金鱼腐皮病病原菌的分离鉴定及药敏试验

Isolation, Identification and Antibiotics Susceptibility of the Pathogenic Bacteria Isolated from the Putrid Skin Diseased Carassius Auratus

从患腐皮病金鱼肝脏中分离获得1株细菌,编号为KL-1,通过细菌形态观察、生理生化特性、16S rDNA克隆测序及构建发育树分析等系统鉴定,并进行动物感染试验、药敏特性研究。结果表明,KL-1菌株为革兰氏阴性短杆菌,可发酵葡萄糖产气,氧化酶、接触酶、赖氨酸脱羧酶等为阳性;进一步采用PCR方法扩增16S rDNA序列,将其亚克隆至p MD18-T载体中,测序获得长度为1 507 bp,与Aeromonas veronii模式菌株ATCC 35624(X74684)序列相似性为99.66%,构建系统进化树分析与其聚为一支,鉴定KL-1菌株为维氏气单胞菌(Aeromones veronii)。动物感染试验表明,对斑马鱼半数致死量LD50为1.4×107 cfu/m L。药敏试验结果显示,该病原菌对头孢克肟、头孢哌酮、头孢噻肟、头孢氨苄、萘啶酸、恩诺沙星、左氟沙星、诺氟沙星、氟苯尼考和氯霉素等药物敏感。

A bacterial strain was isolated from the liver of the Putrid Skin Diseased Carassius auratus, named KL-1. The morphological observation, physiological and biochemical characteristics, the cloning and sequencing of 16S rDNA and phylogenetic tree analysis, animal infection test and the antibiotic susceptibility analysis were carried out in this study. Results showed that KL-1 strain was a rod-shaped, gram-negative bacillus, it can produce acid and gas fromglucose, oxidase, catalase and lysine deearboxylase showed positive reaction; Furthermore, the 16S rDNA sequences was amplified by PCR method, and subcloned into pMD18-T vector, a product of sequencing was 1 507 bp in length. It had a 99.66% similarity of 16S rDNA sequence with Aeromonas veronii type strain ATCC 35624 （X74684） , and clustered into one group with A. veronii, so the KL-1 strain was identified as A. veronii. Artificial infection test showed that the median lethal （LD50）to zebrafish was 1.4 × 10^7 cfu/mL. The susceptibility testing with different antibiotics showed that it was sensitive to cefixime, cefoperazone, cefotaxime, cephalexin, clindamycin, nalidixic acid, enrofloxacin, difloxacin, norfloxacin, and florfenicol, ehloramphenicol etc.