Isolation and Biological Characterization of an Escherichia coli Phage

doi:10.15933/j.cnki.1004-3268.2026.01.014

Abstract

Abstract: To explore the biological characteristics of Escherichia coli phage and evaluate its antibacterial application potential，a strain of Escherichia coli phage was isolated from the lake water samples of Guangdong Medical University，named PE62. Biological characteristics of PE62 were systematically analyzed using methods including spot assay，double‐layer agar plate method，transmission electron microscopy observation，whole‐genome sequencing，one‐step growth curve assay，and time‐kill assay，and its biofilm eradication ability was evaluated via crystal violet staining.The results showed that PE62 could form transparent plaques with a diameter of approximately 1—1.5 mm on the lawn of enteroaggregative Escherichia coli（EAEC）strain E533.Transmission electron microscopy observation revealed that it possessed a head and a tail structure，belonging to the Myoviridae family.The lysis spectrum test showed that PE62 could lyse a variety of pathogenic Escherichia coli（including intestinal agglomeration，intestinal pathogenicity and enterotoxicity），Salmonella and Shigella.The phage maintained activity within the range of 25—55 ℃ and pH 2—11，exhibiting good temperature and acid‐base stability.Its optimal multiplicity of infection（MOI）was 0.1.The one‐step growth curve showed that the latent period was approximately 40 min，the burst period was also 40 min，and the burst size was about 585.Whole‐genome analysis revealed that PE62 had a double‐stranded circular DNA with a G+C content of 35.52% and did not carry known virulence factors or antibiotic resistance genes.In vitro bactericidal assays showed that PE62exhibited excellent bactericidal activity against host bacteria and achieved a biofilm clearance rate of 27.9%.In conclusion，PE62possesses a broad host range，good environmental adaptability，and strong in vitro bactericidal as well as biofilm eradication capabilities，indicating promising potential for application in the prevention and control of Escherichia coli infections.

Key words: Escherichia coli, Bacteriophage, Biological characteristic, Biofilm

摘要： 为探究大肠杆菌噬菌体的生物学特性并评估其抗菌应用潜力，前期从广东医科大学校内湖水样本中分离出一株大肠杆菌噬菌体，命名为PE62。通过点斑法、双层琼脂平板法、透射电镜观察、基因组测序、一步生长曲线和时间-杀伤试验等方法，系统分析PE62的生物学特性，并利用结晶紫染色法评估了其对生物膜的消减能力。结果表明，PE62在肠集聚性大肠杆菌E533菌苔上可形成直径1~1.5 mm的透明噬菌斑；透射电镜显示其具有头部和尾部结构，属于肌尾噬菌体科。裂解谱试验表明，PE62可裂解多种致病性大肠杆菌（包括肠集聚性、肠致病性和肠产毒性等）以及沙门氏菌和志贺氏菌。该噬菌体在25~55 ℃和pH值2~11范围内均能保持活性，表现出良好的温度与酸碱稳定性。其最佳感染复数为0.1；一步生长曲线显示潜伏期约为40 min，暴发期亦为40 min，暴发量约为585。全基因组分析显示PE62为双链环状DNA，G+C含量为35.52%，未携带已知毒力因子和耐药基因。体外杀菌试验显示，PE62对宿主菌体外杀菌效果良好，对生物膜的清除率达27.9%。综上，PE62具有较宽的裂解谱、良好的环境适应性和较强的体外杀菌与生物膜消减能力，在防控大肠杆菌感染方面具备潜在应用价值。

关键词: 大肠杆菌, 噬菌体, 生物学特性, 生物膜

CLC Number:

Q939.48

WANG Xue, YANG Zhenhong, WANG Shihui, LIN Bokun. Isolation and Biological Characterization of an Escherichia coli Phage[J]. Journal of Henan Agricultural Sciences, 2026, 55(1): 151-161.

王雪, 杨振洪, 王世慧, 林伯坤. 一株大肠杆菌噬菌体的分离及其生物学特性分析[J]. 河南农业科学, 2026, 55(1): 151-161.

Figures/Tables 12

References

［1］YANG Y Q，XIAO T S，LI J R，et al. Wild‐type cutoff for apramycin against Escherichia coli［J］. BMC Veterinary Research，2020，16（1）：309.

［2］RILEY L W. Distinguishing pathovars from nonpathovars：Escherichia coli［J］.Microbiology Spectrum，2020，8（4）：10.

［3］WILSON J，ELGOHARI S，LIVERMORE D M，et al.Trends among pathogens reported as causing bacteraemia in England，2004—2008［J］. Clinical Microbiology and Infection，2011，17（3）：451‐458.

［4］李海利，徐引弟，王治方，等. 胶冻样芽孢杆菌对革兰氏阴性菌的抑菌活性及其抗菌次级代谢产物分析［J］. 河南农业科学，2024，53（10）：159‐169.

LI H L，XU Y D，WANG Z F，et al. Analysis of antibacterial activity and secondary metabolites of Paenibacillus kribbensis against gram‐negative bacteria［J］. Journal of Henan Agricultural Sciences，2024，53 （10）：159‐169.

［5］闫安龙，张亚南，王帝，等. 国内部分地区猪禽粪便中产气荚膜梭菌的分离鉴定、耐药性及生物被膜形成能力分析［J/OL］.中国动物传染病学报，2025［2025‐03‐23］.https：//doi. org/10. 19958/j. cnki. cn31‐2031/s.20250126.001.

YAN A L，ZHANG Y N，WANG D，et al. Isolation，identification and biological characteristics of Clostridium perfringens in pig and poultry manure［J/OL］.Chinese Journal of Animal Infectious Diseases，2025［2025‐03‐23］.https：//doi. org/10. 19958/j. cnki.cn31‐2031/s. 20250126. 001.

［6］LIVERMORE D M，HOPE R，BRICK G，et al.Non‐susceptibility trends among Enterobacteriaceae from bacteraemias in the UK and Ireland，2001‐06［J］.Journal of Antimicrobial Chemotherapy，2008，62（S2）：ii41‐ii54.

［7］聂阿真，倪子富，冯先阳，等. 醋糟乳酸菌的筛选、鉴定及其益生特性研究［J］. 河南农业大学学报，2025，59（5）：859‐871.

NIE A Z，NI Z F，FENG X Y，et al. Screening，identification and probiotic properties of lacticacid bacteria from vinegar lees［J］. Journal of Henan Agricultural University，2025，59（5）：859‐871.

［8］张文刚，严凤，姜芹，等. 某地养殖场大肠杆菌耐药率与粪便中抗菌药物残留情况的初步分析［J］.上海畜牧兽医通讯，2019（2）：48‐49.

ZHANG W G，YAN F，JIANG Q，et al. Preliminary analysis on the drug resistance rate of Escherichia coli in a farm and the residue of antibacterial drugs in feces［J］. Shanghai Journal of Animal Husbandry and Veterinary Medicine，2019（2）：48‐49.

［9］BARTON M D. Impact of antibiotic use in the swine industry［J］. Current Opinion in Microbiology，2014，19：9‐15.

［10］HALL C W，MAH T F. Molecular mechanisms of biofilm‐based antibiotic resistance and tolerance in pathogenic bacteria［J］. FEMS Microbiology Reviews，2017，41（3）：276‐301.

［11］LOHSE M B，GULATI M，JOHNSON A D，et al.Development and regulation of single‐and multi‐species Candida albicans biofilms［J］. Nature Reviews Microbiology，2018，16（1）：19‐31.

［12］ROY R，TIWARI M，DONELLI G，et al. Strategies for combating bacterial biofilms：A focus on anti‐biofilm agents and their mechanisms of action［J］. Virulence，2018，9（1）：522‐554.

［13］MORYL M，RÓŻALSKI A，DE FIGUEIREDO J A P，et al. How do phages disrupt the structure of Enterococcus faecalis biofilm？［J］.International Journal of Molecular Sciences，2023，24（24）：17260.

［14］ÖZTÜRK F Y，DARCAN C，KARIPTAŞ E. The determination，monitoring，molecular mechanisms and formation of biofilm in E.coli［J］. Brazilian Journal of Microbiology，2023，54（1）：259‐277.

［15］HØIBY N，BJARNSHOLT T，MOSER C，et al. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014 ［J］. Clinical Microbiology and Infection，2015，21（S1）：S1‐S25.

［16］MITTAL S，SHARMA M，CHAUDHARY U. Biofilm and multidrug resistance in uropathogenic Escherichia coli［J］. Pathogens and Global Health，2015，109（1）：26‐29.

［17］周杰，牟航，王舒博，等. 重庆地区畜禽源大肠杆菌耐药性变化分析［J］. 南方农业学报，2024，55（10）：3179‐3189.
ZHOU J，MOU H，WANG S B，et al. Changes in drug resistance of poultry and livestock sourced Escherichia coli in Chongqing［J］. Journal of Southern Agriculture，2024，55（10）：3179‐3189.

［18］GIBB B P，ADJIARGYROU M. Bacteriophage therapy for bone and joint infections［J］. The Bone & Joint Journal，2021，103‐B（2）：234‐244.

［19］STRATHDEE S A，HATFULL G F，MUTALIK V K，et al. Phage therapy：From biological mechanisms to future directions［J］. Cell，2023，186（1）：17‐31.

［20］CAMARILLO‐GUERRERO L F，ALMEIDA A，RANGEL‐PINEROS G，et al. Massive expansion of human gut bacteriophage diversity［J］.Cell，2021，184 （4）：1098‐1109.

［21］DION M B，OECHSLIN F，MOINEAU S. Phage diversity，genomics and phylogeny［J］. Nature Reviews Microbiology，2020，18（3）：125‐138.

［22］REA M C，DOBSON A，O’SULLIVAN O，et al. Effect of broad‐and narrow‐spectrum antimicrobials on Clostridium difficile and microbial diversity in a model of the distal colon［J］. Proceedings of the National Academy of Sciences of the United States of America，2011，108（S1）：4639‐4644.

［23］于华波，张亮，冯超，等. 噬菌体药代动力学研究进展［J］.中国兽医学报，2022，42（2）：399‐404.
YU H B，ZHANG L，FENG C，et al. Advances in phage pharmacokinetics［J］. Chinese Journal of Veterinary Science，2022，42（2）：399‐404.

［24］王冰，李阳，黄安琪，等. 噬菌体防治食品中食源性致病菌的研究进展［J］. 食品研究与开发，2024，45(15）：204‐209.
WANG B，LI Y，HUANG A Q，et al.Research progress on the use of bacteriophages in controlling foodborne
pathogens in foods［J］.Food Research and Development，2024，45（15）：204‐209.

［25］刘子琛，陈婷，张永亮. 噬菌体在畜牧生产中的研究应用进展［J］. 畜牧与兽医，2024，56（12）：139‐146.
LIU Z C，CHEN T，ZHANG Y L. Progress in research on application of bacteriophages in poultry and livestock production［J］. Animal Husbandry & Veterinary Medicine，2024，56（12）：139‐146.

［26］LU R M，HWANG Y C，LIU I J，et al. Development of therapeutic antibodies for the treatment of diseases ［J］. Journal of Biomedical Science，2020，27（1）：1.

［27］CHEN Q Q，DHARMARAJ T，CAI P C，et al.Bacteriophage and bacterial susceptibility，resistance，and tolerance to antibiotics［J］. Pharmaceutics，2022，14（7）：1425.

［28］陈坤，王成森，钱淼，等. 一株新的防治大肠杆菌O157：H7的烈性噬菌体特性及其应用研究［J］.食品科技，2025，50（9）：28‐35.
CHEN K，WANG C S，QIAN M，et al. Biological characteristic and application of a novel lytic bacteriophage against Escherichia coli O157：H7［J］.Food Science and Technology，2025，50（9）：28‐35.

［29］ALEXYUK P，BOGOYAVLENSKIY A，ALEXYUK M，et al. Isolation and characterization of lytic bacteriophages active against clinical strains of E.coli and development of a phage antimicrobial cocktail［J］.Viruses，2022，14（11）：2381.

［30］SULTAN‐ALOLAMA M I，AMIN A，EL‐TARABILY K A，et al.Characterization and genomic analysis of Escherichia coli O157：H7 phage UAE_MI‐01 isolated from birds［J］.International Journal of Molecular Sciences，2022，23（23）：14846.

［31］KORTRIGHT K E，CHAN B K，KOFF J L，et al. Phage therapy： A renewed approach to combat antibiotic‐resistant bacteria［J］. Cell Host & Microbe，2019，25（2）：219‐232.

［32］JUN J W，GIRI S S，KIM H J，et al. Bacteriophage application to control the contaminated water with Shigella［J］. Scientific Reports，2016，6：22636.

［33］KIM H J，GIRI S S，KIM S G，et al.Isolation and characterization of two bacteriophages and their preventive effects against pathogenic Vibrio coralliilyticus causing mortality of Pacific oyster（Crassostrea gigas）larvae［J］.Microorganisms，2020，8 （6）：926.

［34］BUTTIMER C，LYNCH C，HENDRIX H，et al.Isolationand characterization of Pectobacterium Phage vB_PatM_CB7：New insights into the genus Certrevirus［J］.Antibiotics，2020，9（6）：352.

［35］刘亮. 蛋鸡禽致病性大肠杆菌分离鉴定及噬菌体制剂体外杀菌研究［J］.畜禽业，2024，35（2）：13‐16.
LIU L. Isolation and identification of pathogenic Escherichia coli from laying hens and in vitro sterilization of phage preparation［J］.Livestock and Poultry Industry，2024，35（2）：13‐16.
［36］DENG H W，LI M J，ZHANG Q Y，et al. The broad‐spectrum endolysin LySP2 improves chick survival after Salmonella pullorum infection［J］.Viruses，2023，15（4）：836.

［37］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.
［38］CHAMBLEE J S，RAMSEY J，CHEN Y，et al. Endolysin regulation in phage Mu lysis［J］.mBio，2022，13（3）：e0081322.
［39］RAJAURE M，BERRY J，KONGARI R，et al. Membrane fusion during phage lysis［J］.Proceedings of the National Academy of Sciences of the United States of America，2015，112（17）：5497‐5502.
［40］YOUNG R. Phage lysis：Three steps，three choices，one outcome［J］. Journal of Microbiology，2014，52（3）：243‐258.

［41］PIRES D P，OLIVEIRA H，MELO L D R，et al.Bacteriophage‐encoded depolymerases：Their diversity and biotechnological applications ［J］.Applied Microbiology and Biotechnology，2016，100（5）：2141‐2151.
［42］周玲玲，王宇航，撒俊梦，等. 抗菌肽LL-1体内治疗大肠杆菌感染的效果［J］.河南农业科学，2025，54（5）：142‐148.
ZHOU L L，WANG Y H，SA J M，et al. Treatment effect of antimicrobial peptide LL‐1 on E.coli infection in vivo［J］. Journal of Henan Agricultural Sciences，2025，54（5）：142‐148.

［43］LU H，NI S Q. Review on sterilization techniques，and the application potential of phage lyase and lyase immobilization in fighting drug‐resistant bacteria［J］.Journal of Materials Chemistry B，2024，12（14）：3317‐3335.

［44］BALLÉN V，CEPAS V，RATIA C，et al. Clinical Escherichia coli：From biofilm formation to new antibiofilm strategies［J］. Microorganisms，2022，10（6）：1103.

［45］GONZÁLEZ J F，HAHN M M，GUNN J S. Chronic biofilm‐based infections：Skewing of the immune response［J］. Pathogens and Disease，2018，76（3）：fty023.

［46］JDEED G，KRAVCHUK B，TIKUNOVA N V. Factors affecting phage‐bacteria coevolution dynamics［J］.Viruses，2025，17（2）：235.

［47］KIM B O，KIM E S，YOO Y J，et al. Phage‐derived antibacterials：Harnessing the simplicity，plasticity，and diversity of phages［J］. Viruses，2019，11（3）：268.

［48］刘敏，齐海涛，袁辉，等. 厦门希瓦氏菌噬菌体的分离、表征和应用研究［J］. 动物医学进展，2025，46（2）：47‐53.
LIU M，QI H T，YUAN H，et al. Isolation，characterization and application of a phage from Shewanella xiamenensis［J］. Progress in Veterinary Medicine，2025，46（2）：47‐53.

[1]	ZHANG Changcheng, JIANG Jun, PAN Shouhui, XUE Yuan, ZHANG Quan, LI Zikuo, WANG Hancheng, SHI Caihua, FANG Shouguo, ZHANG Songbai, LI Xiquan. Molecular Identification and Complete Sequence Analysis of Chilli Veinal Mottle Virus GZ‑tobacco Isolate [J]. Journal of Henan Agricultural Sciences, 2025, 54(8): 102-110.
[2]	FENG Lili. Research Progress on Application in Livestock，Poultry and Aquaculture Production of Bacillus subtilis and Its Effect on Intestinal Health of Animals [J]. Journal of Henan Agricultural Sciences, 2025, 54(5): 1-9.
[3]	DU Chenyang, SHEN Fengying, WU Junwei, ZHAO Xinrui, WANG Xue, WU Weigang. Identification，Biological Characterization and Screening of Prevention and Control Agents of the Pathogens Causing the Rot Disease of Malus×robusta（CarriŠre）Rehder [J]. Journal of Henan Agricultural Sciences, 2025, 54(3): 99-109.
[4]	LI Haicheng, WANG Fanzhen, JIANG Yongcheng, SONG Xin, CHEN Zhaorong. Identification and Indoor Fungicide Screening of Pathogen Causing Leaf Spot on Rehmannia glutinosa [J]. Journal of Henan Agricultural Sciences, 2024, 53(4): 92-101.
[5]	LI Haili, XU Yindi, WANG Zhifang, ZHU Wenhao, CHEN Jianhao, LI Bin, DUAN Jin’gang, FENG Lili, YANG Fan, MA Chunjiang, YAN Xiangzhou. Analysis of Antibacterial Activity and Secondary Metabolites of Paenibacillus kribbensis against Gram⁃negative Bacteria [J]. Journal of Henan Agricultural Sciences, 2024, 53(10): 159-169.
[6]	. Construction and Biological Characterization of an Attenuated Salmonella typhimurium Type Ⅲ Secretion System Carrying NDV HN Gene [J]. Journal of Henan Agricultural Sciences, 2021, 50(10): 116-123.
[7]	HAO Liying, WANG Yanwei, SUN Yujie, CAO Hongmei, HUANG Tian, PANG Wenqiang, LI Xiangdong, DENG Junhua, TIAN Kegong. Preparation and Application of Monoclonal Antibodies against P30 Protein of African Swine Fever Virus [J]. Journal of Henan Agricultural Sciences, 2020, 49(10): 124-129.
[8]	JIN Shengnan, SHU Huiping, ZHANG Dongxing, KANG Yuanhuan, SHAN Xiaofeng, QIAN Aidong. Screening,Identification and Probiotic Properties Assessment of Swine Lactic Acid Bacteria with Biofilm [J]. Journal of Henan Agricultural Sciences, 2019, 48(12): 121-127.
[9]	CHEN Yumei, MA Liping, ZHOU Jingming. The Novel Progress of the Biology of Porcine Parvovirus [J]. Journal of Henan Agricultural Sciences, 2019, 48(11): 1-6.
[10]	ZHU Ｒui, WU Lifang, LI Liang, YU Zhe, ZOU Ｒuixing, ZU Xiujie. Ｒesearch Status and Farming Development Prospects of Barbus capito [J]. Journal of Henan Agricultural Sciences, 2019, 48(10): 1-5.