纳米材料抗菌过程中诱导多重抗生素耐受细菌形成及机制研究

Antimicrobial agents are the primary means of water disinfection and the treatment of infectious diseases. But they have some disadvantage, including the rapid development of antibiotic resistance and producing disinfection byproducts which is toxicity to the human body. This has prompted the development of alternative strategies for new green antibacterial materials. Use of nanoparticles is among the most promising strategies to overcome the shortcomings of traditional antimicrobials. However, our previous study found that nanomaterials can not completely kill the bacteria, and even induce persisters formation, which are with the ability to tolerate multiple antibiotics, and seem to indicate the source of chronic infections. The formation of persisters induced by nanomaterials has become a challenge to use of nanomaterial as antimicrobial. Here, we propose a hypothesis that 'Nanomaterials can induce the formation of multiple antibiotic tolerance bacteria in the process of antibacterial'. In this project, we will observe the processes and conditions of persisters formation in the antibacterial process using nanomaterials and determine the resistance performance of persisters to clarify the rules and factors of persisters formation induce by nanomaterials. And using electron microscopy, fluorescence quantitative PCR, Western-blot, whole-genome expression profiling, functional gene construct technology, we will observe the effect of nanomaterials on the microsurrounding, metabolic and gene regulatory processes of bacteia to clarify the mechanisms of persissters formation induced by nanomaterials. After completing this project,we will gain more wealth of knowledge on bacterial resistance and the behaviors of bacteria at environmental pressure，which will provide technical and theoretical support for the prevention and control of drug resistance and treatment of chronic infections. We can provide theoretical and technical support for application of nanomaterials as antimicrobial agents with the results and technicals established in this project.

抗菌剂是饮水消毒和感染性疾病治疗时使用的主要手段，但是消毒副产物的产生及抗生素耐药的迅速发展，迫使人们寻找新型绿色的抗菌材料。利用纳米材料是克服传统抗菌剂缺点最有前途的策略。然而我们前期研究发现纳米材料并不能完全杀死细菌，甚至诱导具有多重抗生素耐受的持留细菌产生。这是细菌获得耐药性的新机制，也是纳米材料作为新型抗菌剂的严峻挑战。本项目提出"纳米材料抗菌过程中诱导多重抗生素耐受细菌形成"的假说，通过观察抗菌过程中持留细菌形成过程和条件及其耐药性能，阐明纳米材料诱导持留细菌形成条件和规律；利用电镜、定量PCR、Western-blot、全基因组表达谱、功能基因构建等技术观察纳米材料对细菌微环境、代谢及基因调控过程的影响，阐明纳米材料诱导多重抗生素耐受细菌形成机制，丰富耐药性获得知识，提高对环境压力下细菌行为的认识，为防控细菌耐药产生、感染性疾病治疗及纳米抗菌剂的研究与应用提供理论与技术支持。

抗菌剂是饮水消毒和感染性疾病治疗时使用的主要手段，但是消毒副产物的产生及抗生素耐药的迅速发展，迫使人们寻找新型绿色的抗菌材料。利用纳米材料是克服传统抗菌剂缺点最有前途的策略。我们前期研究发现纳米材料并不能完全杀死细菌，甚至会诱导细菌向引起持续感染的多重抗生素耐受的持留细菌转变，这是对纳米材料作为新型抗菌剂的严峻挑战。因此本项目提出“纳米材料抗菌过程中诱导多重抗生素耐受细菌形成”的假说，通过观察抗菌过程中持留细菌形成的过程和条件，阐明纳米材料诱导持留细菌形成条件规律及机制。.本项目建立了基于氧氟沙星和氨苄青霉素筛选的持留细菌测定方法和技术，准确测定混合细菌体系中进入持留细菌的数量，并利用该方法研究了纳米材料作用于模式菌株E.Coli K12后迫使细菌进入持留状态的规律和条件。结果表明常见的纳米Ag、纳米Al2O3、纳米TiO2等具有抗菌和抑菌作用的纳米材料在杀灭细菌的同时能够诱导部分细菌进入持留状态而获得对多种抗生素的耐受。其中纳米Al2O3的作用效果最明显，其能够诱导存活细菌成为持留菌的比例1.98e-4，是未经纳米材料处理的15倍多。同时纳米材料的浓度越多，持留细菌占存活细菌的比例越高；持留细菌的数量随着作用时间的延长先增高后降低；而且纳米材料直径越小产生的持留细菌越多。.我们建立了数学模型研究了纳米材料对细菌的抑制过程，发现纳米材料通过诱导细菌进入持留状态而降低生长速率；细菌明显分化成三类细胞：持留细胞、损伤细胞和死亡细胞，而且随着纳米材料的作用增强持留细胞的比例增加；通过形态学分析进入持留状态的细菌其细胞体积变小、细胞质更致密、细胞膜的褶皱变小；全基因组表达谱分析结果显示主要通过抑制细胞代谢和合成过程，节省必需物质的原材料和能量，从而为细菌的生存积累物质和能量。.本项目的研究结果为纳米材料抗菌剂应用提供理论与技术支持，丰富耐药性获得的知识，提高了人们对环境压力下细菌行为的认识，为防控耐药性的产生提供技术与理论支持。

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