1株肠炎沙门菌噬菌体GD01的分离鉴定及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2023.12.037

摘要/Abstract

摘要： 【目的】分析从污水中分离纯化出的1株肠炎沙门菌噬菌体的生物学特性及基因组信息。【方法】以肠炎沙门菌为宿主,从华南农业大学湖水和广州某肉鸡养殖场污水混合样本中分离纯化1株肠炎沙门菌噬菌体,通过电镜观察噬菌体形态,检测噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性和pH稳定性,对该噬菌体的基因组进行高通量测序以及生物信息学分析,并探究该噬菌体在不同温度污染乳中对沙门菌的抑菌能力。【结果】成功分离到1株肠炎沙门菌噬菌体,并命名为GD01,其在双层琼脂平板上能形成圆形、透亮、边缘清晰的噬菌斑,电镜观察显示GD01头部呈正二十面体对称,直径为62 nm,尾部长度为224.4 nm,属于有尾噬菌体目、长尾噬菌体科。噬菌体GD01宿主谱较窄,酸碱耐受力和温度耐受力较强,最适pH为8.0,最适温度为37 ℃,最佳感染复数为0.01;一步生长曲线结果显示,GD01的潜伏期为10 min,裂解期为110 min,平均裂解量为50 PFU/cell。全基因组测序分析结果显示,GD01基因组全长为60 109 bp,GC含量为56.22％,为双链DNA,含有50个开放阅读框(ORF),不存在毒力基因、耐药基因和tRNA基因。体外抑菌试验结果显示,37 ℃作用8 h、4 ℃作用72 h后,噬菌体GD01对沙门菌仍具有良好的抑菌效果。【结论】本研究分离出的肠炎沙门菌噬菌体GD01酸碱耐受力和温度耐受力强,生物信息学特性清晰,且体外抑菌效果良好,具有替代抗生素的潜力和应用前景。

关键词: 沙门菌; 噬菌体; 生物学特性; 全基因组测序

Abstract: 【Objective】 The objective of this study was to analyze the biological characteristics and genomic information of a phage of Salmonella Enteritidis isolated and purified from sewage.【Method】 Using Salmonella Enteritidis as host,a phage of Salmonella Enteritidis was isolated and purified from mixed samples of lake water from South China Agricultural University and sewage from a broiler farm in Guangzhou.The host spectrum,optimal multiplicity of infection,one-step growth curve,thermal stability and pH stability of phage were detected.The genome of the bacteriophage was sequenced and bioinformatics analyzed,and the bacteriostatic ability of the bacteriophage against Salmonella in milk contaminated at different temperatures was investigated.【Result】 A phage of Salmonella Enteritidis was successfully isolated and named GD01,which could form round,transparent and clearly edged plaque on a double-layer agar plate.Under electron microscopy,the head of GD01 was icosahedral symmetry,with a diameter of 62 nm and a tail length of 224.4 nm,belonged to the Caudovirales,Siphoviridae. The host spectrum was narrow,and the optimum pH was 8.0,the optimum temperature was 37 ℃,the optimal multiplicity of infection was 0.01.The one-step growth curve showed that the latency of GD01 was 10 min,the lysis period was 110 min,and the average amount of cleavage was 50 PFU/cell.Whole genome sequencing analysis results showed that the total length of GD01 genome was 60 109 bp,the GC content was 56.22%,it was double-stranded DNA,contained 50 open reading frame (ORF),and did not contain virulence genes,drug resistance genes and tRNA genes.The results of in vitro bacteriostatic test showed that phage GD01 still had good bacteriostatic effect on Salmonella after treatment at 37 ℃ for 8 h and at 4 ℃ for 72 h.【Conclusion】 The isolated bacteriophage GD01 of Salmonella Enteritidis had strong acid-base tolerance and temperature tolerance,clear bioinformatics properties,and good antibacterial effect in vitro,which had the potential and application prospect of replacing antibiotics.

Key words: Salmonella; phage; biological characteristics; whole genome sequencing

中图分类号:

S852.61

高铎, 王晋宇, 李河谕, 蒋祺丰, 陈子涵, 徐禧莹, 贾坤. 1株肠炎沙门菌噬菌体GD01的分离鉴定及生物学特性分析[J]. 中国畜牧兽医, 2023, 50(12): 5160-5169.

GAO Duo, WANG Jinyu, LI Heyu, JIANG Qifeng, CHEN Zihan, XU Xiying, JIA Kun. Isolation,Identification and Biological Characterization Analysis of a Bacteriophage GD01 from Salmonella Enteritidis[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 5160-5169.

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