人类活动影响下的高原湿地四环素类抗生素抗性基因赋存与微生物群落共现性

Co-occurrence of Tetracycline Antibiotic Resistance Genes and Microbial Communities in Plateau Wetlands Under the Influence of Human Activities

抗生素的广泛使用导致抗生素大量进入环境中,进而使微生物产生耐药性.近年来,随着高原区域人类活动的加剧,高原湿地中抗生素耐药基因(ARGs)的赋存和迁移研究备受关注.以地处云贵高原的鹤庆草海国家湿地公园为研究区,分析河流上游(泉眼附近原始生境)和河流下游(居民生活污水排放口)沉积物中的四环素类、磺胺类、喹诺酮类和大环内酯类共4类抗生素的含量分布,其中四环素类抗生素检出含量为103.65~2185μg·kg^(-1),是含量最高的抗生素种类.为进一步探究四环素类抗性基因的赋存特征和影响因素,通过相关性分析和网络分析揭示了人类活动影响下环境因子、细菌群落结构和病原菌对四环素类ARGs的影响.结果发现,上、下游沉积物中共检出15种四环素类抗性基因,其中,上游检出tetPA、tetD和tetPB等7种抗性基因,下游检出tetPA、tetE、tetM和tetX等13种抗性基因,下游新增的8种抗性基因占下游基因丰度的43.44%;四环素类抗生素含量及有效磷、总有机碳、硝态氮和总磷等理化指标是影响四环素类ARGs分布的主要环境因子;上、下游沉积物中检出细菌分属于64个细菌门,其中变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidota)是影响四环素类ARGs丰度的主要菌门;同时,上、下游共检出27种病原菌.网络分析显示下游新增的8种抗性基因与病原菌相关性占网络连接度的70%,且李斯特菌(Listeria monocytogenes)、粪肠球菌(Enterococcus faecalis)和普通拟杆菌(Bacteroides vulgatus)是四环素类ARGs传播的主要潜在宿主.相较于原始生境,居民生活污水的排放在引入大量抗生素的同时也改变了河流湿地微环境和微生物群落结构,并增加了沉积物中的ARGs种类,进而促进ARGs在微生物甚至是病原菌之间的扩散和传播.

The widespread use of antibiotics has led to a large number of antibiotics entering the environment,to which microorganisms have become resistant.In recent years,with the intensification of human activities in the plateau region,the occurrence and migration of antibiotic resistance genes(ARGs)in plateau wetlands have attracted considerable attention.Here,we selected the Caohai National Wetland Park,located in the Yunnan-Guizhou Plateau,as our study area.The contents of tetracyclines,sulfonamides,quinolones,and macrolides in sediments from the upstream(the pristine habitat near the spring eye)and downstream(the sewage discharge outlet of residents)areas of the river in the park were analyzed.Among them,the detection content of tetracycline antibiotics was 103.65-2185μg·kg^(-1),which was the highest antibiotic detection content.To further investigate the occurrence characteristics and influencing factors of tetracycline resistance genes,the influence of environmental factors,bacterial community structure,and pathogenic bacteria on tetracycline ARGs under the influence of human activities were revealed via correlation analysis and network analysis.The results showed that a total of 15 tetracycline resistance genes were detected in the upstream and downstream sediments.Among them,seven resistance genes including tetPA,tetD,and tetPB were detected in the upstream,and 13 resistance genes such as tetPA,tetE,tetM,and tetX were detected in the downstream.The abundance of eight new resistance genes in the downstream accounted for 43.44%of the downstream genes.The tetracycline-like antibiotics and soil physicochemical indicators(i.e.,available phosphorus,total organic carbon,nitrate nitrogen,and total phosphorus)were the main environmental factors affecting the distribution of tetracycline ARGs.Additionally,the bacteria detected in the upstream and downstream sediments belonged to 64 bacterial phyla,among which Proteobacteria,Firmicutes,and Bacteroidota were the main phyla affecting the abundance of tetracycline ARGs;meanwhile,27 pathogenic bacteria were detected in the upstream and downstream sediments.Network analysis showed that the correlation between the eight new resistance genes and pathogens in the downstream area accounted for 70%of the network connectivity,and Listeria monocytogenes,Enterococcus faecalis,and Bacteroides vulgatus were identified as potential hosts for the transmission of tetracycline ARGs.Compared to the pristine habitat,the discharge of domestic sewage introduced large amounts of antibiotics and also changed the microenvironment and microbial community structure of the river wetland.Additionally,it increased the species of ARGs in sediments,which promoted the spread and transmission of ARGs among microorganisms and even pathogens.