Characterization and Host Phage Interaction Analysis of Two Lytic Bacteriophages Targeting Anti-Microbial Resistant Escherichia coli

Abstract

Background: Antimicrobial resistance in Escherichia coli (E. coli), represents a significant and growing public health threat, driven not only by the overuse and misuse of antibiotics but also aggravated by environmental factors such as pollution and insufficient wastewater treatment systems. This current situation highlights the urgent need for alternatives to antibiotics, particularly natural ones like bacteriophages, to combat antimicrobial resistance (AMR). This study reports the characterization of two lytic bacteriophages, EPIMAM01 (gb:PQ493298) and EPIMRB01 (gb:PQ657784), from untreated sewage in Beirut, Lebanon.

Methods: Isolated phages characterized by their bacteriolytic activity, their latency period and burst size determined by a one-step growth assay, their ability to clear bacterial biofilm post-formation, and their stability under different temperatures and pH values, alongside phage sequencing to identify the phage whole genome. We also demonstrated a genomic-driven approach to predict the phage-host susceptibility of the lytic phages EPIMAM01 and EPIMRB01 using a supervised machine learning model employing logistic regression. The model aims to identify E. coli strains susceptible to each phage, ensuring effective and targeted treatment.

Results: Both phages exhibited potent activity against E. coli ATCC 25922. One-step growth curve analysis revealed a relatively short latent period for both phages, with a high burst size for both phages. Stability tests demonstrated optimal viability at pH 7 and temperatures up to 50°C. In bacteriolytic assays, both phages exhibited dose-dependent E. coli lysis, with EPIMAM01 showing faster and more sustained lytic activity compared to EPIMRB01. Interestingly, while EPIMRB01 effectively disrupted mature biofilms, EPIMAM01 displayed no efficacy in this regard. Whole-genome sequencing classified both phages within the Myoviridae family and revealed the absence of lysogeny, virulence, or antimicrobial resistance genes, reinforcing their suitability for therapeutic applications. These complementary characteristics highlight the potential of EPIMAM01 and EPIMRB01 as a synergistic therapeutic duo, combining the high virulence of EPIMAM01 with the biofilm-clearing capacity of EPIMRB01. Host range analysis confirmed the phages’ virulence against clinical E. coli isolates, with efficacy-of-plating (EOP) values supporting their classification as highly virulent. A logistic regression analysis of bacterial genes influencing bacterial susceptibility to both phages identified yfcS_1, yfcS_2, mboIIM, repB and vgrG1as both susceptibility-enhancing and resistance-conferring features when modeling host–phage interactions. Conclusion: These findings underscore the potential of EPIMAM01 and EPIMRB01 lytic bacteriophages as natural alternatives to mitigate AMR in E. coli, particularly in regions with a high prevalence of multidrug-resistant strains.