A weighted and cumulative point system for accurate scoring of intestinal pathology in a piglet model of necrotizing enterocolitis

Offersen, Simone Margaard; Henriksen, Nicole Lind; Brunse, Anders

doi:10.1101/2024.01.05.574327

Cited by 3 publications

(2 citation statements)

References 27 publications

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“…One was hematoxylin and eosin stained prior to histopathological assessment by an experienced investigator blinded to the investigation. Each segment received a microscopic lesion score of 0-8 as described previously [37]. Another was used for goblet cell visualization by Alcian blue and Periodic acid-Schiff (PAS) staining.…”

Section: Lesion Histological Analyses and Cytokine Measurementsmentioning

confidence: 99%

“…The stomach, small intestine, and colon were assessed for macroscopic lesions of hyperemia, hemorrhage, necrosis, and pneumatosis intestinalis (transmural gas) by an experienced person blinded to the investigation. A gross pathology score was given for each intestinal segment according to the weighted and cumulative point system described in [37]. NEC was de ned as the presence of moderate hemorrhage or presence of pneumatosis intestinalis, and/or necrosis.…”

Section: Euthanasia Tissue Collection and Scoring Of Intestinal Lesionsmentioning

confidence: 99%

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Fecal virome is sufficient to reduce necrotizing enterocolitis

Offersen,

Mao,

Spiegelhauer

et al. 2024

Preprint

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Background: Fecal filtrate transfer (FFT, i.e. “sterile filtered” fecal matter) is gaining increasing attention as a safer alternative to traditional fecal microbiota transplantation (FMT) for treating gastrointestinal (GI) complications. Indeed, in the case of necrotizing enterocolitis (NEC), a life-threatening GI emergency occurring in preterm infants, FFT is superior to FMT in terms of both safety and efficacy when investigated in preterm piglets. Since fecal filtrate is virtually devoid of bacteria, prokaryotic viruses (bacteriophages) are assumed to mediate the beneficial effects by modulating the recipient gut microbiota. However, this assumption remains unproven. Results: To address this gap, we isolated the virome of donor feces from the residual postbiotic fluid with no loss of bacteriophage infective potential or bacteriophage spillover to the residual fluid. We then compared clinical and gut microbiota responses to these fractions with the parent FFT solution after transferring to NEC-susceptible preterm piglets. Importantly, transfer of isolated donor virome was equally effective as FFT in reducing the severity of NEC-like pathology, whereas the residual postbiotic fraction was ineffective. The bacterial compositional data corroborated clinical findings as virome transfer reduced the relative abundance of several NEC-associated pathogens e.g. Klebsiella pneumoniae and Clostridium perfringens. A viral metagenomics analysis indicated enrichment and diversification of recipient gut viral communities with concomitant constraining effect on bacterial composition in accordance with lytic phage predation of resident bacteria. Surprisingly, virome transfer but not residual postbiotic fluid was associated with suspected viral gastroenteritis as indicated by diarrhea, intestinal atrophy, and weight loss. Although virome sequencing did not reveal any obvious causative agent, we suggest that unidentified eukaryotic viruses are candidates responsible for these side effects. Conclusion: Using NEC as a relevant case for microbiota-directed therapy, we show that transfer of isolated fecal virome is sufficient to reduce pathogenic bacterial load and overall disease burden. However, we also highlight that receiving exogenous virome increases the risk of enteric virus infection. Despite diarrhea being a minor concern in the human infant context, future work should identify ways of eliminating eukaryotic viruses without losing treatment efficacy.

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Section: Lesion Histological Analyses and Cytokine Measurementsmentioning

confidence: 99%

Section: Euthanasia Tissue Collection and Scoring Of Intestinal Lesionsmentioning

confidence: 99%

Fecal virome is sufficient to reduce necrotizing enterocolitis

Offersen,

Mao,

Spiegelhauer

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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Fecal virus-like particles are sufficient to reduce necrotizing enterocolitis

Offersen,

Mao,

Spiegelhauer

et al. 2024

Gut Microbes

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Fecal filtrate transfer (FFT) is emerging as a safer alternative to traditional fecal microbiota transplantation (FMT) – particularly in the context of necrotizing enterocolitis (NEC), a severe gastrointestinal condition affecting preterm infants. Using a preterm piglet model, FFT has demonstrated superiority over FMT in safety and NEC prevention. Since FFT is virtually devoid of bacteria, prokaryotic viruses (bacteriophages) are assumed to mediate the beneficial effects. However, this assumption remains unproven. To address this gap, we separated virus-like particles (30 kDa to 0.45 µm) of donor feces from the residual postbiotic fluid. We then compared clinical and gut microbiota responses to these fractions with the parent FFT solution after transferring them to NEC-susceptible preterm piglets. Virome transfer was equally effective as FFT in reducing the severity of NEC-like pathology. The bacterial compositional data corroborated clinical findings as virome transfer reduced the relative abundance of several NEC-associated pathogens e.g. Klebsiella pneumoniae and Clostridium perfringens . Virome transfer diversified gut viral communities with concomitant constraining effects on the bacterial composition. Unexpectedly, virome transfer, but not residual postbiotic fluid, led to earlier diarrhea. While diarrhea may be a minor concern in human infants, future work should identify ways of eliminating this side effect without losing treatment efficacy.

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Protection against necrotizing enterocolitis by fecal filtrate transfer requires an active donor virome

Spiegelhauer,

Offersen,

Mao

et al. 2024

Preprint

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Necrotizing enterocolitis (NEC) remains a frequent catastrophic disease in preterm infants, but fecal filtrate transfer (FFT) has been identified as a promising prophylactic therapy in preclinical studies. This study examined the importance of the FFT virome viability on gut colonization and NEC occurrence. We established an ultraviolet irradiation-based viral inactivation protocol and demonstrated total loss of infectivity of a viral mock community. Using this protocol, we inactivated an aliquot of sterile-filtered donor feces and compared the response in preterm piglets subjected to experimental NEC induction. Gut pathology and barrier properties were assessed, and bacterial and viral compositions were determined by 16S rRNA amplicon and viral metagenomics sequencing, respectively. Native FFT decreased NEC severity and proinflammatory cytokines, but inactivated FFT (iFFT) completely abolished these effects. Mild side effects in the form of diarrhea manifested earlier in recipients of native FFT than iFFT or controls. A distinct gut colonization pattern of increased viral heterogeneity increased bacterial homogeneity and reduction in pathobionts like Clostridium perfringens and Escherichia was observed in the group receiving native FFT, but not in the iFFT group . The present study uncovered a clear distinction between active and inactivated transferred viromes in the ability to modulate gut colonization after preterm birth and decrease NEC. FFT efficacy is potentially driven by active bacteriophages targeting pathogenic bacteria.

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A weighted and cumulative point system for accurate scoring of intestinal pathology in a piglet model of necrotizing enterocolitis

Cited by 3 publications

References 27 publications

Fecal virome is sufficient to reduce necrotizing enterocolitis

Fecal virome is sufficient to reduce necrotizing enterocolitis

Fecal virus-like particles are sufficient to reduce necrotizing enterocolitis

Protection against necrotizing enterocolitis by fecal filtrate transfer requires an active donor virome

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