基于高通量共聚焦激光扫描显微镜方法定量分析副溶血性弧菌生物被膜结构

Quantitative analysis of Vibrio parahaemolyticus biofilm structure based on high-throughput confocal laser scanning microscopy

摘要:

[目的] 副溶血性弧菌是水产品中常见的食源性致病菌，生物被膜的形成对副溶血性弧菌的环境生存和传播至关重要。这项工作的目的是评估临床和环境中分离出的44株副溶血性弧菌菌株形成的生物被膜的结构多样性。[方法] 该研究基于共聚焦激光扫描显微镜的高通量方法，使用与高分辨率成像兼容的96孔微量滴定板，结合结构分析软件ISA-2来研究生物被膜形成和结构，分析22株食品与22株临床来源的副溶血性弧菌菌株形成的生物被膜结构参数（生物体积、平均厚度、粗糙系数）。[结果] CLSM图像显示，44株副溶血性弧菌菌株在培养48 h后能够形成3D结构，进一步比较分析了临床来源菌株与环境来源菌株形成的生物被膜结构异同，发现临床菌株生物被膜的变异系数比环境菌株生物被膜的变异系数小，且同时携带tdh和trh两种毒力因子的菌株生物被膜变异性最小。凝聚层次聚类分析结果显示，副溶血性弧菌生物被膜可以分为致密且表面光滑（39%）、斑驳且表面粗糙（27%）、疏松且表面坑洼（34%），临床菌株易形成致密且表面光滑和斑驳且表面粗糙的生物被膜，而环境菌株易形成致密且表面光滑和疏松且表面坑洼的生物被膜。[结论] 该研究深入了解了副溶血性弧菌生物被膜结构差异性，为今后对临床和环境来源的副溶血性弧菌生物被膜采取不同的防控和清除措施提供了理论支撑。

Abstract:

[Objective] Vibrio parahaemolyticus is a common foodborne pathogen in aquatic products. The formation of biofilm is essential for the survival and spread of Vibrio parahaemolyticus. The aim of this study was to evaluate the structural diversity of biofilms formed by 44 strains of Vibrio parahaemolyticus isolated under clinical and environmental conditions. [Methods] This study proposed a high-throughput method based on confocal laser scanning microscopy (CLSM), using 96-well microtiter plates compatible with high-resolution imaging, combined with structural analysis software ISA-2 to study biofilm formation and structure, the biofilm structure parameters (Biovolume, Average thickness, Biofilmroughness) formed by 22 clinical strains and 22 environmental strains were analyzed. [Results] CLSM images showed that 44 strains of Vibrio parahaemolyticus could form 3D structure after 48 h incubation, and further analyzed the similarities and differences of biofilm formation between clinically derived strains and environmentally derived strains, showed that the coefficient of variation in the clinical strains were smaller than that of the environmental strains, and the strains BF with both tdh and trh virulence factors had the smallest variability. Agglomerative hierarchical clustering (AHC) showed that biofilm can be divided into dense and smooth surface (39%), mottled and rough surface (27%) and loose and uneven surface (34%), the clinical strain formed a biofilm which is easy to form dense and smooth surface, mottled and rough surface, whereas the environmental strain formed a biofilm which is easy to dense and smooth surface and loose and uneven surface. [Conclusion] This study provides an in-depth understanding of the biofilm structure of Vibrio parahaemolyticus, it provides theoretical support for different prevention and control measures for Vibrio parahaemolyticus biofilms from clinical and environmental sources.