黄颡鱼暴发性出血病病原菌的分离鉴定及其生物膜形成特性

Isolation, identification and biofilm forming characteristics of pathogen causing fulminant hemorrhagic disease in yellow catfish (Pelteobagrus fulvidraco)

摘要:

【背景】安徽省当涂县某池塘养殖黄颡鱼发生暴发性出血病，而当前对该病的病原存在争议。【目的】确定引起黄颡鱼暴发性出血病的病原菌，并明确分离菌株的生物膜形成特性，为从抗生物膜形成角度防治病原菌感染提供参考。【方法】取濒死期黄颡鱼病变脏器分别接种EPC细胞与培养基(TSB琼脂平板和血琼脂平板)分离病原，并通过人工感染回归试验确定其致病性；采用表型鉴定与16S rRNA基因序列分析相结合的方法鉴定分离菌株，并对其生物膜形成最佳条件、成膜能力及携带的生物膜形成相关基因进行研究。【结果】从病变脏器中分离纯化到一株优势菌株(HSY-2)，对黄颡鱼的半数致死量为1.05×106 CFU/mL。经形态学、生化特性和细菌16S rRNA基因测序等分析确定分离株HSY-2为简达气单胞菌。其形成生物膜的最佳条件是将细菌接种TSB培养基于30 °C培养静置96 h，可形成中等强度的生物膜。同时，分离菌株携带气单胞菌甘油-3-磷酸脱氢酶D编码基因glpD、S-核糖同型半胱氨酸裂解酶基因luxS和LuxI家族蛋白同系物编码基因ahyI三种生物膜形成相关基因，但未检测到甘露糖敏感型血凝素菌毛合成蛋白Q编码基因。【结论】本实验为进一步研究简达气单胞菌生物膜形成的调控机制打下基础，并且从抗生物膜形成角度防治简达气单胞菌感染提供了参考。

Abstract:

[Background] The disease, causing fulminant hemorrhagic of yellow catfish (Pelteobagrus fulvidraco), occurred in a pond in Dangtu County, Anhui Province, and the pathogen is controversial currently. [Objective] Investigate the pathogen causing outbreak hemorrhagic disease of yellow catfish (Pelteobagrus fulvidraco) and the biofilm formation characteristics of the isolated strain, to provide a reference for the prevention and treatment of A. jandaei infection. [Methods] The organs with obvious pathological changes in dying yellow catfish were inoculated on the EPC cell and media (TSB agar plate and blood agar plate) to isolate the pathogen, and the pathogenicity of the isolate was detected by artificial infection test. The isolated strain was identified by phenotypic identification and 16S rRNA gene sequence analysis. Then, the optimal conditions for biofilm formation, biofilm forming ability of the isolated strain and biofilm-forming genes carried by the strain were detected. [Results] A dominant isolated strain named HSY-2 was recovered and purified from organs with obvious pathological changes. In the artificial infection trail, the bacterial isolate HSY-2 exhibited virulence to yellow catfish with an LD50 value of 1.05×106 CFU/mL. The bacterial isolate was identified as Aeromonas jandaei according to morphology, biochemical characteristics and phylogenetic analysis derived from 16S rRNA gene. The optimum condition for forming biofilm was to inoculated on the TSB medium for 96 h at 30 °C, at which the isolated strain might form a moderate intensity biofilm. Three biofilm formation related genes, including glycerol-3-phosphate dehydrogenase D gene glpD, S-ribosylhomocysteine lyase gene luxS and LuxI family protein homolog gene ahyI were detected in the isolated strain, whereas the mannose-sensitive hemagglutinin pili biosynthesis protein Q gene was not detected. [Conclusion] This experiment provides a basis for further study on the regulation mechanism of A. jandaei biofilm formation and a reference for the prevention and treatment of A. jandaei infection from the perspective of anti-biofilm formation.