一株强裂解性大肠杆菌T1样噬菌体新成员的分离与鉴定

Isolation and characterization of an efficient lytic T1-like phage against Escherichia coli

摘要:

【目的】自然界中噬菌体种类繁多，其裂菌功能在针对细菌耐药方面具有潜在应用价值。不同噬菌体也呈现出显著的基因多样性及宿主特异性。从上海某猪场仔猪肠内容物样品中分离、纯化大肠杆菌的裂解性噬菌体，分析其生物学特性和病毒学特征，为探索应用噬菌体治疗细菌性感染提供研究材料。【方法】采用双层琼脂平板法分离、纯化噬菌体，观察噬菌斑特征，通过电镜观察噬菌体形态特征，测定其裂菌谱、最佳感染复数、一步生长曲线和生物学特性，进行噬菌体全基因组测序和遗传进化分析。【结果】分离、纯化获得一株能高效裂解大肠杆菌K-12菌株的噬菌体，命名为vB_EcoS_SH2 (SH2)，噬菌斑呈圆形、大而透明、边缘整齐。电镜观察SH2的头部呈二十面体立体对称，尾部较长。噬菌体的潜伏期为10 min，暴发期为60 min，裂解量高达121 PFU/感染细胞，其最佳感染复数为0.1。基因组测序和比对结果表明，SH2的核酸类型为dsDNA，基因组全长为49 088 bp，G+C%含量为45%，GenBank登录号为KY985004，结合电镜观察及BLASTp分析，确定其属于有尾噬菌体目长尾噬菌体科成员。同源性及进化分析表明，该噬菌体为大肠杆菌T1样噬菌体的新成员。【结论】分离鉴定了一株裂解效率极高的大肠杆菌T1样噬菌体，并确认其为T1样噬菌体新成员，为研究大肠杆菌噬菌体及其抗菌应用提供了新的实验材料。

Abstract:

[Objective] Bacteriophages are recognized as the most abundant microorganisms on earth, and have potential to control antibiotic-resistant bacterial infections for their bactericidal activity. Bacteriophages display remarkable genetic diversity and host specificity. To determine the biological characteristics and sequences of an efficient lytic Escherichia coli phage isolated from swine intestines. [Methods] Potential phages in the samples from piglet intestinal contents were isolated and purified, and plaque characteristics of the phage were observed. Morphological analysis was performed by electron microscope. The host spectrum, multiplicity of infection, one-step growth curve, thermal stability, pH stability, sensitivity to ultraviolet and chloroform sensitivity were determined, and genomic evolution was analyzed. [Results] A new lytic Escherichia coli phage, named vB_EcoS_SH2 (SH2), was isolated. The phage plaque was shown as big, circular, transparent and neat. Morphological analysis by electron microscopy revealed that phage SH2 had an icosahedron head with 50 nm in diameter, and a systolic tail fiber with 8 nm width and 120 nm length. Phage SH2 had a short latent period of 10 min and an outbreak period of 60 min, and a burst size of 121 PFU/infected cell. The optimal MOI is 0.1. Genome sequencing and comparison revealed that the nucleic acid type of SH2 is dsDNA, and the DNA genome of SH2 is composed of 49 088 bp with a G+C% content of 45% (GenBank accession number: KY985004). Phage morphology and BLASTp analysis revealed that SH2 belongs to the family of Caudovirales, Siphoviridae. Genomic and phylogenetic analysis suggested that the phage is closely related to phage T1, with 95% homology to T1 phage. [Conclusion] a novel efficient lytic T1-like phage vB_EcoS_SH2 against Escherichia coli was identified. The microbiological properties of SH2 suggest that it may be useful for controlling bacterial populations.