the gut microbiota as an emerging target … · eubacterium rectale (ci xiva) faecalibacterium...

THE GUT MICROBIOTA

AS AN EMERGING

TARGET FOR THE

HEALTH OF THE HOST

Patrizia Brigidi

Department of Pharmacy and

Biotechnology

THE HUMAN INTESTINAL MICROBIOTA

1013–1014 habits our body and the great majority of these microorganisms is hidden in the gastrointestinal tract

• 1012 CFU/g

• up to 1.5 kg

the most dense bacterial ecosystem on our planet

organized in a ecosystem of highly interconnected

microbes

EUKARYOME AND VIROME: OVERLOOKED

COMPONENTS OF THE GUT MICROBIOTA

Sokol et al., Gut 2015 Rampelli et al., BMC Genomics 2016

HUMAN INTESTINAL MICROBIOTA

• 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

our bacterial counterpart provides essential features we have not evolved

HOW DO WE KNOW THIS?

Culture-based methods allow to recover

20-30% of total microscopic counts

MOSTLY UNCULTURED!

Culture-independent molecular survey:NGS

Profiling intestinal microbiota at high resolution by studying the collective set of genomes

Challenges:

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)

NEXT GENERATION SEQUENCING(NGS)

Strain

Species

Genus

Family

~ 500 bp amplicon are

sufficient for the phylogenetic

analysis (OTU: operational

taxonomic unit)

Sequence similarity

1.5 kb

16S bacterial Small Subunit rRNA gene-

based survey of the gut microbiota

distance

matrixPCA-PCoA

NEXT GENERATION SEQUENCING

β-diversity

between subjects

α-diversity

within subjects

compositional

differences

diversity

index

analysis of

variance

rarefaction

box plots

- Possibility to sequence even 100 samples per run- Obtain a large amount of infos simultaneously, thanks to BIOINFORMATICS

similaritiescorrelation

matrix clustering

Profiling intestinal microbiota at high resolution by studying the collective set of genomes

Challenges:

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)

NEXT GENERATION SEQUENCING(NGS)

MICROBIOTA MOLECULAR

ASSESSMENT

16S rDNAnext generation

sequencingwhole genome

OTU composition

phylogenetic structure

rel. ab. of community members

metagenome sequence

rel. ab. of community

gene pathways

PHYLOGENETIC DIVERSITY

> 1000 species

6 (out of 100) bacterial phyla

• Firmicutes, Bacteroidetes : 90%• Actinobacteria, Proteobacteria, Fusobacteria and

Verrucomicrobia : 10%

MICROBIOME(collective genome of the

microbiota)

106 GENES

58% KNOWN 42% UNKNOWN • carbohydrate metabolism (CAZymes)

• energy metabolism

• amino acid metabolism

• biosynthesis of secondary metabolites

• metabolism of cofactors and vitamins

FUNCTIONAL DIVERSITY

Schloissing et al, Nature 2013

HOW DO WE KNOW THIS?

THE ADOPTION OF GERM-FREE

MICE ALLOWED TO MEASURE

THE IMPACT OF GUT

MICROBIOTA-HOST MUTUALISM

ON SEVERAL PHYSIOLOGICAL

PARAMETERS

- Microbiome sequence analysis- Observation of human gut microbiota in different physiological/pathological

condition- In vitro studies using complex bacterial communities

MICROBIOTA PLASTICITY

THE INDIVIDUAL MICROBIOTA COMPOSITION CONTINUOUSLY CHANGES IN

RESPONSE TO EXTRINSIC AND INTRINSIC VARIABLES

DIET

HOST GENETICS

MICROBIOTA

MAKE-UP

GIT ENVIRONMENT(inflammation-disease)

ENV. FACTORS(env. microbes-geography-climate

sanitization-medication)

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

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

equipped with a real arsenal of CAZymes – absent in human genome –

intestinal microorganisms degrade plant polysaccharides to SCFA

SCFA

key microbiota metabolites

regulating the host nutritional

status

indigestible plant

polysaccharides

SUBSTRATES OF THE GM CAZymes ARSENALthousands of enzymes while we possess only 17

The GM possesses a broad glycobiome complexity, complementing the limited diversity of the human glycobiome and enhancing the superorganism capacity to metabolize

complex polysaccharides

not accessible to the human

glycobiome !

El Kaoutari et al., Nat Rev Microbiol. 2013

HOST

POLYSACCHARIDE

S

STARCH PLANT CELL WALLcellulose

SOLUBLE CELL WALL

POLYSACCHARIDEShemicellulose, xylan, pectin, mannans,

inulin, fructans

Bacteroidetes

Ruminococci

Clostridium clusters

IV and XIVaFaecalibacterium prausnitzii,

Butyrrivibrio, Roseburia,

Eubacterium rectale

acetogensBlautia hydrogenotrophica

methanogensMethanobrevibacter smithii

sulfate-reducing

bacteria Bilophila wadsworthia

ACETATE

PROPIONATE BUTYRATE

SUCCINATE

H2

H2S

CH4

ACETATE

ACETATE

low CO2

DIETARY COMPONENTS: SYNTROPHIC MICROBIAL NETWORKS

Metabolic regulations

SCFA, MICROBIAL METABOLITES WITH A KEY MULTIFACTORIAL ROLE IN HOST PHYSIOLOGY

SCFAs as Signaling Molecules

- HDAC inhibitors

- GPCR (41,43,109A) ligands

CNS activity

Immune homeostasis

release of major

appetite and glucose

regulatory peptides

(PYY, GPL1) (L-cells)

Intestinal

gluconeogenesisenergy source

anti lipolytic

activity (WAT)

leptin production

(WAT)

IEC barrier

fortificationIL-18

biosynthesis

(IEC)

Reduction of TNF-α,

IL-6, IFN-γ (DC)

Enhancement

of IL-10 (DC)

Treg

polarization

IgA

promotion

Modulation of ENS and

CNS function

Serotonin

biosynthesis

GABA

production

in CNS

Nastasi et al., Sci Rep. 2015; Koh et al., Cell. 2016

SIDE PRODUCTS FROM GM PROTEIN FERMENTATION

METHYLAMINESPHENOLIC AND INDOLIC METABOLITES

(indoles; p-cresol; phenols; phenylacetic acid)

AROMATIC

AMINO ACIDS

IMMUNE

ACTIVATION

AND

DIABETES

ALIPHATIC

AMINO ACIDS

LIVER

STEATOSIS,

OBESITY AND

DIABETES

IMPACT ON HOST PHYSIOLOGY

DETRIMENTAL FOR THE HOST HEALTHAMINO ACIDS

proteolytic

clostridia and

Bacteroidetes(Alistipes)

SCFA

BCFA

METHYLAMINES

PHENOLIC AND

INDOLIC

METABOLITES

IMPACT ON HOST PHYSIOLOGY OF THE GM ADAPTATION TO FAT

H2SSECONDARY

BILE ACIDS

DIETARY FATS BILE ACIDS

BIOACTIVE

COMPOUNDS

INCREASE OF

TRANSEPITHELIAL

PERMEABILITY

INFLAMMATION IN THE GUT

reabsorption and

MODULATION OF FXR AND TGR5 RECEPTORS

REGULATION OF DIETARY

FAT ABSORPTION

OBESITY, TYPE II DIABETES

PRO-INFLAMMATORY

DYSBIOSES OF GM

GM-HOST CO-METABOLIC LAYOUTS

diet regulates microbiota composition and metabolic output with a final impact on host physiology

SACCHAROLYTICMETABOLISM

PROTEOLYTIC METABOLISM

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

H2SSECONDARY

BILE ACIDS

SCFA

BCFAMETHYLAMINES

PHENOLIC AND

INDOLIC

METABOLITESSCFA

Healthpromoting

Diseaseassociated

amino acids animal fat

CH4SUCCINATE

complex polysaccharides

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

INFANT-TYPE MICROBIOTA: SIMPLE, READILY CHANGEABLE AND BIFIDOBACTERIUM-DOMINATED

Centanni et al., PLoS ONE. 2013

INFANT-TYPE MICROBIOTRA IS STRUCTURED TOCOPE WITH INFLAMMATION, BEING CO-EVOLVEDTO PRIME THE EARLY IMMUNE SYSTEM IN THECONTEXT OF TRANSIENT INFLAMMATORYRESPONSES; MILK DIGESTION; FOLATEBIOSYNTHESIS

ADULT-TYPE MICROBIOTA: COMPLEX AND ADAPTABLE ECOSYSTEM DOMINATED BY FIRMICUTES AND

BACTEROIDETES

Schnorr et al., Nature Communication, 2014

ELDERLY TYPE MICROBIOTA

• LOW DIVERSITY

• DECREASE IN BIFIDOBACTERIA • INCREASE IN PATHOBIONTS

• REARRANGEMENT OF BUTYRATE PRODUCERS

Ruminococcus obeum (CI XIVa) Roseburia intestinalis (CI XIVa)Eubacterium rectale (CI XIVa)

Faecalibacterium prausnitzii (Cl IV)

(Fusobacteria, Bacillus, Staphylococcus, Enterobacteriaceae)

Anaerotruncuscolihominis (Cl IV)

Eubacterium limosum (Cl XV)

DOMINANT FRACTION

SUBDOMINANTFRACTION

GM

Biagi et al., Cur Biol, 2016

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)

health-promoting GM configurations

disease-associated GM configuration







STRESSORSSTOCHASTIC DISPERSION

All microbiomes are similar; each dysbiotic microbiome is dysbiotic in its own way



health-plan of GM variation health-plan of GM variation

Zaneveld et al., Nat Microbiol 2017

EUBIOTIC AND DYSBIOTIC DISTRIBUTIONS OF THE MAJOR GM FAMILIES

Rel

. Ab

b.

Eubiotic distributions

Dysbiotic distributions

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

MICROBIOTA

DYSBIOSIS

RAISE IN

PATHOBIONTS

PRO-INFLAMMATORY

LOOP

PRO-INFLAMMATORY

INTESTINAL

MICROBIAL

COMMUNITY

DECREASE OF

IMMUNO

MODULATORY

GROUPS

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. H2S 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

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

THE GM OF HADZA HUNTER-GATHERERS AND URBAN

ITALIANS, TWO DICHOTOMOUS MODELS OF HUMAN

LIFESTYLE AND SUBSISTENCE

- 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

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

The Hadza from Tanzania

Urban Italians from Bologna metropolitan area

HADZA AND ITALIANS POSSESS PHYLOGENETICALLY AND FUNCTIONALLY DISTINCTIVE GM

GM COMPOSITIONAL DIFFERENCES

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

HIGH BACTERIAL DIVERSITY

ABSENCE OF BIFIDOBACTERIUM

HARD DIGESTIBLE FOOD

HEAVY PLANT BASED DIET

SEX DIFFERENCES IN DIET COMPOSITION (higher consumption of plant foods in women)

ABSENCE OF AGRO-PASTORAL-DERIVED FOODS

PECULIARITIES OF THE HDZA GM COMPOSITIONAL STRUCTURE NEEDS IN HADZA LAND

ENRICHMENT OF OPPORTUNISTIC BACTERIA Proteobacteria and Spirochaetes

IMMUNE EDUCATION TO THE DIRECT INTERFACE WITH NATURAL ENVIRONMENT

[email protected]

mailto:[email protected]

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

METAGENOME STRUCTURES

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

RESPECTIVE ENVIRONMENTS

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. 2016 Jun 6;26(11):1480-5.

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

THE LONGEST AVAILABLE TRAJECTORY OF THE HUMAN GUT MICROBIOTA ALONG AGING

Comparisonbetween 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

Temporal dynamics of the gut microbiota in people sharing a confined environment, a 520-day ground-

based 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. 2017 Mar 24;5(1):39.

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 microbiotacompositional 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.

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 2013• 30 partners from 15 EU and non-EU countries• EU contribution ≅ € 9 millionGut microbiome influences on diet-related diseases and behaviour

MyNewGut OBJECTIVES

1. Investigating the role of the GM and specificcomponents in nutrient metabolism and, therefore,in energy balance

2. Identifying specific GM components that predictand contribute to metabolic and eating disorders.

3. Understanding the influence of environmentalfactors on the GM in pregnancy and the offspringand its impact on brain, immune system andmetabolic development and health programming.

4. Developing new food ingredients and foodprototypes, by collaborating with EU food industry,targeting the gut ecosystem, to reduce the risk ofmetabolic and brain-related disorders.

The gut microbiota of gilthead sea bream (Sparus

aurata, L.) fed with different diets

Parma et al., Animal Feed Sci Technol 2016

15 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




A TREND TOWARDS SEPARATION ACCORDING TO

DIET




Cyanobacteria Synergistetes

Actinobacteria 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

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




Meconium and amniotic fluid share a peculiar structure, being inhabited by

«cosmopolitan» bacteria and including components from mare microbiomes




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 speciesOTU SHARING

POSSIBLE INTERNAL TRANSMISSION ROUTES, WITH MARE

MICROBIAL FACTORS BEING VERTICALLY TRANSMITTED TO

THE FETUS




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

The gut microbiota of pigs fed with

einkorn vs wheat bread – preliminary data

Gianotti et al., 2018

12 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




BOTH DIETS MODULATE THE FECAL MICROBIOTA BUT ONLY THE EINKORN BREAD FAVORS SPECIFIC HEALTH-PROMOTING BACTERIA (including

Blautia and Faecalibacterium)

T0, wheat

T30, wheat

T0, einkorn

T30, einkorn

Unweighted UniFrac 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

Weighted UniFrac

Pat r izia Br igidi

Full Pr ofessor

Silv ia Tur r oni, PhD

Sar a Quer cia

PhD student

Monica Bar one

PhD student

Simone Rampell i , PhD

Elena Biagi, PhD

Mat teo Sover ini

PhD student

Mar co Candela, PhD

Mic

rob

ial

Eco

log

yo

f H

ea

lth

UN

ITD

ept.

Pharm

acy a

nd B

iote

chnolo

gy,

Univ

ers

ity o

f B

olo

gna, It

aly

Pat r izia Br igidi

Full Pr ofessor


Sar a Quer cia

PhD student

Monica Bar one

PhD student


Elena Biagi, PhD

Mat teo Sover ini

PhD student

Mar co Candela, PhD

Pat r izia Br igidi

Full Pr ofessor


Sar a Quer cia

PhD student

Monica Bar one

PhD student


Elena Biagi, PhD

Mat teo Sover ini

PhD student

Mar co Candela, PhD

Pat r izia Br igidi

Full Pr ofessor


Sar a Quer cia

PhD student

Monica Bar one

PhD student


Elena Biagi, PhD

Mat teo Sover ini

PhD student

Mar co Candela, PhD

Patrizia Brigidi

Full Professor

Silvia Turroni, PhD

Jessica Baldini

Graduate student

Federica D’Amico

PhD student

Monica Barone

PhD student

Simone Rampelli, PhD

Elena Biagi, PhD

Andrea Castagnetti, PhD

Matteo Soverini

PhD student

Marco Candela

Associate Professor

Sara Quercia

PhD student

THANK YOU FOR YOUR ATTENTION !!!

www.unibo.it

Patrizia Brigidi

Department of Pharmacy and Biotechnology

[email protected]

the gut microbiota as an emerging target … · eubacterium rectale (ci xiva) faecalibacterium...

Documents