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      Functional amplification and preservation of human gut microbiota

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          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

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          The archaeal cell envelope.

          Sonja-Verena Albers, Benjamin H Meyer (2011)
          At first glance, archaea and bacteria look alike; however, the composition of the archaeal cell envelope is fundamentally different from the bacterial cell envelope. With just one exception, all archaea characterized to date have only a single membrane and most are covered by a paracrystalline protein layer. This Review discusses our current knowledge of the composition of the archaeal cell surface. We describe the wide range of cell wall polymers, O- and N-glycosylated extracellular proteins and other cell surface structures that archaea use to interact with their environment.
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            Protectants used in the cryopreservation of microorganisms.

            Zdenek Hubalek (2003)
            The cryoprotective additives (CPAs) used in the frozen storage of microorganisms (viruses, bacteria, fungi, algae, and protozoa) include a variety of simple and more complex chemical compounds, but only a few of them have been used widely and with satisfactory results: these include dimethylsulfoxide (Me2SO), glycerol, blood serum or serum albumin, skimmed milk, peptone, yeast extract, saccharose, glucose, methanol, polyvinylpyrrolidone (PVP), sorbitol, and malt extract. Pairwise comparisons of the cryoprotective activity of the more common CPAs used in cryomicrobiology, based on published experimental reports, indicate that the most successful CPAs have been Me2SO, methanol, ethylene glycol, propylene glycol, and serum or serum albumin, while glycerol, polyethylene glycol, PVP, and sucrose are less successful, and other sugars, dextran, hydroxyethyl starch, sorbitol, and milk are the least effective. However, diols (as well as some other CPAs) are toxic for many microbes. Me2SO might be regarded as the most universally useful CPA, although certain other CPAs can sometimes yield better recoveries with particular organisms. The best CPA, or combination of CPAs, and the optimum concentration for a particular cryosensitive microorganism has to be determined empirically. This review aims to provide a summary of the main experimental findings with a wide range of additives and organisms. A brief discussion of mechanisms of CPA action is also included.
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              The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

              Fiona Fouhy, Jennifer Deane, Mary C. Rea … (2015)
              Background High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed. Methods Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed. Results No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples. Conclusions Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Microb Ecol Health Dis
                Journal ID (iso-abbrev): Microb. Ecol. Health Dis
                Journal ID (archive): ZMEH
                Journal ID (publisher-id): zmeh20
                Title: Microbial Ecology in Health and Disease
                Publisher: Taylor & Francis
                ISSN (Print): 0891-060X
                ISSN (Electronic): 1651-2235
                Publication date Collection: 2017
                Publication date (Electronic): 10 April 2017
                Volume: 28
                Issue: 1
                Electronic Location Identifier: 1308070
                Affiliations
                [ a ]EA-4678 CIDAM, Clermont Université, Université d’Auvergne , Clermont-Ferrand, France
                Author notes
                CONTACT Jean-François Brugère jf.brugere@ 123456udamail.fr eEA 4678 CIDAM, 5e CBRV – Facultés de Médecine et de Pharmacie de Clermont-Ferrand, Université d’Auvergne , Place Henri Dunant, F-63000 Clermont-Ferrand, France
                Article
                Publisher ID: 1308070
                DOI: 10.1080/16512235.2017.1308070
                PMC ID: 5443092
                PubMed ID: 28572754
                SO-VID: a681e67b-9e2e-4233-ad14-07d75837ff11
                Copyright © © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 29 December 2016
                Date accepted : 15 March 2017
                Page count
                Figures: 4, Tables: 1, References: 35, Pages: 11
                Funding
                Funded by: French Ministère de l’Enseignement Supérieur et de la Recherche to NG
                Award ID: 2012-MESR
                Funded by: PPC was funded by a grant from the Université d’Auvergne (UdA)
                Award ID: 2014-UdA
                Funded by: the European Union (UE) and the Auvergne Council to WT (FEDER)
                Award ID: 2011-Auv
                This work was supported by two PhD scholarships, one from the French Ministère de l’Enseignement Supérieur etde la Recherche to NG [2012-MESR] and another from the European Union (UE)/the Auvergne Council to WT (FEDER) [2011-Auv]; PPC was funded by a grant from the Université d’Auvergne (UdA) [2014-UdA].
                Categories
                Subject: Research Article
                Subject: Research Article

                ScienceOpen disciplines: Microbiology & Virology
                Keywords: fecal microbiota transplantation (fmt),glycerol,dimethylsulfoxide,polyethylene glycol,environmental control system for intestinal microbiota (ecsim),cryoprotective agent (cpa),gut microbiota preservation
                Data availability:
                ScienceOpen disciplines: Microbiology & Virology
                Keywords: fecal microbiota transplantation (fmt), glycerol, dimethylsulfoxide, polyethylene glycol, environmental control system for intestinal microbiota (ecsim), cryoprotective agent (cpa), gut microbiota preservation

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