海南地区与环渤海湾美人鱼发光杆菌美人鱼亚种水产动物分离株的表型与遗传特征分析

Phenotypic and genetic diversity of Photobacterium damsela subsp.damsela isolated from aquatic animals in Hainan Province and Bohai Sea region

美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp.damselae,PDD)是一种广泛分布于海洋环境内的重要病原菌,可导致多种海洋生物患病死亡,近年来在我国不同养殖区域内均有发现和报道。【目的】通过生理代谢表型、毒力基因分布和分子遗传分析,系统比较我国海南地区和环渤海湾不同株系PDD的表型与遗传多样性特征。【方法】使用选择性培养基测定PDD的蔗糖发酵能力、运动性、溶血性和磷脂酶活性;通过PCR检测质粒pPHDD1复制起始点基因pPHDD1 replication origin和毒力基因dly,hlyA_(pl),hlyA_(ch),plpV;基于toxR基因序列构建系统发育树,分析16株PDD的遗传关系。【结果】4株PDD蔗糖发酵测试呈阳性,12株PDD蔗糖发酵测试呈阴性。不同株系PDD运动能力存在显著差异,其中环渤海湾分离株Pdd1608、Pdd2009和Pdd1704的运动能力较强。仅有环渤海湾分离株Pdd1608表现为强溶血性、强磷脂酶活性;海南地区分离株Pdd1612和Pdd0912表现为弱溶血性、无磷脂酶活性;其余菌株表现为弱溶血性、弱磷脂酶活性。菌株Pdd1608的毒力基因谱为pPHDD1 replication origin-dly-hlyAp-hlyAch-plpV,其余菌株的毒力基因谱为hlyAch-plpV。系统发育分析揭示不同株系PDD具有高度的遗传多样性。【结论】流行于海南地区和环渤海湾的PDD可能都由多克隆种群组成,它们具有复杂的表型特征、相似的毒力基因分布和高度的遗传多样性。

Photobacterium damselae subsp.damselae(PDD)is an important pathogen distributed widely in global marine environment,which could infect multiple marine organisms,and reported in various mariculture regions of China in recent years.[Objective]This study aimed to systematically compare the phenotypic and genetic diversity of PDD isolated from Hainan province and Bohai Sea region base on physiological and metabolic phenotype,virulence gene distribute and molecular genetic analysis.[Methods]The physiological and metabolic phenotypes of sucrose fermentation,moveability,hemolysis,and phospholipase activity were assayed.The profiles of pPHDD1 replication origin,dly,hlyA_(pl),hlyA_(ch) and plpV were investigated by PCR detection.Genetic diversity of PDD was analyzed based on the toxR sequences.[Results]Four PDD strains were positive for sucrose fermentation while other twelve strains were negative.There are significant differences among the motility of PDD,in which strain Pdd1608,Pdd2009,and Pdd1704 showed strong motility.Pdd1608 isolated from Bohai Sea region exhibited strong hemolysis and phospholipase activities,whlie strain Pdd1612 and Pdd0912 isolated from Hainan Province showed weak hemolysis and none phospholipase activities,and 13 strains showed weak hemolysis and phospholipase activities.The virulence gene profile of Pdd1608 was pPHDD1 replication origin-dly-hlyApl-hlyAch-plpV,while other strains were hlyAch-plpV.Phylogenetic analysis suggested remarkable genetic diversity among different kinds of PDD.[Conclusion]PDD isolated from Hainan Province and Bohai Sea region were probably composed of a polyclonal bacterial population with complex phenotypic characterizations,similar virulence gene profiles,and high genetic diversity.