团头鲂源肺炎克雷伯氏菌的分离鉴定  

Isolation and Identification of Pathogenic Klebsiella pneumoniae from Megalobrama amhlycephala

作　　者：滕涛[1,2] 梁利国[2] 谢骏[1,2] 徐跑[1,2] 

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214081 [2]中国水产科学研究院淡水渔业研究中心农业部淡水渔业和种质资源利用重点实验室,江苏无锡214081

出　　处：《水生态学杂志》2016年第6期95-100,共6页Journal of Hydroecology

基　　金：现代农业产业技术体系建设专项(CARS-46-10);江苏省水产三新工程(D2013-5;D2015-13)

摘　　要：建立肺炎克雷伯氏菌感染团头鲂的模型,为团头鲂疾病的有效检验与防治及鱼类病原肺炎克雷伯氏菌的进一步研究提供参考和依据。2014年8月江苏省常州市某养殖场暴发出血病,团头鲂大量死亡,濒死团头鲂呈现轻微细菌性败血症症状,但未见大量充血发红,仅腹部、鳍基部有出血点,鳃丝发白,肠道无食物,肝胰腺轻微充血发红。采用常规的培养特征、理化特性及分子生物学的方法,于濒死团头鲂的肝、脾中分离到优势菌株CZBY-1,进行了形态观察、药敏试验、毒力因子检测等,并对其16S rRNA基因序列(Gen Bank登录号:KF413424)进行分析。构建系统发育树确定供试菌株与肺炎克雷伯氏菌同源性最高,在99%以上,结合表型及分子生物学分析,判定为肺炎克雷伯氏菌,人工回感可导致团头鲂死亡并呈现同检测鱼患病特征。利用设计的特异性引物进行外膜蛋白C(ompC)基因的PCR扩增,发现分离菌在252 bp对应位置有明显扩增条带,证明其具有毒力因子ompC。药敏试验表明,菌株对磷霉素、复方新诺明等27种抗生素敏感,对链霉素、妥布霉素、万古霉素3种抗生素耐药。Megalobrama amhlycephala is an important pond-reared freshwater fish in China. However,large-scale,high density culturing of Megalobrama amhlycephala has resulted in the occurrence of bacterial diseases,especially bacterial septicemia,causing huge losses to farmers. During August 2014,serious mortality of cultured Megalobrama amhlycephala occurred in a farm in Changzhou City,Jiangsu Province and infected fish presented mild symptoms of bacterial septicemia： white gills,blood spots on the abdomen and fin base,no food visible in the gut and slight hepatopancreatic congestion. To identify the pathogen infecting the Megalobrama amhlycephala and find effective inspection and prevention strategies for the disease,the dominant pathogenic strain was isolated from the liver and spleen of diseased Megalobrama amhlycephala and labeled the CZBY-1 strain. The phenotypic characteristics of the pathogenic strain were examined,including the morphological,physiological and biochemical characteristics,virulence factor detection and susceptibility test. The 16 S rRNA gene was amplified and compared with sequences deposited in Gen Bank databases. Simultaneously,an artificial infection study was carried out on healthy Megalobrama amhlycephala by injecting different concentrations of a bacterial suspension of incubated CZBY-1strain. Megalobrama amhlycephala,with body mass（ 100 ± 5） g,were randomly assigned to five test groups（ 1 ×10^9,1 × 10^8,1 × 10^7,1 × 10~6,1 × 10^5 CFU/m L） and a control group,with 10 Megalobrama amhlycephala in each group and each test run in replicate. The Megalobrama amhlycephala in each test group were injected with 100 μL of the appropriate bacterial suspension. Symptoms of the diseased Megalobrama amhlycephala and the mortality rate of each group were recorded daily,and the pathogenic strain from the infected Megalobrama amhlycephala was isolated to confirm the cause was strain CZBY-1. After one week,the mortality rate of Megalobrama amhlycephala in the test groups receiving 1 × 10^9 CFU/m

关 键 词：团头鲂 肺炎克雷伯氏菌 分离鉴定 外膜蛋白基因C 药敏试验 

分 类 号：S941.42[农业科学—水产养殖]