THE GUT MICROBIOTA AS AN EMERGING TARGET FOR THE HEALTH OF THE HOST

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1 THE GUT MICROBIOTA AS AN EMERGING TARGET FOR THE HEALTH OF THE HOST Patrizia Brigidi Department of Pharmacy and Biotechnology

2 THE HUMAN INTESTINAL MICROBIOTA habits our body and the great majority of these microorganisms is hidden in the gastrointestinal tract CFU/g up to 1.5 kg organized in a ecosystem of highly interconnected microbes the most dense bacterial ecosystem on our planet

3 EUKARYOME AND VIROME: OVERLOOKED COMPONENTS OF THE GUT MICROBIOTA Sokol et al., Gut 2015 Rampelli et al., BMC Genomics 2016

4 HUMAN INTESTINAL MICROBIOTA our bacterial counterpart provides essential features we have not evolved enhancement of the digestive efficiency and modulation of energetic homeostasis vitamin synthesis competitive barrier against colonization/invasion development, education and function of the immune system strengthening of the GIT epithelium impermeability detoxification of xenobiotics central nervous system modulation endocrine system modulation

5 HOW DO WE KNOW THIS? Culture-based methods allow to recover 20-30% of total microscopic counts MOSTLY UNCULTURED! Culture-independent molecular survey: NGS

6 Profiling intestinal microbiota at high resolution by studying the collective set of genomes Challenges: NEXT GENERATION SEQUENCING (NGS) Phylogenetic characterization: obtaining accurate microbial identification for thousands of gut microbiota species in a reasonable time and for a reasonable cost (microbial identification via single genetic targets: 16S rrna V3/V4 and V4/V5 regions: 18S rrna; 23S rrna) Functional metagenomics: to identify and annotate diverse arrays of microbial genes that encode many different biochemical and metabolic functions of the gut microbiota (whole-genome methodologies)

7 16S bacterial Small Subunit rrna genebased survey of the gut microbiota 1.5 kb Strain ~ 500 bp amplicon are sufficient for the phylogenetic analysis (OTU: operational taxonomic unit) Species Genus Family Sequence similarity

8 NEXT GENERATION SEQUENCING - Possibility to sequence even 100 samples per run - Obtain a large amount of infos simultaneously, thanks to BIOINFORMATICS α-diversity within subjects diversity index rarefaction β-diversity between subjects distance matrix PCA-PCoA similarities compositional differences correlation matrix analysis of variance clustering box plots

9 Profiling intestinal microbiota at high resolution by studying the collective set of genomes Challenges: NEXT GENERATION SEQUENCING (NGS) Phylogenetic characterization: obtaining accurate microbial identification for thousands of gut microbiota species in a reasonable time and for a reasonable cost (microbial identification via single genetic targets: 16S rrna V3/V4 and V4/V5 regions: 18S rrna; 23S rrna) Functional metagenomics: to identify and annotate diverse arrays of microbial genes that encode many different biochemical and metabolic functions of the gut microbiota (whole-genome methodologies)

10 MICROBIOTA MOLECULAR ASSESSMENT 16S rdna next generation sequencing whole genome OTU composition metagenome sequence phylogenetic structure rel. ab. of community members rel. ab. of community gene pathways

11 PHYLOGENETIC DIVERSITY > 1000 species 6 (out of 100) bacterial phyla Firmicutes, Bacteroidetes : 90% Actinobacteria, Proteobacteria, Fusobacteria and Verrucomicrobia : 10%

12 FUNCTIONAL DIVERSITY MICROBIOME (collective genome of the microbiota) 10 6 GENES 58% KNOWN 42% UNKNOWN carbohydrate metabolism (CAZymes) energy metabolism amino acid metabolism biosynthesis of secondary metabolites metabolism of cofactors and vitamins Schloissing et al, Nature 2013

13 HOW DO WE KNOW THIS? - Microbiome sequence analysis - Observation of human gut microbiota in different physiological/pathological condition - In vitro studies using complex bacterial communities THE ADOPTION OF GERM-FREE MICE ALLOWED TO MEASURE THE IMPACT OF GUT MICROBIOTA-HOST MUTUALISM ON SEVERAL PHYSIOLOGICAL PARAMETERS

14 MICROBIOTA PLASTICITY THE INDIVIDUAL MICROBIOTA COMPOSITION CONTINUOUSLY CHANGES IN RESPONSE TO EXTRINSIC AND INTRINSIC VARIABLES DIET ENV. FACTORS (env. microbes-geography-climate sanitization-medication) MICROBIOTA MAKE-UP GIT ENVIRONMENT (inflammation-disease) HOST GENETICS AGE IN A MUTUALISTIC CONTEXT, THE PLASTICITY OF THE HUMAN MICROBIOTA GUARANTEES A RAPID ADAPTATION OF THE SUPER-ORGANISM IN RESPONSE TO DIET CHANGES, AGE, ETC there is a strong selection towards a readily changeable individual microbiome profile

15 IMPACT ON HOST NUTRITION indigestible plant polysaccharides (xylan, pectin, arabinose containing-dietary carbohydrates as plant-derived pectin, cellulose, hemicellulose, resistant starches) reach unchanged the colon where they are metabolized by the intestinal microorganisms indigestible plant polysaccharides SCFA key microbiota metabolites regulating the host nutritional status equipped with a real arsenal of CAZymes absent in human genome intestinal microorganisms degrade plant polysaccharides to SCFA

16 SUBSTRATES OF THE GM CAZymes ARSENAL thousands of enzymes while we possess only 17 not accessible to the human glycobiome! The GM possesses a broad glycobiome complexity, complementing the limited diversity of the human glycobiome and enhancing the superorganism capacity to metabolize complex polysaccharides El Kaoutari et al., Nat Rev Microbiol. 2013

17 DIETARY COMPONENTS: SYNTROPHIC MICROBIAL NETWORKS HOST POLYSACCHARIDE S Bacteroidetes ACETATE SUCCINATE low CO 2 PROPIONATE STARCH SOLUBLE CELL WALL POLYSACCHARIDES hemicellulose, xylan, pectin, mannans, inulin, fructans PLANT CELL WALL cellulose Clostridium clusters IV and XIVa Faecalibacterium prausnitzii, Butyrrivibrio, Roseburia, Eubacterium rectale BUTYRATE Ruminococci ACETATE methanogens Methanobrevibacter smithii CH 4 H 2 sulfate-reducing bacteria Bilophila wadsworthia H 2 S acetogens Blautia hydrogenotrophica ACETATE

18 SCFA, MICROBIAL METABOLITES WITH A KEY MULTIFACTORIAL ROLE IN HOST PHYSIOLOGY SCFAs as Signaling Molecules - HDAC inhibitors - GPCR (41,43,109A) ligands GABA production in CNS Serotonin biosynthesis CNS activity energy source Intestinal gluconeogenesis Modulation of ENS and CNS function Metabolic regulations release of major appetite and glucose regulatory peptides (PYY, GPL1) (L-cells) anti lipolytic activity (WAT) leptin production (WAT) Enhancement of IL-10 (DC) Reduction of TNF-α, IL-6, IFN-γ (DC) Nastasi et al., Sci Rep. 2015; Koh et al., Cell IL-18 biosynthesis (IEC) Immune homeostasis Treg polarization IEC barrier fortification IgA promotion

19 SIDE PRODUCTS FROM GM PROTEIN FERMENTATION AMINO ACIDS DETRIMENTAL FOR THE HOST HEALTH SCFA BCFA proteolytic clostridia and Bacteroidetes (Alistipes) PHENOLIC AND INDOLIC METABOLITES METHYLAMINES IMPACT ON HOST PHYSIOLOGY AROMATIC AMINO ACIDS PHENOLIC AND INDOLIC METABOLITES (indoles; p-cresol; phenols; phenylacetic acid) LIVER STEATOSIS, OBESITY AND DIABETES ALIPHATIC AMINO ACIDS METHYLAMINES IMMUNE ACTIVATION AND DIABETES

20 IMPACT ON HOST PHYSIOLOGY OF THE GM ADAPTATION TO FAT DIETARY FATS BILE ACIDS PRO-INFLAMMATORY DYSBIOSES OF GM BIOACTIVE COMPOUNDS SECONDARY BILE ACIDS H 2 S INFLAMMATION IN THE GUT reabsorption and MODULATION OF FXR AND TGR5 RECEPTORS REGULATION OF DIETARY FAT ABSORPTION INCREASE OF TRANSEPITHELIAL PERMEABILITY OBESITY, TYPE II DIABETES

21 GM-HOST CO-METABOLIC LAYOUTS diet regulates microbiota composition and metabolic output with a final impact on host physiology complex polysaccharides SACCHAROLYTIC METABOLISM amino acids PROTEOLYTIC METABOLISM animal fat FAT ADAPTATION highly diverse community of polysaccharide-degrading Bacteroidetes and Clostridia establishing syntrophy in the gut low diverse community enriched in specialized proteolytic Alistipes and Clostridia selection of a low diverse community made of bile resistant Erysipelotrichi, Bilophila wadsworthia, Enterobacteriaceae SUCCINATE Health promoting SCFA CH4 SCFA BCFA PHENOLIC AND INDOLIC METABOLITES METHYLAMINES SECONDARY BILE ACIDS Disease associated H 2 S

22 THE GUT MICROBIOTA DESCRIBES AN ADAPTIVE TRAJECTORY ALONG HUMAN AGING AGE RELATED CHANGES OF THE GUT MICROBIOTA PROVIDE THE HOST WITH ECOLOGICAL SERVICES CALIBRATED FOR EACH STAGE OF LIFE Candela et al., Critical Rev Microbiol, 2013

23 INFANT-TYPE MICROBIOTA: SIMPLE, READILY CHANGEABLE AND BIFIDOBACTERIUM-DOMINATED INFANT-TYPE MICROBIOTRA IS STRUCTURED TO COPE WITH INFLAMMATION, BEING CO-EVOLVED TO PRIME THE EARLY IMMUNE SYSTEM IN THE CONTEXT OF TRANSIENT INFLAMMATORY RESPONSES; MILK DIGESTION; FOLATE BIOSYNTHESIS Centanni et al., PLoS ONE. 2013

24 ADULT-TYPE MICROBIOTA: COMPLEX AND ADAPTABLE ECOSYSTEM DOMINATED BY FIRMICUTES AND BACTEROIDETES Schnorr et al., Nature Communication, 2014

25 ELDERLY TYPE MICROBIOTA LOW DIVERSITY DECREASE IN BIFIDOBACTERIA INCREASE IN PATHOBIONTS (Fusobacteria, Bacillus, Staphylococcus, Enterobacteriaceae) REARRANGEMENT OF BUTYRATE PRODUCERS SUBDOMINANT FRACTION DOMINANT FRACTION Biagi et al., Cur Biol, 2016 Ruminococcus obeum (CI XIVa) Roseburia intestinalis (CI XIVa) Eubacterium rectale (CI XIVa) Faecalibacterium prausnitzii (Cl IV) GM Anaerotruncus colihominis (Cl IV) Eubacterium limosum (Cl XV)

26 THE MICROBIAL ECOLOGY OF GM DYSBIOSIS, THE ANNA KARENINA PRINCIPLE All happy families are alike; each unhappy family is unhappy in its own way Leo Tolstoy: Anna Karenina (1878) STRESSORS health-plan of GM variation healthpromoting GM configurations diseaseassociated GM configuration diseaseassociated GM configuration diseaseassociated GM configuration diseaseassociated GM configuration health-plan of GM variation STOCHASTIC DISPERSION diseaseassociated GM configuration diseaseassociated GM configuration diseaseassociated GM configuration diseaseassociated GM configuration diseaseassociated GM configuration All microbiomes are similar; each dysbiotic microbiome is dysbiotic in its own way Zaneveld et al., Nat Microbiol 2017

27 Rel. Abb. EUBIOTIC AND DYSBIOTIC DISTRIBUTIONS OF THE MAJOR GM FAMILIES Eubiotic distributions Dysbiotic distributions

28 INFLAMMATION AND MICROBIOTA A non-controlled pro-inflammatory pathway can dramatically impact on the composition of the intestinal microbiota SUSCEPTIBLE HOST - GENETICS - ENV. FACTORS CHRONIC GIT INFLAMMATION PRO-INFLAMMATORY LOOP MICROBIOTA DYSBIOSIS RAISE IN PATHOBIONTS PRO-INFLAMMATORY INTESTINAL MICROBIAL COMMUNITY DECREASE OF IMMUNO MODULATORY GROUPS

29 COMMON TRAITS OF DYSBIOSIS Reduction of SCFA producing bacteria (butyrate producers such as Faecalibacterium, Roseburia, Lachnospiraceae, Eubacterium) Increased mucus degradation potential by abnormal mucin degraders that displace Akkermansia Reduced hydrogen and methane production combined with increased hydrogen sulphide production. H 2 S is toxic for the epithelium Increase in abundance of bacteria with LPS endotoxins (Proteobacteria) that can drive inflammation Increased potential to manage oxidative stress, i.e. microbes became able to proliferate in close vicinity to the epithelium Le Chatellier et al., Nature 2013

30 GUT MICROBIOTA-ASSOCIATED DISORDERS Nutrition related disorders (obesity, type 2 diabetes, metabolic syndrome) Inflammatory bowel diseases (UC and CD) Functional bowel disorders (IBS) Systemic complications of decompensated liver disease Cardio-vascular diseases Atopy/allergy Colo-rectal Cancer Neuro-developmental conditions (autism, depression) Arthritis

31 THE GM OF HADZA HUNTER-GATHERERS AND URBAN ITALIANS, TWO DICHOTOMOUS MODELS OF HUMAN LIFESTYLE AND SUBSISTENCE The Hadza from Tanzania - direct interface with the natural environment, deriving their wild food - no cultivation or domestication of plants and animals, minimal agricultural products from external sources, < 5% calories Urban Italians from Bologna metropolitan area Heavy plant based diet - 70% of kcal from wild plant foods; 30% from bird and animal meat (dry season) - diet rich in wild plant polysaccharides, starch and protein while lean in fat - typical Western urban environment and lifestyle Mediterranean diet

32 HADZA AND ITALIANS POSSESS PHYLOGENETICALLY AND FUNCTIONALLY DISTINCTIVE GM

33 GM COMPOSITIONAL DIFFERENCES PECULIARITIES OF THE HDZA GM COMPOSITIONAL STRUCTURE NEEDS IN HADZA LAND HIGH BACTERIAL DIVERSITY ENRICHMENT IN FIBROLYTIC BACTERIA high abundance in xylan-degrading Prevotella, Treponema, unclassified Bacteroidetes and Clostridiales SEX-RELATED DIVERGENCE IN GM STRUCTURE higher Treponema in Hadza women HARD DIGESTIBLE FOOD HEAVY PLANT BASED DIET SEX DIFFERENCES IN DIET COMPOSITION (higher consumption of plant foods in women) ABSENCE OF BIFIDOBACTERIUM ENRICHMENT OF OPPORTUNISTIC BACTERIA Proteobacteria and Spirochaetes ABSENCE OF AGRO-PASTORAL-DERIVED FOODS IMMUNE EDUCATION TO THE DIRECT INTERFACE WITH NATURAL ENVIRONMENT

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35 GENE DISTRIBUTION AT KO LEVEL RESULTS IN THE SEPARATION BETWEEN HADZA AND ITALIAN METAGENOME STRUCTURES

36 FUNCTIONAL PECULIARITIES OF THE HADZA AND ITALIAN GUT MICROBIOME FAVORING THE HOST ADAPTATION TO THE RESPECTIVE ENVIRONMENTS

37 GUT MICROBIOTA AND EXTREME LONGEVITY Biagi E, Franceschi C, Rampelli S, Severgnini M, Ostan R, Turroni S, Consolandi C, Quercia S, Scurti M, Monti D, Capri M, Brigidi P, Candela M. Curr Biol Jun 6;26(11):

38 THE LONGEST AVAILABLE TRAJECTORY OF THE HUMAN GUT MICROBIOTA ALONG AGING core microbiota of highly occurring, symbiotic bacterial groups, which remains approximately constant during aging but varies in the cumulative relative abundance of its members. aging-associated microbiota is characterized by an increasing contribution of subdominant species, as well as a rearrangement in their co-occurrence network. the microbial ecosystem found in extremely old people, is enriched in health-associated Akkermansia, Bifidobacterium, and Christensenellacea

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40 Comparison between Italian (I) and Chinese (C) centenarians and young adults Akkermansia and Christensenellaceae can represent a signature of adaptation to the changes associated with the long living, regardless of lifestyle and dietary habits

41 Temporal dynamics of the gut microbiota in people sharing a confined environment, a 520-day groundbased space simulation, MARS500. Turroni S, Rampelli S, Biagi E, Consolandi C, Severgnini M, Peano C, Quercia S, Soverini M, Carbonero FG, Bianconi G, Rettberg P, Canganella F, Brigidi P, Candela M. Microbiome Mar 24;5(1):39.

42 GUT MICROBIOTA DYNAMICS IN THE SIX ASTRONAUTS OF THE LONGEST GROUND-BASED SPACE SIMULATION PROJECT, MARS500 the human intestinal microbiota retains a significant degree of temporal variability even under the strictly controlled conditions of an enclosed environment, oscillating between different configurations typically with rearrangements of autochthonous microorganisms. sharing life in a confined habitat does not compromise the individual specificity of the microbiota compositional layout, even in the long term, confirming the resilience of the individuality of the gut microbial ecosystem a combination of factors, including isolation and stress, force a conserved dynamic response of certain important components of the microbiota eg. Bacteroidetes increase, reduction of F. prausnitzii and R. faecis - with the potential to drive unbalances in the pattern of SCFA production, with cascading implications for the host metabolic and immunological homeostasis.

43 FP7 KBBE MyNewGut Microbiome influence on energy balance and brain development/function put into action to tackle diet-related diseases and behaviour Project duration: 5 years Start date: 1 December partners from 15 EU and non-eu countries EU contribution 9 million Gut microbiome influences on diet-related diseases and behaviour

44 MyNewGut OBJECTIVES 1. Investigating the role of the GM and specific components in nutrient metabolism and, therefore, in energy balance 2. Identifying specific GM components that predict and contribute to metabolic and eating disorders. 3. Understanding the influence of environmental factors on the GM in pregnancy and the offspring and its impact on brain, immune system and metabolic development and health programming. 4. Developing new food ingredients and food prototypes, by collaborating with EU food industry, targeting the gut ecosystem, to reduce the risk of metabolic and brain-related disorders.

45 The gut microbiota of gilthead sea bream (Sparus aurata, L.) fed with different diets Parma et al., Animal Feed Sci Technol tanks with 60 sea bream per tank 100 days of trial with 5 isoproteic, isolipidic experimental diets: - Low fishmeal-based diets with increasing soybean meal levels (0, 100, 200, 300 g/kg) - Fishmeal-based diet, as a control Parameters: - Growth performance (e.g., specific growth rate, feed intake, etc.) - Gut histology - Whole body composition and nutritional indices - Gut microbiota profiling

46 The gut microbiota of gilthead sea bream (Sparus aurata, L.) fed with different diets Parma et al., Animal Feed Sci Technol 2016 A TREND TOWARDS SEPARATION ACCORDING TO DIET

47 The gut microbiota of gilthead sea bream (Sparus aurata, L.) fed with different diets Parma et al., Animal Feed Sci Technol 2016 Cyanobacteria Actinobacteria Synergistetes Lactobacillaceae With increasing soybean meal levels: - Enrichment in Cyanobacteria (providing essential vitamins) and Lactobacillaceae (immunomodulatory) - Decreased relative abundance of Synergistetes (opportunistic pathogens) Increasing soybean meal levels promote specific microbiota components associated with a healthy intestine

48 Early colonization and temporal dynamics of the gut microbial ecosystem in Standardbred foals Quercia et al., 2018 submitted to Equine Vet J 13 Standardbred mare-foal pairs AT DELIVERY: - MARE: amniotic fluid, feces and colostrum - FOAL: meconium AT 1, 3, 5, 7, 10 DAYS POST-PARTUM: - MARE: milk and feces - FOAL: feces

49 Early colonization and temporal dynamics of the gut microbial ecosystem in Standardbred foals Quercia et al., 2018 submitted to Equine Vet J Meconium and amniotic fluid share a peculiar structure, being inhabited by «cosmopolitan» bacteria and including components from mare microbiomes

50 Early colonization and temporal dynamics of the gut microbial ecosystem in Standardbred foals Quercia et al., 2018 submitted to Equine Vet J OTU SHARING The existence of OTUs shared between meconium and the two mare ecosystems suggests that each of them can specifically contribute to the meconium bacterial community in terms of founder microbial species POSSIBLE INTERNAL TRANSMISSION ROUTES, WITH MARE MICROBIAL FACTORS BEING VERTICALLY TRANSMITTED TO THE FETUS

51 Early colonization and temporal dynamics of the gut microbial ecosystem in Standardbred foals Quercia et al., 2018 submitted to Equine Vet J A PECULIAR DEVELOPMENTAL TRAJECTORY OF THE FOAL GUT MICROBIOTA, PROGRESSIVELY APPROACHING THE TYPICAL ADULT-LIKE CONFIGURATION, DRIVEN BY MILK FEEDING AND COPROPHAGY EXTERNAL TRANSMISSION ROUTES OF MARE MICROORGANISMS AFTER BIRTH

52 The gut microbiota of pigs fed with einkorn vs wheat bread preliminary data Gianotti et al., commercial hybrid pigs Landrace x Duroc (6 males, 6 females; 36 kg; 10-wk old) 30 days with 2 experimental diets (1 kg/day of 1:1 mixture of Swine Standard Diet and bread): - Einkorn bread - Wheat bread Gut microbiota profiling at T0, T30 in fecal samples, as well as at T30 in mucosal scrapings from ileum and colon

53 The gut microbiota of pigs fed with einkorn vs wheat bread preliminary data Gianotti et al., 2018 Unweighted UniFrac Weighted UniFrac T0, wheat T30, wheat T0, einkorn T30, einkorn BOTH DIETS MODULATE THE FECAL MICROBIOTA BUT ONLY THE EINKORN BREAD FAVORS SPECIFIC HEALTH-PROMOTING BACTERIA (including Blautia and Faecalibacterium)

54 The gut microbiota of pigs fed with einkorn vs wheat bread preliminary data Gianotti et al., 2018 Weighted UniFrac AFTER THE EINKORN- BASED DIET, THE ILEAL MUCOSA-ASSOCIATED MICROBIOTA IS MORE BIODIVERSE, WITH AN ENRICHMENT IN BIFIDOBACTERIUM SPP. AND A DECREASE IN STREPTOCOCCUS AND LACTOBACILLUS Wheat, ileum Einkorn, ileum Wheat, colon Einkorn, colon

55 Microbial Ecology of Health UNIT Dept. Pharmacy and Biotechnology, University of Bologna, Italy Patrizia Brigidi Full Full Professor Patr izia Br igidi Silvia Full Tur Prr oni, ofessor PhD Silvia Turroni, PhD Jessica Sar a Quer Baldini cia Silvia Patr izia TurBr r oni, igidi PhD PhD student Graduate Full Pr ofessor student Federica Monica Bar one PhD Sar a student Quer D Amico cia PhD Silvia PhD student Tur r oni, PhD Simone Rampelli, PhD Monica Barone one Sar PhD a Quer student cia PhD student PhD student Elena Biagi, PhD Simone Rampelli, PhD Simone Monica Rampelli, Bar onephd PhD student Matteo Sover ini PhD student Elena Biagi, PhD Simone Rampelli, PhD Elena Biagi, PhD THANK YOU FOR YOUR ATTENTION!!! Mar co Candela, PhD Matteo Sover ini Elena Biagi, PhD PhD student Andrea Castagnetti, PhD Matteo Soverini PhD student Patr Matteo izia Sover Br igidi ini Mar co Candela, PhD Full PhD Prstudent ofessor Marco Mar Silvia co Tur Candela, Candela r oni, PhD PhD Associate Professor Sara SarQuercia a cia PhD PhD student

56 Patrizia Brigidi Department of Pharmacy and Biotechnology