urban and natural environment, are receiving increasingly more attention to their role in ARGs dissemination. Recent studies had revealed the effluent of WWTPs could bring about evident ARGs enrichment in the receiving water, however, the microbial mechanism and influential factors of such resistome augmentation have not been sufficiently studied. Additionally, different from a chemical pollutant, ARGs could propagate in the environment not only by the reproduction of carrying microbes but also by horizontal gene transfer (HGT) among different microbial populations. Such “mobility” of ARGs largely determines the likeliness they got transferred into human pathogens in the environment and thus the health risk associated with them. However, effective ARG-HGT identification protocol is still lacking. Nanopore sequencing is a promising technology to solve the problem, not only because it could capture genetic information long enough for direct identification of ARG carriers and their mobile gene elements, but also the DNA methylation pattern provided by nanopore signal could serve as a brand-new tool to reveal the exogenous conjugative plasmids that disseminated among ARG carriers. However, at this very early stage, there are very few applications of nanopore sequencing in studying the environmental behavior of ARGs. Therefore, in the proposed study, we planned to decipher the influential factors as well as the HGT mechanism of ARGs propagation from wastewater treatment plants (WWTPs) to receiving water using nanopore-based metagenomics. We meat to carry out our study in the following aspects:1) establish nanopore-based ARGs identification methodology; 2) reveal environmental factors that may play a determinative role in regulating ARGs increment in receiving water and decipher the metabolic machinery underpin the selective enrichment of particular ARG carrier; 3) use DNA methylation pattern to disclose ARG-HGT enabled by conjugative plasmids transmission during resistome enrichment in receiving water.