Bacteriophage susceptibility in clinical Enterococcus faecalis reflects host genomic background and Epa-associated phage adsorption

Abstract

Cytolysin-positive Enterococcus faecalis has been implicated as a disease-associated pathobiont in alcohol-associated hepatitis, motivating development of selective bacteriophage-based strategies. However, strain-level determinants of phage susceptibility remain incompletely understood. We characterized the host range of three E. faecalis phages, {Phi}Ef2.1, {Phi}Ef2.2, and {Phi}Ef2.3Ev, across 45 clinical isolates and investigated bacterial and phage factors associated with adsorption and productive infection. Isolates were characterized by whole-genome sequencing, cytolysin status, antimicrobial resistance determinants, and predicted anti-phage defense systems. Phage susceptibility was measured by efficiency of plating (EoP) and adsorption assays. Thirty-five of the 45 isolates (77.8%), including all 15 cylLs-positive isolates, were susceptible to at least one of the three phages at EoP [≥]0.01. {Phi}Ef2.1 and {Phi}Ef2.2 showed greater activity against cylLs-positive isolates, which also carried higher burdens of predicted antimicrobial resistance determinants and anti-phage defense system types. Analysis of 367 complete E. faecalis RefSeq genomes classified the variable region of the enterococcal polysaccharide antigen (epa) locus into 15 recurrent architecture groups that were associated with phage-specific adsorption patterns. Efficient adsorption did not always result in productive infection, indicating additional post-adsorption barriers. AbiD emerged as a candidate factor associated with reduced {Phi}Ef2.2 productivity, although this signal was prediction-tool dependent, while unexplained post-adsorption blocks suggest the involvement of additional uncharacterized mechanisms. These findings link naturally occurring epa architecture to phage adsorption and highlight distinct post-adsorption constraints on host range, providing a framework for rational phage selection against clinically relevant E. faecalis populations.