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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