The microbiota: general overview

Les Pensières • October 10, 2012

Joël Doré UMR1319 MICALIS & SU MetaGenoPolis, Jouy-en-Josas, France

Microbiota acquisition and early development

Microbiota composition – a metagenomics view

Dysbiosis – towards risk biomarkers

Microbiota functions – towards new bioactives

The microbiota: general overview

Microbiota acquisition and early development

• Intestinal colonization is affected by : – Mode of delivery (vaginal or Cesarean)

– Higene of neonatal environment

– Maternal microbiome

– Food & feeding mode (breast milk vs formula)

• Early colonisation and hygene hypothesis: early exposure to low microbial diversity would prevent or

delay maturation of the mucosal immune system and thereby promote aberrant responses to allergens or autoantigens and development of associated diseases Bach JF. N Engl J Med. 2002;347:911-920

Okada et al Clin Exp Immunol 2010 ; 160:1-9

Dominguez 2010

Is species richness affected by long term changes?

Gordon Lab, Nature, May 2012

Species richness

Sp

ecie

s r

ich

ne

ss

Impact, over generations, of :

• Nutritional transition?

• Repeated antibiotherapy?

• …

Microbiota composition – a metagenomics view

Whole Genome

shotgun sequencing

The metagenome, made of the combined genomes

of all dominant microbes within a given ecosystem

DNA

extraction

Assembly and annotation

Reference gene catalog

and gene counts

Our second genome - first reference gene catalog

3.3 million genes catalog from a cohort of 124 European individuals

150 X the size of the human genome ; conserved accross continents

Prevalent and abundant genes = a core metagenome

EU US Jp Seq

uen

ce m

ap

pin

g, %

Gen

e n

um

ber

Qin et al, Nature March 2010

Quantitative metagenomics - and the enterotypes

Arumugam, Raes et al,

Nature May 2011

Europeans,

Americans,

Asians.

n=33

Sanger

3 enterotypes observed so-far (n~700)

No link with birth place, sex, disease

Ecological settings linked to diet ?

Humans differ by species, by enterotypes and by gene count

Low High Gene count

n=277

Each column is an individual

Each row is a set of 50 genes

Colors reflect gene abundance

low high

Known

species

n=10

Unknown

species

n=58

Marker species for low/high

gene-count microbiota

Differences will permit stratification, with

potential applications in personalized /

digitized medicine and nutrition

Dysbiosis – towards risk biomarkers

Crohn Seksik 2003; Sokol 2006, 2008, 2009; Frank 2007; Qin 2010;

Dicksved 2010; Kang 2010; Mondot 2010; Joossens 2010;

Ulcerative colitis Sokol 2008; Martinez 2008; Lepage 2011

Pouchitis Lim et al., 2009, Kühbacher et al., 2006

IBS Rajilic-Stojanovic 2011, Saulnier 2011, Talley 2011, Chassard 2012

Obesity Ley 2007; Kalliomäki 2008; Furet 2010;

Type-2 diabetis Cani 2009; Sun 2010

Type-1 diabetis Dessein 2009; Wen 2008

Coeliac disease Nadal et al., 2007; Collado et al., 2009

Allergy Kirjavainen 2002; Björkstén, 2009

Frailty in seniorsVan Tongeren 2005

NAFLD, Rheumatoid arthritis, Colorectal cancer, Cdiff., HIV …

Potential dysbiosis in immune mediated diseases

Metagenomic signatures

of dysbiosis

Diagnostic genes and genomes are specific of the microbiome of patients

BMI

inflammatory bowel diseases and obesity d = 0.1

Canonical weights

d = 0.1

Dorea.f ormicigenerans

Bacteroides.v ulgatus.ATCC.8482

Bacteroides.sp..9_1_42FAA

Faecalibacterium.prausnitzii.SL3.3

Roseburia.intestinalis.M50.1

Bacteroides.unif ormis

Bacteroides.sp..2_1_7

Coprococcus.comes.SL7.1

Clostridium.sp.SS2.1

unknown.sp.SS3.4

Eubacterium.rectale.M104.1

Dorea.longicatena

Bacteriodes.xy lanisolv ens.XB1A

Collinsella.aerof aciens

Alistipes.putredinis Bacteroides.sp..4_3_47FAA

Bacteroides.sp..D4

Bacteroides.dorei

Ruminococcus.sp.SR1.5

Bacteroides.sp..2_2_4

Bif idobacterium.longum.subsp..inf antis.CCUG.52486

Eubacterium.hallii

Bacteroides.ov atus Parabacteroides.merdae

Bif idobacterium.adolescentis

Ruminococcus.torques.L2.14

Bacteroides.thetaiotaomicron.VPI.5482

Bacteroides.sp..D1

Parabacteroides.distasonis.ATCC.8503

Eubacterium.siraeum.70.3

Ruminococcus.obeum.A2.162

Ruminococcus.bromii.L2.63 Bif idobacterium.bif idum.NCIMB.41171

Bacteroides.caccae

Bacteroides.eggerthii Streptococcus.thermophilus.LMD.9 Bacteroides.stercoris

Bacteroides.coprocola

Prev otella.copri

Bacteroides.f ragilis.3_1_12

Clostridium.bolteae Eubacterium.v entriosum

Eubacterium.bif orme

Bacteroides.f inegoldii

Bacteroides.plebeius Clostridium.bartlettii

Escherichia.coli.O157.H7.str..EC4115

Holdemania.f ilif ormis

Clostridium.sp.M62.1

Ruminococcus.gnav us

Ruminococcus.lactaris

Bacteroides.capillosus Clostridium.sp.L2.50

Subdoligranulum.v ariabile

Desulf ov ibrio.piger.ATCC29098

Catenibacterium.mitsuokai

Bif idobacterium.pseudocatenulatum

Clostridium.leptum

Parabacteroides.johnsonii

Methanobrev ibacter.smithii.DSM2375

Anaerotruncus.colihominis

Bif idobacterium.animalis.subsp..lactis.AD011

Bacteroides.cellulosily ticus

Bif idobacterium.catenulatum Clostridium.sy mbiosum

Gordonibacter.pamelaeae.gen.nov .sp.Nov

Akkermansia.muciniphila.ATCC.BAA.835

Mitsuokella.multacida Clostridium.nexile Blautia.hy drogenotrophica

Coprococcus.eutactus

Clostridium.asparagif orme

Blautia.hansenii

Anaerostipes.caccae

Bacteroides.intestinalis

Clostridium.spirof orme

Bacteroides.pectinophilus

Bacteroides.coprophilus

Streptococcus.gordonii.str..Challis.substr..CH1

Clostridium.ramosum

Megamonas.hy permegale.ART12.1

Streptococcus.pneumoniae.Hungary 19A.6

Eubacterium.dolichum

Lactobacillus.delbrueckii.subsp..bulgaricus.ATCC.11842

Clostridium.scindens

Mollicutes.bacterium.D7

Buty riv ibrio.crossotus

Enterococcus.f aecalis.TX0104

Haemophilus.inf luenzae.86.028NP

Enterobacter.cancerogenus

Lactobacillus.sakei.subsp..sakei.23K Lactobacillus.saliv arius.ATCC.11741

Bif idobacterium.brev e

Bif idobacterium.dentium Collinsella.stercoris

Streptococcus.inf antarius

Streptococcus.mutans.UA159

Lactobacillus.acidophilus.NCFM Fusobacterium.nucleatum.subsp..nucleatum.ATCC.25586 Lactobacillus.gasseri.ATCC.33323

Lactobacillus.f ermentum.IFO.3956

Proteus.mirabilis.HI4320

Lactobacillus.casei.BL23

Helicobacter.pullorum.MIT.98.5489 Bif idobacterium.angulatum

Klebsiella.pneumoniae.342

Collinsella.intestinalis

Escherichia.f ergusonii.ATCC.35469 Actinomy ces.odontoly ticus

Lactococcus.lactis.subsp..cremoris.MG1363

Streptococcus.sanguinis.SK36

Campy lobacter.hominis.ATCC.BAA.381

Pediococcus.pentosaceus.ATCC.25745

Clostridium.methy lpentosum

Bry antella.f ormatexigens

Buty riv ibrio.f ibrisolv ens.16.4

Citrobacter.sp..30_2

Methanosphaera.stadtmanae.DSM.3091

Clostridium.hy lemonae

Clostridium.sp..7_2_43FAA

Enterococcus.f aecalis.TX1332

Porphy romonas.gingiv alis.ATCC.33277

Pseudomonas.aeruginosa.LESB58

Citrobacter.sp

Enterobacter.sp..638

Desulf ov ibrio.v ulgaris.str...Miy azaki.F.

Haemophilus.parasuis.SH0165

Pasteurella.multocida.subsp..multocida.str..Pm70

Leuconostoc.mesenteroides.subsp..mesenteroides.ATCC.8293

Citrobacter.koseri.ATCC.BAA.895

Clostridium.perf ringens.ATCC.13124

Campy lobacter.concisus.13826

Enterococcus.sp.7L76 Trophery ma.whipplei.str..Twist

Candidatus.Sulcia.muelleri.GWSS Clostridium.dif f icile.630

Lactobacillus.hilgardii.ATCC.8290

Lactobacillus.reuteri.SD2112

Salmonella.enterica.subsp..enterica.serov ar.Heidelberg.str..SL476

Finegoldia.magna.ATCC.29328

Streptococcus.suis.05ZYH33

Thermoanaerobacter.sp..X514

Actinobacillus.pleuropneumoniae.serov ar.7.str..AP76

Anaerof ustis.stercorihominis Clostridium.phy tof ermentans.ISDg

Proteus.penneri

Streptococcus.py ogenes.MGAS10750 Lactobacillus.ultunensis.DSM.16047 Lactobacillus.helv eticus.DPC.4571

Lactobacillus.johnsonii.NCC.533 Lactobacillus.paracasei.subsp..paracasei.ATCC.25302

Anaerococcus.hy drogenalis Bif idobacterium.gallicum

Enterobacter.sakazakii.ATCC.BAA.894

Staphy lococcus.saprophy ticus.subsp..saprophy ticus.ATCC.15305

Canonical weights

Variables

Dorea.f ormicigenerans

Bacteroides.v ulgatus.ATCC.8482

Bacteroides.sp..9_1_42FAA

Faecalibacterium.prausnitzii.SL3.3

Roseburia.intestinalis.M50.1

Bacteroides.unif ormis

Bacteroides.sp..2_1_7

Coprococcus.comes.SL7.1

Clostridium.sp.SS2.1

unknown.sp.SS3.4

Eubacterium.rectale.M104.1

Dorea.longicatena

Bacteriodes.xy lanisolv ens.XB1A

Collinsella.aerof aciens

Alistipes.putredinis Bacteroides.sp..4_3_47FAA

Bacteroides.sp..D4

Bacteroides.dorei

Ruminococcus.sp.SR1.5

Bacteroides.sp..2_2_4 Bif idobacterium.longum.subsp..inf antis.CCUG.52486

Eubacterium.hallii

Bacteroides.ov atus Parabacteroides.merdae Bif idobacterium.adolescentis

Ruminococcus.torques.L2.14

Bacteroides.thetaiotaomicron.VPI.5482

Bacteroides.sp..D1

Parabacteroides.distasonis.ATCC.8503

Eubacterium.siraeum.70.3

Ruminococcus.obeum.A2.162

Ruminococcus.bromii.L2.63 Bif idobacterium.bif idum.NCIMB.41171

Bacteroides.caccae

Bacteroides.eggerthii Streptococcus.thermophilus.LMD.9

Bacteroides.stercoris

Bacteroides.coprocola

Prev otella.copri

Bacteroides.f ragilis.3_1_12

Clostridium.bolteae Eubacterium.v entriosum

Eubacterium.bif orme

Bacteroides.f inegoldii

Bacteroides.plebeius Clostridium.bartlettii

Escherichia.coli.O157.H7.str..EC4115

Holdemania.f ilif ormis

Clostridium.sp.M62.1

Ruminococcus.gnav us

Ruminococcus.lactaris

Bacteroides.capillosus Clostridium.sp.L2.50

Subdoligranulum.v ariabile

Desulf ov ibrio.piger.ATCC29098 Catenibacterium.mitsuokai

Bif idobacterium.pseudocatenulatum

Clostridium.leptum

Parabacteroides.johnsonii

Methanobrev ibacter.smithii.DSM2375

Anaerotruncus.colihominis

Bif idobacterium.animalis.subsp..lactis.AD011

Bacteroides.cellulosily ticus

Bif idobacterium.catenulatum Clostridium.sy mbiosum Gordonibacter.pamelaeae.gen.nov .sp.Nov

Akkermansia.muciniphila.ATCC.BAA.835

Mitsuokella.multacida Clostridium.nexile

Blautia.hy drogenotrophica

Coprococcus.eutactus

Clostridium.asparagif orme

Blautia.hansenii Anaerostipes.caccae

Bacteroides.intestinalis

Clostridium.spirof orme

Bacteroides.pectinophilus

Bacteroides.coprophilus

Streptococcus.gordonii.str..Challis.substr..CH1

Clostridium.ramosum

Megamonas.hy permegale.ART12.1

Streptococcus.pneumoniae.Hungary 19A.6

Eubacterium.dolichum

Lactobacillus.delbrueckii.subsp..bulgaricus.ATCC.11842

Clostridium.scindens

Mollicutes.bacterium.D7

Buty riv ibrio.crossotus

Enterococcus.f aecalis.TX0104

Haemophilus.inf luenzae.86.028NP

Enterobacter.cancerogenus Lactobacillus.sakei.subsp..sakei.23K Lactobacillus.saliv arius.ATCC.11741

Bif idobacterium.brev e

Bif idobacterium.dentium Collinsella.stercoris

Streptococcus.inf antarius

Streptococcus.mutans.UA159

Lactobacillus.acidophilus.NCFM Fusobacterium.nucleatum.subsp..nucleatum.ATCC.25586 Lactobacillus.gasseri.ATCC.33323

Lactobacillus.f ermentum.IFO.3956

Proteus.mirabilis.HI4320

Lactobacillus.casei.BL23 Helicobacter.pullorum.MIT.98.5489 Bif idobacterium.angulatum

Klebsiella.pneumoniae.342

Collinsella.intestinalis

Escherichia.f ergusonii.ATCC.35469 Actinomy ces.odontoly ticus

Lactococcus.lactis.subsp..cremoris.MG1363

Streptococcus.sanguinis.SK36

Campy lobacter.hominis.ATCC.BAA.381

Pediococcus.pentosaceus.ATCC.25745

Clostridium.methy lpentosum

Bry antella.f ormatexigens

Buty riv ibrio.f ibrisolv ens.16.4

Citrobacter.sp..30_2

Methanosphaera.stadtmanae.DSM.3091

Clostridium.hy lemonae

Clostridium.sp..7_2_43FAA

Enterococcus.f aecalis.TX1332

Porphy romonas.gingiv alis.ATCC.33277

Pseudomonas.aeruginosa.LESB58

Citrobacter.sp

Enterobacter.sp..638

Desulf ov ibrio.v ulgaris.str...Miy azaki.F.

Haemophilus.parasuis.SH0165 Pasteurella.multocida.subsp..multocida.str..Pm70

Leuconostoc.mesenteroides.subsp..mesenteroides.ATCC.8293

Citrobacter.koseri.ATCC.BAA.895

Clostridium.perf ringens.ATCC.13124

Campy lobacter.concisus.13826

Enterococcus.sp.7L76 Trophery ma.whipplei.str..Twist

Candidatus.Sulcia.muelleri.GWSS Clostridium.dif f icile.630

Lactobacillus.hilgardii.ATCC.8290

Lactobacillus.reuteri.SD2112

Salmonella.enterica.subsp..enterica.serov ar.Heidelberg.str..SL476

Finegoldia.magna.ATCC.29328

Streptococcus.suis.05ZYH33

Thermoanaerobacter.sp..X514

Actinobacillus.pleuropneumoniae.serov ar.7.str..AP76

Anaerof ustis.stercorihominis Clostridium.phy tof ermentans.ISDg

Proteus.penneri

Streptococcus.py ogenes.MGAS10750 Lactobacillus.ultunensis.DSM.16047 Lactobacillus.helv eticus.DPC.4571 Lactobacillus.johnsonii.NCC.533 Lactobacillus.paracasei.subsp..paracasei.ATCC.25302

Anaerococcus.hy drogenalis Bif idobacterium.gallicum

Enterobacter.sakazakii.ATCC.BAA.894

Staphy lococcus.saprophy ticus.subsp..saprophy ticus.ATCC.15305

Variables

02

46

Eigenvalues

d = 5

Scores and classes

N:N

Y:CD

Y:UC

Axis1 Axis2

Axis3

Inertia axes

d = 2

Classes

N:N

Y:CD

Y:UC

p-value: 0.031

Crohn

Patients

UC

Patients

Healthy

Controls

Guarner (HUVH, Barcelona)

Wang Jun (BGI, Shenzen)

Ehrlich, Lepage, Tap (INRA)

Pedersen (SDC, Copenhagen)

Wang Jun (BGI, Shenzen)

Ehrlich (INRA, Paris)

Metagenomic signatures

of risk – diversity, a key stratifier?

Prognostic genes and genomes are biomarkers of risks of comorbidities

Low gene count individuals (low bacterial richness, 23%)

have less healthy prognosis

In obesity (O. Pedersen & K. Clément), with:

• less healthy metabolic & inflammatory traits

• higher weight gain over the past 10 years

• more biomarkers of risk of aggravation and comorbidities

• worse response to a calories-restricted diet

In Ulcerative Colitis (F. Guarner), with:

• higher relapse rate of chronic acute phases

• non-responders to probiotic-induced microbiota stabilization

Note: Low bacterial gene count microbiomes are enriched in Bacteroides

enterotype and often lack core-species of dominant firmicutes.

Microbiota functions – towards new bioactives

The metagenome ; a window to unexplored

microbiota functions – acting at interfaces :

• Metabolism (food-microbe)

• Mucosal barrier integrity & beyond (cell-microbe) Mucus ; Tight junctions ; Trophicity of epithelium and cell renewal

Immuno-modulation ; Dendritic cells, Paneth cells and defensins

Gut-brain crosstalk

• Barrier against pathogens – colonization prevention

(microbe-microbe)

Clones genes enzymes..

Large homogenous DNA

library

Metagenomic DNA

Bacterial fraction

Recombinant clones

Fosmid

Vectors

Cloning

DNA extraction

- vitamin production (Riboflavin)

- plant polysaccharides degradation (Tasse GenomRes 2010)

- beta-glucuronidase activity (Gloux et al PNAS 2010)

Microbiota functions – food-microbe

Clones genes actives..

Metagenomic DNA

Bacterial fraction

Recombinant clones

Fosmid

Vectors

Cloning

DNA extraction

Microbiota functions – microbe-host

9 metagenomic libraries ; 338 000 clones Transfected cells with reporter systems : - proliferation - immune response - cell metabolism Blottière and col:

Gloux 2007; Lakhdari 2010, 2011; Madi 2010; Kaci 2011; Rochat 2011

20 screens ; 50,000 clones ; 30 bioactives

Strains from culture collections

(gut commensals, probiotics)

Large homogenous DNA

libraries

Microbiota functions – microbe-host

Collab. with Maria Rescigno et al, IEO - Milan

CTRL

FB62

EPI300 F4

EPI300 + FB62 F4 + FB62

Tissue with control medium control Ecoli clone F4

without

Salmonella

with

Salmonella

FB62

Glue

Sealed cylinder luminal compartment

Tissue specimen

Organ culture inset

Tsilingiri 2012

Bioactive metagenomic clone F4 protects against

Salmonella (FB62)-induced tissue destruction

Microbiota functions – microbe-microbe

This RumC region is part of a 220 kb ICE ; a transferable “colonisation island”

Fons and col. Crost 2010, 2011; Pujols 2011

In vivo elimination of Clostridium perfringens

by Ruminococcus gnavus E1

Faecal dilution Days post inoculation L

og

10

(CF

U / g

of

fec

es

)

0

2

4

6

8

10

0 2 4 6 8

R. gnavus

C. perfringens

Passive

transit

marker

rumC1 rumC2 rumC3 rumC4 rumC5

rumMC1 rumKC rumRC rumIC1 rumTC rumMC2 rumEC rumIC2 rumFC rumGC rumPC

rumYC

Microbiota as an organ

Relevant to the push for personalized and digital medicine

Relevant for health, preventive nutrition and medical applications

Predict responders / non-responders (to nutritional supplements or therapeutics)

Predict relative risk of disease onset in healthy subjects Predict risk of aggravation and co-morbidities in patients

Signatures to assist in diagnosis/prognosis and clinical management of patients

Biomarkers to provide rationale targets and strategies for microbiota modulation

Bioactives with potential for translation in the clinic

INRA Jouy-en-Josas

Christel Béra-Maillet

Hervé Blottière

Marion Leclerc

Patricia Lepage

Catherine Juste

Nicolas Lapaque

Tomas de Wouters

Antonella Cultrone

Malgorsata Nepelska

Elsa Jacouton

ChenHong Zhang

Julien Tap

Stanislas Mondot

Omar Lakhdari

European Community

& ANR-France

Philippe Seksik

Harry Sokol

Philippe Marteau

S Dusko Ehrlich, Peer Borck (EMBL Heidelberg), Francisco

Guarner (Val d’Hebron Hospital Barcelona), Oluf Pedersen (SDC

Copenhagen)

and EU-MetaHIT Consortium

Karine Clément, (INSERM U972, CR des Cordeliers) Denis Le

Paslier & Eric Pelletier, (CEA-Genoscope)

and ANR MicroObese consortium

Micro Obes

Merci de votre attention

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