# Unveiling Novel Lytic Bacteriophages as Natural Biocontrol Agents Against Multidrug-Resistant Escherichia coli: Isolation, Characterization, and In vitro Application

## TL;DR

This study isolates and characterizes four new bacteriophages that effectively target multidrug-resistant Escherichia coli, showing potential for biocontrol applications.

## Contribution

The discovery of novel lytic bacteriophages with strong activity against MDR-E. coli and their classification as new Vectrevirus members.

## Key findings

- Phage Sem4 showed stronger lytic activity than a phage cocktail, fully suppressing MDR-E. coli growth.
- The phages remained stable for up to a year at 4°C and effectively reduced bacterial counts in tap water.
- Genomic analysis revealed novel Vectrevirus members with no genes linked to antibiotic resistance or lysogeny.

## Abstract

The alarming findings presented in the latest WHO report on the global antimicrobial resistance crisis have redirected scientific attention toward phage-based approaches as a renewed line of defense against multidrug-resistant (MDR) bacteria. In this study, four bacteriophages infecting a MDR-Escherichia coli strain were isolated from water sources and subjected to detailed phenotypic and genomic characterization. All phages efficiently inhibited MDR-E. coli at MOIs of 0.1 and 0.01, showing high stability across a broad temperature (4–65 °C) and pH (4–10) range. TEM analysis revealed that all phages exhibited a podovirus-morphotype. At 4 °C, titers remained stable for 6 months, with only a 1–2 log reduction -over a year. Notably, phage Sem4 exhibited markedly stronger lytic activity than the phage cocktail, fully suppressing bacterial growth. In tap water, phage Sem4 treatment reduced bacterial counts from 10⁷ to 7 × 10⁴ CFU/mL within 8 h, with no detectable colonies at 24–48 h. Genomic analysis showed that these phages possess linear dsDNA genomes of 44,244–45,205 bp, with ~ 45% GC content. Phylogenetic and comparative analyses classified them as novel Vectrevirus members within the Molineuxvirinae subfamily of the Autographiviridae family, sharing less than 95% intergenomic similarity with known Vectrevirus species. No genes associated with antibiotic resistance, toxins, or lysogeny were detected. These findings underscore the potential of - phages as a promising candidate for the development of next-generation biocontrol strategies, especially marking the efficiency of Sem4 in water sanitation systems, paving the way for sustainable and targeted interventions against MDR bacterial contamination.

The online version contains supplementary material available at 10.1007/s12560-026-09684-4.