Phage-based suppression of plasmid-mediated antibiotic resistance: A promising approach for antimicrobial stewardship

The global spread of antibiotic resistant bacteria (ARB), particularly plasmid-bearing strains, poses a major threat to public health due to their association with life-threatening infections. As alternatives to conventional antibiotics become increasingly necessary, bacteriophages are recognized as promising tools for controlling ARB. This study recovered three lytic phages, vB_EcoP_LHP, vB_EcoP_IUE, and vB_EcoM_BL from agricultural soil, which specifically target Escherichia coli K12 carrying the multidrug-resistant plasmid RP4. Based on morphological and phylogenetic analyses, phage LHP was classified within the genus Cronosvirus, subfamily Melnykvirinae, phage BL within the species Felixounavirus JLBYU32, genus Felixounavirus, subfamily Ounavirinae, while phage IUE was identified as a novel Caudoviricetes phage. No virulence, antibiotic resistance, or lysogeny-associated genes were detected, supporting their biosafety for application. Phages LHP and IUE exhibited strong lytic activity against E. coli strains carrying RP4 and pKJK5, with optimal multiplicity of infection of 100 and estimated burst sizes of 40 and 600 PFU/cell, respectively, but were more sensitive to high temperatures. In contrast, phage BL displayed a broader host range, an estimated burst size of approximately 45 PFU/cell, and higher thermal stability. In soil microcosms, substantial reductions in E. coli K12 (RP4) and plasmid persistence were achieved through phage treatment, with the cocktail showing the most rapid and sustained suppression. Notably, durable inhibition was conferred by the phage IUE, whereas moderate effects were exerted by phages LHP and BL. Collectively, these findings highlight the potential of strictly lytic putative plasmid-dependent phages, particularly in cocktails, as eco-friendly biocontrol agents for constraining the environmental dissemination of plasmid-mediated antibiotic resistance. SYNOPSIS: This study isolated and characterized three novel lytic phages and demonstrated their potential, individually and in cocktails, to control soil-borne antibiotic resistant bacteria and plasmid-mediated resistance.