A Novel Enterococcus Phage Exhibits Potent Anti-biofilm Activity Against Multidrug Resistant Strains and Protects Mice from Peritonitis Sepsis.

Enterococci are opportunistic pathogens that circulate among humans, animals, and the environment, and multidrug-resistant (MDR) enterococci have emerged as a growing One Health concern. In this study, a lytic Enterococcus phage, PEX, was isolated from horse-stable sewage using an equine Enterococcus faecium strain as the host. The antibacterial activity of PEX against MDR Enterococcus isolates of both human and animal origin was systematically investigated, and its therapeutic efficacy was evaluated in both in vitro and in vivo models. Phage PEX was isolated and purified using the double-layer agar technique. The stability, lytic activity, and host range of PEX were determined, and whole-genome sequencing was performed to characterize its biological features. Furthermore, its antibiofilm and therapeutic effects were evaluated using confocal microscopy, biofilm removal assays, and a mouse peritonitis sepsis model. The results showed that PEX exhibited a siphovirus-like morphology. Genomic analysis revealed that PEX is closely related to previously reported Enterococcus phages but possesses distinct genomic characteristics and lacks virulence, antibiotic resistance, and lysogeny-related genes. EOP assays confirmed that PEX exhibited a broad lytic host range, infecting multiple Enterococcus species, including clinical isolates and strains from diverse animal sources. Phage PEX remained stable at temperatures ranging from 4 °C to 50 °C and across pH values of 5-11. The optimal multiplicity of infection (MOI) was 0.1, and strong inhibition of planktonic bacterial growth was observed across different MOIs. Importantly, phage PEX significantly disrupted biofilms formed by MDR Enterococcus strains in a concentration-dependent manner. In the murine peritoneal septicemia model, PEX significantly reduced bacterial dissemination and alleviated organ damage. Collectively, these findings demonstrate that phage PEX exhibits potent antibacterial activity against MDR Enterococcus strains both in vitro and in vivo, highlighting its potential as a promising candidate for controlling MDR Enterococcus infections.