NDM-1基因在纳污水环境微生物菌群的多态性分布及传播扩散机制

Our study focuses the distribution and the propagation-proliferation mechanism of the NDM-1 genes in water-environment microflora of NDM-1 genes pollution waters, with the approaches of analyzing field samples and building laboratory aquatic microcosm. Firstly, the sampling map is designed according to the standards along the orientation of contaminant diffusion in the waters accepted sewage effluent, and water and sediment samples are collected meanwhile implementing detection of ecological factors. NDM-1 genes can be firstly detected qualitatively by PCR, and then quantified by RTQ-PCR. Some bacteria carrying NDM-1 gene will be isolated, and identified by 16S rDNA sequencing and Biolog system. These species specific sequences of the isolates will be treated as indicators of these resistance strains, which indicator will help us to analyze composition of microflora by RTQ-PCR. Secondly, plasmid is extracted from the isolates, and full sequences of these plasmids are sequenced by pyrosequencing. Molecule analysis is carried out on the plasmid sequences, and classification and transfer mechanism of plasmids are focused on with bioinformatics methods. Finally, several microcosm systems, such as conjugations of same genus bacteria, conjugation of different genus bacteria and transformation system, will applied to detect the horizontal transfer of NDM-1 genes undergoing the influences of ecological factors. An opportunistic pathogen of mankind is even included in the microcosm system to indicate the ecological risk of NDM-1 genes. Our study has a potential theoretical and practical significance preventing the propagation of NDM-1 genes in the waters.

本研究针对NDM-1基因污染水域，拟用野外采样和室内模拟结合的方法，开展NDM-1基因在水环境菌群中的多态性分布和传播扩散机制研究。首先，以排放污水地点为起点，沿着受纳水体污染物扩散方向，布设采样点，采取水和沉积物样本，同时检测分析水生态因子。定性定量分析NDM-1基因，分离携带NDM-1基因的抗性细菌，并通过16S rDNA测序和Biolog鉴定细菌，再进一步通过细菌种特异序列定量细菌，逐步解析菌群结构；然后，从分离细菌中提取质粒DNA，实施质粒测序，对转移等机制进行分子分析；最后，通过实验室构建的各类模拟体系（同属种接合、异属接合、转化等）分析NDM-1基因的水平转移效率，及各因子对NDM-1基因转移机制影响，通过构建含有人类条件致病菌的模拟体系，考察NDM-1基因由环境传至人致病菌的风险。本研究对揭示NDM-1基因在水体中的传播机制，制定措施降低公共健康风险，具有理论和现实意义。

blaNDM-1基因是一种多重耐药基因，作为“超级细菌”的耐药机制之一，其主要检出于临床样本，环境中检出状况和分布还不清楚。因此，本项目开展了环境中blaNDM-1基因的检测、来源与分布及传播扩散的研究。研究发现，在中国北方的多个污水处理厂中从进水到出水的各个处理环节，包括各个处理单元中均能检测到blaNDM-1基因，浓度达3.1*10^3~9.2*10^7 拷贝/毫升水或污泥，并在其下游纳污河中检测到了blaNDM-1基因，数据分析和基于序列的进化分析表明污染来自于污水厂处理后出水。进入纳污河的blaNDM-1基因具备很强的在环境土著菌间传播扩散的能力。环境中blaNDM-1基因主要分布于底泥的胞外DNA和细菌胞内DNA以及河水细菌胞内DNA中，胞外和胞内blaNDM-1基因都具备水平转移的能力。底泥和河水中blaNDM-1基因的增殖扩散都受到有机质和抗生素浓度的显著影响。我们检测到了多个携带blaNDM-1基因的质粒载体，分属不同质粒类群，但都具有较高接合转移能力。同时在研究中我们也检测到了变种基因blaNDM-5基因的出现，其赋予细菌具有更强的耐药性，对致病性亦有一定影响。本研究为后续健康风险研究提供了基础信息，并奠定了可行的检测方法学，为相关管理部门制定污水排放标准和管理政策提供了数据依据。