污泥中抗性细菌和抗性基因的噬菌体微生态控制方法及机制研究

Antibiotic resistance has become a major health concern; wastewater treatment plants (WWTPs) as the main treatment facilities for antibiotic substance, are also considered as important hot-spots for antibiotic resistance spreading under long-term antibiotic selection pressures. The sewage sludge of WWTPs contain large amounts of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) during the wastewater treatment process, which contribute a greater amount of ARB and ARGs released to the environment relative to that of the effluent. Therefore, it is necessary to construct a valid method to remove ARB and ARGs from sludge before being discharged from WWTPs. To date, the ARB and ARGs accumulated in sludge cannot be effectively treated by traditional treatment and disposal ways of sludge. Thus, this proposal intends to utilize the regulatory role of bacteriophage on micro-ecology to realize the regulation and control of ARB and ARGs in activated sludge. Firstly, bacteriophage with different lytic host range will be isolated from sewage sludge and be further mixed with each other or production host into different cocktails, which can increase the probability of ARB infection in complex environments. Secondly, bacteriophage will be conjugated with magnetic nanoparticles to improve its migration capability in sludge. The release and distribution of ARGs under different bacteriophage treatment and various antibiotic pressures will be systematically investigated. The effect of bacteriophage treatment on the distribution mechanism of ARGs from the aspect of horizontal gene transfer, microbial community structure and physicochemical properties of sludge will be analyzed. At last, the complex regulatory mechanism of bacteriophage on ARB and ARGs in sludge will be elucidated. These results will benefit more efficient removal of ARB and ARGs in activated sludge and establish a theoretical basis for the antibiotic resistance spreading control with bacteriophage in WWTPs.

污水处理厂是抗生素类物质处理的主要场所，但在长期的抗生素压力选择下，也成为了抗生素抗性传播热点。针对废水处理系统中污泥内大量累积抗性细菌（ARB）和抗性基因（ARGs）现状及相关处理手段匮乏的问题，本项目拟利用噬菌体在微生态控制中的作用，实现对污泥中ARB/ARGs的调控，并阐明其作用机制。拟通过分离驯化不同宿主范围噬菌体、复配噬菌体制剂及优化转运条件和方法，强化噬菌体在复杂体系的迁移能力和抑菌效果。拟探索在不同噬菌体和抗生素残留等毒性压力下ARGs的释放和分布规律变化，解析噬菌体处理过程对ARGs水平转移的影响，揭示噬菌体制剂对微生物群落结构和污泥生化特性、沉降脱水性能的改变规律，阐明利用噬菌体方法控制污泥中ARB/ARGs的微观机制，为噬菌体微生态技术应用于控制废水处理系统中抗生素抗性的传播提供科学依据和技术支撑。

污水处理厂是抗生素类物质处理的主要场所，但在长期的抗生素压力选择下，也成为了抗生素抗性传播热点。相对于水相，污泥内会大量累积抗性细菌（ARB）和抗性基因（ARGs），如何特异性裂解固相中ARB并释放ARGs到水相，使其更易被出水的深度处理手段消除，是有效控制剩余污泥传播抗生素抗性的关键所在。本项目利用噬菌体微生态控制方法实现抗ARB/ARGs的削减，并探究抗生素共暴露对ARGs的释放及其分布规律的影响，解析噬菌体对抗性基因迁移的作用和噬菌体抗性的产生规律及影响因素。主要研究结果如下：1)通过循序多宿主法从活性污泥中成功分离可侵染泛耐药大肠杆菌NDM-1的多价噬菌体PE1、KNT和KN，这些噬菌体在4℃储存60天仍保持了较好侵染活性，且其对温度、pH具有较宽耐受范围，对多种重金属离子也具有一定的耐受能力；2)氨苄青霉素的长期暴露使得活性污泥微生物群落结构发生了显著变化，敏感菌群减少，与氮循环和颗粒污泥形成相关的菌属相对丰度显著增加有助于抗生素的降解，但相关耐药基因丰度也显著增加；3)多价噬菌体PE1能在16小时内几乎完全抑制NDM-1的增殖，与产生菌复配的噬菌体鸡尾酒可延长该时间至34小时，并显著消减固相抗性基因blaNDM-1；随着处理时间增加，宿主细菌开始产生噬菌体抗性，但噬菌体处理组仍可在一定程度上减少菌量并促进ARGs释放到水相，使其更易被UV/PAA去除；噬菌体与壳聚糖改性Fe3O4磁性材料耦合可以显著促进耐药细菌生物膜的去除；4）细菌耐药能力和环境压力与噬菌体抗性的产生具有一定的相关关系：低浓度抗生素和重金属暴露环境下，敏感型大肠杆菌所形成的噬菌体抗性菌对抗生素的敏感性增强，与体内的溶原性噬菌体和共抗性机制有关，而耐药大肠杆菌TEM和NDM-1噬菌体抗性的产生对其抗生素耐药能力没有明显影响，且耐药能力越强其噬菌体抗性的产生不受抗生素暴露浓度的影响。该项目的研究结果将为噬菌体微生态技术应用于控制废水处理系统中抗生素抗性的传播奠定理论基础。

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