Gut Microbes Are Essential: What to Feed Them? David M. Klurfeld USDA Agricultural Research Service Beltsville, MD

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1 Gut Microbes Are Essential: What to Feed Them? David M. Klurfeld USDA Agricultural Research Service Beltsville, MD

2 The idea of a beneficial microbiome is not new Life would not long remain possible in the absence of microbes. Louis Pasteur, ~1883 Aging is caused by toxic bacteria in the gut; lactic acid could prolong life. Sour milk and yogurt promote health and longevity. Elie Metchnikoff, ~ PubMed Hits for Human Gut Microbiome

3 Is the Definition of Dietary Fiber the Problem? Multiple chemically distinct (mostly) polysaccharides Analyzed by disparate methods that correlate moderately Methods relate to lack of (mammalian) digestion Purpose of fiber in the gut is to be digested by bacteria Bacteroides cellulosyliticus has 424 glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases That is ~25x the number in the human GI tract EC Martens et al, J Mol Biol, 426: , 2014

4 Why Would We Rely on Bacteria for Energy? Allows dietary variation both of sources and over time No single organism has the enzyme repertoire to metabolize the varied foods that arrive in the colon Multiple microbial species allows shifting populations and metabolism to respond to varied dietary sources of fiber Horizontal transfer of genetic material estimated at 40-50% Plasmids, bacteriophage, direct transfer of genes Bacteria, fungi, archaea, viruses Generation time of min allows rapid response to different inputs

5 Is Fiber Bioactive or Essential? Proposed bioactive definition by Federal ad hoc working group: Constituents in foods or dietary supplements, other than those needed to meet basic human nutritional needs, which are responsible for changes in health status. Federal Register 69: , 2004 The microbiome is needed for normal digestion, nutrient absorption, mucosal integrity, host physiology and immunity So, fiber is essential

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7 Microbiota Nutrient Sources Dietary Fiber parts of plants and chemically related compounds not digested in the small intestine (bacterial dinner) May also reduce nutrient absorption from whole grains Fiber- like Stuff (bacterial snacks) Resistant Starch may be as much as fiber in diet Any other unabsorbed macronutrients: protein & fat Mucin Dead Bacteria Sloughed Cells

8 Mammalian Gut Microbiomes Share a Functional Core 16s rrna- derived OTUs KEGG Orthology groups BD Muegge et al, Science 332: , 2011

9 Disparity Between Microbial Composition and Function F Backhed et al, Cell Host Microbe, 12:611-22, 2012

10 Growth of Anaerobic Fungi on Cellulose & Crude Biomass Increased growth resulting from higher gene expression of cellulases, hemicellulases, and pectin degrading enzymes KV Solomon et al, Science 351:1192-5, 2016

11 Seasonal variation in produce consumption by Hutterites FFQ asked about # of servings of fresh, frozen, canned, or pickled F/V per day from a total of 61 food categories ER Davenport et al, PLoS ONE 9:e90731, 2014

12 Phylum Level Taxa Differ in Stool Seasonally in Hutterites 60 individuals replicate DNA extractions/sample ER Davenport et al, PLoS ONE 9:e90731, 2014

13 Decreased Diversity of Fecal Microbiome in Summer ER Davenport et al, PLoS ONE 9:e90731, 2014

14 Bacterial Diversity Increases with Age 316 from US 115 from Malawi 100 from Venezuela T Yatsunenko et al, Nature 2012

15 30 most discriminatory OTUs for weight gain Model explains ~66% of phenotypic Variation (p<0.0001) Red denotes significant effect on weight and lean mass gains Purple indicates species that reflect gut maturation in Malawian children LV Blanton et al, Science, 351:6275, 2016

16 FGFP=1106 Belgians LLDeep=1135 Dutch G Falony et al, Science 352:560, 2016

17 1106 Belgians- covariates pooled in predefined categories G Falony et al, Science 352:560, 2016

18 Transplantation of microbiota from Malawian children into gnotobiotic mice Murine microbiota >50% OTU diversity in donor samples LV Blanton et al, Science, 351:6275, 2016

19 Fecal Butyrate at baseline and after 25g NSP & 22g RS from mixed foods in 40 subjects after 2 & 4 weeks Diet explained 30% of variation: protein 16%, starch 6%, CHO 2%, fiber 4%, fat 2% AL McOrist et al, J Nutr 141:883, 2011

20 Correlation Between Fecal Butyrate and Ammonia AL McOrist, J Nutr 141:883, 2011

21 Amino Acid Levels in Hepatic Portal Vein from Conventional or Germ Free Mice ^ ^ ^ ^ ^ ^ ^ ^ ^ ^= Essential +=Conditionally essential A Mardinoglu et al, Molec Sys Biol 11:834, 2015

22 Whole Body Arginine Bioavailability, Citrulline Flux & Mannitol Excretion in Humans 70 Arginine Bioavailability µmol. kg FFM - 1. h - 1 Whole- Body Citrulline Flux µmol. kg FFM - 1. h Urinary Mannitol, mg/6 h * * * * * Fasting Postprandial 0 Fasting Postprandial 0 Urinary Mannitol Excretion American Indian Jamaican American Indian Jamaican American Indian Jamaican *P<0.001 CC Kao et al, J Nutr, 2015 & 2016

23 CC Kao et al, J Nutr 2016

24 Absence of Enteral Nutrition Decreases Microbial Diversity H Engstrand Lilja et al, Microbiome 3:18, 2015

25 Shannon Diversity Index in Children with SBS still on PN or weaned from PN H Engstrand Lilja et al, Microbiome 3:18, 2015

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27 Diets for Multi- Generation Mouse Study High- MAC LabDiet 5010 Laboratory Autoclavable Rodent Diet Ground corn, dehulled soybean meal, wheat middlings, fish meal, whole wheat, wheat germ, brewers yeast, ground oats, alfalfa meal, beet pulp, many other ingredients 23% protein, 5% fat, 15% Neutral Detergent Fiber, 8% ash Lo- MAC Harlan Purified Diet TD % casein, 32% sucrose, 32% corn starch, 5% corn oil, 5% cellulose ED Sonnenburg et al, Nature, 529:212, 2016

28 Change in Bacterial Species Upon Refeeding High- MAC Diets ED Sonnenburg et al, Nature 529:212, 2016

29 Fecal Transplant and Hi- MAC Diet Restore Microbiota Diversity ED Sonnenburg et al, Nature 529:212, 2016

30 Loss of Glycoside Hydrolase Family Members in Generation 4 High- MAC diet Generation 1 ED Sonnenburg et al, Nature 529:212, 2016

31 B Schnabl & DA Brenner, Gastroenterology, 146:1513, 2014

32 G- Protein Coupled SCFA Receptors in Many Tissues Regulate Host Metabolism Suggests that circulating levels of all 3 SCFAs are important Contradicts that butyrate is fully used by colon and that propionate is extracted by liver in first pass M Kasubuchi et al, Nutrients 7:2839, 2015

33 Trans- Epithelial Resistance and Mucosal- Serosal Transit of Dextran Correlated to loss of tight junction localization of claudin- 4 and tricellulin, reduced expression of claudin- 5 and claudin- 8 FR Demehri et al, Dig Dis Sci, 61:1524, 2016

34 Microbiome Also Affects: Epigenetics of gut stem cells DH Yu et al, Genome Biol 2015 GI pathogens Chronic Disease Risk Fatty Liver CVD Colon Cancer ML Jones et al, Gut Microbes, 5:446, 2014

35 At present there is insufficient evidence to use changes in faecal bacterial metabolite concentrations as markers of prebiotic effectiveness. KA Verbeke et al, Nutr Res Rev 28: 42, 2015

36 Old Observations Relevant to Metabolism of the Microbiome Transit Time Decreased exposure of mucosa to microbial metabolites Gut ph Changes enzyme optima, solubility Stool Volume Increased bacterial mass, decreased concentration of metabolites Stool Hardness Bristol Stool Scale Less water, less diffusion of metabolites But harder stool = greater microbial diversity

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