巯乙磺酸钠对大肠杆菌生物膜早期黏附及胞外聚合物的影响

Effects of mesna on Escherichia coli biofilm adhesion and extracellular polymeric substances in vitro

目的研究巯乙磺酸钠对大肠杆菌生物膜(biofilm,BF)早期黏附及胞外聚合物(extracellular polymeric sub-stances,EPS)的影响。方法采用了平板菌落计数法检测高浓度(2 mg/ml)、低浓度(0.5 mg/ml)巯乙磺酸钠在不同时间点对大肠杆菌黏附的影响;采用免疫荧光技术标记多糖和细菌,利用激光扫描共聚焦显微镜定性观察细菌黏附及胞外多糖变化;利用硫酸-苯酚法定量各组细菌胞外多糖的产量;利用考马斯亮蓝染料结合法(Bradford法)测定胞外蛋白含量。结果巯乙磺酸钠干预2、4、6、8 h后,与生理盐水对照组比较,各组黏附细菌数均有所减少(P<0.05),高浓度组较低浓度组更明显(P<0.05);经巯乙磺酸钠干预8 h,经FITC-ConA和PI双染后,激光扫描共聚焦显微镜观察见菌落稀疏,散在分布,胞外多糖减少,稀薄,以高浓度为甚;胞外多糖定量实验中,高浓度组胞外多糖/细菌干质量(13.57±0.59)μg/mg、低浓度组(27.77±0.77)μg/mg,分别与生理盐水对照组(35.73±0.44)μg/mg比较,均降低了胞外多糖的产生(P<0.05),高浓度组较低浓度组更明显(P<0.05);胞外蛋白定量实验中,高浓度组胞外蛋白/细菌干质量(7.76±0.32)μg/mg、低浓度组(17.59±0.86)μg/mg分别与生理盐水对照组(23.31±1.36)μg/mg比较,均降低了胞外蛋白的产生(P<0.05),高浓度组较低浓度组更明显(P<0.05)。结论巯乙磺酸钠可显著减少大肠杆菌黏附及产EPS的能力。

Objective To determine the effect of mesna on the adhesion of Escherichia coli（E.coli） biofilm at early stage and biosynthesis of extracellular polymeric substances（EPS）.Methods Standard colony counting method was used to determine the effects of mesna（2 and 0.5 mg/ml） on E.coli adhesion at different time points.After polysaccharides and bacteria were labeled by immunofluorescent technique,the adherence and polysaccharides of E.coli were qualitatively observed by confocal laser scanning microscopy（CLSM）.Phenol-sulfuric acid method was used to quantitate the polysaccharides content in each group.Extracellular protein content was determined by Bradford protein assay.Results Compared with normal saline group,the number of adhered bacteria was decreased in 2,4,6 and 8 h after intervention with mesna（P0.05）,especially in the high-dose group（P0.05）.CLSM based on FITC-ConA and PI staining showed that in 8 h after mesna intervention,the colony distribution of E.coli became rarefactive compared with normal saline group.The content of polysaccharides in biofilm was significantly decreased,especially in the high-dose group.Quantification of polysaccharides showed that total polysaccharides（μg）/dry weight of bacterial（mg） was 13.57±0.59 in the high-dose group and 27.77±0.77 in the low-dose group（P0.05）,significantly lower than that of normal saline group（35.73±0.44,P0.05）.Quantification of extracellular protein showed that total extracellular protein（μg）/dry weight of bacterial（mg） in high-dose group（7.76±0.32）and low-dose group（17.59±0.86）were significantly lower than in normal saline group（23.31±1.36,P0.05）,and there was significant difference between high-dose group and low-dose group（P0.05）.Conclusion Mesna decreases the adhesion and EPS formation of E.coli significantly.