Search

The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

The microbes that reside within the equine hindgut create a complex and dynamic ecosystem. The equine hindgut microbiota is intimately associated with health and, as such, represents an area which can be beneficially modified. Synbiotics, supplements that combine probiotic micro-organisms with prebiotic ingredients, are a potential means of influencing the hindgut microbiota to promote health and prevent disease. The objective of the current study was to evaluate the influence of an equine probiotic/prebiotic supplement on characteristics of the microbiota and metabolite production in vitro. Equine cecal fluid and fecal material were collected from an abattoir in QC, CAN. Five hundred milliliters of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (chemostat cecal, N = 11) with either 0 g (control) or 0.44 g of supplement added at 12 h intervals. One hundred milliliters of cecal fluid (anaerobic cecal, N = 15) or 5% fecal slurry (anaerobic fecal, N = 6) were maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment. Samples were taken from vessels at vessel establishment (0), 24, or 48 h of incubation. Illumina sequencing of the V4 region of the 16S rRNA gene and bioinformatics were performed for microbiome analysis. Metabolite data was obtained via NMR spectroscopy. All statistical analyses were run in SAS 9.4. There was no effect of treatment at 24 or 48h on alpha or beta diversity indices and limited taxonomic differences were noted. Acetate, propionate, and butyrate were higher in treated compared to untreated vessels in all methods. A consistent effect of supplementation on the metabolic profile with no discernable impact on the microbiota of these in vitro systems indicates inoculum microbe viability and a utilization of the provided fermentable substrate within the systems. Although no changes within the microbiome were apparent, the consistent changes in metabolites indicates a potential prebiotic effect of the added supplement and merits further exploration., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The development of novel food preservatives based on natural antimicrobials such as phenolic compounds is increasing, but their safety should be established before use, including evaluating their impact on the gut microbiota. This work explored the influence of antimicrobial phenolics presented in different forms on selected human gut microbiota members through in vitro susceptibility tests. The bacteria tested exhibited a wide range of susceptibilities to phenolics depending on the molecule structure and mode of administration. Agathobacter rectalis and Clostridium spiroforme, members of the phylum Firmicutes, were the most sensitive strains. Susceptibility was strain- and species-specific, suggesting that it may not be possible to easily extrapolate results across the human microbiome in general. Species of other phyla including Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia were more resistant than Firmicutes, with growth of some strains even enhanced. Our results provide insights into the biocompatibility of free and immobilized phenolics as potential food additives., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The human gut microbiota is a complex community of prokaryotic and eukaryotic microbes and viral particles that is increasingly associated with many aspects of host physiology and health. However, the classical microbiology approach of axenic culture cannot provide a complete picture of the complex interactions between microbes and their hosts in vivo. As such, recently there has been much interest in the culture of gut microbial ecosystems in the laboratory as a strategy to better understand their compositions and functions. In this review, we discuss the model platforms and methods available in the contemporary microbiology laboratory to study human gut microbiomes, as well as current knowledge surrounding the isolation of human gut microbes for the potential construction of defined communities for use in model systems.