1株大肠杆菌噬菌体EP_H11的分离鉴定及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2023.04.039

摘要/Abstract

摘要： 【目的】拟分离1株对大肠杆菌具有良好抑菌效果且适应性广泛的噬菌体,为开发新的大肠杆菌防治方法提供基础。【方法】以大肠杆菌H11为宿主菌,采用双层琼脂平板法从污水样品中分离纯化1株大肠杆菌噬菌体,电镜下观察噬菌体的形态,测定噬菌体的效价、宿主谱、最佳感染复数、一步生长曲线、pH稳定性与温度敏感性,对噬菌体的全基因组进行分析和序列比对,并探究该噬菌体在液体培养基中对H11的抑制效果。【结果】成功分离到1株裂解性大肠杆菌噬菌体,命名为EP_H11(登录号:OP688485)。双层琼脂平板法结果显示,该噬菌体能够形成大小一致、中间有透明亮点、周围有光晕的噬斑,噬菌体裂解效价可达10⁹~10¹⁰ PFU/mL。电镜观察显示噬菌体EP_H11头部呈规则的多面体状,直径约85 nm,尾部直径约23 nm,长度约109 nm。以大肠杆菌H11为宿主菌测得EP_H11的最佳感染复数是0.1,一步生长曲线结果显示其潜伏期为40 min,爆发期为80 min,裂解量为200 PFU/cell。该噬菌体在pH 4.0~10.0的环境中至少可以存活2 h以上,并能够在10~60 ℃下保持稳定90 min。结合透射电镜观察及全基因组分析结果显示,噬菌体EP_H11属于有尾病毒目、长尾噬菌体科、肌尾噬菌体属。噬菌体基因组总片段长度为111 373 bp,GC含量为44.49%。噬菌体线性对比结果显示,大肠杆菌噬菌体EP_H11的基因组与噬菌体Escherichia phage vB_EcoM_IME392同源性最高。系统发育进化树分析结果表明,噬菌体EP_H11与肌尾属噬菌体Escherichia phage vB_EcoM_IME392位于同一分支,均属于有尾噬菌体目、长尾噬菌体科。经BLAST比对分析显示,噬菌体EP_H11基因组中共有153个CDSs,其中28个CDSs编码功能蛋白。体外抑菌结果显示,8 h内,EP_H11在MOI为10时表现出良好的抑菌效果。【结论】本研究分离到1株裂解性极强,适应性范围较广,体外抑菌效果良好的噬菌体。该噬菌体具有进一步开发成为大肠杆菌抗菌剂的潜力。

关键词: 大肠杆菌; 噬菌体; 全基因组分析

Abstract: 【Objective】 It was planned to isolate a strain of phages with good bacteriostatic effect and wide adaptability for Escherichia coli,which provided a basis for the development of new Escherichia coli control methods.【Method】 Taking Escherichia coli H11 as the host bacteria,a strain of Escherichia coli phage was isolated and purified from sewage samples by double-layer agar plate method.The morphology of the phage was observed under electron microscope,and the titer,host spectrum,optimal multiplicity of infection(MOI),one-step growth curve,pH stability and temperature sensitivity of the phage were determined.The whole phage genome was analyzed and sequenced,and the inhibitory effect of the phage on H11 in liquid medium was explored.【Result】 A lytic Escherichia coli phage was successfully isolated and named EP_H11(accession number:OP688485).The results of double-layer agar culture method showed that the phage EP_H11 could form plaques with uniform size,transparent bright spots in the middle and halo around,and the phage cleavage titer could reach 10⁹-10¹⁰ PFU/mL.Electron microscopy showed that the phage EP_H11 had a regular polyhedral head with a diameter of about 85 nm,a tail with a diameter of about 23 nm and a length of about 109 nm.Using Escherichia coli H11 as host bacteria,the optimal MOI of phage EP_H11 was 0.1.The one-step growth curve showed that the incubation period was 40 min and the outbreak period was 80 min.The amount of cleavage was about 200 PFU/cell.The phage could survive for at least 2 h in the environment of pH 4.0-10.0,and could remain stable for 90 min at 10-60 ℃.Combined with transmission electron microscopy and whole genome analysis,the phage EP_H11 belonged to the Caudovirales,Siphoviridae family,Myoviridae genus.The total length of the phage genome was 111 373 bp and the GC content was 44.49%.According to the results of phage linear comparison,Escherichia coli phage EP_H11 and phage Escherichia phage vB_EcoM_IME392 had the highest homology.Phylogenetic tree analysis showed that the phage EP_H11 and Escherichia phage vB_EcoM_IME392 (Myoviridae genus) were located in the same branch,belonging to the order of Caudovirales and the Siphoviridae family.BLAST sequence analysis showed that there were 153 CDSs in genome of EP_H11,28 of which were predicted to encode functional proteins.In vitro bacteriostasis results showed that within 8 h,EP_H11 showed good bacteriostatic effect when MOI was 10. 【Conclusion】 In this study,a bacteriophage with strong lytic ability,wide adaptability and good bacteriostatic effect in vitro was isolated.The bacteriophage had the potential to be further developed as an antimicrobial agent for Escherichia coli.

Key words: Escherichia coli; phage; whole genome analysis

中图分类号:

S852.61

黄巧辰, 廖彬如, 余春燕, 莫玉鹏, 黄炜乾, 李磊, 韩凯欧, 王乐平, 李珣, 王晓晔. 1株大肠杆菌噬菌体EP_H11的分离鉴定及生物学特性分析[J]. 中国畜牧兽医, 2023, 50(4): 1684-1694.

HUANG Qiaochen, LIAO Binru, YU Chunyan, MO Yupeng, HUANG Weiqian, LI Lei, HAN Kaiou, WANG Leping, LI Xun, WANG Xiaoye. Isolation,Identification and Biological Characteristics Analysis of a Bacteriophage EP_H11 from Escherichia coli[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1684-1694.

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