MiningMining NovelNovel TherapeuticTherapeutic BacterialBacterialStrainsStrains UsingUsing FecalFecal MicrobiotaMicrobiotaStrainsStrains Using Using FecalFecal MicrobiotaMicrobiota

TransplantationTransplantation toto Combat Combat MetabolicMetabolicDiseaseDiseaseDiseaseDisease

Seminar Infectious DiseasesSeminar Infectious DiseasesSeminar Infectious DiseasesSeminar Infectious Diseases2121--0505--20152015

GGGuido Bakker MD, PhD studentGuido Bakker MD, PhD studentDepartmentDepartment of of VascularVascular MedicineMedicine

AcademicAcademic MedicalMedical Center AmsterdamCenter AmsterdamAcademicAcademic MedicalMedical Center, AmsterdamCenter, AmsterdamThe NetherlandsThe Netherlands

Take home messages• Personalized intestinal microbiota

• Diet and antibiotics are major regulators of microbiotacomposition

• Associations microbiota with disease, but causality has onlybeen proven for few diseases!

• Fecal transplantation may be used to find therapeutic/ pathologic strains or metabolites

BackgroundGut microbiotaGut microbiota

• 6000-7000 kilogram feces in a human life produced!6000 7000 kilogram feces in a human life produced!

1 Ley et al, Nature 2006, 444:1022-1023 2 Bäckhed et al, PNAS 2004, 101:15718-15723, ,

BackgroundGut microbiotaGut microbiota

• 6000-7000 kilogram feces in a human life produced!6000 7000 kilogram feces in a human life produced!

• The gut microbiota (60% of the dry mass of feces) content consists of 1014 (!) bacteria

1 Ley et al, Nature 2006, 444:1022-1023 2 Bäckhed et al, PNAS 2004, 101:15718-15723, ,

Microbes Maketh Man

90% of the cells in your body are bacteria!

Gut microbiome in physiology

P li d i t ti l i biPersonalized intestinal microbiome

Growing Up In A Microbial World

The microbiota develops in the first 2 years

(40-50% by parents, rest via lifestyle)Dominguez-Bello, PNAS 2010

Impact of Diet on Microbiota Variation?

• Diet explains 77%

Mice• Diet explains 40-50%

Humansp

Zhang et al. ISME J. 2010 4:2

• Diet explains 81%Faith et al. Science 2011 333(6038)

pMetagenomic study of 17 Europeans

Tap et al. 2009 EM 11:10

Individual Variation Large( )

“Diet has a dominating role in shaping the gut microbiota”

Individual Variation Large

Habitual diet > Intervention

Hildebrandt et al. Gastroenterology 137 (2009) Cani et al. Diabetes 56 (2007)

Wu et al. Science 333 (2011) Walker et al. ISME J 55 (2011)

Mouse & Human Metagenomes Share < 5% Identical Genes

Dogma shift: best infectiousmicrobe is a dead microbe in themicrobe is a dead microbe in the

1950’s..

Relation antibiotic prescriptions and obesity

Antibiotics Impact MicrobiotaD l t i Child

Nature 2012

Development in ChildrenNature 2012

Average child in USA 12 courses AB age of 7

(NL: 2 courses)

Fecal microbiota and obesity/type 2 diabetes mellitus: associations!diabetes mellitus: associations!

Qin, Nature 2012

Le Chatelier, Nature 2013

Karlsson, Nature 2013

Ridaura, Science 2013

Shortcomings of current fecal sample centered approachcentered approach

1. Small intestine is more involved in1. Small intestine is more involved in metabolism than the colon

2. Diet is not taken into account

3. Association is not causality!y

Gut epithelium as barrier

Bacterial translocation

Postprandial endotoxemia parallelsplasma triglyceride curves

Harte, Diabetes Care 2012 ,

Results of cohort based studies i f l lusing fecal samples

Smits/Nieuwdorp, Gastroenterology 2013p, gy

Causal role of microbiota in metabolism and diabetes

Association or causality of intestinalymicrobiota: visceral adipose tissue

• Inflamed visceral adipose tissue is strongly associated with DM2

• Causal driving factors? Bacterial translocation?

Depres JC, Ann Int Medicine 2004

Koch’s postulatespfor causality

• The microorganism must beidentified/isolated from a diseasedorgan(ism).organ(ism).

• The cultured microorganism should induceb fi i l d ff t hbeneficial or adverse effects whenintroduced into an organism (inoculation).

• The microorganism must be reisolated afterinoculation

Manipulating gut microbiota byp g g yfecal transplantation

De Vrieze, Science 2013

Donors

Screening donors:- Questionnaire (bowel habits, travel history,

medication, etc)F ( it Cl t idi diffi il SSYC d- Faeces (parasites, Clostridium difficile, SSYC and viruses (rota etc))

- Bloodborn viruses (Hepatitis, HIV, HTLV, CMV, EBV)Bloodborn viruses (Hepatitis, HIV, HTLV, CMV, EBV)- Checklist day before treatment (been ill? different

sexual contact? travelling? no antibiotics in last 3 th ?)months?)

Effects of fecal transplantations in l t idi diffi il di hclostridium difficile diarrhea

Van Nood, NEJM 2013,

Emerging indications (ESBL andEmerging indications (ESBL andVRE)

Metabolic syndrome

D li id i Insulin

Increased wais

Dyslipidemia Insulin resistance

H t i HyperglycemiaHypertension

Effect donor faeces on peripheralinsulin sensitivityinsulin sensitivity

A.Vrieze, Gastroenterology 2012

Eiseman (Surgery 1958)

• Fecal transplant doesn’t induce a definite cure!• Fecal transplant doesn t induce a definite cure!

• The cultured microorganism should induce beneficial or adverse effects when introduced into a healthy organism (3rd postulate)effects when introduced into a healthy organism (3rd postulate)

Small intestinal gut microbiotacompositioncomposition

A.Vrieze, Gastroenterology 2012, gy

Bacterial strains as novel probiotics:Bacterial strains as novel probiotics: Eubacterium hallii

Eubacterium halliiEubacterium hallii

• Belongs to Firmicutes phylum (spore former)g p y ( p )

• Anaerobic gram positive lactate-utilizing SCFA butyrate-producing bacterial strain natural inhabitant of duodenumproducing bacterial strain, natural inhabitant of duodenum

• Can produce butyrate at pH 4 (small intestine) as well as p y p ( )at pH 6-7 (colon)

Study protocol I

• Db/db male mice (8 weeks old), n=8 per group

• Daily gavage (100ul/mouse) with E. Hallii (storedi 10% l l 80C) i hi 1 hin 10% glycerol at -80C), gavage within 1 hourafter thawing for 4 weeks with:

10^6 CFU/ml- 10^6 CFU/ml- 10^8 CFU/ml- 10^10 CFU/ml- Placebo (dissolvens = 10% glycerol)

Caecal E.hallii levels upon 1 month daily gavage

Udayappan/Manneras submitted

Mean ±SEM

Udayappan/Manneras, submitted

E. Hallii improves insulin sensitivity(insulin tolerance test )

130

140

150placebo (10% glycerol)

106 CFU/ml E.Hallii

1010 CFU/ml E.Halii

108 CFU/ml E.Hallii

100

110

120

70

80

90

*

E. Hallii normalises insulin sensitivity (ITT) compared to placebo

0 10 20 30 40 50 60 70 80 90 100 110 120 13050

60 *

Time (min)

Udayappan/Manneras submittedUdayappan/Manneras, submitted

Study protocol II • Db/db male mice (8 weeks old), n=7-9 per group

• Daily gavage (100ul/mouse) with alive or heat inactivated E. Hallii 10x8CFU/ (stored in 10% glycerol at -80C), gavage within 1 hour after thawing for 4 weeks ), g g gfollowed by:

• - 48h in Metabolic cages (Somedic cages) - hyperinsulinemic normoglycemic clamp

E.hallii treatment metabolism and insulin sensitivity (clamp) in db/dbsensitivity (clamp) in db/db

P=0.06

y

P=0.06

sens

itivi

tyIn

sulin

Udayappan/Manneras submittedUdayappan/Manneras, submitted

Challenges for these novel strains

• Strain stability (storage, glycerol vsfreezedrying) under Nitric oxide for anaerobics

• Industrial level (30-40liters) anaerobic culturing

• Human phase 1-2 dosefinding trial with GMP d d E h llii i M tS t t d i 2014produced E.hallii in MetSyn started in 2014

De Vos WM and Nieuwdorp M Nature 2013; 498(7452):48-9

Eubacterium hallii

De Vos WM and Nieuwdorp M. Nature 2013; 498(7452):48-9

A deeper understanding of the gut microbiota in diseasemicrobiota in disease

• At AMC >300 FMT’s since 2006, predominantly in RCT: long term sidepredominantly in RCT: long term side effects not observed yet

• At AMC ongoing/finished RCT’s with single/multiple FMT using hard clinical g p gendpoints for:- VRE/ESBL POSTIVE - Insulin resistance/DM2 POSITIVE- IBD (Colitis ulcerosa, TURN trial) NEG- Vascular inflammation ONGOING- NAFLD/NASH ONGOING- Type 1 diabetes ONGOING

Smits/Nieuwdorp, Gastroenterology 2013 [; van Nood/Nieuwdorp, NEJM 2013p, gy [; p,

Take home messages• Personalized intestinal microbiota

• Diet and antibiotics are major regulators of microbiotacomposition

• Associations microbiota with disease, but causality has onlybeen proven for few diseases!

• Fecal transplantation may be used to isolate therapeutic/ pathologic strains or metabolites

AcknowledgementsgNieuwdorp group @ Diabetes research centerPhD studentsRuud Kootte MDFle an de Valk MD

Willem de Vos

Fleur van der Valk, MDPim Gilijamse, MDKasper ter Horst, MDLoek Smits, MDSophie Bernelot Moens, MD Erik Stroes

ijMax NieuwdorpWillem de Vos

WUR/Helsinki

p ,Kristien Bouter, MScPieter de Groot, MDAnnick Hartstra MDGuido Bakker, MDMark Smits MD

Erik Stroes AMCHans Romijn

AMC

Max NieuwdorpAMC

Mark Smits, MDMarcel Muskiet MDLennaert Tonneijk, MDShanti Udayappan, MscPostdocsHan Levels PhDDaniel van Raalte, MD PhDHilde Herrema PhDJing Zhao PhDGeesje Dallinga, PhD

Anne VriezeAMC

Fredrik backhedGothenborg

Loek SmitsAMC

Geesje Dallinga-ThieAMC

Geesje Dallinga, PhD