AccScience Publishing / JBM / Online First / DOI: 10.14440/jbm.0436
Submit to JBM
Cite this article
2
Download
15
Views
Journal Browser
Volume | Year
Issue
Search
Forthcoming Issue
Current Issue
View All
News and Announcements
View All
RESEARCH ARTICLE

Isolation of bacteriophage and ESBL-producing Escherichia coli from downstream water samples and examination of phage lytic activity against ESBL-producing E. coli

Masooma Jawad Hussain1 ,  Yawar Abbas1 ,  Hadia Gul2 ,  Sarah Gul2 ,  Sumbal Komal1 ,  Eid Nawaz3
Show Less
1 Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
2 Department of Biotechnology, Faculty of Biological Sciences, International Islamic University, Islamabad 45320, Pakistan
3 National Veterinary Laboratory, National Agriculture Research Center, Islamabad 44000, Pakistan
JBM , e99010103 https://doi.org/10.14440/jbm.0436
Submitted: 14 December 2025 | Revised: 16 February 2026 | Accepted: 5 March 2026 | Published: 30 June 2026
© 2026 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution -Noncommercial 4.0 International License (CC-by the license) ( https://creativecommons.org/licenses/by-nc/4.0/ )
Download PDF
Cite
XML
Abstract

Background: Amid global health concerns, extended-spectrum beta-lactamase (ESBL)-producing bacteria create substantial resistance challenges to current antibiotics. ESBL-producing pathogens are resistant to many antibiotics, including first- to third-generation cephalosporins, monobactams, or other antibiotic classes. Therefore, healthcare relies on carbapenems and colistin as their last options. Alarmingly, resistance to these critical drugs is increasing, emphasizing the urgent need for contemporary solutions, such as phage therapy. Objective:To develop an integrated workflow for identification and phage susceptibility assessment of ESBL-producing E. coli from environmental water samples. Methods: In this study, 15 downstream water samples were collected from five locations in Islamabad and Rawalpindi, including Nala Lai, Chattar Park, Shahdara, Nescom Hospital H-10, and G-10. Results: Of these, 10 samples (66.67%) tested positive for Escherichia coli, while five (33.33%) were negative. All samples from Chattar Park were negative for E. coli. Screening via the double-disk synergy test and combination disk test identified four samples (26.66%) with ESBL-producing E. coli and six (40%) with non-ESBL-producing isolates. Bacteriophages were isolated from Nescom Hospital stream water using the double agar overlay method and purified with a 0.45 μm syringe filter. The phages exhibited lytic activity against ESBL-producing E. coli NLA-3, as indicated in spot test results, and their therapeutic potential was validated through reduction assays. The isolated phage NHE-1 shows promise as an alternative treatment for antibiotic-resistant infections. Conclusion: Further studies are needed to expand its application to diverse bacterial strains and evaluate its clinical efficacy. Utilizing bacteriophages’ natural ability to target bacteria may provide sustainable, effective solutions to combat antibiotic resistance.

Keywords
Extended-spectrum beta-lactamase-producing E. coli
Bacteriophage
Antibiotic resistance
Lytic ability
Carbapenems
Downstream water
Conflict of interest
The authors declare they have no competing interests.
References
  1. Salam MA, Al-Amin MY, Salam MT, et al. Antimicrobial resistance: a growing serious threat for global public health. Healthcare. 2023; 11(13):1946. doi:10.3390/healthcare11131946
  2. Liu H, Fan S, Zhang X, et al. Antibiotic-resistant characteristics and horizontal gene transfer ability analysis of extended-spectrum β-lactamase-producing Escherichia coli isolated from giant pandas. Front Vet Sci. 2024;11:1394814. doi:10.3389/fvets.2024.1394814
  3. Hernandez-Pastor L, Geurtsen J, Baugh B, et al.Clinical burden of invasive Escherichia coli disease among older adult patients treated in hospitals in the United States. BMC Infect Dis. 2023;23:550. doi:10.1186/s12879-023-08479-3
  4. Yoo S, Lee KM, Kim N, Vu TN, Abadie R, Yong D. Designing phage cocktails to combat the emergence of bacteriophage-resistant mutants in multidrug-resistant Klebsiella pneumoniae. Microbiol Spectr. 2024;12:12:e01258-23. doi:10.1128/spectrum.01258-23
  5. Zalewska-Piątek B. Phage therapy—challenges, opportunities and future prospects. Pharmaceuticals. 2023;16(12):1638. doi:10.3390/ph16121638
  6. Longhi C, Maurizi L, Conte AL, et al. Extraintestinal pathogenic Escherichia coli: beta-lactam antibiotic and heavy metal resistance. Antibiotics. 2022;11(3):328. doi:10.3390/antibiotics11030328
  7. Whelan S, Lucey B, Finn K. Uropathogenic Escherichia coli (UPEC)-associated urinary tract infections: the molecular basis for challenges to effective treatment. Microorganisms. 2023;11(9):2169. doi:10.3390/microorganisms11092169
  8. Gashaw M, Gudina EK, Tadesse W, et al. Hospital wastes as potential sources for multi-drug-resistant esbl-producing bacteria at a tertiary hospital in Ethiopia. Antibiotics. 2024;13(4):374. doi:10.3390/antibiotics13040374
  9. Ribeiro LF, Nespolo NM, Rossi GAM, Fairbrother JM. Exploring Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli in Food-Producing Animals and Animal-Derived Foods. Pathogens. 2024;13(4):346. doi:10.3390/pathogens13040346
  10. Chaisaeng S, Chopjitt P, Kasemsiri P, et al. High prevalence of ESBL-producing E. coli phylogroup B2 clinical isolates in northeastern Thailand. BMC Microbiol. 2024;24(1). doi:10.1186/s12866-024-03582-0
  11. Husna A, Rahman MM, Badruzzaman ATM, et al. Extended-spectrum β-lactamases (ESBL): challenges and opportunities. Biomedicines. 2023;11(11):2937. doi:10.3390/biomedicines11112937
  12. Cho S, Jackson CR, Frye JG. Freshwater environment as a reservoir of extended-spectrum β-lactamase-producing Enterobacteriaceae.  J Appl Microbiol. 2023;134(3):lxad034. doi:10.1093/jambio/lxad034
  13. Hitchcock NM, Devequi Gomes Nunes D, Shiach J, et al. Current clinical landscape and global potential of bacteriophage therapy. Viruses. 2023;15(4):1020. doi:10.3390/v15041020
  14. Cui L, Watanabe S, Miyanaga K, et al. A Comprehensive review on phage therapy and phage-based drug development. Antibiotics. 2024;13(9):870. doi:10.3390/antibiotics13090870
  15. Jacob ME, Keelara S, Aidara-Kane A, Matheu Alvarez JR, Fedorka-Cray PJ. Optimizing a screening protocol for potential extended-spectrum β-lactamase Escherichia coli on macconkey agar for use in a global surveillance program. J Clin Microbiol. 2020;58(9):01039-19. doi:10.1128/jcm.01039-19
  16. Wang J, Cahyadi A, Wu B, Pee W, Fane AG, Chew JW. The roles of particles in enhancing membrane filtration: A review.  J. Membr. Sci. 2020;595:117570. doi:10.1016/j.memsci.2019.117570
  17. Tripathi N, Zubair M, Sapra A. Gram staining. In: StatPearls. Treasure Island,

FL: StatPearls Publishing;2026.

  1. Cowan S. Bergey's manual of determinative bacteriology. Nature. 1948:162:833.

doi:10.1038/162833a0

  1. Luo W, He B, Han D, et al. The clinical performance of fetal sex chromosome abnormalities in serum biochemical screening in the second trimester. Sci Rep. 2024;14: 29011. doi:10.1038/s41598-024-78724-5
  2. Ahmeed HW, Al-Khikani FHO, Khudair KK. The antibacterial activity of lactophenol, safranin, and methyl red dyes against clinically isolated Escherichia Coli. Assam J Intern Med. 2024;14(1):3-7. doi:10.4103/ajoim.ajoim_4_24
  3. Mekonnen E, Kebede A, Nigussie A, Kebede G, Tafesse M. Isolation and characterization of urease-producing soil bacteria. Intern J Microbiol. 2021;2021:1-11. doi:10.1155/2021/8888641
  4. Shoaib M, Muzammil I, Hammad M, Bhutta ZA, Yaseen I. A mini-review on commonly used biochemical tests for identification of bacteria. Intern J Res Publ. 2020;54(1):1-6. doi:10.47119/ijrp100541620201224
  5. Artati D, Oman M, Supriyanto S, Sukarta D, Robisalmi A. Validation of analytical method for aeromonas hydrophila identification using analytical profile index (API) 20E KIT method. J Med Vet. 2024;7(1):88-104. doi:10.20473/jmv.vol7.iss1.2024.88-104
  6. Howard A, Hughes DM, Green PL, et al. Personalised antimicrobial susceptibility testing with clinical prediction modelling informs appropriate antibiotic use. Nat Commun. 2024;15:9924. doi:10.1038/s41467-024-54192-3
  7. Das P, Mahapatra D, Mazumder SS. A Guide Towards the phenotypic detection of extended-spectrum β-lactamases production in enterobacteriaceae: alone or in presence of other interfering enzymes. J Pure Appl Microbiol. 2023;17(3):1410-1421. doi:10.22207/jpam.17.3.31
  8. Amro MM, Güler E, Süer K, Güvenir M. Which phenotypic method is the most accurate for detection of extended–spectrum β-lactamases (ESBLs) in Escherichia coli? Clin Exp Health Sci. 2022;12(3): 624-628. doi:10.33808/clinexphealthsci.987433
  9. Zimoń B, Psujek M, Matczak J, Guziński A, Wójcik E, Dastych J. Novel multiplex-PCR test for Escherichia colidetection. Microbiol Spectr. 2024;12(6):e03773-23. doi:10.1128/spectrum.03773-23
  10. Stachurska X, Roszak M, Jabłońska J, Mizielińska M, Nawrotek P. Double-layer agar (DLA) modifications for the first step of the phage-antibiotic synergy (PAS) identification. Antibiotics. 2021;10(11):1306. doi:10.3390/antibiotics10111306
  11. Lapras B, Marchand C, Merienne C, et al. Rationalisation of the purification process for a phage active pharmaceutical ingredient. Eur J Pharm Biopharm. 2024;203:114438. doi:10.1016/j.ejpb.2024.114438
  12. Paranos P, Pournaras S, Meletiadis J. A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens.  J Clin Microbiol. 2024;62(8):e0074324. doi:10.1128/jcm.00743-24
  13. Niazy AA, Alrashed MM, Lambarte RNA, Niazy AA. 5-Fluorouracil inhibits bacterial growth and reduces biofilm in addition to having synergetic effects with gentamicin against pseudomonas aeruginosa. Microorganisms. 2024;12(11):2257. doi:10.3390/microorganisms12112257
  14. Shree S, Suman E, Kotian H, Paul SH, M SS. Effect of lebsiella-specific phage on multidrug-resistant Klebsiella pneumoniae- an experimental study. Indian J Med Microbiol. 2024;47:100515. doi:10.1016/j.ijmmb.2023.100515
  15. Beshiru A, Isokpehi NA, Igbinosa IH, Akinnibosun O, Ogofure AG, Igbinosa EO. Extended-spectrum beta-lactamase (ESBL)- and non-ESBL producing Escherichia coli surveillance in surface water sources in Edo State, Nigeria: a public health concern. Sci Rep. 2024;14(1)::21658. doi:10.1038/s41598-024-72993-w
  16. Yanestria SM, Dameanti F, Musayannah BG, et al. Antibiotic resistance pattern of Extended-Spectrum ?-Lactamase (ESBL) producing Escherichia coli isolated from broiler farm environment in Pasuruan district, Indonesia. Biodiversitas. 2022;23(9):4460-4465. doi:10.13057/biodiv/d230911
  17. Davies DT, Everett M. Designing inhibitors of β-lactamase enzymes to overcome carbapenem resistance in gram-negative bacteria. Acc Chem Res. 2021;54(9):2055-2064. doi:10.1021/acs.accounts.0c00863
  18. Mó I, da Silva GJ. Tackling Carbapenem resistance and the imperative for one health strategies—insights from the portuguese perspective. Antibiotics. 2024;13(6):557. doi:10.3390/antibiotics13060557
  19. Abdullah AA, Al-Taee HA. Phenotypic characterization of uropathogenic Escherichia coli in patients of Nineveh province in Iraq. Malaysian J Microbiol. 2024;20(6):350-355. doi:10.21161/mjm.240051
  20. Alhadlaq MA, Aljurayyad OI, Almansour A, et al. Overview of pathogenic Escherichia coli, with a focus on Shiga toxin-producing serotypes, global outbreaks (1982–2024) and food safety criteria. Gut Pathog. 2024;16(1):57. doi:10.1186/s13099-024-00641-9
  21. Pearse O, Zuza A, Tewesa E, et al. High diversity of Escherichia coli causing invasive disease in neonates in Malawi poses challenges for O-antigen based vaccine approach. Commun Med. 2025;5(1):298. doi:10.1038/s43856-025-01007-1
  22. Siopi M, Skliros D, Paranos P, et al. Pharmacokinetics and pharmacodynamics of bacteriophage therapy: a review with a focus on multidrug-resistant Gram-negative bacterial infections. Clin Microbiol Rev. 2024;37(3):e0004424. doi:10.1128/cmr.00044-24
  23. Zhang K, Pankratz K, Duong H, et al. Interactions between Viral Regulatory Proteins Ensure an MOI-Independent Probability of Lysogeny during Infection by Bacteriophage P1. mBio. 2021;12(5):e0101321. doi:10.1128/mbio.01013-21
Next article in this issue
Share
Back to top
Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here
Accept