禽致病性大肠杆菌噬菌体vB_EcoS_AH50的分离鉴定、生物学特性及全基因组分析  

Isolation, identification,biological characteristics and whole-genome analysis of avian pathogenic Escherichia coli bacteriophage vB_EcoS_AH50

作　　者：姚澜 张贝贝 王芷洋 郭伟奇 王帝 王欣宇 胡剑刚 祁晶晶[2] 田明星 李海花 王少辉 YAO Lan;ZHANG Bei-bei;WANG Zhi-yang;GUO Wei-qi;WANG Di;WANG Xin-yu;HU Jian-gang;QI Jing-jing;TIAN Ming-xing;LI Hai-hua;WANG Shao-hui(Tianjin Key Laboratory of A gricultural Animal Breeding and Healthy Husbandry,College of A nimal Science and Veterinary Medicine,Tianjin A gricultural University,Tianjin 300384,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)

机构地区：[1]天津农学院动物科学与动物医学学院天津市农业动物繁育与健康养殖重点实验室,天津300384 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2022年第12期1568-1577,共10页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31972654,32172856);上海市自然科学基金项目(22ZR1476100)。

摘　　要：为给噬菌体防控禽大肠杆菌病提供参考和依据,以禽致病性大肠杆菌为宿主菌,利用双层琼脂平板法从鸡场粪便样品中分离纯化禽致病性大肠杆菌噬菌体,对其形态、裂解谱、最佳感染复数(MOI)、一步生长曲线、p H和温度稳定性、体外抑菌能力及抑制生物被膜形成能力进行测定,并通过全基因组测序分析其基因组特征。结果显示,分离到1株裂解性禽致病性大肠杆菌噬菌体v B_EcoS_AH50,透射电镜观察显示其属于有尾噬菌体目长尾噬菌体科。v B_EcoS_AH50可特异性裂解O1和O2血清型大肠杆菌;MOI为1;潜伏期和爆发期均为20 min,爆发量约为76 PFU/cell;在4~50℃和pH值在3~11的范围内可保持较强的裂解能力,且在室温下能稳定存活4周。在MOI分别为0.01、0.1和1的条件下,该噬菌体能够有效抑制大肠杆菌生长及形成生物被膜。全基因组测序发现,v B_EcoS_AH50基因组全长为37 268 bp,不含有与毒力因子、毒素、耐药和溶源性相关基因。BLASTn比对显示,该噬菌体全基因组序列与噬菌体PC2最为相近,具有94.56%的一致性和94%的覆盖率。基于噬菌体末端酶大亚基构建的系统发育树显示,v B_EcoS_AH50与噬菌体v B_EcoS_011D5的亲缘关系最近。结果表明,噬菌体v B_EcoS_AH50可特异性裂解大肠杆菌,裂解能力强、耐受性高、安全性好,提示其在防治禽大肠杆菌病方面均有潜在应用价值。In order to provide reference and basis for bacteriophages prevention and controlling avian colibacillosis, avian pathogenic Escherichia coli(APEC) phages were isolated and purified from chicken farm feces samples by double-agar plate method using APEC as the host bacteria.The morphology,host range,optimal multiplicity of infection(MOI),one-step growth curve,pH and temperature stability,in vitro antibacterial ability and biofilm formation inhibition ability of phages were determined,and genomic characteristics were analyzed by whole-genome sequencing.The results showed that a lytic APEC phage v B_Eco S_AH50 was successfully isolated.Transmission electron microscopy observation showed that v B_Eco S_AH50 belonged to the family Siphoviridae and the order Candovirales.v B_Eco S_AH50specifically lysed E.coli of O1 and O2 serotypes.The optimal MOI of v B_Eco S_ AH50 was 1,the latent period and the burst period were both 20 min,and the burst size was about 76 plaque-forming unit(PFU)/cell.v B_Eco S_AH50 can maintain strong lysis ability in the range of 4℃ to 50℃ and pH 3 to 11,and can survive stably for 4 weeks at room temperature.At MOIs of 0.01,0.1 and 1,the phage could efficiently inhibit bacterial planktonic cells growth and biofilm formation.Whole-genome sequencing revealed that the v B_Eco S_AH50 genome was 37 268 bp in length,which did not contain virulence genes,toxin genes,drug resistance genes and integrase genes.Phylogenetic analysis showed that the whole genome sequence of this phage was the homologous to that of phage PC2,with 94.56% percent identity and 94% query coverage.A phylogenetic tree based on the terminase large subunit showed that phage v B_Eco S_AH50 was most closely related to phage v B_Eco S_011D5.These results showed that v B_Eco S_AH50 could specifically lyse E.coli,with strong lysis ability,high tolerance and good safety,suggesting that it has potential application value in the prevention and treatment of avian colibacillosis.

关 键 词：禽致病性大肠杆菌 噬菌体 生物学特性 全基因组分析 

分 类 号：S852.69[农业科学—基础兽医学]