一株新型广谱肠侵袭性大肠杆菌噬菌体ΦEP1生物学特性及基因组分析

Biological and genome characteristics of a novel broad host-range phage ΦEP1 infecting enteroinvasive Escherichia coli

摘要:

为了给肠侵袭性大肠杆菌(enteroinvasive Escherichia coli, EIEC)的预防与治疗提供资源和参考，对EIEC噬菌体进行生物学特性和基因组分析。以实验室冻存的EIEC为宿主菌，从浙江湖州某养鸡场的环境污水样品中分离得到一株噬菌体，命名为ΦEP1。采用双层琼脂平板法测定噬菌体的效价以及最佳感染复数、一步生长曲线、温度、pH值、氯仿和胆盐敏感性等生物学特性，对噬菌体进行透射电镜检查以观察其形态，测试其在不同食品基质中的生物防治效果以及对Caco-2细胞的保护作用。结果表明，ΦEP1的最佳感染复数为0.1，效价为1.3×10¹⁰ PFU/mL。噬菌体对温度、pH、氯仿以及胆盐的耐受性均较强，具有较广的裂解谱，对多株不同血清型的多重耐药致病大肠杆菌和志贺氏菌均表现出裂解活性。噬菌体的潜伏期为10 min，爆发期为80 min，爆发量为48 PFU/cell。透射电镜观察显示噬菌体ΦEP1属于尾病毒目，同时对Caco-2细胞有良好的保护作用。噬菌体ΦEP1的基因组大小为87 182 bp，GC含量为39.80%，含有128个推定的开放阅读框(open reading frame, ORF)，不含耐药基因和毒力因子。ΦEP1能显著抑制人工污染牛奶和牛肉中EIEC的增长，在细胞保护性实验中能有效消杀EIEC，显著提高Caco-2细胞的存活率，减少细胞因子白细胞介素6 (interleukin-6, IL-6)和白细胞介素1β (interleukin-1β, IL-1β)的表达，使得炎症水平下调。本研究所得的肠侵袭性大肠杆菌噬菌体效价较高，对环境的耐受性较强，为噬菌体在食品保鲜等领域应用提供依据。

关键词:大肠杆菌;噬菌体;生物学特性;基因组分析;Caco-2细胞;细胞因子

Abstract:

We analyzed the biological and genome characteristics of a phage infecting enteroinvasive Escherichia coli (EIEC), aiming to provide resources and a reference for the prevention and treatment of EIEC. With the EIEC preserved in our laboratory as the host bacterium, one strain of phage was isolated from the effluent sample from a chicken farm in Huzhou, Zhejiang and named ΦEP1. The titer, optimal multiplicity of infection, one-step growth curve, temperature, pH value, chloroform and bile salt sensitivity of ΦEP1 were determined by the double-layer agar plate method. The morphology of the phage was observed by transmission electron microscopy. The biocontrol effects of ΦEP1 in different food matrixes and the protective effect of this phage on Caco-2 cells were tested. The phage ΦEP1 showed the optimal multiplicity of infection of 0.1, the titer of 1.3×10¹⁰ PFU/mL, strong tolerance to temperature, pH, chloroform, and bile salt, and a broad host spectrum. Furthermore, it expressed lysis activity against multiple strains of multiple antibiotic-resistant pathogenic E. coli and Shigella with different serotypes. Phage ΦEP1 had an incubation period of 10 min, an outbreak period of 80 min, and an outbreak volume of 48 PFU/cell. According to the morphology observed by transmission electron microscopy, phage ΦEP1 belonged to the order of Caudovirales, and it had a good protective effect on Caco-2 cells. Phage ΦEP1 had a genome of 87 182 bp with the GC content of 39.80%, 128 putative open reading frames, and no antibiotic resistance genes or virulence genes. ΦEP1 inhibited the growth of EIEC in artificially contaminated milk and beef and eliminated EIEC in cell protection experiments. It significantly increased the survival rate of Caco-2 cells and down-regulated the expression of interleukin (IL)-6 and IL-1β to reduce inflammation. We obtained an EIEC-targeting phage ΦEP1 with a high titer and strong tolerance to the environment, which provided a basis for the application of phages in food preservation and other fields.

Key words:Escherichia coli;phage;biological characteristics;genome analysis;Caco-2 cells;cytokines