水环境中新兴污染物类抗生素效应（Like-Antibiotic Effects,L-AE）作用机制研究

The wide spread of antibiotic resistance contaminants, such as antibiotic resistance bacteria (ARB) and genes (ARGs), has triggered increasing concerns and research interests on it due to their potential adverse effects to various eco-systems. Aside from the abusive use of antibiotics, there are latest evidences that suggest many substances ubiquitously present in the environment, e.g., heavy metals, nano-materials, and disinfection by-products (DBPs), can also induce effects similar to antibiotics; that is, the presence of these contaminants can facilitate the spread of antibiotic resistance genes (ARGs) in the environment, an effect termed like-antibiotic effect (L-AE). However, much about such L-AE remains unknown. In consideration of the L-AE of environmental emerging contaminations (EECs), the objectives of the proposed research are: (1) to develop methods for evaluating the mode of action for L-AE, including methods for evaluating genetic mutations leading to antibiotic resistance, and within and across species horizontal gene transfer of ARGs; (2) to explore the L-AE mode of action of environmental emerging contaminations, mainly including nano-materials and emerging DBPs, in comparison to antibiotics; and (3) to elucidate the molecular mechanisms of L-AE of EECs by examining the global gene expression profiles and the regulations of key pathways and genes; these would be achieved by using state-of-the-art techniques such as gene expression microarrays and toxicogenomic analysis methods. The proposed research would enhance our understanding on ARGs contamination in the environments and extend research into new areas combining EECs and ARGs in environments, which would improve the safety assessment and risk management of EECs and ARGs in future.

近年来，全球不断出现各类抗生素耐药菌及抗性基因，以此形成的抗性效应污染引起国际社会高度重视，成为迫切需要解决的科学问题。除抗生素外，重金属、纳米材料及消毒副产物等都具有类似于抗生素的效应（Like-Antibiotic Effects，L-AE），即可促进抗性效应污染的形成及扩散，引发严重安全风险。本课题针对这一重要科学发现，研究建立类抗生素效应作用模式（Mode of Action）评价方法，包括诱导抗性基因突变、促进抗性基因种内和跨种属转移评价方法；在此基础上，深入研究典型新兴污染物（重点研究纳米材料和消毒副产物等）类抗生素效应作用模式；针对具有显著类抗生素效应的新兴污染物，利用毒理基因组学和基因芯片等技术，阐明类抗生素效应的分子调控机制。本研究将丰富环境抗性污染理论体系，有助于完善环境化学污染物安全评价及风险管理，也将大为扩展环境新兴污染物和抗性基因安全研究领域。

近年来，全球不断出现各类新型抗生素耐药菌及抗性基因，以此形成的抗性污染引起国际社会的高度重视。除抗生素外，一些环境污染物具有类似于抗生素的效应（Like-Antibiotic Effects, L-AE），即可促进环境中抗性基因产生及转移。针对这些科学问题，本项目研究建立了L-AE作用模式评价方法，包括诱导抗性基因突变、促进抗性基因种内和跨种属转移评价方法；在此基础上，系统研究水环境中典型新兴污染物的类抗生素效应作用模式，并深入解析环境污染物类抗生素效应的分子调控机制。本项目主要取得了以下研究结果：（1）以消毒副产物（DBPs）和纳米金属氧化物（NMOs）为研究对象，探究其对大肠杆菌的抗性突变率及突变菌株抗药能力的影响。结果表明，DBPs和NMOs均可明显诱导大肠杆菌突变为抗生素耐药菌，且突变株具有良好的抗性遗传稳定性。（2）对DBPs和NMOs诱导突变形成抗性基因的机制进行了探究。DBPs和NMOs可明显诱导生成菌内活性氧物种（ROS），进而诱发氧化应激，导致DNA损伤，启动SOS响应，从而提高基因突变速率，形成耐药性突变株。全基因测序组结果显示，DBPs和NMOs诱导形成的突变基因主要在转录和翻译基因、膜结构和转运蛋白基因等。（3）构建了大肠杆菌同种属接合转移模型和从大肠杆菌到鼠伤寒沙门氏菌的跨种属接合转移模型。研究结果表明，环境浓度水平的消毒剂和重金属均可明显促进抗性基因水平转移。（4）亚致死剂量消毒剂和重金属促进抗性基因水平转移的分子机制包括：诱导胞内生成适量胞内ROS，诱导启动SOS响应；促进外膜蛋白量表达，增加细胞膜通透性，形成膜孔和细胞间相互接触与黏附；接合转移全局性调控基因、接合配对形成基因以及质粒转移复制基因的差异性表达。 本研究将丰富环境抗性污染形成及扩散理论体系，有助于完善环境化学污染物安全评价及风险管理，也将扩展环境新兴污染物和抗性基因安全研究领域。