NOVEL INTRADUODENAL FECAL MICROBIOTA TRANSPLANTATION METHODOLOGY AMELIORATES GUT ATROPHY AND CHOLESTASIS IN AN AMBULATORY PEDIATRIC PARENTERAL NUTRITION PIGLET MODEL

Background: Total parenteral nutrition (TPN) provides all nutritional support intravenously, however, this life saving therapy is associated with significant systemic side effects. Using a novel ambulatory TPN piglet model, our lab has demonstrated an alteration in gut microbes and gut-derived signaling driving such injury. Given emerging evidence of the role of gut microbiota as a key player in modulating gut-systemic signaling, we hypothesized that transferring fecal microbiota from healthy control animals to those on TPN would restore the altered gut microbiota and mitigate TPN associated injury.

Methods: Thirty-one, 7-day old piglets were randomly allocated in a 3:1 ratio to either TPN or regular enteral nutrition (EN). The TPN group was sub categorized to receive TPN only, TPN plus antibiotics (TPN-A) or TPN plus fecal microbiota transplant (TPN-FMT) for 14 days. Histological analysis, serum biochemistry, and qPCR were used to assess gut, liver tissues and serum samples. Serial stool samples prior to the study (PS) and at sacrifice (SAC) underwent culture independent 16s rRNA sequencing. Statistical comparison of β-diversity was achieved using PERMANOVA with Jaccard and Bray-Curtis similarity metrics.

Results: Serological analysis revealed a significant elevation in bilirubin in TPN and TPN-A vs EN (p<0.001) with prevention upon FMT. Serum cytokine profiles showed significantly higher IFN-G, TNF-alpha, IL-beta, IL-8, in TPN (p=0.009/0.001/0.043/0.011), with FMT preventing these elevations. Gut and exhibited significant gut atrophy in TPN and TPN-A, but not in TPN-FMT, as indicated by linear gut density and villous/crypt ratio. A cholestasis score (CS) was designated to each group based on hepatic intra-parenchymal bile deposition. CS was significantly lower in FMT treated animals against both TPN (p=0.001) and TPN-A (p<0.0001). CD3 immunostaining also revealed significantly higher levels in TPN groups in comparison to FMT. Microbiota profiles analyzed using Principal Coordinate Analysis (PCA) demonstrated that FMT resulted in a significant overlap with EN, with the largest separation observed in TPN-A followed by TPN, driven primarily by firmicutes and fusobacteria. Gene expression noted upregulation of CYP7A1 and BSEP in TPN and TPN-A, with downregulation of FGFR4, EGF, FXR and TGR5 compared with EN and its prevention with FMT.

Conclusion: Our study provides novel evidence for the preservation of gut atrophy and prevention in liver injury with FMT, as well as the detrimental effects of microbial dysbiosis with antibiotics. These findings challenge current paradigms into mechanisms of injury during TPN and underscore the importance of the gut microbes, as prime targets for further therapeutics and drug discovery.