A safety-prioritized pipeline for therapeutic phage discovery: Isolation of a novel phage NLE251 from soil using Escherichia coli DH5α as a host

Abstract

The relentless spread of antimicrobial-resistant Escherichia coli underscores the critical need for alternative therapeutics, such as bacteriophages. A major translational hurdle is the efficient and safe initial isolation of lytic phages with defined therapeutic potentials. This study established and validated a safety-conscious pipeline using the non-pathogenic laboratory strain E. coliDH5α (BSL-1) for environmental phage screening, a strategy designed for accessibility and to circumvent early biosafety concerns. After continuous screening, we isolated a novel lytic phage, vB_EcoS_NLE251, from the soil. Comprehensive phenotyping revealed robust stability (tolerant up to 60 °C) and efficient replication, with an optimal multiplicity of infection (MOI) of 100, yielding a high titer of 2.1 × 10¹⁰ PFU/mL. Crucially, host-range determination using a panel of 35 clinical E. coliisolates showed that NLE251 only exhibited lytic activity in spot assays against a single biliary tract infection-derived strain (54959124), though subsequent quantitative infection efficiency data indicate extremely low productive infection against this target strain. Genomic analysis (49,336 bp dsDNA, belonging to the class Caudoviricetes) further confirmed the absence of virulence or antibiotic resistance genes, fulfilling a key safety prerequisite for therapeutic use. Although NLE251 is a narrow-spectrum agent, this study provides a validated methodological framework that prioritizes safety and specificity from the initial isolation step, offering a safety-prioritized, standardized pipeline for accelerating phage discovery against multidrug-resistant bacterial infections.

Keywords

Escherichia coliDH5α / Escherichia phage NLE251 / Antimicrobial resistance / Phage screening

References

[1] Brinkac L, Voorhies A, Gomez A, Nelson KE. The Threat of Antimicrobial Resistance on the Human Microbiome. Microb Ecol. 2017; 74(4):1001-1008. doi: 10.1007/s00248-017-0985-z

[2] The Lancet. Antimicrobial resistance: an agenda for all. Lancet . 2024; 403(10442):2349. doi: 10.1016/s0140-6736(24)01076-6

[3] Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent Advances in Understanding Enteric Pathogenic Escherichia coli . Clin Microbiol Rev. 2013; 26(4):822-880. doi: 10.1128/cmr.00022-13

[4] Kolenda R, Burdukiewicz M, Schierack P. A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli. Front Cell Infect Microbiol. 2015; 5:23. doi: 10.3389/fcimb.2015.00023

[5] Tang F, Wang J, Li D, et al. Comparative genomic analysis of 127 Escherichia coli strains isolated from domestic animals with diarrhea in China. BMC Genom. 2019; 20(1):212. doi: 10.1186/s12864-019-5588-2

[6] Zhou Y, Zhou Z, Zheng L, et al. Urinary Tract Infections Caused by Uropathogenic Escherichia coli: Mechanisms of Infection and Treatment Options. Int J Mol Sci. 2023; 24(13):10537. doi: 10.3390/ijms241310537

[7] Mazzariol A, Bazaj A, Cornaglia G. Multi-drug-resistant Gram-negative bacteria causing urinary tract infections: a review. J Chemother. 2017; 29(sup1):2-9. doi: 10.1080/1120009x.2017.1380395

[8] Makvana S, Krilov LR. Escherichia coli Infections. Pediatr Rev. 2015; 36(4):167-171. doi: 10.1542/pir.36.4.167

[9] Hoffman A, Satyavolu S, Muhanna D, et al. Predictors of mortality and severe illness from Escherichia coli sepsis in neonates. J Perinatol. 2024; 44(12):1816-1821. doi: 10.1038/s41372-024-02117-9

[10] Wan S, Li N, Habib S, et al. Biological Characteristics and Whole-Genome Analysis of a Porcine E. coli Phage. Vet Sci. 2025; 12(1):57. doi: 10.3390/vetsci12010057

[11] Leimbach A, Hacker J, Dobrindt U. E. coli as an all-rounder: the thin line between commensalism and pathogenicity. Curr Top Microbiol Immunol. 2013; 358:3-32. doi: 10.1007/82_2012_303

[12] Byrne L, Kaindama L, Bentley M, et al. Investigation into a national outbreak of STEC O157:H7 associated with frozen beef burgers, UK, 2017. Epidemiol Infect. 2020; 148:e215. doi: 10.1017/S0950268820001582

[13] Gobin M, Hawker J, Cleary P, et al. National outbreak of Shiga toxin-producing Escherichia coli O157:H7 linked to mixed salad leaves, United Kingdom, 2016. Euro Surveill. 2018; 23(18):17-00197. doi: 10.2807/1560-7917.Es.2018.23.18.17-00197

[14] Jenkins C, Dallman TJ, Launders N, et al.Public Health Investigation of Two Outbreaks of Shiga Toxin-Producing Escherichia coli O157 Associated with Consumption of Watercress. Appl Environ Microbiol. 2015; 81(12):3946-3952. doi: 10.1128/aem.04188-14

[15] Launders N, Locking ME, Hanson M, et al. A large Great Britain-wide outbreak of STEC O157 phage type 8 linked to handling of raw leeks and potatoes. Epidemiol Infect. 2016; 144(1):171-182. doi: 10.1017/S0950268815001016

[16] Treacy J, Jenkins C, Paranthaman K, et al. Outbreak of Shiga toxin-producing Escherichia coli O157:H7 linked to raw drinking milk resolved by rapid application of advanced pathogen characterisation methods, England, August to October 2017. Euro Surveill. 2019; 24(16):1800191. doi: 10.2807/1560-7917.Es.2019.24.16.1800191

[17] Yang S-C, Lin C-H, Aljuffali IA, Fang J-Y. Current pathogenic Escherichia coli foodborne outbreak cases and therapy development. Arch Microbiol. 2017; 199(6):811-825. doi: 10.1007/s00203-017-1393-y

[18] Sheveleva SA, Kuvaeva IB, Efimochkina NR, Minaeva LP. Mikrobiologicheskaya bezopasnost pishchi: razrabotka normativno-metodicheskoi bazy [Microbiological safety of food: development of normative and methodical base]. Vopr Pitan. 2020; 89(4):125-145. [In Russian]. doi:10.24411/0042-8833-2020-10048

[19] Kortright KE, Chan BK, Koff JL, Turner PE. Phage Therapy: A Renewed Approach to Combat Antibiotic-Resistant Bacteria. Cell Host Microbe. 2019; 25(2):219-232. doi: 10.1016/j.chom.2019.01.014

[20] Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli . Nat Rev Microbiol. 2004;2(2):123-140. doi: 10.1038/nrmicro818

[21] Da Silva GJ, Mendonça N. Association between antimicrobial resistance and virulence in Escherichia coli . Virulence. 2012; 3(1):18-28. doi: 10.4161/viru.3.1.18382

[22] Zhang Y-Z, Singh S. Antibiotic stewardship programmes in intensive care units: Why, how, and where are they leading us. World J Crit Care Med. 2015; 4(1):13-28. doi: 10.5492/wjccm.v4.i1.13

[23] Maffei E, Shaidullina A, Burkolter M, et al. Systematic exploration of Escherichia coli phage-host interactions with the BASEL phage collection. PLoS Biol. 2021; 19(11):e3001424. doi: 10.1371/journal.pbio.3001424

[24] Nikkhahi F, Soltan Dallal MM, Alimohammadi M, et al. Phage therapy: assessment of the efficacy of a bacteriophage isolated in the treatment of salmonellosis induced by Salmonella enteritidis in mice. Gastroenterol Hepatol Bed Bench. 2017; 10(2):131-136.

[25] Lee H, Ku H-J, Lee D-H, et al. Characterization and Genomic Study of the Novel Bacteriophage HY01 Infecting Both Escherichia coli O157:H7 and Shigella flexneri: Potential as a Biocontrol Agent in Food. PLoS ONE. 2016; 11(12):e0168985. doi: 10.1371/journal.pone.0168985

[26] Chang H-C, Chen C-R, Lin J-W, et al. Isolation and characterization of novel giant Stenotrophomonas maltophilia phage phiSMA5. Appl Environ Microbiol. 2005; 71(3):1387-1393. doi: 10.1128/AEM.71.3.1387-1393.2005

[27] Sillankorva S, Hyman P. Isolation of Bacteriophages for Clinically Relevant Bacteria. Methods Mol Biol. 2024; 2734:3-12. doi: 10.1007/978-1-0716-3523-0_1

[28] Satish R, Desouza A. Study of characteristics of mycobacteriophage - A novel tool to treat Mycobacterium spp. Int J Mycobacteriol. 2019; 8(2):170-174. doi: 10.4103/ijmy.ijmy_42_19

[29] Chen L, Yuan S, Liu Q, et al. In Vitro Design and Evaluation of Phage Cocktails Against Aeromonas salmonicida . Front Microbiol. 2018; 9:1476. doi: 10.3389/fmicb.2018.01476

[30] Wang W, Tan C, Li L, et al. The China National GeneBank Sequence Archive (CNSA) 2024 update. Hortic Res. 2025; 12(5):uhaf036. doi: 10.1093/hr/uhaf036

[31] Turner D, Adriaenssens EM, Amann RI, et al. Summary of taxonomy changes ratified by the International Committee on Taxonomy of Viruses (ICTV) from the Bacterial Viruses Subcommittee, 2025. J Gen Virol. 2025; 106(7):002111. doi: 10.1099/jgv.0.002111

[32] Meier-Kolthoff JP, Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017; 33(21):3396-3404. doi: 10.1093/bioinformatics/btx440

[33] Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform. 2013; 14:60. doi: 10.1186/1471-2105-14-60

[34] Lefort V, Desper R, Gascuel O. FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program. Mol Biol Evol. 2015; 32(10):2798-2800. doi: 10.1093/molbev/msv150

[35] Krupovic M, Turner D, Morozova V, et al. Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021. Arch Virol. 2021; 166(11):3239-3244. doi: 10.1007/s00705-021-05205-9

[36] Costanzo ML, D’Andrea V, Lauro A, Bellini MI. Acute Cholecystitis from Biliary Lithiasis: Diagnosis, Management and Treatment. Antibiotics. 2023; 12(3):482. doi: 10.3390/antibiotics12030482

[37] Navaneethan U, Jayanthi V, Mohan P. Pathogenesis of cholangitis in obstructive jaundice-revisited. Minerva Gastroenterol Dietol. 2011; 57(1):97-104.

[38] Mirzaei MK, Nilsson AS. Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy. Plos ONE. 2015; 10(3):e0118557. doi: 10.1371/journal.pone.0118557

[39] Gibson SB, Green SI, Liu CG, et al. Constructing and Characterizing Bacteriophage Libraries for Phage Therapy of Human Infections. Front Microbiol. 2019; 10:2537. doi: 10.3389/fmicb.2019.02537

[40] Nasrollahian S, Graham JP, Halaji M. A review of the mechanisms that confer antibiotic resistance in pathotypes of E. coli . Front Cell Infect Microbiol. 2024; 14:1387497. doi: 10.3389/fcimb.2024.1387497

[41] Roth N, Käsbohrer A, Mayrhofer S, Zitz U, Hofacre C, Domig KJ. The application of antibiotics in broiler production and the resulting antibiotic resistance in Escherichia coli: A global overview. Poult Sci. 2019; 98(4):1791-1804. doi: 10.3382/ps/pey539

[42] Arbab S, Ullah H, Wang W, Zhang J. Antimicrobial drug resistance against Escherichia coli and its harmful effect on animal health. Vet Med Sci. 2022; 8(4):1780-1786. doi: 10.1002/vms3.825

[43] Laird TJ, Abraham S, Jordan D, et al. Porcine enterotoxigenic Escherichia coli: Antimicrobial resistance and development of microbial-based alternative control strategies. Vet Microbiol. 2021; 258:109117. doi: 10.1016/j.vetmic.2021.109117

[44] Boucher D, Barnich N. Phage Therapy Against Adherent-invasive E. coli: Towards a Promising Treatment of Crohn’s Disease Patients? J Crohn’s Colitis. 2022; 16(10):1509-1510. doi: 10.1093/ecco-jcc/jjac070

[45] Chegini Z, Khoshbayan A, Vesal S, Moradabadi A, Hashemi A, Shariati A. Bacteriophage therapy for inhibition of multi drug‐resistant uropathogenic bacteria: a narrative review. Ann Clin Microbiol Antimicrob. 2021; 20(1):30. doi: 10.1186/s12941-021-00433-y

[46] Biswas SK, Sumon MMH, Ahmed S, et al. Beyond Antibiotics: Exploring the Potential of Bacteriophages and Phage Therapy. Phage Ther Appl Res. 2024; 5(4):186-202. doi: 10.1089/phage.2024.0005

[47] Duan X, Liu W, Xiao Y, et al. Exploration of the feasibility of clinical application of phage treatment for multidrug-resistant Serratia marcescens-induced pulmonary infection. Emerg Microbes Infect. 2025; 14(1):2451048. doi: 10.1080/22221751.2025.2451048

[48] Peng X, Chang J, Zhang H, et al. Isolation, characterization, and genomic analysis of a novel bacteriophage vB_Kp_XP4 targeting hypervirulent and multidrug-resistant Klebsiella pneumoniae . Front Microbiol. 2025; 16:1491961. doi: 10.3389/fmicb.2025.1491961

[49] Abebe AA, Birhanu AG, Tessema TS. Isolation, purification, and phenotypic characterization of virulent Klebsiella pneumoniaephages from environmental samples in Addis Ababa, Ethiopia: A synergistic approach combining spot assay and streak plating. PLoS ONE. 2025; 20(9):e0331955. doi: 10.1371/journal.pone.0331955

[50] Ghatbale P, Blanc A, Sue A, et al. Experimental phage evolution results in expanded host ranges against antibiotic resistant Klebsiella pneumoniae isolates. Nat Commun. 2025; 16(1):9903. doi: 10.1038/s41467-025-66062-7

[51] Ando H, Lemire S, Pires DP, Lu TK. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Syst. 2015; 1(3):187-196. doi: 10.1016/j.cels.2015.08.013