冷激条件下预形成副溶血性弧菌生物被膜的发展变化

Fate of pre-formed Vibrio parahaemolyticus biofilm under cold shock

摘要:

[目的] 生物被膜可附着在食品或医药生物材料表面引起持续性的感染，而现今极少有关于温度变化对预形成生物被膜影响的研究。本文分析了冷激条件下副溶血性弧菌预形成生物被膜的发展变化。[方法] 以改进的结晶紫染色法检测生物被膜总量，以改进的超声法和Lowry法量化胞外多糖和蛋白质，以RNA试剂盒提取并纯化生物被膜形成的相关基因。同时，激光共聚焦扫描显微镜直观显示了冷激条件下预形成生物被膜形态结构的变化，并深入分析了生物被膜结构的变化，以及生物被膜结构参数和基因转录的相关性。[结果] 在冷激条件下，副溶血性弧菌生物被膜总量增加，同时，副溶血性弧菌预形成的生物被膜胞外多聚物的主要成分胞外多糖和蛋白质也逐渐增加，被膜形成相关鞭毛基因和毒力基因的转录活跃。生物被膜的平均厚度（MT）、平均扩散距离（ADD）、孔隙率（P）、生物被膜粗糙度（BR）和均匀性（H）在冷激过程中也发生了变化，同时这些参数之间存在显著相关性（P<0.01），而生物被膜结构参数与生物被膜相关基因转录的相关性较弱（P<0.05）。[结论] 因此，4℃和10℃冷激不能完全抑制副溶血性弧菌预形成生物被膜的生长，风险评估人员在制定控制食源性感染风险的战略时应考虑到这一因素。

Abstract:

[Objective] The aim of the study was to analyze changes of pre-formed Vibrio parahaemolyticus biofilm under cold shock. The source of persistent infections can be biofilms that occur naturally on food surfaces and medical biomaterials. The change of pre-formed Vibrio parahaemolyticus biofilm under cold shock was studied.[Methods] The changes of biofilm biomass were checked by crystal violet staining, exopolysaccharides and proteins were quantified by modified sonication method and Lowry method, and genes related to the expression of biofilm were extracted and purified by RNA extraction kit. The Confocal laser scanning microscopy (CLSM) images reveal biofilm morphological structure, the correlation analysis shows inner relationship among biofilm structure parameters and biofilm related genes.[Results] V. parahaemolyticus biofilm biomass increased significantly during this cold shock period. Polysaccharides and proteins increased gradually in the extracellular polymeric matrix (EPS). In addition, the expression of flagella and virulence-related genes were differentially regulated. The architecture of the biofilm, quantified using mean thickness, average diffusion distance, porosity, biofilm roughness and homogeneity also changed during the cold shock and these parameters were correlated (P < 0.01). However, the correlation between biofilm architecture and biofilm-related gene expression was relatively weak (P<0.05). [Conclusion] Cold shock at 4℃ and 10℃ is not sufficient to reduce V. parahaemolyticus biofilm formation.