Genetically distant bacteriophages elicit unique genomic changes in Enterococcus faecalis

The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages promote genetic alterations in medically relevant bacteria is important as phages continue to become established biologics for the treatment of multidrug-resistant (MDR) bacterial infections. Before phages are used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages, to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole genome sequencing revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in several genes previously unreported to be associated with phage infection in E. faecalis. Intriguingly, there was only one shared mutation in the E. faecalis genome in response to each of the two phages tested, demonstrating that infection by genetically distinct phages results in different host responses. This study shows that infection of the same host by disparate phages leads to evolutionary trajectories that result in distinct genetic changes. This implies that bacteria respond to phage pressure through host responses that are tailored to specific phages. This work serves as the basis for the study of E. faecalis genome evolution during phage infection and will inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis. IMPORTANCEStudies characterizing the genome evolution of bacterial pathogens following phage selective pressure are lacking. Phage therapy is experiencing a rebirth in Western medicine. Such studies are critical for understanding how bacteria subvert phage infection and how phages evolve to counter such mutations. This study utilizes comparative genomic analyses to demonstrate how a pathogenic strain of Enterococcus faecalis responds to infection by two genetically distant phages. We show that genetic alterations in the E. faecalis genome accumulate in a manner that is specific to the infecting phage with little to no overlap in shared fixed mutations. This suggests that bacterial genome evolution in response to phage infection is uniquely tied to phage genotype, and sets a precedence for investigations into how phages drive bacterial genome evolution relevant to phage therapeutic applications.