amarcos@ictan.csic.es

PROF. ASCENSIÓN MARCOS

PROFESORA DE INVESTIGACION

MICROBIOTA: CIENCIA Y TECNOLOGÍA

DE ALIMENTOS PARA NUESTROS

PEQUEÑOS INQUILINOS

90% MICROBES

10% HUMAN CELLS

HOST DIET

HUMAN GUT MICROBIOTA

Host metabolites

Nutrients

Bacterial metabolites

SCHEMATIC ECOLOGICAL INTERACTIONS OCCURRING IN THE HUMAN COLON

Chassard &Lacroix. Curr Opin Clin Nutr Metab Care 2013;16:453-60

billions of microbes

extracting nutrient and

energy from complex polysaccharides

polysaccharides not degraded by host enzymes

polysaccharides

fermented by the

colonic gut microbiota

caracterización

Chassard &Lacroix. Curr Opin Clin Nutr Metab Care 2013;16:453-60

FATE OF INGESTED DIETARY FOOD COMPONENTS

IN THE SMALL AND LARGE INTESTINE

Scott, Gratz, Sheridan, Flint, Duncan. Pharmacol Res 2010;61:52–60

Adapted from Topping and Clifton

CHO – carbohydrate, RS – resistant starch, NSP – non-starch polysaccharide, OS –

oligosaccharides, SCFA – short chain fatty acids, BCFA – branched chain fatty acids.

IMPACT OF BACTERIAL CROSS-FEEDING IN THE CONVERSION OF LACTATE OR

RELEASED OLIGOSACCHARIDE DEGRADATION PRODUCTS INTO BUTYRATE

Scott, Gratz, Sheridan, Flint, Duncan. Pharmacol Res 2010;61:52–60

Chassard &Lacroix. Curr Opin Clin Nutr Metab Care 2013, 16:453–460

OVERVIEW OF CARBOHYDRATES AVAILABLE FOR THE COLONIC GUT MICROBIOTA

AND THEIR MAJOR BACTERIAL UTILIZERS

Short chain fatty acid concentrations in fermenta after 24 h of fecal

fermentation with chlorogenic acid, caffeic acid, rutin and quercetin at 10, 30

and 100 μg/mL respectively and inulin at 2, 10 and 20 mg/mL

PHYTOCHEMICALS AND PHYSIOLOGICAL FUNCTION

Laparra & Sanz. Pharmacol Res 2010;61:219-25

Obesity in humans leads to changes in the composition of gut

microbiota, some of those changes being reversed upon dieting

and changes in dietary habits.

SCHEMATIC VIEW OF THE

POSSIBLE MECHANISMS

LINKING GUT

MICROBIOTA TO OBESITY

Bacterial LPS derived from intestinal microbiota may act as a

triggering factor linking inflammation to high-fat diet-induced obesity

The composition of the microbiota can shape a healthy

immune response or predispose to disease

PA

Deviations from the normal development of the microbiota may alter the

outcome of immune development and potentially predispose individuals to

various inflammatory diseases later in life

THE ENTERIC MICROBIOTA-GUT-BRAIN AXIS

(BI-DIRECTIONAL COMMUNICATION SYSTEM)

Brain modulates

gastrointestinal function

Any alteration in gastrointestinal

function is communicated to

the brain with the perception

of visceral events (nausea,

satiety, pain)

NEURAL PATHWAYS AS WELL AS IMMUNE AND ENDOCRINE MECHANISMS

Cryan JF & O'Mahony SM. Neurogastroenterol Motil. 2011;23(3):187-92

MICROBIOTA–GUT–BRAIN COMMUNICATION IN HEALTH AND DISEASE

COMPOSITION IN THE GUT

MICROBES MAY BE ASSOCIATED

WITH CHANGES IN THE NORMAL

FUNCTIONING OF THE NERVOUS

SYSTEM

PSYCHIATRIC DISORDERS ?

Grenham et al. Frontiers in Physiology. 2011;2:94

Studies evaluate new substances which could be used to

body weight control or to reduce the risk of associated

pathologies and to improve the health of obese patients

Nutritional strategies could contribute to reduce risk of

diseases throughout modifications in the microbiota

FAT STORAGE

METABOLISM

GUT

MICROBES

(Bäckhed et al., 2004; Cani and Delzenne, 2009) (Ley et al., 2006; Santacruz et al., 2009;

Delzenne et al., 2011; Jumpertz et al., 2011)

FIRMICUTES

LEAN

OBESE

BACTEROIDETES

PROGRAMA INTEGRAL EVASYON

(DIETA + ACTIVIDAD FÍSICA) PARA ADOLESCENTES

(13-16 AÑOS)

< 2 KG

> 4 KG

FASE INTENSIVA

PÉRDIDA PESO

PÉRDIDA

PESO

Correlaciones entre el recuento de bacterias fecales y

la pérdida de peso después de la intervención en el

grupo de adolescentes con mayor pérdida ponderal

(n=23; >4.0 kg pérdida de peso)

CONCLUSION

FAT STORAGE

METABOLISM

GUT

MICROBES

Gut microbiota composition is more important

to achieve weight loss than expected

EVASYON intervention was only successful in one group (HWL),

maybe due to a different microbiota between both groups

OBJECTIVE

The aim of this study was to evaluate

the effects of two dietary treatments to

lose weight on the gut microbiota

composition in female ICR mice.

Changes in gut microbiota due to supplemented fatty acids in diet-induced obese mice

JR. Mujico, GC. Baccan, A Gheorghe, LE. Díaz & A Marcos. BJN (in press)

EXPERIMENTAL DESIGN

1. Induction of obesity:

High fat diet (HF)/ 8 weeks

2. Effects of treatments on weight and gut

microbiota

HF+T1 / 6 weeks

HF+T2 / 6 weeks

TREATMENTS

Treatment 1 (T1): compound derived

from oleic acid (1500mg/kg/day)

Treatment 2 (T2): omega-3 fatty

acids

(EPA + DHA, 3000mg/kg/day)

MATERIALS AND METHODS

Table 1- Composition of the diets Maintenance

diet High fat diet

Protein (%) 14.3 23.5 Carbohydrate (%) 48.0 27.3 Fat (%) 4.0 34.3 Saturated (%) 0.6 37 Monounsaturated (%) 0.7 47 Polyunsaturated (%) 2.1 16 Energy density (Kcal/g) 2.9 5.1 Calories from Protein (%) 20.0 18.4 Calories from Carbohydrate (%) 67.0 21.3 Calories from Fat(%) 13.0 60.3

MATERIALS AND METHODS

The real-time PCR analysis was used together with the

group-specific primers* to detect the populations of:

PCR assay Oligonucleotide sequence (5'–3') Amplicon

size (bp) Reference

Total bacteria F: 5'-ACTCCTACGGGAGGCAGCAG-3' R: 5'-ATTACCGCGGCTGCTGG-3'

200 Fierer_2005

Firmicutes Phylum F: 5'-GGAGYATGTGGTTTAATTCGAAGCA-3' R: 5'-AGCTGACGACAACCATGCAC-3'

126 Guo_2008

Clostridial Cluster XIVa F: 5'-GCGGTRCGGCAAGTCTGA-3' R: 5'-CCTCCGACACTCTAGTMCGAC-3'

81 Ramírez-Farias_2009

Lactobacillus Group F: 5'-AGCAGTAGGGAATCTTCCA-3' R: 5'-CACCGCTACACATGGAG-3'

341 Rinttila_2004

Enterobacteriales Order F: 5'-ATGGCTGTCGTCAGCTCGT-3' R: 5'-CCTACTTCTTTTGCAACCCACTC-3'

177 Castillo_2006

Bacteroidetes Phylum F: 5'-GGARCATGTGGTTTAATTCGATGAT-3' R: 5'-AGCTGACGACAACCATGCAG-3'

126 Guo_2008

Bifidobacterium spp. F: 5'-TCGCGTCYGGTGTGAAAG-3' R: 5'-RCCACATCCAGCRTCCAC-3'

243 Rinttila_2004

*These primers were commercially synthesized by Isogen

INDUCTION OF OBESITY

0 7 14 21 28 35 42 49 560

10

20

30

40

50Control

Obese

Treatment 1

Treatment 2

Days

Weig

ht

(g)

Contr

ol

Obes

e

Treat

men

t 1

Treat

men

t 2

0

1000

2000

3000 **

*

Are

a u

nd

er

cu

rve

Effects of diets on body weight (8 weeks)

HF

HF+T1

HF+T2

HF C HF+T1 HF+T2

EFFECTS OF TREATMENTS

0 7 14 21 28 35 42 490

10

20

30

40

50

60Control

Obese

Treatment 1

Treatment 2

Days

Weig

ht

(g)

Contr

ol

Obes

e

Treat

men

t 1

Treat

men

t 2

0

500

1000

1500

2000

2500

3000

3500

*** *

Are

a u

nd

er

cu

rve

Effects of treatment on body weight

HF C HF+T1 HF+T2

HF

HF+T1

HF+T2

8 wk (weeks: 8-14)

BODY FAT SCANNER

Control Obese Treatment 1 Treatment 2

EFFECTS OF TREATMENTS

HF HF+T1 HF+T2

EFFECTS OF TREATMENTS

Effects of diets on visceral fat

Contr

ol

Obes

e

Treat

men

t 1

Treat

men

t 2

0

2

4

6

***

**W

eig

ht

(g)

HF C HF+T1 HF+T2

EFFECTS OF TREATMENTS

Effects of treatment 1 and 2 on triglycerides

0 6

0

50

100

150

200

250Control

Obese

Treatment 1

Treatment 2

*

**

Weeks of treatment

Tri

gly

ceri

des (

mg

/dL

)

HF

HF+T1

HF+T2

8 wk 6 wk

TOTAL DNA (ng DNA / mg faeces)

Spearman r = -0.8810

p = 0.0072**

• HF and HF+T2 decreased total DNA

content in faeces

• HF+T1 increased total DNA content up to

similar level than control group (NS)

Significant negative correlation

was observed between total

DNA content and body weight

Kruskal-Wallis test

(Dunn's Multiple Comparison Test)

HF C HF+T1 HF+T2

GUT MICROBIOTA

PCR assay Slope

PCR

efficiency

(%)

Correlation

coefficient

Total bacteria -3.288 101.4 0.999

Firmicutes Phylum -3.382 97.6 0.999

Clostridial Cluster XIVa -3.439 95.3 0.999

Lactobacillus Group -3.709 86.0 0.999

Enterobacteriales Order -3.379 97.7 0.990

Bacteroidetes Phylum -3.401 96.9 0.999

Bifidobacterium spp. -3.613 90.2 0.997

• Standard curves had correlation coefficient values

between 0.990-0.999

• Using the formula E = [10(-1/slope) - 1], the efficiencies

for the individual assays were between 86.0-101.4%

Total Bacteria (Pan-Bacteria)

Spearman r = -0.7143

p = 0.0576 (NS)

Tendency to negative correlation

was observed between total bacteria in faeces and body weight

Firmicutes Phylum

Significant positive correlations were

found between Firmicutes &

Clostridium and body weight

Spearman r = 0.8333

p = 0.0154*

Spearman r = 0.9286

p = 0.0022**

Firmicutes Phylum

Clostridial Cluster XIVa

Lactobacillus Group

Lactobacillus Group

Lactobacillus Group (Relative Units)

Bo

dy W

eig

ht

(g)

0 100 200 300 4000

20

40

60

80

Control Obese Treatment 1 Treatment 2

Spearman r = 0.6429

p = 0.0962 (NS)

The Lactobacilliales order of the Bacilli class from the Firmicutes phylum

No significant correlation was found between Lactobacillus and body weight

Enterobacteriales Order

Enterobacteriales Order

Enterobacteriales Order (Relative Units)

Bo

dy W

eig

ht

(g)

0 100 200 300 400 5000

20

40

60

80

Control Obese Treatment 1 Treatment 2

Spearman r = 0.5952

p = 0.1323 (NS)

The Enterobacteriales order of the γ-Proteobacteria class from the

Proteobacteria phylum

No significant correlations between Enterobacteriales and body weight

Bacteroidetes Phylum Bacteroidetes Phyla

Bacteroidetes Phyla (Relative Units)

Bo

dy W

eig

ht

(g)

0 100 200 300 4000

20

40

60

80

Control Obese Treatment 1 Treatment 2

Spearman r = -0.7857

p = 0.0279*

Significant negative correlation between Bacteroidetes and body weight

Bifidobacterium spp. Bifidobacterium spp.

Bifidobacterium spp. (Relative Units)

Bo

dy W

eig

ht

(g)

0 20 40 60 80 1000

20

40

60

80

Control Obese Treatment 1 Treatment 2

Spearman r = -0.5538

p = 0.1966 (NS)

No significant correlation between Bifidobacterium and body weight

SUMMARY

High fat feeding induced important changes in the

microbial community structure:

Bacteroidetes Phylum

Bifidobacterium spp.

Firmicutes Phylum

Clostridial Cluster XIVa

Lactobacillus group

Enterobacteriales Order

SUMMARY

• HF+T1 restored (partially or completely) the analyzed microbial

groups to proportions similar to the control group

• HF+T2 significantly increased the quantities of Firmicutes

• Significant positive correlations were observed between body weight

and Firmicutes Phylum / Clostridial Cluster XIVa

• Significant negative correlations were observed between body weight

and Bacteroidetes Phylum

HF

HF+T2

HF+T1

INTERACTIONS BETWEEN FUNCTIONAL FOOD COMPONENTS

AND THE GUT MICROBIOTA

Laparra & Sanz. Pharmacol Res 2010;61:219-25

LOS COMPUESTOS BIOACTIVOS TAMBIÉN EJERCEN EFECTOS

SIGNIFICATIVOS SOBRE EL AMBIENTE INTESTINAL MODULANDO LA

COMPOSICIÓN DE LA MICROBIOTA INTESTINAL CON REPERCUSIÓN A

NIVEL TISULAR

LA MICROBIOTA INTESTINAL EJERCE UN GRAN IMPACTO SOBRE EL

ESTADO NUTRICIONAL Y LA SALUD DEL HUESPED MEDIANTE LA

MODULACIÓN DEL SISTEMA INMUNITARIO Y LAS FUNCIONES

METABÓLICAS.

EL MICROBIOMA PROPORCIONA ACTIVIDADES ENZIMÁTICAS

ADICIONALES IMPLICADAS EN LA TRANSFORMACIÓN DE LOS

COMPUESTOS DIETARIOS.

www.ellecenter.it

Esther

Nova

L.Esperanza

Díaz

Sonia

Gómez

Jorge R.

Mujico

Aurora

Hernández

Ana M.

Veses

Belén

Zapatera

Alina

Gheorghe Noemí

Redondo

Ascensión Marcos

Julia Wärnberg Fátima Pérez de Heredia Tamara Pozo

David Martínez-Gómez