Stepwise Development of an in vitro Continuous Fermentation Model for the Murine Caecal Microbiota

Abstract

Murine models are valuable tools to study the role of gut microbiota in health or disease. However, murine and human microbiota differ in species composition, so further investigation of the murine gut microbiota is important to gain a better mechanistic understanding. Continuous in vitro fermentation models are powerful tools to investigate microbe-microbe interactions while circumventing animal testing and host confounding factors, but are lacking for murine gut microbiota. We therefore developed a novel continuous fermentation model based on the PolyFermS platform adapted to the murine caecum and inoculated with immobilized caecal microbiota. We followed a stepwise model development approach by adjusting parameters [pH, retention time (RT), growth medium] to reach fermentation metabolite profiles and marker bacterial levels similar to the inoculum. The final model had a stable and inoculum-alike fermentation profile during continuous operation. A lower pH during startup and continuous operation stimulated bacterial fermentation (115 mM short-chain fatty acids at pH 7 to 159 mM at pH 6.5). Adjustments to nutritive medium, a decreased pH and increased RT helped control the in vitro Enterobacteriaceae levels, which often bloom in fermentation models, to 6.6 log gene copies/mL in final model. In parallel, the Lactobacillus , Lachnospiraceae , and Ruminococcaceae levels were better maintained in vitro with concentrations of 8.5 log gene copies/mL, 8.8 log gene copies/mL and 7.5 log gene copies/mL, respectively, in the final model. An independent repetition with final model parameters showed reproducible results in maintaining the inoculum fermentation metabolite profile and its marker bacterial levels. Microbiota community analysis of the final model showed a decreased bacterial diversity and compositional differences compared to caecal inoculum microbiota. Most of the caecal bacterial families were represented in vitro , but taxa of the Muribaculaceae family were not maintained. Functional metagenomics prediction showed conserved metabolic and functional KEGG pathways between in vitro and caecal inoculum microbiota. To conclude, we showed that a rational and stepwise approach allowed us to model in vitro the murine caecal microbiota and functions. Our model is a first step to develop murine microbiota model systems and offers the potential to study microbiota functionality and structure ex vivo .

Impact of manipulation of glycerol/diol dehydratase activity on intestinal microbiota ecology and metabolism

“…Both the European Food Safety Authority and the US Food and Drug Administration support the use of the gut simulators to provide mechanistic evidence of proposed effects (Venema and Van den Abbeele, 2013), which can be combined with batch fermentations to investigate the potential of intestinal microbiota to respond to manipulation strategies on metabolism and composition (Reichardt et al ., 2018; Bircher et al ., 2018a; Bircher et al ., 2018b). In this study, we used intestinal microbiota cultivated on the PolyFermS fermentation system originally collected from different hosts (Poeker et al ., 2018; Asare, 2019; Poeker et al ., 2019) because it offered several advantages over the use of faecal samples such as inoculum stability, control on the experimental procedure (Zihler Berner et al ., 2013; Fehlbaum et al ., 2015; Poeker et al ., 2019) and low inoculum variation between days. The PolyFermS‐derived intestinal model microbiota differed in overall gdh abundance and diversity which allowed us to concurrently study the impact of two parameters in a highly controlled setting.…”

“…In order to assess GDH activity of intestinal microbiota (IM1–IM9, numbered in chronological order of analysis), fermentation effluent microbiota were obtained from the inoculum reactors of nine independent PolyFermS in vitro continuous fermentation systems that were run at the Laboratory of Food Biotechnology during the time of the study (Poeker et al ., 2018; Asare, 2019; Poeker et al ., 2019). This fermentation system harbours gellan‐xanthan beads that are colonized by fecal or cecal microbiota.…”

In vitrogut modeling as a tool for adaptive evolutionary engineering ofLactiplantibacillus plantarum

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted October 23, 2020. ; https://doi.org/10.1101/2020.10.21.349795 doi: bioRxiv preprint Continuous fermentation models are best suited for in vitro cultivation of gut microbiota in conditions akin to the gut (19,20). Different PolyFermS models were successfully developed for cultivating colonic microbiota of humans of different age and conditions, swine, murine and chicken cecum microbiota (21)(22)(23)(24)(25). The continuous PolyFermS model allows testing several treatments in parallel in second-stage treatment reactors (TRs) seeded with the same gut microbiota produced in the inoculum reactor (IR) containing immobilized microbiota (21).…”

“…Continuous fermentation models are best suited for in vitro cultivation of gut microbiota in conditions akin to the gut (19,20). Different PolyFermS models were successfully developed for cultivating colonic microbiota of humans of different ages and conditions, swine, murine, and chicken cecum microbiota (21)(22)(23)(24)(25). The continuous PolyFermS model allows testing several treatments in parallel in second-stage treatment reactors (TRs) seeded with the same gut microbiota produced in the inoculum reactor (IR) containing immobilized microbiota (21).…”

Prediction Correction Topic Evolution Research for Metabolic Pathways of the Gut Microbiota

“…In recent years, scientific production on the metabolic pathways of gut microbiota and its metabolites have produced datasets of extreme great interests and has expanded the physiology field of microbiome concepts. It is increasingly necessary to recognize emerging links, both theoretically and empirically (Poeker et al, 2019). This is momentous for the comprehensive assessment of gut Microbiota metabolism pathways.…”