环境浓度抗生素和纳米抗菌剂对生物膜和细菌抗药性的联合作用与机制

Antibiotics in the environment are generally characterized by low concentration, which can induce the formation of biofilm and thus facilitate the bacterial resistance mutation and the horizontal genes transfer. Nano-antibacterial agents are promising alternatives to antibiotics and have been extensively used. However, it remains unclear how the combined exposure of antibiotics and nano-antibacterial agents would influence the biofilm and the emergence and dissemination of antibiotic resistance genes (ARGs). Therefore, this research will focus on the joint effects of low-concentration antibiotics and nano-antibacterial agents on the biofilm formation and the ARGs of biofilm bacteria. Three commonly used antibiotics, i.e. tetracycline, quinolone and β-lactam, and several typical nano-antibacterial agents, i.e. nano-Ag, CuO and ZnO, are chosen as the research subjects; crystal violet staining, fluctuation analysis, and plasmid junction transfer test are employed to investigate the joint effects on the biofilm formation, the bacterial resistance mutation and the horizontal genes transfer, respectively. The aim of this research is to figure out whether the combined exposure of antibiotics and nano-antibacterial agents would influence the biofilm formation and if so how, and to reveal the effects of their combined exposure on the resistance mutation and horizontal genes transfer of the biofilm bacteria. This research will give a more comprehensive understanding on the environmental and health risks of combined exposure to antibiotics and nano-antibacterial agents, provide a theoretical guide on the effective control of the emergence and dissemination of ARGs and the drug-resistant bacteria in the natural environment.

环境中普遍以低浓度形式存在的抗生素能够诱导生物膜的形成，从而促进细菌抗药性突变和抗性基因的水平转移。纳米抗菌剂作为抗生素的理想替代物，已经得到了广泛的应用。但抗生素和纳米抗菌剂的联合暴露对生物膜以及抗性基因产生和传播的影响尚缺乏充分认识。因此，本课题拟选取四环素、喹诺酮、β-内酰胺等常用抗生素和纳米Ag、CuO、ZnO等典型抗菌剂作为研究对象，选用大肠杆菌作为模式细菌，采用结晶紫染色、波动分析和质粒接合转移实验等方法，开展二者低浓度联合暴露对细菌生物膜以及抗性基因产生和传播的影响与机制研究；通过研究，探明低浓度抗生素和纳米抗菌剂是否影响生物膜形成以及如何影响，揭示二者联合暴露下生物膜细菌的抗药性突变和抗性基因水平转移的规律。本课题的研究有助于更加全面地认识抗生素和纳米抗菌剂联合暴露的环境和健康风险，为有效防控抗性基因和耐药细菌在自然环境中的传播和扩散提供理论指导。

细菌耐药性严重威胁人类健康，而抗生素暴露是细菌耐药性产生的主要原因之一。此外，以纳米抗菌剂和群体感应抑制剂作为代表的抗生素替代药物已被广泛应用，造成在人体和真实环境中抗生素及其替代物普遍共存，但是二者的联合抑菌效应，特别是对细菌耐药性演化的联合诱导效应，所受关注较少。本项目以抗生素及其替代物（纳米抗菌剂和群体感应抑制剂）作为目标化合物，开展不同化合物联合作用时对大肠杆菌的抑菌效应和耐药性突变诱导效应研究，结合DNA提取、基因测序和比对、荧光定量PCR等分子生物学技术对细菌的突变位点和突变诱导机制进行探讨。本项目的主要研究结果和关键数据归纳如下：（1）获取了氯霉素、红霉素、四环素和氟喹诺酮等抗生素及相关降解产物对生物膜形成的剂量效应关系，以及对细菌的耐药性突变率，揭示了抗生素在低浓度下对生物形成和耐药性突变的促进作用；（2）获取了纳米银和氧化锌与抗生素的联合抑菌效应和耐药性突变诱导效应的数据，发现了纳米抗菌剂对耐药性突变的促进作用，并且联合作用时，纳米抗菌剂减少了浮游菌的突变但是增加了生物膜的突变；（3）发现了以肉桂醛和4-硝基吡啶-N-氧化物为代表的群体感应抑制剂能够遏制细菌的耐药性突变，根据PCR的结果推断，群体感应抑制剂通过促进细菌DNA修复系统的表达从而使细菌的耐药性突变率稳定在较低水平；（4）通过DNA测序确定了抗生素、纳米抗菌剂和群体感应抑制剂诱导的利福平耐药性突变位点，结果表明这些位点均位于利福平抗性决定区域；同时，本项目还搜集了文献和数据库中细菌耐药性突变的实验数据，借助机器学习构建了细菌耐药性突变的预测模型。本项目的研究结果为抗生素及其替代物在真实环境中的联合生态和健康风险评估提供了数据基础，为探索细菌耐药性突变的发生机制以及防控细菌耐药性产生开辟了新的思路，同时，构建的机器学习模型在预测细菌耐药性和新药研发等领域具有很好的应用前景。