肠出血性大肠埃希菌O157:H7噬菌体FEC14和FEC19特性及污染牛肉杀菌潜在应用探究

Characterization of enterohaemorrhagic Escherichia coli O157:H7 phages FEC14 and FEC19 and their potential use in contaminated beef

摘要:

【背景】肠出血性大肠埃希菌O157:H7是重要的食源性致病菌之一，并且其耐药性越来越严重，寻找裂解性强噬菌体用于防治大肠埃希菌感染具有广阔的应用前景。【目的】从环境中分离鉴定能特异裂解大肠埃希菌O157:H7的噬菌体，通过对生物学特性及裂解细菌功效的探究，为其在食品安全防控中提供理论依据和研究基础。【方法】通过双层平板法分离并纯化噬菌体，透射电镜观察噬菌体形态，测定最佳感染复数、一步生长曲线、pH稳定性、温度稳定性，对噬菌体进行全基因组测序及噬菌体裂解细菌功效。【结果】 2株大肠埃希菌O157:H7噬菌体FEC14和FEC19的头部皆呈二十面体立体对称，FEC14头部直径约80 nm，尾丝呈星形，FEC19头部直径约58 nm，尾丝呈针形；噬菌体FEC14的最佳感染复数为0.001，潜伏期为15 min，裂解期为65 min，平均暴发量为156 PFU/cell，FEC19的最佳感染复数为0.1，潜伏期为10 min，裂解期为80 min，平均暴发量为800 PFU/cell；噬菌体FEC14能在60℃、pH 4.0-11.0条件下存活，噬菌体FEC19在70℃、pH 5.0-9.0条件下存活； FEC14归类于有尾目(Caudovirales) Ackermannviridae科Kuttervirus属，FEC19归类于有尾目(Caudovirales)肌尾科(Myoviridae) Wifcevirus属；单一噬菌体或混合鸡尾酒法均能降低染菌牛肉中的细菌数量，并且未发现已知毒力基因和耐药基因，能控制大肠埃希菌O157:H7的污染。【结论】 2株大肠杆菌O157:H7噬菌体FEC14和FEC19特异性高、稳定性强、裂解效率高，在控制食品中大肠埃希菌O157:H7污染的应用中具有很大潜力，将来可以开发为食品防控制剂用于食品加工各个领域。

关键词:大肠埃希菌O157;噬菌体;生物防控;鸡尾酒法

Abstract:

[Background] Escherichia coli O157:H7 is one of the most important foodborne pathogens. The accelerated drug resistance of E. coli O157:H7 invokes the selection of phages with strong lytic properties to achieve the purpose of disease prevention and treatment with broad application prospects. [Objective] To isolate and identify lytic phages against E. coli O157:H7 and analyze their biological characteristics and bacteria-killing efficiency, which may provide insights into the research and application of phages. [Methods] Phages were isolated and purified using double-layer plate method, and their morphology was observed by transmission electron microscope (TEM). The optimal multiplicity of infection (MOI), one-step growth curve, pH and temperature stability were determined, and the whole genome of phages was sequenced. Lysis efficiency was determined by a single phage or cocktail method in contaminated beef. [Results] Two strains of lytic E. coli O157:H7 phages FEC14 and FEC19 were isolated and purified. According to TEM results, the heads of the two phages were both icosahedral, and FEC14 presented a head diameter of about 80 nm and star-shaped tail filaments, while FEC19 had a head diameter of about 58 nm and needle-shaped tail filaments. Moreover, FEC14 showed an optimal MOI of 0.001, the latent period of 15 min and the lysis period of 65 min with an average burst size of 156 PFU/cell, and FEC19 displayed an optimal MOI of 0.1, the latent period of 10 min and the lysis period of 80 min with an average burst size of 800 PFU/cell. FEC14 stabilized at 60 ℃ and pH 4.0–11.0, while FEC19 survived at 70 ℃ and pH 5.0–9.0. FEC14 belongs to the genus Kuttervirus, family Ackermannviridae, order Caudovirales, and FEC19 falls into the genus Wifcevirus, family Myoviridae, and order Caudovirales. The single phage or phage cocktail effectively inhibited the growth of host bacteria in the contaminated beef, and the two phages had no known virulence genes and drug resistance genes, showing great potential efficacy in the control of E. coli O157:H7 contamination. [Conclusion] Two strains of lytic E. coli O157:H7 phages FEC14 and FEC19 with high specificity, stability and lysis efficiency reveal their potential to be an alternative antimicrobial agent for food safety in the future.

Key words:Escherichia coli O157;bacteriophage;biological control;cocktail method