Characterization of a virulent bacteriophage consortium targeting <i>Enterobacteriaceae</i> from inflamed preterm gut mucosa

Aims: Preterm infants are susceptible to necrotizing enterocolitis (NEC), which is associated with gut microbial dysbiosis with increased Pseudomonadota abundance. Bacteriophages (phages) are essential components of the gut microbiome, but their role in intestinal protection remains unclear. This study aims to isolate and characterize phages targeting Enterobacteriaceae with potential to use as a NEC-preventive therapy. Materials and methods: Enterobacteriaceae strains were isolated from the ileal mucosa of preterm piglets with NEC-like pathology. Fecal filtrates from healthy donors were screened for growth-inhibitory capacity against these strains, and isolated phages were characterized by whole-genome sequencing, electron microscopy, host receptor profiling, and immunogenic response in THP-1 macrophages. A selected phage was orally administered to two-day-old piglets to evaluate its safety and gastrointestinal survival. Results: A consortium of 10 phages targeting NEC-associated bacteria was defined, spanning five Caudoviricetes genera, and displaying three morphologies. The phages modulated both pro- and anti-inflammatory responses in THP-1 cells. In vivo administration of a selected phage was well tolerated in newborn piglets, with viable phages and their DNA recovered from ileum and colon. Conclusion: This study demonstrates the isolation of phages targeting NEC-associated bacteria, and supports further exploration of phage therapy as a prophylactic strategy against NEC.