典型耐药菌及耐药基因在光和矿物作用下的环境地球化学转化机制研究

The presence of antibiotic-resistant bacteria and antibiotic-resistance genes in aquatic environment has gained increasing concern recently, due to their ability to potentially transmit resistance to other organisms, disturb the natural ecological balance and shade health risks to humans and animals. This project aims to have a full insight of the environmental geochemical transformation mechanism of typical antibiotic-resistant bacteria and antibiotic-resistance genes onto the mineral interface under the light irradiation. Firstly, typical antibiotic-resistant bacteria and antibiotic-resistance genes were isolated from antibiotics heavily polluted water. Secondly, the damage kinetics and inactivation mechanism of typical antibiotic-resistant bacteria and antibiotic-resistance genes were investigated under the irradiation of light particularly under sun light. Thirdly, the adsorption/desorption and enrichment/inhibition of antibiotic-resistant bacteria and antibiotic-resistance genes onto the mineral interface were studied to understand their migration and transformation, damage as well as the elimination mechanism in the presence of minerals. Furthermore, the transformation, inhibition and inactivation mechanisms were studied more detailed in the presence of both light and minerals by tracking the damage of antibiotic-resistant bacteria and by monitoring the antioxidative enzyme activities, photoreactivation and dark repair, antibiotic resistance, the transfer of antibiotic resistance genes between different antibiotic resistant bacteria as well as the evolution of bacterial communities. Finally, the environmental geochemical transformation mechanism of typical antibiotic-resistant bacteria and antibiotic-resistance genes were also investigated and further validated by knocking out some special antibiotic-resistance genes. The successful accomplishment of the project will provide a fundamental knowledge of the transportation, transformation, environmental fate and elimination mechanism of typical antibiotic-resistant bacteria and antibiotic-resistance genes in water environments.

抗生素耐药菌和耐药基因对水体的污染严重，危害大众健康，从而引起了全球广泛关注。本项目首先拟从环境污染水体中分离出耐药大肠杆菌和耐药基因，研究不同光照特别是太阳光照射下对它们的损伤动力学和灭活机制；然后研究它们在矿物尤其是在天然矿物界面的吸附/解吸、富集/抑制作用，探讨它们在矿物界面的迁移转化、损伤和消除机制；进一步研究典型耐药基因和耐药菌在光和矿物同时存在条件下的环境损伤、抑制、迁移转化和灭活机制；通过追踪耐药菌的损伤、酶应激系统启动、复活机制、耐药性变化检测、基因的转化和耐药菌群落演替及种群间耐药基因的转移等一系列生物和物理化学表征，阐明它们在光照下的矿物界面环境地球化学过程，进一步通过耐药基因的敲除技术来定向阐明并验证特殊耐药基因的迁移转化及其对耐药菌灭活的影响作用机制。本项目的顺利实施可以为环境水体中典型耐药基因和耐药菌的迁移、转化和归趋及其环境消除提供翔实的理论和实验依据。

抗生素耐药菌(ARB)和耐药基因(ARGs)在多种环境介质中持久存在并广泛传播，对社会卫生安全造成潜在威胁，而目前其在环境中的迁移转化机制还有待进一步明晰。光、矿物及细菌在环境中常常同时存在。因此，本项目探求了光和矿物作用在ARGs传播过程中的作用机理。主要的研究内容和结果如下：.(1)在光照刺激下，发现VL光照对接合转移频率没有明显的影响，而在SS和UV照射下，接合转移频率分别有2~10倍和高达100倍的增加。同时发现VL光照下即不影响细菌的生理生化条件，也不诱导氧化应激和基因表达。在SS照射下，氧化应激发生缓慢，相关的基因在细菌细胞中的表达略有增加。而在紫外照射下，细菌迅速失活，氧化应激程度非常严重，相关的基因表达明显上调。.(2)在矿物作用下，发现天然闪锌矿(NS)暴露可以引起细菌应激反应并引发细胞膜通透性改变，改变接合相关基因表达和导致接合转移效率的增加，其中ARGs在环境系统中表现出更强的持久性。与商业ZnS相比，其对耐药基因接合转移效率的变化随矿物浓度的增加而增加。NS促进ARGs转移的机制可能涉及氧化应激对细胞膜的损伤、应激相关基因表达的增强以及膜蛋白基因mRNA表达的改变。.(3)此外，研究了NS在光照射下对ARGs转移机制的影响。结果显示，NS在VL和UV254nm光照下，耐药基因的接合转移频率分别有2~12和2~10倍的增加，而在SS和UV365nm光照下，耐药基因的接合转移频率没有明显的变化。ARGs转移与ROSs形成，氧化应激反应，ARB的功能基因的表达等应激有较强的联系。NS在VL光照下对细菌的ROSs形成、诱导氧化应激和基因表达均有轻微影响。NS在SS光照下，氧化应激发生缓慢，细胞内目标基因的表达略有增加。而NS在UV254nm照射下能迅速灭活细菌，氧化应激增加，目标基因表达明显上调，这仅取决于UV254nm光照的影响。此外，NS在UV365nm光照下对细菌的灭活较强，氧化应激程度增加，目标基因表达明显上调。.本项目的顺利实施可以为环境水体中典型耐药基因和耐药菌的迁移、转化和归趋及其环境消除提供翔实的理论和实验依据。