1株多重耐药大肠杆菌噬菌体BP32的分离鉴定及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2024.08.031

摘要/Abstract

摘要： 【目的】拟分离1株可裂解多重耐药大肠杆菌的噬菌体,明确其生物学特性及基因组特征。【方法】本研究从病死山羊肺脏组织中分离大肠杆菌,并通过药敏试验分析其耐药性。以大肠杆菌ER2738菌株为宿主菌,从养殖场污水中进行噬菌体的初步增殖,通过点滴法确定分离噬菌体的宿主谱,采用双层琼脂平板法分离纯化特异性裂解大肠杆菌的噬菌体,进一步对其形态、最佳感染复数(MOI)、生长曲线、酸碱度和温度耐受性等生物学特性进行研究;通过高通量测序平台测定其全基因组序列,并对其基因组及遗传进化特征进行分析。【结果】从病死山羊肺脏中分离到1株多重耐药性大肠杆菌,命名为YNEC001。从养殖场污水中分离到1株可特异性裂解YNEC001菌株的噬菌体BP32,电镜观察显示噬菌体BP32属于肌尾科噬菌体,最佳MOI为0.1,潜伏期约为30 min,爆发期持续约60 min,裂解量约为660 PFU/cell,在30~50 ℃和pH 4.0~10.0条件下可保持稳定的活性。噬菌体BP32基因组长度为166 346 bp,GC含量偏低(35.42%),未发现耐药基因和毒力因子,基因组共包含261个开放阅读框(ORF),其中125个为已知功能基因,含有8个tRNA。在全基因组水平上,噬菌体BP32与肠杆菌噬菌体Escherichia phage BF15(MW822007)核苷酸序列相似性最高(98.25%),但基于末端酶大亚基序列的进化分析则表明BP32与大肠杆菌噬菌体Escherichia phage vB_EcoM_SP1(MT682709)的亲缘关系最近,而与Escherichia phage BF15(MW822007)的关系较远。【结论】本研究分离到1株多重耐药大肠杆菌YNEC001,并分离到可特异性裂解YNEC001菌株的噬菌体BP32,该噬菌体具有较强的裂解能力和良好的稳定性,具备开发为耐药性大肠杆菌治疗制剂的潜力。

关键词: 多重耐药大肠杆菌; 噬菌体; 生物学特征; 全基因组分析

Abstract: 【Objective】 The aim of this study was to isolate a phages that specifically lysed multidrug-resistant Escherichia coli and clarify the biological properties and genomic characteristics of the isolated phages furtherly.【Method】 In this study,Escherichia coli was isolated from the lung tissue of dead goats and its drug resistance was analyzed by drug sensitivity test.Using Escherichia coli ER2738 as host bacteria,the phage was initially propagated from farm sewage,and the host spectrum of the phage was determined by drop method.The phage of lytic Escherichia coli was isolated and purified by double agar plate method,and its morphology,optimal multiplicity of infection (MOI),growth curve,pH and temperature tolerance were further studied.Finally,the whole genome sequence was determined by high-throughput sequencing platform,and the characteristics of the genome and genetic evolution were analyzed.【Result】 A multidrug-resistant Escherichia coli strain named YNEC001 was isolated from the lung of dead goats.A phage (BP32) that could specifically lyse YNEC001 strain was isolated.Electron microscopic observation showed that phage BP32 belonged to Myoviridae,with the optimal MOI of 0.1,the incubation period was about 30 min,and the lysis period lasted about 60 min,and the average amount of lysis was about 660 PFU/cell.Phage BP32 could maintain stable activity at 30-50 ℃ and pH 4.0-10.0.The genome length of phage BP32 was 166 346 bp,the GC content was low (35.42%),no drug resistance genes and virulence factors were found,and the genome contained 261 open reading frame (ORF),of which 125 were known functional genes and contained 8 tRNA.At the genome-wide level,the nucleotide similarity between phage BP32 and enterobacter phage Escherichia BF15 (MW822007) was the highest (98.25%). Based on the phylogenetic analysis of the large subunit of the terminal enzyme,BP32 was most closely related to Escherichia vB_EcoM_SP1 (MT682709),but more distant to Escherichia BF15 (MW822007).【Conclusion】 In this study,a strain of multidrug-resistant Escherichia coli YNEC001 was isolated,and phage BP32,which could specifically lyse the YNEC001 strain,was isolated.This phage had strong lytic ability and good stability,and had the potential to be developed as a therapeutic agent for multidrug-resistant Escherichia coli.

Key words: multidrug-resistant Escherichia coli; phage; biological characteristics; whole genome analysis

中图分类号:

S852.61

吴静楠, 李占鸿, 李富祥, 张振兴, 张以芳, 宋建领. 1株多重耐药大肠杆菌噬菌体BP32的分离鉴定及生物学特性分析[J]. 中国畜牧兽医, 2024, 51(8): 3540-3551.

WU Jingnan, LI Zhanhong, LI Fuxiang, ZHANG Zhenxing, ZHANG Yifang, SONG Jianling. Isolation,Identification and Biological Characterization Analysis of Phage BP32 from Multidrug-Resistant Escherichia coli[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3540-3551.

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