文章摘要
两株溶藻弧菌烈性噬菌体的分离鉴定及其生物学特性
Isolation, identification and biological properties of 2 lytic phages against Vibrio alginolyticus
投稿时间:2021-01-07  修订日期:2021-02-07
DOI:
中文关键词: 溶藻弧菌  噬菌体  分离  生物学特性  微生态抗菌剂
英文关键词: Vibrio alginolyticus  Bacteriophage  Isolation  Biological characteristics  Antimicrobial agents
基金项目:
作者单位邮编
乔欢 菲吉乐科(南京)生物科技有限公司 210000
陈海 菲吉乐科(南京)生物科技有限公司 
徐旭凌 菲吉乐科(南京)生物科技有限公司 210000
丛郁 菲吉乐科(南京)生物科技有限公司 
刘墨 菲吉乐科(南京)生物科技有限公司 
何四龙 菲吉乐科(南京)生物科技有限公司 
费文斌 菲吉乐科(南京)生物科技有限公司 
黄杰 菲吉乐科(南京)生物科技有限公司 
胡怿林 菲吉乐科(南京)生物科技有限公司 
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中文摘要:
      溶藻弧菌是海洋经济动物的主要病原菌之一。噬菌体作为一种新兴的抗细菌感染制剂,在水产养殖方面具有较大潜力。为寻找并丰富溶藻弧菌噬菌体资源,本研究以溶藻弧菌VAHN1为宿主菌,采用双层平板法从海南虾塘水样及福建海产品样本中对溶藻弧菌噬菌体进行分离。通过透射电镜、限制性内切酶及构建发育树等方法对所获溶藻弧菌噬菌体进行了分类鉴定;同时对其生理生化性能进行了研究。结果显示,本研究分离获得2株溶藻弧菌噬菌体VAP9与VAP21,其噬菌斑均清晰透亮,直径约1.5~2.0 mm。2株噬菌体核酸均为双链DNA,于透射电镜下可见其头部均呈正二十面体结构,2株噬菌体均属肌尾噬菌体科。噬菌体VAP9与VAP21对理化环境具有良好的耐受性;VAP9最适pH为6~8,VAP21最适pH为7~11;2株噬菌体可耐受通用杀菌浓度的过氧乙酸,且对氯仿与乙醚不敏感,同时对紫外线具有一定耐受性。噬菌体VAP9与VAP21的最佳感染复数均为0.001;2株噬菌体对供试溶藻弧菌的裂解率达95.2%,可裂解部分副溶血性弧菌,但无法裂解除溶藻弧菌与副溶血性弧菌外的弧菌属、葡萄球菌属、假单胞菌属等其他种属的供试细菌;噬菌体VAP9与VAP21可高效抑制溶藻弧菌VAHN1的生长,且2株噬菌体的混合制剂对溶藻弧菌的抑制效果好于单株溶藻弧菌噬菌体。将噬菌体VAP9及VAP21保守蛋白序列于NCBI上比对后发现2株供试噬菌体与其他噬菌体间同源性较低,因此噬菌体VAP9及VAP21很可能为2株新发现的肌尾科噬菌体。
英文摘要:
      Vibrio alginolyticus is one of the main pathogens that causes mass mortality in aquatic animals and infects humans. To reduce the application of antibiotic, alternative therapies have been proposed. One of the promising possibilities is through the use of lytic bacteriophages to control diseases. As few current bacteriophages against V. alginolyticus have been reported, the main objective of this study was to develop effective bacteriophages of controlling pathogenic bacteria. This study describes the isolation and characterization of lytic bacteriophages against a V. alginolyticus strain (VAHN1). The bacteriophages were isolated from Hainan shrimp pond and Fujian marine products by double-layer agar culture method, and V. alginolyticus VAHN1 was used as the host strain for bacteriophage isolation. The phages were classified and identified by transmission electron microscope (TEM), restriction endonuclease and phylogenetic tree analysis.The?physiological and biochemical?characteristics?of bacteriophages were determined, including optimal multiplicity of infection (MOI), lysis spectrum detection, pH stability, thermal stability, resistance to UV light, and sensitivity to chloroform and ether. In addition, the?effect?of?bacteriostasis were also measured. The results showed that 2 lytic bacteriophages against V. alginolyticus were isolated, named as VAP9 and VAP21. The plaques of VAP9 and VAP21 were neat and transparent, with a diameter of 1.5~2 mm. The nucleotides of the phage VAP9 and VAP21 were all dsDNA, and all their heads were shown as icosahedral shape with about 55 nm and 65 nm diameter respectively under TEM. The tail of the phage VAP9 was approximately 65-70 nm in length and 15 nm in width, while the tail of the phage VAP21 was about 75-80 nm in length and 18 nm in width. The 2 bacteriophages are grouped under the Myoviridae family. The phage VAP9 and VAP21 had good tolerance to different physical and chemical environment. The survival?rate of 2 bacteriophages was greater than 43% at 60℃ for 2 h. The optimal pH of VAP9 was 6-8 and VAP21`s was 7-11. The 2 bacteriophages tolerated peracetic acid in universal bactericidal concentration. The phage VAP9 and VAP21 were insensitive to chloroform and ether, with a certain resistance to UV light. Both of optimal MOIs of the VAP9 and VAP21 were 0.001. The cocktail of VAP9 and VAP21 was able to infect 95.2% (157 strains among 165 strains) of the V. alginolyticus strains and 50% (3 strains among 6 strains) of V. parahaemolyticus strains used in this study. And they could not infect other species of tested bacteria except V. alginolyticus and V. parahaemolyticus. The phage VAP9 and VAP21 could effectively inhibit the growth of V. alginolyticus VAHN1 and have the same inhibitory trend on VAHN1, but the VAP21 strain had a stronger bacteriostatic effect than that of the VAP9 strain. Moreover, the inhibitory effect involved the cocktail of 2 bacteriophages on V. alginolyticus were better than that of a single phage. Alignment with the sequences of the conserved protein amino acid sequences of the phage VAP9 and VAP21 on National Center for Biotechnology Information?(NCBI), it was showed that the 2 bacteriophages had low homology with other bacteriophages. Furthermore, phylogenetic and genome analysis revealed that the phage VAP9 and VAP21 have low homology with other bacteriophages and may be 2 novel Myoviridae bacteriophages that infect bacteria related Vibrio spp. This study did not only enrich the species resources of bacteriophages against V. alginolyticus, but it also laid a theoretical foundation for the development and application of Vibrio bacteriophages as microecological antimicrobial agents.
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