Isolation and characterization of lytic Escherichia coli phages from Al-Zarqa River and evaluation of their in vitro antibiofilm activity

Pharmacia 73: e188523 DOI 10.3897/pharmacia.73.e188523 Research Article Isolation and characterization of lytic Escherichia coli phages from Al-Zarqa River and evaluation of their in vitro antibiofilm activity Yara Al Tall1 , Amal Al-qasem1, Nid’A Alshraiedeh1 , Mohammad Alsaggar1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid 22110, Jordan Corresponding author: Yara Al Tall () Received 12 February 2026 ♦ Accepted 7 April 2026 ♦ Published 5 May 2026 Citation: Al Tall Y, Al-qasem A, Alshraiedeh N, Alsaggar M (2026) Isolation and characterization of lytic Escherichia coli phages from Al-Zarqa River and evaluation of their in vitro antibiofilm activity. Pharmacia 73: e188523. https://doi.org/10.3897/pharmacia.73.e188523 Abstract The increasing prevalence of antibiotic-resistant Escherichia coli and its ability to form biofilms necessitate alternative antimicrobial strategies. In this study, two lytic bacteriophages infecting E. coli were isolated from freshwater samples collected from the Al-Zarqa River in Jordan and characterized phenotypically. Transmission electron microscopy identified both phages as tailed viruses within the order Caudovirales, provisionally classified as Podoviridae-like and Myoviridae-like phages. Both phages exhibited efficient lytic activity against the reference host strain, with latent periods of 20–30 min and burst sizes of 5–9 plaque-forming units per infected cell. The phages remained stable across a wide range of temperatures and pH values, retaining infectivity during storage at 4 °C. Host-range analysis demonstrated narrow strain specificity. Both phages significantly inhibited biofilm formation and reduced established biofilms in a dose-dependent manner. These results support the potential of the isolated phages as promising antibiofilm agents for further development. Keywords Antibiofilm agents, biofilm inhibition, Caudovirales, freshwater isolate, lytic bacteriophages, phage therapy Introduction Infectious diseases continue to pose a serious threat to public health and global economic stability (Qu et al. 2025). Throughout history, they have ranked among the leading causes of mortality and disability, and in the 21st century, this threat has intensified due to the emergence and re-emergence of novel pathogens (Bloom and Cadarette 2019; De Gaetano et al. 2025). Over the past three decades, at least 30 new infectious agents affecting humans have been identified, most of which are zoonotic in origin and influenced by socioeconomic, ecological, and environmental factors (Nii-Trebi 2017; Carlson et al. 2022). These conditions have increased human exposure to infectious agents, underscoring the need for effective and adaptable strategies to combat infectious disease threats (Ortiz-Millan 2025). The advent of antibiotics in the 20th century revolutionized modern medicine, drastically reducing mortality from bacterial infections (Hutchings et al. 2019). However, the widespread and often inappropriate use of antibiotics has led to the global crisis of antimicrobial resistance (AMR) (Estany-Gestal et al. 2024). Bacteria can develop resistance through spontaneous mutations or by acquiring resistance genes from other organisms (Ghosh et al. 2019). Resistant Copyright Al Tall Y et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 organisms are now found in humans, animals, food, water, and the environment, making AMR a universal challenge (Sharma et al. 2024). Of particular concern are multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) bacterial strains, which severely limit treatment options (Tacconelli et al. 2018). MDR is defined as non-susceptibility to at least one agent in three or more antimicrobial categories, XDR as non-susceptibility to at least one agent in all but two or fewer categories, and PDR as non-susceptibility to all agents in all antimicrobial categories tested (Magiorakos et al. 2012). The World Health Organization (WHO) ranks antimicrobial resistance among the top global health threats, with recent estimates highlighting its increasing burden and projected impact if left unaddressed (Estany-Gestal et al. 2024). The growing ineffectiveness of current antibiotics underscores the urgent need for novel antibacterial strategies and alternative therapeutic options. Among these, bacteriophage therapy has regained attention as a promising solution to combat resistant infections (Gordillo Altamirano and Barr 2019). Bacteriophages are viruses that infect bacteria and can follow either a lytic or lysogenic lifecycle, with only lytic phages causing immediate bacterial cell lysis (Kortright et al. 2019; Shang et al. 2025). They can be readily isolated from natural sources, such as sewage and rivers, especially in regions with high bacterial loads (Nale and Clokie 2021). Phages are highly specific, self-amplifying, relatively inexpensive to isolate, and can be formulated in multiple delivery formats, including liquids, creams, and solid supports (Olawade et al. 2024). Their ability to target biofilm-forming bacteria is particularly relevant, given that biofilms contribute to chronic and recalcitrant infections (Mayorga-Ramos et al. 2024). In this study, the aim was to isolate and characterize bacteriophages from the Al-Zarqa River in Jordan with lytic activity against Escherichia coli, a clinically significant pathogen known for its AMR potential and biofilm-forming capacity (Ramatla et al. 2023). The morphology, lytic activity, growth characteristics, environmental stability, host range, biofilm-disrupting capabilities, and long-term storage stability of the isolated phages were investigated to assess their therapeutic potential. Materials and methods Bacterial hosts Four reference strains obtained from the American Type Culture Collection (ATCC) were used in this study: Escherichia coli (ATCC 25922), E. coli (ATCC BAA-2452), Staphylococcus aureus (ATCC 29213), and S. aureus (ATCC BAA-44). All bacterial strains were cultured in 8 mL of Luria-Bertani (LB) broth at 37 °C with shaking at 110 rpm for 3–4 h to reach the logarithmic growth phase. Each batch included parallel subcultures of the inoculum on LB agar plates and media controls, followed by 48-hour incubation at 37 °C to confirm the purity of the bacterial cultures and sterility of the media. Al Tall Y et al.: Lytic E. coli phages from Al-Zarqa River Sample collection Surface water samples were collected from the AlZarqa River (ZR) at Jerash Bridge (Jordan) using sterile 500 mL polypropylene containers. The precise GPS location of the collection site was recorded (32°13’03.7”N, 35°52'54.1"E). Samples were transported to the laboratory within 2 h of collection in (...truncated)