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      Establishing a mucosal gut microbial community in vitro using an artificial simulator

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          Abstract

          The Twin Simulator of the Human Intestinal Microbial Ecosystem (TWINSHIME ®) was initially developed to study the luminal gut microbiota of the ascending (AC), transverse (TC), and descending (DC) colon regions. Given the unique composition and potential importance of the mucosal microbiota for human health, the TWINSHIME was recently adapted to simulate the mucosal microbiota as well as the luminal community. It has been previously demonstrated that the luminal community in the TWINSHIME reaches a steady state within two weeks post inoculation, and is able to differentiate into region specific communities. However, less is known regarding the mucosal community structure and dynamics. During the current study, the luminal and mucosal communities in each region of the TWINSHIME were evaluated over the course of six weeks. Based on 16S rRNA gene sequencing and short chain fatty acid analysis, it was determined that both the luminal and mucosal communities reached stability 10–20 days after inoculation, and remained stable until the end of the experiment. Bioinformatics analysis revealed the formation of unique community structures between the mucosal and luminal phases in all three colon regions, yet these communities were similar to the inoculum. Specific colonizers of the mucus mainly belonged to the Firmicutes phylum and included Lachnospiraceae (AC/TC/DC), Ruminococcaceae and Eubacteriaceae (AC), Lactobacillaceae (AC/TC), Clostridiaceae and Erysipelotrichaceae (TC/DC). In contrast, Bacteroidaceae were enriched in the gut lumen of all three colon regions. The unique profile of short chain fatty acid (SCFA) production further demonstrated system stability, but also proved to be an area of marked differences between the in vitro system and in vivo reports. Results of this study demonstrate that it is possible to replicate the community structure and composition of the gut microbiota in vitro. Through implementation of this system, the human gut microbiota can be studied in a dynamic and continuous fashion.

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          Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing

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            Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota.

            The gut microbiota is a complex and densely populated community in a dynamic environment determined by host physiology. We investigated how intestinal oxygen levels affect the composition of the fecal and mucosally adherent microbiota. We used the phosphorescence quenching method and a specially designed intraluminal oxygen probe to dynamically quantify gut luminal oxygen levels in mice. 16S ribosomal RNA gene sequencing was used to characterize the microbiota in intestines of mice exposed to hyperbaric oxygen, human rectal biopsy and mucosal swab samples, and paired human stool samples. Average Po2 values in the lumen of the cecum were extremely low (<1 mm Hg). In altering oxygenation of mouse intestines, we observed that oxygen diffused from intestinal tissue and established a radial gradient that extended from the tissue interface into the lumen. Increasing tissue oxygenation with hyperbaric oxygen altered the composition of the gut microbiota in mice. In human beings, 16S ribosomal RNA gene analyses showed an increased proportion of oxygen-tolerant organisms of the Proteobacteria and Actinobacteria phyla associated with rectal mucosa, compared with feces. A consortium of asaccharolytic bacteria of the Firmicute and Bacteroidetes phyla, which primarily metabolize peptones and amino acids, was associated primarily with mucus. This could be owing to the presence of proteinaceous substrates provided by mucus and the shedding of the intestinal epithelium. In an analysis of intestinal microbiota of mice and human beings, we observed a radial gradient of microbes linked to the distribution of oxygen and nutrients provided by host tissue. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.
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              The role of pH in determining the species composition of the human colonic microbiota.

              The pH of the colonic lumen varies with anatomical site and microbial fermentation of dietary residue. We have investigated the impact of mildly acidic pH, which occurs in the proximal colon, on the growth of different species of human colonic bacteria in pure culture and in the complete microbial community. Growth was determined for 33 representative human colonic bacteria at three initial pH values (approximately 5.5, 6.2 and 6.7) in anaerobic YCFA medium, which includes a mixture of short-chain fatty acids (SCFA) with 0.2% glucose as energy source. Representatives of all eight Bacteroides species tested grew poorly at pH 5.5, as did Escherichia coli, whereas 19 of the 23 gram-positive anaerobes tested gave growth rates at pH 5.5 that were at least 50% of those at pH 6.7. Growth inhibition of B. thetaiotaomicron at pH 5.5 was increased by the presence of the SCFA mix (33 mM acetate, 9 mM propionate and 1 mM each of iso-valerate, valerate and iso-butyrate). Analysis of amplified 16S rRNA sequences demonstrated a major pH-driven shift within a human faecal bacterial community in a continuous flow fermentor. Bacteroides spp. accounted for 27% of 16S rRNA sequences detected at pH 5.5, but 86% of sequences at pH 6.7. Conversely, butyrate-producing gram-positive bacteria related to Eubacterium rectale represented 50% of all 16S rRNA sequences at pH 5.5, but were not detected at pH 6.7. Inhibition of the growth of a major group of gram-negative bacteria at mildly acidic pH apparently creates niches that can be exploited by more low pH-tolerant microorganisms.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Methodology
                Role: ConceptualizationRole: Formal analysisRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Writing – original draftRole: Writing – review & editing
                Role: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                17 July 2018
                2018
                : 13
                : 7
                : e0197692
                Affiliations
                [1 ] Dairy and Functional Foods Research Unit, Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, Wyndmoor, Pennsylvania, United States of America
                [2 ] Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America
                [3 ] Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
                [4 ] ProDigest, Ghent, Belgium
                National Institute for Agronomic Research, FRANCE
                Author notes

                Competing Interests: The authors have read the journal’s policy and have the following conflicts: author Pieter Van den Abbeele is employed by ProDigest. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0003-3962-290X
                Article
                PONE-D-18-02051
                10.1371/journal.pone.0197692
                6050037
                30016326
                c8bf8762-f335-41e0-9cfe-644dbf44552d

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 19 January 2018
                : 7 May 2018
                Page count
                Figures: 8, Tables: 1, Pages: 20
                Funding
                This work was supported by funds from the US Congress. The company ProDigest provided support in the form of salary for author Pieter Van den Abbeele. The specific role of author Pieter Van den Abbeele is articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Colon
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Colon
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                Microbiology
                Medical Microbiology
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                Microbiology
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                Biochemistry
                Proteins
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