Functional genomic characterization of anti-phage defense systems in vancomycin-resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VREfm) is a significant public health threat, demonstrating high rates of antibiotic resistance and frequently causing hospital-acquired infections. Bacteriophages (phages) are a promising adjunctive therapy to reduce bacterial load and manage VREfm infections. Many bacteria encode anti-phage defense systems—groups of genes that can counteract phage infection—but these systems have not been characterized in VREfm. Here we explored the diversity of anti-phage defense systems within clinical VREfm isolate genomes and examined their impact on phage susceptibility. We first used two available bioinformatic tools (DefenseFinder and PADLOC) to identify putative anti-phage defense systems in 21 hybrid-assembled VREfm clinical isolate genomes, and observed that most anti-phage defense systems were located on plasmids, defense islands, and prophages. Combining this information with functional testing of phage susceptibility did not reveal robust associations between specific defense systems and resistance to four VREfm-targeting lytic phages. Due to inherent limitations of phage susceptibility testing, we next developed an assay to assess phage survival after co-incubation with bacteria, and identified over two dozen genes associated with phage killing, including a potentially novel defense island. Lastly, we attempted to functionally validate 10 candidate anti-phage defense systems, only four of which were ultimately tested in enterococci. Expression of one of these candidates, AbiE, decreased bacterial susceptibility to two Enterococcus -targeting phages. As VREfm- targeting phage therapy becomes more widely available, identification of genes contributing to phage resistance can aid in streamlining the provision of phages to patients who could benefit from this new therapeutic modality.