大肠杆菌K88噬菌体的分离鉴定及其生物学特性

Isolation and Characterization of a Lytic Bacteriophage of Enterotoxigenic Escherichia coli K88

分离鉴定裂解性产肠毒素性大肠杆菌(ETEC)K88噬菌体,并研究其生物学特性及裂菌效力。用双层平板法从猪场污水中分离噬菌体,通过透射电镜和酶切基因组对获得的噬菌体进行鉴定;同时测定了噬菌体最佳感染复数、一步生长曲线、酸碱稳定性、热稳定性等噬菌体的生物学特性及其体外裂菌效力。结果表明分离获得了一株产肠毒素性大肠杆菌K88强裂解性噬菌体,命名为PK88-4;其噬菌斑清晰透亮,周围无晕环;电镜显示:其头部呈正多面体对称,有可伸缩性尾部;核酸类型为双链DNA(基因组大小约60 kb);该噬菌体能耐受60℃左右高温、在pH值为5～10内效价稳定;最佳感染复数为0.01;潜伏期为10 min,暴发期为40 min,裂解量为40;在5 h内对培养液中的大肠杆菌杀灭率将近100%。PK88-4是一株强裂解性肌尾科大肠杆菌K88噬菌体,具有裂解周期短、杀菌能力强等特点,为畜牧业防治产肠毒素性大肠杆菌K88感染提供了新的思路。

To isolate and characterize the lytic bacteriophage that might be used in prevention and treatment of porcine postweaning diarrhea due to enterotoxigenic E. coli K88 （ETEC-K88）. E. coli expressing the K88 adhesin on their surface is a common cause of diarrhea in newborn and weaned piglets. Mixtures of 3 strains of ETEC K88 were used as hosts for isolation of phages in sewage from 22 pig farms. The isolated phage was characterized at the microbiological and molecular levels. The optimal multiplicity of infection （MOI） , one-step growth curve, Thermo- and pH stability,lytic effective in vitro of the isolated bacteriophage were investigated. One phages that lysed ETEC K88 was isolated by double-layer agar plate method and named PK88-4. The phage produced large, clear plaques. The electron microscope showed the phage had necks and contractile tails and therefore belonged to the Myoviridae. The estimated genome size was about 60 kb and the restriction enzyme fragments suggested that it was dsDNA. The optimal MOI was 0. 01. The phage was stable at pH 5 - 10 and under 60℃ , and could kill 100% host strain within 2 hours with MOI higher than 0. 001. The new isolated phage, named PK88-4, are potential candidates for prophy- laxis and therapy of porcine post-weaning diarrhea due to ETEC K88.