Novel strategies for vancomycin-resistant Enterococcus faecalis bioflm control: bacteriophage (vB_EfaS_ZC1), propolis, and their combined efects in an ex vivo endodontic model

Abstract

Background: Endodontic treatment failures are predominantly attributed to Enterococcus faecalis (E. faecalis) infection, a Gram-positive coccus. E. faecalis forms biofilms, resist multiple antibiotics, and can withstand endodontic disinfection protocols. Vancomycin-resistant strains, in particular, are challenging to treat and are associated with serious medical complications.

Methods: A novel phage, vB_EfaS_ZC1, was isolated and characterized. Its lytic activity against E. faecalis was assessed in vitro through time-killing and biofilm assays. The phage's stability under various conditions was determined. Genomic analysis was conducted to characterize the phage and its virulence. The phage, propolis, and their combination were evaluated as an intracanal irrigation solution against a 4-week E. faecalis mature biofilm, using an ex vivo infected human dentin model. The antibiofilm activity was analyzed using a colony-forming unit assay, field emission scanning electron microscopy, and confocal laser scanning microscopy.

Results: The isolated phage, vB_EfaS_ZC1, a siphovirus with prolate capsid, exhibited strong lytic activity against Vancomycin-resistant strains. In vitro assays indicated its effectiveness in inhibiting planktonic growth and disrupting mature biofilms. The phage remained stable under wide range of temperatures (- 80 to 60 °C), tolerated pH levels from 4 to 11; however the phage viability significantly reduced after UV exposure. Genomic analysis strongly suggests the phage's virulence and suitability for therapeutic applications; neither lysogeny markers nor antibiotic resistance markers were identified. Phylogenetic analysis clustered vB_EfaS_ZC1 within the genus Saphexavirus. The phage, both alone and in combination with propolis, demonstrated potent antibiofilm effects compared to conventional root canal irrigation.

Conclusion: Phage vB_EfaS_ZC1 demonstrates a promising therapy, either individually or in combination with propolis, for addressing challenging endodontic infections caused by E. faecalis.