黄颡鱼拟态弧菌的鉴定、毒力相关因子及药敏特性

IDENTIFICATION, VIRULENCE-RELATED FACTORS, AND ANTIMICROBIAL SUSCEPTIBILITY OF VIBRIO MIMICUS FROM YELLOW CATFISH, PELTEOBAGRUS FULVIDRACO

  • 摘要: 2018年浙江省黄颡鱼主养区暴发了严重的溃疡病, 为查明该病的病原特点, 了解病原的致病因子及药物敏感性, 对溃疡黄颡鱼(Pelteobagrus fulvidraco)上分离的多株优势菌, 分别采用生理生化特性和管家基因16S rRNA、pyrH的系统进化树进行分类鉴定, 通过人工回归感染试验分析菌株的病原性, 利用平板法测定相关的毒力因子并采用PCR法对毒力相关基因进行检测, 应用微量二倍稀释法测定14种抗菌药物的最小抑菌浓度。结果表明, 11株优势菌皆为拟态弧菌, 2株代表菌腹腔注射感染黄颡鱼后均可引发溃疡症状; 11株拟态弧菌均具有溶血活性、蛋白酶、明胶酶和DNA酶活性, 不具有卵磷脂酶活性, 可分泌产生铁载体; 所有菌株均携带vmhompUtoxRmatCDB-iucABCD-iutA等毒力相关基因, tcpActxAstzotacetdh等毒力基因均未检出。各菌株对氨苄青霉素、磺胺嘧啶、磺胺甲噁唑、磺胺间甲氧嘧啶高度耐药, 对环丙沙星、恩诺沙星、氟苯尼考、多西环素、土霉素、甲砜霉素、红霉素、交沙霉素、磺胺二甲氧嘧啶较敏感, 个别菌株对新霉素和甲砜霉素耐药。以上研究结果表明, 拟态弧菌感染可引起黄颡鱼溃疡病, 不同发病养殖场的分离株具有相同的表型特征及毒力基因型, 并具有较一致的生理生化特性和耐药谱型, 说明它们具有相同的遗传背景或传染源。

     

    Abstract: In 2018, a severe disease characterized by serious and shaped ulcers in the skins were broke out in the main culture area of yellow catfish (Pelteobagrus fulvidraco) in Zhejiang Province. In order to elucidate the characterization of the causative agent and analyze virulence factors and antimicrobial susceptibility of the pathogen, several bacterial strains were isolated from the lesions and visceral organs of diseased fish. These isolates were subjected to physiology and biochemistry characteristics analyses by using bacterial biochemical identification instrument and biochemical identification micro-tubes. For molecular identification, the 16S rRNA and pyrH were amplified by PCR and compared with sequences deposited in databases, and molecular phylogenetic trees were constructed. Based on the morphological characteristics, phenotypic characteristics, 16S rRNA and pyrH gene sequence analysis, the 11 predominant bacterial strains were identified as Vibrio mimicus. Challenge trials were conducted by intraperitoneal injection, and the results showed that the two representative isolates exhibited high virulence in yellow catfish, skin ulcers occurred in both changed group. The potential virulence of the V. mimicus isolates were examined by phenotypic and PCR assays. All the studied strains exhibited hemolytic activity, and were positive for extracellular protease and gelatinase and DNA degrading activity, but negative for lecithinase activity. Siderophore activity were observed in all V. mimicus isolates. The vmh, ompU and toxR genes were found in all the isolated V. mimicus, whereas none possessed the tcpA, ctxA, st, zot, ace or tdh gene. Antimicrobial susceptibility of the isolated V. mimicus were determined by trace double dilution method and the results showed that the isolates were resistant to ampicillin, sulfadiazine, sulfamethoxazole, sulfamonomethoxine, were sensitive to ciprofloxacin, enrofloxacin, florfenicol, doxycycline, oxytetracycline, thiamphenicol, erythromycin thiocyanate, josamycin, sulfamethoxine, while resistance to neomycin sulphate and thiamphenicol were found in two isolates. Therefore, our study demonstrated the virulence of V. mimicus in yellow catfish as the causative agent of the enzootic. The isolated V. mimicus exhibited the same phenotypic traits and virulence-related genotype, also the biochemical characteristics and antibiotic resistance profile were similar, indicating that they had the same genetic background or source of infection.

     

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