水生态学杂志2016，Vol.37Issue(6)：95-100,6.DOI:10.15928/j.1674-3075.2016.06.014
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
摘要
Abstract
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 Megalobra-ma amhlycephala occurred in a farm in Changzhou City,Jiangsu Province and infected fish presented mild symp-toms 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 ef-fective inspection and prevention strategies for the disease,the dominant pathogenic strain was isolated from the liv-er and spleen of diseased Megalobrama amhlycephala and labeled the CZBY-1 strain.The phenotypic characteris-tics of the pathogenic strain were examined,including the morphological,physiological and biochemical character-istics,virulence factor detection and susceptibility test.The 16S rRNA gene was amplified and compared with se-quences deposited in GenBank databases.Simultaneously,an artificial infection study was carried out on healthy Megalobrama amhlycephala by injecting different concentrations of a bacterial suspension of incubated CZBY-1 strain.Megalobrama amhlycephala,with body mass (100 ±5)g,were randomly assigned to five test groups (1 × 109 ,1 ×108 ,1 ×107 ,1 ×106 ,1 ×105 CFU /mL)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 iso-lated to confirm the cause was strain CZBY-1.After one week,the mortality rate of Megalobrama amhlycephala in the test groups receiving 1 ×109 CFU /mL,1 ×108 CFU /mL and 1 ×107 CFU /mL were 100%,70% and 40%,re-spectively,and no deaths were observed in the 1 ×106 CFU /mL,1 ×105 CFU /mL or control groups.Clinical symp-toms in artificially infected Megalobrama amhlycephala were similar to those observed in naturally infected fish and the morphological,physiological and biochemical characteristics of the pathogenic strain isolated from the artificial test study were identical with strain CZBY-1.Bacterial strain CZBY-1 is smooth,opaque,round,gray-white color and the16S rDNA sequence is 1 440 bp (GenBank accession No.:KF413424).The phylogenetic tree analysis, based on 16S rRNA gene sequences,shows that CZBY-1 strain clusters with a strain of Klebsiella pneumoniae with homology more than 99%.The phenotypic characteristics and molecular biological analyses also show that CZBY-1 strain belongs to Klebsiella pneumoniae.By using specific primers for the PCR amplification of ompC (Outer mem-brane protein C)gene,the 252 bp gene fragments were amplified,indicating that CZBY-1 strain carried virulence factors of ompC.The drug susceptibility of the CZBY-1 strain was tested by paper extension methods using 44 test antibiotics and the results show that Klebsiella pneumoniae was highly sensitive to 27 antibiotics including Fosfomy-cin and Cotrimoxazole,but has strong resistance to Streptomycin,Vancomycin and Tobramycin.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Key words
Megalobrama amhlycephala/Klebsiella pneumoniae/isolation and identification/ompC/drug sensitiv-ity test分类
农业科技引用本文复制引用
滕涛,梁利国,谢骏,徐跑..团头鲂源肺炎克雷伯氏菌的分离鉴定[J].水生态学杂志,2016,37(6):95-100,6.基金项目
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）。 （）
