克氏原螯虾病原弗氏柠檬酸杆菌的分离鉴定及其药敏与黏附特性
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安徽农业大学动物科技学院人兽共患病重点实验室,安徽农业大学动物科技学院人兽共患病重点实验室,安徽农业大学动物科技学院人兽共患病重点实验室,安徽农业大学动物科技学院人兽共患病重点实验室,安徽农业大学动物科技学院人兽共患病重点实验室,安徽农业大学动物科技学院人兽共患病重点实验室,安徽农业大学动物科技学院人兽共患病重点实验室

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Q785; S941

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国家自然科学基金(31272696)


Isolation and identification of Citrobacter freundii from diseased Procambarus clarkii and its antibiotics susceptibility and adhesion characteristics
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Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China,,,,Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University,,

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    摘要:

    为了明确安徽省当涂县淡水养殖克氏原螯虾暴发性疾病的病原,取濒临死亡的病虾分离病原。从肝胰腺中分离到一株优势细菌(命名为XLX1分离株),人工感染试验证实其具有较强的致病性;采用形态学检查、生理生化特性测定和细菌16S rRNA基因序列分析,确定XLX1分离株为弗氏柠檬酸杆菌。进一步对XLX1分离株进行药物敏感性、携带黏附素基因情况和细胞黏附性进行检测。结果显示:该分离株对头孢噻肟、庆大霉素、阿米卡星、硫酸新霉素、恩诺沙星、诺氟沙星、替考拉宁和米诺环素敏感或中敏,对其他7种测试药物呈现耐药。该分离株携带黏附素基因cfaure基因簇;序列分析发现ureABC结构基因和ureD关键辅助基因高度保守,在虾源分离株与人源参考株间前者的核苷酸和氨基酸序列同源性分别在93.2%~98.3%和91.7%~97.4%,后者的核苷酸和氨基酸序列同源性分别在90.8%~98.3%和94.7%~98.7%;与人源参考株相比,虾源分离株UreABC结构蛋白中第294、600和608位氨基酸,以及UreD蛋白中的第62和122位氨基酸发生了有意突变。XLX1分离株可以聚集方式黏附于EPC细胞周围,平均黏附菌数为29.8±5.3,但随黏附时间延长,EPC细胞出现病变。上述研究结果可为防控弗氏柠檬酸杆菌引起的水产动物疾病提供理论依据。

    Abstract:

    In order to define the potential pathogens causing fulminant illness of Procambarus Clarkii in Dangtu County, Anhui Province, pathogens were isolated from the moribund Procambarus Clarkii. A dominant strain named XLX1 was isolated from the hepatopancreas and was confirmed to be pathogenic to Procambarus Clarkii by artificial challenge test. The XLX1 isolate was identified as Citrobacter freundii by means of the morphology, physiological and biochemical characteristics and 16S rRNA gene sequencing. The antibiotics sensitivity, adhesin-carrying genes and cell adhesion of XLX1 isolate were further detected. The results showed that the XLX1 isolate was sensitive or medium sensitive to cefotaxime, gentamicin, amikacin, neomycin sulfate, enrofloxacin, norfloxacin and teicoplanin, but resistant to other 7 kinds of antibiotics. The XLX1 isolate carried adhesin genes cfa and ure clusters. It was found that ureABC structure gene and ureD key accessory gene was relatively conservative by the sequences analysis. The nucleotide and amino acid homology of former respectively ranged from 93.2% to 98.3% and 91.7% to 97.4%, and the latter ranged from 90.8% to 98.3% and 94.7% to 98.7% between XLX1 isolate and reference strains. Compared with reference strains derived from patient, the 294th, 600th, 608th amino acids in UreABC structure protein and the 62 nd and 122 nd amino acids in UreD key accessory protein generated sense mutation amino acids from XLX1 isolate. XLX1 isolate adhered to the epithelioma papulosum cyprinid(EPC) cells mainly in aggregative pattern, and the average adhesive bacterial number was 29.8±5.3, which caused EPC cells lesion with the extension of adhesion time. This study may contribute to providing a theoretical basis for controlling aquatic animal disease caused by Citrobacter freundii.

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肖宁,孔令严,周昊,曹际,李槿年,相翊卿,王蒙蒙.克氏原螯虾病原弗氏柠檬酸杆菌的分离鉴定及其药敏与黏附特性[J].水产学报,2016,40(6):946~955

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  • 收稿日期:2015-09-26
  • 最后修改日期:2016-03-23
  • 录用日期:2016-06-24
  • 在线发布日期: 2016-06-29
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