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      Altered microbiota, fecal lactate, and fecal bile acids in dogs with gastrointestinal disease

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          Abstract

          The intestinal microbiota plays an important role in health and disease and produces, through fermentative reactions, several metabolic products, such as lactate, that can affect the host. The microbiota also interacts with and metabolizes compounds produced by the host, such as primary bile acids. Lactate and bile acids (BA) are of particular interest in gastrointestinal diseases because they have been associated with metabolic acidosis and bile acid diarrhea, respectively. The objectives of this study were to validate an enzymatic assay to quantify D-, L-, and total lactate in canine feces, and to characterize fecal lactate and BA concentrations as well as bacterial abundances in healthy dogs and dogs with gastrointestinal diseases. Fecal samples were collected from 34 healthy dogs, 15 dogs with chronic enteropathy (CE), and 36 dogs with exocrine pancreatic insufficiency (EPI). Lactate was quantified with an enzymatic assay, BA with gas chromatography-mass spectrometry, and 11 bacterial groups with qPCR. A fecal lactate reference interval was established from 34 healthy dogs and was 0.7–1.4 mM, 0.3–6.0 mM, and 1.0–7.0 mM for D-, L-, and total lactate, respectively. The assay to measure D-, L-, and total lactate in canine fecal samples was linear, accurate, precise, and reproducible. Significant increases in fecal lactate and decreases in secondary BA concentrations were observed in dogs with CE and dogs with EPI. Dogs with EPI had an increased abundance of Escherichia coli, Lactobacillus, and Bifidobacterium; a decreased abundance of Fusobacterium and Clostridium hiranonis; and a higher Dysbiosis Index when compared to healthy dogs. Further studies are necessary to determine the clinical utility of lactate and BA quantification in canine feces. These metabolites suggest functional alterations of intestinal dysbiosis and may become promising targets for further elucidating the role of the microbiota in health and disease.

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          Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases.

          Gut microbiota metabolises bile acids (BA). As dysbiosis has been reported in inflammatory bowel diseases (IBD), we aim to investigate the impact of IBD-associated dysbiosis on BA metabolism and its influence on the epithelial cell inflammation response. Faecal and serum BA rates, expressed as a proportion of total BA, were assessed by high-performance liquid chromatography tandem mass spectrometry in colonic IBD patients (42) and healthy subjects (29). The faecal microbiota composition was assessed by quantitative real-time PCR. Using BA profiles and microbiota composition, cluster formation between groups was generated by ranking models. The faecal BA profiles in germ-free and conventional mice were compared. Direct enzymatic activities of BA biotransformation were measured in faeces. The impact of BA on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1β. IBD-associated dysbiosis was characterised by a decrease in the ratio between Faecalibacterium prausntizii and Escherichia coli. Faecal-conjugated BA rates were significantly higher in active IBD, whereas, secondary BA rates were significantly lower. Interestingly, active IBD patients exhibited higher levels of faecal 3-OH-sulphated BA. The deconjugation, transformation and desulphation activities of the microbiota were impaired in IBD patients. In vitro, secondary BA exerted anti-inflammatory effects, but sulphation of secondary BAs abolished their anti-inflammatory properties. Impaired microbiota enzymatic activity observed in IBD-associated dysbiosis leads to modifications in the luminal BA pool composition. Altered BA transformation in the gut lumen can erase the anti-inflammatory effects of some BA species on gut epithelial cells and could participate in the chronic inflammation loop of IBD.
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            Utilization of nutrients by isolated epithelial cells of the rat colon.

            Isolated suspensions of colonocytes from the rat were used to assess utilization, interaction, and fate of metabolic substrates normally obtained from colonic bacteria (acetate, propionate, butyrate) or derived from the blood circulation to the colonic mucosa (D-glucose, acetoacetate, L-glutamine). The short-chain fatty acid n-butyrate (10 mM), on its own, accounted for 86% of the total oxygen consumption and suppressed oxidation of endogenous fuel by 82%. Ths value was not altered by the addition of acetoacetate (5 mM), of L-glutamine (5 mM), or of D-glucose (10 mM). Activation of short-chain fatty acids by colonocytes proceeded in the order of butyrate greater than acetate greater than propionate. D-Glucose on its own accounted for 30% of the oxygen consumption by colonocytes and hardly suppressed utilization of endogenous fuels. Colonocytes utilized ketone bodies (acetoacetate) and produced them (acetoacetate and beta-hydroxybutyrate) from short-chain fatty acids. Considering the interaction of substrates, isolated colonic epithelial cells utilized respiratory fuels in the preferential order of butyrate greater than acetoacetate greater than glutamine greater than glucose. The high rate of CO2 production from butyrate should be a worthwhile means of examining the functional activity of the colonic mucosa clinically and in vivo.
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              Lactic acid bacteria of foods and their current taxonomy.

              Application of molecular genetic techniques to determine the relatedness of food-associated lactic acid bacteria has resulted in significant changes in their taxonomic classification. During the 1980s the genus Streptococcus was separated into the three genera Enterococcus, Lactococcus and Streptococcus. The lactic acid bacteria associated with foods now include species of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. The genus Lactobacillus remains heterogeneous with over 60 species (ymol% G+C content ranging from 33 to 55), of which about one-third are strictly heterofermentative. However, many changes have been made and reorganization of the genus along lines that do not follow previous morphological or phenotypic differentiation from Leuconostoc and Pediococcus is being studied. Phylogenetically belonging to the Actinomyces branch of the bacteria, Lactobacillus bifidus has been moved to the genus Bifidobacterium also on account of its greater than 50 mol% G+C content. It is therefore no longer considered one of the lactic acid bacteria senso strictu, which form part of the Clostridium branch of the bacteria. The new genus Weissella has been established to include one member of the genus Leuconostoc (Leuc, paramesenteroides) and heterofermentative lactobacilli with unusual interpeptide bridges in the peptidoglycan. Contrary to the clear-cut division of the streptococci, morphological and physiological features of Weissella do not directly support this grouping which now incorporates species that produce D(-)- as well as DL-lactate. The new genus Carnobacterium is morphologically similar to the lactobacilli, but it shares some physiological similarities (e.g. growth at pH 9.5) and a common phylogenetic branch with the genus Enterococcus. The review includes information on the taxonomic changes and the relationship of the bacteria of food fermentation and spoilage.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: MethodologyRole: Software
                Role: Data curationRole: InvestigationRole: MethodologyRole: Software
                Role: Funding acquisitionRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Funding acquisitionRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                31 October 2019
                2019
                : 14
                : 10
                : e0224454
                Affiliations
                [001]Gastrointestinal Laboratory, Texas A&M University, Texas, United States of America
                University of Minnesota Twin Cities, UNITED STATES
                Author notes

                Competing Interests: Jan Suchodolski is a member of the PLOS ONE editorial board. The authors have declared that no other competing interests exist. Furthermore, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0002-0866-6662
                http://orcid.org/0000-0002-2176-6932
                Article
                PONE-D-19-09395
                10.1371/journal.pone.0224454
                6822739
                31671166
                61390a57-d7d8-45d5-8ae2-20ec3ac2f569
                © 2019 Blake et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 April 2019
                : 14 October 2019
                Page count
                Figures: 5, Tables: 4, Pages: 21
                Funding
                The authors received no specific funding for this work.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Mammals
                Dogs
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Bile
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Bile
                Biology and Life Sciences
                Physiology
                Body Fluids
                Bile
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Bile
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbiome
                Biology and Life Sciences
                Genetics
                Genomics
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Microbiology
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Biology and Life Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Metabolites
                Biology and Life Sciences
                Nutrition
                Diet
                Medicine and Health Sciences
                Nutrition
                Diet
                Research and Analysis Methods
                Bioassays and Physiological Analysis
                Biochemical Analysis
                Enzyme Assays
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                All relevant data are within the manuscript and its Supporting Information files.

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