解译驱动抗生素抗性产生的关键环境诱变因子及抗性产生机制研究

The mechanisms of emergence and spread of antibiotic resistance genes (ARGs) in environment have recently drawn great attention all over the world. In this study, antibiotics, heavy metals, detergents, biocides, antiseptics and disinfection byproducts which were frequently detected in environments were selected as the potential mutagenic agents of ARGs to conduct the identification of the key environmental factors resulting in antibiotic resistance generation and the corresponding selection mechanisms exploration. High-throughput sequencing based genomics/metagenomics and transcriptomics/metatranscriptomics in combination with flow cytometry were utilized in the present study and the tasks mainly include (1) the decipher of antibiotic resistance generation mechanisms under single mutagenic factor; (2) the explanation of synergistic effect and antagonistic effect of multiple mutagenic factors for antibiotic resistance development; (3) the revelation of the ARGs elimination and the corresponding molecular mechanisms without mutagenic agents; (4) the identification of the key mutagenic agents leading to antibiotic resistance generation by determining the minimal selective concentration and (5) the elucidation of antibiotic resistance generation mechanisms in activated sludge samples. Via investigating the ARGs formation process driven by antibiotics and non-antibiotic pollutants systematically, the rationale of antibiotic resistance mechanisms will be further improved. Additionally, this study could provide scientific evidences for the environment risk assessment of antibiotic resistance dissemination as well as the elimination and management of ARGs.

抗生素抗性基因（ARGs）在环境中的产生和传播机制已成为研究热点。本项目选取多种抗生素、重金属、清洁剂、杀菌剂、防腐剂和消毒副产物作为潜在的ARGs诱变因子，率先联合应用基于高通量测序技术的（宏）基因组学与（宏）转录组学手段及流式细胞技术，开展如下研究：（1）阐明单一诱变因子驱动抗生素耐药性形成的机理；（2）解析多重诱变因子驱动抗生素耐药性形成的协同效应和拮抗效应；（3）揭示失去诱变压力后耐药性的存在、消除行为及相关分子机制；（4）确定不同诱变因子导致细菌产生耐药性的最小选择浓度，甄别出驱动抗生素耐药性产生的关键环境污染物种类；（5）阐释诱变因子导致活性污泥混合菌群耐药性产生的分子机制。通过系统阐释抗生素类和非抗生素类污染物驱动抗生素抗性产生的诱变机理，可进一步完善抗生素抗性产生的理论基础，为针对细菌耐药性产生及传播的环境风险评估及ARGs的削减与管控提供科学依据。

抗生素抗性的出现能够显著降低现有抗生素药效，其中抗生素抗性基因（ARG）介导的抗生素抗性能够促进其在环境中的传播与扩散，严重威胁公共健康和生态安全。本项目以模式菌株大肠杆菌K12（Escherichia coli K12）和金黄色葡萄球菌ATCC 25922（Staphylococcus aureus ATCC 25922）作为研究对象，在个体水平上开展了不同浓度抗生素暴露实验，识别了不同抗生素诱变细菌耐药性形成的最小选择浓度，并基于基因组学和转录组学技术分析了细菌在表型、基因型以及转录水平的抗生素胁迫响应，明确了gyrA和rpoB基因突变分别是E.coli K12和S.aureus ATCC 25922获得氟喹诺酮和利福平类抗生素抗性的主要机制，且细菌耐药性在撤销诱变压力后并没有发生变化，表明菌株获得了可稳定遗传的抗生素抗性。抗生素和重金属的复合诱变实验表明在一定金属离子浓度阈值内，恩诺沙星和重金属对细菌耐药性产生表现为协同效应，反之则表现为拮抗效应。此外，项目联合应用宏基因组学、宏转录组学和纯培养技术，以活性污泥为研究对象，在群落水平上探究了抗生素和消毒副产物选择压力下微生物菌群的相互作用、应答过程及耐药基因元件功能，明确了微生物群落主要通过高表达抗生素灭活酶和钝化酶来产生耐药性。本研究为进一步明确环境诱变因子驱动细菌耐药性的形成机理，有效管控细菌耐药性在环境中的产生与扩散提供了技术支持和理论依据。