胆盐胁迫下不同毒力基因型副溶血性弧菌混合生物被膜异质性

Study on the heterogeneity of mixed biofilms of Vibrio parahaemolyticus with different virulence genotypes under bile salts stress

摘要:

为了解在纯培养条件及胆盐胁迫下来源于淡水产品的不同毒力基因型副溶血性弧菌（Vibrio parahaemolyticus）单个及混合菌株生物被膜的形成特征，通过结晶紫染色法和CCK-8法检测生物被膜的形成量和细胞活力，并借助共聚焦激光扫描显微镜（Confocal laser scanning microscopy，CLSM）对被膜结构进行可视化观察，最后通过二喹啉甲酸（Bicinchoninic acid，BCA）法及苯酚-硫酸法对胆盐处理前后的被膜胞外蛋白和胞外多糖含量进行定量分析。结果表明，胆盐处理抑制副溶血性弧菌生物被膜的形成，使被膜形成量和细胞活力显著下降，影响被膜系统的聚集程度和混乱程度，破坏被膜结构的同时影响胞外蛋白和胞外多糖的合成和分泌。含有tdh基因的菌株能够形成较多且较稳定的生物被膜，混合菌株在胆盐刺激下表现出更强的适应性，能够迅速做出响应以抵御这种胁迫，从而保证菌体更加稳定地存活。这一研究结果为副溶血性弧菌混合生物被膜的形成和控制提供了新的理论支持，对于探讨淡水产品中携带副溶血性弧菌对食品安全和人体健康造成的影响具有重要意义，为进一步研究其在人体肠道中的适应机制和防控提供了重要参考依据。

Abstract:

In this study， the characteristics of biofilm formation of single and mixed strains of Vibrio parahaemolyticus with different virulence genotypes from freshwater products under pure culture conditions and bile salts stress were investigated. The amount of biofilm formation and cell viability were detected by crystal violet staining and CCK-8 method， and the structure of biofilm was visually observed by confocal laser scanning microscopy （CLSM）. Bicinchoninic acid （BCA） method and phenol-sulfuric acid method were used to quantitatively analyze the contents of extracellular protein and extracellular polysaccharide before and after bile salts treatment. The results showed that bile salts treatment inhibited the formation of V. parahaemolyticus biofilm， significantly decreased the amount of biofilm formation and cell viability， and affected the degree of aggregation and disorganization of biofilm system， and disrupted the biofilm structure， while affecting the synthesis and secretion of extracellular protein and extracellular polysaccharide. Strains containing tdh gene can form more and more stable biofilms， and the mixed strains showed greater adaptability to bile salts stimulation and were able to respond rapidly to counteract this stress， thus ensuring more stable survival of the strains. This study provides a new theoretical support for the formation and control of V. parahaemolyticus mixed biofilms， which is of great significance for exploring the impact of V. parahaemolyticus carriage in freshwater products on food safety and human health， and provides an important reference for further research on its adaptive mechanism and prevention and control in the human gut.