长期施猪粪农田土壤中抗生素抗性基因的污染特征及扩散机制研究

Antibiotic resistance genes (ARGs) have become one of the topics of great importance in global research, which had caused serious environmental problems. The emission of livestock manure is an important source of environmental ARGs, and the containing ARGs could be transferred into human pathogens via horizontal gene transfer, which may pose a potential human health risk. It has great significance to study the impact of pig manure application on ARGs in farmland soil for a comprehensive assessment of environmental ARGs reservoir. In this proposal, by using high through-put quantitative PCR and Illumina Miseq sequencing approach, we aim to, (1) examine the types and abundances of ARGs and mobile genetic elements (MGEs) in manure-amended soil; (2) investigate the bacterial community composition and diversity in manure-amended soil; (3) assess the persistence of ARGs in manure-amended soil by analyzing the abundance of ARGs at different time after manure application and the distribution of ARGs between rhizosphere soil and non-rhizosphere soil. By comparing the genetic diversity of ARGs and MGEs, and bacterial community, and statistical analysis of correlation with environmental factors, we will try to elucidate the characterization of contamination and dissemination of ARGs in long-term manure-amended soil, and provide a scientific basis for the ARGs contamination in farmland soil.

抗生素抗性基因（ARGs）所导致的环境污染问题日趋严重，已成为国际上的研究热点。畜禽粪便的排放是环境中ARGs的重要来源，ARGs可通过基因水平转移进入人类致病菌中，对人类健康存在潜在危害。因此研究猪粪对农田土壤中ARGs的影响，对全面评估环境中ARGs库具有重要意义。本项目以长期施猪粪的农田土壤为研究区域，采用高通量定量PCR技术探讨施猪粪农田土壤中ARGs和基因水平转移元件的丰度和分布；采用Illumina Miseq测序手段揭示猪粪对农田土壤中微生物群落结构的影响；通过分析施加猪粪后不同时间点土壤中ARGs的丰度，以及ARGs在根际区与非根际区土壤中的分布，探究ARGs在农田土壤中的持续性。进一步从ARGs丰度、微生物群落结构、基因水平转移机制等方面，同时结合环境因子进行统计分析，全面系统地阐释农田土壤ARGs的污染特征及扩散机制，为探明施用猪粪对农田土壤中ARGs污染提供科学依据。

抗生素抗性基因（ARGs）在全球范围内的传播扩散严重危害了人类健康。农田土壤生态系统作为ARGs的源与汇，因与人类健康密切相关而受到广泛关注，其中畜禽粪便施用是农田土壤ARGs的主要来源。因此研究畜禽粪便对农田土壤中ARGs影响，对全面评估环境中ARG库具有重要意义。基于此，依托中国科学院栾城农业生态系统试验站，利用高通量定量PCR技术和高通量测序技术，解析了长期施猪粪农田土壤中ARGs的分布特征及影响因素。结果表明，连续15年施加猪粪增加了土壤中ARGs的多样性和丰度。在施加猪粪土壤中共检出81种ARGs，相对丰度高达0.23拷贝数/16S rRNA基因拷贝数。施加猪粪主要是增加了本底土壤ARGs的丰度。施加猪粪也增加了土壤中可移动基因元件的丰度，促进了ARGs在土壤中的传播扩散潜力。同时，施加猪粪显著改变了土壤微生物群落结构，而微生物群落结构的变化是影响土壤ARGs分布的主导因子。此外，在施加猪粪后，连续一年采集0-1 m土壤样品进行分析。结果表明，施加猪粪后，ARGs随施肥时间呈现递减的趋势，但ARGs丰度依然高于未施猪粪的对照土壤样品。说明ARGs在土壤中具有长时间的持续性。此外土壤中的ARGs主要集中在0-30 cm土层，且与土壤微生物群落结构变化有着密切关系。本项目研究结果为阐释长期施畜禽粪便农田土壤中ARGs的污染特征以及ARGs在土壤中的持续性具有重要意义。