大黄鱼假单胞菌病病原的分离鉴定及药物敏感试验

Isolation, identification and the sensitivity to antimicrobial agents of Pseudomonassis pathogen from Great yellow croaker (Pseudosciaena crocea)

于2009、2010年分别从浙江温州南麂岛网箱养殖的患病大黄鱼体内分离出优势菌株ZSH和DHY10051103K,人工感染试验证实其为大黄鱼的病原菌,其半数致死量(LD50)分别为2.5×106、3.2×106CFU/mL。对分离菌株进行了形态、生理生化特性的测定及16S rRNA分子鉴定,结果显示:分离菌株为革兰氏阴性短杆菌,氧化酶阳性;利用柠檬酸盐和麦芽糖,降解硝酸盐,精氨酸双水解酶阳性,甲基红试验阴性,明胶酶阴性,鸟氨酸脱羧酶阴性;在16S rRNA系统发育树中,分离菌株与恶臭假单胞菌(Pseudomonas putida)聚为一支,GenBank中序列登录号分别为HQ007288、GQ449379。由此可知,分离菌株属于恶臭假单胞菌(P.putida)。药物敏感性试验结果表明,分离菌株对强力霉素、卡那霉素、庆大霉素、妥布霉素、左旋氧氟沙星、氧氟沙星、阿米卡星、诺氟沙星、恩诺沙星、四环素等10种药物敏感;五倍子和石榴皮对其抑菌效果最好,MIC值分别为1.57、3.13 mg/mL,其次为黄连和黄芩,而大黄、白头翁、苦参、黄柏对其抑菌效果较弱。药物敏感性试验结果可以为大黄鱼假单胞菌病的防治提供参考。

The bacterium stain ZSH, DHY10051103K were isolated from the diseased great yellow croaker （Pseudosciaena crocea） in Wenzhou, Zhejiang province, in 2009 and 2010 respectively. Artificial infection proved that the isolated bacterium were the pathogen of the disease, and the LDs0 to great yellow croaker were 2.5x106 CFU/mL and 3.2x106 CFU/mL respectively. The two isolated bacterium had the characteristics of gram-negative bacilli, oxidase-positive, utilization of Simmons citrate and maltose, degradation-nitrate, lysine deearboxylase-positive, methyl-red-negative, gelatinase-negative, ornithine decarboxylase-negative. A phylogenetic tree was constructed by comparing the 16S rRNA gene sequence of the bacterium with other relative bacteria species in the RDP and GenBank databases. In the phylogenetic tree, the isolated bacterium and some Pseudomonas putida strains converged and constituted a branch. According to the morphological, physiological, biochemical characteristics and 16S rRNA gene analysis, the isolated bacterium is identified as P. putida. The sensitivity test to drugs revealed that the pathogen was sensitive to Doxycycline, Kanamycin, Gentamycin, Tobramycin, Levofloxacin, Ofloxacin, Amikacin, Norfloxacin, Enrofloxacine, Tetracycline. And it was found that Galla chinensis and Pericarpium granat had best bacteriostatic effect on the two isolated bacterium, the MIC were 1.57 mg/mL and 3.13 mg/mL respectively, followed by Rhizoma coptidis and Radix scutellariae, and Radix et rhizoma Rhei, Radix pulsatiUae, Radix sophorae Flavescentis and Cortex phellodendri had the weaker effect.