Characterization of a virulent bacteriophage consortium targeting Enterobacteriaceae from inflamed preterm gut mucosa

Preterm infants have a high risk of intestinal inflammation which can progress to necrotizing enterocolitis (NEC). The gut microbial colonization commencing at birth is essential for proper intestinal maturation, but this process is often disrupted in preterm infants, leading to dysbiosis and increased risk of developing NEC. Bacteriophages (phages), viruses that specifically infect bacteria, are an important constituent of the gut microbiome and protects the gut epithelium against invading bacteria. This study aimed to isolate and characterize phages for use as a preventive measure against NEC-associated bacteria. We initially cultured Enterobacteriaceae from ileal mucosa of preterm piglets that exhibited severe NEC-like pathology. We then screened 23 donor fecal samples for inhibition of bacterial growth and isolated a collection of unique phages to use further. The phages were characterized by whole genome sequencing, host receptor binding determination, and immune cell activation in vitro. The final phage collection consisted of ten virulent phages within five genera, representing myovirus, podovirus and siphovirus morphologies. All phages in the collection induced expression of both pro-and anti-inflammatory genes in co-culture with macrophage-like THP-1 cells, but to different extents than Escherichia coli. Ultimately, we selected one phage for a high-dose oral administration to newborn piglets and assessed its infectivity and presence in different gut segments. This intervention did not result in any direct side effects, while both infective phages and signatures of phage DNA were detected in the intestinal content and mucosa. Having characterized a set of rationally selected virulent phages, we support the advancement of phage therapy as a potential protection against NEC.