Microbiota and Metabolite Profiles Linked to Severity in Irritable Bowel Syndrome
Conditions: Irritable Bowel Syndrome, Microbial Colonization, Dietary Habits
Sponsor: Wageningen University

NL64950.081.18  
IBSQUtrition 
 
 
 
 
 
 
 
 
 
IBSQUtrition: Microbiota and Metabolite 
Profiles Linked to Severity in Irritable 
Bowel Syndrome 
 
RESEARCH PROTOCOL 
Version 2 Click her e to enter  a date. 
13 March 2018 
 

NL64950.081.18  
IBSQUtrition 
 
 
 
PROTOCOL TITLE 
Protocol ID 
NL64950.081.18 
Short title 
Microbiota profiling in IBS 
EudraCT number  
Not applicable 
Version 
2 
Date 
16-03-2018 
Coordinating investigator / 
project leader 
 
Iris Rijnaarts, PhD Candidate 
Division of Human Nutrition, Wageningen 
University & Research 
Stippeneng 4, 6708 WE Wageningen 
Phone: +31 (0)6 42 50 65 44 
E-mail: Iris.rijnaarts@wur.nl  
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
Prof. Ben Witteman, PhD MD  
Endowed Professor in Nutrition and Intestinal 
Health Transitional Care and Gastro-enterologist 
Wageningen University & Research (WUR) 
Division of Human Nutrition  
Stippeneng 4, 6708 WE Wageningen 
E-mail: Ben.witteman@wur.nl  
 
Erwin Zoetendal, PhD,  
Associate Professor 
Wageningen University & Research (WUR) 
Laboratory of Microbiology 
Stippeneng 4, 6708 WE Wageningen 
E-mail: Erwin.zoetendal@wur.nl  
 
Nicole de Wit, PhD  
Senior investigator  
Wageningen University & Research (WUR) 
Food and Biobased Research (FBR) 
Bornse Weilanden 9, 6708 WG Wageningen 
E-mail: Nicole.dewit@wur.nl 
  
Nicole de Roos, PhD  

NL64950.081.18  
IBSQUtrition 
 
 
 
Assistent Professor  
Wageningen University & Research (WUR) 
Division of Human Nutrition  
Stippeneng 4 
6708 WE Wageningen 
E-mail: Nicole.deroos@wur.nl 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
Wageningen University  
Division of Human Nutrition (Bode 62) 
PO Box 8129 
6700 EV Wageningen 
Phone: 0317-485302 
Subsidising party 
Ministry of Economic affairs (PPS project) and nine 
different industrial partners: Ingredia, Nexira, 
Naturex, Bíoiberica, Wecare, Roquette, Darling, 
Ingredion and Winclove. Because three partners 
(Darling, Naturex and Nexira) were just included 
(November 2017), only 6 partners have signed the 
contract. This will be updated soon.  
Independent expert (s) 
Dr. Naguib Muhsen 
Phone: +31 (0)20 354 777 / +31 (0)6 16963517 
E-mail: nmuhsen@hotmail.com  
Medical supervision (in Dutch: 
onderzoeksarts) 
  
 
Prof. Ben Witteman PhD MD  
Endowed Professor in Nutrition and Intestinal 
Health Transitional Care and Gastro-enterologist  
Wageningen University & Research (WUR) 
Division of Human Nutrition  
Stippeneng 4 
6708 WE Wageningen 
E-mail: ben.witteman@wur.nl  
Phone: +31 (0)317 48 51 86 
Study sites 
 
Wageningen University & Research (WUR) 
Division of Human Nutrition  
Stippeneng 4 
6708 WE Wageningen 
Phone: 0317-485302 
Pharmacy 
Not applicable  

NL64950.081.18  
IBSQUtrition 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor or legal representative: 
 
Head of Department: 
Dr R.J. Bino 
Managing Director 
Wageningen University, Department 
Agrotechnology and Food Sciences 
 
 
 
Coordinating Investigator / Project 
leader: 
Iris Rijnaarts, PhD Candidate 
Division of Human Nutrition 
Wageningen University & Research 
 
 
 
Principal investigator 
Prof. Ben Witteman, PhD MD 
Division of Human Nutrition 
Wageningen University & Research 
 
 
 
Medical supervision 
Dr. Ineke Klopping-Ketelaar 
Research Physician 
Division of Human Nutrition 
Wageningen University & Research 
 
 
 
 
 
 
 

NL64950.081.18  
IBSQUtrition 
 
 
 
TABLE OF CONTENTS 
1. 
INTRODUCTION AND RATIONALE ..................................................................10 
2. 
OBJECTIVES ...............................................................................................12 
3. 
STUDY DESIGN ...........................................................................................13 
4. 
STUDY POPULATION ....................................................................................15 
4.1 
IBS patients ............................................................................................15 
4.2 
Healthy controls .......................................................................................16 
4.3 
Screening visit .........................................................................................17 
4.4 
Sample size calculation .............................................................................17 
5. 
TREATMENT OF SUBJECTS ............................................................................19 
6. 
INVESTIGATIONAL PRODUCT ........................................................................20 
7. 
NON-INVESTIGATIONAL PRODUCT ................................................................21 
8. 
METHODS ...................................................................................................22 
8.1 
Study parameters/endpoints ......................................................................22 
8.1.1 
Main study parameter/endpoint ...........................................................22 
8.1.2 
Secondary study parameters/endpoints (if applicable) ............................22 
8.1.3 
Other study parameters (if applicable) ..................................................22 
8.2 
Randomisation, blinding and treatment allocation .........................................22 
8.3 
Study procedures .....................................................................................22 
8.4 
Withdrawal of individual subjects................................................................24 
8.4.1 
Specific criteria for withdrawal (if applicable) .........................................24 
8.5 
Replacement of individual subjects after withdrawal .....................................24 
8.6 
Follow-up of subjects withdrawn from treatment ..........................................24 
8.7 
Premature termination of the study ............................................................24 
9. 
SAFETY REPORTING .....................................................................................25 
9.1 
Section 10 WMO event ..............................................................................25 
9.2 
AEs, SAEs and SUSARs .............................................................................25 
9.2.1 
Adverse events (AEs) .........................................................................25 
9.2.2 
Serious adverse events (SAEs) ............................................................25 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .....................26 
9.3 
Annual safety report .................................................................................26 
9.4 
Follow-up of adverse events ......................................................................26 
9.5 
Data Safety Monitoring Board (DSMB) / Safety Committee ............................26 
10. 
STATISTICAL ANALYSIS ...............................................................................27 
10.1 
Primary study parameter(s) ....................................................................27 
10.2 
Secondary study parameter(s) ................................................................27 
10.3 
Other study parameters .........................................................................28 
10.4 
Interim analysis (if applicable) ................................................................28 
11. 
ETHICAL CONSIDERATIONS ..........................................................................29 
11.1 
Regulation statement .............................................................................29 
11.2 
Recruitment and consent ........................................................................29 
11.3 
Objection by minors or incapacitated subjects (if applicable) ......................29 
11.4 
Benefits and risks assessment, group relatedness .....................................29 

NL64950.081.18  
IBSQUtrition 
 
 
 
11.5 
Compensation for injury .........................................................................30 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ..........................31 
12.1 
Handling and storage of data and documents ............................................31 
12.2 
Monitoring and Quality Assurance (if applicable) .......................................31 
12.3 
Amendments ........................................................................................31 
12.4 
Annual progress report ..........................................................................32 
12.5 
End of study report................................................................................32 
12.6 
Public disclosure and publication policy ....................................................32 
13. 
STRUCTURED RISK ANALYSIS .......................................................................33 
13.1 
Potential issues of concern .....................................................................33 
13.2 
Synthesis .............................................................................................33 
14. 
REFERENCES ..............................................................................................34 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
7 of 35 
LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS 
 
ABR 
ABR form, General Assessment and Registration form, is the application form 
that is required for submission to the accredited Ethics Committee (In Dutch, 
ABR = Algemene Beoordeling en Registratie) 
AE 
Adverse Event 
ANOVA 
Analysis of Variance 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
EU 
European Union 
EudraCT 
European drug regulatory affairs Clinical Trials  
FFQ 
Food Frequency Questionnaire 
FODMAP Fermentable, Oligo-, Di-, Monosaccharides And Polyols: short chain 
carbohydrates that are poorly absorbed in the small intestine  
GCP 
Good Clinical Practice 
HADS 
Hospital Anxiety and Depression Score 
IB 
Investigator’s Brochure 
IBS 
Irritable Bowel Syndrome 
IBS-C 
Irritable Bowel Syndrome type constipation 
IBS-D 
Irritable Bowel Syndrome type diarrhoea  
IBS-M 
Irritable Bowel Syndrome type mixed: alternating between diarrhoea and 
constipation 
IBS-QoL 
Irritable Bowel Syndrome Quality of Life Questionnaire 
IBS-SSS 
Irritable Bowel Syndrome Symptom Severity Score 
IBS-U 
Irritable Bowel Syndrome: type unspecified: IBS with no specific alterations 
in stool pattern.  
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
METC  
Medical research ethics committee (MREC); in Dutch: medisch ethische 
toetsing commissie (METC) 
MLDS 
Maag Lever Darm Stichting: Dutch Association for gastro-intestinal diseases 
OTU 
Operational Taxonomic Units 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
8 of 35 
PDSB 
Prikkelbare Darm Syndroom Belangenvereniging: Dutch association voor IBS 
patients  
QoL 
Quality of Life 
RCT 
Randomized Controlled Trial 
(S)AE 
(Serious) Adverse Event  
SCFA 
Short-Chain Fatty Acids 
SPC 
Summary of Product Characteristics (in Dutch: officiële productinfomatie 
IB1-tekst) 
Sponsor 
The sponsor is the party that commissions the organisation or performance 
of the research, for example a pharmaceutical company, academic hospital, 
scientific organisation or investigator. A party that provides funding for a 
study but does not commission it is not regarded as the sponsor, but referred 
to as a subsidising party. 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
Wbp 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 
 
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
9 of 35 
SUMMARY 
Rationale: Irritable Bowel Syndrome (IBS) is a gastro-intestinal disorder that strongly 
affects Quality of Life and impairs daily functioning. However, the aetiology and 
pathophysiology has been poorly understood. Studies suggest that intestinal microbiota in 
IBS is altered, however a general consensus remains elusive. This may be due to the large 
individual variation in microbiota and IBS symptoms, and the cross-sectional designs. 
Moreover, other factors like diet, wellbeing and metabolite profiles are often not taken into 
account. New evidence is suggesting that IBS severity may be an important factor in 
microbiota composition.  
Objective: To determine faecal microbiota composition and metabolite production (such 
as acetate, propionate and butyrate), and investigate differences between healthy controls 
and mild or severe patients IBS. Moreover, to investigate whether (clinical) parameters 
such as symptom severity fluctuated, and if these fluctuations are associated with an 
alteration in faecal microbiota composition and metabolite production, one month after 
baseline, compared to healthy controls.  
Study design: This study is a longitudinal study, with two data collection points (baseline 
and after one month). 
Study population: For this study, 100 IBS patients and 30 healthy controls will be 
recruited. After the first data collection point (T1), the 30 most mild and 30 most severe 
IBS patients will be included for data collection at T2 (after one month). All subjects will 
be aged between 18 and 65 years. 
Intervention (if applicable): Not applicable.  
Main study parameters/endpoints: primary endpoint of this study is microbiota 
composition and metabolite profiles, and the difference between groups and possible 
change after one month. These are assessed two faecal samples, which are analysed by 
16S rRNA gene-based microbiota profiling. Secondary parameters are dietary intake, 
Quality of Life, depression and anxiety scores, and stool consistency and frequency, which 
are assessed by validated questionnaires.  
Nature and extent of the burden and risks associated with participation, benefit 
and group relatedness: This study has a relatively low burden and risk, since it is an 
observational study. Subjects only have to visit the study site once (at the information 
event). All data collection will be done from home, and will be done twice, which include 
faecal samples online questionnaire. 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
10 of 35 
1. INTRODUCTION AND RATIONALE 
Irritable Bowel Syndrome (IBS) is a functional gastrointestinal disorder, which affects 10 
to 20% of adults, with a female predominance [1-3]. Although IBS it not a life-threatening 
disorder, or no histological abnormalities are seen in the intestine, it strongly affects quality 
of life (QoL) and impairs daily functioning [1]. IBS is characterized by altered bowel habits, 
abdominal pain and discomfort, and is often diagnosed using the Rome IV criteria [4-6]. 
Based on predominant stool patterns, IBS can be roughly divided into four subtypes: IBS 
with constipation (IBS-C), IBS with diarrhoea (IBS-D), Mixed IBS (IBS-M) or Unsubtyped 
IBS (IBS-U) [4].  
 
The aetiology and pathophysiology of IBS is poorly understood. IBS has been associated 
with increased intestinal permeability, low grade inflammation, abnormal gut motility and 
increased visceral hypersensitivity [7, 8]. Evidence is indicating an important role for gut 
microbes in the pathophysiology of IBS. A meta-analysis pooling 18 studies found an 
increased risk for developing IBS after bacterial gastroenteritis after 1 year and even 36 
months [9]. Moreover, double blind randomized controlled trials (RCT) found improvement 
in symptoms after short-term treatment with antibiotics such as rifaximin and probiotics 
[8, 10-14].  
 
Several studies showed differences between microbiota of IBS patients, or subtypes, and 
healthy controls [13, 15, 16]. The Rome Team Working group (Simren et al 2013) 
concluded that there is good evidence supporting the concept that intestinal microbiota in 
IBS is altered. However, these studies lack general consensus and the specific microbial 
signature in IBS, even within IBS subtypes, remains elusive [17]. These conflicting results 
may be due to the cross-sectional design, the large individual variation in microbiota 
composition and relatively small sample size in many studies. Moreover, IBS is a chronic 
disorder with relapses and remissions, and large variation in severity of symptoms and 
type of defecation, even within the subtypes [5].  
 
Possibly, microbiota clusters may not match the traditional IBS subtypes, as found by 
Jeffrey et al (2012) [12]. Recently, Tap et al (2017) defined a microbiota signature which 
identified 90 operational taxonomic units (OTU’s). This signature showed associations with 
stool consistency, transit time, microbial richness, exhaled CH4, presence of 
Methanobacteriales, and enterotype distribution. IBS severity was found to be the a strong 
factor that was associated with this microbial signature. Other (clinical) parameters, like 
IBS subtypes, diet, medication, anxiety and depression scores were not strongly associated 
with the microbial signature [18]. Because this is the first study to define a microbiota 
signature that is linked to severity, and this microbial signature had a relatively low 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
11 of 35 
sensitivity, results should be interpreted with caution, the more since the set-up of the 
study was cross-sectional. Moreover, Tap et al (2017) did not find associations between 
microbiota profile and anxiety and depression scores, while Jeffrey et al (2012) found that 
IBS patients with normal-like microbiota had an increased score of depression and anxiety, 
again highlighting the lack of consensus observations [12]. Further research is thus needed 
to elucidate the microbiota signature in IBS and its link with other factors like severity, 
QoL, depression, anxiety and diet over time.  
 
Among IBS patients, 91% report symptom onset related to diet. Food groups such as 
cereals, spicy foods, vegetables, fatty foods, animal protein, dairy, fruits, nuts, seeds and 
caffeine are reported as most problematic [19]. FODMAPS (Fermentable, Oligo, Di, 
Monosaccharides and Polyols; a specific group of carbohydrates) are often problematic in 
IBS patients as they are fermented by intestinal microbes, thus increasing gas production 
and possibly provoking IBS symptoms. These effects of diet, and especially consumption 
of dietary fibre, on intestinal microbiota composition have been demonstrated in studies. 
Fibres are largely resistant against digestion in the human intestine, and are therefore able 
to reach the colon, where they increase viscosity and faecal mass [20]. The fibres are 
fermented by microbiota in the colon, which results in groups of metabolites [21]. Short-
Chain Fatty Acids (SCFA), including acetate, propionate and butyrate, are the major group 
[20, 22]. Recent research indicates that faecal SCFA levels may be altered in bowel 
disorders [23], and that SCFA can play a role in prevention and treatment of bowel 
disorders. Studies show that SCFA administration positively influenced Ulcerative Colitis, 
Crohn’s disease and antibiotic-associated diarrhoea [24-26]. In IBS patients, several 
studies suggest that faecal SCFA levels are altered, but these are limited and inconsistent 
[27-30]. Moreover, SCFAs are a major product of FODMAP fermentation, and since IBS 
patients often limit FODMAP intake, the reduction of faecal SCFA may just rather reflect 
diet than be a feature of disease [27]. 
 
To date, much remains unclear about the role of microbiota in IBS, and the association 
with other parameters like diet and wellbeing. Categorizing IBS patients into the traditional 
IBS subtypes did not reveal subtype-specific microbiota compositions. Moreover, SCFA 
levels in IBS have been limited investigated, and controlled for factors like diet. In our 
study, we aim to investigate microbiota composition and SCFA in IBS patients, categorized 
by severity of symptoms, and investigate associations with diet, depression, anxiety and 
QoL, as well as defecation type at time of sampling. Moreover, we aim to assess to what 
extent severity and other parameters change after one month, and if, and to what extent 
we see changes in microbiota and SCFA profiles.  

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
12 of 35 
2. OBJECTIVES 
Primary Objective:  
To determine faecal microbiota composition and metabolite production (SCFA such as 
acetate, propionate and butyrate), and investigate differences mild or severe patients IBS.  
 
Moreover, this study aims to investigate whether (clinical) parameters such as symptom 
severity fluctuated, and if these fluctuations are associated with an alteration in faecal 
microbiota composition and metabolite production, one month after baseline, and if this is 
different between mild or severe IBS patients.  
 
Secondary Objective(s):  
- 
To investigate whether microbiota composition and metabolite production are 
associated with dietary intake, depression & anxiety and QoL, and whether this 
differs between mild IBS and severe IBS.  
- 
To investigate if one month after baseline measurements, dietary intake, QoL and 
depression & anxiety changed, and if they are associated with possible alterations 
in IBS severity, microbiota composition or metabolite profiles.   
 
 
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
13 of 35 
3. STUDY DESIGN 
The acronym IBSQUtrition stands for Irritable Bowel Syndrome, Questionnaire, QoL, and 
Nutrition. This research project is in collaboration with three departments of Wageningen 
University & Research: the division of Human Nutrition, Food and Biobased Research (FBR) 
and the Laboratory of Microbiology. IBSQUtrition consist of several studies, and aims to 
investigate the effects of nutrition on IBS symptoms, in order to improve QoL. The first 
study conducted within this research project was a large online questionnaire-study, which 
assessed associations between nutrition, IBS symptoms and QoL.   
 
This study focusing on composition and metabolite production of the microbiota has an 
observational longitudinal design. IBS subjects will be recruited from the online 
questionnaire-study, which has been conducted previously. In order to understand 
intestinal microbiota in IBS and its association to other parameters, such as severity and 
nutrition, IBS patients that completed the online questionnaire and gave permission to 
receive more information on follow-up studies, will be invited for an information event. We 
aim to recruit 100 IBS patients. After the first data collection (T1), assessment of IBS 
severity will be done according to the IBS Severity Symptom Score (IBS-SSS). IBS patients 
who are classified either as most mild (n=30) or most severe (n=30) will be included for 
follow-up at T2, which is one month later. Figure 1 shows this schematically.  
 
As mentioned before, IBS is a chronic disorder with relapses and remissions, and large 
variation in severity of symptoms and type of defecation [31, 32]. Moreover, studies 
suggest that microbiota can differ over time [33]. Therefore, we will assess parameters at 
two time points, with one month in between, to have a better understanding of this 
variability. Since IBS severity is also highly variable, but we would like to specify groups 
based on IBS severity (mild or severe), we will assess a larger group at T1 (n=100). After 
T1, we will select IBS patients who are the most mild or most severe. This study design 
will give us the opportunity to do a cross-sectional analysis at T1, where data is collected 
at the same moment to reduce variability, and to elucidate whether (and to what extent) 
there is a change in severity and other parameters after one month (T2), and if microbiota 
and metabolite profiles also changed, and to what extent. Healthy controls will be tested 
at T1 and T2 for comparison.   
 
There will be two moments of data collection in this study (T1 and T2). At T1 and T2, all 
subjects (both IBS subjects and healthy controls) will fill out several online questionnaires 
on dietary intake, symptom severity, stool form and wellbeing. Moreover, they will collect 
a faecal sample for analysis. More detailed information on specific methods for data 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
14 of 35 
collection can be found at chapter 8 Methods. Figure 2 gives a schematic overview of the 
data collection.  
 
Figure 1. Flowchart of the selection of study population 
 
 
Figure 2. Overview of the data collection 
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
15 of 35 
4. STUDY POPULATION 
4.1 IBS patients 
This study will be performed in IBS patients who already participated in a previous cross-
sectional questionnaire study. For the questionnaire study, patients were recruited online, 
in close cooperation with the Dutch Association for IBS (Prikkelbare Darm Syndroom 
Belangenvereniging, PDSB), the Dutch Assocation for gastro-intestinal diseases (Maag 
Lever Darm Stichting, MLDS), and hospital the Gelderse Vallei Ede, where our PI MD Ben 
Witteman has an outpatient clinic for IBS patients. After completion of the online 
questionnaire, IBS patients could choose to receive more information on this study. When 
IBS patients are willing to participate, they will be invited for an information meeting, 
where they can receive more information. If recruitment from the subjects of the online 
questionnaire is not successful, there will be an open call for recruitment of IBS patients. 
This will be done in collaboration with the PDSB and online using the website 
http://voedingsonderzoek.wur.nl.  
 
In order to be eligible, IBS patients must fulfil the following criteria:  
- 
Adults, aged 18-65 years. Research shows that microbiota composition and activity 
can change in elderly [34].  
- 
IBS patients that meet the Rome IV criteria [5], as shown in table 1. This will be 
evaluated by the principal and/or medical investigator.  
- 
In close proximity of Wageningen (max. 50 km), for practical reasons: collection of 
the faecal samples.  
- 
Have an Body Mass Index (BMI) between 18.5 and 30 kg/m2. Microbiota was found 
to be altered in obese subjects, therefore these subjects will be excluded. [35].  
- 
Signed informed consent. 
- 
After T1, IBS severity will be checked using the IBS-SSS. Subjects will be included 
for follow-up at T2, when they have the most mild symptoms (n=30) or most severe 
(n=30) symptoms.  
 
IBS patients will be excluded from participation of this study, when:  
- 
Presence of gastro-intestinal diseases, such as celiac disease, Crohn’s disease, or 
Ulcerative colitis. 
- 
Have a history of intestinal surgery that might interfere with study outcomes. This 
does not include an appendectomy or cholecystectomy.  
- 
Presence of significant systemic diseases, such as diabetes mellitus, cancer, 
cardiovascular disease or respiratory disease. 
- 
Female subjects: currently pregnant or breast-feeding.  

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
16 of 35 
- 
Use of antibiotic treatment less than 3 months before start of the study and no use 
of antibiotics during the study. 
- 
Are a subject in another study during this study.  
- 
Are an employee or student of the Division of Human Nutrition, Food and Biobased 
Research or Laboratory of Microbiology, of Wageningen University & Research.   
- 
Unwilling or unable to fulfil study criteria. 
- 
If they are not selected in the most mild or most severe group at T2.  
 
Table 1. Rome IV criteria 
                                                                                                                                                         
4.2 Healthy controls 
Healthy volunteers will be recruited through the database of the division of Human 
Nutrition, Wageningen University & Research. They will be matched for age (±5 years), 
gender and Body Mass Index (±1 kg/m2). Healthy controls will be matched with the mild 
IBS patients, since the smallest difference is expected between those groups. An e-mail 
will be sent out explaining the study and a call for recruitment, and will be posted on 
http://voedingsonderzoek.wur.nl. Healthy controls will be recruited after the IBS subjects 
finished the study, in order to be sufficiently matched. Subjects will be invited for an 
information event, where eligibly will be checked. Controls will be eligible for participation 
in this study when: 
Rome IV criteria for IBS 
Patient has recurrent abdominal pain (≥1 day per week, on average, in the previous 
3 months), with an onset of ≥6 months before diagnosis. 
Abdominal pain is associated with: 
- 
Pain related to defecation 
- 
Change in frequency of stool 
- 
Change in form (appearance) of stool 
Patient has none of the warning signs: 
- 
Age ≥50 years, no previous colon cancer screening and presence of symptoms 
- 
Recent change in bowel habit 
- 
Evidence of overt GI bleeding (i.e., melena or haematochezia) 
- 
Nocturnal pain or passage of stools 
- 
Unintentional weight loss 
- 
Family history of colorectal cancer or inflammatory bowel disease 
- 
Palpable abdominal mass or lymphadenopathy 
- 
Evidence of iron-deficiency anaemia on blood testing 
- 
Positive test for faecal occult blood 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
17 of 35 
- 
Adults, aged 18-65 years. 
- 
No history of IBS, as assessed by the Rome IV criteria.  
- 
Has a score <75 from the IBS-SSS.  
- 
Are age (±5 years), gender and Body Mass Index (±1 kg/m2) matched with IBS 
patients. 
- 
In close proximity of Wageningen (max. 50 km), for practical reasons: collection of 
the faecal samples.  
- 
Signed informed consent. 
 
Subjects will be excluded from participation, when: 
- 
Presence of gastro-intestinal diseases, such as celiac disease, Crohn’s disease, or 
Ulcerative colitis. 
- 
Have a history of intestinal surgery that might interfere with study outcomes. This 
does not include an appendectomy or cholecystectomy.  
- 
Presence of significant systemic diseases, such as diabetes mellitus, cancer, 
cardiovascular disease or respiratory disease. 
- 
Female subjects: currently pregnant or breast-feeding. 
- 
Use of antibiotic treatment less than 3 months before start of the study. 
- 
Are an employee of the Division of Human Nutrition, Food and Biobased Research 
or Laboratory of Microbiology, of Wageningen University & Research.  
- 
Are subjects in another study during this study.  
- 
Unwilling or unable to fulfil study criteria. 
 
4.3 Screening visit 
Possible subjects will be invited for an information event, where additional information on 
the study will be given and subjects have an opportunity to ask questions. If willing to 
participate, subjects will sign an informed consent (see Appendix E2). After this, they will 
receive a questionnaire to assess eligibility. If the informed consent is signed and subjects 
are eligible, the intended start of date of the study is within 4 weeks. This study will first 
start with assessment of IBS patients. When all IBS patients have finished the study, 
recruitment for healthy controls will be open, in order to match controls to the IBS patients.  
 
4.4 Sample size calculation 
We used a somewhat comparable study of Mättö et al (2005), which investigated the 
stability of microbiota composition in faecal samples of IBS patients and controls at 
baseline, 3 months and 6 months [33]. Based on their study, we expect to find a difference 
of 3.6 (with a standard deviation of 4.9) in similarity of intestinal microbiota between IBS 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
18 of 35 
patients and controls over time. In our calculation, we used an α=0.05 (probability of Type 
I error) and a β=0.2 (probability of Type II power).  
We used the following formula: 𝑛= 2 ×
(𝑍𝛼+𝑍𝛽)2
(𝜎
𝛿
⁄ )2 . When filled in, this results in the following 
formula: 𝑛= 2 ×
7.9
(4.9 3.6
⁄
)2 = 29.3 
Therefore, our aim is to include 30 research subjects per group: 30 most mild IBS patients, 
30 most severe IBS patients, and 30 healthy controls.  
 
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
19 of 35 
5. TREATMENT OF SUBJECTS 
Not applicable.  

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
20 of 35 
6. INVESTIGATIONAL PRODUCT  
Not applicable.  

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
21 of 35 
7. NON-INVESTIGATIONAL PRODUCT 
Not applicable.  

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
22 of 35 
8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 
Main study parameter/endpoint 
The main study endpoint is microbiota composition, metabolite production and 
other faecal markers. This will be assessed with 16S rRNA gene-based microbiota 
profiling. At T1 and T2, subjects will collect a faecal sample for microbiota profiling. 
Faecal samples will be used for 16S rRNA gene-based profiling and subsequent 
analyses of a variety of microbial ecological characteristics, including microbiota 
composition, α-diversity (diversity within the sample), β-diversity (diversity 
between the samples) and group differences. Moreover, metabolites such as 
propionate, butyrate and acetate will be determined, which are an end product of 
bacterial metabolism in the large intestine [28]. 
8.1.2 
Secondary study parameters/endpoints (if applicable) 
Secondary endpoints are assessed at T1 and T2, and include QoL, depression & 
anxiety scores, symptom severity, stool frequency and form. Dietary intake will 
only be assessed at T1. All questionnaires will be equal for IBS patients and healthy 
controls.  
8.1.3 
Other study parameters (if applicable) 
All subjects fill in a general screening questionnaire to assess characteristics such 
as age, gender, educational level, ethnicity, body weight, height, physical activity 
and smoking history. In order to check eligibility, the screening questionnaire is 
different for healthy controls and IBS patients: for the healthy controls the IBS-
SSS is added, for the IBS patients the ROME IV criteria is added. At T2, subjects 
will be asked whether they changed their diet during the last month, and yes, what 
changes they made.   
8.2 Randomisation, blinding and treatment allocation 
Since this is an observational study, this is not applicable.  
 
8.3 Study procedures 
A general overview is shown in table 2. This study consists of two data collection 
time points (T1 and T2), which are separated by one month. All questionnaires can 
be found in Appendix F1. The following parameters will be assessed:  
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
23 of 35 
Microbiota and metabolite profiling: Faecal material will be collected at T1 and T2. 
After collection, faecal material will be immediately frozen at the home freezers of 
the subjects, transported on dry ice to the laboratory and subsequently frozen at -
80 °C until analysis. DNA will be isolated from part of the faecal samples and 
subsequently be used for 16S rRNA gene-based microbiota profiling using NG-tax 
as described earlier [36, 37]. Metabolite concentrations (including SCFAs) will be 
determined by HPLC as described earlier [38, 39]. A variety of multivariate 
analytical tools available as R scripts will be used to determine differences between 
microbiota composition and diversity between subjects, as well as determining 
microbial taxa that are associated with parameters derived from the different 
questionnaires.  
 
IBS severity: will be assessed using the validated score IBS-SSS [40]. Maximum 
achievable score is 500. A score <75 indicates no IBS or in remission, 75-175 is 
mild IBS, 175-300 indicated moderate IBS, and >300 indicates severe IBS. The 
IBS-SSS score will be assessed at T1 and T2. Since we only have a relatively small 
group, these cut-off points will not be used, but the most mild IBS patients and 
most severe IBS patients will be selected as the study population for T2.  
 
Dietary intake: will be assessed using a validated Food Frequency Questionnaire 
(FFQ). An FFQ is a valid way to assess habitual dietary intake [41]. Subjects will 
receive an online invitation with login to fill in their FFQ. From this FFQ, energy 
content (kcal) and carbohydrate, protein, fat, saturated fat, fibre intake (g and 
en%) will be assessed. Moreover, water content (L) and food groups such as 
vegetable and fruit intake, will be assessed. If subjects indicate in the FFQ that they 
follow the FODMAP diet, a dietician will call them to ask what specific groups they 
are excluding from their diet. This gives more insight when subjects data who follow 
the FODMAP diet differs significantly from those who are not following the diet.   
 
Quality of Life: quality of life will be at T1 and T2, using the validated 34-item 
Irritable Bowel Syndrome Quality of Life questionnaire (IBS-QoL). This 
questionnaire gives an overall score of QoL and holds different subscales, such as 
dysphoria, interference with activities, body image, health worry, food avoidance, 
social reaction, sexual and relationships [42].  
 
Depression and anxiety: will be assessed at T1 and T2 using the validated 
questionnaire Hospital Anxiety and Depression score (HADS) [43]. This is a 14-item 
questionnaire, which gives a screening score for anxiety and depression. 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
24 of 35 
 
Stool frequency and form: will be assessed using the validated Bristol Stool Chart. 
Stool frequency will be assessed using general questions, such as: “Please think of 
the week month. How many days did you have defecation?” “Please think of an 
average day on which you have defecation. During this day, how many times did 
you have defecation?” “Did you feel relief of abdominal pain after defecation?” 
 
Table 2. Schematic overview of the study procedure 
 
 
T1 
 
T2 
Collection of faecal samples 
X 
X 
IBS Symptom Severity Score (IBS-SSS) 
X 
X 
IBS Quality of Life Questionnaire (IBS-QoL) 
X 
X 
Hospital Anxiety and Depression Scale (HADS) 
X 
X 
Stool frequency and form 
X 
X 
Food Frequency Questionnaire (FFQ) 
X 
 
IBS: Irritable Bowel Syndrome 
 
8.4 Withdrawal of individual subjects 
Subjects may discontinue the trial at any moment without the obligation to state 
the reason for discontinuation. Subjects may be withdrawn from the study by the 
principal investigator if they do not comply with the rules and regulations of the 
study. Subjects may be withdrawn from the study by the medical supervisor in 
case of reported serious adverse events or in case of other medical, social or 
psychological events as evaluated by the medical investigator and discussed with 
the principal investigator. 
8.4.1 
Specific criteria for withdrawal (if applicable) 
 
         Not applicable. 
8.5 Replacement of individual subjects after withdrawal 
If more than five subjects per group withdraw to participate at the beginning of 
the study, new subjects will be recruited in order to obtain sufficient power. 
 
8.6 Follow-up of subjects withdrawn from treatment 
 
        Not applicable.  
 
8.7    Premature termination of the study 
A premature termination of the study is not expected, since this is an observational 
study. If the study is premature terminated, data which was collected before the 
termination will be analysed.  

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
25 of 35 
9. SAFETY REPORTING 
9.1 Section 10 WMO event 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety.  The sponsor will notify the accredited METC with undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending further review by the accredited METC. The investigator will take care that all 
subjects are kept informed. 
 
9.2 AEs, SAEs and SUSARs 
9.2.1 
Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to the study. All adverse events 
reported spontaneously by the subject or observed by the investigator or his staff 
will be recorded. Adverse events will be reported to the reviewing METC. 
9.2.2 
Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that at any 
dose:  
- 
Results in death; 
- 
Is life threatening (at the time of the event); 
- 
Requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
Results in persistent or significant disability or incapacity; 
- 
Is a congenital anomaly or birth defect; 
- 
Any other important medical event that may not result in death, be life 
threatening, or require hospitalization, may be considered a serious adverse 
experience when, based upon appropriate medical judgement, the event may 
jeopardize the subject or may require an intervention to prevent one of the 
outcomes listed above. 
 
The investigator will report all SAEs to the sponsor without undue delay after 
obtaining knowledge of the events. 
The sponsor will report the SAEs through the web portal ToetsingOnline to the 
accredited METC that approved the protocol, within 7 days of first knowledge for 
SAEs that result in death or are life threatening followed by a period of maximum 
8 days to complete the initial preliminary report. All other SAEs will be reported 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
26 of 35 
within a period of maximum 15 days after the sponsor has first knowledge of 
the SAE.  
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) 
Not applicable.  
 
9.3 Annual safety report 
 
Not applicable. 
 
9.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported till end of study within the Netherlands, as defined in the 
protocol.  The follow-up of AE’s will also be reported in the annual progress report. 
 
9.5 Data Safety Monitoring Board (DSMB) / Safety Committee 
Not applicable. 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
27 of 35 
10. STATISTICAL ANALYSIS 
Data will be presented as mean ± standard deviation (SD) if normally distributed, or 
median (interquartile range) when skewed. Normal distribution will be verified by the 
Kolmogorov-Smirnov test. To test associations between parameters, multiple linear 
regression will be used. Data will be tested for confounding or effect mediators, and 
confounding factors will be added to the regression. Microbiota data will be assessed 
using the statistical program R. Other data will be assessed using SPSS statistics. A p-
value of <0.05 will be considered significant.   
 
10.1  Primary study parameter(s) 
A variety of in house R-scripts and the Phyloseq package will be used for microbiota 
composition analyses using R v3.4.0 software (The R Foundation for Statistical 
Computing, Vienna, Austria). To determine the abundance differences of microbial 
groups at various phylogenetic levels within individuals a paired Wilcoxon test will be 
used. 
 
To contrast between the different IBS groups (over time effect), a linear mixed model 
taking into account the effects of repeated measurements using the lme4 package will 
be used. Variables that will be added to the model are time of sampling, group, gender, 
age, and BMI. Diversity of the microbiota will be quantified at different levels (taking 
into account the impact of abundance)  using the Vegan package (https://cran.r-
project.org/web/packages/vegan/vegan.pdf) and picante package (http://picante.r-
forge.r-project.org/picante-intro.pdf). Analysis of variance with the Tukey Honest 
Significant post hoc analysis will be applied to compare beta diversity between and 
within groups. P values will be corrected for multiple comparisons using the Benjamini–
Hochberg procedure. To correct for a false discovery rate (FDR), FDR adjusted p-values 
will be computed, a so called q-value [44]. A corrected q-value of less than 0.2 was 
considered significant. Student T-tests and ANOVA will be used to evaluate differences 
between faecal metabolite concentration over time and between groups. 
 
10.2  Secondary study parameter(s)  
Secondary parameters are dietary intake, QoL, depression and anxiety scores, 
symptom severity, stool frequency and form (Bristol Stool score). These will be 
assessed using various validated questionnaires. For the Bristol stool score, the top 3 
type of stools will be determined, ranging from 1 (hard to pass) to 7 (entirely liquid).   

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
28 of 35 
Differences between T1 and T2 will be assessed using a repeated measure Analysis of 
Variance (ANOVA) if normally distributed, otherwise a Kruskall-Wallis test when 
skewed. Variables such as age, gender, BMI and group will be added to the model.  
 
10.3  Other study parameters 
Other study parameters include age, gender, ethnicity, educational level, body weight, 
height, and lifestyle factors such as physical activity and smoking. These variables will 
be checked if they differ across the groups using an ANOVA, or when skewed a Kruskall-
Wallis test. Moreover, these variables will be assessed if they are a confounder or effect 
mediator, and if they have to be in the multiple linear regression model.  
 
10.4  Interim analysis (if applicable) 
When all IBS patients completed the study, a short interim analysis will be done to 
check for distribution of age, gender and BMI. These data will be used to recruit age, 
gender and BMI matched healthy controls. All other data will not be analysed until full 
completion of the study. 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
29 of 35 
11. ETHICAL CONSIDERATIONS 
11.1  Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki 
(64th WMA Assembly, October 2013), and in accordance with the Medical Research 
Involving Human Subjects Act (WMO 1998) and other guidelines, regulations and Acts.  
 
11.2  Recruitment and consent 
IBS patients will be recruited through a previous online questionnaire study. Subjects 
who participated in that study can choose at the end of the questionnaire if they would 
like to receive more information on follow-up studies like this microbiota profiling study. 
If they indicated this, they will receive a patient information folder (see Appendix E1). 
When willing to participate, subjects will be invited for an information meeting. Subjects 
will receive additional information, and there is time to answer questions. Within 4 
weeks after the information meeting, the subject has to sign the informed consent if 
they wish to enter the study. After this, eligibility will be checked. Healthy subjects will 
be recruited using the online database the division of Human Nutrition of Wageningen 
University & Research. If willing and eligible, subjects will be invited for an information 
meeting (separate meeting than for IBS patients). There, additional information will be 
given and subjects have the chance to ask questions. Within 4 weeks after the 
information meeting, subjects have to sign the informed consent if they wish to enter 
the study.  
 
Subjects will sign the informed consent first, followed directly by the investigator, and 
the subject will receive a copy of the signed informed consent directly after signing. 
The informed consent is enclosed in Appendix E2. 
 
11.3  Objection by minors or incapacitated subjects (if applicable) 
 
Not applicable.  
 
11.4  Benefits and risks assessment, group relatedness 
This study can help elucidate the association between IBS severity, microbiota 
composition and diet, QoL, depression and anxiety. There is no additional risk for the 
subjects, since this is an observational study. Subjects will have to invest time at three 
points: information meeting (screening), and T1 and T2, where they will fill in online 
questionnaires and collect a faecal sample. They will only have to visit the study site 
once, for the information meeting. After screening, all data collection will be done 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
30 of 35 
online. Subjects can collect and store faecal samples at home, and will be picked up by 
the investigator.  
 
11.5  Compensation for injury 
Since this is an observational study with no invasive measurements, there is no 
additional risk for the subject. Therefore, an exemption for insurance will be asked of 
the METC. This letter can be found in Appendix G1.  
 
11.6 Incentives (if applicable) 
Subjects will receive a financial compensation of €25,- after completion of this study. 
If IBS patients completed T1, but are not selected for T2, they will receive a financial 
compensation of €15,-. Subjects are free to withdraw from further participation for any 
reason and at any time during the study and will receive a proportional repayment for 
the effort they made.  
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
31 of 35 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1  Handling and storage of data and documents 
Before the start of the study, subjects will be assigned a study code that will remain 
the same during the study. The code list with both the codes and the names of the 
subjects will only be accessible for researchers of this study in a password protected 
file. For all other purposes, the study code will be used for subjects identification. The 
informed consent will be stored separately from all other information. No names of the 
patients will be used in the publication of this study. All researchers have signed a 
confidentially statement. The handling of personal data will be done according to the 
General Data Protection Regulation (GDPR), which is enforced at 25th of May 2018, and 
is registered on the website of the Dutch Data Protection Authority (AP). According to 
standard data management procedures, all research data are stored for a period of at 
least 15 years after collection. Thereafter, data and documents will be deleted and 
destroyed (e.g. by a shedder). Biological samples will be disposed at the end of the 
study according to biomedical waste procedures. 
 
12.2  Monitoring and Quality Assurance (if applicable) 
Monitoring will not be done, this is a relatively short study with minimum risks due to 
the observational study design. 
 
12.3  Amendments  
Amendments are changes made to the research after a favourable opinion by the 
accredited METC has been given. Non-substantial changes (such as typing errors, 
administrative changes like changes in names, telephone numbers and other contact 
details of involved persons mentioned in the submitted study documentation) will not 
be notified to the reviewing METC.  Substantial amendments will be notified to the 
METC that gave a favourable opinion. The documentation that will be included in the 
submission should cover the following information: 
1. Covering letter, including the reasons for the amendment in one or two 
sentences, a brief description of the changes that are included in the 
amendment and the name of the documents that are modified; 
2. An extract of the modified documents, where applicable, showing both the 
previous and new wording, where applicable. 
3. The new version of the modified documents, where applicable, identified with 
updated number of version and date. 
 
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
32 of 35 
12.4  Annual progress report 
Because the study period is less than a year, the investigator will submit a final study 
report within one year after the end of the study to the accredited METC.  
 
12.5  End of study report 
The investigator will notify the accredited METC of the end of the study within a period 
of 8 weeks. The end of the study is defined as the last data collection of the last subject. 
In case the study is ended prematurely, the investigator will notify the accredited METC 
within 15 days, including the reasons for the premature termination. Within one year 
after the end of the study, the investigator/sponsor will submit a final study report with 
the results of the study, including any publications/abstracts of the study, to the 
accredited METC. 
 
12.6  Public disclosure and publication policy 
All trial results, both positive and negative, will be disclosed in agreement with the CCMO 
statement on publication policy. Regardless of the outcome of the study, the results will 
be submitted for publication to a peer-reviewed scientific journal. The authorship of the 
article shall be determined in appropriate consultations based on a considerable 
contribution to set-up and execution of the study and an active participation in 
publication. The nine industrial partners are entitled to examine the manuscript prior to 
publication and make comments on it. None of the partied concerned has the right of 
veto considering publication.  
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
33 of 35 
13. 
STRUCTURED RISK ANALYSIS  
13.1  Potential issues of concern 
Not applicable.  
 
13.2  Synthesis 
Not applicable.  
 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
34 of 35 
14. 
REFERENCES 
1. 
Hungin, A., et al., The prevalence, patterns and impact of irritable bowel syndrome: an 
international survey of 40 000 subjects. Alimentary pharmacology & therapeutics, 2003. 
17(5): p. 643-650. 
2. 
Saito, Y.A., P. Schoenfeld, and G.R. Locke III, The epidemiology of irritable bowel 
syndrome in North America: a systematic review. The American journal of 
gastroenterology, 2002. 97(8): p. 1910-1915. 
3. 
Canavan, C., J. West, and T. Card, The epidemiology of irritable bowel syndrome. Clinical 
epidemiology, 2014. 6: p. 71. 
4. 
Longstreth, G.F., et al., Functional bowel disorders. Gastroenterology, 2006. 130(5): p. 
1480-1491. 
5. 
Ford, A.C., B.E. Lacy, and N.J. Talley, Irritable Bowel Syndrome. New England Journal of 
Medicine, 2017. 376(26): p. 2566-2578. 
6. 
Drossman, D.A., Functional gastrointestinal disorders: history, pathophysiology, clinical 
features, and Rome IV. Gastroenterology, 2016. 150(6): p. 1262-1279. e2. 
7. 
Ramsay, D.B., et al., Mast cells in gastrointestinal disease. Gastroenterology & hepatology, 
2010. 6(12): p. 772. 
8. 
Kassinen, A., et al., The fecal microbiota of irritable bowel syndrome patients differs 
significantly from that of healthy subjects. Gastroenterology, 2007. 133(1): p. 24-33. 
9. 
Thabane, M., D. Kottachchi, and J. Marshall, Systematic review and meta‐analysis: the 
incidence and prognosis of post‐infectious irritable bowel syndrome. Alimentary 
pharmacology & therapeutics, 2007. 26(4): p. 535-544. 
10. 
Pimentel, M., et al., The effect of a nonabsorbed oral antibiotic (rifaximin) on the symptoms 
of the irritable bowel syndrome. Ann Intern Med, 2006. 145(8): p. 557-563. 
11. 
Sharara, A.I., et al., A randomized double-blind placebo-controlled trial of rifaximin in 
patients with abdominal bloating and flatulence. The American journal of gastroenterology, 
2006. 101(2): p. 326-333. 
12. 
Jeffery, I.B., et al., An irritable bowel syndrome subtype defined by species-specific 
alterations in faecal microbiota. Gut, 2012. 61(7): p. 997-1006. 
13. 
Krogius-Kurikka, L., et al., Microbial community analysis reveals high level phylogenetic 
alterations in the overall gastrointestinal microbiota of diarrhoea-predominant irritable 
bowel syndrome sufferers. BMC gastroenterology, 2009. 9(1): p. 95. 
14. 
Lee, C., et al., The Increased Level of Depression and Anxiety in Irritable Bowel Syndrome 
Patients Compared with Healthy Controls: Systematic Review and Meta-analysis. Journal of 
neurogastroenterology and motility, 2017. 23(3): p. 349. 
15. 
Rajilić–Stojanović, M., et al., Global and deep molecular analysis of microbiota signatures in 
fecal samples from patients with irritable bowel syndrome. Gastroenterology, 2011. 
141(5): p. 1792-1801. 
16. 
Chassard, C., et al., Functional dysbiosis within the gut microbiota of patients with 
constipated‐irritable bowel syndrome. Alimentary pharmacology & therapeutics, 2012. 
35(7): p. 828-838. 
17. 
Simrén, M., et al., Intestinal microbiota in functional bowel disorders: a Rome foundation 
report. Gut, 2012: p. gutjnl-2012-302167. 
18. 
Tap, J., et al., Identification of an intestinal microbiota signature associated with severity of 
irritable bowel syndrome. Gastroenterology, 2017. 152(1): p. 111-123. e8. 
19. 
Hayes, P., et al., A dietary survey of patients with irritable bowel syndrome. Journal of 
human nutrition and dietetics, 2014. 27(s2): p. 36-47. 
20. 
Simpson, H. and B. Campbell, dietary fibre–microbiota interactions. Alimentary 
pharmacology & therapeutics, 2015. 42(2): p. 158-179. 
21. 
den Besten, G., et al., The role of short-chain fatty acids in the interplay between diet, gut 
microbiota, and host energy metabolism. Journal of lipid research, 2013. 54(9): p. 2325-
2340. 
22. 
Salonen, A. and W.M. de Vos, Impact of diet on human intestinal microbiota and health. 
Annual review of food science and technology, 2014. 5: p. 239-262. 

NL64950.081.18  
IBSQUtrition 
Version number 2, 16-03-2018 
 
35 of 35 
23. 
Huda-Faujan, N., et al., The impact of the level of the intestinal short chain fatty acids in 
inflammatory bowel disease patients versus healthy subjects. The open biochemistry 
journal, 2010. 4: p. 53. 
24. 
Sabatino, A., et al., Oral butyrate for mildly to moderately active Crohn's disease. 
Alimentary pharmacology & therapeutics, 2005. 22(9): p. 789-794. 
25. 
Scheppach, W. and G.-A.S.S. Group, Treatment of distal ulcerative colitis with short-chain 
fatty acid enemas a placebo-controlled trial. Digestive diseases and sciences, 1996. 
41(11): p. 2254-2259. 
26. 
Binder, H.J., Role of colonic short-chain fatty acid transport in diarrhea. Annual review of 
physiology, 2010. 72: p. 297-313. 
27. 
Rajilić-Stojanović, M., et al., Intestinal microbiota and diet in IBS: causes, consequences, 
or epiphenomena? The American journal of gastroenterology, 2015. 110(2): p. 278-287. 
28. 
Tana, C., et al., Altered profiles of intestinal microbiota and organic acids may be the origin 
of symptoms in irritable bowel syndrome. Neurogastroenterology & Motility, 2010. 22(5): 
p. 512. 
29. 
Mortensen, P., et al., Short-chain fatty acids and the irritable bowel syndrome: the effect of 
wheat bran. Scandinavian journal of gastroenterology, 1987. 22(2): p. 185-192. 
30. 
Treem, W.R., et al., Fecal short-chain fatty acids in patients with diarrhea-predominant 
irritable bowel syndrome: in vitro studies of carbohydrate fermentation. Journal of pediatric 
gastroenterology and nutrition, 1996. 23(3): p. 280-286. 
31. 
Palsson, O.S., et al., IBS patients show frequent fluctuations between loose/watery and 
hard/lumpy stools: implications for treatment. The American journal of gastroenterology, 
2012. 107(2): p. 286-295. 
32. 
Drossman, D.A., et al., Severity in irritable bowel syndrome: a Rome Foundation Working 
Team report. The American journal of gastroenterology, 2011. 106(10): p. 1749-1759. 
33. 
Mättö, J., et al., Composition and temporal stability of gastrointestinal microbiota in 
irritable bowel syndrome—a longitudinal study in IBS and control subjects. FEMS 
Immunology & Medical Microbiology, 2005. 43(2): p. 213-222. 
34. 
Ottman, N., et al., The function of our microbiota: who is out there and what do they do? 
Frontiers in cellular and infection microbiology, 2012. 2. 
35. 
Verdam, F.J., et al., Human intestinal microbiota composition is associated with local and 
systemic inflammation in obesity. Obesity, 2013. 21(12). 
36. 
Salonen, A., et al., Comparative analysis of fecal DNA extraction methods with phylogenetic 
microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. 
Journal of microbiological methods, 2010. 81(2): p. 127-134. 
37. 
Ramiro-Garcia, J., et al., awaiting peer review. 2016. 
38. 
Stams, A.J., et al., Growth of syntrophic propionate-oxidizing bacteria with fumarate in the 
absence of methanogenic bacteria. Applied and environmental microbiology, 1993. 59(4): 
p. 1114-1119. 
39. 
O’Keefe, S.J., et al., Fat, fibre and cancer risk in African Americans and rural Africans. 
Nature communications, 2015. 6: p. 6342. 
40. 
Francis, C.Y., J. Morris, and P.J. Whorwell, The irritable bowel severity scoring system: a 
simple method of monitoring irritable bowel syndrome and its progress. Alimentary 
pharmacology & therapeutics, 1997. 11(2): p. 395-402. 
41. 
Streppel, M.T., et al., Relative validity of the food frequency questionnaire used to assess 
dietary intake in the Leiden Longevity Study. Nutrition journal, 2013. 12(1): p. 75. 
42. 
Patrick, D.L., et al., Quality of life in persons with irritable bowel syndrome (development 
and validation of a new measure). Digestive diseases and sciences, 1998. 43(2): p. 400-
411. 
43. 
Zigmond, A.S. and R.P. Snaith, The hospital anxiety and depression scale. Acta psychiatrica 
scandinavica, 1983. 67(6): p. 361-370. 
44. 
Yang, Q., et al. Power and type I error rate of false discovery rate approaches in genome-
wide association studies. in BMC genetics. 2005. BioMed Central.