Discussion Group 4

Identifying biomarkers linking the composition and function of

the gut microbiome to health status –how close are we?

Karen Scott, Seppo Salminen

Rapporteur - Carlos Gomez-Gallego

The Group

Karen Scott, Seppo SalminenRapporteur - Carlos Gomez-Gallego

Emma Allen-Vercoe Ardythe Morrow Hermie Harmsen Anne SalonenRan Blekhman Edwin Abeln Suzan WopereisLesley Hoyles

Maria Collado

Industry experts Sylvie Binda (Danone) Stephan Theis (Beneo)

Karen Goehring (Abbott Laboratories) Danielle de Montigny (BioK+ Int)

David Hayashi (Mondelez Int) Shintaro Yui (Yakult Honsha Co. Ltd)

Christopher Martoni (UAS labs) Sarmauli Manurung (Mead Johnson Nutrition)

General Topics for discussion1. What are currently available/accepted biomarkers related to the gut

• calprotectin• Short chain fatty acids (SCFAs), other bacterial metabolites• Microbiome for dysbiosis (aberrant microbiome) - low diversity,

richness• Age-specific biomarkers

2. Currently available and validated/accepted health biomarkers (not only gut/microbiome related, but broader or transferable– e.g. cardiovascular, bone health, infection, etc.). Consider site specific aspects.

3. Recent developments in the field (e.g. newly developed biomarkers)

4. Validation of biomarkers in a robust way

Challenges for a biomarker:

A – Biomarker has to be a quick testage specific, niche specific, sample specific

B – easier to define a biomarker for disease than health

C- focus normally on bacterial composition but functionality and metabolite production (more) important

SCFA – ratio in faecesurine TMA

D- Microbial resilience or dysbiosis as a measure of health of microbiome - how

Dysbiosis is any change to the composition (or

activity) of resident commensal communities relative to the community found in healthy individuals

Petersen et al. 2014 Cell Microbiol

How could this be used as a marker?

Need accurate and consistent ways to measure this

HITChip microbiota profiling Next generation

sequencing

microbialcommunity DNA

RNA

hybridisation

Nucleic acids extraction & labelling

Data analysis- Profiling

- Identification- Quantification

imagingConsistent comparable data

Method continually Upgraded

Hard to compare datasetsProcedures differ

When, who and where do you measure microbial dysbiosis?

Effects of AgeDietGeographyGenetics (inc Gender)Interindividual variation

What about ecosystem dysbiosis?Change in redox potentialchange in pH

How does dysbiosis link to resilience? How could you measure this? Specific challenge? ETEC?

Bacteriodes

Parabactero

ides

Alistipes

Bacteriodes

Parabactero

ides

Alistipes

Anaerostipes

Blautia

CoprococcusDorea

EubacteriumRoseburia

Faecalibacterium

SubdoligranulumAkkermansia

Escherichia coliEnterobacteria

VeillonellaDialister

Anaerotruncus

Bryantella

Gordonibacter

HoldemaniaButyrivibrio

Catenibacterium

+

-

EnterococcusStreptococcusLactobacillus

Methanobrevibacter

Rum. gnavuset rel.

Fusobacterium

Desulfovibrio

Prevotella

Collinsella

EggerthellaAtopobium

Ruminococcus

Cl. perfringens

Cl. dificile

Cl. ramnosum

Bifidobacterium

Gemmiger

• Fusobacterium nucleatum – emerging pathogen in Colorectal cancer

• May co-occur with other signature microbes which may provide a stronger biomarker (Subject of a patent)

HOWEVER – not all strains/species are equal

• highly heterogeneous at the species level

• Not all strains are aggressive (similar to the H.pylori story)

Ruminococcus gnavus

• Enriched in: IBD, Spondyloarthritis, Infantile eczema

• HOWEVER – not all strains/species are equal

• Present in >90% of humans,

• some strains contain more mucin breakdown pathways than others

• Strains may have beneficial activities – eg. anti C. perfringens activity

Faecalibacterium prausnitzii

Depleted in CD, anti-inflammatory effect

HOWEVER – not all strains/species are equal

Could signature genes be an alternative option? Not easy to define specifically, or detect

Can a single bacterial species be a biomarker?

• Reduced flatulence: by hydrogen breath test after flatulence triggering meal or substance (beans or lactulose) (reduced intestinal discomfort)

• Increase in faecal bulk (improved bowel function) (number of bowel movements)

• Normal function of digestive enzymes: by measuring activity of digestive enzymes such as lipase

• Lactose digestion: breath hydrogen concentration.

(measurement of breath hydrogen concentration after ingestion of a certain amount of lactose)

• Reduction of postprandial glycemic responses & LDL-cholesterol: by measuring 2h postmeal glucose & insulin concentrationsby measuring total cholesterol, HDL and LDL concentrations

EFSA accepted markers used for claims in area of gut health

Molecules as biomarkers

Measure of functionality of microbiotaWhat to detect, how to detect it,Where to detect – blood, faeces, urine?

diarrhea, stool consistency

gut permeability markers, Tight junction marker (occluding, zonulin..)Metabolites (urine, blood, feces) organic acidsLPS

Gut function markers.IFAB, Calprotectin, Citrulin

Microbial productsSingle metabolite – eg TMA/TMAOMixture of metabolites – SCFA, HPLC/NMR total metabolomic profile

HOW – to get regulatory bodies to accept newMethods or new markers

Do they need to change as quickly as the methodology?

Online sensory tool (EMA, Ecological Momentary Assessment )

Phenflex challenge/ OGTT(response to specific nutritional load)

ETEC challenge (response to attenuated E.coli challenge)Inactivated virus challenge?

Adapt to antibiotic challenge

What about impact of host genetics?On microbial compositionon response to intervention

• LCT (lactase) locus (chr2) linked to Bifidobacterial population (in western)

Known that responders/nonresponders in some dietary interventions depend on starting number of Bifids

FUT-2 status

affects glycosylation of mucosal surface

important human milk oligosaccharide (up to 40% of HMOs)

influences microbiota architechture

GOS intervention

Davis et al. PLoS One. 2011; 6(9): e25200.

Human genetics can be used as a predictor of microbial composition

FUT2 in disease: Balancing selection

FUT2+SeSe, Sese (secretors)

Risks

• Diarrheal disease• Norovirus• Rotavirus• (Other)

FUT2-sese (non-secretors)

Risks

• Crohn’s Disease• Primary sclerosing

cholangitis• Type 1 diabetes

77% 23%428 G>A mutation, rs601338MAF=.48

But 2’-FL in secretor Mum’s milk acts as a receptor decoy less diarrhea

Non-secretors (mother, infant?) at increased risk of infant malnutrition

Responders

Non-Responders

Defining Dietary Responders – helps stratify data

Korpela et al. PLoS One 2014

Microbiota Cholesterol

ΔC

ho

lest

ero

l

Baseline cholesterol level

(correlation)

Responders

>10% decrease

Best prospects for gut microbiome biomarkers: Consensus

• Challenge test (ETEC/inactivated viruses/ antibiotic challenge/ travellers diarhoea) – have definable outcomes at outset

• HitChip (for compositional analysis until sequencing more reproducible between labs/samples)

• target specific metabolite of interest

• integrated systems biology

How can we proceed?

get accepted in studies

get accepted as valid outcomes/measures by regulatory bodies

(difficult as all regulatory bodies have different requirements)