1株大肠杆菌噬菌体vB_EcoP_GN07的分离鉴定及全基因组分析

doi:10.16431/j.cnki.1671-7236.2023.04.024

摘要/Abstract

摘要： 【目的】探究大肠杆菌噬菌体vB_EcoP_GN07的生物学特性和全基因组特征,为开发噬菌体"鸡尾酒"制剂防治致病性大肠杆菌病提供生物学材料。【方法】采用双层平板法从广西南宁某养鸡场分离到1株大肠杆菌裂解性噬菌体vB_EcoP_GN07;通过透射电镜观察、宿主谱测定、pH和温度稳定性评估、最佳感染复数(MOI)测定、一步生长曲线测定、全基因组测序等方法了解噬菌体的形态学、生物学特性和全基因组特性。【结果】透射电镜显示,vB_EcoP_GN07头部直径为45 nm±5 nm,尾部长约25 nm,属于短尾噬菌体科,宿主特异性强;在4~60 ℃可以保持效价稳定,在pH 3.0~11.0可以保持噬菌体活性;该噬菌体的最佳MOI为10^-2;一步生长曲线显示该噬菌体潜伏期为60 min,裂解量为560 PFU/cell;测序结果显示其属于双链线型DNA,长度为70 120 bp,GC含量为42.8%;噬菌体vB_EcoP_GN07共有81个开放阅读框,其中23个为已知功能蛋白。经BLASTN比对显示,vB_EcoP_GN07与NCBI数据库中其他噬菌体相似性较低,符合N4-like属噬菌体的特点,没有发现与耐药性、毒力因子、毒素和溶源性相关蛋白。【结论】试验成功分离到1株大肠杆菌噬菌体vB_EcoP_GN07,其具有裂解能力强、特异性强、耐酸碱的特点,与其他噬菌体相似性较低,为1株新的N4-like噬菌体。结果可为未来应用大肠杆菌噬菌体vB_EcoP_GN07开发新型杀菌剂提供理论依据。

关键词: 大肠杆菌; 噬菌体; 生物学特性; 全基因组分析; 比较基因组学

Abstract: 【Objective】 This study was aimed to explore the biological and genome characteristics of Escherichia coli (E.coli) phage vB_EcoP_GN07,and provide biological materials for the development of phage cocktail formulations against pathogenic E.coli disease.【Method】 A strain of E.coli lytic phage vB_EcoP_GN07 was isolated from a chicken farm in Nanning,Guangxi using the double-layer plate method.Transmission electron microscope observation,host spectrum determination,pH and temperature stability evaluation,the optimal multiplicity of infection (MOI) determination,one-step growth curve determination and whole genome sequencing were used to understand the morphology,biological characteristics and genome-wide characteristics of the phage.【Result】 Transmission electron microscopy showed vB_EcoP_GN07 had a head diameter of about 45 nm±5 nm and tail length of about 25 nm,belonging to the Podoviridae.which had strong host specificity.The phage titer could be kept stable at 4 to 60 ℃,and the phage activity could be maintained at pH 3.0-11.0.The optimal MOI of the phage was 10^-2.The one-step growth curve showed that the incubation period of the phage was 60 min,and the burst size was 560 PFU/cell.Sequencing results showed that it belonged to double stranded DNA,with a length of 70 120 bp and a GC content of 42.8%.The phage vB_EcoP_GN07 had 81 open reading frames,of which 23 were known functional proteins.BLASTN comparison showed that the phage vB_EcoP_GN07 had low similarity with other phages in NCBI database,conformed to the characteristics of N4-like phage,and lacked virulence genes and drug resistance genes associated with antibiotic resistance,toxins and virulence factors.【Conclusion】 An E.coli phage vB_ EcoP_ GN07 was successfully isolated in this study,which had strong cracking ability,host specificity and acid and alkali resistance.vB_ EcoP_ GN07 was less similar to other phages,and determined to be a new N4-like phage.This results provided a theoretical basis for the development of new fungicides with application E.coli phage vB_EcoP_GN07.

Key words: Escherichia coli; phage; biological characteristics; whole genome analysis; comparative genomics

中图分类号:

S852.61⁺2

王乐平, 潘艳, 谭奕舟, 李磊, 韩凯欧, 曹雅洁, 李军, 周雨晴, 廖玉英, 马东鑫, 龚俞, 彭昊, 王晓晔. 1株大肠杆菌噬菌体vB_EcoP_GN07的分离鉴定及全基因组分析[J]. 中国畜牧兽医, 2023, 50(4): 1522-1531.

WANG Leping, PAN Yan, TAN Yizhou, LI Lei, HAN Kaiou, CAO Yajie, LI Jun, ZHOU Yuqing, LIAO Yuying, MA Dongxin, GONG Yu, PENG Hao, WANG Xiaoye. Isolation,Identification and Genome Analysis of Escherichia coli Phage vB_EcoP_GN07[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1522-1531.

参考文献

[1] ALLOCATI N,MASULLI M,ALEXEYEV M F,et al.Escherichia coli in Europe:An overview[J].International Journal of Environmental Research and Public Health,2013,10(12):6235-6254.
[2] WIRTH T,FALUSH D,LAN R,et al.Sex and virulence in Escherichia coli:An evolutionary perspective[J].Molecular Microbiology,2006,60(5):1136-1151.
[3] ROTH N,KÄSBOHRER A,MAYRHOFER S,et al.The application of antibiotics in broiler production and the resulting antibiotic resistance in Escherichia coli:A global overview[J].Poultry Science,2019,98(4):1791-1804.
[4] GOLKAR Z,BAGASRA O,PACE D G.Bacteriophage therapy:A potential solution for the antibiotic resistance crisis[J].Journal of Infection in Developing Countries,2014,8(2):129-136.
[5] RIPP S.Bacteriophage-based pathogen detection[J].Advances in Biochemical Engineering/Biotechnology,2010,118:65-83.
[6] MINMIN Y,CAIRNS L S,CAMILLI A.A cocktail of three virulent bacteriophages prevents Vibrio cholerae infection in animal models[J].Nature Communications,2017,8:14187.
[7] CARVALHO C,COSTA A R,SILVA F,et al.Bacteriophages and their derivatives for the treatment and control of food-producing animal infections[J].Critical Reviews in Microbiology,2017,43(5):583-601.
[8] FERRIOL-GONZÁLEZ C,DOMINGO-CALAP P.Phage therapy in livestock and companion animals[J].Antibiotics (Basel,Switzerland),2021,10(5):559.
[9] MOROZOVA V,BABKIN I,KOZLOVA Y,et al.Isolation and characterization of a novel Klebsiella pneumoniae N4-like bacteriophage KP8[J].Viruses,2019,11(12):1115.
[10] KLEINHEINZ K A,JOENSEN K G,LARSEN M V.Applying the ResFinder and VirulenceFinder web-services for easy identification of acquired antibiotic resistance and E.coli virulence genes in bacteriophage and prophage nucleotide sequences[J].Bacteriophage,2014,4(1):e27943.
[11] PERERA M N,ABULADZE T,MANRONG L,et al.Bacteriophage cocktail significantly reduces or eliminates Listeria monocytogenes contamination on lettuce,apples,cheese,smoked salmon and frozen foods[J].Food Microbiology,2015,52:42-48.
[12] LI F,XING S Z,FU K F,et al.Genomic and biological characterization of the Vibrio alginolyticus-infecting 'Podoviridae' bacteriophage,vB_ValP_IME271[J].Virus Genes,2019,55(2):218-226.
[13] ZHANG L L,BAO H D,WEI C R,et al.Characterization and partial genomic analysis of a lytic Myoviridae bacteriophage against Staphylococcus aureus isolated from dairy cows with mastitis in Mid-east of China[J].Virus Genes,2015,50(1):111-117.
[14] COLOM J,CANO-SARABIA M,OTERO J,et al.Microencapsulation with alginate/CaCO3:A strategy for improved phage therapy[J].Scientific Reports,2017,7:41441.
[15] YAZDI M,BOUZARI M,GHAEMI E A.Isolation and characterization of a potentially novel Siphoviridae phage (vB_SsapS-104) with lytic activity against Staphylococcus saprophyticus isolated from urinary tract infection[J].Folia Microbiologica,2019,64(3):283-294.
[16] LENNEMAN B R,ROTHMAN-DENES L B.Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases[J].Biomolecules,2015,5(2):647-667.
[17] MCGAUGHEY K M,WHEELER L J,MOORE J T,et al.Protein-protein interactions involving T4 phage-coded deoxycytidylate deaminase and thymidylate synthase[J].The Journal of Biological Chemistry,1996,271(38):23037-23042.
[18] BELCAID M,BERGERON A,POISSON G.The evolution of the tape measure protein:Units,duplications and losses[J].BMC Bioinformatics,2011,12(Suppl9):S10.
[19] PREVELIGE P E,CORTINES J R.Phage assembly and the special role of the portal protein[J].Current Opinion in Virology,2018,31:66-73.
[20] KANAMARU S,UCHIDA K,NEMOTO M,et al.Structure and function of the T4 spackle protein Gp61.3[J].Viruses,2020,12(10):1070.
[21] WU Z F,ZHANG Y,XU X Y,et al.The holin-endolysin lysis system of the OP2-like phage X2 infecting Xanthomonas oryzae pv.oryzae[J].Viruses,2021,13(10):1949.
[22] CAHILL J,YOUNG R.Phage lysis:Multiple genes for multiple barriers[J].Advances in Virus Research,2019,103:33-70.
[23] JAKOBSSON E,SCHWARZER D,JOKILAMMI A,et al.Endosialidases:Versatile tools for the study of polysialic acid[J].Topics in Current Chemistry,2015,367:29-73.
[24] MORGADO S,VICENTE A C.Global in-silico scenario of tRNA genes and their organization in virus genomes[J].Viruses,2019,11(2):180.
[25] MAVRICH T N,HATFULL G F.Bacteriophage evolution differs by host,lifestyle and genome[J].Nature Microbiology,2017,2:17112.
[26] JAIN C,RODRIGUEZ-R L M,PHILLIPPY A M,et al.High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J].Nature Communications,2018,9(1):5114.
[27] ADRIAENSSENS E,BRISTER J R.How to name and classify your phage:An informal guide[J].Viruses,2017,9(4):70.
[28] WITTMANN J,TURNER D,MILLARD A D,et al.From orphan phage to a proposed new family-The diversity of N4-like viruses[J].Antibiotics (Basel,Switzerland),2020,9(10):663.