Isolation and characterization of a therapeutic lytic phage cocktail against mastitis-associated Enterococcus faecalis with antibiofilm activity and in vivo efficacy.

BACKGROUND

Antimicrobial-resistant (AMR) mastitis-associated Enterococcus faecalis and Enterococcus faecium threaten dairy production, animal welfare, and public health due to economic losses and zoonotic risk. Environmental mastitis is increasingly linked to E. faecalis, which is frequently detected in milk and animal-derived products.

METHODS

Three novel lytic bacteriophages-vB_Ef3_HMKU_24 (vB_Ef3), vB_Ef5_HMKU_24 (vB_Ef5), and vB_Ef32_HMKU_24 (vB_Ef32)-were isolated from wastewater in Türkiye and characterized for host range, stability, replication kinetics, antibiofilm activity, in vivo efficacy (Galleria mellonella), and genomic features.

RESULTS

The phages vB_Ef3, vB_Ef5, and vB_Ef32 exhibited strong and specific lytic activity against E. faecalis (up to 69.1% individually; 72.7% as a cocktail) with no activity against other species. They remained active across pH 4-10 and up to 70 °C. Optimal MOIs were 10 (vB_Ef3), 0.1 (vB_Ef5), and 0.001 (vB_Ef32). vB_Ef5 and vB_Ef32 fully suppressed bacterial growth for 24 h, whereas vB_Ef3 caused transient inhibition. Burst sizes were ∼2019, ∼153, and ∼287 PFU/cell for vB_Ef3, vB_Ef5, and vB_Ef32, respectively. All phages significantly inhibited and eradicated biofilms; vB_Ef5 was the most effective single agent (71% inhibition, 68% eradication), while the cocktail achieved the highest activity (82% and 73%). In the G. mellonella infection model, phage treatment improved larval survival, with the cocktail reaching ∼90% and showing no toxicity. Genomic analyses (40.3-58.6 kb; 34.9-39.5% GC; 78-118 ORFs) confirmed strictly lytic phages encoding endolysins and lacking virulence or AMR genes, classified within the Herelleviridae.

CONCLUSION

Collectively, these findings demonstrate that the combined use of vB_Ef3, vB_Ef5, and vB_Ef32 represents a safe, stable, and highly effective phage cocktail with strong potential as a precision therapeutic strategy against multidrug-resistant E. faecalis-associated bovine mastitis.