环境因子对鳗弧菌生物膜形成的影响

Characteristics of biofilm formation of pathogenic Vibrio anguillarum

摘要:

【背景】鳗弧菌是海产动物弧菌病的主要病原，在海水水域中广泛存在。鳗弧菌为了适应环境变化会生成生物膜，形成自我保护，对其防治是水产养殖行业的一大难题。【目的】探讨致病性鳗弧菌(Vibrio anguillarum) BYK0638生物膜的形成特性，为进一步研究鳗弧菌生物膜形成机制和致病机理提供参考。【方法】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。【结果】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24 h达到峰值，60 h后趋于稳定；在107?108 CFU/mL范围内，鳗弧菌生物膜的OD450值显著高于其他试验组(P<0.05)；25 °C时的生物膜OD450值显著高于其他温度生物膜的形成量；在pH 4.0?11.0范围内，当pH值为7.0时鳗弧菌形成的生物膜量最高，在pH值为3.0和12.0时鳗弧菌几乎不形成生物膜；在TSB培养基中加入0.03?2.00 mmol/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0.03 mmol/L MgCl2，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(P<0.05)。【结论】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子变化有密切的关系，培养时间、初始菌浓度、温度、pH、Mg2+、盐度及不同组织和黏液等各种环境因子均能显著影响鳗弧菌生物膜的形成。

Abstract:

[Background] Vibrio anguillarum is the main pathogen of marine animal vibriosis, which exists widely in seawater. It can form biofilm to adapt to environmental changes and form self-protection, the prevention and treatment of Vibrio anguillarum is a major challenge to aquaculture industry. [Objective] To study the characteristics of biofilm formation of pathogenic Vibrio anguillarum BYK0638, and to provide a reference for further study on the mechanism and pathogenesis of biofilm formation of V. anguillarum. [Methods] The characteristics of biofilm formation of V. anguillarum BYK0638 were studied by modified microtiter-plate test, and the viability of V. anguillarum in the biofilms was detected by CCK-8. [Results] V. anguillarum BYK0638 developed stable and evident biofilm on the surface of the polystyrene microtiter plate. OD450 value of V. anguillarum biofilm reached the peak after 24 h and stabilized after 60 h. Within the 107?108 CFU/mL, OD450 vaule of biofilm was significantly higher than the others (P<0.05). OD450 value of biofilm recorded at 25 °C was significantly higher than those recorded at other temperatures (P<0.05). In the range of pH 4.0?11.0, when pH value is 7.0, the amounts of bacterial biofilm was the highest, and at pH 3.0 and 12.0, no evident biofilm was found. Biofilm formation of V. anguillarum played no significant role when 0.03 to 2.00 mmol/L CaCl2 was added. Addition of 0.03% MgCl2 significantly promoted the bacterial biofilm formation while CaCl2 played no significantly role. At 5% NaCl, the OD450 value of biofilm was the highest. The biofilms developed on the surface of the microtiter plate coated by the skin mucus, liver, foregut and hindgut tissue extract of large yellow croaker was significantly higher than the others (P<0.05). [Conclusion] Pathogenic V. anguillarum BYK0638 can develop stable and obvious biofilm. The biofilm formation is closely related to the change of environmental factors, pH, temperature, Mg2+, salinity and other environmental factors can significantly affect the formation of biofilm of V. anguillarum.