基于“质粒宏基因组”研究饲料中铜、锌、砷对猪粪细菌耐药质粒的选择与转移

Due to excessive antibiotics used in pig feeds, higher antibiotic-resistant bacteria could be selected in the swine manure which has become a huge reservoirs of antibiotic resistance genes in environment. Through horizontal gene transfer (HGT), ARGs of the antibiotics-resistant bacteria in swine manure might transfer and spread among the microorganisms in soil, plants, animals and human, posing a great threat to human public health and the environment. Except for antibiotics, heavy metals (Cu, Zn, As) were also added in pig feeds. The recent studies showed that heavy metals could also select the antibiotic-resistant bacteria in environment. Then, could heavy metals in feed affect the selection and transfer of drug resistance plasmids in swine manure? If it is the case, what is the mechanisms? There is no similar study at present. In this project, the plasmids metagenome of bacteria in swine manure from heavy metals in-feed pigs was constructed by TRACA and High-throughput sequencing approachs. The transfer frequencies (to Escherichia coli CV601 and environmental bacteria) and the main types of transferable antibiotic resistance plasmids are analyzed, which will clarify the mechanism of the effect of heavy metals in-feed on the transfer of the drug resistance plasmid. The possible selection mechanisms of ARGs by the heavy metals will be conjectured based on the sequence information of the co-resistant plasmids in plasmids metagenome, and the mechanisms will be verified by analyzing the distribution of plasmids and the expression of resistance gene (including ARGs and heavy metal resistant genes) in resistant bacteria on the strain and community levels. The implementation of the project will be helpful for controlling the dissemination of ARGs in swine manure and the spread of superbugs.

抗生素在猪饲料中大量使用，使猪粪细菌成为重要的抗生素抗性基因（ARGs）储存库，ARGs主要通过质粒介导的水平转移对人类健康和生态环境造成极大威胁。而重金属（如铜、锌、砷等）也广泛添加在猪饲料中，最近研究发现重金属对环境耐药细菌也具选择作用，那么，饲料中重金属对猪粪细菌中携带ARGs的耐药质粒是否也有选择作用并影响其转移？如果是，其机制如何？目前还未见类似研究。本项目拟采用TRACA及高通量测序等方法构建饲用含重金属饲料的猪粪细菌总质粒的宏基因组文库，分析猪粪ARGs的整体水平转移效率，参与ARGs转移的主要质粒类型及其向环境细菌的转移能力，阐明重金属对猪粪耐药质粒转移的影响机制；进一步分析共抗性质粒的序列信息来推测重金属对猪粪ARGs的选择机制，并在菌株和群落水平上分析耐药质粒分布和抗性基因的表达来验证。项目的实施将为控制猪粪ARGs在环境中的扩散与传播，解决超级细菌的流行提供理论基础。

抗生素在猪饲料中大量使用，使猪粪细菌成为重要的抗生素抗性基因（ARGs）储存库，ARGs主要通过质粒介导的水平转移对人类健康和生态环境造成极大威胁。而重金属也广泛添加在猪饲料中，最近研究发现重金属对环境耐药细菌也具选择作用，但目前很少关于重金属对猪粪耐药细菌及其携带耐药基因的影响研究。本项目拟利用宏基因组技术研究饲料中重金属的添加对猪粪耐药细菌及其携带耐药基因的选择与转移的影响，阐明其选择机理及其转移机制，所得结果将为控制猪粪细菌ARGs 在环境中的传播与扩散，解决超级细菌的流行奠定基础。.本项目通过宏基因组测序分析发现重金属处理组猪粪菌群多样性要高于对照组，且猪粪优势菌群也发生明显改变：对照组优势菌门为厚壁菌门（Firmicutes，55.7%）和拟杆菌门（Bacteroidetes，占37.3%），而重金属处理组的优势菌门为厚壁菌门（Firmicutes），占了92.12%；重金属处理组猪粪细菌的抗性菌的分离率，抗性基因数量及其丰度均显著高于对照组，这表明重金属的添加可以促进猪粪耐药细菌及其携带耐药基因的水平转移和扩散。进一步发现规模化养猪场的猪粪样本共抗性菌（同时具有重金属和抗生素抗性）具有较高的分离率，并从中分离到多种携带共抗性质粒，这表明共抗性耐药质粒可能是重金属对猪粪耐药细菌及其携带耐药基因选择与转移的重要机制之一。考虑到重金属对环境毒理的作用及其对耐药性的影响，我们还对具重金属抗性微生物在重金属污染修复的潜在作用进行了研究，结果表明重金属抗性微生物可以通过促进植物生长或促进植物对重金属的吸收与富集进而提高植物修复土壤重金属污染修复效率。以上研究结果阐明了重金属对猪粪耐药细菌及其携带耐药基因的选择与转移的影响及其相关机制，这将为控制猪粪细菌ARGs在环境中的传播与扩散，解决超级细菌的流行奠定基础，相关结果发表论文10篇，其中SCI 6篇，核心期刊2篇，并建立了一支较为的稳定的医药环境微生物研究团队。