Impact of diet upon intestinal microbiota and microbial metabolites

Harry J Flint

Microbiology GroupRowett Institute of Nutrition and Health

University of Aberdeen, UK

metabolism of non-digestible dietary components

immune function, inflammation

pathogenesis

modification of host secretions (mucin, bile, gut receptors..)

Gut function, gut disorders: Colitis; Irritable bowel syndromeColorectal cancer Infections

Systemic effects: Energy supply, satiety DiabetesHeart diseaseAutoimmune disorders

barrier function

supply of additional energy, vitamins

Impact of the gut microbiota on human nutrition and health

release of phytochemicals, enterohepatic circulation of xenobiotics, drugs

Modulation of the human gut microbiota by dietary fibres occurs at the species level [Chung WSF et al BMC Biology, 2016]

In vitro chemostat studies

[Separate experiments with 3 fecal microbiota]

Bacteroides spp.

Modulation of the human gut microbiota by dietary fibres occurs at the species level [Chung WSF et al BMC Biology, 2016]

In vitro chemostat studies

[Separate experiments with 3 fecal microbiota]

Microbiota composition (family level)

Modulation of the human gut microbiota by dietary fibres occurs at the species level [Chung WSF et al BMC Biology, 2016]

In vitro chemostat studies

[Separate experiments with 3 fecal microbiota]

Firmicutes

14 obese male volunteers with metabolic syndrome

(mean age 54 years, mean BMI 39.4 kg/m2)

M: maintenance diet

NSP: high non-starch polysaccharide, low RS

RS: high resistant starch, low NSP

WL: high protein, moderate carbohydrate

M NSP RS WL1 wk 3 wks 3 wks 3 wks

Collection of faeces

M NSPRS WL

Mean dietary intake [g/d]:

M 427 230 5 28 103 126

NSP 427 138 2 42 102 136

RS 434 275 26 13 109 127

WL 201 110 3 22 144 63

CHO: carbohydrate

Diet CHO starch RS NSP protein fat

Walker AW et al. ISME J 2011

Does the type of dietary carbohydrate alter the colonic

Weight maintenance

Weight loss

added wheat bran

added type 3 resistant starch

Impact of dietary non-digestible carbohydrates in vivo

Walker AW et al (2011) ISME J 5, 220-230

Salonen A et al (2014) ISME J 8, 2218-2230

Lower diversity with RS than WB NSPSome but not all species are diet-responsive

Increase with RSIncrease with wheat bran NSP

[Hitchip]

[16S rRNA gene sequences]

M NSP RS WL

350

300

250

200

150

100

Inv

ers

e S

imp

so

n in

dex

Walker AW et al (2011) ISME J 5, 220-230

Salonen A et al (2014) ISME J 8, 2218-2230

Lower diversity with RS than WB NSPSome but not all species are diet-responsive

Increase with RSIncrease with wheat bran NSP

[Hitchip]

[16S rRNA gene sequences]

Volunteer

No.

1-P

ears

on

co

rrela

tio

n

Individual variation still dominates

M NSP RS WL

350

300

250

200

150

100

Inv

ers

e S

imp

so

n in

dex

Very extreme dietary changes can lead to wide-ranging shifts in the gut

bacterial community in humans

‘animal based’ – 69.5 % (!) cals from fat, 30% from protein, <1% (!) from carbs/fibre

‘plant based’ - 32.5% from fat, 16.5% from protein, 50% from carbs/fibre

Rise in bile acids with high fat + lack of fibre considered key factors in ‘animal-based’ diet

[David LA et al Nature 2014]

Walker AW et al. ISME J (2011); Salonen A et al. ISME J (2014)

0

10

20

30

40

50

0 10 20 30 40 50 60 70

Volunteer 16

M RS NSP WL

0

10

20

30

40

50

0 10 20 30 40 50 60 70

Volunteer 19

M NSP RS WL

0

10

20

30

40

50

0 10 20 30 40 50 60 70

time [days]

Volunteer 24

M NSP RS WL

0

10

20

30

40

50

0 10 20 30 40 50 60 70

time [days]

Volunteer 20

M RS NSP WL

R. bromii

R. albus

+ R. bicirculans

R. flavefaciens

+ R. champanellensis

+ R. callidus

R. champanellensis

Responses occur rapidly and tend to be species-specific

Cluster IV Ruminococcus species - qPCR

% 1

6S

rR

NA

ge

ne

% 1

6S

rR

NA

ge

ne

Response on

RS is due to

R. bromii

Nutritional specialization among Ruminococcus spp.

- selected glycoside hydrolase families encoded by genomes

Rumen (cellulolytic)

Human colon

Human colon

0

2

4

6

8

10

12

14

16

GH2 GH3 GH5 GH26 GH44 GH74 GH16 GH48 GH9 GH10 GH11 GH43 GH51 GH13 GH31 GH77

R. bicirculans 80/3

R. champanellensis 18P13

R. flavefaciens FD1

R. bromii L2-63

Human colon

amylase/ a-glucosidase/ amylomaltasecellulase

b-glucanase/ mannanase/ xyloglucanase

xylanase/ xylosidase/ arabino-furanosidase

Ruminococcal cellulosome systems from rumento human. Ben David et al (2015) Environ Microbiol 17:3407-3426

Ruminococcus champanellensis

The only human colonic anaerobe able to degrade filter paper (crystalline) cellulose

“Ruminococcus bicirculans”

Able to utilize xyloglucan, beta-glucan for growth, but not cellulolytic

Complete genome of a new Firmicutes speciesbelonging to the dominant human colonic microbiota(‘Ruminococcus bicirculans’) reveals twochromosomes and a selective capacity to utilizeplant glucans. Wegmann U et al (2014) Environ Microbiol 16:2879-2890

Ruminococcus bromii

Specialist starch-degrader

Exceptional starch-degrading activity in the human colonic anaerobe Ruminococcus bromii coincides with unique organization of its extracellular enzymes into ‘amylosomes’. Ze X et al (2015) MBio 6 (5) e01058-15.

Ruminococcus bromii

Specialist starch-degrader

Exceptional starch-degrading activity in the human colonic anaerobe Ruminococcus bromii coincides with unique organization of its extracellular enzymes into ‘amylosomes’. Ze X et al (2015) MBio 6 (5) e01058-15.

C

D G E F

B

A

TonBcomplex

OM

CM

susR susA susB susC susD susE susF susG

Contrast - Bacteroides thetaiotaomicronstarch utilization (sus) system (Salyers)

- soluble starches

Mainly periplasmic amylases

Degradation of corn starches by four amylolytic human gut bacteria

Ruminococcus bromii

Specialist starch-degrader

0%

20%

40%

60%

80%

100%

S1 S2 S3 S4

starches (boiled 10 mins)

Ruminococcus bromii(Firmicutes)

Bifidobacterium adolescentis(Actinobacteria)

Eubacterium rectale(Firmicutes)

Bacteroides thetaiotaomicron(Bacteroidetes)

Gram-positive

Gram-negative

Resistant starches (S2, S3 (RS2) S4 (RS3))

[Ze X et al 2012 ISME J]

Digestible starch (S1)

Exceptional starch-degrading activity in the human colonic anaerobe Ruminococcus bromii coincides with unique organization of its extracellular enzymes into ‘amylosomes’. Ze X et al (2015) MBio 6 (5) e01058-15.

[Walker AW et al. ISME J 2011Ze X et al. ISME J 2012]

qPCR analysis – all timepointsqPCR - % Ruminococcus (cl IV)

16S rRNA gene copies

Residual starch in faeces

11 12 17 18 19 23 240

20

40

60

80

100

14 15 16 20 22 25 26

volunteer% r

es

idu

al fa

ec

al s

tarc

h

RS diet

NSP diet

Incomplete starch fermentation

R. bromii as a ‘keystone’ species in degradation of resistant starch

0

10

20

30

40

50

60

11 12 17 18 19 23 24 14 15 16 20 22 25 26

Rum % (RS diet) Rum % (NSP)

v low R. bromii

Diet

Insoluble complex carbohydrates

Soluble polysaccharides

Oligosaccharides, sugars

Primary degraders

Polysaccharide utilisers

Oligosaccharide/ sugar utilisers

H-utilisers (acetogens, methano-gens, SRB); lactate utilisers

Metabolic products

Microbial ecology of carbohydrate utilization in the gut: functional groups

(Flint HJ et al Env Micro 2007)

ruminococci on faecal fibre

0

10

20

30

40

50

60

2h 4h 8h24h48h 2h 4h 8h24h48h 2h 4h 8h24h48h 2h 4h 8h24h48h

D1 D2 D3 D4

Otu039

Otu030

Otu029

Otu028

Otu024

Otu017

Otu005

Otu006

Otu007

Oscillospira guilliermondii (94% similarity)

Coprococcus sp. ART55/1/eutactus (97% similarity)

Clostridium lactatifermentans

Butyrate-producer A2-166

Lachnospiraceae bacterium A4 (C. hathewayi - 96% similarity)

Eubacterium siraeum (97% similarity)

Roseburia faecis/hominis/intestinalis

Butyrate-producer T2-145 (96% similarity)

Eubacterium xylanophilum (97% similarity)

Enrichment of Firmicutes bacteria on wheat bran in vitro*

% s

equ

ence

s

* Duncan SH et al. Environ Microbiol 2016 online medium

pH- controlled, anaerobicin vitro fermentor system -

inflow

outflow

100% CO2 atmosphere

% s

eq

ue

nce

s

0

10

20

30

40

50

60

2h

4h

8h

24

h4

8h

2h

4h

8h

24

h4

8h

2h

4h

8h

24

h4

8h

2h

4h

8h

24

h4

8h

D1 D2 D3 D4

Otu039

Otu030

Otu029

Otu028

Otu024

Otu017

Otu005

Otu006

Otu007

Duncan SH et al Environ Microbiol -2016 online

Action of isolated bacteria on wheat bran

weight loss (degradation)

phenolics

Otu005-relatedOtu017-relatedOtu006-related

“Wheat bran promotes enrichment within the human colonic microbiota of butyrate-producing bacteria that release ferulicacid”

ferulicacid

ferulic acid metabolite 1

Co

nce

ntr

atio

n (

g cm

-3)

metabolite 2 metabolite 3

5

10

15

20

25

P < 0.001

P = 0.007

P < 0.001

demethylation dehydroxylationhydrogenation

OH

OOH

OH

OOH

OOH

OOH

OOH

OOHO

OOH

O

OOH

OH

OOH

OOH

OOH

OOH

H

[Russell WR et al AJCN 2011]

ferulic acid

Faecal concentrations

(means, 8 overweight male volunteers)

Impact of diet on plant-derived phenolic acids and microbial metabolites in human volunteers

Maintenance (carbs 400g, NSP fibre 28 g/day)

High Protein WL (carbs 170g, NSP fibre 12 g/day)

High Protein WL (carbs 23g, NSP fibre 6 g/day)

diets

4OH-3OMe-PPA 3,4OH-PPA 3OH-PPA

[Louis P, Hold GL & Flint HJ 2014 NRM]

The gut microbiota, bacterial metabolites and colorectal cancer

oxaloacetate

fumarate

succinate

propionyl-CoA

fucose, rhamnose

propane-1,2-diol

propionyl-CoA

DHAP + L-lactaldehydePEP

CO2

lactate

lactoyl-CoA

propionyl-CoA

pyruvate

propionate

hexoses & pentoses

acetyl-CoA

acetoacetyl-CoA

butyryl-CoAacetate

acetyl-CoA

butyrate

acetate

butyryl-P

H2 + CO2

formate

Roseburia inulinivoransRuminococcus obeumSalmonella enterica

methane

Bacteroidetessome Negativicutes

Coprococcus catusMegasphaera elsdenii

Ruminococcusbromii

Eubacterium rectaleEubacterium halliiRoseburia spp.Anaerostipes spp.Coprococcus catusFaecalibacterium prausnitzii

Coprococcus eutactusCoprococcus comes

ethanol

Blautiahydrogenotrophica

Eubacterium halliiAnaerostipes spp.

methanogenicarchaea

hydrogensulphide

sulfate

Desulfovibrio spp.

Major fermentation pathways

[propionate pathways -Reichardt N et al (2014) ISME J]

NSP starch protein

propionate

H2 + CO2

oligo-saccharides, sugars

lactate

butyrate

acetate

succinate

formate

CH4

B10B9

B7

B2, B4, B5B3, B6 B1

B5, B6 B8

PEPpyruvate

acetyl CoA

B1-B9

B1

B1-B9

B3, B1

Kettle H, Louis P, Duncan SH, Holtrop G, Flint HJ (2015) “Modelling the emergent dynamics of communities of human colonic microbiota: response to pH and peptide”Environ Microbiol 15: 1615-1630

Bacterial functional groups

B1 = BacteroidetesB2 = Firmicutes (eg. R. bromii)B3 = Firmicutes (eg. E. eligens)B4 = ActinobacteriaB5 = Roseburia groupB6 = F. prausnitziiB7 = NegativutesB8 = E. hallii, AnaerostipesB9 = acetogensB10 = methanogens

Changes in butyrate-producing bacteria within faecal microbiota in disease states/ extreme diets –

F. prausnitzii in ileal Crohn’s patients Sokol H et al PNAS (2008)

F. prausnitzii in frail elderly Claesson et al Nature (2012)

Roseburia, E. rectale, F. prausnitzii in type 2 diabetics Qin J et al Nature (2012)

Butyrate- producers in type 1 diabetics de Goffau MC et al Diabetes (2013)

Roseburia, butyrate-producers in CR cancer Wang T et al ISME J (2012)

Roseburia in constipation-type-IBS Chassard C et al Alim Ph Th (2012)

Roseburia in hepatic encephalopathy (mucosal flora) Bajaj JS et al Am J Clin Nutr (2012)

Butyrate-producers with improved insulin sensitivity Vrieze A et al Gastroenterol (2012)

Roseburia, E. rectale with low carb WL diets Duncan SH et al AEM (2007)

Roseburia, Faecalibacterium, Coprococcus in LGC individuals (increased metabolic syndrome) Le Chatelier et al Nature (2013)

Dietary intake, medication, inter-individual variation

GUT ENVIRONMENT

GUT MICROBIOTA

metabolic pathways community structure

cross-feeding

METABOLIC PRODUCTS

How do changes in gut microbiota composition impact on metabolite production?

Clone libraries from 1 volunteer

05

10152025303540

% o

f to

tal

clo

ne

s mmdA

16S rRNA

% Bacteroidetes (qPCR)

% p

rop

ion

ate

M

NS

P

RS

[14 male volunteers]

Relative abundance of Bacteroidetes correlates with % propionate

- in faecal samples - and in fermentor studies

R² = 0,558

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100

Pro

port

ion

al

ab

un

dan

ce o

f

pro

pio

nate

(%

of

tota

l S

CF

A)

Bacteroides spp. and Prevotella spp. % of total 16S

rRNA genes

Salonen A et al. ISME J (2014) Chung WSF et al. BMC Biology (2016)

• Total faecal SCFA , especially butyrate, decrease with lower carbohydrate intake

Obese male subjects (UK)

1. Duncan SH et al AEM 2007

2. Russell WR et al Am J Cl Nutr 2011

Diets:M – maintenance (50-52% cals as CHO)HPMC – Moderate carbohydrate (35%)HPLC – Low carbohydrate (4-5%)

0

20

40

60

80

100

120

M1 HPMC1 LC1 M2 HPMC2 HPLC2

isobutyrate

isovalerate

valerate

butyrate

propionate

acetate

mM

study 1 study 2

Faecal SCFA:

• Total faecal SCFA, including butyrate, can be very high in populations that have high intakes of plant-derived foodstuffs

Ou J et al Am J Clin Nutr 2013

Native Africans - Ace: Prop: But 72: 27: 17 mM (total > 100 mM) African Americans - Ace: Prop: But 27: 7: 8 mM (total < 50 mM)

Model describing the relationship between substrate abundance and gut microbiota product

formation and its dependence on gut microbiota composition using the consumption of

fermentable carbohydrates and the production of short chain fatty acids (SCFAs) as an example.

Gary D Wu et al. Gut 2016;65:63-72

Copyright © BMJ Publishing Group Ltd & British Society of Gastroenterology. All rights reserved.

Do ‘restrictive’ communities lack certain keystone species?

GENERAL CONCLUSIONS

Changes in diet composition change the representation of certain ‘diet-responsive’ species within the microbiota

This reflects the fact that many species (especially Firmicutes?) are highly specialised in their substrate utilization

‘Some are more equal than others’ (Ze X et al Gut Microbes 2013) (after George Orwell) - certain ‘keystone species’ play primary roles in degradation of recalcitrant substrates

Changes in microbiota composition (due to diet or individual variation) impact on metabolite formation – especially loss of keystone species

Rowett Institute (U Aberdeen, UK)

Alan Walker

Sylvia Duncan

Petra Louis

Karen Scott

Xiaolei Ze

Faith Chung

Jenny Laverde

Nicole Reichardt

Alexandra Johnstone

Biomathematics and Statistics Scotland

Grietje Holtrop, Helen Kettle

Institute of Food Research, UK

Nathalie Juge, Emmanuelle Crost

Wellcome Trust Sanger Institute, UK

Trevor Lawley, Julian Parkhill

U Groningen, Netherlands

Hermie Harmsen

U Wageningen, Netherlands

Jerry Wells

Weizmann Institute, Israel

Ed Bayer, Sarah Morais

U Helsinki, Finland

Anne Salonen, Willem de Vos

U. Girona, Spain

Mireia Lopez-Siles, Jesus Garcia-Gill

Support from :–

Acknowledgements -

Commercial collaborators

Rowett Institute of Nutrition and Health