体外构建的细菌生物膜反应器仿人体膀胱尿液紊流切应力系统

Urine turbulent shear stress system of bionic human bladder based on bacterial biofilm reactor: in vitro construction

背景:材料表面细菌生物膜形成是导管相关尿路感染的核心问题,已有研究多着眼于静态或简单流体力学条件下感染机制及防治,构建接近真实人体疾病状态下的动态膀胱细菌生物膜模型为研究疾病机制、开发抗生物膜感染新技术的关键。目的:提出人体膀胱尿液紊流切应力概念,体外构建基于仿人体膀胱细菌生物膜反应器的尿液紊流切应力系统,探讨不同应力刺激大肠埃希菌生物膜形成。方法:自行设计体外仿人体动态膀胱尿流模型,以大肠埃希菌标准株ATCC25922为模式菌、医用硅胶膜片为载体,模拟4种人工尿流应力:静水压、恒流切应力、生理切应力及病理切应力(模拟尿潴留环境),建立加载紊流切应力细菌生物膜反应器。24,72,120,168 h时检测生物膜细菌悬液吸光度值、菌落计数与生物膜表面积。结果与结论:①细菌悬液吸光度值:不同组间、不同时间之间存在显著差异(F=110.84,187.96,P均<0.000 1),时间与应力之间存在交互作用(F=50.05,P <0.000 1),从静态应力、动态持续应力力、生理尿流应力到尿潴留病理应力,生物膜细菌菌落数增加;②细菌菌悬液涂片菌落计数:不同时间之间菌落计数有显著差异(F=6.30,P=0.002 9),不同尿流应力组间无差异(F=1.11,P=0.400 1),时间与应力不存在交互作用(F=0.85,P=0.581 4);但随着施加应力时间延长,复杂应力组菌落计数呈增加趋势,尤以病理切应力组显著;③生物膜细菌扫描电镜表征:各组及不同时间点之间定性比较,细菌生物膜形成从稀疏片段、成块到大片成团形状,具有差异;不同尿流应力组间、不同时间之间菌膜表面积存在显著差异(F=505.72,1 201.84,P均<0.000 1),时间与应力之间存在交互作用(F=78.14,P <0.000 1);从静态应力、动态持续应力、生理尿流应力到尿潴留病理应力,细菌生物膜生成显著增加;④结果提示,该仿人体膀胱模型尿液紊流切应力系统可明显刺激体外大肠埃希菌生物膜形成,其功能变化及机制有待进一步探讨。

BACKGROUND: The formation of bacterial biofilm on the material surface is the core problem of catheter-related urinary tract infection. Many researches have focused on the mechanism and prevention of such category of infection under static or simple hydrodynamic stimulation. The construction of dynamic model of bacterial biofilm of bladder urine flow close to real human diseases is the key to study the pathological mechanism and develop new technology of antibiofilm infection.OBJECTIVE: To put forward the concept of turbulent flow shear stress of human bladder urine flow, construct this turbulent shear stress system based on the bacterial biofilm reactor of in vitro bionic human bladder, and explore the formation of E. coli biofilm stimulated by different stresses.METHODS: An in vitro dynamic bionic bladder urine flow model was designed. E. coli standard strain ATCC25922 was used as research object, and the medical silica gel was used as bacterial biofilm forming carrier. Four artificial urine flow stresses were simulated: hydrostatic pressure, constant turbulent flow shear stress, physiological turbulent flow shear stress and pathological turbulent flow shear stress(simulated urine retention environment). A bacterial biofilm reactor loaded with turbulent flow shear stress was established. Optical density value, colony count, and biofilm surface area of bacterial biofilm suspension were detected 24, 72, 120, and 168 hours. RESULTS AND CONCLUSION:(1) Optical density value of bacterial membrane suspension: there was significant difference between different urinary stress groups and different test time points(F=110.84, 187.96, all P < 0.000 1), and there was interaction effect between time and stress(F=50.05, P < 0.000 1). From hydrostatic pressure, constant turbulent flow shear stress, physiological turbulent flow shear stress, to pathological turbulent flow shear stress, the number of biofilm bacterial colonies increased.(2) Colony count of biofilm bacterial suspension smear: there was significant difference between different time(F=6.30, P=0.002 9);no difference was found between different urinary stress groups(F=1.11, P=0.400 1);and there was no interaction effect between time and stress(F=0.85, P=0.581 4). However, with the time extension of stress action, the colony count of complex stress group showed an increasing tendency, especially in the pathological turbulent shear stress.(3) Scanning electron microscopic characterization of biofilm bacteria: qualitative comparison between each group and different time points showed that the formation of bacterial biofilm was different from sparse fragments, lumps to large lumps. There were significant differences in the bacterial biofilm surface area between different urinary stress groups and at different times(F=505.72, 1 201.84, all P < 0.000 1), and there was interaction effect between time and stress(F=78.14, P < 0.000 1). From hydrostatic pressure, constant turbulent flow shear stress, physiological turbulent flow shear stress, to pathological turbulent flow shear stress, the biofilm formation increased significantly.(4) The results showed that this turbulent flow shear stress of human bladder urine flow can obviously stimulate E. coli biofilm formation in vitro. Its functional changes and pathogenic mechanism need to be further explored.