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

Preterm infants are susceptible to necrotizing enterocolitis (NEC), which is associated with gut microbial dysbiosis with increased <i>Pseudomonadota</i> 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 <i>Enterobacteriaceae</i> with potential to use as a NEC-preventive therapy. 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. 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. This study demonstrates the isolation of phages targeting NEC-associated bacteria, and supports further exploration of phage therapy as a prophylactic strategy against NEC.