Simulating the human bladder to construct a bacterial biofilm dynamic incubation system under biomimetic urinary flow stress in vitro

Abstract:

Objective To construct a dynamic incubation system of biomimetic urinary flow stress bacteria biofilm by simulating human bladder in vitro，and to explore the application effect of this system.Methods For the dynamic incubation system to construct bacterial biofilm in vitro，we used Escherichia coli as the experimental bacteria and silica gel membrane as the carrier of membrane formation to establish five bionic effects of urinary flow stress： physiological secretion during the day and night，physiological urination during the day， and pathological urination during the day and night. In addition， we set up two circulation scheme groups： physiological group and pathological group to observe the characteristics of biofilm formation of Escherichia coli under the stimulation of bionic urinary flow stress. The detection methods included： counting the living bacteria of biofilm bacteria， fluorescence staining combined with laser confocal microscope observation，determining the average light density value of the fluorescence image of biofilm bacteria，and constructing biofilm stereogram to measure the thickness of biofilm. The average values among multiple groups were detected by using one-way analysis of variance（ANOVA），and LSD-t method was used to compare the average values within groups in pairs.Results Through the comparison of data in each group，with the prolongation of incubation time，the number of viable bacteria of biofilm bacteria increased gradually，and the number of viable bacteria in pathological group at 48 h，static group and physiological group at 60 h increased significantly and reached the peak（^cP=0.000）；The measured values of average light density and biofilm thickness in the fluorescence image of biofilm bacteria increased gradually，and the measured values in the pathological group at 48 h，the static group and the physiological group at 72 h increased significantly and reached the peak（^cP=0.000）；There was no significant difference in other measured values in each group（P>0.01）. Through the comparison of data between groups，compared with the static group，the viable counts of biofilm bacteria in the physiological group and the pathological group at 36 h，48 h，60 h，84 h and 96 h were significantly increased（^aP=0.000）. Compared with the physiological group， the increase in the pathological group was more significant（^bP=0.000）. Compared with the static group， the fluorescence image average light density of biofilm bacteria in the physiological group at 96 h and the pathological group at 48 h and 96 h increased significantly（^aP=0.000），and the measured values of biofilm thickness in the physiological group at 72 h， pathological group at 48 h and 96 h increased significantly（^aP=0.000）. Compared with the physiological group， the measured values of the fluorescence image average light density and biofilm thickness of bacteria in the pathological group at 48 h and 96 h were significantly increased（^bP=0.000）. There was no significant difference in other measured values among the groups（P>0.01）.Conclusion In this study，a dynamic incubation system of bacterial biofilm has been successfully constructed in vitro to simulate human bladder， which has been verified that the biomimetic urinary flow stress can significantly promote the formation of Escherichia coli biofilm.