杀鲑气单胞菌一新亚种的生物学特性及系统发育学分析

Biological characterization and phylogenetic analysis of pathogenic new subspecies of Aeromonas salmonicida isolated from stone flounder(Kareius bicoloratus L.)

从石鲽(Kareius bicoloratusL.)细菌性败血感染症的病鱼(濒死及死亡不久)中分离到相应病原菌,进行形态特征、理化特性等较系统的表观分类学指征鉴定及代表菌株DNA中G+C mol%的测定。同时,选择代表菌株进行16S rRNA基因的分子鉴定,测定16S rRNA基因序列、分析相关细菌相应序列的同源性,构建系统发生树。结果表明,分离鉴定的60株菌为杀鲑气单胞菌的一个新亚种(subsp.nov.),定名为杀鲑气单胞菌杀鲽亚种(Aeromonas salmonicidasubsp.flounderacidasubsp.nov.)。代表菌株HQ010320-1及HQ010320-5的16S rRNA基因序列与GenBank数据库中的杀鲑气单胞菌的同源性在99%和100%

Stone flounder（Kareius bicoloratus L. ） is a marine fish with high economic value in the aquaculture industry of northern China. Continuous high-level mortalities occurred in stone flounder. The diseased samples were collected from wild stocks and cultured in farms during March 2001 and April 2002 in Qinhuangdao, Hebei Province. Examination of diseased stone flounder showed that the fish belonged to septicaemia infection. The biological characteristics of pathogenic bacteria were studied, including morphological characteristics, physiological and biochemical characteristics, G ＋ C mol% of DNA, and plasmid content of isolates. In addition, the molecular identification of representative strains was conducted, and the 16S rRNA gene were partially sequenced and compared with sequences deposited in databases; molecular phylogenetic trees were constructed. The results showed that the isolates were Gram-negative, were arranged singly or in pairs, round-ends, medium-sized short rods, 0.4 - 1.0 μm × 1.0 - 2.0 μm in size, no-endospores, electron micrograp by negatively stained revealed that the cultures have no flagella; the physiological and biochemical traits showed that the isolates were positive reaction for oxidase, catalase, gelatin hydrolysis, maltose, Dmannitol, produce acid but no gas from glucose, reduce nitrate, tween 80, adonitol, D-raffinose, malonate utilization, and negative reaction for indole, methyl red, esculin, glycerol, urease, trehalose, sucrose reduction, arabinose, Voges-Proskauer reaction, produce H2 S, D-sorbitol, D-arabitol, i-erythritol, m-inositol, L-rhamnose, salicin, have no motility, can not growth at 37 ℃, G ＋ C mol% of DNA were 62.6 % ;the sequenced 16S rRNA gene of strain HQ010320-1（GenBank accession no. AY786177） is 1 425 bp in length,and the sequenced 16S rRNA gene of strain HQ010320-5（GenBank accession no. AY786178） is 1 423 bp in length. The 16S rRNA gene sequence of representative strains（HQ010320-1 and HQ010320-5） exhibited high similarity（99% and 100% ） with the 16S rRNA gene of A. salmonicida from GenBank database. The results showed that the identified 60 strains belonged to new subspecies of Aeromonas salmonicida, and named as Aeromonas salmonicida subsp. JTounderacida subsp, nov. by their morphological characteristics, physiological and biochemical characteristics, and molecular identification by 16S rRNA partial sequence.