耐药型反硝化菌对抗生素的共代谢降解与作用机制研究

The removal efficiency for some antibiotics in municipal wastewater is relative low through the conventional treatment processes, which may cause potential ecological risk of increased antibiotic resistance of aquatic microbe. A co-metabolism technology is proposed to biodegrade antibiotics by antibiotic-resistant denitrifying bacteria in this project. The characteristics and mechanism at genetic and transcriptional level of antibiotics biodegradation will be investigated after the selection of co-metabolic substance and acclimatization of denitrifying bacteria. The co-metabolic degradation pathway of antibiotics by denitrifying bacteria will be deduced based on the continuous identification of degradation product. Through metagenomics sequencing, the metabolic pathways of biodegradation of antibiotics and co-metabolic substance will be deduced, and the denitrifying bacteria community structure will be revealed. The functional genes of antibiotics and co-metabolic substance biodegradation could be identified through the variation analysis in genetic expression. Some inherent regularities will be revealed including the relevance between antibiotics biodegradation and co-metabolic substance biodegradation, the mechanism of effect of antibiotic resistance genes on antibiotics biodegradation, and response of genetic expressions to antibiotic concentration. The stabilities of antibiotic resistance gene abundance and bacteria community structure will be evaluated under a low concentration of antibiotics condition. Through this project, a technology that antibiotic biodegradation through co-metabolism by antibiotic-resistant denitrifying bacteria will be developed. More understanding on antibiotics biodegradation will be unveiled.

城市污水中部分抗生素通过常规工艺处理的去除率较低，导致自然水环境中微生物耐药性增强的生态风险。本项目提出利用耐药型反硝化菌的共代谢作用降解微量抗生素的风险控制技术。基于最适共基质的筛选和反硝化菌群的构建，开展抗生素降解特征以及基因水平和转录水平的降解机理研究。通过降解产物的连续分析鉴定，推断反硝化菌对抗生素的共代谢降解途径；采用宏基因组测序分析，预测抗生素和共基质降解过程的代谢通路，解析抗生素降解反硝化菌群物种组成；通过基因表达的差异化分析，识别出抗生素和共基质降解的关键功能基因，揭示抗生素降解与共基质降解过程之间的关联规律、抗性基因在抗生素降解中的作用机制以及三类基因表达对抗生素浓度的响应关系；评价耐药性反硝化菌长期接触低浓度抗生素时抗性基因丰度和菌群结构的稳定性特征。基于以上研究，开发出共基质调控耐药型反硝化菌促进抗生素降解的风险控制技术，扩展抗生素分解代谢理论的认识。

城市污水中部分抗生素通过常规工艺处理的去除率较低，导致自然水环境中微生物耐药性增强的生态风险。利用特异外碳源对反硝化菌群落进行调控，通过反硝化共代谢过程强化抗生素的降解，是去除微量抗生素的潜在技术途径之一。本课题以水源地水库为研究对象，考察水环境中化学指标、17种代表性抗生素、285种抗生素抗性基因（ARGs）和12种可移动元件在不同位置以及季节的赋存特征。结果表明生态净化工程可以削减水染物、抗生素的ARGs的绝对丰度；同时发现，ARGs的相对丰度与硝态氮和亚硝态氮变化有显著相关性，揭示了反硝化过程对于抗生素抗性在微生物群落中的传播具备潜在的关键作用；以工程上主要应用的乙酸钠和甲醇作为特异外碳源，同时以接近污水中抗生素实际浓度的氧氟沙星作为胁迫条件，培养反硝化菌。利用驯化稳定的反硝化污泥研究不同浓度抗生素对反硝化过程的影响，以及抗生素的吸附降解效果。结果发现：以甲醇为特异碳源无生素胁迫条件下培养的反硝化菌具有一定的抗生素耐药性，说明抗生素抗性可以通过选择碳源调控微生物群落的共选择过程实现传播；长期接触微量抗生素和特异碳源的反硝化菌可能对高浓度抗生素产生耐药性；经过特异碳源培养和微量抗生素胁迫的反硝化菌对抗生素具有显著的吸附和降解效果。以上结果说明长期投加外碳源可以对污水处理系统的微生物群落结构进行调控，筛选特异外碳源驯化稳定的污泥具有较高的耐药性，并可以通过共代谢增强降解抗生素的效果。采用16S rDNA扩增子和宏基因组的高通量测序技术对特异碳源和不同抗生素胁迫条件下培养反硝化菌以及多处长期投加外碳源的污水处理厂活性污泥的微生物群落和功能基因进行分析，揭示外碳源对微生物组成的影响规律。