抗生素抗性基因（Antibiotic Resistance Genes，ARGs）作为一种新兴污染物，不仅具有一般污染物的污染特性，还可以通过水平基因转移（Horizontal gene transfer，HGT）进入新的环境、微生物甚至人体内，对环境以及人体健康构成威胁。再生水补给河湖在解决水资源短缺的同时，也造成了ARGs的生态风险。为了解补水中ARGs的分布及转移，分别在不同季节采集潮白河顺义段补水场地的地表水和再生水、土壤和地下水样品，分析了ARGs（sul1、sul2、dfrA1、dfrA12、tetB、tetO、tetW、tetM）分布及环境因素的影响，通过土柱模拟及U型管研究了ARGs在介质中的HGT及影响因素。 对地表水样的分析发现，除了tetO及tetB外，ARGs的检出率均为100%；ARGs的丰度表现为磺胺类>甲氧苄氨嘧啶类>四环素类；sul2在研究区内相对丰度最高。夏季和秋季样品中ARGs的丰度显著高于春季和冬季。不同介质样品分析发现，地下水中ARGs丰度排序与再生水一致。总体而言，sul2、sul1和I型整合子（Class 1 integron，intI1）应为本研究区内首要关注的ARGs。 地表水样品的常规指标分析发现，再生水补水对地表水水质产生了一定的影响。高通量测序结果表明地表水中以假单胞菌属（Pseudomonas）的相对丰度最高，且夏秋两季的物种多样性高于春季和冬季。统计学分析表明，intI1与ARGs普遍具有显著的相关关系，R2最高可达0.974。多种微生物种属与ARGs相对丰度之间存在相关性，特别是夏季和秋季的不动杆菌（Acinetobacter）。主成分分析表明，不同环境介质中ARGs分布的主要影响因素包括intI1、磺胺甲恶唑、甲氧苄氨嘧啶以及重金属Cu、Zn。 室内土柱模拟结果发现细胞外ARGs更容易穿过包气带进入地下水系统，对于ARGs在再生水补水入渗中可能发生的HGT需要深入研究。利用U型管实验获得了细胞外ARGs在再生水环境中的转移接合子，其转移率占总的接合转移率的7.29%。多重选择压力可以促进细胞外ARGs的水平基因转移，对接合子的测序发现质粒RP4体系产生接合子的群落丰度更高，转移的风险更大。同时还发现土壤表面对ARGs的吸附可以增加ARGs基因转移的几率。
As a kind of emerging pollutants, Antibiotic resistance genes (ARGs) can spread through horizontal gene transfer (HGT) which is distinguish it with normal pollutants. HGT is the main reason for the prevalence of ARGs in the natural environment. With HGT the ARGs can transfer between microorganisms and even from microorganisms to humans, which might threaten safety of the environment and human health. ARGs might enter the groundwater during the process of reclaimed water applied in water supply. To develop a better understanding of the distribution and transmission of ARGs during the recharge process, a field study on the seasonal variations occurrence and concentration of ARGs was conducted in Chaobai River of Beijing. Surface water samples collected, together with soil and groundwater samples were analyzed for the distribution of ARGs (sul1, sul2, dfrA1, dfrA12, tetB, tetO, tetW, tetM). The impacts of the principal selection pressure on the transmission of ARGs during the process of reclaimed water recharging were studied using soil columns and U-tube. Overall, the detection rates of the target genes were 100% except tetO and tetB. The abundant order of ARGs followed sulfonamides resistance genes > methoprim resistance genes > Tetracycline resistance genes in the surface water samples. Gene sul2 was detected with the highest relative abundance in the study area. Seasonal variations appeared in all water samples, followed summer > autumn > spring > winter. Analysis of the ARGs distribution along the vertical direction showed that the order of ARGs in groundwater was consistent within reclaimed water. In general, sul2, sul1, and intI1should be the primary controlled genes in the study area. Pass synthesises analysis of surface water samples indicated that the recharge of reclaimed water caused a certain influence on the surface water. The results of high-throughput sequencing indicated that the relative abundance of Pseudomonas was highest in surface water, and the species diversity was higher in summer and autumn than in spring and winter. Statistical analysis showed that intI1 had a significant correlation with most of ARGs, R2 up to 0.974. And there was a correlation between various microbial species and abundance of ARGs, especially Acinetobacter in summer and autumn. Principal component analysis in different environment medium showed that the main factor of ARGs is intI1, Sulfamethoxazole, Trimethoprim and Cu, Zn in the studied area. The soil column simulation results showed that the possibility of extracellular ARGs (eARGs) enters into the groundwater system through the soil layer might be greater than antibiotic resistance bacteria (ARB). Meanwhile, the soil column results also pointed out that, as a key factor in the propagation ARGs in environment, HGT may occur in the process of reclaimed water recharging and needs further in-depth study. Transconjugations in the system with eARGs (plasmid RP4) as donor were obtained using modified U-tube experiments. The transfer rate caused by eARGs accounted for 7.29% of the total conjugate transfer. HGT under different environmental selective pressures were studied through this device, which suggested that multiple selection stress promoted the HGT of eARGs. The sequencing analysis indicated that the community abundance tansconjugations in RP4 system was higher than the ARB system. Furthermore, the adsorption of exogenous ARGs in soil can increased the probability of ARGs in environments.
