Dietary fat and the human gut microbiome

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feeding rather than genetically induced obesity (Murphy et al., 2010; Liu et ..... the microbial ecosystem within the changing contexts of the human host (Ley ...... [411] Zoetendal, E. G., A. von Wright, T. Vilpponen-Salmela, K. Ben-Amor, A. D. L..
Overall,
the
results
of
this
doctoral
research
demonstrate
that
an
increased
 delivery
of
fat
to
the
colon
may
significantly
impact
health‐related
microbial
 processes.
These
novel
findings
underpin
the
need
for
further
in vivo
research
 concerning
the
impact
of
colonic
fat
for
human
health.


Dietary fat and the human gut microbiome

The
aim
of
this
doctoral
work
was
to
inves4gate
the
effect
of
an
increased
level
 of
fat
in
the
Western
diet
on
the
composi4on
and
metabolic
ac4vity
of
the
 colon
microbiota.
Specific
interest
thereby
went
to
two
‘fa?y’
compounds:
 glycerol
and
the
omega‐6
polyunsaturated
fa?y
acid
linoleic
acid. In vitro
 experiments
were
performed
with
various
models
of
the
human
gut
 microbiota.
In
addi4on,
a
model
of
the
colon
epithelium
was
used
to
study
the
 effect
of
glycerol
fermenta4on
on
infec4on
by
Salmonella.
Using
these
models,
 it
was
demonstrated
that
glycerol
and
linoleic
acid
may
significantly
impact
 microbial
processes
and
species
that
are
associated
with
human
health.
 Glycerol
fermenta4on
was
found
to
protect
against
pathogenic
infec4on,
while
 high
levels
of
linoleic
acid
were
considered
a
threat
for
the
prevalence
and
 ac4vity
of
beneficial
microbes.
These
detrimental
effects
were
dependent
on
 the
presence
of
a
simulated
mucus
layer.



ir. Rosemarie De Weirdt

2013

ISBN 978-90-5989-586-7

Dietary fat and the human gut microbiome

ir. Rosemarie De Weirdt

Promoters Em. Prof. Dr. ir. Willy Verstraete Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Prof. Dr. ir. Tom Van de Wiele Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

Members of the examination committee Prof. Dr. ir. Frank Devlieghere (voorzitter) Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Prof. Dr. ir. Veerle Fievez (secretaris) Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Prof. Dr. Stefan Roos Department of Microbiology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden Prof. Dr. Geert Huys Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. Martine De Vos Department of Internal Medicine, University Hospital Ghent, Ghent, Belgium

Dean Prof. Dr. ir. Guido Van Huylenbroeck

Rector Prof. Dr. Paul Van Cauwenberghe 


ir. Rosemarie De Weirdt

Dietary fat and the human gut microbiome

Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences 


Titel van het doctoraat in het Nederlands: Voedingsvet en het humaan darmmicrobioom

Cover illustration: Schematic representation of dietary triglycerides (glycerol + linoleic acid) reaching the gut microbiota in the lumen and the mucosa of the colon.

De Weirdt, R. (2013). Dietary fat and the human gut microbiome. PhD thesis, Ghent University, Belgium.

ISBN 978-90-5989-586-7

This work was supported by a Special Research Fund (BOF) of Ghent University. The author and the promoters give the authorisation to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. 


Notation Index

Notation Index ALA

alpha-linolenic acid

BATH

bacterial adhesion to hydrocarbons

CFU

colony-forming unit

CLA

conjugated linoleic acid

CR

colonization resistance

CVD

cardiovascular disease

DGGE

denaturing gradient gel electrophoresis

ESS

evolutionary stable strategy

FAME

fatty acid methyl esters

FISH

fluorescent in situ hybridization

HDL/LDL

high-/low-density lipoprotein

HFA

hydroxy-fatty acid

HMI

host-microbe interaction

HPA

hydroxypropanal

HPLC

high-performance liquid chromatography

IBD

inflammatory bowel disease

IW

intestinal water

LA

linoleic acid

LAB

lactic acid bacteria

LC/IRMS

liquid chromatography/isotope-ratio mass spectrometry

LCFA

long-chain fatty acid

LPS

lipopolysaccharide

LTA

lipoteichoic acid

MAMC

mucosa-associated microbial community

MAMP/PAMP

microbe-/pathogen-associated molecular pattern

MCFA

medium-chain fatty acid

MIC/MBC

minimal inhibitory/bactericidal concentration

MOI

multiplicity of infection

MucBP

mucin-binding protein

MUFA/PUFA

mono/polyunsaturated fatty acid

NLR

nucleotide-binding oligomerisation domain-like receptor

NOD

nucleotide-binding oligomerisation domain



i


Notation Index (D)PBS

(Dulbecco's) phosphate buffered saline

PCA

principal component analysis

(q)PCR

(quantitative) polymerase chain reaction

PDO

propanediol

PI

propidium iodide

PRR

pathogen recognition receptor

PSA

polysaccharide A

RA

rumenic acid

RWV

rotating wall vessel

SA

stearic acid

SCFA

short-chain fatty acid

SFA

saturated fatty acid

(L-/M-)SHIME

luminal/mucosal simulator of the human intestinal microbial ecosystem

SPI

Salmonella pathogenicity island

T3SS

type three secretion system

TFA

trans unsaturated fatty acid

Th1/2

T-helper cell type 1/2

TLR

Toll-like receptor

UC

ulcerative colitis

VA

vaccenic acid

VBNC

viable but nonculturable



ii


Table of contents

Table of contents Chapter 1 Introduction

1

1 Disturbance of gut microbiome homeostasis by diet

1

2 Dietary fat

7

2.1 Introduction

7

2.2 Glycerol

9

2.3 Western-style dietary lipid consumption

10

2.4 Fat losses to the colon

13

3 The human microbial ecosystem

14

4 Homeostasis of the gut microbiome

17

5 Gut microbiome homeostasis and human health

18

5.1 Colonization resistance

18

5.2 Bacterial fermentation products

19

5.3 Microbial interactions with the human immune system

21

5.4 Managing the human gut microbiome

22

5.4.1 Pre- and probiotics

22

5.4.2 Lactic acid bacteria

23

5.4.3 Lactobacillus reuteri

24

6 The gut microbiota and fat

27

6.1 Anaerobic glycerol metabolism by the colon microbiota

27

6.2 LCFA and antimicrobial effects

31

6.3 PUFA biohydrogenation

33

7 Objectives

37

Chapter 2 Human faecal microbiota display variable patterns of glycerol metabolism

39

1 Introduction

40

2 Materials and methods

42

2.1 Chemicals

42

2.2 Donors and sample preparation

42

2.3 Incubations of faecal samples

42


 iii


Table of contents 2.4 Incubations of in vitro cultivated colon suspension

43

2.5 Chemical analyses

43

2.6 Microbial community analysis

44

2.7 Statistical tools

45

3 Results

45

3.1 Interindividual variability

45

3.2 Temporal stability

48

3.3 Linear regression analysis

49

3.4 Microbial community analysis

50

3.5 Stable isotope enrichment

51

4 Discussion

53

5 Conclusions

56

6 Acknowledgements

56

Chapter 3 Glycerol enhances Lactobacillus reuteri’s protective effect against Salmonella Typhimurium colonization in a 3-D model of the colon epithelium 57 1 Introduction

58

2 Materials and methods

61

2.1 Bacterial strains, media and growth conditions

61

2.2 Reuterin production and quantification of glycerol metabolites

64

2.3 Growth inhibition of Salmonella Typhimurium by 3-HPA and supernatant from the Lactobacillus reuteri ferments

64

2.4 3-D model of colonic epithelium

65

2.5 Adherence and invasion, intracellular survival and intracellular growth assays

65

2.6 Statistical tools

66

3 Results

67

3.1 Characterization of the supernatant from the Lactobacillus reuteri ferments

67

3.2 Growth effects of supernatant from the Lactobacillus reuteri ferments and pure 3-HPA on a Salmonella Typhimurium population

67


 iv


Table of contents 3.3 Infection of 3-D intestinal cells with Salmonella Typhimurium in the presence of supernatant from the Lactobacillus reuteri ferments or pure 3-HPA (approach 1)

69

3.4 Infection of 3-D intestinal cells with Salmonella Typhimurium in the presence of an established Lactobacillus reuteri population (approach 2)

71

3.5 3-D HT-29 aggregate morphology and cell viability

72

4 Discussion

74

5 Acknowledgements

78

Chapter 4 A simulated mucus layer protects Lactobacillus reuteri from the inhibitory effects of linoleic acid

79

1 Introduction

80

2 Materials and methods

81

2.1 Chemicals and preparation of intestinal water

81

2.2. Mucin characterization and quantification

82

2.3 Preparation of Lactobacillus reuteri suspensions

82

2.4 Growth and survival assays

83

2.5 Microtiter plate mucin(-agar) adhesion assay

83

2.6 BATH assay: measuring bacterial hydrophobicity

84

2.7 Dynamic gut model (SHIME)

84

2.8 Molecular analysis

85

2.9 Statistical tools

86

3 Results

86

3.1 LA rapidly decreased Lactobacillus reuteri survival

86

3.2 Mucin protected Lactobacillus reuteri against LA inhibition

87

3.3 Mucin sugar monomers did not protect against LA inhibition

89

3.4 Mucin protected the bacterial cell membrane against disruption by LA and daptomycin

90

3.5 Lactobacillus reuteri was protected against LA by specifically colonizing the mucin-agar compartment of the M-SHIME

92

4 Discussion

93

5 Acknowledgements

96


 v


Table of contents

Chapter 5 Inefficient linoleic acid biohydrogenation impairs butyrate production in the simulated gut microbiome

97

1 Introduction

98

2 Materials and methods

99

2.1 Chemicals and preparation of nutritional media

99

2.2 Donors and faecal sample preparations

100

2.3 Batch incubations

100

2.4 Dynamic gut model (SHIME)

101

2.5 Chemical analyses

101

2.6 Molecular analyses

103

2.7 Statistical tools

104

3 Results

105

3.1 Batch incubations – Butyrate production was positively correlated with the level of LA biohydrogenation

105

3.2 SHIME – Incorporation of a mucin-agar environment supported growth of butyrate-producing Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii

107

3.3 SHIME – Incorporation of a mucin-agar environment warranted LA biohydrogenation and butyrate production

109

3.4 SHIME – The mucin-agar layer fortified Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii in the presence of LA

111

4 Discussion

113

5 Acknowledgements

115

Chapter 6 Discussion

117

1 Positioning of this research

117

2 Main research outcomes

120

3 General discussion and future perspectives

123

3.1 Glycerol fermentation by the colon microbiota

123

3.2 LA biohydrogenation and butyrate production

126


 vi


Table of contents 3.3 The importance of the mucus layer for Lactobacillus reuteri and Roseburia spp. to escape LA stress

131

3.4 Modelling gut microbial processes and host-microbe interactions

132

4 Conclusions

141

Summary

143

Samenvatting

146

Bibliography

149

Curriculum Vitae

177

Dankwoord

181


 vii


Chapter 1: Introduction

Chapter 1: Introduction 1 Disturbance of gut microbiome homeostasis by diet The human gut microbiota can be viewed as a structured, microbial community that operates like a microbial organ within the human host (see Chapter 1.3 and Chapter 1.4). While this organ provides us with functional features - the gut microbiome1 - we did not have to evolve ourselves, gut microbial homeostasis2 is considered crucial for our health (see Chapter 1.5). However, aspects of modern life may influence the ancient human host-microbe cooperation in ways that are unprecedented in our co-evolutionary history. These factors include urbanization, rapid global mobility, the consumption of manipulated and processed foods, reduced physical activity, improved sanitation and hygiene, and medical therapies (Bengmark, 1998; Dethlefsen et al., 2007). Moreover, there is a vast amount of literature reporting that modern life makes us more prone to certain diseases, such as allergy, inflammation diseases, cardiovascular diseases, cancer, diabetes and obesity (Björkstén, 1994; Lindeberg, 1994; Martinez, 2005; Gilbert & Khokhar, 2008; Ehlers & Kaufmann, 2010; Cheng, 2012; Garduno-Diaz & Khokhar, 2012). For this reason, these are named ‘prosperity diseases’ or ‘post-modern diseases’ (Blaser et al., 2006). Several observational studies have linked the prevalence of these diseases with the abundance of specific microbial species or phyla in our gut, while others have associated inflammatory bowel disease and obesity with a decreased gut microbial diversity – a sign of a dysfunctional ecosystem (Ott et al., 2004; Ley et al., 2005). In addition, an increasing amount of well-designed animal studies and human intervention studies have been performed in an attempt to elucidate the role of gut microbiota in the onset of these diseases. However, it remains difficult to establish causal relationships and to identify the mechanisms involved. In industrialized countries, food habits have changed so drastically that the current diet is now typified as a ‘Western-style diet’. These changes include a high intake of red meat, an 























































 1 Gut microbiome: the totality of gut microbes, their genes and interactions with the host 2 Homeostasis: property of a biological system to maintain stability while adjusting to conditions that are optimal for survival. If homeostasis is successful, life continues; if unsuccessful, disaster or death ensues. The stability attained is actually a dynamic equilibrium, in which continuous change occurs yet relatively uniform conditions prevail (after Encyclopedia Britannica online)
 


1


Chapter 1: Introduction increasing sodium consumption, a doubled consumption of fat (including cholesterol), an increased consumption of refined sugar and a much-decreased consumption of vegetable fibres, minerals, vitamins and antioxidants (Bengmark, 1998; WHO/FAO, 2002; Cordain et al., 2005). It is generally accepted that among these changes, two play a crucial role in the development of gut-associated prosperity diseases: the decrease in fibre consumption and the increased consumption of fats. These dramatic dietary changes have been evidenced to significantly affect the gut microbiota and hypothesized to disturb gut (microbial) homeostasis (O’Keefe et al., 2009; De Filippo et al., 2010). De Filippo et al. (2010) compared the faecal microbiota from European children on a Western diet and those from children from a rural African village of Burkina Faso consuming a rural diet, rich in fibre and low in animal fat and protein. African children typically harboured unique bacteria from the Prevotella and Xylanibacter group, which are known to own genes for cellulose and xylan breakdown. The European children, on the other hand, had significantly higher numbers of Enterobacteriaceae, among which the potentially pathogenic genera Shigella and Escherichia. In addition, the authors also observed significant differences in the microbial composition at the more general level of phyla (i.e. Actinobacteria, Bacteroidetes, Proteobacteria and Firmicutes) between the two groups of children. The results of two recent studies confirmed that dietary patterns may indeed correlate with the characteristic composition of the faecal microbiota. In a first study, Arumugam et al. (2011) demonstrated that the faecal metagenomes of 22 European, 13 Japanese and 4 Americans could be classified within three robust enterotypes that are mainly driven by species composition. They are referred to as the Bacteroides, Prevotella and Ruminococcus enterotype and were characterized by distinct functionalities. While these enterotypes were not nation or continent specific and did not correlate with host properties such as body mass index (BMI), age or gender, Wu et al. (2011) demonstrated their relation with dietary intake on the long-term. In this regard, consumption of protein and animal fat was associated with the Bacteroides enterotype and carbohydrate (fibre) consumption with the Prevotella enterotype. Interestingly, they found that a short-term (10 days) switch to a high-fat/low-fibre or low-fat/high-fibre diet could not alter the enterotype identity, despite its rapid (within 24h) and significant impact on the overall microbial composition. Although the phylogenetic and functional differences among the enterotypes have not yet been causally related to disease, the results of this study are crucial for our understanding of the relation between diet and hostmicrobe cooperation. 


2


Chapter 1: Introduction Other relevant data concerning the effects of a high-fat sugar-rich/fibre-depleted diet on the gut microbial community come from studies investigating the role of gut microbiota in (dietinduced) obesity. Firstly, Bäckhed et al. (2007) demonstrated that exposure of both germfree and conventional mice to a high-fat sugar-rich diet did not result in obesity in germfree mice. Hence, the presence of a gut microbiota seemed to be required in order to induce obesity by diet. Secondly, it was found that transplantation of the gut microbiota from obese, but not lean, conventional mice to lean, germ-free mice increased their body fat, without any increase in food consumption (Turnbaugh et al., 2006). Therefore, it appeared that not only the presence, but also the specific composition of the gut microbial community (in other words, the gut microbiome) plays a role in the development of obesity by affecting the amount of energy harvested from diet. Mice studies demonstrated that high-fat diet-induced obesity is associated with a decreased faecal number of Bifidobacterium spp., Lactobacillus ssp., Bacteroides-related bacteria and the Eubacterium rectale-Clostridium coccoides cluster (Cani et al., 2007a&b; Neyrinck et al., 2011&2012). In addition, supplementation of prebiotic fibres that specifically target bifidobacteria or bacteria from Clostridium cluster XIVa was shown to effectively lower high-fat diet-induced obesity (Cani et al., 2007b; Neyrinck et al., 2011&2012). Other studies using genetically and diet-induced obese mice demonstrated that the obese state is positively correlated with an unusual high level of Firmicutes and a lower level of Bacteroidetes (Ley et al., 2005; Turnbaugh et al., 2006&2008; Hildebrandt et al., 2009). These proportional changes in the Firmicutes to Bacteroidetes ratio were shown to be independent of the obese state (Hildebrandt et al., 2009) and primarily a feature of high-fat feeding rather than genetically induced obesity (Murphy et al., 2010; Liu et al., 2012). In humans, similar observations of increased Firmicutes and decreased Bacteroidetes levels were made in faeces of obese subjects (Ley et al., 2006) and lean subjects overfeeding on a high caloric diet (3400 kcal.day-1 compared to 2400 kcal.day-1) (Jumpertz et al., 2011). Others did not observe changes in the Firmicutes:Bacteroidetes ratio or even demonstrated an opposite change of the ratio in obese individuals (Collado et al., 2008; Duncan et al., 2008; Santacruz et al., 2009; Turnbaugh et al., 2009; Schwiertz et al., 2010). Turnbaugh & Gordon (2009) suggested that these discrepant findings might be due to the use of different molecular tools (16S rRNA sequencing, qPCR, FISH) and the selection of different study subjects (pigs, humans, patients of gastric bypass surgery, pregnant women). It was furthermore proposed that discrepancies might have come from differences in storage conditions and DNAextraction procedures (Maukonen et al., 2012). In addition, I would like to point out that



3


Chapter 1: Introduction different types of diets are used to study obesity, with a varying fat concentration and composition as summarized in Table 1.1. Despite the inconsistency in the data, these studies have in common that specific phyla, classes or bacterial species, or bacterial functionalities are suggested to play a role in obesity, and more importantly, that their presence or activity can be manipulated by changing diet. However, these in vivo studies did not allow studying the effect of specific dietary components (fat versus fibre) on the gut microbial composition and activity. In order to understand how the dietary effects are mediated, it is advisable to perform welldesigned in vitro studies, distinguishing microbial effects from host physiological processes.



4


Chapter 1: Introduction

Table 1.1 Dietary intervention in various animal and human studies investigating the effect of a Western-style diet on gut microbial composition in the context of obesity.



Experimental Diet set-up mice Western diet TD.96132 (Harlan Teklad, Madison, USA)

Caloric composition (% kcal) 4.49 kcal.g-1 18.7 % protein 40.7 % carbohydrate 40.6 % fat

Fat fraction

Caloric intake

mice

FAT-R diet TD.05633 (Harlan Teklad, Madison, USA)

hydrogenated vegetable shortening and beef tallow 41% SFA; 17% TFA; 35% MUFA (cis); 7% PUFA (cis)

open

Bäckhed et al., 2007; Turnbaugh et al., 2008

3.95 kcal.g-1 18.7 % protein 60.0 % carbohydrate 21.3 % fat

hydrogenated vegetable shortening and beef tallow 41% SFA; 17% TFA; 35% MUFA (cis); 7% PUFA (cis)

open

Turnbaugh et al., 2008

mice

CARB-R diet TD.05634 4.31 kcal.g-1 (Harlan Teklad, 48.3 % protein Madison, USA) 11.2 % carbohydrate 40.5 % fat

hydrogenated vegetable shortening and beef tallow 41% SFA; 17% TFA; 35% MUFA (cis); 7% PUFA (cis)

open

Turnbaugh et al., 2008

mice

high-fat diet 5.98 kcal.g-1 (*) (UAR, Epinay-sur-Orge, 27 % protein France) 1 % carbohydrate 72 % fat

corn oil (29%) and lard (71%)

open

Cani et al., 2007a&b

mice

high-fat diet D12492 (Research Diets Inc., New Jersey, USA)

soybean oil (9%) and lard (91%)

open

Neyrinck et al., 2011&2012;

5.24 kcal.g-1 20 % protein 20 % carbohydrate 60 % fat

Reference

5


Chapter 1: Introduction mice

high-fat diet D12451 (Research Diets Inc., New Jersey, USA)

4.73 kcal.g-1 20 % protein 35 % carbohydrate 45 % fat

soybean oil (12%) and lard (88%)

dietary intervention lean and obese individuals

fat-restricted (FAT-R) diet carbohydrate-restricted (CARB-R) diet

30 % fat 10-15 g.day-1 fibre 25 % carbohydrate 10-15 g.day-1 fibre

not controlled

dietary intervention lean and obese individuals

normal- and high-caloric 20 % protein diet 50 % carbohydrate 30 % fat

not controlled

2400 kcal.day-1 vs 3400 kcal.day-1

Jumpertz et al., 2011

dietary intervention obese individuals

high protein, low carbohydrate, ketogenic diet

1.31 kcal.g-1 30 % protein 4 % carbohydrate 66 % fat 1.31 kcal.g-1 30 % protein 35 % carbohydrate 35 % fat

limited control

fixed according to energy requirements

Duncan et al., 2008

20 % protein 50 % carbohydrate 30 % fat

not controlled

men: max 1800 kcal.day-1 women: 2200 kcal.day-1

not controlled

not controlled

not controlled

high protein, moderate carbohydrate, non-ketogenic diet dietary intervention obese individuals

energy-restricted diet

observational not controlled study in human subjects (*)



not controlled

open

Hildebrandt et al., 2009; Murphy et al., 2010

men: 1500-1800 Ley et al., 2006 kcal.day-1 women: 1200-1500 kcal.day-1

limited control

Santacruz et al., 2009

Ley et al., 2006; Collado et al., 2008; Turnbaugh et al., 2009; Schwiertz et al., 2010

calculated value based on weight % macronutrients (protein, carbohydrate, fat)

6


Chapter 1: Introduction

2 Dietary fat 2.1 Introduction Fat belongs to the broad group of lipids, which are defined as hydrophobic or amphipathic3 molecules and are divided into eight categories, depending on their biochemical structure: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterols and prenols (Fahy et al., 2011). In the human body (and elsewhere), glycerophospholipids are omnipresent as double layers that form the membranes of both eukaryotic and microbial cells. Human dietary lipids, on the other hand, consist for 90% (or more) of triglycerides and smaller amounts of phospholipids, saccharolipids, sterols (cholesterol) and fat-soluble vitamins A, D, E and K (Mu & Høy, 2004; Binder & Reuben, 2009). Triglycerides are tri-substituted esters of a glycerol molecule with 3 fatty acids and group within the category of glycerolipids (Fig. 1.1). They are referred to as fats and oils, depending on their fluidity at room temperature, which in its turn is determined by the linearity of the fatty acids that are esterified on the glycerol backbone molecule.

Figure 1.1 Chemical structure of glycerol (left) and a saturated triglyceride (right) Fatty acids are characterized by their chain length and the presence of branches and/or double bonds. Depending on the number of carbon atoms, they are indicated as short-chain (C3-C7; SCFA), medium-chain (C8-C13; MCFA) or long-chain (>C14; LCFA) fatty acids. The presence of double bonds further distinguishes saturated from mono- and polyunsaturated 























































 3 amphipathic: containing both a hydrophobic and a hydrophilic end 


7


Chapter 1: Introduction fatty acids, respectively abbreviated as SFA, MUFA and PUFA (Fig. 1.2). In contrast to the single C-C bonds in SFA, the double C-C bonds in MUFA and PUFA cannot rotate freely, which results in a fixed configuration of the chains at each side of the double bond. When the chains are bended towards each other, the configuration is indicated as cis, or Z(usammen) (Fig. 1.2). If the chains are bended opposite from the double bond, this is indicated as trans, or E(ntgegen) (Fig. 1.2). PUFA containing at least one pair of double bonds separated by only one single bond are indicated as conjugated fatty acids (Fig. 1.2). OH

saturated stearic acid

O

OH O

mono-unsaturated vaccenic acid (trans or E-configuration)

mono-unsaturated oleic acid (cis or Z-configuration) OH O

OH O

poly-unsaturated rumenic acid (conjugated configuration)

Figure 1.2 Chemical structures of the C18 fatty acids stearic acid, vaccenic acid, oleic acid and rumenic acid In this work, fatty acids are noted as Cn:x, with n being the number of carbon atoms and x being the amount of double bonds. The position of the double bonds is indicated according to the International Union of Pure and Applied Chemistry (IUPAC) by adding in suffix the symbol Δ and the position of the first carbon atom of the double bond counted from the carboxyl carbon atom (C1) (Fig. 1.3). Then, the configuration of the double bond is indicated



8


Chapter 1: Introduction with c(is) or t(rans). MUFA and PUFA can furthermore be indicated as omega-x fatty acids, for which x is the position of the first double bond, now counted from the last methyl carbon atom. According to this method, one can distinguish omega-3 (ω-3 or n-3) from omega-6 (ω-6 or n-6) fatty acids, which have their first double bond starting on the third and sixth carbon atom respectively (Fig. 1.3).

!6" !"

C9 C1 Figure 1.3 Chemical structure and nomenclature of linoleic acid according to IUPAC (C18:2Δ9c,12c) and ‘the omega-method’ (C18:2 ω-6)

2.2 Glycerol Glycerol is a water-soluble polyalcohol, which consists of three carbon atoms that are covalently bound to an OH-group (Fig. 1.1). In the human body, it plays a fundamental role in several vital physiological processes and is an important intermediate of energy metabolism (reviewed by Brisson et al., 2001). In the liver, glycerol can be used as precursor for glucose biosynthesis, a process indicated as gluconeogenesis. Under normal conditions of health and diet, this process accounts for less than 5% of the glucose production. However, under conditions of starvation, its share may increase to 20% or more (Bortz et al., 1972; Baba et al., 1995). In addition, obese subjects were observed to have higher glycerol blood levels, increased turnover rates and a higher contribution to glucose formation than lean subjects (Bortz et al., 1972). Hence, it seems probable that homeostasis of glycerol levels is essential for good health. (Dietary) glycerol sources are numerous and involve (1) its direct consumption as an additive (E422) in numerous pharmaceutical and food products, (2) its production from glucose, proteins, pyruvate… in eukaryotic and microbial glycerolipid metabolic pathways, and (3) its release from dietary triglycerides.



9


Chapter 1: Introduction

2.3 Western-style dietary lipid consumption Lipids (triglycerides), together with carbohydrates and proteins, are one of the three energy substrates available in diet. They contain more than double the amount of energy (9 kcal.g-1) when compared to the other two substrates (4 kcal.g-1) (Merrill & Watt, 1955). When absorbed into the blood stream, fat is transported to the liver (hepatocytes), the fat tissue (adipocytes) or muscle fibres, where it can be either stored or immediately oxidized to acetylCoA for energy delivery. In addition, fats play a role in maintaining human health, e.g. by delivering essential fatty acids4 and fat-soluble vitamins to the body cells, protecting the body organs against (temperature) shocks, encapsulating harmful substances, etc. The daily consumption of triglycerides is recommended to take up 20-35% of one’s total energy requirement (FAO/WHO, 2008). For a reference diet of 2500 kcal.day-1, this is equal to a triglyceride consumption of 56-97 g.day-1. However, the Western-style diet contains approximately twice as much triglycerides as recommended. In 2007, the daily triglyceride consumption in Western Europe and the USA was 145 g per capita and 161 g per capita respectively (FAOSTAT, 2010; Fig. 1.4). recommended daily fat intake World USA Western Europe

Figure 1.4 Daily fat intakes (in gram per person) in 2007 for the World, the United States of America (USA) and several countries of Western Europe according to FAOSTAT (2010). The recommended daily fat intake lies between 45 and 97 g.day-1, as calculated for reference diets of 2000 kcal and 2500 kcal consisting of 20 energy% fat and 35 energy% fat, respectively.

UK Switzerland Sweden Spain Portugal Norway Netherlands Malta Luxembourg Italy Ireland Iceland Greece Germany France Finland Denmark Belgium Austria 0

20

40

60

80

100

120

140

160

180

Actual daily fat consumption (g per person)

























































 4 Essential fatty acids: PUFA that are required for good health but cannot be synthesized by the body as humans lack the desaturase enzymes required for their production. Only two PUFA are truly essential: n-6 linoleic acid (C18:2Δ9c,12c) and n-3 alpha-linolenic acid (C18:3Δ9c,12c,15c). 


10


Chapter 1: Introduction The Western dietary triglyceride fraction furthermore deviates from the acceptable macronutrient distribution range depicted in Table 1.2. Firstly, it typically contains high levels of SFA from meat and milk products of which palmitic acid (C16:0) is the most abundant, followed by stearic acid (C18:0) and myristic acid (C14:0) (Allison et al., 1999; Chong et al., 2006). Intake of SFA (C12:0, C14:0 and C16:0) was shown to increase lowdensity lipoprotein (LDL) cholesterol and total cholesterol concentrations (Clarke et al., 1997) and has therefore long been associated with an increased risk on cardiovascular diseases (CVD). However, this relation has been contradicted by several studies and is therefore not recognized by the Joint FAO/WHO Expert Panel (2008). Yet, this panel recommends to replace SFA with PUFA in order to decrease the risk on CVD, and the total SFA intake should not exceed 10 energy% (FAO/WHO, 2008). In 1998, average SFA consumption was estimated to constitute 13 energy% of the dietary intake in the USA (Allison et al., 1999) and 11-16 energy% in Western Europe (Lloyd-Williams et al., 2008). Table 1.2 Acceptable macronutrient distribution ranges as energy percentage of dietary intake (FAO/WHO, 2008). SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids, LA: linoleic acid, TFA: trans-unsaturated fatty acids Total triglycerides SFA MUFA Total PUFA n-6 PUFA (LA) n-3 PUFA TFA

Energy % 20-35 < 10 6-11 2.5-9 0.5-2 95%) and D-mannose (>95%) were purchased from 


81


Chapter 4: Mucin protects Lactobacillus reuteri against LA  Sigma (Bornem, Belgium). Daptomycin (purity >98%) was derived from Biotang Inc. (Waltham, USA). Intestinal water (IW) was prepared by harvesting suspension from the colon ascendens (pH 5.6-5.9) of the simulator of the human intestinal microbial ecosystem (SHIME®; ProDigestGhent University, Ghent, Belgium; Molly et al., 1993; Van den Abbeele et al., 2010) followed by centrifugation (10 min, 1500 rcf) and autoclavation of the supernatant. A 0.1 M phosphate buffered saline (PBS) was prepared by mixing 0.1 M K2HPO4 and 0.1 M KH2PO4 to obtain pH 5.9. Anaerobic PBS (0.1 M) contained 1.0 g.L-1 sodium thioglycolate.

2.2 Mucin characterization and quantification Porcine mucin type II (lot n° 100M0187V) was analysed for its protein and total sugar contents according to Damen et al. (2011). Fatty acids (0.05) and total SCFA production (-3.2 ± 3.5 mM; p>0.05). Interestingly, butyrate, acetate and total SCFA production correlated with relative LA levels at 48h, which is a measure of LA biohydrogenation (Fig. 5.1). More specifically, conversion of LA to VA and SA correlated with higher levels of butyrate, acetate and total SCFA. Propionate production was not correlated with the LA levels.

LA after 48h (g/100 g LA+CLA+VA+SA)

total SCFA

acetate

100

100

75

75

50

50

25

25

R2 = 0.781 slope = -1.7 p < 0.001

R2 = 0.647 slope = -6.2 p = 0.002

0

0 0

50

100

0

150

LA after 48h (g/100 g LA+CLA+VA+SA)

propionate

20

40

60

80

butyrate

100

100

75

75

50

FS11 herh FS22 herh

R2 = 0.825 slope = -3.5 p < 0.001

50

herh FS33 herh FS44

25

FS55 herh

25

R2 = 0.297 slope = 4.4 p = 0.125

0

0 0

10

20

30

40

0

10

20

30

40

Absolute SCFA production (mM)

Figure 5.1 Inefficient LA biohydrogenation correlated with lower acetate, butyrate and total SCFA production. LA concentration in function of the absolute SCFA production after 48h incubation of faecal slurries supplemented with 1.0 g.L-1 LA. Each datapoint represents a technical replicate within a total of five independent experiments (Faecal Sample 1-5)



105


Chapter 5: Linoleic acid impairs butyrate production The relation between LA biohydrogenation and butyrate production was investigated in greater detail by incubating the faecal slurries in the presence of LA or its biohydrogenation products VA or SA (Fig. 5.2). In contrast to LA, its more saturated derivatives (VA and SA) had no inhibitory effect on the butyrate production. Also the propionate levels remained unaffected by VA and SA. For acetate and total SCFA, differences indicated as significant were negligible (maximal decrease of 3.3 ± 1.7 mM SCFA observed for total SCFA upon VA supplementation). No significant relation was found between the supplementation of LA, VA or SA and total gas production or hydrogen partial pressure in the headspace. In general, 48h incubations with LA, VA and SA resulted in a total gas production of 39.7 ± 4.9 kPa and

Relative net SCFA production after 48h (mM treatment – mM control)/mM control

hydrogen partial pressures of 11.0 ± 1.5%.

1,0

0,6

* 0,2

LA b-glucan + LA oat

* *

VA b-glucan + VA oat

*

-0,2

SA b-glucan + SA oat

-0,6

* -1,0

total SCFA SCFA-net

acetate AC-net

propionate PROP-net

butyrate

BUT-net

Figure 5.2 Addition of LA stimulated propionate production and decreased butyrate production, while VA and SA had no effect on the SCFA profile. Relative difference in SCFA production from faecal slurries incubated during 48h with 1 g.L-1 LA, VA or SA, compared to the control. Averages are based on the replicates of different faecal slurries and error bars represent standard errors. Values significantly different from 0 are indicated as * (p < 0.05).

For all batch incubations, the nutritional medium was buffered and the pH dropped from 6.9 ± 0.1 to 6.2 ± 0.1 after 48h of incubation.



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Chapter 5: Linoleic acid impairs butyrate production

3.2 SHIME – Incorporation of a mucin-agar environment supported growth of butyrate-producing Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii A simplified, dynamic model mimicking colon ascendens microbial fermentation processes was run two times during 48h and exposed to 0, 0.1 or 1.0 g.L-1 LA. The model was applied in parallel without (L-SHIME) and with a mucin-agar compartment (M-SHIME), which was simulated by the incorporation of mucin-agar coated microcosms. The presence of such a mucin-agar compartment within the M-SHIME model was previously shown to increase the colonization by butyrate-producing species (Van den Abbeele et al., 2012a), when compared to the L-SHIME model (Van den Abbeele et al., 2010). More specifically, the mucin-agar was demonstrated to be significantly enriched in clostridia belonging to cluster XIVa (59% of total bacteria) and IV (19% of total bacteria), with Roseburia intestinalis (7% of total bacteria) and Eubacterium rectale (9% of total bacteria) being the most dominant species present (Van den Abbeele et al., 2012a). The results of the present study confirmed the importance of the mucin-agar environment for butyrate-producing Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii species. Firstly, both groups were equally or more abundant in the lumen of M-SHIME compartments compared to the lumen of L-SHIME compartments (Table 5.2). Secondly, Eubacterium rectale/Roseburia spp. more specifically colonized the mucin-agar compartment of the M-SHIME as opposed to Faecalibacterium prausnitzii. In general, the mucin-agar layer harboured 1.0 ± 0.2 log unit (copies.mL-1) more Eubacterium rectale/Roseburia spp. than the M-SHIME lumen. In contrast, Faecalibacterium prausnitzii counts were 1.1 ± 0.6 log units (copies.mL-1) higher in the M-SHIME lumen than in the mucin-agar. Finally, the mucin-agar compartment counted 2.8 ± 0.3 log units (copies.mL-1) more Eubacterium rectale/Roseburia spp. than Faecalibacterium prausnitzii, supporting the previously reported preferential stimulation of clostridial cluster XIVa species.



107


Chapter 5: Linoleic acid impairs butyrate production Table 5.2 The mucin-agar compartment of the M-SHIME protects butyrate-producing Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii against a daily dose of 1.0 g.L-1 LA. Log units of total Bacteria, Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii qPCR counts (copies.mL-1) in the lumen and mucin-agar of L- and M-SHIME vessels after 2 days and a daily exposure to 0, 0.1 or 1.0 g.L-1 LA. On day 0, averages are shown for the lumen of the different L- and M-SHIME vessels and the mucin-agar was not sampled (nd). Values are averages of technical replicates ± standard deviations for 2 independent SHIME-runs inoculated with faecal slurries from the same donor. Light and dark grey values deviate from the 0 g.L-1 LA control with more than half a log and one log unit, respectively.

Total Bacteria

Eubacterium rectale/Roseburia spp.

Faecalibacterium prausnitzii



SHIME-run 1 lumen lumen mucus L-SHIME M-SHIME M-SHIME

SHIME-run 2 lumen lumen mucus L-SHIME M-SHIME M-SHIME

day

LA (g.L-1)

0

average

2

0

8.55 ± 0.30

8.34 ± 0.15

8.81 ± 0.09

8.60 ± 0.00

8.44 ± 0.03

7.97 ± 0.07

2

0.1

8.29 ± 0.10

8.36 ± 0.17

8.83 ± 0.05

8.55 ± 0.02

8.52 ± 0.04

7.83 ± 0.04

2

1.0

8.38 ± 0.16

8.18 ± 0.16

8.56 ± 0.05

7.53 ± 0.09

8.05 ± 0.04

8.51 ± 0.06

0

average

2

0

6.91 ± 0.16

7.47 ± 0.35

8.59 ± 0.08

5.15 ± 0.02

6.49 ± 0.04

7.08 ± 0.02

2

0.1

7.14 ± 0.00

7.44 ± 0.09

8.37 ± 0.08

5.83 ± 0.06

6.52 ± 0.08

7.43 ± 0.12

2

1.0

6.20 ± 0.03

7.13 ± 0.04

8.20 ± 0.09

4.93 ± 0.09

6.78 ± 0.08

8.02 ± 0.00

0

average

2

0

6.44 ± 0.07

6.96 ± 0.22

5.50 ± 0.01

5.43 ± 0.04

5.43 ± 0.03

4.56 ± 0.07

2

0.1

6.40 ± 0.09

6.68 ± 0.03

5.35 ± 0.03

6.09 ± 0.23

6.48 ± 0.04

4.55 ± 0.04

2

1.0

5.38 ± 0.01

6.12 ± 0.14

5.25 ± 0.05

4.23 ± 0.01

5.97 ± 0.12

5.63 ± 0.04

8.47 ± 0.39

8.62 ± 0.14

6.55 ± 0.29

nd

nd

nd

8.80 ± 0.16

8.59 ± 0.09

6.29 ± 0.17

nd

nd

nd

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Chapter 5: Linoleic acid impairs butyrate production

3.3 SHIME – Incorporation of a mucin-agar environment warranted LA biohydrogenation and butyrate production Daily supplementation of 1.0 g.L-1 LA to the L- and M-SHIME was shown to affect butyrate production in relation to the efficiency of LA biohydrogenation (Fig. 5.3, Table 5.3). In the LSHIME vessels, LA biohydrogenation was limited and butyrate production was decreased in SHIME-run 1 or unaffected in SHIME-run 2. Incorporation of a mucin-agar compartment in the M-SHIME allowed for a remarkably better biohydrogenation of LA, which was highest in SHIME-run 2. The main biohydrogenation product was VA. In SHIME-run 1, no SA was formed, while in SHIME-run 2 limited biohydrogenation to SA was observed upon incorporation of the mucin-agar environment (± 8 g SA/100 g LA+CLA+VA+SA). This coincided with restored levels of butyrate production in SHIME-run 1, while in SHIME-run 2 butyrate production was stimulated above the levels found in the control vessel without LA. SHIME-run 1

SHIME-run 2

LA consumption after 24h (g/100 g LA+CLA+VA+SA)

A1

A2

10

10

0

0

-10

-10

-20

-20

-30

-30

-40

-40

-50

0

0

0.1

1.0

0,1

-50

1

L-SHIME M-SHIME

0

Net SCFA production (mM 1 g.L-1 LA – mM control)

LA supplemented (g.L-1)

20

B1

20 15

10

10

5

5

0

0

-5

-5 total SCFA net SCFA

acetate net AC

propionate net PROP

butyrate net BUT

0,1

1.0 1

LA supplemented (g.L-1)

15

-10

0.1

0

-10

B2

total SCFA net SCFA

acetate net AC

propionate net PROP

butyrate net BUT

Figure 5.3 The incorporation of a mucin-agar layer in the M-SHIME stimulated LA biohydrogenation and butyrate production upon daily exposure to 1 g.L-1 LA. (A) LA consumption during the first 24h in L- and M-SHIME vessels exposed to 0, 0.1 or 1.0 g.L-1 LA. (B) Net SCFA production on day 2 in L- and M-SHIME vessels exposed to 1.0 g.L-1 LA, when compared to the untreated control (0 g.L-1 LA). 


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Chapter 5: Linoleic acid impairs butyrate production

Table 5.3 Absolute SCFA production over time in the lumen of L- and M-SHIME vessels exposed to 0, 0.1 or 1.0 g.L-1 LA. Values on day 0 are averages ± standard deviations. Light and dark grey values deviate from the 0 g.L-1 LA control with more than 25% and 50%, respectively (based on relative net SCFA-production).

LA g.L-1 L-SHIME 1

44.3

37.5

1.0

43.6

0

day0

day1

Butyrate (mM)

day2

5.8

6.5

7.3

6.8

37.4

8.6

42.0

34.3

33.2

25.7

1.0

35.8

0

day0

day1

Total SCFA (mM)

day2

16.1

19.3

19.3

18.2

12.1

17.2

6.7

6.1

4.7

4.1

44.8

6.4

39.8

38.1

45.2

35.3

1.0

61.8

0 0.1 1.0



day2 40.7

0.1 M-SHIME 2

day1

Propionate (mM)

41.6

0.1 L-SHIME 2

day0

0 0.1

M-SHIME 1

Acetate (mM)

2.0±0.3

2.0±0.2

3.1±0.4

3.0±0.8

day0

day1

day2

64.9

67.9

72.5

64.0

11.4

70.2

61.5

16.6

16.2

67.0

58.1

13.7

11.8

53.0

42.6

8.2

15.3

16.4

58.9

70.6

2.6

7.0

2.6

8.8

45.1

53.9

2.9

4.1

14.5

16.1

62.6

55.6

36.6

2.6

7.0

11.9

7.8

76.2

51.4

40.0

36.9

2.5

3.2

11.5

13.2

54.0

53.3

44.3

40.2

2.6

4.6

11.4

14.2

58.3

59.0

63.8

40.1

6.8

8.3

17.0

17.8

87.5

66.2

0.5±0.1

0.5±0.01

0.3±0.2

0.3±0.3

0.1±0.1

0.1±0.1

0.5±0.1

0.4±0.1

2.5±0.5

2.6±0.2

3.8±0.5

3.7±1.0

110


Chapter 5: Linoleic acid impairs butyrate production In general, the SCFA profiles demonstrated that the bacterial community of SHIME-run 1 was more sensitive to 1.0 g.L-1 LA supplementation than that of SHIME-run 2 (Fig. 5.3B, Table 5.3). Moreover, the presence of a mucin-agar layer in SHIME-run 1 mostly stimulated acetate production, while in SHIME-run 2 higher total SCFA production was the result of a stimulated acetate, propionate and butyrate production. Daily supplementation of 0.1 g.L-1 LA resulted in higher levels of SCFA production in the L-SHIME vessels when compared to the M-SHIME vessels (Table 5.3). In SHIME-run 1, this was due to a decreased SCFA production in the M-SHIME vessels, while in SHIME-run 2 LA addition stimulated butyrate production in the L-SHIME vessels.

3.4 SHIME – The mucin-agar layer fortified Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii in the presence of high levels of LA In concordance with the butyrate production levels, exposure to 1.0 g.L-1 LA resulted in decreased Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii communities on day 2 in the lumen of both L-SHIME’s (Table 5.2). In contrast, both butyrate-producing communities were better able to cope with the daily addition of 1.0 g.L-1 LA in the M-SHIME vessels (Table 5.2). For both SHIME-runs, the presence of mucin-agar eliminated or decreased the inhibitory effect of 1.0 g.L-1 LA on both communities in the lumen. There, addition of 1.0 g.L-1 LA did not affect both communities in SHIME-run 1, and in the case of SHIME-run 2, it even stimulated them with 1 log unit (copies.mL-1) when compared to the control. Exposure to 0.1 g.L-1 LA did not affect the butyrate-producers in SHIME-run 1. However, in SHIME-run 2, this concentration stimulated growth of both communities in the lumen of the L-SHIME and growth of Faecalibacterium prausnitzii in the lumen of the MSHIME. The PCA of both metabolic data and qPCR data confirmed a clear difference between the Land M-SHIME vessels exposed to 1.0 g.L-1 LA and the other vessels exposed to 0 or 0.1 g.L-1 LA (Fig. 5.4). Moreover, it demonstrated that the observed effects of a daily exposure to 1.0 g.L-1 LA on the butyrate production process, LA biohydrogenation and the Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii communities were significantly different between L- and M-SHIME vessels (Fig. 5.4).



111


Chapter 5: Linoleic acid impairs butyrate production

SHIME-run 2

2

2

VA 1 acetate SCFA

1

propionate, CLA SA

0 -2

-1

0

1

2

Roseburia ssp. butyrate, F. prausnitzii -1

3

29.5 % of variability

39.5 % of variability

SHIME-run 1

propionate SA acetate SCFA

CLA 0 -3

-2

-1

0

1

LA -1

Bacteria

2

Roseburia ssp. butyrate, F. prausnitzii Bacteria, VA

-2

-2

60.3% of variability

65.4% of variability

-1 L-SHIME 00 g.L L-SHIME g/L LA

L-SHIME 0.1 L-SHIME 0.1g.L g/LLA

L-SHIME 1.0 L-SHIME 1.0g.L g/LLA

M-SHIME 00g.L M-SHIME g/L-1 LA

-1 LA M-SHIME g.Lg/L M-SHIME0.1 0.1

-1 LA M-SHIME 1.0 M-SHIME 1.0g.L g/L

-1

-1

variables objects

Figure 5.4 Addition of 1 g.L-1 LA significantly affected colon ascendens microbial processes and its effects were significantly different in L- and M-SHIME vessels. PCA incorporating total Bacteria, Eubacterium rectale/Roseburia spp. and Faecalibacterium prausnitzii qPCR counts (copies.mL-1, day 2), SCFA production (mM, day 2) and LA, VA, SA and CLA levels (g/100 g LA+CLA+VA+SA, day 1) for L- and M-SHIME vessels of SHIME-run 1 and 2 exposed to 0, 0.1 or 1.0 g.L-1 LA.



112


Chapter 5: Linoleic acid impairs butyrate production

4 Discussion The relation between LA biohydrogenation to VA and SA and the butyrate production process was established by repeated batch incubation of a faecal microbiome. This microbiome was sampled from the same individual at five different moments within a relatively short period of time (2 months). Interestingly, a high variation in butyrate production and LA biohydrogenation activity was observed between the different faecal inocula (Fig. 5.1). While stool microbiomes of the same individual were found to be robust to perturbation and generally stable over time (Costello et al., 2009, Parfrey & Knight, 2012), it is speculated that the observed variation was not due to significant differences in the composition of the microbiome but rather originated in its altered activity. More specifically, butyrate-producing, biohydrogenating species may have been affected by various environmental parameters. Thereby, hydrogen partial pressure should not have played a role, because 48h of incubation consistently resulted in a hydrogen partial pressure of 11.0 ± 1.5% for all conditions and inocula. When considering the effect of pH in the batch incubations, it should be mentioned that pH was not optimal for butyrate-producing species (6.9 ± 0.1 at 0h and 6.2 ± 0.1 after 48h). Walker et al. (2005) demonstrated that butyrate production by faecal microbiomes significantly increased at an initial pH of 5.5 when compared to a pH of 6.5. Later, it was indicated that the success of butyrate producers at pH 5.5 was mainly due to the inhibition of Gram-negative bacteria (Bacteroides ssp.) under mild acidic conditions, which created niches for low pH-tolerant microorganisms (Duncan et al., 2009). Hence, in this study an initial pH of 6.9 ± 0.1 may have been a stress factor for butyrate-producing, biohydrogenating species, which increased their sensitivity towards LA and lowered their biohydrogenating activity. In this regard, Kim et al. (2000) demonstrated that actively growing and metabolically active species are less inhibited by LA and do not only isomerise LA to CLA but also produce the hydrogenated products VA and SA. However, a general increased sensitivity towards LA due to pH would not explain for the observed variation between the different inocula, indicating that (an)other environmental parameter(s) should be involved. One parameter could be the particle contents of the faecal slurries, because food particles were previously demonstrated to provide an important site for adsorption and biohydrogenation of LA (Harfoot & Hazlewood, 1997). In this regard, a low or insufficient presence of particles in faecal slurries 1, 4 and 5 may have inhibited LA biohydrogenation, leading to a decrease in LA-sensitive butyrate-producing species and butyrate production (Fig. 5.1). 


113


Chapter 5: Linoleic acid impairs butyrate production The importance of an adherence site for LA biohydrogenating species was supported using the more dynamic and validated in vitro SHIME-model of colonic fermentation processes (Molly et al., 1993, Van den Abbeele et al., 2010). Recently, we upgraded this model by incorporation of a mucin environment (M-SHIME) establishing a representative surfaceattached, mucosal microbiome (Van den Abbeele et al., 2012a&b). Mucin-adhered bacteria consisted for almost 60% out of Clostridium cluster XIVa species, among which Roseburia intestinalis and Eubacterium rectale were most prominent (Van den Abbeele et al., 2012a. This was reflected in a shift from acetate towards butyrate when compared to the conventional L-SHIME model. Considering the high biohydrogenating activity of Roseburia spp. (Devillard et al., 2007), the M-SHIME set-up was considered ideal to study the importance of Roseburia spp. for butyrate production upon supplementation of LA within a complex microbial community of the gut. The results of the present study support our previous findings because (i) Eubacterium rectale/Roseburia spp. specifically colonized the mucin compartment of the M-SHIME with 1-2 log units (copies.mL-1) more than found in the lumen of both L- and M-SHIME, and (ii) Faecalibacterium prausnitzii qPCR counts were generally higher in the M-SHIME. Moreover, incorporation of the mucin environment stimulated LA biohydrogenation to VA (Fig. 5.3A) and lowered the inhibition of butyrate production by the daily addition of 1.0 g.L-1 LA (Fig. 5.3B). Hence, it was concluded that enrichment of biohydrogenating Roseburia spp. and Faecalibacterium prausnitzii allowed for butyrate production in the presence of LA. Thereby, Roseburia spp. are considered most important as these species were most abundant (Table 5.2; 2.8 ± 0.3 log units more than Faecalibacterium prausnitzii). While these findings suggest that LA may be a threat for colonic butyrate levels, future research should elucidate its impact within a variety of gut microbial communities. In this regard, it is crucial to not only study its effects for healthy individuals but also in the case of IBD, when the abundance of Roseburia spp. is low (Swidsinski et al., 2005; Sokol et al., 2009). Therefore, the M-SHIME model can be used to study the effect of LA supplementation on multiple microbiomes of healthy individuals and UC patients (Vermeiren et al., 2012). In these studies, the effect of various LA concentrations should be monitored. In this regard, a mild LA stress might stimulate microbial metabolism and Faecalibacterium prausnitzii levels, as was seen in SHIME-run 2 (Table 5.2&5.3).



114


Chapter 5: Linoleic acid impairs butyrate production To our knowledge, this is the first study demonstrating an inverse relation between LA biohydrogenation and butyrate production by the human gut microbiome. Moreover, enrichment of biohydrogenating Roseburia spp. in a model of the colon microbiota was found to stimulate LA biohydrogenation and improve butyrate production in the presence of high levels of LA. While this indicated that Roseburia spp. may be important species to warrant colonic butyrate production in the presence of inhibitory levels of LA, it should be mentioned that also other (unknown) gut microbes may be important for the situation in vivo. In view of the increasing LA concentrations in the Western diet, further research concerning the effects of LA on colonic butyrate production, is warranted

5 Acknowledgements We thank Jessica Benner and Annelies Geirnaert for critical revision of this manuscript. BV, PVDA and TVDW benefited from an FWO-Vlaanderen postdoctoral grant. JV was financially supported by a Concerted Research Action of the Flemish Community (GOA) (BOF12/GOA/008).



115


Chapter 5: Linoleic acid impairs butyrate production



116


Chapter 6: Discussion

Chapter 6: Discussion 1 Positioning of this research Gut microbes are important regulators of human health. They challenge and stimulate our immune system and provide extra nutrients and vitamins for the host. Moreover, the presence of a well-balanced gut microbial community protects the host against (potentially harmful) intruders. Recently, several aspects of modern life were found to affect gut microbial processes or species (Fig. 6.1) (De Filippo et al., 2010; Rook et al., 2010; Dethlefsen & Relman, 2011). Moreover, these aspects seem to substantially affect human health, because modern societies are characterized by the increasing incidence of ‘prosperity diseases’ such as allergy, inflammation diseases, cancer and obesity (Fig. 6.1).

Modern life environment: -! Diet: high in fat, refined sugar, red meat and sodium; low in fibres, minerals, antioxidants and vitamins -! Antibiotics -! Increased hygiene and sanitation -! Lower physical activity -! Global mobility -! Urbanization -! …

HOST HEALTH:

GUT MICROBIOTA:

-! Inflammation -! Allergy -! Pathogenic infection -! Energy status: weight, Adiposity, diabetes -! Cancer -! Cardiovascular health

-! Colonization resistance -! SCFA production (energy delivery) -! Stimulation of the immune system -! Production of vitamins and other micronutrients -! Pathogenic overgrowth…

Figure 6.1 Modern life affects the gut microbial composition and activity and is characterized by an increased incidence of ‘prosperity diseases’. While modern life can target gut microbiota and human health in various ways, three parameters were evidenced to exert significant effects. A first parameter involves the high standards for hygiene and sanitation, which is increasingly correlated with the prevalence of 


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Chapter 6: Discussion allergies and inflammation diseases. In 1989, Strachan was the first to put forward the hygiene hypothesis, suggesting that a decreased exposure to infectious agents during early life could be responsible for the rise in hay fever. It was proposed that a reduced exposure to early childhood infections decreases the maturation of T-lymphocytes into type 1 T-helper (Th1) cells, leading to the overexpression of Th2 cells, which cause allergy (reviewed by Romagnani, 1994; Shirakawa et al., 1997). Over the past years, the hygiene hypothesis has been regularly updated and expanded. In addition to infectious agents, changes in the normal commensal microbiota and the loss of interaction with ‘old friends’, such as pseudocommensals and parasites (helminths), were also suggested to affect the immunological response of the host (Blaser, 2006; Rook, 2009; Rook, 2012). Moreover, the immunoregulatory mechanisms involved were demonstrated to be more complex than the Th1/Th2 balance and proposed to originate in a reduced stimulation and activity of regulatory T cells (Romagnani, 2004; Rook, 2009). For these reasons, the hygiene hypothesis was reformulated as the Old Friends Hypothesis (Rook, 2007; Rook, 2010). This hypothesis suggests that one reason for the increasing incidence of chronic inflammatory disorders (and not only allergies) in high-income countries is the depletion from the urban environment of organisms that accompanied mammalian evolution and had to be tolerated by the host. A second important aspect of modern life is the consumption of antibiotics. Since the development of the first naturally derived antibiotic gramicidin (René Dubos) at the start of World War II in 1939, antibiotics have been used intensively to treat bacterial infections in humans and animals. Although they were designed to target pathogens, most of them have a broad-spectrum activity affecting other related, non-pathogenic microorganisms. Moreover, the decrease in directly targeted microbiota may indirectly disturb other species that depend on the former for the production of nutrients. Single exposure to antibiotics typically results in a temporary decline of the community diversity, followed by a rapid recovery of the community (De La Cochetière et al., 2005; Dethlefsen et al., 2008). However, the recovery is never complete, with persistent long-term impacts that remain for up to 2 years (Jernberg et al., 2007). Furthermore, repeated exposure to antibiotics, both at therapeutic and subtherapeutic (e.g. in animal products) levels, was shown to cause stable and lasting alterations in the gut microbiome (Cho et al., 2009; Dethlefsen & Relman, 2011) that went along with an increased adiposity in young mice (Cho et al., 2012). The changes in the microbial homeostasis were described to affect host immunity by the loss of bacterial ligands and signals, or by changes in bacterial metabolites (Willing et al., 2011). Both the loss of immune 


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Chapter 6: Discussion and microbial homeostasis, or ‘colonization resistance’, furthermore creates opportunities for pathogens to infect and worsen disease (Brandl et al., 2008; Sekirov et al., 2008; Stecher & Hardt, 2011). One of the most striking examples is antibiotic-induced infection by Clostridium difficile, which may result in life-threatening diarrhoea and colitis (Chen et al., 2008; De La Cochetière et al., 2008). Although it has not been unequivocally established whether Clostridium difficile-associated diseases are caused by disturbances in immune or microbial homeostasis, the reported beneficial effects of faecal transplantation in severely diseased patients demonstrate the importance of a well-balanced microbial ecosystem for human health (Khoruts et al., 2010; Surawicz & Alexander, 2011). In this work, we investigated the impact of dietary habits, as a third crucial parameter of modern life. These habits, typified as a ‘Western-style diet’, include a low intake of fibre and a high consumption of fat, and have been evidenced to significantly affect the gut microbiota in vivo (O’Keefe et al., 2009; De Filippo et al., 2010). Furthermore, a low-fibre/high-fat diet was found to result in a specific gut microbial community, which may induce obesity in mice (Turnbaugh et al., 2006; Bäckhed et al., 2007). Several studies support these observations demonstrating the importance of dietary fibres for human health by stimulating the presence or activity of beneficial microbes (i.e. the prebiotic concept) (Gibson et al., 1995; Grootaert et al., 2007; Macfarlane et al., 2008). However, little to no information is available on the impact of fat on colonic microbial processes. Therefore, the aim of the present work was to investigate how colonic fat may indirectly affect human health by interfering with colonic microbial processes and host-microbe interactions. The emphasis was thereby placed on glycerol, the central molecule of triglycerides, and the n-6 PUFA LA (C18:2Δ9c,12c), which is increasingly consumed in Western societies. The results of this work demonstrate that these compounds may target various gut processes associated with human health. Hence, (an increased) fat load to the colon should be carefully considered as one of the aspects of modern life that may affect microbial processes and overall human health.



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Chapter 6: Discussion

2 Main research outcomes The aim of this research was to investigate how glycerol and LA may affect gut microbial processes associated with human health. The latter involved the protection against pathogenic infection by the production of reuterin from glycerol (Chapter 2 and Chapter 3), the interaction of beneficial microbes with a simulated mucosal environment of the gut (Chapter 4 and Chapter 5) and the production of the health-promoting SCFA butyrate (Chapter 5). Figure 6.2 provides an overview of the main findings. Chapter 2 describes how glycerol is fermented in batch incubations of mixed cultures of the human faecal microbiota. Although it was found that glycerol fermentation is prone to high inter- and intraindividual variability, clear correlations were established between glycerol consumption and microbial conversion to 1,3-PDO on the one hand, and SCFA production on the other hand. More specifically, efficient glycerol conversion to 1,3-PDO correlated positively with higher acetate production and negatively with propionate production. By using a molecular fingerprinting technique (DGGE) and performing batch fermentations with 13

C-labelled glycerol, it furthermore seemed plausible that glycerol-degrading lactobacilli

and/or

enterococci

were

responsible

for

efficient

1,3-PDO

formation.

These

lactobacilli/enterococci were proposed to have gained a competitive growth advantage over other species that oxidize glycerol for their growth because the former metabolise less reduced carbohydrates, such as glucose. Thus, for the fixation of 1 mol carbon, these lactobacilli/enterococci need to deposit 1 mol H2 less than other glycerol-degrading species, thereby converting glycerol more rapidly to 1,3-PDO (see Fig. 2.5). This finding holds physiological importance as it may involve the formation and possible accumulation of intermediary reuterin, a strong and wide-spectrum antimicrobial aldehyde. In Chapter 3, we assessed whether in situ reuterin formation from glycerol by Lactobacillus reuteri (ATCC PTA 6475) was able to protect a 3-D model of colonic epithelium against pathogenic infection by Salmonella (enterica serovar) Typhimurium. In this work, for the first time a model of colonic epithelium was combined with both a pathogenic and a commensal gut microbe, allowing direct host-microbe and microbe-microbe interactions. Using this model, it was found that the mere presence of Lactobacillus reuteri protected the model of colonic epithelium against Salmonella colonization. Moreover, this effect was substantially enhanced when glycerol was supplemented and Lactobacillus reuteri was producing reuterin in situ,



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Chapter 6: Discussion supporting the combined application of glycerol and Lactobacillus reuteri to combat intestinal infection by Salmonella Typhimurium. In a second part of this research, the focus was put on LA and its capacity to inhibit (beneficial) gut microbial processes and species. In Chapter 4, LA was found to inhibit a logphase culture of the potentially probiotic Lactobacillus reuteri ATCC PTA 6475 within 1h of exposure. In addition, within faecal and simulated colonic microbial communities (SHIME®), LA appeared to specifically inhibit butyrate production (Chapter 5). In Chapter 4 and 5, incorporation of a mucin-agar environment within a dynamic model of the colon microbiota (M-SHIME®) was found to protect Lactobacillus reuteri and butyrate production from the inhibitory effects of high levels of LA. In Chapter 4, mechanistic studies with Lactobacillus reuteri revealed that LA toxicity was lowered by the combined action of a viscous mucin-agar matrix and the release of mucin from this agar to the lumen. The protective effect of mucin was not a nutritional effect, because mucin monosaccharides did not counteract the bactericidal effects of LA. Based on cell hydrophobicity measurements, it was proposed that the observed effects originated in the viscous nature of mucin, which physically impaired the access of LA to the bacterial cells. In Chapter 5, the mucin-agar layer was discussed to warrant LA biohydrogenation by stimulating the prevalence and growth of butyrateproducing, biohydrogenating Roseburia spp.



121


Chapter 2: Glycerol fermentation by the faecal microbiota

LINOLEIC ACID Chapter 5: Inefficient biohydrogenation of LA impaired butyrate production.

GLYCEROL

Chapter 4: A simulated mucus layer protected Lactobacillus reuteri from the inhibitory effects of LA.

host-microbe interactions

mucosal, microbial processes

luminal, microbial processes

Chapter 6: Discussion

Chapter 3: Glycerol conversion to reuterin increased Lactobacillus reuteri’s protective effects against Salmonella Typhimurium colonization in a model of colon epithelium.

Lactobacillus reuteri commensal colon microbes Salmonella Typhimurium butyrate-producing colon microbes

Chapter 5: Enrichment of Roseburia spp. in the M-SHIME warranted butyrate production and LA biohydrogenation.

intestinal epithelial cell

Figure 6.2 Schematic overview and positioning of the main conclusions within the colon physiology



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Chapter 6: Discussion

3 General discussion and future perspectives 3.1 Glycerol fermentation by the colon microbiota In Chapter 2, it was discussed that glycerol supplementation to the faecal microbiota may provide a growth benefit for glycerol-fermenting lactobacilli/enterococci as these species use glycerol as an electron sink by reducing it to 1,3-PDO. This microbial process is of particular importance, because it involves the formation of intermediary 3-HPA (reuterin), which is a strong broad-spectrum antimicrobial (see Chapter 1.6.1). Accumulation and secretion of reuterin was suggested for strains of Enterococcus faecium isolated from human faeces (Vanhaecke et al., 2008b) and demonstrated for strains of Lactobacillus brevis and Lactobacillus buchneri (Schütz & Radler, 1984), strains of Lactobacillus collinoides and Lactobacillus diolivorans isolated from fermented apple juice (Sauvageot et al., 2000; GaraiIbabe et al., 2008), a strain of Lactobacillus coryniformis isolated from goat’s milk cheese (Martin et al., 2005), and multiple strains of Lactobacillus reuteri isolated from birds and mammals (Walter et al., 2011). In the human intestine, it is anticipated that Lactobacillus reuteri strains are the most important glycerol consumers and reuterin producers, because of their predominance within the autochthonous Lactobacillus community (Reuter, 2001) and their capacity to produce and tolerate high concentrations of reuterin (Talarico et al., 1988; Vollenweider et al., 2003). However, it should be acknowledged that in Chapter 2 of this work glycerol-effects were only studies for one microbial group and that future research should clarify whether glycerol may affect other (unknown) glycerol-consuming microbial groups, including the genera Klebsiella, Citrobacter, Clostridium and Enterobacter (see Chapter 1.6.1). Microbial reuterin formation may be hazardous for human health for several reasons. First, 3HPA can be spontaneously dehydrated to the toxic and mutagenic acrolein (Vollenweider et al., 2003). Secondly, its presence was demonstrated to stimulate bioactivation of the carcinogenic food contaminant PhIP (Vanhaecke et al., 2008a&b). Thirdly, in Chapter 3 of this work, 3-HPA was shown to destroy cell-cell contact and cell viability within a highly differentiated, in vivo-like model of colon epithelium. On the other hand, formation of 3-HPA by Lactobacillus reuteri was demonstrated to be favourable because it increased the protective behaviour of Lactobacillus reuteri ATCC PTA 6475 against Salmonella Typhimurium colonization in a model of colon epithelium (see Chapter 3). Moreover, Cleusix



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Chapter 6: Discussion et al. (2008) showed that this microbial process decreased Escherichia coli numbers in an in vitro model of colonic fermentation. Finally, many in vivo trials support the safe administration of high doses of reuterin-producing Lactobacillus reuteri cells (see Table 1.3). Hence, glycerol fermentation by Lactobacillus reuteri may be considered as a healthpromoting microbial process, provided that it does not result in the formation of acrolein or other carcinogenic compounds nor harms the colonic epithelial tissue. Glycerol fermentation and 3-HPA formation by strains of Lactobacillus reuteri depends on the concentration of less reduced carbohydrates required for growth (Vollenweider & Lacroix, 2004). More specifically, a molar ratio of glucose to glycerol below 0.33 was found to be essential for 3-HPA accumulation (Lüthi-Peng et al., 2002a; Tobajas et al., 2009). The nutritional composition of the media in Chapter 2 and 3 was designed to allow for reuterin formation. More specifically, in Chapter 2 a maximum of 1 g.L-1 degradable carbohydrates was present in the medium and 40 or 140 mM glycerol was added. Assuming a complete conversion of the former carbohydrates to glucose, molar ratio’s of 0.14 or 0.03 were provided. In Chapter 3, the GTSF-2 medium contained 10 mM glucose (together with 4.7 mM galactose and fructose) (Lelkes et al., 1997) and was supplemented with 40 mM glycerol (approach 2), resulting in a glucose to glycerol ratio of 0.25. To get an idea about the in vivo relevance of microbial reuterin production and its protective effect against Salmonella infection, some theoretical calculations can be performed. First, a normal intake of carbohydrates is considered. Levitt et al. (1987) estimated that 40 g of unabsorbed carbohydrates reach the colon daily. Assuming the consumption of 3 meals a day resulting in 13 g of colonic carbohydrates each and the complete conversion of these carbohydrates to glucose, this implies a colonic glucose load of 72 mmol. In order for 3-HPA accumulation to occur at these glucose levels, colonic glycerol levels should be at least 218 mmol or 20 g. Considering the glycerol consumption by the route of dietary fat, following a recommended daily intake of 70 g triglycerides per day (see Fig. 1.4; FAOSTAT, 2010) or 23 g per meal, and considering their 95% digestion and absorption in the small intestine (Hill, 1995), this means that 1.2 g of triglycerides will reach the colon. Assuming that 1 mol of triglycerides consist of 1 mol of glycerol, 1 mol of palmitic acid, 1 mol of stearic acid and 1 mol of oleic acid, this means that the glycerol contents of this triglyceride fraction is 10% (w/w) (Table 6.1). Hence, the colonic glycerol load derived from dietary triglycerides will be 0.12 g or 1.3 mmol, which is 168 times less than what is required for 3-HPA accumulation at the above levels of carbohydrate formation. Therefore, it can be concluded that for balanced dietary 


124


Chapter 6: Discussion intakes of carbohydrate and fat, the impact of reuterin formation from dietary ‘fatty’ glycerol on microbial homeostasis in vivo can be neglected. However, colonic glycerol levels and reuterin accumulation may become significant under specific conditions. These involve a decreased intake of resistant starch or dietary fibres and an increased consumption of glycerol e.g. by fat - dietary patterns which are typical for a Western-style diet. In this regard, the consumption of a high-fat, fibre-depleted meal should be favourable for reuterin accumulation in the colon. Yet, dietary fat is not the only source of glycerol. In vivo, colonic glycerol levels will be the result of glycerol consumption by various pharmaceutical and food products (E422) and its in situ microbial synthesis, release from desquamated epithelial cells and intestinal clearing of endogenous plasma glycerol (Casas & Dobrogosz, 2000). To better evaluate the impact of dietary triglycerides on glycerol fermentation and 3-HPA formation, future research efforts should target the quantification of colonic glycerol concentrations in vivo. Table 6.1 Molar weights and weight percentages of glycerol and fatty acids within a theoretical triglyceride molecule, resembling the typical composition of lard (pork fat) 1 mol glycerol 1 mol palmitic acid 1 mol stearic acid 1 mol oleic acid 1 mol triglyceride

Molar weight (g/mol) 92.1 256.4 284.5 282.5 915.5

% (w/w) 10.1 28.0 31.1 30.1 100

An additional point of attention is the fact that reuterin production by Lactobacillus reuteri is strain-dependent (Cadieux et al., 2009; Oh et al., 2010; Walter et al., 2011). Walter et al. (2011) highlighted that most human-derived Lactobacillus reuteri strains are able to produce reuterin. However, it was demonstrated that these differ significantly in their quantities of reuterin secreted (Spinler et al., 2008; Jones & Versalovic, 2009). In this regard, it should be mentioned that the strain used in Chapter 3 and 4 (ATCC PTA 6475) was found to produce three times less reuterin when cultured in a rich medium than the established probiotic strain ATCC 55730 (Spinler et al., 2008; Jones & Versalovic, 2009). This was probably due to the up-regulation of the gene responsible for reuterin conversion to 1,3-PDO in strain 6475 (Saulnier et al., 2011). Therefore, it is anticipated that the impact of reuterin production from glycerol may be higher for other reuterin-producing Lactobacillus reuteri strains.



125


Chapter 6: Discussion

The results of the present study encourage future research towards the application of glycerol as a ‘prebiotic’ food supplement to stimulate beneficial Lactobacillus reuteri numbers in the colon. These research efforts should utilize a dynamic in vitro model of the colonic microbiota and later, go for in vivo trials with various animal models. While in the former in vitro models colonic glycerol levels can be easily controlled, its supplementation as a colonic supplement in vivo should consider digestion and absorption in the small intestine. Therefore, the application of encapsulated glycerol appears advisable so that it remains unaffected during its passage through the stomach and small intestine, and is released in the colon under specific conditions of pH. While colonic Lactobacillus reuteri numbers can also be increased upon supplementation of encapsulated, live Lactobacillus reuteri (Zhao et al., 2012), the preparation, storage and application of glycerol capsules should be less cumbersome. In view of the current European guidelines for the assignment of health claims to pre- and probiotic products, study end points should relate to a measurable clinical outcome (EFSA, 2010; Guarner et al., 2011). In this regard, the experimental design should allow to determine a dose-response relationship between encapsulated glycerol supplementation and a decrease in (potentially) pathogenic bacteria, such as Escherichia coli, Salmonella Typhimurium, etc… In addition, the formation of carcinogenic acrolein and bioactivation of PhIP should be monitored.

3.2 LA biohydrogenation and butyrate production Previous studies indicated that LA may inhibit a variety of species, among which Grampositive are typically more sensitive than Gram-negative species (Dilika et al., 2000; Thormar & Hilmarsson, 2007). However, within a mixed microbial community, LA was shown to specifically inhibit the production of butyrate (see Chapter 5). This is supported by Maia et al. (2007) who showed an increased sensitivity of butyrate-producing rumen species towards LA, when compared to other rumen species. Moreover, while the antibacterial activity of fatty acids is mostly explained to originate from the disruption of the bacterial membrane (Desbois & Smith, 2010), LA toxicity in butyrate-producers was related to metabolism (Maia et al., 2010). In this work, the impact of LA on butyrate production was further demonstrated to depend on its efficient biohydrogenation to VA. Hence, it is postulated that LA toxicity occurs at the interface of the biohydrogenation reaction and butyrate production. This relation is intriguing and suggests that one might consider protecting beneficial butyrate producers



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Chapter 6: Discussion from LA toxicity by stimulating biohydrogenation. Future research for a better understanding of this relation could involve (1) the use of (meta)transcriptomics to identify the pathways involved, and (2) the analysis of the thermodynamics to unravel the driving force of the biohydrogenation reaction.

(Meta)transcriptomics In order to gain information about the expression (down- or upregulation) of genes, one can study the mRNA of a given bacterial species (transcriptomics). This involves the mapping of sequence reads onto a reference genome. For identifying the genes and pathways involved in LA biohydrogenation and butyrate production, various reference genomes are available for Roseburia spp. (www.genomesonline.org). While transcriptomics could provide valuable mechanistic information about the relation between butyrate production and LA biohydrogenation/toxicity within a specific bacterial population, there is no guarantee for these pathways to be relevant within the more complex microbial community of the gut. Therefore, it is recommended to additionally study the effect of LA supplementation on the transcriptome of a whole microbial community (metatranscriptomics). These data should allow identifying key functional effects within the community and can be combined with metagenomic data to reveal the main bacterial species involved. In this regard, it is important to identify risk biomarkers, such as the presence or absence of a certain species or the expression of a certain gene, associated with inefficient LA biohydrogenation and the inhibition of butyrate production. Once such biomarker genes or species have been identified, metatranscriptomics and -genomics can be used to screen various population groups and individuals for their sensitivity towards LA consumption. The possibility to differentiate between LA-sensitive and -non-sensitive individuals using metatransciptomics, is supported by the recent identification of a uniform functional pattern in the gut microbiomes of ten healthy individuals (Gosalbes et al., 2011). In addition, Booijink et al. (2010) successfully used metatransciptomics to compare gene expression profiles over time. The usefulness of metagenomics, on the other hand, is demonstrated by the work of Willing et al. (2010). By performing pyrosequencing on faecal samples, these authors revealed the importance of butyrate-producing species to differentiate between healthy individuals and patients suffering from the IBD Crohn’s disease and UC. More specifically, patients suffering from ileal Crohn’s disease were characterized by decreased abundances of Faecalibacterium prausnitzii and Roseburia spp. and increased amounts of Escherichia coli and Ruminococcus gnavus.



127


Chapter 6: Discussion Considering the biohydrogenation and butyrate production potential of the former organisms, it appears plausible that sequencing studies could help identifying the role of LA toxicity/biohydrogenation for the aetiology of IBD.

Thermodynamics of LA biohydrogenation Biohydrogenation is the microbial process in which an unsaturated fatty acid is combined with hydrogen (H2), resulting in the formation of a more saturated fatty acid. Thermodynamically, biohydrogenation of LA to VA can be written as a redox reaction as follows:

C18 H 32O2 + 2H + + 2e" # C18 H 34 O2 H 2 # 2H + + 2e" C18 H 32O2 + H 2 # C18 H 34 O2

(reaction 6.1)

While most microbial processes are dependent on a variety of environmental parameters (pH, temperature, salt, etc…), limited attention is given to their thermodynamic requirements.

!

However, thermodynamic limits represent the most fundamental limits of life (Hoehler et al., 2007). To investigate whether a microbial redox process is thermodynamically favourable, one can calculate the change in Gibbs free energy (ΔGprocess), which represents the available energy that is derived from the process (Equation 6.1).

"G process = # nF"E

(Eq. 6.1)

n represents the number of exchanged electrons per mol of product and F is Faraday’s constant (C/mol). ΔE is the difference between the potentials of the donor and acceptor reactions. In order for the microbial conversion to occur, ΔG should be negative and thus ΔE

!

should be positive. In practice, the microbial catalysis is energy dissipating, requiring a more negative ΔG than predicted by the theoretical thermodynamic approach.

Provided that LA biohydrogenation is a respiration process, it would become thermodynamically favourable when the electrical potential of reaction 1 is positive. This potential is dependent on the temperature, the concentration of the reactants and the pH and



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Chapter 6: Discussion can be calculated from the standard potential E° using the Nernst equation (Equation 6.2). In the gut, the major parameters affecting this potential will be the LA concentration, the hydrogen partial pressure and the (local) pH. + n

[ox ][H ] RT E = E° " log nF [red]

(Eq. 6.2)

To the author’s knowledge, the standard potential of LA saturation to VA is unknown. However, the importance of a suitable oxidation-reduction potential for LA biohydrogenation

!

was previously demonstrated by Eyssen & Parmentier (1974). These authors were able to stimulate a Eubacterium species to hydrogenate LA under specific conditions. Monocultures of this species were unable to perform biohydrogenation and isomerized LA to an unknown octadecadienoic acid. However, when co-incubated with other species (Proteus, E. coli, Clostridium etc.) that showed no effect on LA or the octadecadienoic isomer, LA hydrogenation occurred. Interestingly, a similar effect on LA hydrogenation was found when monocultures of the Eubacterium species were cultivated in an atmosphere of 95% H2 and 5% CO2 in the presence of a palladium catalyst, resulting in a redox potential below -300 mV (pH 7.0). Further characterization of the electrical potential for LA biohydrogenation, e.g. in a rotating disk electrode coated with a nickel on silica catalyst (Jovanovic et al., 1997; Mondal & Lalvani, 2003), could enable to calculate the theoretical concentration for this microbial process to occur in the colon. Specific interest goes thereby to the identification of a maximum LA concentration for the pH range of the colon above which LA conversion to VA is thermodynamically not feasible and butyrate production is most likely to be affected. Considering their need for hydrogen, biohydrogenating species are likely to prefer high hydrogen partial pressures. However, when considering thermodynamics, hydrogen formation becomes more favourable at low hydrogen partial pressures (Thauer et al., 1977). Therefore, hydrogen formation is warranted by its efficient elimination indicating that biohydrogenation may depend on hydrogen production and consumption processes. In the colon, major sources of hydrogen are fermentation processes, in which carbohydrates (polysaccharides) are consumed by anaerobic bacteria and degraded to yield SCFA (acetate, propionate and butyrate), CO2 and H2 (Cummings, 1983; Nakamura et al., 2010). Considering an availability of 40 g of unabsorbed carbohydrates per day and a hydrogen production of 340 mL per gram 


129


Chapter 6: Discussion of carbohydrate, approximately 13.6 L of hydrogen would be generated in the colon daily (Nakamura et al., 2010). Removal of this hydrogen can happen via flatus and/or absorption into the systemic circulation and subsequent respiratory excretion (Strocchi & Levitt, 1992). However, the most important hydrogen sink is its consumption by colonic microbiota in three major processes: (1) the production of methane by methanogens, (2) the reduction of sulphate to sulphide by sulphate reducing bacteria (SRB) or (3) the reduction of CO2 to acetate by homoacetogens (Strocchi & Levitt, 1992; Nakaruma et al., 2010). In humans, methanogenesis and sulphate reduction are the two major pathways of H2 consumption as these are thermodynamically more favourable. In order to study the impact of microbial hydrogen consumption and production processes on biohydrogenation, two research strategies are proposed. First, batch incubations can be used to assess the effect of the major hydrogen-consuming processes: methane production and sulphate reduction. While hydrogen consumption for biohydrogenation of C22:6n-3 and C20:5n-3 was previously indicated to be negligible when compared to methanogenesis in the rumen (Fievez et al., 2003), the impact of LA biohydrogenation on methane production and sulphate reduction has not been investigated for the human colon microbiota. This can easily be done by incubating LA in the presence of faecal inocula that either produce CH4 or H2S. Subjects can be selected to deliver inocula that produce CH4 by performing a simple breathtest, because the exhalation of detectable levels CH4 is correlated with the prevalence of methanogens in the colon. Although methanogens and SRB were demonstrated to coexist in the gut, detectable CH4 levels were shown to only occur when densities of hydrogenotrophic methanogens are higher than 107-108 CFU.g-1 wet faecal matter (Lewis & Cochrane, 2007). Inocula of apparent non-producers harbour approximately 104 CFU.g-1 wet faecal matter and most probably rely on SRB to consume hydrogen by reducing sulphate to H2S (Pochart et al., 1992). The importance of these processes for biohydrogenation by human gut microbiota can further be studied by applying 2-bromoethanesulfonate or chloroform and sodium molybdate to respectively block methanogenesis (Xu et al., 2010) and sulphate reduction (Ranade et al., 1999; Rubin et al., submitted), respectively. Secondly, the impact of various (prebiotic) fibres on the biohydrogenation process can be assessed. In this regard, in vitro batch and continuous SHIME experiments can be performed in order to screen for a fibre that warrants butyrate production in the presence of LA by stimulating biohydrogenation.



130


Chapter 6: Discussion

3.3 The importance of the mucus layer for Lactobacillus reuteri and Roseburia spp. to escape LA stress In Chapter 4 and 5, the presence of a mucin-agar layer in the M-SHIME was demonstrated to protect Lactobacillus reuteri ATCC PTA 6475 and Roseburia spp. against LA stress. While this resulted in their increased colonization in both the luminal and mucin-agar compartment, both species were shown to preferentially inhabit the mucin-agar layer. For Lactobacillus reuteri, the protective effect of the mucin-agar compartment was discussed to originate in the viscous nature of mucin(-agar), which may restrict the diffusion of LA towards the bacterial cell membrane. In contrast, for Roseburia spp., the protective effect was related to a remarkable increase of LA biohydrogenation to VA. Considering the fact that these are major biohydrogenating species in the human gut microbiome, it seems unlikely that they were protected due to a decreased contact with LA. On the other hand, LA biohydrogenation by Roseburia spp. appeared not to be responsible for Lactobacillus reuteri survival in the presence of a simulated mucus layer, because protection also occurred in monoculture experiments, i.e. in the absence of Roseburia spp. Moreover, in the M-SHIME efficient biohydrogenation was observed in the lumen, while Lactobacillus reuteri levels were only maintained in the mucin-agar layer. Hence, it is concluded that the simulated mucus layer protected Lactobacillus reuteri and Roseburia spp. from the inhibitory effects of LA in different ways. Both species are thereby proposed to differ in their interaction with the mucinagar environment, with Lactobacillus reuteri cells being protected from exposure to LA and Roseburia spp. having full access to LA. In order to restrict its interaction with LA, it is plausible that Lactobacillus reuteri ATCC PTA 6475 binds mucin chains on its cell surface by the secretion of (a) mucin-binding protein(s) (MucBP) (Mackenzie et al., 2010) or multifunctional mucus adhesins like the collagen-binding protein discovered in Lactobacillus reuteri NCIB11951 (Roos et al., 1996). This should be investigated in future experiments by developing mutants and/or antibodies that block the mucin-binding site, as was previously done for the mucus-binding protein of Lactobacillus reuteri ATCC 53608 (Roos & Jonsson, 2002; Mackenzie et al., 2010). If the protective effect against LA is indeed due to the secretion of such adhesion proteins, additional studies should elucidate whether this is a random effect or an active process regulated by the bacterial genome upon exposure to LA. In this regard, qPCR and ELISA can be used to determine whether LA supplementation can stimulate the expression of specific



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Chapter 6: Discussion genes involved in the production of adhesion proteins and their secretion on the cell surface, respectively. In addition, fluorescence microscope time-laps videos may help to elucidate whether bacteria may actively migrate towards a fixed mucin-layer upon exposure to LA. The latter would require the development of a chemical coating procedure that covalently binds mucin to a synthetic or glass surface (Gabriel et al., 2005) without affecting its protective characteristics. Considering the protective effect of the simulated mucus layer for Roseburia spp. in the presence of high concentrations of LA, it is proposed that these originated in the stimulation of LA biohydrogenation. This implies that the biohydrogenation process is stimulated by the presence of an adhesion site, which was previously shown to be important for biohydrogenation of LA (Harfoot & Hazlewood, 1997). In this regard, Roseburia spp. may bind to mucins using their flagellin proteins (Louis & Flint, 2009) as has been demonstrated for pathogens such as enterohemorrhagic and enteropathogenic E. coli and Clostridium difficile (reviewed by Juge, 2012). In addition, it is plausible that Roseburia spp. levels were maintained upon exposure to LA due to the protection of mucin-degrading and/or acetateproducing species within the mucin-agar layer (cross-feeding) (Louis & Flint, 2009), such as Akkermansia muciniphila (Derrien et al., 2004; Belzer & de Vos, 2012) and Lactobacillus reuteri.

3.4 Modelling gut microbial processes and host-microbe interactions When trying to make hard statements about the relation between the gut microbiota and the health status of the host, three difficulties arise. Firstly, the gut is a highly complex environment in which a huge set of interconnected bacterial and eukaryotic processes take place. Secondly, the morphology of the gut complicates sampling of eukaryotic and bacterial samples, especially in the proximal areas of the colon. Finally, the gut microbial community composition is prone to a high inter- and intra-individual variability. To overcome these problems, a set of in vitro techniques have been developed that allow to simulate simplified microbial processes and host-microbe interactions of the gut. In this work, in vitro experiments have been performed simulating microbial processes within the luminal (Chapter 2) and the mucosal environment of the gut (Chapter 4 and 5) or combining microbial processes with a 3-D model of the colon epithelium (Chapter 3).



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Chapter 6: Discussion

Batch incubations In Chapter 2 and 5, batch incubations of human faecal microbiota allowed to easily and rapidly screen for metabolic effects of glycerol and LA supplementation. This type of experimental set-up was used because it is a well-suited tool to rapidly screen for the effect of a dietary compound on metabolic processes in mixed microbial communities (Vanhaecke et al., 2006; Vigsnaes et al., 2011) and to elucidate inter- and intra-individual variability (Possemiers et al., 2007, Gross et al., 2010). Batch cultures follow the typical bacterial growth curve, which results in changes of pH, redox potential and community structure. Therefore, they are only suitable when short incubation times (usually 24-48h) are respected (Gibson & Fuller, 2000). This was the case for all batch incubations, with the exception of one series in Chapter 2. In the latter, ten human faecal microbial communities were incubated during 72h, with intermediate sampling after 4, 24 and 48h. DNA from the 72h sampling point was used to perform microbial community analysis by means of DGGE. At this time, the communities had reached the end of their stationary growth phase (see Fig. 2.1) where nutrients had become limited and surviving species had started to degrade the DNA of dead bacterial species. Hence, analysis of the microbial community DNA was considered relevant to detect specific glycerol effects. However, when trying to relate the presence of specific species (lactobacilli-enterococci or Lactobacillus reuteri) with the conversion rates of glycerol to 1,3-PDO, DNA-based molecular tools yielded no relevant results (unpublished results). Therefore, for future batch experiments targeting to relate functionality with specific bacterial species, it is recommended to sample RNA.

The M-SHIME, a dynamic gut model In Chapter 4 and 5, a more dynamic gut model was used to study the impact of LA on gut microbial metabolism and the prevalence of beneficial microbes within the mucosal environment. The SHIME® (simulator of the human intestinal microbial ecosystem) model, which was developed at LabMET (Molly et al., 1993, Van den Abbeele et al., 2010) and commercialized by ProDigest-Ghent University (Ghent, Belgium), allows for a representative simulation of colonic microbial processes. Typically, this model is built to simulate the luminal microbiota of the three different regions of the colon (colon ascendens, colon transversum and colon descendens) by stabilizing human faecal inocula under specific conditions of pH, retention time and nutrient availability during two weeks (Molly et al., 1993, Van den Abbeele et al., 2010). Recently, this model was expanded with mucin-agar



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Chapter 6: Discussion covered microcosms, allowing for the simulation of a mucosal environment and the development of a mucosal microbial community (Van den Abbeele et al., 2012b). These microcosms are placed in the vessels immediately upon inoculation with the faecal sample to avoid washout of gut microbes that cannot survive luminal conditions. Moreover, as they are quickly colonized and degraded by the microbiota, the microcosms should be renewed regularly (2-3 days). While this M-SHIME model can be used for typical SHIMEexperiments of several weeks (Van den Abbeele et al., 2012b; Van den Abbeele et al., submitted), it also facilitates short-term colonization experiments assessing the preference of gut microbes for a luminal or a mucosal environment (Van den Abbeele et al., 2012a&b). In this work, such short-term M-SHIME runs were performed in which only the proximal (ascending) colon was simulated. This experimental set-up was found suitable for several reasons. Firstly, simulating only one part of the colon allowed increasing the amount of test conditions using the same faecal inoculum. Secondly, LA can be rapidly biohydrogenated by the gut microbiota. Therefore, this process was studied under conditions of the ascending colon. Thirdly, LA is a fatty compound that does not homogeneously mix within an aqueous environment and easily sticks to the surface of plastic recipients (http://gestis-en.itrust.de; Mabrouk & Dugan, 1961). Hence, short-term experiments with limited transfers between vessels seemed advisable. Using this set-up, we consistently demonstrated a protective effect of the mucin environment for Lactobacillus reuteri against LA stress in a run of six days (Chapter 4). In Chapter 5, studies of two days demonstrated a similar protective effect of the mucin environment for butyrate producing Roseburia spp. and Faecalibacterium prausnitzii against LA stress by allowing for biohydrogenation. In this study, one run was continued for six days (unpublished data). Then, the butyrate production had collapsed after two days (Fig. 6.3). Although this was a one-time observation, one can speculate that this was caused because conditions of the colon ascendens were simulated. More specifically, the frequent arrival of carbohydrate-rich nutritional medium in this vessel may have stimulated hydrolysing and acetate/lactateproducing bacteria to a level that was unfavourable for butyrate-producing organisms. In this regard, temporary and local pH drops below 5.6 (colon ascendens pH range 5.6-5.9) may have played a role. Previously, it was established that an initial pH of 5.5 is more favourable for butyrate-producing Firmicutes (Eubacterium rectale/Roseburia spp.) within a mixed microbial community when compared to pH 6.5 (Walker et al., 2005; Duncan et al., 2009). However, an initial pH below 5.5 was found to slow down growth of the former butyrate 


134


Chapter 6: Discussion producers (Duncan et al., 2009) and an initial pH of 5.2 was demonstrated to inhibit butyrate production within a mixed microbial community while lactate was accumulating (Belenguer et al., 2007). Hence, it is proposed that butyrate production was impaired in our colon ascendens set-up due to (short-term) acidification and lactate accumulation. Therefore, future research regarding butyrate production in the M-SHIME should be improved by applying a nutritional medium that is less rich in carbohydrates or by adding a second colon vessel to the set-up simulating distal conditions (pH 6.2-6.9).

14

Butyrate (mM)

12 10

L-SHIME

8

M-SHIME

6 4 2 0 0

1

2

3

4

5

6

Time (days) Figure 6.3 Butyrate production in the L- and M-SHIME simulating conditions of the colon ascendens had collapsed after 3 days (unpublished observation regarding Chapter 5). In addition, the SHIME-model should be further optimized to more easily study the effect of fatty compounds, allowing for long-term studies of different regions of the colon. Thereby, one engineering strategy could target the elimination of plastic tubing for transfer between the colon vessels so that fat accumulation is avoided. In this regard, it is proposed to use overflow connections between the vessels that are closed with computer-programmed valves maintaining a representative residence time for each colon vessel (Fig. 6.4). This would not only facilitate fat studies, but would also allow for studies with (food) particles to better simulate the shear forces (Marzorati et al., 2011) and particle-associated microbiota (Walker et al., 2008) in the colon. Moreover, dynamic long-term studies with encapsulated products or bacteria would become feasible. Thus far, (food) particles in the SHIME-model can only be renewed or transferred between vessels by opening the colon vessels under a continuous flow of nitrogen gas - a delicate and time-consuming procedure which may disturb the microbial 


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Chapter 6: Discussion community structure. Hence, revision of the transfer between the colon compartments of the SHIME-model would increase its in vivo-relevance and application area. A final improvement of the SHIME-model for fat studies would involve the development of a representative microbial community of the small intestine, e.g. through inoculation with ileostomy effluents (Zoetendal et al., 2012). While relatively low concentrations of fat may affect the colonic microbial community (see Chapter 4 and 5), it is expected that fat effects in the small intestine can be substantial because in this region, fat concentrations are higher and the microbial community is less dense (104-107 cells.g-1 compared to 1010-1012 cell.g-1 in the colon; Payne et al., 2012).

SHIME feed

Pancreatic juice

pH

pH

N2 Stomach

Small intestine

Proximal colon

Distal colon

Effluent

pump overflow connection with valve

Figure 6.4 Schematic overview of a proposed SHIME set-up allowing for fat and particle transfer between the colon vessels

Colon epithelium models to study host-microbe interactions Gut microbes interact with the host at the interface of the gastrointestinal mucosa, which is constituted by the epithelium, lamina propria, glycocalyx and secreted mucus (Patsos & Corfield, 2009). To study the host-microbe interactions at this interface, several in vitro 


136


Chapter 6: Discussion models of the colon epithelium can be used. An appropriate in vitro cell model should have predictive power and be easy to handle (Cencič & Langerholc, 2010). In this regard, primary cells isolated from human or animal tissue retain most of their in vivo functionality, yet, they survive only few days in cell culture and the reproducibility of results may vary, depending on the donor (Quaroni & Beaulieu, 1997; Panja, 2000). In order to avoid these limitations, immortalized cell lines have been developed from cancer tumours (Cencič & Langerholc, 2010). These are cultivated as mono- and co-cultures that are grown on various substrates such as collagen (Bracke et al., 2001a) or mucin (Marzorati et al., 2010 - patent WO 2010/118857 A2) and characterized by different levels of cell-cell contact and differentiation (Fig. 6.5). In 2-D in vitro models, cells are typically cultivated as monolayers on flat surfaces. However, in contrast to the native in vivo polarised structure of the gut epithelial tissues, 2-D cell monolayers are typically not or little differentiated and this affects their functionality (Bentrup et al., 2006; Radtke et al., 2010; Hakanson et al., 2011; Tung et al., 2011). Moreover, the cancer origin of these cell lines has been criticized because they possess altered cell properties, such as specific glycosylation profiles, that may affect their proliferation and behaviour under environmental stimuli. Hence, in order to determine the model most suitable for a particular study, critical consideration of several physiological aspects is required, such as the differentiation and activation state, mucus production and/or the level of expression of specific receptors (Chantret et al., 1988; Cencič & Langerholc, 2010; Leonard et al., 2010). During this doctoral work, several in vitro models of the colon epithelium have been used to study host-effects of the glycerol fermentation intermediate reuterin. First, undifferentiated 2-D models were used to explore the effect of microbial reuterin production on the viability and metabolic activity of the colon epithelial cancer cell lines Caco-2 and HT-29 (unpublished data). Although these models have previously been applied to study the effect of microbial products on cell viability and metabolic activity (Vanhaecke et al., 2008a; Grootaert et al., 2011), they appeared unsuitable to study reuterin effects because of a high variability and inconsistency among the biological replicates. However, unpublished results of the chick heart invasion assay revealed a reproducible effect of microbial reuterin on the viability of highly differentiated tissue. In this assay, aggregates of breast cancer cells (MCF7/6) (Fig. 6.5) are combined with biopsies of chick heart tissue to study invasion (Bracke et al., 2001b). In contrast to what was expected, low concentrations of reuterin (0.1 – 1 µM) did not affect survival or the invasive behaviour of the MCF7/6 cancer cells but significantly harmed the chick heart tissue. Therefore, we postulated that microbial reuterin effects on colonic 


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Chapter 6: Discussion epithelial processes are better studied in highly differentiated, in vivo-like colonic epithelial tissue.

Figure 6.5 In vitro models of the colon epithelium derived from immortalized colon cancer cell lines are characterized by different levels of cell-cell contact and differentiation. Consequently, in Chapter 3, a 3-D model of colonic epithelium was used to study reuterin’s potential to protect against Salmonella Typhimurium colonization. While this model was previously validated to respond to Salmonella Typhimurium infection in key ways that reflect the infection process in vivo (Bentrup et al., 2006; Radtke et al., 2010), it was found suitable to study reuterin effects on host-pathogen interactions. Indeed, we were able to demonstrate the effects of in situ reuterin production by Lactobacillus reuteri ATCC PTA 6475 on



138


Chapter 6: Discussion Salmonella Typhimurium colonization of the tissue. However, 24h-exposure to reuterin was found to harm the structure and viability of this colon epithelium model. While many in vivo trials support the safe administration of high doses of Lactobacillus reuteri strains (see Table 1.3), the in vivo relevance of the latter observation was questioned. Considering the reactive nature of reuterin, it was proposed that, in vivo, this microbial product interacts first with compounds of the lumen and outer mucus layer before it can affect the host. Therefore, further research considering the effect of reuterin formation on colonic epithelial health would benefit from the inclusion of a more complex microbial environment. When increasing the complexity of the microbial environment to better study host-microbe interactions, the challenge lies in finding the optimal conditions for successful aerobic growth of human cells in the presence of (facultative) anaerobic gut bacteria. This is particularly difficult because cell cultures require a rich medium that easily allows bacterial overgrowth and/or contamination. Therefore, a first strategy could be the addition of sterile SHIMEsupernatant to the cell medium. The concentration of this supernatant should be maximal without affecting the morphology or viability of the colon epithelium model. In a second strategy, the colon epithelium model can be physically separated from the complex microbial community by a mucin-covered membrane in a Host-Microbe Interaction (HMI) module as developed by Marzorati et al. (2010 - patent WO 2010/118857 A2). A schematic overview of the HMI-module is given in Figure 6.6. The microbial compartment of this module is coupled to the colon vessels of the (M-)SHIME under a representative, continuous low-shear flow, which allows for the development of a bacterial biofilm on the mucin-covered membrane. While this model has been validated demonstrating the anti-inflammatory effect of a prebiotic compound on differentiated Caco-2 colon epithelial cells (Marzorati et al., in prep), its future simulation of the host compartment can be improved in various ways. Firstly, the Caco-2 monolayer can be replaced with a 3-D model of the colon epithelium. This model can be obtained by growing highly differentiated unilayers of HT-29 colon cancer cells (1) on porous collagen-coated microcarrier beads in a rotating wall vessel (RWV) bioreactor as was done in Chapter 3 (Bentrup et al., 2006; Radtke et al., 2010; Barrila et al., 2010) or (2) on microscale finger-shaped hydrogels that mimick the intestinal villi according to Sung et al. (2011). Secondly, the intestinal epithelial models should be combined with immune-competent cells (primary or macrophage cell lines) (Cencič & Langerholc, 2010; Leonard et al., 2010 and references therein) for a better simulation of gastrointestinal immune reactions. Finally, one



139


Chapter 6: Discussion could realize the complete simulation of the gastrointestinal tissue by applying human-derived biopsies of the colon.

Figure 6.6 HMI-module separating anaerobic, microbial processes from colon epithelial cells cultivated under aerobic conditions via a functional double-layer (Marzorati et al., 2010 patent WO 2010/118857 A2)



140


Chapter 6: Discussion

4 Conclusions A variety of in vitro models of the colon microbiome were used to study the effect of a highfat diet (glycerol and LA) on gut microbial processes and species associated with human health. Using these models, it was found that: -

glycerol may specifically stimulate reuterin-accumulating lactobacilli-enterococci, such as strains of Lactobacillus reuteri;

-

reuterin production by Lactobacillus reuteri ATCC PTA 6475 may protect against colonization by Salmonella Typhimurium, but might also harm the viability and structure of the colon epithelium;

-

LA may decrease luminal Lactobacillus reuteri ATCC PTA 6475 levels and butyrate production;

-

Lactobacillus reuteri ATCC PTA 6475 and butyrate production are protected from LA stress by the presence of a simulated mucus layer.

It was discussed that colonic glycerol effects due to an increased consumption of triglycerides are negligible, but may become significant in terms of reuterin accumulation when the diet is depleted in fibre. For LA, on the other hand, significant effects were observed at a relatively low concentration, which are representative for the LA levels in the Western diet. Future in vitro research regarding the impact of colonic fat on human health, requires the optimization of existing host-microbe interaction models that allow for a better simulation of the luminal and mucosal colon microbiota in the presence of an in vivo-like model of the colon epithelium.



141


Chapter 6: Discussion



142


Summary The colon microbiota provide us with features we did not have to evolve ourselves and are therefore crucial for human health. They digest dietary fibres and supply us with up to 10% of our energy requirements; produce vitamins and minerals; stimulate the immune system and offer protection against pathogenic infection. Modern life may disturb the ancient human host-microbe cooperation in ways that are unprecedented in our co-evolutionary history, e.g. by the consumption of antibiotics and the increased standards for hygiene. Modern life also includes a drastic change in dietary habits: the Western-style diet. This is characterized by a doubled consumption of fat and a much-decreased consumption of fibres. Several studies have demonstrated the effect of a high-fat/low fibre diet on the composition of the gut microbiota. These effects have even been associated with gut inflammation and the aetiology of type-2 diabetes and obesity. The specific effect of fat has thereby barely been investigated. In contrast to fibres, dietary fat is well digested and absorbed in the small intestine. However, this process is never complete and results in the loss of 1-5% of dietary fat to the colon. For a Western diet, several grams of fat may thus reach the colon microbiota daily. Moreover, colonic fat effects may be enhanced in case of disturbed digestion and absorption in the small intestine due to disease or the consumption of anti-obesity drugs such as orlistat. The aim of this doctoral work was therefore to investigate the effect of (an increased level of) colonic fat on the composition and metabolic activity of the colon microbiome. Specific interest thereby went to gut microbial processes and species that are associated with human health. These included the production of health-promoting short-chain fatty acids (SCFA), the protection against infection of the colon epithelium by pathogens and the prevalence of beneficial microbes within the mucus layer where host-microbe crosstalk is more likely to occur. For this purpose, a variety of in vitro models of the colon microbiome were used. Two ‘fatty’ compounds were considered: glycerol, which is the backbone molecule of all triglycerides, and the n-6 poly-unsaturated fatty acid linoleic acid (LA), because of its tripled consumption in Western countries over the past 100 years. Using batch incubations of faecal microbiota derived from ten healthy individuals, it was found that the rate of glycerol fermentation is prone to a high inter- and intra-individual variability. Efficient and rapid glycerol fermentation thereby related with high levels of acetate and a high yield of the fermentation product 1,3-propanediol (1,3-PDO). Experiments with

13

C-labelled glycerol and shifts in the lactobacilli-enterococci community DGGE

fingerprints indicated that rapid consumption of glycerol may be due to the activity of 


143


Summary glycerol fermenting lactobacilli-enterococci, such as strains of Lactobacillus reuteri. This holds physiological importance because the latter may accumulate and secrete relatively high levels of reuterin, which is a strong and wide-spectrum antimicrobial that is formed as an intermediary product during the reduction of glycerol to 1,3-PDO. The importance of reuterin formation by Lactobacillus reuteri for host health was investigated using a 3-D model of the colon epithelium. This model was previously utilized and validated to study the Salmonella (enterica serovar) Typhimurium infection process in vitro. In this doctoral work this model was for the first time expanded with a commensal gut microbe to simulate host-microbe and microbe-microbe interactions in the gut. Using this model, we demonstrated the importance of reuterin formation – and hence the presence of glycerol – for Lactobacillus reuteri (ATCC PTA 6475) to protect against pathogenic infection by Salmonella Typhimurium. In a second part of this work, a dynamic model of the luminal colon microbiota (L-SHIME) was used to investigate the effect of increased colonic loads of LA on the colonization potential of Lactobacillus reuteri (ATCC PTA 6475) and the production of the healthpromoting SCFA butyrate. Upon exposure to a high concentration of LA (1 g.L-1), the Lactobacillus reuteri levels were decreased and butyrate production was inhibited. A ‘normal’ (0.1 g.L-1) level of LA seemed not have an effect. The high level of LA was thereby representative of a colonic load of 1 g or a LA consumption of about 20 g per day. Interestingly, the inhibitory effects of high concentrations of LA were neutralized upon the incorporation of mucin-agar covered microcosms, simulating the mucus layer that covers the gut epithelium in vivo (M-SHIME). In the case of Lactobacillus reuteri, the protective effect of the mucin-agar layer was demonstrated to originate in the viscous nature of mucin(-agar), which physically lowered the access of LA to the bacterial cell membrane. Butyrate production, on the other hand, was warranted by the increased prevalence of LAbiohydrogenating and butyrate-producing Roseburia spp. in the M-SHIME, when compared to the L-SHIME. In fact, in the L-SHIME, high levels of LA remained, while in the MSHIME efficient biohydrogenation of LA to the less antimicrobial vaccenic acid was observed. The correlation between LA-biohydrogenation and butyrate production was further established by repeated batch incubation of human faecal microbiota. Hence, an increased delivery of glycerol and LA to the colon may significantly affect microbial processes and species that are associated with human health. The impact of glycerol, i.e. reuterin accumulation, was thereby discussed to become significant when the 


144


Summary diet is depleted in dietary fibre. In addition, LA effects were demonstrated to depend on the presence of a simulated mucus layer, which suggests that a high consumption of LA may worsen overall human health in case of a damaged mucus layer, e.g. in patients suffering from inflammatory bowel disease. In order to validate the impact of our observations for human health, future in vitro and in vivo research is warranted. Particular interest thereby goes to the development of a validated in vitro model that simulates the host-microbiota interactions in the gut in vivo.



145


Samenvatting Darmbacteriën voorzien ons van functies die we zelf niet hebben ontwikkeld en zijn daarom van cruciaal belang voor de gezondheid van de mens. Ze verteren voedingsvezels en leveren daarbij tot 10% van onze totale energiebehoefte; ze produceren vitaminen en mineralen, ze stimuleren het immuunsysteem en bieden bescherming tegen infectie door pathogenen. Onze moderne levensstijl grijpt in op de samenwerking tussen mens en darmbacterie op een manier die ongekend is in onze co-evolutionaire geschiedenis. Zo gebruiken we antibiotica en zijn de normen voor hygiëne sterk toegenomen. Deze levensstijl wordt eveneens gekenmerkt door een drastische wijziging in ons voedingspatroon: het ‘westers dieet’. Dit kenmerkt zich door een verdubbeling van de consumptie van vet en een sterk verminderde consumptie van vezels. Verschillende studies hebben aangetoond dat dergelijk dieet de samenstelling van de darmmicrobiota beïnvloedt. De effecten ervan worden zelfs geassocieerd met darmontsteking en de ontwikkeling van type-2 diabetes en obesiteit. De specifieke invloed van een verhoogde vetconsumptie is daarbij nauwelijks onderzocht. Vet wordt, in tegenstelling tot voedingsvezels, goed verteerd en geabsorbeerd in de dunne darm. Dit proces is echter nooit volledig waarbij 1-5% van het voedingsvet doorstroomt naar de dikke darm. Met ons westers voedingspatroon kunnen dus dagelijks verschillende grammen vet de bacteriën van de dikke darm bereiken. Bovendien kan de doorstroom van vet naar de dikke darm sterk toenemen in geval van een verstoorde vertering en absorptie in de dunne darm door ziekte of het gebruik van anti-obesiteit medicijnen, zoals orlistat. In dit doctoraatswerk werd daarom het effect van (een verhoogde hoeveelheid) vet op de samenstelling en de metabolische activiteit van de dikke darmbacteriën onderzocht. Specifieke interesse ging daarbij naar microbiële processen en species die worden geassocieerd

met

de

menselijke

gezondheid.

Deze

omvatten

de

productie

van

gezondheidsbevorderende korte keten vetzuren, de bescherming tegen infectie van het dikke darmepitheel door pathogenen en de aanwezigheid van gezondheidsbevorderende darmbacteriën in de mucuslaag waar gastheer-bacterie interactie waarschijnlijker zijn. Hiervoor werden verschillende in vitro modellen van het dikke darm microbioom gebruikt. Daarbij werd het effect van twee vetmoleculen onderzocht: glycerol, als centrale molecule in alle triglyceriden, en het n-6 poly-onverzadigd vetzuur linolzuur (LA), wegens zijn verdrievoudigde consumptie in westerse landen over de afgelopen 100 jaar. Aan de hand van batch incubaties van fecale microbiota afkomstig van tien gezonde individuen, werd gevonden dat de snelheid van glycerolfermentatie onderhevig is aan een 


146


Samenvatting hoge inter- en intra-individuele variabiliteit. Efficiënte en snelle glycerolfermentatie was daarbij gerelateerd aan een hoge acetaatproductie en een hoge opbrengst van het fermentatieproduct 1,3-propaandiol (1,3-PDO). Experimenten met

13

C-gelabeld glycerol en

verschuivingen in de DGGE-patronen van de lactobacilli-enterococci gemeenschappen deden vermoeden dat een snelle glycerolconsumptie het gevolg was van de activiteit van glycerolfermenterende lactobacilli-enterococci, zoals stammen van Lactobacillus reuteri. Dit heeft fysiologisch belang omdat deze species relatief hoge concentraties van reuterine kunnen accumuleren en uitscheiden. Reuterine is een sterk en breed-spectrum antimicrobieel product dat intermediair wordt gevormd tijdens de reductie van glycerol tot 1,3-PDO. Het belang van reuterinevorming door Lactobacillus reuteri voor de gezondheid van de gastheer werd onderzocht met behulp van een 3-D model van het dikke darmepitheel. Dit model werd eerder gebruikt en gevalideerd voor de in vitro studie van het Salmonella (enterica serovar) Typhimurium infectieproces. In dit doctoraat werd dit model voor het eerst uitgebreid met een commensale darmbacterie om gastheer-bacterie en bacterie-bacterie interacties in de darm te simuleren. Met behulp van dit model werd aangetoond dat reuterinevorming – en dus de aanwezigheid van glycerol – belangrijk is voor Lactobacillus reuteri (ATCC PTA 6475) om het darmepitheel te beschermen tegen Salmonella Typhimurium infectie. In een tweede deel werd een dynamisch model van de luminale dikke darmbacteriën (LSHIME) gebruikt om het effect van een verhoogde hoeveelheid LA in de dikke darm te onderzoeken. Daarbij werd in het bijzonder gekeken naar de overleving van Lactobacillus reuteri (ATCC PTA 6475) en de productie van het gezondheidsbevorderende korte keten vetzuur butyraat. Na blootstelling aan een hoge concentratie LA (1 g.L-1) namen de Lactobacillus reuteri niveaus af en werd de butyraatproductie geïnhibeerd. Een 'normale' (0.1 g.L-1) hoeveelheid LA leek geen effect te hebben. De hoge LA concentraties waren representatief voor een toelevering van 1 g LA in de dikke darm of een consumptie van ongeveer 20 g per dag. Interessant was dat de inhiberende effecten van hoge concentraties LA geneutraliseerd werden door het toevoegen van een mucine-agar omgeving, die de mucuslaag van het darmepitheel in vivo simuleerde (M-SHIME). Voor Lactobacillus reuteri werd aangetoond dat het beschermend effect van de mucine-agar omgeving veroorzaakt werd door de viscositeit van de mucine(-agar) en daarbij de vrije diffusie van LA naar de bacteriële celmembraan verhinderde. Butyraatproductie, anderzijds, werd gewaarborgd door de aanrijking van LA-biohydrogenerende en butyraatproducerende Roseburia spp. in de MSHIME ten opzichte van de L-SHIME. Daarbij werd in de M-SHIME efficiënte omzetting 


147


Samenvatting van LA naar het minder antimicrobiële vacceenzuur waargenomen, terwijl in de L-SHIME de LA-concentratie hoog bleef. De correlatie tussen LA-biohydrogenatie en butyraatproductie werd verder bevestigd door middel van herhaalde batch incubatie van fecale microbiota. Een verhoogde toelevering van glycerol en LA naar de dikke darm kan dus een significante impact hebben op microbiële processen en species die worden geassocieerd met de menselijke gezondheid. Daarbij werd besproken dat de impact van glycerol, i.e. accumulatie van reuterine, significant wordt bij een lage consumptie van voedingsvezels. Daarnaast werd aangetoond dat de LA-effecten afhankelijk zijn van de aanwezigheid van een gesimuleerde mucuslaag, wat suggereert dat een hoge consumptie van LA de gezondheid kan verslechteren in geval van een beschadigde mucuslaag, e.g. bij patiënten met een inflammatoire darmziekte. Toekomstig in vitro en in vivo onderzoek is nodig om de impact van onze waarnemingen voor de menselijke gezondheid te valideren. Bijzondere aandacht moet daarbij uitgaan naar de ontwikkeling van een gevalideerd in vitro model dat de in vivo gastheer-microbiota interacties in de darm simuleert.



148


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Curriculum Vitae

Curriculum Vitae Personal details Full name

Rosemarie De Weirdt

Place and date of birth

Ghent (Belgium) - May 4, 1985

Nationality

Belgian

Phone

+32 478 96 67 26

E-mail

[email protected] [email protected]

Education 2008 - 2012

Doctoral Schools of Life Sciences and Medicine (Ghent University)

2005 – 2008

Master of Bioscience Engineering option Environmental Technology (Ghent University) Greatest Distinction => Master thesis (2007-2008): Anaerobic metabolism of glycerol by the colon microbiota Promoter: Prof. Dr. ir. Willy Verstraete (LabMET) => Exchange program ‘Erasmus’ (2006-2007): Institute of Chemical Technology (ICT), Prague

2003 – 2005

Candidate Bioscience Engineer (Ghent University) Distinction

1997 – 2003

Secondary education Science-Mathematics (Sint-Bavo Humaniora, Ghent)

Research, teaching and working experience 2008 – now

Scientific collaborator at the Laboratory of Microbial Ecology and Technology (LabMET) Coupure Links 653, 9000 Ghent, Belgium Phone: +32 9 264 59 76 Fax: +32 9 264 62 48 Ph.D-research Dietary fat and the human gut microbiome Promoters: Prof. Dr. ir. Willy Verstraete - Prof. Dr. ir. Tom Van de Wiele



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Curriculum Vitae Teaching activities Practical exercises of the Master course ‘Microbial Ecology & Environmental Sanitation’ Tutor of 4 Master thesis students and 1 Bachelor project April-July 2011

Scientific collaborator at The Biodesign Institute, Center of Infectious Diseases and Vaccinology (Arizona State University, USA) Use and optimization of the 3-D organotypic model of the colon epithelium to study interactions between Lactobacillus reuteri and its fermentation products with the host Supervisors: Prof. Dr. Cheryl A Nickerson & Dr. Aurélie Crabbé

May 2009

Scientific collaborator at the Laboratory for Food Biotechnology (Swiss Federal Institute of Technology (ETH), Zürich, Switzerland) Production and purification of reuterin, a glycerol fermentation intermediate of the gut commensal Lactobacillus reuteri Supervisors: Prof. Dr. ir. Christophe Lacroix & Dr. Sabine Vollenweider

Awards Poster Presentation Price, Gut Day 2009 in Vlaardingen (The Netherlands) Belgian Alpro Foundation Award for Masters 2008 for master thesis research ‘Anaerobic metabolism of glycerol by the colon microbiota’

Publications A1 publications De Weirdt R, Possemiers S, Vermeulen G, Moerdijk-Poortvliet TCW, Boschker HTS, Verstraete W, Van de Wiele T (2010). Human faecal microbiota display variable patterns of glycerol metabolism. FEMS Microbiology Ecology 74: 601-611. De Weirdt R, Crabbé A, Roos S, Vollenweider S, Lacroix C, Sarker S, Van de Wiele T, Nickerson CA (2012). Glycerol supplementation enhances Lactobacillus reuteri’s protective effect against Salmonella Typhimurium colonization in a 3-D model of colonic epithelium. Plos One 7:11. Van den Abbeele P, Belzer C, Goossens M, Kleerebezem M, De Vos WM, Thas O, De Weirdt R, Kerckhof FM, Van de Wiele T (2012). Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model. The ISME Journal: in press.



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Curriculum Vitae Van den Abbeele P, Derde M, De Weirdt R, Vermeiren J, Possemiers S, Verstraete W, Van de Wiele T. Arabinoxylans, inulin and Lactobacillus reuteri repress the adherent-invasive Escherichia coli from mucus in a mucosa-comprising gut model. Microbial Biotechnology: accepted manuscript. De Weirdt R, Coenen E, Van den Abbeele P, Van de Wiele T. A simulated mucus layer protects Lactobacillus reuteri from the inhibitory effects of the PUFA linoleic acid. Submitted. De Weirdt R, Vlaeminck B, Mees E, Van den Abbeele P, Eeckhaut V, Fievez V, Vermeiren J, Verstraete W, Van de Wiele T. Inefficient linoleic acid biohydrogenation impairs butyrate production in the simulated gut microbiome. In preparation.

B2 Book chapters Marzorati M, Van den Abbeele P, Grootaert C, De Weirdt R, Marcos Carcavilla A, Vermeiren J, Van de Wiele T. In vitro models of the Human Microbiome. In: Julian Marchesi, The Human Microbiota and Microbiome. Wallingford: CABI. In press

Presentations De Weirdt R, Vermeulen G, Possemiers S, Van de Wiele T, Verstraete W. Glycerol metabolism by the human colonic microbiota. 11th International Gut Day, Vlaardingen, The Netherlands, 12th November 2009, Poster presentation. De Weirdt R, Vermeulen G, Possemiers S, Van de Wiele T, Verstraete W. Glycerol metabolism by the human colonic microbiota. Meeting of the Belgian Society for Microbiology, Brussels, Belgium, 11th December 2009, Poster presentation. De Weirdt R, Possemiers S, Vermeulen G, Moerdijk-Poortvliet T, Boschker HTS, Verstraete W, Van de Wiele T. Human faecal microbiota display variable patterns of glycerol metabolism. 7th Joint Rowett/INRA Symposium, University of Aberdeen, Scotland (UK), 22nd-25th June 2010, Poster presentation. De Weirdt R, Crabbé A, Roos S, Lacroix C, Verstraete W, Vanhoecke B, Bracke M, Nickerson CA, Van de Wiele T. Microbial reuterin production and its effects on a 3-D model of colonic epithelium. 1st International Symposium on Microbial Resource Management in Biotechnology, Ghent, Belgium, 30th June and 1st July 2011, Oral presentation. De Weirdt R, Vlaeminck B, Fievez V, Eeckhaut V, Vermeiren J, Possemiers S, Verstraete W, Van de Wiele T. Linoleic acid biohydrogenation and butyrate production upon rolled oats addition to a fed-batch reactor simulating conditions of the colon. 1st International Symposium on Microbial Resource Management in Biotechnology, Ghent, Belgium, 30th June and 1st July 2011, Poster presentation.



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Curriculum Vitae

De Weirdt R, Crabbé A, Roos S, Vollenweider C, Lacroix C, Sarker S, Van de Wiele T, Nickerson CA. Lactobacillus reuteri protects better against Salmonella Typhimurium infection of a model of human colonic epithelium when supplemented with glycerol. 13th International Gut Day, Wageningen, The Netherlands, 20th October 2011, Poster presentation. De Weirdt R, Vlaeminck B, Mees E, Fievez V, Eeckhaut V, Vermeiren J, Possemiers S, Verstraete W, Van de Wiele T. The PUFA linoleic acid reduced butyrate production while its biohydrogenation products are inert. 13th International Gut Day, Wageningen, The Netherlands, 20th October 2011, Poster presentation. De Weirdt R, Crabbé A, Roos S, Vollenweider S, Lacroix C, Sarker S, Van de Wiele T, Nickerson CA. Glycerol supplementation enhances Lactobacillus reuteri’s protective effect against Salmonella Typhimurium infection in the colon. 2nd International Symposium on Microbes for Health, Paris, France, 1st and 2nd December 2011, Poster presentation. De Weirdt R, Crabbé A, Roos S, Vollenweider S, Lacroix C, van Pijkeren JP, Britton RA, Sarker S, Van de Wiele T, Nickerson CA. Glycerol supplementation boosts Lactobacillus reuteri’s protective effect against Salmonella Typhimurium infection in a 3-D organotypic model of colon epithelium. 3rd TNO Conference on Beneficial Microbes, Noordwijkerhout, The Netherlands, 26th-28th March 2012, Oral presentation. De Weirdt R, Vlaeminck B, Mees E, Fievez V, Eeckhaut V, Vermeiren J, Possemiers S, Verstraete W, Van de Wiele T. Faecal butyrate production is inhibited by the PUFA linoleic acid but not by its biohydrogenation products. 3rd TNO Conference on Beneficial Microbes, Noordwijkerhout, The Netherlands, 26th-28th March 2012, Poster presentation.



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Dankwoord 

Zoveel geschreven, en toch is het belangrijkste nog niet gezegd. Dit doctoraat zou niet geworden zijn wat het is zonder de bijdrage en energie van heel wat mensen. Een woord van oprechte dank vind ik dan ook onontbeerlijk in dit werk.

Bedankt – Thank you – Merci – Danke – Tack!

Professor Verstraete, voor uw enthousiasme en waardevolle input van begin tot eind! Hoewel ik het in de eerste jaren niet altijd kon appreciëren dat u nog maar eens met een nieuw idee kwam aandraven, ben ik nu tevreden met dit resultaat dat hierdoor meerdere niveau’tjes is gestegen. Als u me toestaat dat ik even plagiaat pleeg, onthoud ik van u dat het gemakkelijker is om de dingen positief te bekijken (dan om inhibities te bestuderen). Tom, voor de verbeteringen van al mijn teksten en omdat ik op elk moment aan jouw deur kon komen kloppen met dringende vragen en problemen! Een bijzonder dankjewel ook om nog vlug mijn BOF-voorstel te gaan binnensteken op de dag van de deadline en voor de kans die je me biedt om nog wat op LabMET te blijven als postdoc op het GOA-project. Nico, omdat ik steeds bij jou terecht kon met vragen en ideeën over moleculair werk. Korneel, voor de tijd die je nam om mij wat thermodynamica bij te brengen. Professor Geert Huys, Professor Veerle Fievez, Professor Martine De Vos en Professor Frank Devlieghere, voor de tijd die jullie hebben genomen om dit werk te evalueren. Professor Stefan Roos, for evaluating this work, for our collaboration and for everything I’ve learnt from you about Lactobacillus reuteri. Professor Lacroix and Dr. Sabine Vollenweider from the Institute of Food, Nutrition and Health at the ETH in Zürich, for guiding me with those first steps in the science of reuterin. Sabine, Pierre et Coralie, merci de votre gentillesse et votre hospitalité pendant mon séjour à Zürich. Tanja Moerdijk, voor jouw vriendelijkheid en behulpzaamheid bij de HPLC-IRMS analyses. Barbara Vanhoecke en Professor Marc Bracke van het Laboratorium voor Experimenteel Kankeronderzoek van het Gentse Universitair Ziekenhuis en Kim Van Deun van de Vakgroep Pathologie, Bacteriologie en Pluimveeziekten van de Faculteit Diergeneeskunde te Gent, voor jullie geduld en expertise tijdens die frustrerende maanden van onherhaalbaar celwerk. aan de mensen van Lanupro: Veerle en Bruno voor jullie interesse en denkwerk in het biohydrogenatie-verhaal. Sjaraï en Charlotte, voor jullie hulp bij de extracties. to the collaborators of the Center of Infectious Diseases and Vaccinology at the Biodesign Institute of Arizona State University: Cheryl, Aurélie and Shameema, for your enthousiasm and for helping me grow in science! Jen and Rebecca, for your kindness and your cheerful chatter. Those were the days!



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Dankwoord  Krystal, Aurélie and Benny, for your friendship. You made my days in Phoenix like fresh strawberries make cheesecake and an episode of True Blood makes a Sunday evening! mijn thesisstudenten Ann, David, Els en Eva: voor jullie inzet en doorzettingsvermogen tijdens onze samenwerking. Bedankt voor alles wat ik van jullie heb mogen leren! Een speciaal woordje voor Eva en Els: merci voor jullie hun harde werk en zelfstandigheid toen ik met mijn dikke buik geen labo-werk meer kon doen. Jan, Jo, Eva, Tan, Annelies en Linde: de huidige generatie A2.086-bewoners: voor de aangename sfeer in ons bureau en op onze avondjes uit! Voor de hilarische quotes en de absurde gesprekken die de stress binnen de perken hielden. En ook voor jullie kussen-onderde-buik solidariteit in de laatste weken van mijn zwangerschap! For your company on congresses: Pieter en Peter, voor de voetbalwedstrijden en het ongedwongen netwerken in Aberdeen; Massimo, Sam en Tom, voor het plaatsje aan tafel bij ‘de groten’ in het Théâtre National de Chaillot in Parijs; Sahar and Noriko, for the wonderful diner and the walk back to my hotel in Paris. Annelies, voor de lange wandeling naar het strand, het extra kussen en het ochtendlijk zwemmen in Noordwijkerhout. Joan, Tim, Sam, Jessica, Griet, Yu, Annelies, Pieter VdC, Varvara, Beatriz, Jan A, Pieter VdA, Karen, David, Liesje and Jan S for the entertaining lunch breaks and the superb winter weekends! Carlos, for everything I’ve learnt from you about evaluating students and for the template of your cover. Thanks to you, I saved a lot of precious time! Kris en Regine, voor jullie hulp bij al die administratieve vragen en problemen die opduiken bij het afronden van een doctoraat. Tim, LabMET’s IT-redder-in-nood, voor het oplossen van al mijn computerproblemen en de prachtige overzichtsfiguur van hoofdstuk 3. Griet, voor jouw hulp bij de glycerolfermentaties – ook al had je daar niet voor getekend. Ellen en Jana, om steeds bij te springen als ik het praktische werk niet meer alleen aankon. Sam, Lynn, Selin, Charlotte, Pieter en Joan, de reeds-gedoctoreerde garde van de HAMcluster, voor de tips en tricks en het hart onder de riem, als ik het even niet meer zag zitten. Supervriendin en dokter in nood Nathalie voor onze 12-jarige vriendschap in goede en kwade dagen en de bio-ingenieurmeisjes Joni, Elien, Annabel, Elke, Sofie, Charlotte voor jullie interesse en de ontspannende brunchkes, koffiekletskes, kerstfeestjes... Barrah, voor onze dwalingen door de vallei van de Oude Kale die me toelieten om eens grondig door te denken op hypotheses en resultaten die op het eerste zicht niet logisch waren. Marleen en Johan, voor jullie interesse en al die goede zorgen, en natuurlijk ook voor de dinokoeken en de Inexkes. Marjolein, bijna Dr. Zus, voor het ‘Geef maar gaze’ briefje dat me telkens weer in gang deed schieten.



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Dankwoord 

Papa, om op tijd en stond het onderzoeksvuur terug aan te wakkeren en voor de ‘p’, want daar is het tenslotte allemaal mee begonnen. Mama, voor de rust en regelmaat waar je altijd voor zorgde tijdens mijn studies en het schrijven van dit doctoraat. Nu ik zelf mama ben, begrijp ik dat dit niet altijd zo evident moet zijn geweest. Stella, lieve meid, omdat je mama toch al af en toe eens goed laat doorslapen. Ik hoop dat je mag opgroeien tot een gelukkige jongedame (- maar ook dat je nog even klein mag blijven)! Tenslotte, Wim, voor al die kleine en grote dingen die je deed om dit doctoraat samen met mij af te leggen. Je was – en bent – er altijd: met verse fruitsla en een goed biefstuk op tijd en stond, maar ook om platen te helpen tellen in Phoenix en stalen te nemen van mijn reactor toen mijn arm in het gips zat. Er zijn geen woorden voor wat jij voor mij betekent. Nakupenda.



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