Novel Lytic Enterococcus Phage as a Biocontrol Strategy for Multidrug-Resistant Enterococcus faecalis Biofilms on Food-Contact Surfaces: Genomic and Functional Insights

Abstract Enterococcus faecalis is one of the most prevalent biofilm-forming bacteria that persists on dairy processing surfaces, posing significant food safety and health concern. In this study, two lytic bacteriophages, BV26 and BV34, were isolated and characterized from sewage for use as biocontrol for E. faecalis . Both types of phages belong to the family of Siphoviridae , as observed by transmission electron microscopy. The level of stability for the phage BV26 was evaluated to determine its potential biocontrol effectiveness at a wide range of pH levels (3 to 12), UV resistance, and thermal stability up to 50°C. Whole-genome sequencing of BV26 revealed their predicted coding sequences, with no detectable virulence or antimicrobial resistance genes, supporting its safety for biocontrol applications. Comparative genomic and phylogenetic analyses indicated that BV26 represents a distinct lineage among Enterococcus phages. BV26 was tested for anti-biofilm activity against planktonic and biofilm-bound E. faecalis . In microtiter-plate biofilm formation assays, the anti-biofilm effect of phage BV26 was most pronounced when BV26 was added at a multiplicity of infection of 100. In a dairy processing surface biofilm formation assay, BV26 decreased E. faecalis biofilm formation by more than 3 log₁₀ CFU/coupon after five days. These findings highlight the novelty and potential of phage BV26 as a safe, stable, and effective biocontrol agent for preventing and eliminating E. faecalis biofilms in dairy processing facilities.