水族箱气单胞菌的鉴定及致病特性分析

为了确定南京市某渔场发病鱼感染的病原,本研究采集患鱼脏器,采用平板培养、生化实验和特异性gyrB基因扩增测序等方法进行细菌的分离鉴定;利用PCR技术检测分离株毒力基因的分布,分析其生物学特性,并进一步通过动物实验确定菌株致病力.结果分离到1株水族箱气单胞菌,命名为LK-25.该菌株携带5种主要毒力基因:气溶素(aer)、细胞毒性肠毒素(act)、细胞兴奋性肠毒素(alt)、温敏胞外蛋白酶(epr)和丝氨酸蛋白酶(ahp),其溶血性和溶蛋白能力较强,对斑马鱼的半数致死量为1.02×103 CFU/尾,确定为强毒株.进化树分析表明,水族箱气单胞菌与嗜水气单胞菌达卡亚种亲缘关系较近.本研究在国内首次报道发现水族箱气单胞菌,为进一步预防该菌所引起的相关疾病的发生和传播提供理论基础.

Identification and pathogenic characteristics of Aeromonas aquariorum, a new member of the genus Aeromonas

Aeromonas is widely distributed in freshwater environments.Since the late 1980s, Aeromonas has caused serious economic losses to the aquaculture industry in our country.The published species names are increasing in the genus Aeromonas.In order to determine the disease-causing bacterium at one fish farm of Nanjing, some tissue samples from diseased fish were collected and the pathogenic bacteria were identified by means of bacterial culture, biochemical characterizations and gyrB gene sequencing.One strain of bacterium was isolated and named LK-25.The results of biochemical tests showed that the bacterial strain has many similarities compared with A.hydrophila.Then the strain was subjected to sequencing of the gyrB gene for species identification, and then it demonstrated that the strain shared 99% homology with the A.aquariorum strain MDC 47.The corresponding gyrB phylogenetic analysis including representative strains from some known Aeromonas species showed that A.aquariorum LK-25 was very closely related to the A.hydrophila ssp.dhakensis.The detection result of virulence factors indicated that A.aquariorum LK-25 strain carried five virulent genes, including act, ahp, epr, hcp and alt genes, which encoded the cytotoxin, serine proteases, extracellular protease, hemolysin co-mediate protein and cytotonic enterotoxins, respectively.A.aquariorum LK-25 also showed strong hemolytic and proteolytic activities.The median lethal dose(LD50)with 1.02×10³ CFU/ml in zebrafish indicated that A.aquariorum LK-25 is a highly virulent strain.A.aquariorum was a new species originally isolated from ornamental fish in 2008, and widely distributed in clinical and environmental specimens.Recent reports highlighted the importance of this species in cases of gastroenteritis, soft tissue infections, bacteremia and peritoneal dialysates in human, suggesting its public health significance.To our knowledge, this was the first report of A.aquariorum in China.The study will lay a good foundation for a better understanding of the occurrence and spread of A.aquariorum infection.