Post on 31-Jan-2021
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,
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
http://www.nih.gov/http://www.nih.gov/