牛蛙源达卡气单胞菌的分离鉴定及毒力特性分析

采用细菌形态与生化鉴定方法并结合16S rRNA及gyrA基因测序分析,对海南省文昌市某养殖场死亡牛蛙病变组织中分离获得的1株优势菌株WC0803进行鉴定,并对其进行致病性试验、药物敏感性试验及耐药基因和毒力基因检测。结果显示,该菌株为革兰阴性短杆菌,培养特性、生化反应与达卡气单胞菌(Aeromonas dhakensis)基本一致;16S rRNA和gyrA基因同源性及系统进化分析显示,WC0803菌株与达卡气单胞菌参考菌株同源性大于99%,且为同一进化分支。该菌对头孢噻肟、环丙沙星、恩诺沙星等7种抗生素敏感,对头孢氨苄、万古霉素、磺胺异恶唑和链霉素等10种抗生素耐药;携带耐药基因qnrA、qnrS、ant3、tem、ctxM,毒力基因ascV、aer、act、ast、lip、ela和fla;对牛蛙的半数致死量(LD50)为4.68×10⁶CFU/mL,具有较强的致病性。本研究首次从患病牛蛙体内分离出一株强致病性达卡气单胞菌,为该菌感染的防治提供科学依据。

Isolation,identification and virulence analysis of Aeromonas dhakensis from Rana catesbiana

In this study,a dominant strain WC0803 was isolated from the diseased tissues of dead Rana catesbiana in Wenchang of Hainan Province and was identified by morphological and biochemical identification combined with 16 S rRNA and gyrA gene sequencing.Pathogenicity analysis and drug sensitivity test were performed on the strain,and its drug-resistance and virulence genes were tested.The results showed that the isolated strain was a gram-negative bacillus,and its culture characteristics and biochemical reaction were consistent with those of Aeromonas dhakensis.According to 16 S rRNA and gyrA gene homology and phylogenetic analysis,the isolates WC0803 and CP023141.1 of the Aeromonas dhakensis KN-Mc-6 U21 strain were of more than 99%homology and they belonged to the same phylogenetic branch.The isolate was sensitive to seven drugs,such as cefotaxime,ciprofloxacin and enrofloxacin,and so on,and was resistant to 10 drugs,such as cefalexin,vancomycin,sulfisoxazole and streptomycin,and so on.The isolated strain carried drug-resistant genes including qnrA,qnrS,ant3,tem,ctxM,and virulence genes,including ascV,aer,act,ast,lip,ela and fla.The LD50 of the isolate to Rana catesbiana was 4.68×10⁶CFU/mL,indicating that the strain was a highly pathogenic bacterium.To sum up,a highly pathogenic bacterium A.dhakensis was first found in the infected Rana catesbiana in the present study,which provided scientific reference for prevention and treatment of the related diseases.