1株棕熊源多重耐药大肠杆菌噬菌体生物学特性及比较基因组学研究

doi:10.16431/j.cnki.1671-7236.2024.10.031

中国畜牧兽医 ›› 2024, Vol. 51 ›› Issue (10): 4510-4521.doi: 10.16431/j.cnki.1671-7236.2024.10.031

• 预防兽医 • 上一篇

1株棕熊源多重耐药大肠杆菌噬菌体生物学特性及比较基因组学研究

郭志良^1,3, 朱秋艳², 王雪³, 曾君³, 季芳³, 郑迎春⁴, 左君豪^1,3, 张乐怡³, 郭常春⁵, 王学静⁶, 徐莉莉⁷, 赵瑞利¹, 王承民³

1. 天津农学院动物科学与动物医学学院, 天津市农业动物繁育与健康养殖重点实验室, 天津 300392;
2. 乐亭县农业农村局, 乐亭 063600;
3. 广东省科学院动物研究所, 广东省动物保护与资源利用重点实验室, 广州 510260;
4. 河北省承德县农业农村局, 承德 067400;
5. 河北滦牧农业开发股份有限公司, 滦州 063700;
6. 河北省畜牧兽医研究所, 保定 071000;
7. 优宜邦生物科技(上海)有限公司, 上海 201199

收稿日期:2024-02-03 发布日期:2024-09-30
通讯作者: 赵瑞利, 王承民 E-mail:Zhaoruili1109@126.com;wangchm@giz.gd.cn
作者简介:郭志良，E-mail:jygzl252727@163.com;朱秋艳，E-mail:ltdj123@126.com。
基金资助:
优宜邦生物科技(上海)有限公司产业化项目(UYB20220001-01、UYB20230001-02)

Biological Characteristics and Comparative Genomics Analysis of a Phage of Multi-drug Resistant Escherichia coli from Brown Bear

GUO Zhiliang^1,3, ZHU Qiuyan², WANG Xue³, ZENGJun³, JI Fang³, ZHENG Yingchun⁴, ZUO Junhao^1,3, ZHANG Leyi³, GUO Changchun⁵, WANG Xuejing⁶, XU Lili⁷, ZHAO Ruili¹, WANG Chengmin³

1. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China;
2. Agricultural and Rural Bureau of Laoting County, Laoting 063600, China;
3. Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou 510260, China;
4. Agricultural and Rural Bureau of Chengde County, Chengde 067400, China;
5. Hebei Luanmu Agricultural Development Co., Ltd., Luanzhou 063700, China;
6. Hebei Institute of Animal Husbandry and Veterinary Medicine, Baoding 071000, China;
7. Union Biotechnology (Shanghai) Co., Ltd., Shanghai 201199, China

Received:2024-02-03 Published:2024-09-30

摘要/Abstract

摘要： 【目的】探究1株多重耐药大肠杆菌裂解性噬菌体的生物学特性及基因组特征，为多重耐药菌株的科学防控提供参考。【方法】试验以棕熊源大肠杆菌M719-6WT为宿主菌，使用双层平板法从养殖场污水中分离纯化出噬菌体，通过透射电镜观察其形态，利用空斑法测定噬菌体宿主谱，并探究其生物学特性。基于噬菌体全基因组测序数据进行噬菌体生物信息学分析，采用体外、体内抑菌试验评估噬菌体抑菌效果。【结果】通过双层平板法分离并纯化得到1株大肠杆菌噬菌体，命名为pEC-M719-6WT.2，其噬菌斑边缘清晰且透亮。结合透射电镜观察和全基因组分析结果显示，噬菌体pEC-M719-6WT.2属于Tevenvirinae亚科肌尾状噬菌体。宿主谱测定结果表明，噬菌体pEC-M719-6WT.2可裂解11株大肠杆菌。该噬菌体最佳感染复数(MOI)为0.1，最适温度为25 ℃，最适pH为7.0，潜伏期＜10 min，裂解周期为70 min，裂解量约为190 PFU/cell。基因组分析结果显示，噬菌体pEC-M719-6WT.2基因组大小为170.441 kb，未发现携带已知的耐药、溶原和毒力相关基因。受体结合试验结果显示，噬菌体pEC-M719-6WT.2可以结合宿主菌表面的脂多糖受体。体外抑菌试验结果显示，当MOI为100时，噬菌体pEC-M719-6WT.2与8 μg/mL四环素联合应用，抑菌时间可延长至24 h；体内保护试验结果表明，2.8×10⁵ CFU/mL M719-6WT感染大蜡螟幼虫后，注射2×10⁷ PFU/mL噬菌体pEC-M719-6WT.2可在24和48 h内将大蜡螟幼虫存活率分别提高至100%和70%。【结论】本研究分离获得1株多重耐药大肠杆菌噬菌体，其具有良好的环境稳定性和抑菌活性，生物信息学特征明确，体内外抑菌效果较好，与抗生素联合使用可产生协同作用，具有治疗临床耐药大肠杆菌感染的潜在应用价值。

关键词: 噬菌体; 大肠杆菌; 多重耐药; 生物学特性; 比较基因组学

Abstract: 【Objective】 The aim of this study was to explore the biological and genomic characteristics of a lytic phage of multi-drug resistant Escherichia coli，so as to provide reference for the scientific prevention and control of multi-drug resistant strains. 【Method】 The phage was isolated and purified from farm sewage by double-plate method with Escherichia coli M719-6WT as host bacteria from Brown bear. The morphology of phage was observed by transmission electron microscopy. The host spectrum of phage was determined by plaque method and its biological characteristics were investigated. Phage bioinformatics analysis was conducted based on phage whole genome sequencing data，and bacteriostatic effects of phage were evaluated by in vitro and in vivo bacteriostatic tests.【Result】 Through double-plate method，a phage specific to Escherichia coli was isolated and purified，named pEC-M719-6WT.2，with clear and transparent plaques. Transmission electron microscopy and whole-genome analysis revealed that phage pEC-M719-6WT.2 belonged to the subfamily Tevenvirinae of the Myoviridae family. Host range determination showed pEC-M719-6WT.2 could lyse 11 strains of Escherichia coli. The optimum multiplicity of infection (MOI) was 0.1，the optimum temperature was 25 ℃，the optimum pH was 7.0，the incubation period was less than 10 min，the lysis period was 70 min，and the cleavage volume was about 190 PFU/cell. Genomic analysis showed that phage pEC-M719-6WT.2 had a genome size of 170.441 kb and no known genes related to drug resistance，lysogen or virulence were found. The results of receptor binding tests showed that phage pEC-M719-6WT.2 could bind to lipopolysaccharide receptors on the surface of host bacteria. In vitro bacteriostatic results showed，when MOI was 100，phage pEC-M719-6WT.2 combined with 8 μg/mL tetracycline could prolong the bacteriostasis time to 24 h. The results of in vivo protection test showed that after 2.8×10⁵ CFU/mL M719-6WT was infected with Galleria mellonella larvae，the survival rate of the larvae was increased to 100% and 70% within 24 and 48 h after injection of 2×10⁷ PFU/mL phage pEC-M719-6WT.2.【Conclusion】 A phage against multi-drug resistant Escherichia coli was isolated. The phage had good environmental stability and antibacterial activity，clear bioinformatics characteristics，and good antibacterial effect in vivo and in vitro. It could produce synergistic effect when combined with antibiotics，and had potential application value in the treatment of clinical drug-resistant Escherichia coli infection.

Key words: phage; Escherichia coli; multi-drug resistance; biological characteristics; comparative genomics

中图分类号:

S852.61

郭志良, 朱秋艳, 王雪, 曾君, 季芳, 郑迎春, 左君豪, 张乐怡, 郭常春, 王学静, 徐莉莉, 赵瑞利, 王承民. 1株棕熊源多重耐药大肠杆菌噬菌体生物学特性及比较基因组学研究[J]. 中国畜牧兽医, 2024, 51(10): 4510-4521.

GUO Zhiliang, ZHU Qiuyan, WANG Xue, ZENGJun, JI Fang, ZHENG Yingchun, ZUO Junhao, ZHANG Leyi, GUO Changchun, WANG Xuejing, XU Lili, ZHAO Ruili, WANG Chengmin. Biological Characteristics and Comparative Genomics Analysis of a Phage of Multi-drug Resistant Escherichia coli from Brown Bear[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4510-4521.

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1株棕熊源多重耐药大肠杆菌噬菌体生物学特性及比较基因组学研究

Biological Characteristics and Comparative Genomics Analysis of a Phage of Multi-drug Resistant Escherichia coli from Brown Bear

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