郑州市区河流水系接合质粒介导的碳青霉烯类及多粘菌素类抗性基因污染特征及分子传播机制研究

The spread of carbapenem resistance genes and polymyxin resistance genes posed serious threat to human health. Therefore, several studies on carbapenem resistance genes and polymyxin resistance genes have been conducted in urban river system of Zhengzhou city. First, real-time quantitative PCR were used to determine the spatial distribution and abundance of carbapenem resistance genes and polymyxin resistance genes. Second, carbapenem resistant conjugative plasmids and polymyxin resistant conjugative plasmids were isolated through filter mating assays. And then, the phenotypes, genotypes, conjugative frequency of these obtained plasmids were determined by antibiotic susceptibility testing, qualitative PCR and broth mating assays to investigate the diversity and transferability of these plasmids in urban river system of Zhengzhou city. In the meantime, representative plasmids were completely sequenced using high-throughput sequencing techniques to preliminarily reveal the molecular dissemination mechanism of carbapenem resistance genes and polymyxin resistance genes in urban river system of Zhengzhou city. At last, 16S rDNA high-throughput sequencing techniques were applied to study the community structure and abundance of microbes in urban river system of Zhengzhou city, which aimed to clarify the dominant bacteria in these areas. Results of this research will help to provide basic data and a scientific basis for the control and removal of antibiotic resistance genes in urban river system, which is of theoretical significance and practical significance.

碳青霉烯类抗性基因和多粘菌素类抗性基因对人类健康构成巨大威胁，本项目拟通过荧光定量PCR技术，研究碳青霉烯类抗性基因和多粘菌素类抗性基因在郑州市区河流水系中的分布及污染情况；通过滤膜接合实验，分离碳青霉烯类抗性接合质粒和多粘菌素类抗性接合质粒，并采用药物敏感性实验、定性PCR、肉汤接合实验等方法检测分离到的接合质粒的抗生素抗性表型、基因型和接合转移能力，以研究郑州市区河流水系中碳青霉烯类抗性接合质粒和多粘菌素类抗性接合质粒的多样性和迁移扩散能力；同时，选取代表性接合质粒，采用高通量测序方法进行质粒全序列测定，初步揭示碳青霉烯类抗性基因和多粘菌素类抗性基因传播扩散的分子机制；最后，采用16S rDNA高通量测序方法，研究郑州市区河流水系微生物群落组成及相对丰度，阐明其中的优势菌群。本项目将为城市河流中抗生素抗性基因污染的控制和消减提供基础数据和科学依据，具有重要理论意义及现实意义。

随着抗生素抗性问题的发展和恶化，碳青霉烯类抗生素和多粘菌素类抗生素在对抗耐药菌感染中发挥着越来越重要的作用。城市河流与人类活动关系密切，是抗生素抗性基因传播扩散的一个重要环节，因此对碳青霉烯类抗性基因和多粘菌素类抗性基因在城市河流中的污染特征及传播扩散的分子机制进行综合研究并制定相关防控消减对策刻不容缓。本项目首先通过16S rRNA高通量测序研究了郑州市区河流水系底泥样品的微生物组成，发现不动杆菌属在大部分样品中含量比较高，为优势菌属。本项目进而通过定量PCR系统地研究了碳青霉烯类抗性基因和多粘菌素类抗性基因在郑州市河流水系中的污染特征，发现blaNDM、blaIMI、blaOXA-58-like、blaOXA-48-like、blaOXA-51-like等碳青霉烯类抗性基因的含量在河流的上游普遍低于下游，有些基因甚至在上游检测不到；多粘菌素类抗性基因mcr-1的含量也是在河流的上游普遍低于下游。最后本项目全面地研究了郑州市区河流中碳青霉烯类抗性菌和多粘菌素类抗性菌，并对有代表性的多株菌进行了基因组测序，初步阐明了碳青霉烯类抗性基因传播扩散的分子机制。本项目的研究成果一方面可以为郑州市区河流环境污染提供风险预警，另外一方面可以为抗生素抗性基因这种新型污染物的防控提供科学依据和决策支撑。