一株新型旱獭源性宽谱大肠杆菌噬菌体的分离鉴定及基因组分析

Characterization and complete genomic analysis of a novel broad host-range Escherichia coli phage isolated from Marmota himalayana

摘要:

[目的]分离喜马拉雅旱獭肠内容物样本中的噬菌体,并研究其生物学特性和基因组特征。[方法]以大肠杆菌为宿主菌,利用双层琼脂平板法从喜马拉雅旱獭肠内容物样本中分离噬菌体;用透射电镜观察形态特征;测定其最佳感染复数、一步生长曲线、酸碱耐受度及宿主裂解谱等生物学特性,并进行全基因组测序。[结果]从喜马拉雅旱獭肠内容物样本中分离得到一株裂解性大肠杆菌噬菌体,命名为vB_EcoM_TH18,其噬菌斑呈无晕环的透亮圆形,透射电镜观察发现该噬菌体头部直径为(90±5) nm,尾部长度为(115±5) nm;最佳感染复数为1;一步生长曲线显示其潜伏期为10 min,110 min后进入平台期,平均裂解量为15 PFU/mL;在pH 4.5-9.5的范围内具有稳定活性;可裂解多种致病型和血清型大肠杆菌和宋内志贺氏菌,无法裂解沙门氏菌、屎肠球菌、金黄色葡萄球菌、肺炎克雷伯杆菌及鲍曼不动杆菌;基因组测序结果表明,其基因组长度为133 882 bp,GC含量为39.95%。基因组共注释到210个编码序列(CDS)和13个tRNAs,不含毒力基因及耐药基因。BLASTn比对结果表明该基因组与Avunavirus属噬菌体Av-05同源性为95.17%。基于噬菌体全基因组、主要衣壳蛋白和终止酶大亚基分别构建系统进化树,结果表明vB_EcoM_TH18是一株肌尾噬菌体科(Myoviridae) Avunavirus属的新型噬菌体。[结论]从喜马拉雅旱獭肠内容物中成功分离并鉴定了一株新型宽谱大肠杆菌噬菌体vB_EcoM_TH18,可裂解多种致病型和血清型的大肠杆菌及宋内志贺菌。

Abstract:

[Objective] To isolate and characterize the bacteriophage from the intestinal contents of Marmota himalayan.[Methods]A lytic phage was isolated from the host Escherichia coli in intestinal contents of M.himalayan with the double-layer agar method.The morphological characteristics were observed by transmission electron microscopy (TEM).Meanwhile,the optimal multiplicity of infection (MOI),one-step growth curve,pH stability,and the host range of the phage were investigated,and the genome was analyzed as well.[Results] The lytic bacteriophage was named vB_EcoM_TH18 and the phage plaque was transparent and round with a clear boundary.According to TEM,the phage had the head of (90±5) nm in diameter and tail of (115±5) nm in length.The optimal MOI was 1.According to the one-step growth curve,vB_EcoM_TH18 had a latent period of 20 min and the lysis period of about 110 min with the average burst size of about 15 PFU/mL.vB_EcoM_TH18 can keep stable titer at pH 4.5-9.5.Additionally,it can lyse various pathotypes and serotypes of E.coli and Shigella sonnei,while Salmonella,Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumonia,and Acinetobacter baumannii can't be lysed.The genome of the vB_EcoM_TH18 was 133 882 bp with the G+C content of 39.95%.It comprised 210 coding sequences (CDS) and 13 tRNAs genes,without any virulence genes or antibiotic-resistant genes.BLASTn alignment showed that genome of vB_EcoM_TH18 shared 95.17% identity to E.coli phage Av-05.According to the polygenetic trees for the whole genomes,major capsid proteins,and terminase large subunits of vB_EcoM_TH18 and otherMyoviridae phages,respectively,vB_EcoM_TH18 was a novel phage in Avunavirus of Myoviridae.[Conclusion] In this study,we isolated and identified a novel broad host-range E.coli phage vB_EcoM_TH18.It can lyse various pathotypes and serotypes E.coli and S.sonnei.