鳗弧菌（Vibrio anguillarum）生物膜的形成及三种中草药对其干预的研究

发布时间：2018-05-11 09:03

本文选题：鳗弧菌 + 生物膜　；参考：《上海海洋大学》2017年硕士论文

【摘要】：采用激光共聚焦显微镜技术观察鳗弧菌生物膜形成过程,体外构建法构建鳗弧菌生物膜模型,并分析结构特征。本实验以致病性鳗弧菌BYK0638为研究对象,使用盖玻片生物膜培养法体外构建鳗弧菌的生物膜,并在生物膜形成过程中,采用CCK-8法定量检测生物膜的形成量;在培养不同时间后,用免疫荧光剂碘化吡啶(PI)染色,用激光共聚焦显微镜(Confocal laser scanning microscope,CLSM)观察鳗弧菌生物膜的形成过程和结构特征。结果显示,鳗弧菌能够在玻片上形成较致密的生物膜,鳗弧菌生物膜的形成量在培养前24 h升高较快,60 h后趋于稳定。获得了生物膜形成过程不同时间节点的CLSM图像,观察到鳗弧菌一般在24 h后开始逐渐形成生物膜,在72 h形成稳定的生物膜。探讨致病性鳗弧菌(V.anguillarum)BYK0638生物膜的形成特性,为进一步研究鳗弧菌生物膜的形成机制和致病机理提供参考。采用改良的微孔板法研究静置培养条件下鳗弧菌(V.anguillarum)BYK0638在96孔酶标板上的成膜情况,CCK-8法(Cell Counting Kit-8)定量检测生物膜中鳗弧菌的活力。鳗弧菌BYK0638能够在聚苯乙烯酶标板上形成稳定而明显的生物膜,其生物膜的OD450值在24h达到峰值,60h后趋于稳定;在107-108CFU/mL范围内,鳗弧菌生物膜的OD450值显著高于其他试验组(P0.05);25℃时的生物膜OD450值显著高于其他温度生物膜的形成量(P0.05);在pH4-11范围内,当pH值为7时鳗弧菌形成的生物膜量最高,在pH值为3和12时,鳗弧菌几乎不形成生物膜;在TSB培养基中加入0.03-2.00mmol/L CaCl2,鳗弧菌生物膜形成量与未添加CaCl2对照组无显著性差异;在TSB培养基中加入0.03mmol/L MgCl_2,可促进鳗弧菌生物膜形成;NaCl浓度为5%时,形成的生物膜OD450值最高;鳗弧菌在大黄鱼表皮黏液、肝脏、前肠、后肠组织提取液包被的96孔酶标板上形成的生物膜显著高于其他黏液和组织提取液包被组(P0.05)。致病性鳗弧菌BYK0638能形成稳定而明显的生物膜,其生物膜形成与外界环境因素变化有密切的关系,培养时间、温度、初始菌浓度、pH、盐度、Mg~(2+)、鱼体黏液及组织等各种环境因素均能显著影响鳗弧菌生物膜的形成。为探索大黄、穿心莲与五倍子对鳗弧菌及其生物膜活性的影响。采用微量二倍稀释法分别测定大黄、穿心莲及五倍子对鳗弧菌游离菌的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)。用1/8、1/4、1/2、1、2、4、8倍MIC的大黄、穿心莲与五倍子分别作用于鳗弧菌生物膜,CCK-8法测定药物干预后生物膜活力变化。结果表明,五倍子能较好地干预鳗弧菌生物膜形成,在2倍MIC浓度时,在培养4 h时即可完全抑制BYK0638菌株生物膜的生长,且其SMIC50浓度为2MIC。推荐生产防治用量为6.25mg/mL。
[Abstract]:The formation process of Vibrio anguilis biofilm was observed by laser confocal microscopy. The model of Vibrio eel biofilm was constructed in vitro, and the structure characteristics were analyzed. This experiment was made to study the BYK0638 of Vibrio Anguilla. The biofilm of Vibrio Anguilla was constructed in vitro by means of cover glass biofilm culture, and C was used in the process of biofilm formation. CK-8 method was used to quantify the formation of biofilm. After different times of culture, the formation and structural characteristics of the biofilm of Vibrio Anguilla were observed with the immunofluorescent iodipyridine (PI) staining and the Confocal laser scanning microscope (CLSM). The results showed that Vibrio Anguilla was able to form a dense biofilm on the slide. The formation of Vibrio anguilis biofilm increased rapidly at 24 h before culture and stabilized after 60 h. The CLSM image of different time nodes in the biofilm formation process was obtained. It was observed that Vibrio Anguilla formed a biofilm gradually after 24 h and formed a stable biofilm at 72 h. The formation of pathogenic Vibrio anguilis (V.anguillarum) BYK0638 biofilm was formed. In order to provide reference for further study on the formation mechanism and pathogenesis of Vibrio anguilis biofilm, the membrane formation of Vibrio Anguilla (V.anguillarum) BYK0638 on the 96 hole enzyme labeled plate was studied by modified microplate method. The activity of Vibrio Anguilla in the biofilm was quantified by CCK-8 (Cell Counting Kit-8). The BYK0638 energy of Vibrio Anguilla was determined by the CCK-8 method. A stable and obvious biofilm was formed on the polystyrene label plate. The OD450 value of the biofilm reached its peak value at 24h and then stabilized after 60H; in the 107-108CFU/mL range, the OD450 value of the biofilm of Vibrio Anguilla was significantly higher than that of the other experimental groups (P0.05), and the OD450 value of the biofilm at 25 C was significantly higher than that of the other temperature biofilms (P0.05); In the range of pH4-11, when the value of pH was 7, the biofilm formed by Vibrio anguilis was the highest. When the value of pH was 3 and 12, Vibrio eel almost did not form a biofilm. The formation of Vibrio anguilis biofilm in the TSB medium was not significantly different from that of the non CaCl2 control group, and the addition of 0.03mmol/L MgCl_2 to the TSB medium could promote the eel arc. Bacterial biofilm was formed; when the concentration of NaCl was 5%, the OD450 value of the biofilm was highest, and the biofilm formed on the 96 orifice of the epidermal mucus, the liver, the foregut and the posterior intestinal tissue was significantly higher than that of the other mucus and tissue extracts (P0.05). The pathogenic Vibrio anguilis BYK0638 could form a stable and obvious birth. Membrane formation has a close relationship with the changes of external environmental factors. The culture time, temperature, initial bacteria concentration, pH, salinity, Mg~ (2+), fish body mucus and tissue can significantly affect the formation of the biofilm of Vibrio Anguilla. The effects of rhubarb on the activity of Vibrio Anguilla and its biofilm are explored. The minimum bacteriostasis concentration (MIC) and minimum bactericidal concentration (MBC) of rhubarb, andrographolic and Galla chinensis were measured by two times dilution method respectively. Using 1/8,1/4,1/2,1,2,4,8 times MIC of rhubarb, Andrographis paniculata and Galla chinensis were respectively acted on the biofilm of Vibrio Anguilla, and the activity of biofilm after drug intervention was determined by CCK-8 method. The results showed that the Chinese gall was the Chinese gall. It can interfere with the formation of the biofilm of Vibrio anguilis. At the concentration of 2 times MIC, the growth of the biofilm of the BYK0638 strain can be completely suppressed when the culture of 4 h is cultured, and the concentration of SMIC50 is 2MIC. recommended for the production of 6.25mg/mL..

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S941.4;S948

【参考文献】