摘要

       抗生素的使用和滥用加速了耐药基因(ARGs)和耐药菌(ARB)的传播,严重威胁人类健康和生态环境。城市污水处理厂是ARGs和ARB的重要环境库,受到越来越多的关注。本研究旨在明确污水处理工艺对环境ARGs丰度和多样性的影响；进一步阐明污水处理系统中ARGs分布、丰度与微生物群落结构之间关系；在全面了解可移动遗传元件(MGEs)的基础上探索污水处理系统中ARGs水平转移的潜在分子机制,并为控制污水处理系统中ARGs传播提供理论参考依据。污水处理厂中四环素耐药基因的分布特征：使用定性PCR、定量PCR等常规分子生物学的方法检测污水处理厂中四环素耐药基因(四环素ARGs)的分布情况。PCR定性检测表明15种四环素ARGs在污水处理厂的各工艺段中普遍存在。其中8种四环素ARGs定量分析表明活性污泥过程可以降低四环素ARGs的相对丰度(以16Sr RNA基因为对照)。同样,经过活性污泥过程并沉淀处理后单位体积水样中四环素ARGs去除率达95%以上。说明经过污水处理后单位体积水样中四环素ARGs被明显的去除,但处理出水中四环素ARGs丰度仍然高于环境水体。通过tetG分子克隆、DNA测序、系统进化分析发现,污水处理系统中tetG的OTU(操作分类单元)相似度较高,其宿主可分为两组(进水样品组、活性污泥／出水样品组)污水处理系统中ARGs和MGEs的分布及其相关性：为了研究污水处理系统中ARGs和MGEs的分布特征以及两者之间的关系,本文采用宏基因组学的方法,对四座城市生活污水处理系统中的ARGs和MGEs进行了分析。宏基因组数据与耐药基因数据库(ARDB)比对注释筛选表明,四座城市生活污水处理系统中进水中的ARGs的相对丰度均高于活性污泥与出水,活性污泥处理可去除72%以上ARGs。同时,本文采用宏基因组学的方法分析了质粒、整合子和插入序列,结果表明活性污泥工艺可有效降低MGEs丰度。统计分析表明城市生活污水处理系统中总ARGs相对丰度与总整合酶基因、质粒和插入序列的相对丰度之间具有显著的正线性相关性(P0.001)。污水处理系统群落结构及其与ARGs的相关性：采用16S rRNA基因扩增片段焦磷酸测序与宏基因组学分析方法阐明污水处理系统中微生物多样性和结构、污水处理系统ARGs分布之间的相关性。OTU、Chao 1和Shannon指数以及稀释曲线分析结果表明活性污泥过程可增加生物多样性。在门、纲、目、科、属水平的系统分类基础上进一步进行聚类分析,结果表明活性污泥过程显著改变了菌群结构。菌群结构的变化会影响ARGs的分布,对菌群结构与ARGs之间的相关性进行CCA分析,结果显示tetE和tetM显著地影响城市生活污水处理系统中细菌在属水平的分布(P0.05)。Pearson相关性系数揭示了每种ARG至少与一种菌属具有显著的正相关关系(R0.5；P0.05),经过活性污泥处理后潜在ARB的相对丰度低于33%。综上所述,本文研究结果表明,活性污泥过程可显著改变废水中的菌群结构,并显著影响ARGs的分布。由于与ARGs显著正相关的细菌种属在活性污泥中丰度降低,从而降低了ARGs在活性污泥中的相对丰度。在ARGs降低的同时MGEs丰度也被降低,分析发现ARGs与MGEs丰度具有显著的正相关关系,暗示MGEs、 ARGs丰度随着菌群更替时共有宿主丰度变化而变化。

       The use and abuse of antibiotics has accelerated the spread of drug-resistant genes (ARGs) and drug-resistant bacteria (ARB), seriously threatening human health and the ecological environment. Urban sewage treatment plants are important environmental reservoirs of ARGs and ARBs, which have received more and more attention. This study aims to clarify the impact of wastewater treatment technology on the abundance and diversity of environmental ARGs; to further clarify the relationship between the distribution and abundance of ARGs in the wastewater treatment system and the structure of microbial community; to fully understand the basis of mobile genetic elements (MGEs) To explore the potential molecular mechanism of the horizontal transfer of ARGs in the sewage treatment system, and to provide a theoretical reference for controlling the spread of ARGs in the sewage treatment system. Distribution characteristics of tetracycline resistance genes in sewage treatment plants: use conventional molecular biology methods such as qualitative PCR and quantitative PCR to detect the distribution of tetracycline resistance genes (tetracycline ARGs) in sewage treatment plants. PCR qualitative testing showed that 15 kinds of tetracycline ARGs are widespread in various process sections of sewage treatment plants. The quantitative analysis of 8 tetracycline ARGs showed that the activated sludge process can reduce the relative abundance of tetracycline ARGs (using 16Sr RNA gene as a control). Similarly, after the activated sludge process and precipitation treatment, the removal rate of tetracycline ARGs per unit volume of water samples reached more than 95%. It shows that the tetracycline ARGs in the unit volume of water samples are obviously removed after the sewage treatment, but the abundance of tetracycline ARGs in the treated effluent is still higher than that of environmental waters. Through tetG molecular cloning, DNA sequencing, and systematic evolution analysis, it is found that the OTU (Operational Taxonomy Unit) of tetG in the sewage treatment system is relatively similar, and its hosts can be divided into two groups (influent sample group, activated sludge/effluent sample group) ) The distribution of ARGs and MGEs in sewage treatment systems and their correlation: In order to study the distribution characteristics of ARGs and MGEs in sewage treatment systems and the relationship between the two, this paper adopts metagenomics methods to analyze domestic sewage treatment systems in four cities. The ARGs and MGEs in the paper were analyzed. Comparison of metagenomic data and ARDB (ARDB) annotation screening shows that the relative abundance of ARGs in the influent water in the domestic sewage treatment systems of the four cities is higher than that of activated sludge and effluent, and activated sludge treatment can remove 72% Above ARGs. At the same time, this article uses metagenomics method to analyze the plasmid, integron and insert sequence, and the results show that the activated sludge process can effectively reduce the abundance of MGEs. Statistical analysis showed that there was a significant positive linear correlation between the relative abundance of total ARGs and the relative abundance of total integrase genes, plasmids and insert sequences in the urban domestic sewage treatment system (P0.001). The community structure of sewage treatment system and its correlation with ARGs: 16S rRNA gene amplification fragment pyrosequencing and metagenomics analysis methods are used to clarify the correlation between the microbial diversity and structure in the sewage treatment system and the distribution of ARGs in the sewage treatment system . OTU, Chao 1 and Shannon index and dilution curve analysis results show that the activated sludge process can increase biodiversity. Based on the systematic classification of phylum, class, order, family, and genus level, the cluster analysis was further carried out, and the results showed that the activated sludge process significantly changed the flora structure. Changes in the structure of the flora will affect the distribution of ARGs. CCA analysis of the correlation between the structure of the flora and ARGs shows that tetE and tetM significantly affect the distribution of bacteria at the genus level in the urban sewage treatment system (P0.05). ). Pearson correlation coefficient reveals that each ARG has a significant positive correlation with at least one genus (R0.5; P0.05), and the relative abundance of potential ARB after activated sludge treatment is less than 33%. In summary, the results of this study show that the activated sludge process can significantly change the structure of the bacterial community in wastewater and significantly affect the distribution of ARGs. Because the bacterial species that are significantly positively correlated with ARGs are reduced in abundance in activated sludge, the relative abundance of ARGs in activated sludge is reduced. When ARGs decrease, the abundance of MGEs is also reduced. The analysis found that ARGs and MGEs abundance have a significant positive correlation, suggesting that the abundance of MGEs and ARGs changes with the change of the shared host abundance when the bacterial flora changes.

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