1株鼠疫耶尔森菌噬菌体的分离鉴定及基因组学分析

Genome analysis and identification of a Yersinia pestis phage

目的 分离鉴定一株喜马拉雅旱獭携带鼠疫噬菌体,并对其基本生物学特性及基因组学特征进行分析。方法 采集鼠疫自然疫源地宿主动物喜马拉雅旱獭盲肠标本,采用双层琼脂平板法,以鼠疫菌疫苗株EV76为宿主菌,分离纯化噬菌体,利用透射电子显微镜观察其形态,研究生物学特性,提取该噬菌体的DNA进行二代测序,并利用生物信息学软件分析其基因组的特征与功能。结果 在喜马拉雅旱獭鼠疫自然疫源地宿主动物喜马拉雅旱獭盲肠中分离获得一株鼠疫噬菌体,命名为vB_YpM_QH041,电镜观察其具有正二十面体头部和收缩性的尾部,为肌尾噬菌体。噬菌体生物学特性研究显示,最佳感染复数为0.1,潜伏期为40 min,暴发期为40 min,暴发量为10蚀斑形成单位(PFU)/cell。该噬菌体对来自10个自然疫源地的33株鼠疫菌均裂解,对耶尔森菌属内1株假结核耶尔森菌裂解;对肠杆菌科内1株非致病大肠埃希菌(MG1655)具有裂解性,对其他大肠埃希菌、沙门菌、痢疾杆菌没有明显的裂解性。该噬菌体基因组长度为31 940 bp,GC含量为51.52%,有42个开放阅读框,开放阅读框总长度28.99 kb,其中有35个为已知编码序列,不含毒力基因和抗生素耐药基因。噬菌体全基因组系统发育树同源性分析结果显示,该噬菌体与鼠疫噬菌体vB_YpM_22和VB-YpM-18亲缘关系较近,为P2类噬菌体,但宿主谱存在不同。结论 该噬菌体对鼠疫菌裂解性好,同时能裂解其他肠道菌,有助于了解鼠疫菌在自然疫源地中的保存问题,具有一定的潜在应用价值。

Objective A Yersinia pestis phage from a Himalayan marmot was isolated and identified,and its basic biological characteristics and genomics characteristics were analyzed.Methods The cecum specimens of Marmota himalayan,a host animal in the natural focus of pestis,were collected,and the Yersinia pestis vaccine strain EV76 was used as the host bacteria to isolate and purify the phage.The morphology of the phage was observed by transmission electron microscopy,and its biological characteristics were studied.The DNA of the pestis phage was extracted for second-generation sequencing,and its genome characteristics and functions were analyzed by bioinformatics software.Results The phage named vB_YpM_QH041 was isolated from the cecum of a Himalayan marmot from natural plague focus belonged to Myoviridae with a icosahedral structure and a typical retractable tail.Studies on the biological characteristics showed that the multiplicity of infection was 0.1,the latent period was 40 minutes,the outbreak period was about 40 minutes,and the burst size was 10 PFU/cell(plaque forming unit,PFU).The bacteriophage cracked all the 33 strains in the 10 natural focus of pestis tested,and cracked one Yersinia pseudotuberculosis in the genus Yersinia;It has lytic activity against a non-pathogenic Escherichia coli(MG1655)strain within the Enterobacteriaceae family,but does not have significant lytic activity against other Escherichia coli,Salmonella,or Shigella strains.The genome length of this phage was 31940 bp,with a GC content of 51.52%.There are 42 open reading frames,with a total length of 28.99 k,of which 35 are known coding sequences.No virulence genes or antibiotic resistance genes are present.The result of homology analysis of the phylogenetic tree of the whole genome of the bacteriophage shows that this bacteriophage and the pestis bacteriophage vB_YpM_22 and VB_YPM_18 are closely related and belong to the P2 phage group.Conclusion This phage vB_YpM_QH041 has good lytic ability to Yersinia pestis and can also lyse other intestinal bacteria,which is helpful for us to understand the preservation problem of Yersinia pestis in the natural foci.