宽谱大肠杆菌O157∶H7 CVCC4050噬菌体vB_EcoM_GXBP08的分离鉴定及特性分析

doi:10.16431/j.cnki.1671-7236.2022.07.032

摘要/Abstract

摘要： 【目的】探究宽谱大肠杆菌O157∶H7噬菌体的生物学特性和全基因组特征。【方法】采用双层琼脂平板法从广西南宁某猪场污水样品中分离宽谱大肠杆菌O157∶H7裂解性噬菌体，通过空斑试验、噬斑试验对噬菌体进行宿主谱和裂解效价的测定；利用透射电镜观察、最佳感染复数测定、一步生长曲线测定、热稳定性与酸碱稳定性评估、杀菌试验及全基因组测序的方法分析噬菌体的形态学、生物学和全基因组特征。【结果】试验成功分离1株宽谱大肠杆菌O157∶H7裂解性噬菌体，命名为vB_EcoM_GXBP08。噬菌体vB_EcoM_GXBP08能高效裂解14株大肠杆菌，裂解效价可达10⁹~10¹⁰ PFU/mL。以大肠杆菌O157∶H7 CVCC4050为宿主菌测得噬菌体的最佳感染复数（multiplicity of infection，MOI）为1。一步生长曲线显示，该噬菌体的潜伏期和爆发期分别为20和50 min，爆发量为154 PFU/cell，耐受温度范围为30~70 ℃，在pH为4.0~10.0的环境中能维持其活性。在宿主菌CVCC4050中的杀菌试验结果显示，MOI为1时噬菌体vB_EcoM_GXBP08具有良好的杀菌效果。根据透射电镜观察和全基因组序列分析，该噬菌体属于有尾噬菌体目肌尾噬菌体科T4-like噬菌体属，基因组片段总长为108 114 bp，GC含量为36.23%。噬菌体vB_EcoM_GXBP08基因组中含有8个CDS，编码与噬菌体裂解有关的蛋白，未发现与抗生素耐药性、毒力因子、毒素和溶源性相关的基因。【结论】噬菌体vB_EcoM_GXBP08宿主谱宽、效价高，且温度、酸碱稳定性良好，在液体培养基中的杀菌能力强，本研究结果为后续研制宽谱噬菌体制剂以及在食品业、养殖业防治大肠杆菌O157∶H7感染中的应用提供参考。

关键词: 大肠杆菌O157∶H7; 噬菌体; 生物学特性; 全基因组分析

Abstract: 【Objective】 This study was aimed to explore the biological characteristics and genome characteristics of broad-spectrum Escherichia coli O157∶H7 phage.【Method】 The broad-spectrum Escherichia coli O157∶H7 lytic phage was seperated from a swine farm sewage in Nanning of Guangxi using the double-layer agar culture method.The host range and the corresponding titers of phage were determined through the methods of spot tests and plaque tests.The methods of transmission electron microscope observation,determination of the best multiplicity of infection in the plural,one-step growth curve drawing,thermal sensitivity and pH stability evaluation,sterilization experiment and the whole genome sequencing were used to analyze the morphological features,biological characteristics and the whole genomic characteristics of phage.【Result】 A broad-spectrum lytic phage against Escherichia coli O157∶H7 was successfully isolated and purified which was named as vB_EcoM_GXBP08.Phage vB_EcoM_GXBP08 could lyse 14 Escherichia coli strains with high efficiency,and the phage titers could reach 10⁹ to 10¹⁰ PFU/mL.Using Escherichia coli O157∶H7 CVCC4050 as host bacteria,the optimal multiplicity of infection (MOI) of phage vB_EcoM_GXBP08 was 1.One-step growth curve indicated that the latent period of vB_EcoM_GXBP08 was 20 min,the outbreak period was 50 min,and the outbreak quantity was 154 PFU/cell. The tolerable temperature range of phage vB_EcoM_GXBP08 was 30 to 70 ℃,and it could maintain activity at pH 4.0 to pH 10.0.The results of bactericidal experiment against CVCC4050 showed that phage vB_EcoM_GXBP08 had good germicidal efficacy when MOI was 1.According to transmission electron microscope observation and the whole genome analysis,phage vB_EcoM_GXBP08 belonged to Caudovirales order,Myoviridae family,T4-like Phagus,which had a genome consisting of 108 114 bp with a GC content of 36.23%.Phage vB_EcoM_GXBP08 genome harbored 8 CDS associated with lysis proteins and lacked virulence genes and drug resistance genes associated with antibiotic resistance,toxins and virulence factors.【Conclusion】 Phage vB_EcoM_GXBP08 had a broad host range,high titer,good thermal stability and acid-base stability,and a strong bactericidal effect in liquid medium.The results provided reference for the development of broad-spectrum phage and its application in the prevention and treatment of Escherichia coli O157∶H7 infection in food industry and breeding industry.

Key words: Escherichia coli O157∶H7; phage; biological characteristics; the whole genomic analysis

中图分类号:

S852.61⁺2

周雨晴, 马东鑫, 吴洁, 王晓晔. 宽谱大肠杆菌O157∶H7 CVCC4050噬菌体vB_EcoM_GXBP08的分离鉴定及特性分析[J]. 中国畜牧兽医, 2022, 49(7): 2735-2745.

ZHOU Yuqing, MA Dongxin, WU Jie, WANG Xiaoye. Isolation,Identification and Characterization Analysis of Broad-spectrum Escherichia coli O157∶H7 CVCC4050 Phage vB_EcoM_GXBP08[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2735-2745.

参考文献

[1] O'FLYNN G,ROSS R P,FITZGERALD G F,et al.Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7[J].Applied and Environmental Microbiology,2004,70(6):3417-3424.
[2] RANGEL J M,SPARLING P H,CROWE C,et al.Epidemiology of Escherichia coli O157:H7 outbreaks,United States,1982-2002[J].Emerging Infectious Diseases,2005,11(4):603-609.
[3] BAJPAI V K,RAHMAN A,KANG S C.Chemical composition and inhibitory parameters of essential oil and extracts of Nandina domestica Thunb.to control food-borne pathogenic and spoilage bacteria[J].International Journal of Food Microbiology,2008,125(2):117-122.
[4] PERERA M N,ABULADZE T,LI M,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.
[5] JAMALLUDEEN N,JOHNSON R P,FRIENDSHIP R,et al.Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli[J].Veterinary Microbiology,2007,124(1-2):47-57.
[6] 汪祥燕,辛国芹,董佩佩,等.1株宽噬菌谱鸡白痢沙门氏菌噬菌体的分离及生物学特性分析[J].中国畜牧兽医,2018,45(9):2575-2581. WANG X Y,XIN G Q,DONG P P,et al.Isolation and biological properties analysis of a Salmonella Pullorum lytic phage with wide host range[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2575-2581.(in Chinese)
[7] WANG S P,GAO Y L,LIU G,et al.Molecular cloning,characterization and expression of the energy homeostasis-associated gene in piglet[J].Journal of Zhejiang University Science B,2015,16(6):524-532.
[8] 包红朵,张辉,王冉.宽噬菌谱肠炎沙门氏菌噬菌体的生物学特性[J].江苏农业学报,2011,27(5):1117-1121. BAO H D,ZHANG H,WANG R.Biological properties of a Salmonella Enteritidis lytic phage with wide host range[J].Jiangsu Journal of Agricultural Sciences,2011,27(5):1117-1121.(in Chinese)
[9] 张辉,王冉,包红朵.裂解性福氏志贺氏菌噬菌体SF-A2的生物学特性及其在巴氏杀菌牛奶中的灭菌效果[J].食品科学,2010,31(23):214-218. ZHANG H,WANG R,BAO H D.Biological characteristics of lytic SF-A2 phage and its sterilization effect in milk[J].Food Science,2010,31(23):214-218.(in Chinese)
[10] CAREY-SMITH G V,BILLINGTON C,CORNELIUS A J,et al.Isolation and characterization of bacteriophages infecting Salmonella spp.[J].FEMS Microbiology Letters,2006,258(2):182-186.
[11] SHENDE R K,HIRPURKAR S D,SANNAT C,et al.Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens[J].Veterinary World,2017,10(8):973-978.
[12] ZHANG L,BAO H,WEI C,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.
[13] ZHANG C,YUAN J,GUO C,et al.Identification and complete genome of lytic "Kp34likevirus" phage vB_KpnP_Bp5 and therapeutic potency in the treatment of lethal Klebsiella pneumoniae infections in mice[J].Virus Research,2021,297:198348.
[14] LOWE T M,CHAN P P.tRNAscan-SE On-line:Integrating search and context for analysis of transfer RNA genes[J].Nucleic Acids Research,2016,44(W1):W54-W57.
[15] LIU B,ZHENG D,JIN Q,et al.VFDB 2019:A comparative pathogenomic platform with an interactive web interface[J].Nucleic Acids Research,2019,47(D1):D687-D692.
[16] JIA B,RAPHENYA A R,ALCOCK B,et al.CARD 2017:Expansion and model-centric curation of the comprehensive antibiotic resistance database[J].Nucleic Acids Research,2017,45(D1):D566-D573.
[17] GRANT J R,STOTHARD P.The CGView Server:A comparative genomics tool for circular genomes[J].Nucleic Acids Research,2008,36(Web Server issue):W181-W184.
[18] SULLIVAN M J,PETTY N K,BEATSON S A.Easyfig:A genome comparison visualizer[J].Bioinformatics,2011,27(7):1009-1010.
[19] DUC H M,SON H M,YI H,et al.Isolation,characterization and application of a polyvalent phage capable of controlling Salmonella and Escherichia coli O157:H7 in different food matrices[J].Food Research International,2020,131:108977.
[20] MAGNONE J P,MAREK P J,SULAKVELIDZE A,et al.Additive approach for inactivation of Escherichia coli O157:H7,Salmonella,and Shigella spp.on contaminated fresh fruits and vegetables using bacteriophage cocktail and produce wash[J].Journal of Food Protection,2013,76(8):1336-1341.
[21] KIM J H,KIM H J,JUNG S J,et al.Characterization of Salmonella spp.--Specific bacteriophages and their biocontrol application in chicken breast meat[J].Journal of Food Science,2020,85(3):526-534.
[22] 唐宁,殷茵,任慧英,等.沙门氏菌高效价宽谱噬菌体的分离纯化及生物学特性测定[J].中国畜牧兽医,2021,48(7):2577-2583. TANG N,YIN Y,REN H Y,et al.Isolation,purification and detection of biological characteristics of a high-titer and wide-spectrum Salmonella phage[J].China Animal Husbandry & Veterinary Medicine,2021,48(7):2577-2583.(in Chinese)
[23] CASJENS S R,THUMAN-COMMIKE P A.Evolution of mosaically related tailed bacteriophage genomes seen through the lens of phage P22 virion assembly[J].Virology,2011,411(2):393-415.
[24] SORENSEN A N,WOUDSTRA C,SORENSEN M,et al.Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages[J].Computational and Structural Biotechnology Journal,2021,19:4854-4867.
[25] YOUNG R.Phage lysis:Do we have the hole story yet?[J].Current Opinion in Microbiology 2013,16(6):790-797.
[26] PIRES D P,OLIVEIRA H,MELO L D,et al.Bacteriophage-encoded depolymerases:Their diversity and biotechnological applications[J].Applied Microbiology and Biotechnology,2016,100(5):2141-2151.
[27] PARK M,LEE J H,SHIN H,et al.Characterization and comparative genomic analysis of a novel bacteriophage,SFP10,simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7[J].Applied and Environmental Microbiology,2012,78(1):58-69.
[28] BAO H,ZHANG P,ZHANG H,et al.Bio-control of Salmonella Enteritidis in foods using bacteriophages[J].Viruses,2015,7(8):4836-4853.
[29] LI Z,MA W,LI W,et al.A broad-spectrum phage controls multidrug-resistant Salmonella in liquid eggs[J].Food Research International,2020,132:109011.