BACKGROUND

The global emergence of vancomycin-resistant Enterococci (VRE) represents a growing threat to public health worldwide. To address this critical challenge, we isolated and characterized a novel lytic bacteriophage, ENP2309, from agricultural wastewater. Comprehensive analysis revealed distinct morphological features, biological properties, and genomic characteristics of ENP2309. Most notably, systematic evaluation in a mice infection model demonstrated significant in vivo therapeutic efficacy.

METHODS

Bacteriophage isolation was performed using the double-layer agar method with the Enterococcus faecalis strain. Phage morphology was characterized by transmission electron microscopy (TEM), The host range was determined via plaque assays and the plating efficiency of multiple bacterial isolates. was evaluated double-layer agar method was systematically employed to evaluate thermal stability, pH tolerance, one-step growth kinetics, and the optimal multiplicity of infection (MOI) through plaque-forming unit (PFU) enumeration. The genomic features were analysed using next-generation sequencing. Furthermore, the therapeutic efficacy of phage ENP2309 against Enterococcal infection in mice was systematically evaluated through a comprehensive assessment of multiple parameters including body weight dynamics, survival rates, histopathological analysis, peripheral blood cytokine profiles, and bacterial loads in the spleen and liver tissues, demonstrating its multidimensional therapeutic effects.

RESULTS

The phage ENP2309 showed broad-spectrum lytic capability, effectively targeting 13 distinct Enterococcus clinical isolates. TEM revealed the morphology of ENP2309, featuring an icosahedral capsid (70 ± 1 nm in diameter) and a contractile tail structure (145 ± 2 nm in length). Comprehensive biological characterization revealed optimal infection parameters including an exceptionally low multiplicity of infection (MOI = 0.001), a 40-minute latent period, and an extended 40-120 min burst period resulting in a burst size of 920 PFU/cell. The phage exhibited environmental stability, maintaining infectivity across broad temperature (10-60 °C) and pH (3-12) ranges, with optimal activity observed at 37 °C and neutral pH (7.0-7.5). Genomic analysis revealed a 148,806 bp linear dsDNA (35.9% GC content) containing 153 putative ORFs. Phylogenetic classification revealed that ENP2309 a member of the Kochikohdavirus genus, is closely related to the Enterococcus phage PBEF129. In vivo studies demonstrated exceptional therapeutic potential: a single dose (200 µL 2.0 × 10⁸ PFU/mL) of phage ENP2309 achieved 100% survival in mice models, completely clearing VRE from the spleen and liver while significantly improving physiological parameters, reducing organ damage, and attenuating systemic inflammation.

CONCLUSIONS

These comprehensive findings establish ENP2309 as a highly promising therapeutic alternative to conventional antibiotics for VRE infections with distinct advantages.