Novel lytic phages improve the antibiofilm activity of dalbavancin, daptomycin, and fosfomycin against vancomycin-resistant enterococci

ABSTRACT Periprosthetic joint infections (PJI) caused by Enterococcus spp., especially vancomycin-resistant strains (VRE), are challenging to treat due to biofilm tolerance and limited antibiotic options. Bacteriophages offer a promising adjunct through targeted and biofilm-disrupting activity. This study evaluated two novel lytic phages, alone and combined with last-line antibiotics, for their ability to eradicate VRE biofilms in vitro . Two novel lytic phages, CUB-FM ( E. faecium ) and CUB-FS ( E. faecalis ), were isolated from hospital sewage and characterized via whole-genome sequencing and transmission electron microscopy. Antibiofilm efficacy of phages alone and in combination with dalbavancin, daptomycin, and fosfomycin was assessed against biofilm-embedded VRE strains using isothermal microcalorimetry. Synergy was defined as a combined effect exceeding the sum of individual activities. Genomic analysis confirmed both phages as strictly lytic and free of lysogeny, virulence, or resistance genes. TEM classified CUB-FM within Salasmaviridae and CUB-FS within Herelleviridae . Both exhibited dose-dependent antibiofilm activity, with optimal efficacy at 10¹² (CUB-FM) and 10⁸ PFU/mL (CUB-FS). While antibiotic monotherapies showed limited antibiofilm effects, phage-antibiotic combinations markedly enhanced activity. CUB-FM with dalbavancin achieved the strongest suppression against E. faecium (t Max 22.3 h vs. 5.2 h control, P < 0.001), and CUB-FS with dalbavancin or fosfomycin at ≥10 × MIC completely eradicated E. faecalis biofilms. Daptomycin-phage combinations produced additive to synergistic effects. Novel phages CUB-FM and CUB-FS exhibit potent antibiofilm activity and synergize with last-line antibiotics against VRE. Phage-antibiotic combinations, particularly with dalbavancin and fosfomycin, represent a promising strategy for treating biofilm-associated enterococcal PJIs. IMPORTANCE Vancomycin-resistant enterococci (VRE) are increasingly implicated in biofilm-associated periprosthetic joint infections, where treatment options are limited, and clinical outcomes are poor. Conventional antibiotics often fail due to reduced biofilm penetration and bacterial tolerance, highlighting the need for novel therapeutic strategies. Our study introduces two newly characterized lytic phages, CUB-FM and CUB-FS, which demonstrated strong antibiofilm activity and synergistic interactions with last-line antibiotics. Notably, phage-antibiotic combinations achieved either additive or synergistic effects, with dalbavancin and fosfomycin-phage therapy leading to a complete eradication of E. faecalis biofilms. These findings provide proof of concept that combining phages with antibiotics enhances efficacy against multidrug-resistant Enterococcus biofilms, offering a translational pathway for personalized, adjunctive therapies in complex orthopedic infections. By bridging the gap between genomic phage safety validation and functional synergy testing, this work supports further preclinical and clinical development of phage-antibiotic strategies for refractory implant-associated infections.