饮用水消毒影响微生物抗药性的分子生态学机理研究

Extensive use of antibiotics stimulates the spread of antibioc resistant bacteria and resistance genes in aquatic environments including drinking water and source water, which induces potential public health risks. Disinfection is a crucial step in drinking water treatment process for the removal of pathogenic bacteria, but recent studies indicate that disinfection can promote microbial resistance to antibiotics. Currently, concerns are focused upon isolation and screening of pathogens in drinking water by using culture-dependent methods or conventional molecular biological techniques. These methods are low-throughput and time-consuming and often have systematic bias, which makes it impossible to comprehensively investigate the microbial resistance situation in dirnking water. In this study, based on metagenomics investigation, high-throughput sequencing techniques including Illumina and 454-pyrosequencing will be applied to assess the effects of chlorination, ultraviolet and ozone disinfection on the occurrence and abundance of antibiotic resistant bacteria and resistance genes in drinking water. Some molecular methods (e.g. molecular cloning and quantitative real-time PCR) will also be included to reveal the correlations between the abundances of antibiotic resistance genes and mobile genetic elements, as well as between gene abundance and microbial community structure. The underlying molecular ecological methanisms that disinfection affect microbial resistance will be explored in this study to help theoretically and practically to develop monitoring and control techniques for antibiotic resistant bacteria and resistance genes in drinking water.

抗生素的滥用导致耐药菌与耐药基因广泛传播于水环境中，包括饮用水源和自来水，易引发环境健康风险。消毒工艺是水处理过程中控制致病菌传播的关键步骤，但近期研究表明饮用水消毒可增强微生物耐药性。目前研究工作集中于采用微生物培养和常规分子生物学等方法筛检饮用水中致病菌，该方法存在通量低、系统误差大、操作繁琐等不足，导致无法全面分析饮用水中微生物耐药特征。因此，本项目拟应用Illumina 与454-Pyrosequencing高通量测序技术，从宏基因组学层面研究氯、紫外和臭氧消毒对饮用水中耐药性致病菌和耐药基因分布的影响，分析耐药菌群结构和耐药基因丰度对消毒工艺调控的响应机制；并结合分子克隆、实时定量PCR等技术，研究耐药基因丰度与可移动遗传元件水平的关系、及与菌群结构之间关系；探索消毒影响饮用水中微生物耐药性的潜在分子生态学机理，从而为研发饮用水中耐药致病菌和耐药基因检测和控制技术提供理论指导。

抗生素药物的广泛使用甚至滥用，加速了抗生素耐药基因的传播，增加了环境中耐药基因及耐药细菌的多样性与丰度，严重威胁到人类健康和生态安全。消毒工艺是水处理过程中控制致病菌传播的关键步骤，但近期研究表明饮用水消毒可增强微生物耐药性，并且关于消毒对耐药基因多样性和丰度的影响及其分子机理研究报道较少。本项目应用高通量测序技术和宏基因组学方法，系统研究了氯化消毒和紫外消毒过程中抗生素耐药基因、可移动遗传元件和微生物群落结构的变化特征及其相互关系，揭示了两种消毒方式影响饮用水中微生物耐药的分子生态学机理。研究发现氯化消毒可提升饮用水中假单胞菌属、食酸菌属及鞘脂单胞菌属等相对丰度，饮用水样品中检测到150余种（15类）耐药基因，氯化和紫外消毒均可提升耐药基因的相对丰度，降低耐药基因的多样性。假单胞菌属和食酸菌属中携带的多重耐药类抗性基因和杆菌肽类抗性基因是导致耐药基因总体丰度增加的主要原因。基于微生物群落结构改变和抗生素抗性组变化间的显著相关性，本项目揭示了氯化消毒影响抗生素耐药性的微生物学机理，发现了细菌群落结构和可移动遗传元件的复制可能是导致耐药基因组变化的主要原因。阐明了紫外辐照诱导外排泵相关基因mexR、nfxB和mexZ突变，导致RND外排泵系统mexAB-oprM、mexCD-oprJ和mexXY-oprM等过量表达，从而提升微生物耐药性的分子机理。提出了假单胞菌属和芽孢杆菌属是影响氯化、紫外消毒过程中微生物耐药基因组改变的关键菌属，铜绿假单胞菌对消毒过程中微生物多样性变化起主要作用。项目研究结果为研发饮用水中耐药致病菌和耐药基因检测和控制技术提供了理论指导和数据支撑。