蚯蚓肠道消化功能区对污泥中抗生素抗性基因转归的作用机制

Diverse antibiotic resistance genes (ARGs) present in vermicomposting system lower the values of vermicompost products. The gut digestion process of earthworms is considered as the most important step during the generation of vermicompost, thus affecting the abundance and dissemination of ARGs in the vermicompost. Hence, this program proposes to reveal the effects and mechanisms of gut digestive process of earthworms on the fate of ARGs and their mobile genetic elements (MGEs), with the focus on investigating the gut functional zone of earthworms. For this, the high throughput sequencing and qualitative PCR combined with a multiple resistance plasmids of RP4 and a reporter gene of Green fluorescent protein (gfp) are employed to analyze the fate of ARGs, MGEs and their possible hosts in sludge during gut passage process of two epigenic and endogenic earthworms, respectively. The aims of this study are (1) to separately reveal the effects of foregut physical digestion, middle gut chemical digestion, hindgut microbial digestion and cast associated digestion on the fate of the ARGs, MGEs and their possible hosts, (2) to clarify the underlying mechanisms of gut digestive process on the fate of ARGs through investigating the linkages of different gut functional zones, (3) to seek the dominate ARGs that could be reduced after the gut digestion process, and (4) to ascertain the differences between two ecological niches of earthworms affecting on the ARGs and MGEs during the digestion process. This work will provide a rationale for reducing the abundance of ARGs and controlling their dissemination in the environment and promoting the vendibility of final vermicompost of sludge.

污泥蚯蚓堆肥体中含有多种高丰度的抗生素抗性基因（ARGs）降低了蚯蚓粪肥的利用价值。蚯蚓肠道消化是污泥蚯蚓粪肥生产过程中最关键的一环，亦决定了蚯蚓粪肥中ARGs的归趋和传播。本项目以蚯蚓肠道为研究对象，揭示蚯蚓肠道消化过程对污泥中ARGs转归和传播的作用机制。研究拟选用表居型和内居型的蚯蚓，区分其肠道消化功能区；借助多元耐药质粒RP4和报告基因gfp，运用高通量测序、荧光定量PCR等分子生物学手法，对污泥蚯蚓堆肥中的典型ARGs和遗传元件及潜在的宿主进行剖析。分别揭示蚯蚓肠道消化各功能区（前肠器官、中肠消化液、后肠微生物和排粪协同消化）对污泥中ARGs转归的影响机理，探讨其各功能区之间的关联，阐明肠道消化过程对ARGs影响的机制；查明肠道消化阻控的典型ARGs，探知不同生态位蚯蚓对ARGs作用的差异。本研究为消减和控制污泥蚯蚓堆肥体中ARGs及提升蚯蚓粪肥的市场价值提供科学的依据。

污泥蚯蚓堆肥产物中高含量的抗生素抗性基因（ARGs）降低了蚯蚓粪肥的利用价值。蚯蚓肠道消化为蚯蚓堆肥最为关键一步，决定了污泥蚯蚓粪中ARGs的归趋。本研究选用表居型赤子爱胜蚓（Eisenia fetida）进行蚯蚓堆肥，利用宏基因组学揭示了蚯蚓堆肥体中β-内酰胺类、磺胺类和四环素类为主要的ARGs大类，蚯蚓可降低污泥中约45%的ARGs。基于宏基因组学和PMA标记的活体微生物研究发现，蚯蚓肠道消化对ARGs去除具有一定的选择性。蚯蚓前肠和后肠均能降低污泥中ARGs。通过接种RP4供菌体发现，ARGs和MGEs及其宿主多稳态分布在蚯蚓的后肠与新鲜排泄物中。污泥蚯蚓堆肥过程中有机质、硝氮、细菌数量的变化与ARGs丰度存在显著性正相关关系(P<0.01)。携带MGEs的质粒介导的水平转移是蚯蚓堆肥中ARGs难以削减的的主要原因。增加蚯蚓密度和肠道停留时间能提高堆肥效率，但亦能增加污泥蚯蚓中的ARGs丰度。与添加生物质炭相比，添加中药渣能有效去除污泥蚯蚓堆肥体中的ARGs，但协同干化工艺并不能削减ARGs。本研究结果为消减和控制污泥蚯蚓堆肥体中ARGs及提升蚯蚓粪的市场价值提供科学的依据。