1株禽大肠杆菌噬菌体的分离及杀菌效果评估

doi:10.16431/j.cnki.1671-7236.2022.02.041

摘要/Abstract

摘要： 【目的】探索噬菌体作为控制养殖环境致病性大肠杆菌的新手段。【方法】本研究进行了大肠杆菌噬菌体的分离及相应指标评估。通过双层平板法从养殖环境中分离禽致病性大肠杆菌裂解性噬菌体。通过电镜及酶切鉴定、温度及酸碱稳定性、最佳感染复数、一步生长曲线及其在模拟环境中的杀菌效果对该噬菌体进行综合性评估。【结果】分离得到的噬菌体具有正多面体的头部和细而长的尾部结构, 头部直径约98 nm, 尾部长约123 nm, 结合酶切鉴定结果初步判定该噬菌体为肌尾科双链DNA噬菌体。该噬菌体的温度耐受范围为37~50 ℃; 酸碱耐受范围为pH 3.0~11.0。当感染复数为0.00001时产生的子代噬菌体滴度最高; 一步生长曲线测定结果显示, 该噬菌体潜伏期为20~40 min, 裂解时间为80~100 min, 裂解量为4 133 PFU/cell。从该噬菌体对模拟环境中宿主大肠杆菌的杀菌效果可看出, 浓度为1×10⁴~1×10⁶ PFU/mL的噬菌体ФECP2-1对液体中目标大肠杆菌作用1~6 h, 杀菌率均在99.9%以上; 浓度为2×10⁴~2×10⁶ PFU/g的噬菌体ФECP2-1对鸡粪中目标大肠杆菌作用5~10 h, 杀菌率均在99%以上, 该噬菌体对鸡粪中目标大肠杆菌杀菌效果略低于对液体中目标大肠杆菌的杀菌效果。【结论】 ФECP2-1符合噬菌体类消毒剂相关特征, 是一株具有良好应用前景的噬菌体, 可作为一种生物消毒剂应用于养殖环境中禽大肠杆菌的防控。

关键词: 禽大肠杆菌; 裂解性噬菌体; 生物学特性; 杀菌效果

Abstract: 【Objective】 The study was aimed to explore a new way to control environmental pathogenic Escherichia coli (E.coli).【Method】 The isolation and corresponding index evaluation of Escherichia coli phages were carried out in this study.The lytic phages of avian pathogenic E.coli were isolated from breeding environment by double-layer plate method.The phage was comprehensively evaluated through electron microscopy and enzyme digestion identification, stability, optimal multiplicity of infection, one-step growth curve and its bactericidal effect in simulated environment.【Result】 The isolated phage had a regular polyhedron head and a slender tail structure, with a head diameter of about 98 nm and a tail length of about 123 nm.Combined with the results of enzyme digestion, it was preliminarily identified as a double-stranded DNA phage of Cauda muscularis.Its temperature tolerance range was 37-50 ℃, pH tolerance range was 3.0-11.0.When the multiplicity of infection was 0.00001, the titer of progeny phage was the highest.One-step growth curve showed that the incubation period of the phage was about 20-40 min, the lysis time was about 80-100 min, and the lysis amount was 4 133 PFU/cell.It could be seen from the bactericidal effect of the phage on the host E.coli in the simulated environment that the bactericidal rate of phage ФECP2-1 with the concentration of 1×10⁴-1×10⁶ PFU/mL against the target E.coli in the liquid for 1-6 h was above 99.9%.The phage ФECP2-1 with the concentration of 2×10⁴-2×10⁶ PFU/g could kill more than 99% of the target E.coli in chicken manure for 5-10 h.The phage ФECP2-1 had a slightly lower bactericidal effect on the target E.coli in chicken manure than on the target E.coli in liquid.【Conclusion】 ФECP2-1 conformed to the characteristics of phage disinfectants, and was a phage with good application prospect.It could be used as a biological disinfectant for the prevention and control of avian E.coli in aquaculture environment.

Key words: avian Escherichia coli; pyrolytic phages; biological characteristics; sterilization effect

中图分类号:

S852.65

李金敏, 高绪娜, 桑瑞新, 宫本芝, 徐海燕, 谷巍, 郝木强, 兰江华, 王红. 1株禽大肠杆菌噬菌体的分离及杀菌效果评估[J]. 中国畜牧兽医, 2022, 49(2): 783-190.

LI Jinmin, GAO Xuna, SANG Ruixin, GONG Benzhi, XU Haiyan, GU Wei, HAO Muqiang, LAN Jianghua, WANG Hong. Isolation and Evaluation of Sterilization Effect of an Avian Escherichia coli Phage[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 783-190.

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