农药对农田土壤抗生素耐药性的影响机制研究

Antibiotic resistance has become a major global public health issue in recent years. Dissemination of antibiotic resistance genes (ARGs) in the environments are increasingly regarded as a growing concern. Pesticides are among the largest usage and residue of agricultural chemicals in agricultural soils. However, influence of pesticides on the development of soil antibiotic resistance remains unknown. This project aims to study the influence of pesticides on the development of antibiotic resistance in agricultural soil by regulated microcosm experiments. Soils will be amended with frequently-used herbicides, insecticides, or bactericides at series of concentrations. The variation of microbial activity, bacteria community tolerance to antibiotics and pesticides, abundance of ARGs or pesticide-related genes and broad host range plasmids will be characterized in the soil microbiomes of the microcosm system. Metagenomic profiles of ARGs, mobile genetic elements, microbial community, and the connection of bacterial host and ARGs will be revealed by Illumina high-throughput sequencing. The prevalence and types of broad-host transferable antibiotic resistance plasmids will be further exogenously captured by cultivation-independent isolation. The characteristics of plasmids will be analyzed by phenotyping (MICs of antibiotics and pesticides), genotyping (presence of ARGs and pesticides-relative genes), PCR-based replicon typing, restriction endonuclease analysis and complete nucleotide sequencing. Thus, we will further reveal the molecular mechanism of the influence of pesticides on the development of soil antibiotic resistance by metagenomics sequencing and plasmid analysis. The aim of this project is to explore the mechanisms of the impact of pesticides on the development of soil antibiotic resistance by using research strategy of Environmental Science and Molecular Biology. The results will enhance our understanding of soil antibiotic resistance in the contaminated environments and will provide scientific basis for our national action plan to antibiotic resistance.

抗生素耐药性是全球公共卫生的重大威胁，耐药基因环境污染与扩散是环境科学研究的热点。农药是农田中使用量最大、残留最多的农用化学品之一，然而农药是否对土壤细菌耐药性的形成与扩散产生影响及其可能机制知之甚少。本项目拟通过土壤实验研究农药对土壤细菌耐药性的影响机制。通过添加不同浓度除草剂、杀虫剂和杀菌剂，考察土壤微生物基本活性、菌群耐受性、ARGs丰度变化。采用宏基因组测序从耐药基因谱系、移动元件、微生物群落结构以及物种与基因关联性等角度探讨农药对土壤耐药性的影响机理。捕获土壤中的广宿主质粒，分析其耐药表型和基因型、复制子类型、酶切图谱和全序列等特征。结合宏基因组测序和质粒特征分析，揭示农药对土壤抗生素耐药性影响的分子机制。项目结合环境科学和分子生物学等学科研究手段，旨在阐明农药对农田土壤抗生素耐药性的影响机制。研究结果有助于认识土壤环境细菌耐药机制，为我国遏制细菌耐药性国家行动计划提供科学参考。

抗生素耐药性是全球公共卫生的重大威胁之一，环境中耐药基因污染与扩散是环境科学研究的热点。农药是农田中使用量最大、残留最多的农用化学品之一，然而农药对土壤细菌耐药性的影响知之甚少。本项目利用环境微生物与宏基因测序技术研究农药长期作用对土壤细菌抗生素耐药性的影响，主要研究结论如下：（1）采用荧光定量PCR考察了长期使用农药农田土壤中耐药基因的分布特征，结果表明农田土壤中含有多种耐药基因，作物类型与土壤耐药基因的丰度有一定关系。（2）采用长期农药添加的田间实验深入分析了农药长期作用对土壤细菌耐药性的影响机制。通过挖掘相关功能基因的多样性来反映土壤细菌结构与功能在农药长期作用下产生的变化。利用生物信息学手段分析了土壤细菌群落的总体变化、抗生素耐药基因的丰度变化，以及农药相关基因及其宿主菌与抗生素耐药性之间的关联。分析获得了关键基因的多样性及其系统发育树，并构建了关键基因隐马尔可夫分析模型和靶向组装模型。宏基因分析结果发现，农药长期施用减少了土壤细菌总量，同时使得土壤中农药相关基因得到富集，说明农药长期作用下激发了与农药相关的微生物活动。农药的长期使用改变了土壤细菌组成结构，使得伯克氏菌属、假单胞菌属等细菌得到了富集。与此同时，土壤中耐药基因丰度显著增高，说明农药长期施用刺激了土壤细菌耐药性的发展。（3）进一步分析发现农药长期施用的土壤含有丰富的农药降解细菌，这些细菌含有由广宿主质粒编码的降解酶。这些广宿主质粒不仅同时携带多种外源物质的水解或抗性基因，如重金属耐受基因和抗生素耐药基因，而且能够在不同种属细菌之间转移。项目研究结果表明农药长期使用对土壤抗生素耐药基因进行了富集，多药外排泵等多种耐药基因出现在相同的基因片段上或共存于相同细菌中，可导致多重耐药细菌的形成，农药对土壤细菌耐药性的影响不容忽视。