草鱼铜绿假单胞菌的鉴定及药物敏感性分析

Identification and antibiotic sensitivity of pathogenic bacterium Pseudomonas aeruginosa isolated from diseased grass carp

从患病草鱼C tenopharyngodon idella体表病灶上分离到一株优势菌,经分离纯化培养制成细菌悬液,分别以腹腔注射和浸泡两种方式感染草鱼。结果表明:该菌株能感染草鱼,且具较强毒力,腹腔注射0.2mL该菌液(3×109cfu/mL),可导致受试草鱼100%死亡;创伤浸泡感染(3×108cfu/mL),死亡率也达100%,受感染鱼出现与自然感染相似的类似赤皮病症状。对该病菌进行形态特征观察和主要理化特性分析,初步鉴定为铜绿假单胞菌Pseudom onas aeruginosa。进一步对该菌进行分子鉴定,共选用16S rRNA、RNA聚合酶β亚单位(rpoB)和促旋酶亚单位蛋白(gyrB)3个基因进行鉴定分析。序列克隆与BLAST分析结果显示,这3个基因均与GenBank上登录的铜绿假单胞菌的相应序列具有很高的同源性,16S rRNA、rpoB和gyrB基因与已知铜绿假单胞菌相应序列的同源性分别在99%、98%和93%以上。综合生理生化与分子鉴定结果,可判定所分离的菌株为铜绿假单胞菌。药敏试验结果显示:该菌对阿米卡星、菌必治、氟哌酸和妥布霉素等11种药物高度敏感,对链霉素和庆大霉素中度敏感,对罗红霉素、红霉素、四环素、苯唑青霉素、新霉素和头孢氨苄等16种药物具有耐药性。

A pathogenic bacterium was isolated from skin of diseased grass carp Ctenophyaryngodon idellus showing pathological signs similar to those observed on fish suffered from ＂red skin disease＂,including significant external lesions of inflammation,and haemorrhagic and exfoliation scales as well as rot or decay fins.Artificial infection experiment by intraperitoneal injection of the bacterium（0.2 mL of bacterium,3×109 cfu/mL） or immersion of the injured fish with the bacterium（3×108cfu/mL） showed that 100% mortality of the tested grass carp,with similar symptoms found in those natural infection and that the isolated bacteria possessed strong virulence.The bacterial morphology observation,physiology and biochemistry assays suggested that the isolated bacterium was found to be Pseudomonas aeruginosa.The three genes,16S rRNA,rpoB（RNA polymerase beta subunit） and gyrB（DNA gyrase beta subunit） were cloned,sequenced and analyzed in the bacterium by a molecular method.BLAST showed that all the three genes shared high similarities with their counterparts of P.aeruginosa found in GenBank.Similarities of the genes of the isolated bacterium with those in GenBank were found to be above 99% for 16S rRNA,98% for rpoB and 93% for gyrB genes.Both physiological and biochemical assays and molecular identification suggested that the bacterium isolated was a strain of P.aeruginosa.The drug sensitivity test show that the isolated bacterium was highly sensitive to eleven kinds of antibiotics such as Amikacin,Rocephin,Fupaisuan and Tobramycin,intermediately sensitive to Streptomycin and Gentamicin,and resistant to sixteen kinds of antibiotics including Roxithromycin,Erythromycin,Tetracycline,Oxazocilline,Neomycin and Cephalexin.