【作者】 张浩；

【导师】 罗晓； 屈卫泉；

【作者基本信息】 河北科技大学 ， 建筑与土木工程（专业学位）， 2020， 硕士

【摘要】 随着抗生素的发展和使用,近些年抗生素抗性基因（Antibiotic Resistance Genes,ARGs）已经成为一种倍受关注的新型污染物。并且微生物的繁衍和信息交流往往会对抗性基因的传播提供媒介,导致环境中携带抗性基因的致病菌增多,威胁到人们身体健康。本研究选择石家庄饮用水水源地岗南水库水体和沉积物作为研究对象,使用高通量测序和荧光定量PCR等手段,研究水体和沉积物中微生物群落的分布和迁移情况,选定并追踪了8种抗生素抗性基因（sul1、sul2、tet A、tet B、qnr B、qnr C、aad A-1和erm B）和第一类整合子（int I1）的丰度分布和迁移情况。本人采用Mothur,SPSS和R软件等软件进行大量的数据分析,得到了不同季节微生物群落结构和特征与抗生素抗性基因的相关性关系,为水源地水库的ARGs污染情况和水质安全奠定了技术基础和数据支撑。本课题以岗南水库为例研究了ARGs在华北地区水源地空间分布和季节变化,并对其影响因素进行了深入分析。采样和监测覆盖了冬夏两季,采样点位包括入流、库中、出流。荧光定量PCR结果显示,除tet B、qnr B和qnr C抗性基因在部分点位未检出,其他被检测的ARGs在不同采样点的水体和沉积物中均被检出。Sul1、int I1、tet A、erm B和aad A-1基因丰度较高,且在水体和沉积物抗性基因分布规律相似。水体中和沉积物中大部分ARGs入库后呈先降低再增加的变化趋势,出库丰度低于入库丰度,部分水体和沉积物中ARGs出现逐渐升高或逐渐下降的趋势。检测结果显示,岗南水库中水体和沉积物ARGs丰度均不超过10⁶ copies·L^-1和10⁶ copies·g^-1,与上海市饮用水水源地和江苏省代表性水源地相比ARGs丰度低2-3个数量级,现阶段抗生素抗性基因风度处于较低水平。岗南水库中优势菌门为变形菌门、放线菌门和拟杆菌门,沉积物中菌群种类多水体,水库内和水库外菌群结构差异性显著。同时,在冬季2种菌属对温度呈极显著的正相关性;6种菌属对总氮,6种菌属对总磷呈极显著的负相关性。5种菌属对aad A-1呈显著的正相关性;3种菌属对sul1、tet A呈显著的正相关性;1种菌属对sul2呈显著的正相关性;1种菌属对qnr C呈显著的正相关性;5种菌属对tet B呈显著和极显著正相关性;7种菌属对qnr B呈显著的正相关性;5种对sul2和erm B呈显著的负相关性。夏季中,3种菌属对总磷呈显著的正相关;1种菌属对溶解氧呈极显著的正相关性。1种菌属对sul1呈显著的正相关性;2种菌属对tet B呈显著的正相关性;4种菌属对int I1呈显著的正相关性;1种菌属对qnr B呈显著的正相关性;7种菌属对aad A-1呈显著的正相关性。为人为影响和控制抗性基因的传播的可行性提供了数据支持。从季节对比可知在门水平下水源地水库冬季的菌门丰度均高于夏季,两个季节沉积物中菌门丰度均高于水体。通过对冬季和夏季的对比,发现变形菌门是水体和沉积物中最优势菌门。大部分被检测的抗性基因在冬季和夏季中均被检出,水体中tet A、tet B、qnr C、aad A-1和erm B抗性基因表现出冬季高于夏季的规律,而sul1和sul2和int I1基因丰度表现出冬季低于夏季的规律。沉积物中tet A、tet B、qnr C、erm B表现出冬季高于夏季的分布规律,而sul1、sul2、int I1和aad A-1基因丰度表现出冬季低于夏季的分布规律。抗性基因qnr B分布情况受水库环境的影响较大。本研究确定了水源地ARGs的影响因素及每种ARGs的扩散与菌群和环境条件,为控制ARGs扩散提供了技术思路。更多还原

【Abstract】 With the development and use of antibiotics,Antibiotic Resistance Genes（ARGs）have become a new type of pollutant that has attracted much attention in recent years.In addition,the propagation and information exchange of microorganisms often provide media for the transmission of resistance genes,leading to an increase in pathogenic bacteria carrying resistance genes in the environment and threatening people’s health.In this study,the water and sediments of the Gangnan Reservoir where the source of drinking water in Shijiazhuang,were selected as the research objects.High-throughput sequencing and fluorescent quantitative PCR were used to study the distribution and migration of microbial communities in the water and sediments,abundance distribution and migration of antibiotic resistance genes（sul1,sul2,tet A,tet B,qnr B,qnr C,aad A-1,erm B,int I1）.I used Mothur,SPSS,R software,etc.to carry out a large amount of data analysis,and obtained the correlation between the structure and characteristics of the microbial community in different seasons and antibiotic resistance genes.It laid the technical foundation and data support for the ARGs pollution situation and water quality safety improvement of the water source reservoirs.The sampling and monitoring of the Gangnan Reservoir in this study covered the winter and summer,and the sampling points included inflow,reservoir and outflow.Fluorescence quantitative PCR results showed that,except for tet B,qnr B and qnr C resistance genes were not detected at some points,others ARGs all were detected both in water and sediment points.Sul1,int I1,tet A,erm B and aad A-1 genes are relatively abundant,and the distribution of resistance genes in water and sediment were similar.Most ARGs in water bodies and sediments will decrease and then increase after entering the reservoir the outflow abundance is lower than the reservor abundance,and the ARGs abundance exhibited the gradually increase or gradually decrease tendencies in some water and sediment sites.The abundance of ARGs in the water and sediments of Gangnan Reservoir does not exceed 10⁶ copies·L^-1and 10⁶ copies·g^-1,which is 100～1000 times lower than that of drinking water sources in Shanghai and representative water sources in Jiangsu Province Magnitude.This study found that the dominant bacteria phylum in Gangnan Reservoir are Proteobacteria,Actinomycetes and Bacteroides.The types of bacteria in the sediments are higher than those in the water body,and there are differences in the structure of the bacteria in and outside the reservoir.At the same time,in winter,two species of bacteria have a very significant positive correlation with temperature;six species of bacteria have a very significant negative correlation with total nitrogen;six species of bacteria have a very significant negative correlation with total phosphorus;five species of bacteria have a significant positive correlation with aad A-1;three species of bacteria have a significant positive correlation with sul1 and tet A;one species of bacteria have a significant positive correlation with sul2;one species of bacteria has a significant positive correlation with qnr C;five species of bacteria have a significant positive correlation with tet B;seven species of bacteria have a significant positive correlation with qnr B;five species of bacteria have a significant negative correlation with sul2 and erm B.In summer,three species of bacteria have a significant positive correlation to total phosphorus;one species of bacteria has a very significant positive correlation to dissolved oxygen;one species of bacteria has a significant positive correlation with sul1;two species of bacteria have a significant positive correlation with tet B;four species of bacteria have a significant positive correlation with int I1;one species of bacteria has a significant positive correlation with qnr B;seven species of bacteria have a significant positive correlation with aad A-1.It provides data support for the feasibility of man-made influence and control of the spread of resistance genes.From the seasonal comparison,it can be seen that the abundance of bacteria in the water source reservoirs at the phylum level in winter is higher than that in summer,and the abundance of bacteria in the sediments of the two seasons is higher than that in the water body.Compared the detection results in winter and summer,it is found that the Proteobacteria is the most dominant bacteria in the water and sediment.Most of the tested resistance genes were detected in winter and summer.The tet A,tet B,qnr C,aad A-1 and erm B resistance genes in the water body showed a rule that winter was higher than that in summer.And the sul1,sul2 and int I1 genes abundance in winter is lower than summer.The tet A,tet B,qnr C and erm B in the sediments is higher in winter than in summer,while the sul1,sul2,int I1 and aad A-1 genes showed an opposite distribution pattern.The distribution of resistance gene qnr B is greatly affected by environment of the reservoir.This study determined the influencing factors of ARGs in water sources,the spread of each ARGs,the flora and environmental conditions,and provided technical ideas for controlling the spread of ARGs.更多还原