Characterization of bacteriophage Henu4_2 lytic for Escherichia coli and its therapeutic efficacy in infection models

An Escherichia phage, designated Henu4_2, was isolated from hospital sewage sludge. This phage exhibits strict lytic specificity against certain Escherichia coli strains and demonstrates potent antimicrobial and antibiofilm activities in both in vitro and in vivo experiments. It shows an optimal multiplicity of infection (MOI) of 0.1 and displays broad stability across a wide range of temperature (4-55 °C) and pH values (3-12), although it remains sensitive to UV irradiation. The phage features a latent period of 20 min and a lytic cycle of 60 min, with a burst size of 354 PFU per infected cell Genomic analysis revealed a circular double-stranded DNA genome of 50,260 bp with a G + C content of 43%. Phylogenetically, Henu4_2 clusters within the Tlsvirus genus, showing 95.61% sequence identity with Escherichia phage TLS and 93.1% with phage U136B. The genome encodes multiple functional proteins involved in viral replication and maturation, including an endolysin (ORF16), a holin (ORF17), a terminase large subunit (ORF54), and a terminase small subunit (ORF55). Notably, treatment with Henu4_2 at higher MOIs significantly improved the survival of Galleria mellonella larvae upon bacterial challenge. Concurrently, we observed that Henu4_2 rapidly lysed bacterial cells, thereby reducing the bacterial load within infected hosts. These findings indicate that Henu4_2 is a promising candidate for phage therapy against E. coli infections. Moreover, its comprehensive characterization and genomic analysis contribute valuable insights into phage diversity and biological function.