Therapeutic potential of three virulent bacteriophages targeting clinical Enterobacter hormaechei strains: comprehensive biological properties and genomic functional analysis

The emergence of multidrug-resistant Enterobacter cloacae complex (ECC) strains, particularly Enterobacter hormaechei, underscores the urgent need for alternative antimicrobial strategies. This study reports the isolation and comprehensive characterization of three virulent bacteriophages - Enterobacter phage Entb_46, Enterobacter phage Entb_47, and Enterobacter phage Entb_48 - targeting clinical E. hormaechei strains. The phages were isolated from municipal wastewater and exhibited lytic activity against a broad panel of clinical ECC isolates, with Enterobacter phage Entb_46 demonstrating the widest host range. Morphological evaluation using transmission electron microscopy identified all three phages as myoviruses. Genomic sequencing confirmed dsDNA genomes ranging from 11.6 to 179.3 kb and revealed the absence of genes associated with lysogeny, antibiotic resistance, or bacterial virulence, supporting their strictly lytic replication cycle. Functional annotation identified genes involved in host lysis, structural assembly, replication, and DNA packaging. Comparative analyses further classified Enterobacter phage Entb_46 within the Pseudotevenvirus genus, Enterobacter phage Entb_48 within the Slopekvirinae subfamily as a new genus candidate, and Enterobacter phage Entb_47 as a representative of a novel family and genus. Biological assays demonstrated efficient adsorption (89% after 12.5 min for Entb_46; 47% after 7.5 min for Entb_47; 67% after 10 min for Entb_48), relatively short latency periods (25, 20, and 10 min, respectively), and burst sizes ranging from 4 to 43 PFU/cell, along with strong antibacterial activity among a wide range of multiplicity of infection rates (from 0.001 to 1000). All phages exhibited stability under various temperature (from -20 to 60 °C) and pH conditions (from 3 to 11), although long-term exposure to extreme values significantly reduced their titers. However, Entb_48 phage remained stable at 70 and 80 °C and at pH 2 after 1 h of incubation. Together, these findings preliminarily highlight the therapeutic potential of newly isolated Enterobacter-specific phages and support their future development for phage therapy applications targeting ECC-associated infections.