耐药基因在大型底栖无脊椎动物肠道内的时空分布规律及水平转移机制研究

Antibiotic resistance genes are emerging pollutants, and water is a huge reservoir of resistance genes. Investigation and analysis on the present situations of resistance genes in water environment and their diffusion are two of the major research focus in the whole world, but the role of benthic macroinvertebrates feeding on bacteria in diffusion and transfer of resistance genes is unclear. Our prior study have found that there are a variety of resistance genes in aquatic animal, on the basis we put forward the hypothesis "Transfer and diffusion of resistant genes can take place in the intestines of benthic macroinvertebrates", which will be verified by the following contents: detection of temporal and spatial distribution of different resistance genes in benthic macroinvertebrates by resistance selection and PCR; homologous of resistance genes in water, sediment and macroinvertebrates by gene sequencing; analysis of the colonization and proliferation of resistance genes in vivo by conjugation, transformation and transduction model; clarifying the resistance gene transfer mechanism in vivo at the cellular and molecular level by fluorescence tracing technique, scanning electron microscopy, transmission electron microscopy, 3D high resolution fluorescence microscopy and qPCR. This project will supplement the important link of food chain by which resistance genes transfer and diffuse in water environment, and it will be proved that water-bacteria-benthic macroinvertebrates-fish constitute the complete system of resistance gene transfer. It will provide theoretical and technical foundation for detection and control of resistance genes.

耐药基因是一新的环境污染物，且水体是其巨大储存库。水中耐药基因的现状调查和污染规律是目前研究的热点，但以细菌为食的大型底栖无脊椎动物在耐药基因扩散及转移中的作用尚不清楚。我们前期研究发现耐药基因在水生动物体内有分布，结合文献提出“耐药基因能够在大型底栖无脊椎动物的肠道内转移和扩散”的假设，并通过以下研究内容验证：抗性筛选和PCR检测不同耐药基因在大型底栖无脊椎动物肠道内的时空分布；基因测序分析水中、底泥中及动物体内耐药基因的同源性；接合转移、转化和转导模型分析耐药基因在体内的定植和扩增规律；应用荧光示踪、扫描电镜、透射电镜、3D高分辨荧光显微镜和qPCR等研究技术，从细胞和分子水平阐明耐药基因在体内的转移机制。本项目将补齐水环境中耐药基因通过食物链转移和扩散的重要环节，证明水-细菌-大型底栖无脊椎动物-鱼构成了完整的耐药基因转移体系，为耐药基因的检测及防控奠定理论及技术基础。

水体和底泥是环境中耐药基因的巨大储存库，而底栖动物肠道是细菌生长繁殖的优良场所，基于此我们提出本项目的学术假说：作为鱼类重要饵料的大型底栖无脊椎动物能够作为耐药基因的传播媒介，其肠道能够作为耐药基因水平转移的场所。我们采用选择培养、PCR和qPCR等技术，定性、定量检测Tet、Sul2、和Km等ARGs在大型底栖无脊椎动物体内的时空分布；季节、人为活动、金属离子等环境因素对耐药基因分布具有重要影响；基因测序分析拟杆菌门中Proteobacteria和Flavobacterium的Escherichia是摇蚊幼虫体内的主要耐药基因易感菌；使用qPCR技术分析耐药基因转移的分子机制。耐药基因接合转移在小鼠肠道内发生，小肠是耐药基因供体菌和耐药基因定植的主要部位。在没有宿主细菌的情况下，eARGs能够进入肠道微生物。此外，水环境中纳米颗粒特别是纳米铝颗粒能够转导耐药基因进入细菌，且能够是耐药基因避免核酸酶的切割。本项目补齐水环境中耐药基因通过食物链转移和扩散的重要环节，证明水-细菌-大型底栖无脊椎动物-鱼类构成了完整的耐药基因转移体系；增加人们对耐药基因在水环境中的分布及转移扩增规律的认识，丰富相关的理论基础知识；指导污水及河流沉积物的处理，减少耐药基因的传播，有效防控耐药基因产生的危害，促进环境的可持续发展。