一株犬源Ackermannviridae噬菌体LHE287生物学特性和全基因组序列分析

Biological Characteristics and Genomic Analysis of an Ackermannviridae LHE287 Isolated from Beagle Farm

目的:从犬舍环境中分离噬菌体,对其进行生物学特性测定和全基因组序列分析。方法:以96株大肠杆菌为宿主菌,利用双层平板法从犬粪样分离纯化噬菌体;使用透射电子显微镜观察噬菌体形态特征;通过双层平板法测定其裂解谱、最佳感染复数、理化因子耐受性、一步生长曲线及体外抑菌活性,确定噬菌体的生物学特性;通过全基因组测序和分析确定其基因组序列特征。结果:分离到一株Ackermannviridae科噬菌体LHE287,正二十面体头部直径约为86 nm,可收缩尾部呈伞状,长约为107 nm。该噬菌体不仅可裂解大肠杆菌,还可以裂解沙门菌。当感染复数为0.0001时,噬菌体效价最高达1.72×10^(9) PFU/mL,且在24 h后仍可有效抑制细菌增殖,表现出良好抑菌效果。噬菌体LHE287在50℃以下,pH 5~10范围内较稳定,对紫外线具有一定抵抗能力。以E.coli 287为宿主菌增殖,其潜伏期为30 min,爆发期为90 min,爆发量为100 PFU/cell。该噬菌体基因组全长为149485 bp,包含168个开放阅读框和3个tRNA,无毒力基因和耐药基因。结论:成功分离并鉴定1株Ackermannviridae噬菌体LHE287,表现出良好的应用潜力,将为进一步开发为噬菌体制剂防控犬源大肠杆菌病提供支持。

Objective:To isolate a phage from environmental samples of the beagle kennel,and identify its biological characteristics and genomic characterization.Methods:96 clinical isolates of Escherichia coli(E.coli)were used as host bacteria.Phage LHE287 was isolated by the double-layer plate method.Its morphology was observed bytransmission electron microscope.The host range,multiplicity of infection(MOI),stability,one-step growth curve,and in vitro antibacterial activity of phage LHE287 were determined by the double-layer plate method.The genomic characteristics of phage LHE287 were analyzed.Results:A bacteriophage LHE287 with a lysis ability against E.coli and Salmonella was isolated from canine feces.It has an icosahedral head(~86 nm)and a retractable tail(~107 nm)with a forked umbrella-like tip,and was determined to be a member of the Ackermannviridae family.The genome of phage LHE287 was 149485 bp,including 168 open reading frames(ORFs)and three tRNAs,and no virulence or resistance gene was found.E.coli 287 was used as a host strain for the proliferation of phage LHE287 with an incubation time of 30 min and a burst size of 100 PFU/cell.Phage LHE287 had the highest titer of 1.72×10^(9) PFU/mL at a MOI of 0.0001.It remained stable up to 50℃and the range of pH 5-10,and showed a resistance to ultraviolet light.Conclusions:An Ackermannviridae phage,LHE287,was successfully isolated,showing considerable potential for further application.