Clostridia in the Gut Microbiota and Their Implication in Food Allergies and Foodborne Diseases

Dr Bernard Taminiau Dr Cristina Rodriguez Pr Georges Daube CLOSTRIDIA

80’s 2005 16S NGS taxonomy

Cato, E.P. and Collins et al 1994 SILVA DB 2016 Stackebrandt, E 1989 82141 non redundant 30 known sp 110 k n ow n a n d unamed sp 16S

Clostridium is a Cluster I (Clostridium ss) to 34 Families composite group Cluster XIX (Fusobacterium) Majority unknown species

Lack of phenotypic Lack of culture availability discriminating properties Deficit in wet lab compared to sequences

ULg – Faculté de Médecine Vétérinaire - FARAH 2 Microbiota - the new organ

Cultivable

Unknown or non cultivable

ULg – Faculté de Médecine Vétérinaire - FARAH 3 Key determinants

Pre-birth contact Colonization and Increasing sensitivity with bacteria tolerance building to disorder

Kerr et al Crit Rev Microbiol 2014 online 19 March 2014 Microbiota and host

Outside the Immune system relationship, microbiota is a key player

Bile acid Obesity metabolism

Behavior diabetes

Thermogenesis atherosclerose

Intestinal Metabolic disease gluconeogenesis

Appetite Asthma

Hormone autism expression

Schroeder et al 2016 ULg – Faculté de Médecine Vétérinaire - FARAH 5 Infant-type microbiota and Clostridia

Bacterial flora Colonisation by heterogeneous, Clostridium spp. Sterile independent of and other obligate GI tract feeding habits anaerobes

Birth First few First month days

Gradual Bacteria from consumption the mother of oxygen by and the aerobic environment bacteria colonize de GI (conditions tract of for a more neonates diversified flora)

Jost et al., 2012 ULg – Faculté de Médecine Vétérinaire - FARAH Lopetuso et al., 2013 Elderly-type microbiota

The aging process challenges the stability of microbiota and can also affect the presence of Clostridium spp. • Decrease in the stability and in the diversity of the gut microbiota

• Increase in the number of facultative anaerobes (streptococci, staphylococi; enterococci, enterobacteriaceae)

• Decrease in the total number of strict anaerobes (caused by a lower count of bifidobacteria and bacteroides)

Clostridium sensu stricto genus significantly increased

Indispensable and key role of Clostridia in modulating gut homeostasis during the entire lifespan

Drago et al., 2012 ULg – Faculté de Médecine Vétérinaire - FARAH Mariat et al., 20097 Diversity of microbes in the colon PREDOMINANT ORGANISMS: Three groups of strict anaerobes

Bacteroides

Clostridium cluster IV Clostridium, Eubacterium, Ruminococcus, Anaerofilum Clostridium cluster XIVa Clostridium, Eubacterium, Ruminococcus, Coprococcus, Dorea, Lachnospira, Roseburia, Butyrivibrio

12 1x10 organisms per gram of feces Hold et al., 2002 Lopetuso et al., 2013 ULg – Faculté de Médecine Vétérinaire - FARAH Spatial organisation and diversity of microbes across the intestinal lumen

Regions of the central lumen are populated with Bacteroidaceae, Enterococcaceae and Lactobacillaceae

Areas between the mucosal folds are populated with Clostridium cluster XIVa and IV Commensal Clostridia populate a specific region in the intestinal mucosa, establishing a close relationship with gut cells (physiological functions in a cooperative manner)

ULg – Faculté de Médecine Vétérinaire - FARAH Lopetuso et al., 2013 Commensal Clostridia and gut homeostasis

Commensal Clostridia play an important role in the metabolic welfare of colonocytes by releasing butyrate as an end-product of fermentation

Short chain fatty acids Energy source for Butyrate colonocytes

Gene expression (hyperacetylation of chromatin)

Butyrate production by Clostridial Anti-inflammatory effect clusters XIVa and IV (Decreases the expression of (i.e. Roseburia sp. and F. prausnitzii) proinflammatory cytokines)

Protection against colitis, Butyrate producers that display Butyryl colorectal cancer and acetate CoA transferase activity ulcerative colitis (Induces apoptosis in tumor cells in vitro)

Duncan et al., 2002 ULg – Faculté de Médecine Vétérinaire - FARAH Lopetuso et al., 2013 Food allergies Constant increase in infant since 15 years (about 10% population)

Immune system tolerance state

IgE mediated Gut microbiota allergy

Food allergies is linked with dysbiosis in human and mice models

Blazquez and Berin , 2017

ULg – Faculté de Médecine Vétérinaire - FARAH 11 Commensal Clostridia and host immune system

Clostridia can promote the development of αβ T cell receptor intraepithelial lymphocytes (IEL) and IEL Immunoglobulin A (IgA)-producing cells in the large IgA intestine

Key players in determining the nature of the immunological response to antigens or pathogens ingested

Short chain fatty acids and secondary bili acids produce by Clostridia are associated with the initiation of immunological signaling

Elevated levels of Clostridial clusters XIVa and IV in mice leads to resistance to allergy and intestinal inflammation in experimental models

Clostridium spp. can induce the differentiation of naïve cells into antigen-specific colonic T regulatory cells

ULg – Faculté de Médecine Vétérinaire - FARAH Atarashi t al., 2011 Lopetuso et al., 2013 Clostridia as probiotics for food allergies

Commensal bacteria containing Stefka et al , 2014 Clostridia protect against food allergen sensitization in mice

Oral administration of Clostridium butyricum CGMCC0313-1 inhibits Open Access Zhang et al. 2017 β-lactoglobulin-induced intestinal anaphylaxis in a mouse model of food allergy

Promising studies that need to be exported in humans

ULg – Faculté de Médecine Vétérinaire - FARAH 13 Improve microbiota knowledge METAGENETICS aka amplicon sequencing

Establish the profile of target taxonomical identity in the sample

16S rRNA 26S rRNA or ITS Others

Bacteria Micro-eucaryotes

www.arb-silva.de https://unite.ut.ee/ http://fungene.cme.msu.edu/ Bioinformatics

Vic$vallaceae) 100.000%$ Vibrionaceae) Verrucomicrobiaceae) Veillonellaceae) unclassified) 90.000%$ Synergistaceae) Streptococcaceae) Staphylococcaceae) Sphingomonadaceae) 80.000%$ Ruminococcaceae) Rikenellaceae) Rhizobiaceae) Pseudomonadaceae) 70.000%$ Pseudoalteromonadaceae) Propionibacteriaceae) Prevotellaceae) 60.000%$ Porphyromonadaceae) Peptostreptococcaceae* Pasteurellaceae) Oxalobacteraceae) 50.000%$ Moraxellaceae) Micrococcaceae) Microbacteriaceae) Lactobacillaceae) 40.000%$ Lachnospiraceae) Fusobacteriaceae) Flavobacteriaceae) Family_XIII_Incertae_Sedis) 30.000%$ Family_XII_Incertae_Sedis) P16_week_06 Family_XI_Incertae_Sedis) P5_week_01 Eubacteriaceae) P2_week_07 P2_week_01 Erysipelotrichaceae) P2_week_03 P2_week_05 20.000%$ Enterococcaceae) P2_week_09 Enterobacteriaceae) P2_week_11 Desulfovibrionaceae) P2_week_13 Corynebacteriaceae) P2_week_15 10.000%$ Coriobacteriaceae)

P1_week_17 P1_week_10 P1_week_07 P1_week_04 Comamonadaceae) P2_week_17 P1_week_02 P1_week_08 Clostridiaceae) P1_week_12 P1_week_11 Carnobacteriaceae) P5_week_03 P1_week_01 0.000%$ Campylobacteraceae) P1_week_16 P5_week_12 P1_week_14 Bifidobacteriaceae) P1_week_05 Bacteroidaceae) P5_week_16 Alcaligenaceae) Ac$nomycetaceae) $P1_week01$ $P1_week05$ $P1_week07$ $P1_week08$ $P2_week01$ $P2_week05$ $P2_week07$ $P2_week09$ $P1_week02$ $P1_week04$ $P1_week10$ $P2_week03$

P17_week_05 P19_week_15 $P13_week01$ $P13_week02$ $P13_week03$ $P13_week04$ $P13_week06$ $P13_week08$ $P13_week10$ $P15_week01$ $P15_week02$ $P15_week03$ $P15_week04$ $P15_week06$ $P15_week07$ $P15_week08$ $P15_week09$ $P18_week05$ $P18_week07$ $P18_week09$ $P19_week01$ $P19_week02$ $P19_week03$ $P19_week07$ $P19_week09$ $P19_week10$ $P24_week03$ $P24_week05$ $P24_week06$ $P24_week08$ P17_week_06 P19_week_13 P19_week_10 P17_week_07 P19_week_07 P19_week_09 P17_week_08 P19_week_11 P19_week_01 P19_week_03 P15_week_08 P15_week_09 P24_week_17 P24_week_10 P15_week_07 P24_week_08 P15_week_01 P24_week_06 P24_week_05 P15_week_02 P24_week_03 P15_week_04 P15_week_03 P24_week_12 P15_week_06 P18_week_17 HTS P15_week_11 P18_week_15 P18_week_12 P15_week_12 P18_week_09 P18_week_07 P15_week_13 P18_week_05 P15_week_14 P12_week_11 P12_week_10 P15_week_17 P12_week_03 P12_week_07

P13_week_14

P13_week_10 P13_week_12

P19_week_02 P24_week_14

P13_week_01 P13_week_02 P13_week_03 P13_week_04 P13_week_06 P13_week_08

0.05

Group&young& Group&old,CT& Group&old,SP& NMDS2

NMDS3 16S rDNA Amplification NMDS1 LONGITUDINAL STUDY OF FECES MICROBIOTA IN SENIORS AND LINK WITH CLOSTRIDIUM DIFFICILE PRESENCE

FOUR MONTHS FOLLOW-UP IN A SINGLE BELGIAN NURSING HOME

Pr Georges Daube Pr Michel delmée Dr Bernard Taminiau Véronique Avesani Dr Cristina Rodriguez J. Van Broeck Dr Nicolas Korsak

ULg – Faculté de Médecine Vétérinaire - FARAH 17 Objectives

• To evaluate and follow the prevalence of C. difficile in a Belgian nursing home

• To establish a relationship between other intestinal bacterial populations and C. difficile colonization

• To evaluate the global evolutions of the total microflora and the relation with the C. difficile presence Study design WEEKLY stool samples recovery from a group of 13 >65 ears ol elderly care home residents

C. Difficile detection Direct // enrichment Toxin detection Ribotyping

Microbiota profiling V1V3 16S sequencing Community structure and composition Dynamics Microbiota structure and composition A B Bacterial Diversity 40

30

20

10

0 Inverse simpson biodiversity

P01 P02 P04 P05 P10 P12 P13 P15 P17 P18 P19 P21 P24 resident Bacterial Richness 400

300

200

100 Chao1 richness index 0

P01 P02 P04 P05 P10 P12 P13 P15 P17 P18 P19 P21 P24 resident Bacterial Evenness 0.4

0.3

0.2

0.1

Simpson Evenness Index 0.0 Braycurtis dissimilarity index P01 P02 P04 P05 P10 P12 P13 P15 P17 P18 P19 P21 P24 resident ULg – Faculté de Médecine Vétérinaire - FARAH 20 Family

P24 Betaproteobacteria_unclassified Enterococcaceae P21 Clostridia_unclassified Burkholderiales_unclassified Lentisphaeria_unclassified P19 Opitutae_unclassified Streptococcaceae P18 Firmicutes_unclassified Family_XIII_Incertae_Sedis Peptostreptococcaceae P17 Family_XI_Incertae_Sedis Pseudomonadaceae P15 Bifidobacteriaceae Victivallaceae Prevotellaceae P13 Desulfovibrionaceae Veillonellaceae P12 Alcaligenaceae Bacteroidales_unclassified Erysipelotrichaceae P10 Synergistaceae Coriobacteriaceae P05 Bacteroidetes_unclassified Bacteria_unclassified P04 Clostridiales_unclassified Enterobacteriaceae Rikenellaceae P02 Porphyromonadaceae Verrucomicrobiaceae P01 Lachnospiraceae Ruminococcaceae

0 20 40 60 80 Bacteroidaceae 100 relative population abundance (%)

ULg – Faculté de Médecine Vétérinaire - FARAH 21 Clostridiales - Genus Subdoligranulum P24 Ruminococcus Ruminococcaceae_unclassified P21 Oscillibacter P19 Hydrogenoanaerobacterium Flavonifractor P18 Faecalibacterium P17 Acetanaerobacterium Ruminococcaceae Clostridioides Peptostreptococcaceae P15 Roseburia P13 Lachnospiraceae_unclassified Lachnospira P12 Dorea P10 Coprococcus Blautia Lachnospiraceae P05 Family_XIII_Incertae_Sedis_unclassified P04 Anaerovorax Family XIII Sporanaerobacter P02 Peptoniphilus P01 Finegoldia Family XI Eubacterium Eubacteriaceae 0

20 40 60 Clostridium Clostridiaceae relative population abundance (%)

ULg – Faculté de Médecine Vétérinaire - FARAH 22 Link with C. difficile

Others Resident Flavonifractor P01 P02 P04 P05 P10 P12 P13 P15 P17 P18 P19 P21 P24 Genus Desulfovibrio Week01 2 0 0 0 0 0 3 3 0 n 2 n n 100 Pseudomonas Week02 2 n n n n n 3 3 0 n 2 0 n Roseburia Week03 n 0 0 0 0 0 1 3 0 n 3 n 2 Acidaminococcus Week04 0 n 0 n n n 0 1 n n n 0 n Bifidobacterium Week05 0 0 0 n 0 n n n 0 3 n 0 2 Week06 n n 0 0 n n 0 3 0 n n 0 0 Victivallis

Week07 0 0 n n n 0 n 3 0 3 2 n n 50 Sutterella Week08 0 n 0 n 0 n 0 3 2 n n 0 2 Barnesiella Week09 n 0 n 0 n n n 3 n 2 0 n n Collinsella Week10 2 n 0 0 0 0 0 n n n 0 0 1 Faecalibacterium Week11 2 0 n n n 0 n 3 n n 2 n n Subdoligranulum Week12 2 n n 0 0 0 1 3 0 3 n n 1 Week13 n 0 0 n n n n 3 n n 2 n n Blautia

Relative population abundance (%) 0 Week14 2 n n 0 0 0 1 3 0 n n n 0 Escherichia Week15 n 0 0 n n n n n n 2 0 0 n Oscillibacter Week16 2 n n 0 n n n n n n n n n Alistipes Week17 0 0 0 n n 0 n 3 n 2 n 0 0 Parabacteroides C. Difficile positive : both Akkermansia culture Metagenetics C. difficile negativeC. difficile positive Lachnospiraceae_unclassified Ruminococcaceae_unclassified C. Difficile negative : Bacteroides

ULg – Faculté de Médecine Vétérinaire - FARAH 23 Link microbiota – Diarrhea – C. difficile

Bacterial Richness Bacterial Diversity 400 40

300 30

200 20

100 10 Chao1 richness index 0 0 Inverse Simpson Biodiversity Index

Diarrheic C. difficileDiarrheic negative C. difficile positive Diarrheic C. difficileDiarrheic negative C. difficile positive

Non Diarrheic C.Non difficile Diarrheic negative C. difficile positive Non Diarrheic C.Non difficile Diarrheic negative C. difficile positive

Non Diarrheic C. difficile negative a Non Diarrheic C. difficile negative a Non Diarrheic C. difficile positive bc Non Diarrheic C. difficile positive b Diarrheic C. difficile negative b Diarrheic C. difficile negative b Diarrheic C. difficile positive ac Diarrheic C. difficile positive a

ULg – Faculté de Médecine Vétérinaire - FARAH 24 Link microbiota – Diarrhea – C. difficile

Lachnospiraceae relative abundance Genus - patient

40 a Diarrheic C. difficile positive Bifidobacterium Diarrheic C. difficile negative Victivallis 30 100 b Non Diarrheic C. difficile positive Sutterella b Non Diarrheic C. difficile negative Barnesiella 20 b Collinsella Erysipelotrichaceae_unclassified 10 Synergistaceae_unclassified 50 Enterobacteriaceae_unclassified 0

Faecalibacterium Relative population abundance (%) Subdoligranulum Blautia Escherichia 0 Clostridiales_unclassified Relative population abundance (%) Oscillibacter Lachnospiraceae Alistipes Parabacteroides Akkermansia

Diarrhea negativeDiarrhea positive Ruminococcaceae_unclassified Lachnospiraceae_unclassified Bacteroides Link between Lachnospiraceae and C. difficile in diarrheic context

ULg – Faculté de Médecine Vétérinaire - FARAH 25 Link Clostridia – C. difficile

Statistical differences - genus

Lachnospiraceae Lachnospiraceae_unclassified C. difficile negative C. difficile positive

Flavonifractor Ruminococcaceae Genus p value Lachnospiraceae_unclassified 4.1012E-08 Flavonifractor 0.00017038 Blautia Blautia 2.6499E-06 Lachnospiraceae Akkermansia 2.9244E-05

Akkermansia

0 1 2 3 4 10 20 30 40 relative population abundance (%)

Link between Blautia and Clostridioides

ULg – Faculté de Médecine Vétérinaire - FARAH 26 Take home messages

• Clostridia are a key and important player in human gut microbiota

• Clostridia is a complex taxonomical group – even based on 16S phylogenetic • There’s a lot of work to do in wet lab microbiology to further characterize them

• NGS and omic revolution can be used to effectively track down Clostridia in the gut, providing that the bacterial population is abundant enough • It can also highlight possible relations between Clostridia in the GIT

ULg – Faculté de Médecine Vétérinaire - FARAH 27 Fundamental and Applied Research Center for Animal and Health

ONE HEALTH Animal Health Animal Production Feed

Food Focus on relations Environ- ment Many microbiota Waste Human health

Our interest : To improve and broaden our perspective on microbiota dynamic