耐药基因在斑马鱼体内转移和扩增规律及机制研究

Currently, WHO claimed the drug-resistant bacteria and antibiotic resistance genes (ARGs) to be one of the most significant challenges to human health during the twenty-first century as emerging new type of environmental pollutants. Aquatic ecosystem especially surface water is a recognized reservoir for ARGs..ARGs are the genetic basis of drug-resistant bacteria. Its pollution, transfer and spread have become one of the major public health issues and ecological safety threats concerned in the globe. It is reported that ARGs and/or drug-resistant bacteria have been detected in different water (including the hospital sewage, aquaculture wastewater, urban sewage, surface water or even from drinking water), multiple soil and stool. The surface water not only receive the ARGs in various sewages but collect them from the animals excrements in water..The mechanisms of ARGs transfer and amplification have been concerned, and generally believed that bacteria obtain the drug-resistance through two pathways: first, some genes mutation occurred in bacteria under antibiotic stress, and can be passed to the offspring; the other, bacteria got exogenous antibiotic resistance genes by horizontal transfer which is considered as the main way of obtaining ARGs. Horizontal gene transfers include three pathways, conjugation, transformation and transduction..By analyzing literatures and our preliminary experiments results, we believe that the ARGs seldom come from the transfer and amplification in vitro but mainly stem from the transfer and amplification in vivo. However, the mechanisms of ARGs transfer and amplification in vivo have not been fully elucidated, primarily because there are too many kinds of bacteria in animal guts, including both culturable and non-culturable bacteria; beyond that the (micro-) environment in vivo are also extremely complex. Transfer and amplification of ARGs require specific conditions: including the donor strains carried ARGs, recipient strains being competent and appropriate niche. Therefore, it is very difficult to make a perfect experimental design to elucidate the mechanisms of ARGs transfer and amplification under such complex conditions..Here, we proposed the hypothesis that ARGs in surface water mainly come from the transfer and amplification in aquatic animal guts. And we plan to explore the principles of ARGs transfer and amplification in fish using molecular biological, cell biological, and proteomics and (macro-) genomic technologies combined with the typical horizontal transfer models. More importantly, we will identify the genus of recipient bacteria by target genes labeled with double fluorescence, flow cytometry sorting and cluster analysis of conserved genes (16S rDNA). And elucidate the mechanisms of ARGs transfer and amplification in fish from the molecular, cellular and metabolic levels. These achievements will provide a theoretical basis and technical supports for further research and effective control of ARGs contamination in water.

耐药基因是一种新型环境污染物，成为人类健康又一重大威胁。地表水是耐药基因的巨大储存库。地表水中的耐药基因除了接纳各种污水中存在的耐药基因（耐药菌）外，更有可能来源于其在水生动物体内的转移和扩增。本项目在提出“水生动物(肠道)是地表水中耐药基因转移和扩增的重要场所”假说的基础上，利用定量PCR技术、选择性分离培养、双荧光分子标记、流式细胞分选、保守基因聚类分析和宏基因组等技术，摸清耐药基因在模式动物-斑马鱼肠道内的定植、转移和扩增规律；明确耐药基因在斑马鱼体内转移过程中各种受体菌的种类；结合耐药基因水平转移模型，并利用各种分子生物学和细胞生物学技术研究耐药基因转移和扩增过程中关键基因表达调控、供体菌和受体菌表型以及代谢调控等变化情况，阐明耐药基因在斑马鱼体内转移和扩增机制，为水环境中耐药基因污染的评估与控制提供理论依据和技术支持。

水环境是抗生素耐药性基因(ARGs)的储存库之一，鱼类作为重要的水生动物，具有适宜细菌生存的肠道生态环境，也能为耐药基因水平转移提供合适环境，然而，关于抗生素耐药菌(ARB)或ARGs进入肠道后，对肠道微生物的影响我们知之甚少。在本研究中，我们应用培养方法、qPCR、分子标记和16S rDNA扩增序列测定技术，建立了斑马鱼质粒介导的ARG转移模型。此外，我们的目标是研究在供体菌携带编码ARGS的自转移质粒的进入斑马鱼肠道后，ARGS在肠道微生物群落中的转移扩增情况。结果显示，15%的粪便细菌通过Rp4介导的水平转移获得ARG，后肠是ARG扩散的最重要的肠道区域，供体细胞和转接细胞的浓度几乎是其他肠段的25倍。此外，在发生转移最活跃的后肠，Rp4质粒调控基因trbBp和trfAp的mRNA表达显著上调。与对照组相比，外源性细菌似乎通过增加大肠杆菌和类杆菌的种类，而减少气单胞菌，从而改变了细菌群落。我们鉴定了接合子的组成和可培养细菌和不可培养细菌的丰度(后者占总接合子的90.4%-97.2%)。我们的研究表明，水生动物的肠道有助于ARG在水环境中的传播。本研究旨在通过建立耐药基因在水生动物体内定植和转移的模型，在采用细菌培养和分子生物学等技术方法的基础上，定性、定量地研究耐药菌及耐药基因在水体动物体内定植和转移的规律，阐明耐药基因在水生动物肠道内的转移和扩增机制，摸清耐药基因发生定植和转移的消化道部位与转移发生机理，以及明确肠道内接受耐药基因的接合子的种类和数量。课题研究成果为进一步丰富人们对耐药基因在动物体内转移和扩增的规律和机制的认知及提供了充分的理论依据，同时也为水产养殖用药，水源水保护以及生态环境的污染和管理提供了数据依据和技术支撑。