一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析

Isolation and characterization of a lytic phage infecting Escherichia coli from bovine mastitis

摘要:

【目的】从新疆石河子地区奶牛粪样中分离裂解性大肠杆菌噬菌体(Escherichia coli phage)，对其进行纯化及生物学特性分析。【方法】利用双层平板法从奶牛粪样中分离、纯化噬菌体，将纯化后的噬菌体浓缩液用醋酸双氧铀负染后通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析，同时测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性及酸碱稳定性。【结果】分离并纯化出一株裂解性噬菌体vB_EcoM_XJ2，噬菌斑圆形不透明，直径0.7 mm–1.2 mm；电镜显示其头部呈正多面体对称，有可伸缩性尾部；核酸类型为双链DNA，基因组大小为75.617 kb，G+C%含量为42.09%；其核酸序列与大肠杆菌噬菌体NJ01和vB_EcoP_SU10相似性高达94%。生物学特性研究显示该噬菌体能裂解多株临床分离的大肠杆菌；能耐受60 °C左右高温，在pH 5.0–11.0范围内效价稳定；最佳感染复数为0.1，潜伏期为15 min，暴发期为95 min，裂解量约为10.6 PFU/cell。【结论】vB_EcoM_XJ2是一株在不同温度、不同酸碱性环境中有较强适应能力的裂解性肌尾科大肠杆菌噬菌体。

Abstract:

[Objective] To isolate and characterize the lytic phage from fecal samples of dairy cattle in Shihezi, Xinjiang. [Methods] The phage was isolated and purified from dairy cattle fecal samples by double-layer agar plate method. The purified phage was concentrated negatively stained with uranyl acetate and observed by transmission electron microscopy. Meanwhile, the genome of the isolated phage was sequenced. Its genetic and evolutional history were analyzed. In addition, the host range, optimal multiplicity of infection (MOI), one-step growth curve, temperature and pH stability of the phage were investigated. [Results] One strain of phage that lysed Escherichia coli was isolated and named vB_EcoM_XJ2. The phage produced circular, not clear plaques with 0.7 mm–1.2 mm diameter. The electron microscope observation showed that the phage had a symmetrical Polyhedra the head and a contractile tails. The genome of the phage was comprised of double strand DNA, and the size was 75.617 kb with G+C% content of 42.09%. The homology of amino acid sequence of vB_EcoM_XJ2 was 94% with Escherichia coli phage NJ01 and vB_EcoP_SU10. The isolated phage was lytic to many strains of Escherichia coli, isolated from bovine mastitis milk samples. The phage could withstand the temperature up to 60 °C and keep stable titer under pH 5.0–11.0. The optimal MOI was 0.1, the incubation period was 15 min, the burst phase time was 95 min, and the burst size was 10.6 PFU/cell. [Conclusion] The newly isolated phage, named vB_EcoM_XJ2, belongs to lytic phage of Myoviridae, which shows strong capacity to adapt different environments conditions such as temperatures and acid-base values.