Establishment and evaluation of an in vitro model of the small intestinal microbiota

Authors: Firrman, Jenni; Liu, Lin; Mahalak, Karley; Scarino Lemons, Johanna; Narrowe, Adrienne; FRIEDMAN, ELLIOT - University Of Pennsylvania; WU, GARY - University Of Pennsylvania; VAN DE WEILE, TOM - Ghent University

Submitted to: Microbiological Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2026
Publication Date: 2/26/2026
Citation: Firrman, J., Liu, L.S., Mahalak, K.K., Scarino Lemons, J.M., Narrowe, A.B., Friedman, E., Wu, G., Van De Weile, T. 2026. Establishment and evaluation of an in vitro model of the small intestinal microbiota. Microbiological Research. https://doi.org/10.1128/msystems.01373-25.
DOI: https://doi.org/10.1128/msystems.01373-25

Interpretive Summary: The gastrointestinal tract contains a large community of bacteria that aid in digestion and interact with human cells, referred to collectively as the gut microbiota. While the gut microbiota within the colon region has been extensively studied, there is little information available on the gut microbiota in the small intestine and there is no standard model representing this intestinal region. To fill in this gap in knowledge, a bench-top model was developed and used to examine the small intestinal microbiota of 4 different donors. The results showed that the model was able to produce microbial communities that represented each donor, and that while each donor had a unique gut microbiota in terms of which bacteria were present, they all produced similar types of byproducts. In conclusion, this study described and evaluated a model system that can be applied to study the small intestinal gut microbiota in nutrition and health, and identified key differences and similarities that occur between donors.

Technical Abstract: Research continues to evidence the important contribution of the small intestinal microbiota to nutrient utilization within the upper gastrointestinal tract, driving a need for in vitro platforms to elucidate the functional role of this community. An in vitro model of the small intestinal microbiota (SIM) was designed and developed to mimic the terminal ileum with inocula generated from ileostomy effluent. Shotgun sequencing was combined with metabolomics to profile communities that developed from four individual donors and perform comparative analyses. Structurally, the communities were highly variable between donors, and this was maintained in vitro, as was the presence of typical facultative taxa associated with the small intestine, such as Klebsiella, Escherichia, Streptococcus, and Enterococcus. Although the composition was variable between donors, all communities reached stability after 5 days using a 4-hour residence time. Functionally, acetic acid was the most prominent short chain fatty acid (SCFA) detected, and preservation of primary bile acids was observed for three of four communities. A high degree of similarity between communities was found using untargeted metabolomics. Together, metagenomics and metabolomics demonstrated that, although variable, a core set of genes and metabolites were shared across all donors. In conclusion, the SIM model recapitulated the structure and function of the small intestinal gut microbiota found in vivo, with a core set of shared features identified, yet maintained the interindividual variability that is a hallmark of the small intestinal microbiota. This model system can be applied to address the role of the small intestinal microbiota in nutrition and health.