黄河鲤水产细菌的分离及其毒力因子和群体感应信号分子的检测

【背景】水产细菌病害制约水产养殖业健康发展,群体感应与细菌毒力因子的产生密切相关,群体感应调控细菌的毒力因子特性值得进一步研究。【目的】探究群体感应与黄河鲤细菌病害的关系,明确群体感应对细菌毒力因子特性的影响。【方法】通过16S rRNA基因测序并构建系统进化树确定筛选菌株的进化地位,通过脱脂牛奶平板法和偶氮酪蛋白法检测菌株胞外蛋白酶活力,采用结晶紫染色法对菌株的生物膜形成能力进行测定,通过报告菌株BB170和CV026分别测定菌株产信号分子AI-2和高丝氨酸内酯的能力,外源添加高丝氨酸内酯检测信号分子对菌株胞外蛋白酶活力和生物膜形成能力的影响。【结果】哈夫尼亚菌(Hafnia sp.) Z11和气单胞菌(Aeromonas sp.) Z12具有高水平的胞外蛋白酶活力和生物膜形成能力,能够分泌AHLs信号分子且具有菌体密度依赖性。外源添加HSL对菌株毒力因子特性有不同程度的影响,外源添加高浓度的N-丁酰基高丝氨酸内酯(C4-HSL)和N-己酰基高丝氨酸内酯(C6-HSL)能够分别提高菌株Z11和Z12的胞外蛋白酶活力和生物膜形成能力。【结论】高浓度群体感应信号分子AHLs对哈夫尼亚菌和气单胞菌胞外蛋白酶活性有促进作用,说明该2种菌的群体感应现象可能会影响其毒力。

Detection of virulence factors and quorum sensing signal molecules of aquatic bacteria from Yellow River carp

Background] Aquatic pathogens restrict the healthy development of aquaculture; quorum sensing is closely related to the production of virulence factors. The characteristics of virulence factors regulated by quorum sensing is worthy of further study. Objective] To explore the relationship between quorum sensing and bacterial diseases of Yellow River carp, and clarify the influence of quorum sensing on the characteristics of virulence factors. Methods] The 16S rRNA gene was sequenced and the phylogenetic tree was constructed to determine the evolutionary status of the selected strains. The activity of extracellular protease was detected by skim milk plate method and azo casein method. The bacterial biofilm forming ability was determined by crystal violet staining. The producing ability of AI-2 and AHLs was determined by reporter strain BB170 and CV026, respectively. The effects of exogenous HSLs on extracellular protease ability and biofilm formation ability were detected. Results] Hafnia sp. Z11 and Aeromonas sp. Z12 were evidenced with high levels of extracellular protease activity and biofilm formation ability, and could secrete AHLs signal molecules, which were density dependent. High concentration of C4-HSL and C6-HSL could increase the extracellular protease activity and biofilm formation activity of strain Z11 and Z12, respectively. Conclusion] High concentration of quorum sensing factors (AHLs) can promote the extracellular protease activity of Hafnia sp. Z11 and Aeromonas sp. Z12, suggesting that the quorum sensing of these two bacteria may affect their virulence.