屎肠球菌噬菌体vB_EfaS_29生物学特性及基因组分析

Biological characteristics and genome analysis of a virulent Enterococcus faecium bacteriophage vB_EfaS_29

摘要:

[背景] 随着屎肠球菌耐药性越来越严重，亟需筛选获得新的、裂解效率高且遗传背景清晰的裂解性噬菌体，丰富噬菌体资源，为噬菌体疗法提供可用的菌株。[目的] 从江苏省南京市西岗奶牛场粪便样品中分离出一株裂解性屎肠球菌噬菌体，研究其生物学特性及分析基因组学特征。[方法] 通过双层平板法对其进行纯化，观察噬菌斑特征，透射电镜观察噬菌体形态，测定一步生长曲线、pH耐受性、温度耐受性以及最佳感染复数，对噬菌体进行全基因组测序及遗传进化分析。[结果] 分离并纯化的一株裂解性屎肠球菌噬菌体命名为vB_EfaS_29，其噬菌斑圆形透亮，外周无晕环。该噬菌体头部呈二十面体，头部直径约52.4 nm，尾长约157.1 nm，为长尾噬菌体科。该噬菌体的最佳感染复数为0.1，最高耐受温度为70℃左右，当pH值在6.0-9.0时其效价稳定。一步生长曲线表明，其潜伏期为10 min，暴发期约为50 min，暴发量为80。基因组全长41 014 bp，GC含量为34.99%，共有63个开放阅读框（Open Reading Frame，ORF），其中36个被注释出已知基因，基因组中不含毒力基因、耐药基因及整合基因。进化分析显示以裂解酶编码氨基酸为靶标对比，该噬菌体与GenBank上的粪肠球菌噬菌体vB_EfaS_DELF1相似性大于99%，但是以噬菌体衣壳蛋白编码氨基酸为靶标比对发现，该噬菌体肠球菌与其他噬菌体遗传距离均较远。[结论] 分离出一株裂解性屎肠球菌噬菌体，对其进行生物学特性及基因组分析，为噬菌体研究和应用提供理论依据及研究基础。

关键词:屎肠球菌;噬菌体;生物学特性;基因组分析

Abstract:

[Background] With the increasingly serious drug resistance of Enterococcus faecium, it is urgent to screen and obtain new lytic bacteriophages with high lysis efficiency and clear genetic background, to enrich phage resources and provide appropriate strains for phage therapy. [Objective] To evaluate the biological characteristics and genome analysis of an Enterococcus faecium bacteriophage isolated from the fecal sample of Nanjing Xigang Dairy Farm. [Methods] The bacteriophage was purified by double-layer agar plate. The purified bacteriophage was negatively stained with phosphotungstic acid and observed by the transmission electron microscope (TEM). Then, one-step growth curve, pH tolerance, temperature tolerance, and optimal multiplicity of infection were detected. Also, its genomics characteristics and evolutionary history were analyzed. [Results] The results are as follows:A lytic Enterococcus faecium bacteriophage, named as vB_EfaS_29, was isolated and purified. Its plaques were round, bright, and clear. TEM analysis revealed that bacteriophage vB_EfaS_29 had a twenty-side head and a long tail. The head diameter was about 52.4 nm, and the tail length was about 157.1 nm. It belongs to the Siphoviridae family. The optimal multiplicity of infection of bacteriophage vB_EfaS_29 was 0.1; the maximum tolerable temperature was about 70℃; during pH 6.0-9.0, it was stable. The complete genome sequences of bacteriophage vB_EfaS_29 comprised of 41 014 bp with a GC content of 34.99% and contained 63 putative open reading frame (ORF). Only 36 of the ORF were well predicted for functions. Undesirable genes such as lysogenic, antibiotic resistance, or virulence were not detected in the genome. The phylogenetic analysis of vB_EfaS_29 based on amino acid of the endolysin, demonstrated that vB_EfaS_29 had more than 99% of sequence homology to FelixO1-like phages. However, using the amino acid of capsid as the targeted protein, vB_EfaS_29 had a long genetic distance with other Enterococcuss bacteriophages. [Conclusion] The above results provided theoretical data and resources for bacteriophage research and bacteriophage therapy.

Key words:Enterococcus faecium;bacteriophage;biological characteristics;genome analysis