基于纤维素酶和葡萄糖氧化酶级联效应的Mn-Zn-ZIF抗生物膜和抗菌效果评价

Evaluation of the anti-biofilm and antibacterial effects of Mn-Zn-ZIF based on the cascade effect of cellulase and glucose oxidase

摘要:

背景 细菌耐药性严重威胁着人和动物的健康，生物膜的形成是引起细菌耐药的重要原因之一，常规抗菌药物很难渗透进入生物膜内而且细菌容易产生耐药性。本研究基于纤维素酶(cellulase, CL)和葡萄糖氧化酶(glucose oxidase, GOX)的级联效应，构建了包载CL和GOX的Mn-Zn-ZIF材料，目的 实现对生物膜胞外基质的降解作用及活性氧的高效杀菌作用。方法 通过“一锅水合法”制备负载CL和GOX的Mn-Zn-ZIF复合材料(CL&GOX@Mn-Zn-ZIF)，并对该复合材料进行表征，通过倍比稀释法测定其对金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌(Escherichia coli)的最小抑菌浓度(minimum inhibitory concentration, MIC)和抑菌率；考察该复合材料产活性氧(reactive oxygen species, ROS)的能力及对细菌细胞膜完整性的影响；通过结晶紫染色测定其对成熟生物膜的清除情况，考察其对纤维素的降解能力。结果 CL&GOX@Mn-Zn-ZIF有效负载了CL和GOX，其对大肠杆菌和金黄色葡萄球菌的MIC均为160 μg/mL；CL&GOX@Mn-Zn-ZIF加β-D-葡萄糖处理后，其产ROS能力显著增加，金黄色葡萄球菌和大肠杆菌eDNA的泄漏情况显著增加(P<0.05)；CL&GOX@Mn-Zn-ZIF对金黄色葡萄球菌和大肠杆菌成熟生物膜的清除情况也显著性增加(P<0.05)，可以明显降解纤维素以破坏生物膜。结论 CL&GOX@Mn-Zn-ZIF能够抑制大肠杆菌和金黄色葡萄球菌的生长，破坏细胞膜结构，清除成熟生物膜，从而有望作为一种新型有效的抗菌策略。

Abstract:

Background Bacterial resistance poses a serious threat to human and animal health. The formation of biofilms is one of the main reasons for bacterial resistance. Conventional antibacterial drugs have difficulty in penetrating into biofilms and bacteria are prone to developing resistance. In this study, the Mn-Zn-ZIF material loaded with cellulase (CL) and glucose oxidase (GOX) was constructed based on the cascade effect of CL and GOX.Objective To achieve the degradation of extracellular matrix in biofilms and the efficient bactericidal effect of reactive oxygen species (ROS).Methods The CL and GOX-loaded Mn-Zn-ZIF composite material (CL&GOX@Mn-Zn-ZIF) was prepared by a one-pot hydrothermal method and then characterized. The minimum inhibitory concentrations (MICs) and inhibition rates of CL&GOX@Mn-Zn-ZIF against Staphylococcus aureus and Escherichia coli were determined by the serial dilution method. The ability of CL&GOX@Mn-Zn-ZIF to produce ROS and its effect on the integrity of bacterial cell membranes were investigated. The clearance of mature biofilms by CL&GOX@Mn-Zn-ZIF was determined by crystal violet staining, and its cellulose degradation ability was examined.Results CL&GOX@Mn-Zn-ZIF effectively loaded CL and GOX, and its MICs against E. coli and S. aureus were both 160 μg/mL. The treatment with CL&GOX@Mn-Zn-ZIF and β-D-glucose increased the ROS production and promoted the leakage of eDNA from S. aureus and E. coli (P<0.05). In addition, CL&GOX@Mn-Zn-ZIF showed enhanced performance in clearing the mature biofilms of S. aureus and E. coli (P<0.05), and it could significantly degrade cellulose to destroy biofilms.Conclusion CL&GOX@Mn-Zn-ZIF can inhibit growth, disrupt the cell membrane structure, and clear the mature biofilms of E. coli and S. aureus, thus being expected to be a new and effective antibacterial strategy.