一株新型杀鲑气单胞菌烈性类T4噬菌体Asfd-1的分离鉴定和生物学特性分析

Isolation and Characterization of a Novel Lytic T4-Like Bacteriophage Asfd-1 Infecting Aeromonas salmonicide

噬菌体是自然界中最普遍的生物之一,其专一性裂解细菌的能力在针对耐药菌感染治疗方面具有很大的应用前景。杀鲑气单胞菌(Aeromonas salmonicide)是水产养殖业中一种主要病原菌,其引发的疖疮病是一种常见的水产养殖动物疾病,具有高死亡率和高发病率,一旦爆发,将给水产养殖业带来重大的经济损失。该研究以杀鲑气单胞菌MF663675.1为宿主菌,从深圳海鲜市场的污水中,采用双层琼脂平板法分离纯化得到单个噬菌体,通过电镜对其表观形态特征进行观察,并运用生物信息的方法进一步确定其基因型特征及遗传分类地位。研究结果表明,分离出的烈性噬菌体命名为Asfd-1,噬菌斑呈圆形,轮廓分明、透明,直径约1mm。电镜观察显示,Asfd-1具有对称的二十面体头部,以及长度为51 nm的收缩尾部。基因组测序和基因对比分析表明,Asfd-1含双链脱氧核糖核酸(dsDNA),全基因组大小为168 962 bp,鸟嘌呤(G)和胞嘧啶(C)所占的比例为41.53%,共编码263个开放阅读框和16个转移核糖核酸(tRNA)序列,Genbank登录号为MK577502。根据国际病毒分类委员会规定的病毒分类方法,与同源的Aeromonas phage 31 GCA_000862645.1和Aeromonas phiSA4GCA_002710125.1全基因组对比结果显示,Asfd-1是新型噬菌体。综合电镜观察结果及系统进化树分析,Asfd-1属于肌尾科下的T4噬菌体属、肌尾噬菌体科、有尾噬菌体目的新成员。该研究可为应用噬菌体治疗细菌感染提供进一步的参考。

Bacteriophages are one of the most common microorganisms on the earth,and are potential biocontrol agents for multidrug-resistant bacteria.Aeromonas salmonicide is a major bacterial pathogen in aquaculture.It can be responsible for furunculosis,which is a ubiquitous disease in aquaculture operation and featured by high mortality and morbidity,causing significant economic loss of aquaculture.In this study,Aeromonas salmonicida MF663675.1 was used as host to isolate bacteriophage from the sewage of a seafood market in Shenzhen,China.A novel lytic phage Asfd-1 was isolated and purified by double-layer agar plate.The morphological characteristics were observed by transmission electron microscope(TEM).The plaque characteristics were observed as well.The whole genome and phylogenetic analysis of phage Asfd-1 were carried out by bioinformatics methods.The results show that plaque of the phage was round and transparent with size of about 1 mm in diameter.TEM shows that Asfd-1 has an isometric,elongated and icosahedral head of 100 nm in diameter by 71 nm in width and a 51 nm contracted tail.Comparative genomics analysis indicates that Asfd-1 has a double-stranded DNA of 168 962 bp in length,and the content ratio of guanine and cytosine is 41.53%.Asfd-1 genome encodes 263 open reading frames and 16 tRNA genes,indicating that Asfd-1 is not totally dependent on host cell transcription machinery.Combined with TEM and phylogenetic analysis,it proposes that Asfd-1 belongs a new member of the T4 virus,Myoviridae family,Caudovirales order.Based on its microbiological traits,phage Asfd-1 is a promising agent that can be used for controlling Aeromonas salmonicida infection.