Bacteriophages for Neonatal Gut Health: Viral Infectivity and Specificity in a Preclinical Model of Necrotizing Enterocolitis

PhD Student: Malene Roed Spiegelhauer

Date defended: 28 May 2025

Contact: malene.r.s@sund.ku.dk 

THE PROJECT
Preterm infants are at high risk of developing necrotizing enterocolitis (NEC), a severe intestinal inflammatory condition associated with high mortality and long-term morbidities. Infants with NEC often exhibit gut bacterial dysbiosis, characterized by an overgrowth of pathogenic bacteria and a reduction of beneficial commensals. In this project, preterm piglets were applied as a preclinical model of preterm infants, as they display severe intestinal immaturity at birth and increased susceptibility to NEC when administered formula. Prior studies demonstrated a protective role of fecal filtrates from healthy donors against intestinal pathology and mucosal colonization by pathogenic bacteria in preterm formula-fed piglets. Fecal filtrates, prepared by removing bacteria from the fecal material, retain the highly abundant and diverse virome, predominantly consisting of bacteriophages (phages, viruses that specifically infect bacteria).

It remains unclear whether the protective effects of fecal filtrates are driven solely by the presence of viral particles (primarily phages) or if their activity is required. Moreover, the potential transmission of pathogenic eukaryotic viruses from the donor poses an infective risk for the recipient, underscoring the need for a safe treatment. Phage therapy, utilizing defined single phages or multi-phage cocktails, presents a targeted approach to modulate microbial communities and mitigate intestinal inflammation associated with dysbiosis.  

THE PURPOSE
This project aimed to investigate the role of phage infectivity and specificity in preventing NEC using a preterm piglet model. A combination of animal studies and laboratory work was employed to elucidate the potential of phage-based interventions.

THE RESULTS
The thesis involved three manuscripts.

• The first manuscript compared the effects of fecal filtrates with an active or inactivated virome in formula-fed preterm piglets susceptible to NEC. The fecal filtrate containing an active virome significantly reduced intestinal pathology, inflammation, and bacterial pathogen abundance. In contrast, viral inactivation abolished these effects. This study suggests that the protective effects of the fecal filtrate were mediated by active viruses, particularly phages.
• The second paper described a consortium of ten unique phages isolated from fecal filtrates of healthy donors, with the potential to infect bacterial strains from inflamed lesions of preterm formula-fed piglets.
• In the third paper, the phage consortium was applied as a phage cocktail in formula-fed preterm piglets. Due to low levels of intestinal pathology, the effects of the phage cocktail on NEC could not be assessed. However, the phage cocktail demonstrated a safe profile, with no adverse effects or induction of intestinal inflammation. Phage signatures were detected throughout the intestine and in the gut-draining lymph nodes, but without inducing apparent immunity. Overall, the phage consortium was safe for administration in newborn preterm piglets.

In conclusion, these findings indicate that phage activity within fecal filtrates is critical for modulating microbiome dysbiosis and protecting the intestine in a preterm piglet model susceptible to NEC. The application of a specific phage cocktail was safely tolerated in preterm newborn piglets, but further optimization is required to observe a therapeutic effect.