Functional amplification and preservation of human gut microbiota

Abstract

Background: The availability of fresh stool samples is a prerequisite in most gut microbiota functional studies. Objective: Strategies for amplification and long-term gut microbiota preservation from fecal samples would favor sample sharing, help comparisons and reproducibility over time and between laboratories, and improve the safety and ethical issues surrounding fecal microbiota transplantations. Design: Taking advantage of in vitro gut-simulating systems, we amplified the microbial repertoire of a fresh fecal sample and assessed the viability and resuscitation of microbes after preservation with some common intracellular and extracellular acting cryoprotective agents (CPAs), alone and in different combinations. Preservation efficiencies were determined after 3 and 6 months and compared with the fresh initial microbiota diversity and metabolic activity, using the chemostat-based Environmental Control System for Intestinal Microbiota (ECSIM) in vitro model of the gut environment. Microbial populations were tested for fermentation gas, short-chain fatty acids, and composition of amplified and resuscitated microbiota, encompassing methanogenic archaea. Results: Amplification of the microbial repertoire from a fresh fecal sample was achieved with high fidelity. Dimethylsulfoxide, alone or mixed with other CPAs, showed the best efficiency for functional preservation, and the duration of preservation had little effect. Conclusions: The amplification and resuscitation of fecal microbiota can be performed using specialized in vitro gut models. Correct amplification of the initial microbes should ease the sharing of clinical samples and improve the safety of fecal microbiota transplantation. Abbreviations: CDI, Clostridium difficile infection; CPA, cryoprotective agent; D, DMSO, dimethylsulfoxide; FMT, fecal microbiota transplantation; G, glycerol; IBD, inflammatory bowel disease; P, PEG-4000, polyethylene glycol 4000 g.mol−1; SCFA, short-chain fatty acid; SNR, signal-to-noise ratio

Selection of fast and slow growing bacteria from fecal microbiota using continuous culture with changing dilution rate

“…To inoculate the repeated/parallel fermentation experiments, we used the frozen (−80°C) stock suspensions/cultures of fecal microbiota containing dimethylsulfoxide as protectant, also suggested by Gaci et al [31] Functional preservation of the fecal samples has several advantages over using fresh fecal material, as composition of the colon/fecal microbiota varies from day to day. Therefore, use of the same inoculum would afford better reproducibility of the fermentation experiments.…”

“…Therefore, use of the same inoculum would afford better reproducibility of the fermentation experiments. In different from Gaci et al [31], we preserved the diluted feces with added protective agent without any batch cultivations to save the original microbial balance and functionality.…”

Artificial Gastrointestinal Models for Nutraceuticals Research—Achievements and Challenges: A Practical Review

“…Evaluation of the viability and resuscitability of microorganisms after preservation with certain cryoprotective agents (CPAs) Tottey et al [82] 4.6. EnteroMix…”

In vitro conversion of ellagic acid to urolithin A by different gut microbiota of urolithin metabotype A

“…Intestinal microbes have also established critical links with various physiological and pathological conditions, including extraintestinal diseases, cardiac metabolism, and cardiovascular diseases (Wu et al 2015 ). Feces are believed to reflect this intestinal microbial diversity and can be regarded as a reservoir of microbes associated with the human gastrointestinal tract (GIT) (Gaci et al 2017 ). The sources of metabolic substrates for the human gut microbiota are exogenous and endogenous, and the metabolic functions of this microbiota depend on the nature of colonic substrates available for fermentation (Duarte et al 2021 ).…”