PhD Thesis20Chlo%E9%20Mirsepasi... · Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, traditionally divided into Crohn’s disease

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PhD Thesis

Inflammatory bowel disease associated with virulence

factors in Escherichia coli

Hengameh Chloé Mirsepasi-Lauridsen, M.Sc. (Eng)

Department of Microbiology and Infection Control, Statens Serum Institut

Faculty of Sciences, University of Copenhagen

Supervisors:

Professor Anders Løbner-Olesen, M. Sc., PhD

Professor Karen A. Krogfelt, M. Sc. (Eng), PhD

Clinical Associate Professor, Andreas Munk Petersen, MD, Ph.D.

Submitted: 01-06-2015

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Table of Contents

Acknowledgements ...................................................................................................................................... 5

Summary ......................................................................................................................................................... 6

English ............................................................................................................................................................ 6

Danish (Dansk sammendrag) ......................................................................................................................... 7

List of Manuscripts ...................................................................................................................................... 9

Abbreviations .............................................................................................................................................. 10

1. Introduction .......................................................................................................................................... 12

1.1 Research Objectives ........................................................................................................................ 13

2 Background .......................................................................................................................................... 14

2.1 IBD and the gut microbiota ............................................................................................................. 14

2.2 Escherichia coli in intestinal disorders ............................................................................................ 15

2.2.1 Enteropathogenic Escherichia coli (EPEC) ............................................................................... 15

2.2.2 Enterotoxinogenic Escherichia coli (ETEC) ............................................................................... 16

2.2.3 Entroinvasive Escherichia coli (EIEC)........................................................................................ 16

2.2.4 Enteroaggregative Escherichia coli (EAEC) .............................................................................. 17

2.2.5 Verocytotoxigenic Escherichia coli (VTEC) ............................................................................... 17

2.2.6 Extra-intestinal Pathogenic Escherichia coli (ExPEC) ............................................................... 18

2.3 Escherichia coli strains associated with Crohn’s disease ................................................................ 18

2.4 Escherichia coli strains associated with Ulcerative Colitis .............................................................. 19

2.5 Probiotic and antibiotic treatment in IBD ....................................................................................... 20

2.6 IBD and Escherichia coli in vivo models ........................................................................................... 22

2.7 Suggested mechanisms in IBD associated Escherichia coli pathogenesis ....................................... 23

3 Materials and Methods ...................................................................................................................... 26

3.1 General Protocols ............................................................................................................................ 26

3.1.1 Research Ethical Approvals ...................................................................................................... 27

3.2 Aim I: Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA

extraction compared to QIAamp DNA stool mini kit extraction ................................................................. 27

3.2.1 DNA extraction by the QIAamp DNA stool minikit .................................................................. 27

3.2.2 DNA extraction by NucliSENS® easyMag ................................................................................. 27

3.2.3 PCR amplification for denaturing gradient gel electrophoresis .............................................. 27

3.2.4 Denaturing gradient gel electrophoresis ................................................................................. 28

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3.3 AIM II: Secretion of alpha-hemolysin by Escherichia coli disrupts tight junctions in inflammatory

bowel disease patients ................................................................................................................................ 29

3.3.1 Cell infection assay and measurement of transepithelial electric resistance (TER) ................ 29

3.3.2 Detection of occludin by immunofluorescence and Western blotting ................................... 29

3.3.3 Hemolysis determination by titration assay ............................................................................ 30

3.3.4 Quantification of hemolysin expression .................................................................................. 30

3.4 AIM III: Comparison of three commercial fecal calprotectin ELISA test kits in patients with

Inflammatory Bowel Disease ....................................................................................................................... 31

3.4.1 Study material ......................................................................................................................... 31

3.4.2 Study design............................................................................................................................. 31

3.4.3 Statistical approach ................................................................................................................. 31

3.5 AIM IV: Extra intestinal pathogenic Escherichia coli are associated with intestinal inflammation in

patients with Ulcerative Colitis.................................................................................................................... 32

3.5.1 Study material ......................................................................................................................... 32

3.5.2 Statistical Analysis ................................................................................................................... 32

4 Results and Conclusion .................................................................................................................... 33

4.1 Aim I: Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA

extraction compared to QIAamp DNA stool mini kit extraction ................................................................. 33

4.1.1 Aim and Hypothesis ................................................................................................................. 33

4.1.2 Results and Conclusion ............................................................................................................ 33

4.2 Aim II: Secretion of alpha-hemolysin by Escherichia coli ................................................................ 37

disrupts tight junctions in inflammatory bowel disease patients ............................................................... 37



4.3 Aim III: Comparison of three commercial fecal calprotectin ELISA test kits used in patients with

inflammatory Bowel Disease ....................................................................................................................... 40



4.4 Aim IV: Extra intestinal pathogenetic Escherichia coli are associated with intestinal inflammation

in patients with Ulcerative Colitis ................................................................................................................ 44



5. Discussion and Perspectives .......................................................................................................... 48

6 Conclusion ........................................................................................................................................... 51

7 References ............................................................................................................................................ 52

Manuscript I: ................................................................................................................................................ 66

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Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA extraction

compared to QIAamp DNA stool mini kit extraction ................................................................................... 66

Manuscript II: ............................................................................................................................................... 67

Secretion of alpha-hemolysin by Escherichia coli disrupts tight junctions in inflammatory bowel disease

patients ........................................................................................................................................................ 67

Manuscript III: .............................................................................................................................................. 68

Comparison of three commercial fecal calprotectin ELISA test kits in patients with Inflammatory Bowel

Disease ......................................................................................................................................................... 68

Manuscript IV:.............................................................................................................................................. 69

Extra intestinal pathogenic Escherichia coli are associated with intestinal inflammation in patients with

Ulcerative Colitis .......................................................................................................................................... 69

Appendices .................................................................................................................................................. 70

Conferences and Poster Presentations ....................................................................................................... 70

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Acknowledgements This thesis will would never have become reality without the help and suggestions of many

supportive family, friends and colleagues. The work presented in this thesis was carried out at the

Department of Microbiology and Infection Control at Statens Serum Institut (SSI) in the

collaboration with Faculty of Sciences, University of Copenhagen (KU); Department of

Gastroenterology, Hvidovre Hospital of Denmark (KAS Hvidovre) and Host Micorbe Interactomics

group, Wageningen University of Netherland.

I would Hereby like to express my deepest gratitude to everyone, who has contributed to the being

of this thesis. Firstly, my phD supervisor Anders Løbner-Olesen (KU) and Karen A. Krogfelt (SSI)

are thanked for offering me the great opportunity to work very independently throughout the project

and for encouraging me and given me moral support through the project, when needed. The

freedom I was given to pursue my ideas truly learned me about project management and how to

generate new ideas. My greatest appreciation goes to my supervisor Andreas Munk Petersen (KAS

Hvidovre), who introduced me, to inflammatory bowel disease. Most of the work performed in this

thesis would not have succeeded without Andreas Munk Petersen guidance, inspiration and support.

I am also very thankful to Betina Hebbelstrup Jensen, Carsten Struve, Tine Dalby and Erik Juncker

Boll, who have been a great support during my phD and always maintaining a positive spirit and

especially for fruitful scientific discussions. I would like to direct a warm and broad thank you to all

colleagues in “Karens lab” and building 211, specifically to Susanne Jespersen for an informal,

friendly and helpful working environment. In this line I should mention Marian Jørgensen for her

linguistic review of the manuscripts included in this thesis beside all her other responsibilities.

I am also very thankful to prof. Bruce A. Vallance from British Columbia University hospital and to

prof. Jerry M. Wells from Wageningen University, for giving me the opportunity to working in their

lab and learning new skills, for their guidance and inspiration.

I am also thankful to my partner Erik Juhl, for his constant support, when I most needed it.

Last, by not least, I am indebted, beyond words, to my mother, sisters and my 2 brothers, I am very

thankful for their thoughtfulness, support and encouragement in any and everything I do. I dedicate

this thesis to my father, who passed away 3 years ago and who always believed in me and

encouraged me.

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Summary

English

Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract,

traditionally divided into Crohn’s disease (CD) and ulcerative colitis (UC). UC is a relapsing non-

transmural chronic inflammatory disease that is restricted to the colon and during flares the disease

is characterised by bloody diarrhoea. CD is a chronic, segmental localised granulomatous disease

that can affect any part of the entire gastrointestinal tract from the mouth to the anus. The aetiology

of IBD is still unknown, but studies indicate several possible aetiologies such as the host immune

system and influence of the gastrointestinal microbiota.

The gut microbiota of IBD patients contributes to initiation and/ or maintaining the inflammatory

state by providing antigens or co-stimulatory factors that drive the immune response in a

misdirection in these genetically susceptible hosts. Alterations in the makeup of the intestinal

microbiota in IBD patients include a reduced diversity of intestinal microbial species in comparison

to healthy controls, which is linked to the pathogenesis of IBD (Manuscript I). In addition, we

showed that highest amount of DNA from the stool was extracted using manual Qiagen in

comparison to semi-automated easyMag® method (Manuscript I). Bacteriological analysis of

intestinal biopsies and faecal samples from UC patient show an increased number of Escherichia

coli (E. coli) species in the B2 and D phylogenetic groups, harbouring O1, O2, O6, O18 and O75

antigens. These antigens are often expressed by the Extra-intestinal Pathogenic E. coli (ExPEC).

Faecal calprotectin is a marker of gastrointestinal inflammation and is used in order to determine

mucosal healing and disease relapses/ remission in IBD patients. Our study showed that CALPRO

faecal calprotectin kit is most optimal method to analyse inflammation/ infection in IBD patients

(Manuscript III). Patients colonized with E. coli from the B2 phylogenetic group display increased

burdens of inflammation, as measured by colitis activity index (CAI) scores and faecal calprotectin

levels, in comparison to patients not colonized with B2 E. coli (Manuscript IV). Previous studies

have shown that the UC-associated E. coli strain p19A, which belongs to the B2 phylogenetic

group and harbours ExPEC genes, induces cell death in dendritic cells, as well as stimulates the

TNF-α, IL-6 and IL-23 cytokine production (Poster 1). p19A harbours two alpha-hemolysin genes,

which causes rapid loss of tight-junction integrity to monolayers of differentiated Caco2 cells

(manuscript II). These results suggest that IBD-associated E. coli might play a role in the

pathophysiology of IBD.

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In summary, this thesis presents original experimental data characterising IBD-associated E. coli in

hopeful anticipation of inciting further interesting studies.

Danish (Dansk sammendrag)

Inflammatorisk tarmsygdom (IBD) er en kronisk inflammatorisk sygdom, og den opdeles

traditionelt i Crohn’s sygdom (CD) og Colitis ulcerosa (UC). UC er en ikke-transmural kronisk

inflammatorisk sygdom, der er afgrænset til tyktarmen, og som ved opblussen kendetegnes ved

blodig diarré. CD er en kronisk, segmentært lokaliseret granulomatøs sygdom, der kan påvirke hele

mavetarmkanalen fra mund til anus. IBD rammer især børn og yngre voksne og giver anledning til

betydelig forringet livskvalitet allerede i en tidlig alder. Ætiologien bag denne sygdom er fortsat

ukendt, men IBD er karakteriseret ved et opreguleret immunrespons i tarmen, som formodes at være

udløst af ændringer i sammensætningen af den mikrobielle tarmflora.

Tarmbakterier opretholder inflammatoriske processer i IBD patienter ved at agerer som antigener

eller co-stimulerende faktorer, som driver immunresponset i en uhensigtsmæssig retning i disse

modtagelige værter. Sammenlignet med raske individer, ses der i IBD-patienter en ændring i

sammensætning af tarmfloraen samt en reduceret mangfoldighed af de -mikrobielle arter i tarmen,

som er knyttet til patogenesen af IBD (manuscript I). Desuden viste vi, at største mængde DNA fra

afføringen blev ekstraheret ved hjælp af manuel Qiagen sammenlignet med halvautomatisk

easyMag® metoden (manuscript I). Internationale studier og vores egne undersøgelser har vist en

øget forekomst af specifikke virulente Escherichia coli (E. coli), der tilhører de fylogenetiske

grupper B2 og D, og som udtrykker O1, O2, O6, O18 og O75 antigener. Disse overfladmarkører er

kendetegnende for Extra-intestinal Pathogenic E. coli (ExPEC). Fæces calprotectin er en markør for

aktiviteten i mave-tarm inflammationen/ infektion, og denne markør anvendes for at monitorere

slimhinde-heling og sygdoms / remission i IBD-patienter (manuscript III). Vores undersøgelse

viste, at CALPRO faecal calprotectin er mest optimale metode til at analysere inflammation /

infektion i IBD patienter (manuscript III). IBD-patienter, som ikke er koloniseret med B2 E. coli,

sammenlignet med patienter der er koloniseret med E. coli fra fylogenetiske gruppe B2 ses der øget

inflammation i tarmen. Denne øgede inflammation er bestemt ved colitis aktivitet indeks (CAI)

score og faecal calprotectin (manuscript IV). Tidligere undersøgelser har vist, at den UC-

associerede E. coli stamme p19A, som tilhører fylogenetiske gruppe B2 og som udtrykker ExPEC

gener, inducerer celledød i dendritiske celler, samt stimulerer TNF-α, IL-6 og IL-23 cytokin-

produktion (Poster 1). p19A udtrykker to alpha-hæmolysin gener, som forårsager hurtigt tab af

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tight-junction integritet i monolaget af differentierede Caco2-celler (manuskript II). Disse resultater

antyder, at IBD associerede E. coli kan spille en rolle i patofysiologien i IBD.

Sammenfattende præsenterer denne afhandling originale eksperimentelle data inkluderende

karakterisering af IBD associerede E. coli stammer, og det er forfatterens håb, at denne afhandling

kan danne grundlag for yderligere interessante studier inden for dette område.

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List of Manuscripts

I Hengameh Mirsepasi, Søren Persson, Carsten Struve, Lee O B Andersen,

Andreas M Petersen and Karen A Krogfelt. Microbial diversity in fecal samples

depends on DNA extraction method: easyMag DNA extraction compared to QIAamp

DNA stool mini kit extraction. BMC Research Notes 2014, 7:50.

II Hengameh Mirsepasi, Zhengyu Du, Carsten Struve, Godefroid Charbon, Jurgen

Karczewski, Karen Angeliki Krogfelt, Andreas Munk Petersen, Jerry M. Wells.

Secretion of alpha-hemolysin by Escherichia coli disrupts tight junctions in

inflammatory bowel disease patients. Submitted to American Journal of

Gastroenterology.

III Hengameh Chloé Mirsepasi-Lauridsen, Ulla Bachmann Holmetoft, Sofie Ingdam

Halkjær, Karen Angeliki Krogfelt, Andreas Munk Petersen. Comparison of three

commercial fecal calprotectin ELISA test kits in patients with Inflammatory Bowel

Disease. Revision submitted for publication in Scandinavian Journal of

Gastroenterology.

IV Hengameh Chloé Mirsepasi-Lauridsen, Sofie Ingdam Halkjaer, Esben Munk

Mortensen, Magnus Christian Lydolph, Inge Nordgaard-Lassen, Karen Angeliki

Krogfelt and Andreas Munk Petersen. Extra intestinal pathogenic Escherichia coli

are associated with intestinal inflammation in patients with Ulcerative Colitis.

(Manuscript for submission in Journal of PLOS pathogens).

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Krogfelt%20KA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Petersen%20AM%22%5BAuthor%5D

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Abbreviations

AIEC: Adherent-Invasive E. coli

Caco-2 cells: Heterogeneous human epithelial colorectal adenocarcinoma cells

CAI: Colitis Activity Index

CEACAM6: Carcinoembryonic Antigen-related Cell Adhesion Molecules 6

Cipro: Ciprofloxacin

CD: Crohn’s disease

CDA: Active Crohn’s disease

CFU: Colony forming unit

cnf1: Cytotoxic necrotizing factor type 1

DAEC: Diffusely Adherent E. coli

DGGE: Denaturing Gradient Gel Electrophoresis

DMEM: Dulbecco’s modified Eagle medium

EcN: E. coli Nissle 1917

ExPEC: Extra Pathogenic E. coli

E. coli: Escherichia coli

ELISA: Enzyme-linked immunoassay method

GIT: Gastro Intestinal Tract

HBI: Harvey-Bradshaw Index

hly: Hemolysin

IBD: Inflammatory Bowel Disease

IL: Interleukin

LB: Louria broth

MOI: Multiplicity of infection

MDS: Multidimensional scaling

MPDAI: Modified Pouchitis Disease Activity Index

OD: Optical density

PA: Pouchitis active

PI: Pouchitis inactive

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RBC: Red blood cells

ROC: Receiver Operating Characteristic

SCCAI: Simple Clinical Colitis Activity Index

TJ: Tight junction

TNF: Tumour-necrosis factor

UC: Ulcerative Colitis

UCA: Active Ulcerative Colitis

UTI: Urinary Tract Infection

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1. Introduction

Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract.

IBD has traditionally been divided into crohn’s disease (CD) and ulcerative colitis(UC) 1. Crohn’s

disease and Ulcerative Colitis are differentiated by their clinical manifestations and hypothesized

pathogenic mechanisms1. UC is a relapsing non-transmural chronic inflammatory disease that is

restricted to the colon and during disease flares characterised by bloody diarrhoea 1. CD is a

chronic, segmental localised granulomatous disease that can affect anywhere in the entire

gastrointestinal tract from the mouth to the anus. Ulcerative Colitis and Crohn’s disease can appear

at any age, but most patients are diagnosed in their third decade of life2. The prevalence of IBD in

Northern Europe varies from 35 to 50 per 100,000 inhabitants for Ulcerative Colitis and 30 to100

per 100,000 inhabitants for Crohn’s disease3.

The aetiology of IBD is still unknown but studies indicate several possible causes such as genetics3–

6, immunology7–9, nutrition10,11, bacteria12, viruses13,14, and other environmental factors15,16. Animal

model studies suggest that inflammation in IBD patients likely occurs as a result of either excessive

effector T-cell function or deficient regulatory T-cell function, associated with the overproduction

of pro-inflammatory cytokines, such as Tumour-necrosis factor (TNF), interleukin-12 (IL 12) or a

deficiency in the production or function of known regulatory/immunosuppressive cytokines such as

IL-107,17. A special composition of gut microbiota is required to initiate or maintain the intestinal

inflammatory process by providing antigens or co-stimulatory factors, which can drive the immune

response to react in a genetically susceptible individual 7. However, so far there is no specific

pathogenic microorganism directly linked to IBD7. Experimental models reveal that intestinal

microbiota play a role in IBD while placebo-controlled studies of antibiotic treatment in IBD

patients show some effect in promoting remission in IBD. However many of the trials reported to

date have involved small numbers of patients, treated for a short time. Therefore, to determine the

effectiveness of antibiotic therapy in the treatment of IBD, larger RCT of antibiotics need to be

undertaken 18,19. Studies have also noted an aberrant faecal microbiota in IBD patients, as compared

to that found in healthy controls, while a reduced diversity of conventional intestinal microbiota has

been linked to IBD20–22. Interestingly, bacteriological analysis of biopsies and faecal samples from

ulcerative colitis patients shows an increased number of Escherichia coli (E. coli) species belonging

to the B2 phylogenetic groups, that harbour Extra-intestinal Pathogenic E. coli (ExPEC ) genes 23,24.

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1.1 Research Objectives

The Overall objectives of the present dissertation were to understand host-bacterial interaction

during intestinal colonization process in IBD patients and to classify IBD-associated E. coli.

The main objectives were therefore:

I To study bacterial colonization in IBD patients in comparison to healthy controls.

(Manuscript I)

II Using a combination of classical microbiology with gene characterization, in order to

classify IBD-associated E. coli and cellular response. (Manuscript II)

III Optimization for measuring faecal calprotectin by ELISA. (Manuscript III)

IV To study host-bacterial interaction during intestinal colonization process in IBD

patients specifically in development of disease relapses and remission. Clinical

outcome will be assessed using faecal calprotectin, clinical activity indices and

statistical tools. (Manuscript IV)

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2 Background

2.1 IBD and the gut microbiota

Because IBD is an inflammatory disease of the gastrointestinal tract, it has been speculated that

luminal factors are involved. Therefore, gastrointestinal bacteria have frequently been suspected as

the cause of IBD relapses. Some IBD patients experience clinical improvement when they receive

antibiotics such as ciprofloxacin or rifaximin25. Microbiological findings in IBD patients show a

reduction of the resident aerobic and anaerobic microbiota (as compared to healthy controls) and an

increase in potentially pathogenic microorganisms such as Klebsiella, Enterobacter, Proteus and

fungi19. Studies also show a significant reduction in the bacterial species Lactobacilli and

Bifidobacteria as well as an increase in Bacteroides in the intestinal mucosa of IBD patients 21, 22.

The decreased prevalence of Lactobacilli and Bifidobacteria might have an important role in the

aetiology of IBD, since these bacteria have immunoregulatory effects, and therefore contribute to

intestinal host defences through their interactions with the immune system26.

Several microorganisms have been suggested to play a role in the pathogenesis of IBD, but none

convincingly. Mycoplasma27, Mycobacteria species28, Clostridium difficile29, Salmonella spp.30,

Listeria monocytogenes31, Aeromonas hydrophila32, Proteus dysbioses33, E. coli and viruses13 have

all been isolated from the intestines of IBD patients and have been suspected as the reason for

relapses of the disease.

M. paratuberculosis is suspected as an etiologic factor for CD, because the disease’s histology

includes epithelioid granulomas as well as macroscopic lesions with segmental and fibrosing

stenotic areas. One of the reasons for considering M. paratuberculosis as an etiologic agent of CD is

CD’s similarity to Johne´s disease34, a chronic ileitis in cattle caused by M. paratuberculosis. M.

paratuberculosis have been isolated from 3% of the patients with CD, nevertheless, detection of

acid-fast M. paratuberculosis in granuloma and other tissues is very difficult. Additionally, in one

study, remission was shown to occur in the majority of patients with CD after treatment by anti-

mycobacterial antibiotics35.

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2.2 Escherichia coli in intestinal disorders

E. coli is a predominant facultative anaerobic Gram-negative bacterium which colonizes the

gastrointestinal tract of human infants within a few hours of birth and helps maintain normal

intestinal homeostasis36. E. coli strains are - on the basis of genetic and clinical criteria, classified

into 3 major groups: (1) commensal strains found in the human and animal GIT (lacking specialized

virulence factor), (2) intestinal pathogenic strains (diarrheagenic), and (3) extra-intestinal

pathogenic E. coli (ExPEC)37. Six well known intestinal pathogenic E. coli strains are:

Enteropathogenic E. coli (EPEC), Enterohemorrhagic E. coli (EHEC), Enterotoxinogenic E. coli

(ETEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Diffusely adherent

E. coli (DAEC)38. These E. coli strains can cause gastrointestinal disease, ranging from self-limiting

diarrhoea to hemorrhagic colitis38.

E. coli consists of many different serotypes. Serotyping of E. coli utilizes somatic (O) antigen,

capsular (K) antigen, and flagellar (H) antigens, which can all be detected by agglutination assays

using specific rabbit antibodies39. E. coli as well as other enterobacteriaceae carries fimbrial/pilus

structures, which mediate adhesion to a wide range of human and animal cells containing

mannose40.

2.2.1 Enteropathogenic Escherichia coli (EPEC)

EPEC was the first pathotype of E. coli discovered in 1945, and was isolated from the intestines of

infants with diarrhoea in the UK38. EPEC adheres to epithelial cells via the BFP type IV pilus41,42,

followed by activation of its type III secretion system. As a result of its activation, protein kinase C,

protein tyrosine kinases(s), phospholipase Cγ, myosin light-chain kinase and mitogen-activated

protein (MAP) accumulate under the attached bacteria41. Hereafter various effector proteins-

including EspF, Map, EspG, EspH and Tir are translocated into the infected host cell, which

increases intracellular calcium (Ca2+) and triggers depolymerisation of microvilli actin, leading to

the formation of the characteristic pedestal complex43. This leads to increased permeability due to

loosened tight junctions and the activation of Nuclear Factor (NF)-κβ followed by production of

interleukin (IL) 8 and the transmigration of polymorphonuclear leukocytes (PMNs) across the

epithelium and into the intestinal lumen38. Diarrhoea usually results from increased ion secretion,

increased intestinal permeability, intestinal inflammation and loss of absorptive surface area

resulting from microvillus effacement38.

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2.2.2 Enterotoxinogenic Escherichia coli (ETEC)

ETEC is mostly associated with high mortality in children under 5 years of age, as well as being a

frequent cause of diarrhoea in tourists visiting developing countries44. The reasons for ETEC

infections predominantly occurring in countries with warm climate are still unknown, but it is likely

that water contaminated by human or animal sewage may be an important means of spreading the

infection44. ETEC strains produce a heat-stable enterotoxin (ST) and a heat-labile (LT) cholera

toxin-like enterotoxin38. The heat-labile toxin consists of a single A subunit and five identical B

subunits. B subunits mediate binding of the holotoxin to the cell surface gangliosides GM1 and

GD1b, while the A subunit is responsible for the enzymatic activity of the toxin38. LT

regulates/activate adenylate cyclase, which leads to increased levels of intracellular cAMP and

activation of cAMP-dependent kinesis followed by activation of the main chloride channel of

epithelial cells- the cystic fibrosis trans-membrane conductance regulator (CTFR)45. Increased

phosphorylation of CFTR leads to increased secretion of Cl- from secretary crypt cells, which

culminates in diarrhoea45. Heat-stable enterotoxin STb has also been associated with human

disease38. It activates the intestinal brush border guanylate-cyclase-C (GC-C) receptor, which

increases intracellular messenger cyclic GMP44. Cyclic GMP mediates decreased absorption of

sodium and chloride ions and increased secretion of bicarbonate and chloride ions, ultimately

resulting in watery diarrhoea44.

2.2.3 Entroinvasive Escherichia coli (EIEC)

Entroinvasive E. coli (EIEC) shares many properties with Shigella including virulence

mechanisms46. They both possess a large invasion plasmid encoding the Mxi-Spa type III secretion

system and invasion plasmid antigen (Ipa) effectors, which enable bacterial invasion of eukaryotic

cells46. The plasmid also encodes IcsA, which make it possible for bacteria to travel from cell to cell

in vivo, while eluding the immune system46. EIEC/Shigella pathogenesis mechanisms initially

involve epithelial cell penetration, followed by lysis of the endocytic vacuole, intracellular

multiplication, directional movement through the cytoplasm, extension into adjacent epithelial

cells47 and finally they induce the apoptosis in infected macrophages and the release of IL-148.

Enteroinvasive E. coli (EIEC) may on occasion cause inflammatory colitis, and occasionally

dysentery but in most cases it causes watery diarrhoea48.

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2.2.4 Enteroaggregative Escherichia coli (EAEC)

EAEC is mostly associated with chronic diarrhoea in developing countries and in

immunocompromised patients such as AIDS/HIV patients48. EAEC was first defined in 198749 as

an E. coli strain with the ability to adhere to Hep-2 cells in a stacked-brick like configuration50.

EAEC pathogenic mechanisms initially involve bacterial adherence to the intestinal mucosa via

fimbriae known as aggregative adherence fimbriae (AAFs), which are associated with the Dr-family

of adhesins. EAEC surface structures along with its release of flagellin causes inflammation, by

inducing the release of IL-8, thereby stimulating neutrophil transmigration across the epithelium,

which then leads to tissue disruption and fluid secretion51. Additionally, several toxins have been

associated with EAEC:

Pic: an autotransporter protease with mucinase activity52.

EAST1: homologue to STa toxin, which can cause watery diarrhoea53.

Pet: an autotransporter with enterotoxic activity. Potentially it can also lead to cytoskeletal

changes and epithelial-cell rounding by cleavage of the cytoskeletal protein spectrin54.

2.2.5 Verocytotoxigenic Escherichia coli (VTEC)

VTEC is known as the cause of haemorrhagic colitis and haemolytic uraemic syndrome, where

contaminated food is an important source of infection55. Escherichia coli producing a toxin similar

to shiga toxin (Stx, produced by Shigella dysenteriae) is the common cause of the haemolytic

uraemia syndrome in children and often manifests with bloody diarrhoea and acute renal failure56,57.

First discovered in 1982, Karmali58 found increased Stx activity in the f

ecal filtrates of children with haemolytic uraemia syndrome infected with E. coli serotype O157:H7.

Thereafter, additional VTEC serotypes have been discovered such as serotype O145 and O12156,59.

The pathogenic mechanisms of VTEC start when the Stx-E. coli are ingested and then adhere to

gastrointestinal epithelial cells via the bacterial outer membrane protein, intimin60,61, similar to the

adherence mechanisms of EPEC. Thereafter, the VTEC toxin is transported into the kidney via

blood, or by transmigration of neutrophils (PMN)62,63 or it binds to blood platelets64 and

erythrocytes65.

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2.2.6 Extra-intestinal Pathogenic Escherichia coli (ExPEC)

ExPEC cause diseases outside of the GIT, such as in the urinary tract, as well as causing central

nervous system infections, circulatory system infections, and respiratory tract infections37,66–68.

Clermont et al divided E. coli into four main phylogenetic groups: A, B1, B2, and D69 based on the

following three genes; ChuA (heme transport gene), yjaA (function is unknown) and TspE4.C2

(anonymous DNA fragments). The most virulent extra-intestinal E. coli strains belong to the

phylogenetic group B2 (harbouring chuA and yjaA). The E. coli strains displaying less virulence

during extra-intestinal infections belong to phylogenetic group D (harbouring yjaA) and finally, the

various commensal strains of E. coli belong to groups A (harbour none of the above mentioned

genes) and B1 (harbouring TspE4.C2)37,70. Recently E. coli phylogenetic groups E (harbouring

arpA (function is unknown), chuA and TspE4.C2 genes) and F (harbouring chuA), known as a sister

groups to phylogenetic group B2, and phylogenetic groups C (harbouring arpA and yjaA genes),

which are closely related to phylogenetic group B1, have been documented71.

Johnson et al72 defined E. coli isolates harboring at least 2 of the following virulence genes: sfa/foc

(S and FIC fimbriae subunit), papA or papC (P fimbriae), afa/dra (Dr-antigen-binding adhesins),

iutA (aerobactin, iron-acquisition system) and kpsMT II (capsule, host defense-avoidance

mechanisms) as ExPEC strains 70,73. Virulence markers such as Hly (toxin, hemolysin) and/or OmpT

(Outer membrane protease T subunit) genes have also been linked to ExPEC strains74.

2.3 Escherichia coli strains associated with Crohn’s disease

Since the 1970´s E. coli strains have been suspected as a possible reason for the onset of disease in

IBD76. Several studies have found increased numbers of E. coli species with virulence properties

isolated from IBD patients in comparison to healthy control group, especially when one focused on

IBD patients undergoing disease relapses75,76. In 1978 Keighley77 observed a modification of

luminal bacteria concentrations in CD patients with evidence of a dramatic increase in E. coli

species. In 1988 Burke75 showed a significantly higher proportion of adhesive E. coli species

present in active CD (CDA) patients in comparison to a control group76. In 1997 Ilnyckyj78

published a case report study, in which infection with E. coli O157:H7 79mimicked right-sided

colonic CD. Darfeuille-Michaud80 in 1998 showed high prevalence of E. coli isolated from ileal

biopsies of CD patients, i.e. 100 % prevalence in early lesions and 65 % in chronic lesions. These

19

findings suggested that E. coli might participate in the initiation as well as a chronic inducer of the

inflammatory processes in CD.

Martin et al81 showed increased mucosal associated Gram-negative bacteria in colonic biopsies

obtained from patients with CD, of which 73% were identified as E. coli spp.. A number of studies

indicate that there is a link between the prevalence of E. coli spp. and IBD relapses82,83. AIEC has

been associated with CD. One of the histological characteristics of CD is the presence of epithelioid

granulomatous inflammation of the intestine84,85. An in vitro model of human granulomas showed

that AIEC (strain LF82) has the ability to survive and replicate within infected macrophages,

inducing aggregation of infected macrophages as well as their fusion to form multinucleated giant

cells, along with the subsequent recruitment of lymphocytes86, which are histologically linked to

CD.

Epithelium-associated invasive E. coli have been isolated from the ileal and colonic mucosa of CD

patients, and have been shown to often possess the ability to bind to intestinal epithelial cell

monolayers as well as synthesize alpha-hemolysin87,88. Colonic biopsies from the patients with CD

shows specific pathogenic strains of E. coli with the ability to infect and invade host cells, where

they multiply as well as damage the host tissues82,83.

In 1976 Schussler88 showed a significant elevation of the lipid A and O antibody titers against E.

coli in CD patient groups, in comparison to control, UC and acute enteritis groups and suggested it

as a potential marker to differentiate between CD and UC. Antigens against E. coli have been

detected in 57% of CD patient biopsies or resection specimens31 while polyclonal antibodies against

E. coli were detected in macrophages within the lamina propria, in the germinal centres of

mesenteric lymph nodes, and in giant cells along fissures, below ulcers and in granulomas.

Additionally, increased numbers of antibodies against the E. coli outer-membrane protein C were

detected in 37-55% of patients with CD89,90. However, Petersen et al 2012 showed no difference in

anti-Omp C levels in patients with active CD compared with controls and thus this observation did

not support the use of Omp C serology testing, either in disease activity assessment, or in screening

for active CD91.

2.4 Escherichia coli strains associated with Ulcerative Colitis

Tabaqchal et al in 197892 showed that the majority of IBD patients have increased positive antibody

reactions to a variety of Escherichia coli O-antigens, such as O1, O2, O6, O18(O18ac, O18ab) and

20

O75 serotypes when compared to a control group. These serotypes are mostly associated with

urinary tract infections and originate from the faecal microbiota79,92. Serotyping of E. coli strains

isolated from IBD patients24 shows that: 83% of E. coli isolates from active UC (UCA), 33% from

inactive UC (UCI), 50% from CDA and 33% of E. coli isolates from inactive CD (CDI), harbours

O1, O2, O6, O18 (O18ab,O18ac) or O75 genes, which are inked to urinary tract infection.

However, only 22% of E. coli isolates from control groups harboured one of the above-mentioned

O-antigen.

Burke et al in 198794 showed that isolated E. coli strains from the stool of UC patients were

predominantly diffusely adherent E. coli (median adhesion index= 43.5 (range 18-68)) with both

enterotoxigenic95 and enteropathogeneic96 abilities, in comparison to healthy persons (median

adhesion index=2.0, rang 0-15)94. DAEC has been linked to UC95. Bacteriological analysis of rectal

biopsies from active UC (UCA) patients showed a characteristic individual variability amongst the

mucosal bacteria and a greater number of E. coli species belonging to the B2 and D phylogenetic

groups23,24. Our study showed that UCA patients colonized with B2 E. coli have increased burden

of inflammation as measured using the CAI score and faecal calprotectin levels (Manuscript IV). A

previous study showed that UC associated E. coli p19A belongs to the B2 phylogenetic group

harbouring alpha-hemolysin, dissolves occludin and thereby disrupts tight junction (TJ) function in

Caco-2 cells in vitro, followed by increasing barrier permeability97 (Manuscript II). Additionally,

the UC associated E. coli strain p19A induces cell death in DC98 and stimulates the release of the

cytokines TNF-α, IL-6 and IL-23 (poster).

2.5 Probiotic and antibiotic treatment in IBD

Several studies in the past decade suggest both environmental factors as well as genetic

susceptibility influence the abnormal immune responses in IBD. Luminal bacteria are suspected to

have a central role in the pathogenesis of IBD99. Therefore, antibiotics have often been used as a

therapeutic option18,100,101 despite the fact that antibiotic therapy might induce disease relapse due to

Clostridium difficile infection. Some of the antibiotic combinations used as a therapeutic option in

CD are clofazimine together with clarithromycin and rifabutin, because of its activity against

Mycobacterium paratuberculosis (MAP, known from Johne´s disease in animals), which is

considered a potential cause of CD102. Conclusion of this antibiotic trial showed that it was effective

in inducing remission when used concurrently with a course of corticosteroids. Nevertheless usage

of this combination is very limited, since there are several disadvantages103 using corticosteroids,

21

such as the masking of the infection by suppressing the symptoms and signs of inflammation as

well as the increased risk of bleeding. However, a meta-analysis performed on the efficacy of broad

spectrum antibiotics in CD patients showed the most effective antibiotic therapies are metronidazole

and ciprofloxacin 104, which enables clinical improvement 105 .

Placebo-controlled studies in IBD or irritable bowel syndrome patients indicate that probiotic

treatment significantly reduced small bowel permeability (lactulose/mannitol ratio) and induced

remission in IBD patients103,106. The probiotic E. coli strain Nissle 191719,107 was isolated from the

faeces of a German soldier who seemed protected from infectious diarrhoeal disease108. Genomic

studies of E. coli strain Nissle 1917 have shown that in contrast to other non-pathogenic E. coli

strains, it exhibits microcins, adhesins and at least six different iron-uptake system (enterobactin,

salmochelin, aerobactin, yersiniabactin, EfeU) for the generation of energy through ATP.

Nevertheless it lacks prominent virulence factors such as HlyA and P-fimbrial adhesions109–113.

Studies have shown that E. coli Nissle 1917 has immunoregulatory properties such as decreasing

the number of T-cells within the intestinal mucosa, as well as reducing the secretion of pro-

inflammatory cytokines such as IL-2, IFN-γ and TNF-α. It also stimulates the secretion of

regulatory proteins such as IL-10 and IL-1 β113–115. Schlee et al116 showed that E. coli Nissle 1917

induces β-defensin 2 (hBD-2 human antimicrobial peptide), which helps reinforce the intestinal

mucosal barrier, limiting bacterial adherence, as well as invasion of the gut mucosa. In vivo models

have shown that E. coli Nissle 1917 protects against infections with Salmonella117 and Candida

albicans118. In vitro models using various cell have shown that E. coli Nissle 1917 inhibits invasion

by Salmonella, Yersinia enterocolitica, Shigella flexneri, Listeria pneumophila, L.

monocytogenes119, and invasion of host cells by AIEC120. A number of clinical trials have

demonstrated that E. coli Nissle 1917 is equally as effective as mesalazine at maintaining

remission121–125 in UC patients. It has been also demonstrated that E. coli Nissle 1917 is effective at

preventing colitis in different murine models of colitis122,125–127, yet larger studies are needed to

confirm the potential beneficial effects of E. coli Nissle 1917 in IBD13,33,128. However, a

randomized double blind study of E. coli Nissle 1917 given as an add-on treatment to patients with

active UC showed that significantly fewer of the patients treated with E. coli Nissle 1917 reached

symptomatic remission and that significantly more patients treated with E. coli Nissle 1917

withdrew from the study129(Manuscript IV).

22

2.6 IBD and Escherichia coli in vivo models

The most important aspects discovered through IBD animal models are: germ-free animals

generally do not develop intestinal inflammation, spontaneous gut inflammation requires a certain

genetic background, T-cells are involved in most IBD animal models and interactions between T-

cells and dendritic cells seems to be crucial for the initiation and perpetuation of inflammation130.

Overall, IBD animal models can be divided into 5 different categories130;

(1) Antigen-induced colitis and colitis induced by microbiota (e.g. ovalbumin-specific T- cell

receptor transgenic model)131.

(2) Other inducible forms of colitis (e.g. Chemical: Dextran sulfate sodium (DSS) model)132.

(3) Genetic colitis models (e.g. mice genetically deficient in IL-10)133.

(4) Adaptive transfer models134 (e.g. EPEC causes UC in Cotton-top tamarin (CTT: Saguinus

oedipus135)).

(5) Spontaneous colitis models (e.g. NFIL3 (a transcription factor that regulates multiple

immunologic function) deficiency, akin to IL-10 deficiency, results in the development of

spontaneous colitis in mice)136.

Ulceration of lymphoid follicles and the involvement of Peyer's patches have been reported in CD

137,138, which might be caused by the passage of particulate-matter/bacteria from the bowel lumen

into the lymphoid tissue of the mucosa and thence into the lymphatic system of the gut139. Benoit

Chassaing et al 2011140 showed that AIEC harbouring long polar fimbriae (lpf) colonize Peyer's

patches, using lpf expressing type 1 pili to adhere. Experimental animal models have revealed that

intestinal microbiota play a major role in IBD. Genetically susceptible IBD mouse models indicate

a key role for dysfunctional, unregulated, T-cell-mediated immune responses in IBD

pathogenesis141,142. IL-10 promotes development of humoral Th2 cytokine-driven immune

responses and it inhibits the development of Th1 immune responses by reducing the capacity of

macrophages to produce IL-12, which is a key inducer of Th1 immune responses143. In vivo models

have also shown that interleukin 10 deficient mice colonised with non-pathogenic conventional E.

coli strains develop distal colitis and produce high levels of interferon γ and interleukin 4 as a

result144. E. coli HlyA impairs intestinal barrier function by causing focal leaks in the intestinal

epithelium, thereby intensifying antigen uptake and triggering intestinal inflammation in vulnerable

mouse models, as well as disrupting epithelial tight junctions via an HlyA-dependent mechanism145.

Disrupted intestinal epithelial tight junctions in IBD patients with active disease have been

23

associated with a reduction in several tight junction proteins including claudins 1 and 4, occludin

and tricellulin 146. Moreover the synthetic octapeptide (AT1001), which prevents the opening of

tight junctions improves colitis in susceptible IL-10-/- mice 147 145,148. IBD animal models have so far

given us a better understanding of IBD pathogenesis, such as the involvement of intestinal

microbiota and the importance of T-helper cells in driving GI inflammation in vulnerable hosts.

2.7 Suggested mechanisms in IBD associated Escherichia coli

pathogenesis

AIEC has been isolated in 36.4% of CD patients and is associated specifically with the ileal

mucosa149. It interferes with host cell processes including protein synthesis, signal transduction, cell

division, ion secretion, transcription, cytoskeletal function, and mitochondrial function38,150. It is

able to adhere to the intestinal mucosa, invade intestinal epithelial cells, replicate intracellularly,

translocate across the human intestinal barrier and move into deeper tissues151,152.

AIEC invasion of host epithelial cells is initiated by its binding to the Carcinoembryonic Antigen-

related Cell Adhesion Molecules 6 (CEACAM6)153, which is up-regulated in CD patient’s tissues in

response to inflammation. AIEC use their type 1 pilus to adhere, and via a macropinocytosis-like

process enter, survive, and replicate inside the host cell-cytoplasm (Figure 1). AIEC uses host cell-

actin-microfilaments and microtubules to invade the host cell and translocate to other cells154,155. It

is able to survive extensively within macrophages, induce the secretion of large amounts of TNF-α

and promote a granulomatous inflammatory response86,155.

24

Figure 1 Invasion of host cell by AIEC in CD. Abnormal colonisation of the ileal mucosa is initiated by the

interaction of AIEC with intestinal epithelial cells. 1) AIEC binds to CEACAM6, which is up-regulated in the CD

patient’s ileum. 2) By using a macropinocytosis-like process AIEC enter, survive, and replicate inside the host cell

cytoplasm after lysis of the endocytic vacuole. 3) By using its invasive ability and host cell-actin microfilaments and

microtubules, it crosses the intestinal barrier through intestinal epithelial cells or through M cells, invades host cells and

translocates to other cells. 4) AIEC is able to survive extensively within resident macrophages and dendritic cells and it

induces the secretion of high amounts of TNF-α and granulomatous inflammation.

DAEC expressing the Afimbrial adhesins gene (Afa)/Dr causes intestinal as well as Urinary Tract

Infections (UTI) and might impact the pathogenesis of UC24,95,156. Involvement of DAEC

harbouring Afa/Dr in diarrhoea has been controversially demonstrated in polarized monolayers of

intestinal T84 cells157,158. DAEC have also been shown to adhere to the mucosa of UC patients and

promote pro-inflammatory responses via interaction of bacterial adhesins with membrane-bound

receptors158.

DAEC harbouring the Afa/Dr gene initiate their interactions with fully differentiated epithelial cells

through bacterial recognition of decay/accelerating factor (DAF), CEACAM1 or CEACAM6 (by

afa/DrCEA adhesins) (figure 2). DAEC interferes with host cell-signalling pathways, involving

protein tyrosine kinases(s), phospholipase Cγ, phosphatidylinositol 3-kinase, and protein kinase

C156. As a result Ca2+ levels increase in infected host cells, inducing the rearrangement of brush

border/associated F-actin and villin cytoskeletal proteins and the loss of the epithelial cell

microvilli156. Activated MAP kinase-dependent signalling pathways in the host cell induce the

ActA

Luminal capture by dendritic cell?

Translocation through M cell

M cell

Paracellular entry

Trans-epithelial migrationSpacious vacuole

Survival and growth in macrophages

TNFα

NF-κβ

LPS

IL-8CEACAM6

AIEC

Polymorphonuclear Leukocyte

1

3

2

4

ActA

Luminal capture by dendritic cell?

Translocation through M cell

M cell

Paracellular entry

Trans-epithelial migrationSpacious vacuole

Survival and growth in macrophages

TNFα

NF-κβ

LPS

IL-8CEACAM6

AIEC


1

3

2

4

25

secretion of cytokines including IL-8, TNF-α, and IL-1β followed by the up-regulation of DAF and

MHC class I chain/like gene-A. Additionally, DAEC induce changes in the distribution of tight

junction/associated proteins that leads to increased paracellular permeability156. Interactions with

and internalization of afa/Dr DAEC into undifferentiated cells occurs by mechanisms involving

lipid rafts and dynamic microtubules. They start by recognition of DAF via Afa/Dr adhesins and

invasion of DAEC into undifferentiated cells, followed by the survival of DAEC within a large

vacuole and the spreading of DAEC into other epithelial cells after the host cell undergoes

apoptosis156.

Figure 2 Infection with DAEC in UC. Infection with Afa/Dr DAEC starts by bacterial interaction with fully

differentiated epithelial cells via 1) Bacterial recognition of DAF, CEACAM1 or CEACAM6. 2) DAEC interferes with

host cell-signalling pathways involving protein tyrosine kinases(s), phospholipase Cγ, phosphatidylinositol 3-kinase,

and protein kinase C followed by an increase of Ca2+ in the host cell. Increased Ca2+ in the host cell induces

rearrangements of brush border/associated F-actin and villin cytoskeletal proteins, which results in the loss of the

epithelial cell-microvilli. 3) Changes in the distribution of tight junction/associated proteins leads to paracellular

permeability 4) Activated MAP kinase-dependent signalling pathways induce secretion of the cytokines such as

interleukin 8 (IL8), TNF-α, and IL-1β that causes an up-regulation of DAF and MHC class I chain/like gene-A. 5)

DAEC interacts with undifferentiated cells via recognition of DAF by Afa/Dr adhesins followed by invasion of the host

cell. DAEC survives within a large vacuole and spreads to other epithelial cells after host cell apoptosis.

Paracellular entry

Survival within

a large vacuole

Macrophages

TNFα

IL-8DAF, CEACAM6, CEACAM1

DAEC


Luminal capture by dendritic cell

MAP

kinasese

NF- κβ

Distribution of

thight junction

proteinsLPS

Interaction with

undifferentiated cell

PLCγ, Ca 2+,

PI3 kinase

12 3

4

5

Paracellular entry

Survival within

a large vacuole

Macrophages

TNFα


DAEC



MAP

kinasese

NF- κβ

Distribution of

thight junction

proteinsLPS

Interaction with


PLCγ, Ca 2+,

PI3 kinase

Paracellular entry

Survival within

a large vacuole

Macrophages

TNFα


DAEC



MAP

kinasese

NF- κβ

Distribution of

thight junction

proteinsLPS

Interaction with


PLCγ, Ca 2+,

PI3 kinase

12 3

4

5

26

3 Materials and Methods

Methods are described in detail in each individual manuscript. This section is a short description of

all the methods used in Manuscript I, II, III and IV (figure 3).

Figure 3 Schematic outline of four project parts in this thesis. Manuscripts (MS)

3.1 General Protocols

Work performed in the laboratory was according to current guidelines at Statens Serum Institut and

University of Wageningen, Netherlands as well as stated by manufacturers of material and products.

The media and product were stored according to manufacturer’s recommendations. Guidelines were

followed in agreement with previously published papers and current literature.

27

3.1.1 Research Ethical Approvals

For this study, research ethical approvals were obtained from Government institutions. Collection of

patient samples and data were approved by; the Danish Health and Medicines Authority, statistic

Denmark and the Scientific Ethics Committee for Copenhagen Regional Hospitals (Permission no.

KA-20060159), (Permission no. H-1-2009-110) and (Permission no. KA03019).

3.2 Aim I: Microbial diversity in fecal samples depends on DNA

extraction method: easyMag DNA extraction compared to

QIAamp DNA stool mini kit extraction

3.2.1 DNA extraction by the QIAamp DNA stool minikit

DNA extraction was performed according to the instructions of the manufacturer with the following

modification: 100 mg faecal sample was mixed with 1.4 ml ASL buffer in a 2 ml tube and vortexed

until the sample was thoroughly homogenized. Samples were subsequently mixed with 0.2 g

steriale zirconia/silica beads. Hereafter, the samples were processed on a TissueLyser for 6 minutes

at 30 Hz. Lysis was completed at a temperature of 95° C for 5 minutes. Finally, DNA was extracted

according to the instruction of the QIAamp DNA stool MiniKit and eluted in 100 µl elution buffer

provided in the kit.

3.2.2 DNA extraction by NucliSENS® easyMag

DNA extraction was performed according to the manufacturer’s instructions with some

modifications. Briefly, 100 mg faecal sample was mixed with 400 µl Lysis Buffer 1 and vortexed

using Mylab for 10 minutes until the faecal sample was thoroughly homogenized. The samples

were subsequently centrifuged for 5 minutes at 13.000 rpm. Hereafter, 140 µl magnetic silica was

added to each tube and thoroughly mixed with the sample. The remaining steps of the DNA

extraction process were performed by the robot according to protocol A and eluted in 110 µl elution

buffer.

3.2.3 PCR amplification for denaturing gradient gel electrophoresis

The V2-V3 region of the 16S rDNA gene was amplified by universal primer set HDA 1 position

338-357: (5′ACT CCT ACG GGA GGC AGC AGT′3) and HDA 2 position 539–561: (5′GTA TTA

CCG CGG CTG CTG GCA C–′3) [8]. The forward primer, HDA 1, was at the 5′end labeled with

28

GC clamp (5′CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGGGGG G ′3).

PCRs were performed in a total volume of 50 μL containing 20 μL of 5 PRIME Mastermix, 0.8 μM

primer HDA 1-GC, 0.8 μM primer HDA 2, 10 μL of DNA template and, finally, 4 μL RNase free

water. The PCR was performed using the following conditions: preheating at 94°C for 4 minutes

proceeded by 30 cycles of denaturing at 94°C for 30 sec, annealing at 56°C for 30 sec, elongation at

68°C for 45 sec, and finally a single step of 68°C for 7 minutes; the PCR products were run on a

0.8% agarose gel.

3.2.4 Denaturing gradient gel electrophoresis

PCR fragments were separated by Denaturing Gradient Gel Electrophoresis (DGGE) with DCode

System according to the manufacturer’s instructions. 8% Polyacrylamide (vol/vol) (ratio of

acrylamid:bisacrylamide (37.5:1)) were diluted in 0.5xTAE buffer with pH 8.0 using a gradient

ranging from 35% to 65% (100% acrylamide corresponds to 7 M urea and 40% (vol/vol)

formamide) [17]. Gels were cast using a gradient maker and a pump with a flow speed of 5 mL per

minute. After polymerization of the gel (2 hours), a 3% stacking gel without denaturing chemicals

was cast, and an appropriate comb was subsequently inserted and left for 30 minutes for

polymerization. Gels were run at 60°C for 16 hours at a constant voltage of 70 V in 0.5 × TAE

buffer. After electrophoresis, gels were stained with GelRED

for 45 minutes and analysed using an ultraviolet trans-illuminator.

29

3.3 AIM II: Secretion of alpha-hemolysin by Escherichia coli

disrupts tight junctions in inflammatory bowel disease patients

3.3.1 Cell infection assay and measurement of transepithelial electric

resistance (TER)

In order to characterize the role of hlyA in damaging TJ the following mutants were created (by

Carsten struve) from the p19A E. coli isolate:

1: CAS965: hly cluster II upstream cytotoxic necrotizing factor type 1 (cnf1)

2: CAS954: hly cluster I mutant

3: CAS956: knock out both loci of hly

4: CAS964: cnf1 mutant

and 2 wild types "CAS963" and "CAS939" used to create the mutants.

E. coli clinical isolates from from 3 healthy controls and 9 IBD patients were tested in this study.

Transepithelial/-endothelial electric resistance (TER) was used to monitor establishment of

modulation of barrier-forming cell-to-cell contact. Caco-2 cells were grown in 24-well/ transwell

for 14 days and exposed to E. coli clinical isolates, at MOI 10 for 24 h. Cytotoxicity of p19A,

mutants and commensally E. coli isolates was studied as neutral red uptake in Caco-2 cells co-

cultured with p19A WT and mutants at MOI 10 for 4 hrs compared to Caco-2cells without added

bacteria.

3.3.2 Detection of occludin by immunofluorescence and Western blotting

In order to visualize the effect of bacteria on Caco-2 cells, occludin was detected by

immunofluorescence microscopy and Western blotting. Caco-2 cell monolayers were grown as

described above and infected with bacteria for up to15 h at an MOI 100. Caco2 cell monolayers

were either fixed for immunofluorescence or lysed in 100 µl of lysis buffer (Promega) on ice for 5

to 10 min. The cell lysate was centrifuged at 13.000 g for 12 min. to pellet debris, and the

supernatant was used for Western blotting. 50 µg Caco-2 cell proteins were resolved by 10% SDS-

PAGE and transferred to 0.2 µm polyvinylidene fluoride (PVDF) membranes. Membranes were

blocked for 1 h. with 3% non-fat milk powder diluted in 0.05 % Tween 20 (TBST), then incubated

with primary antibody in 3% non-fat milk powder diluted in TBST overnight at 4°C. Hereafter,

30

membranes were visualized with secondary (SIGMA-ALDRICH ) anti-body for 1 h. at room

temperature. Rabbit polyclonal anti-actin antibody (SIGMA-ALDRICH, A2066) and anti-Occludin

antibody (Merck KGaA, Darmstadt, Germany, ABT146) was used in this study.

3.3.3 Hemolysis determination by titration assay

Defibrinated horse blood (Statens Serum Institute Diagnostica, SSI nr. 23699) was washed twice in

hemolysis buffer (0.0077 M Tris-HCl, 0.137M NaCl and 0.02M CaCl pH 7.4) and centrifuged at

3000 g for 5 min. Washed red blood cells (RBC) were re-suspend in hemolysis buffer to a final

concentration of 2 % RBC suspension, 150 µl of RBC suspension was added to each well in 96 well

plates. Overnight bacteria pellet (grown in LB 37 °C) was diluted in 5 ml hemolysis buffer.

Hereafter, 150 µl of bacteria pellet suspension and /or supernatant was re-suspended in the first well

containing 2% RBC suspension, two-fold serial dilutions (1:2 to 1:1024) in microtiter plates were

done and incubated for 1 hr at 37 °C. After incubation the plate was centrifuged 10 min at 700 g,

150 µl of supernatant was transferred to a new microtiter plate and optical density measured at 562

nm. Hemolytic titration assays were performed twice.

3.3.4 Quantification of hemolysin expression

Total RNA was phenol and chloroform extracted from LB growing cultures at OD600 of 0.8 x 106

followed by DNase I digestion (Thermoscientific # EN0525). The RNA was then purified using

Qiagen column (cat. no 74104) treated with a dsDNase (Thermoscientific # EN0771) and directly

used for cDNA preparation using First Strand cDNA synthesis kit (Thermoscientific # K0702).

Primers: hlyA forward and hlyA reverse (table 1), were used 159. The gene RpoA was used as

housekeeping/reference gene; RrpoA1 RrpoA2 (table 1) 160. The Quantitative-PCR was performed

using Takara SYBR Premix Ex Taq II (RR820A) in a BioRAD CFX96. The PCR was performed

using manufacturer recommendation conditions: preheating at 95°C for 30 sec followed by 40

cycles of 95°C for 5 sec and 60°C for 30 sec for elongation.

31

3.4 AIM III: Comparison of three commercial fecal calprotectin

ELISA test kits in patients with Inflammatory Bowel Disease

3.4.1 Study material

This study includes 148 faecal samples, 96 from patients with a previously confirmed IBD

diagnosis and 52 from healthy controls, aged between 25 to 86 and 18 to 67 years, respectively.

Disease activity was measured using the following clinical activity indices: the Simple Clinical

Colitis Activity Index (SCCAI), the Harvey Bradshaw Index (HBI) and the Modified Pouchitis

Disease Activity Index (MPDAI). Three ELISA calprotectin tests (EK-CAL, CALPRO and HK325)

were compared.

3.4.2 Study design

In order to evaluate the ELISA HK325 method in comparison to ELISA CALPRO method, the first

part of the study was designed, including 29 healthy controls and 21 UCA patients from an, at that

time, ongoing study (Experiment 1). Results of this evaluation showed pour performance of

HK325. Therefore, in the second part of the study ELISA CALPRO method was evaluated in

comparison to the newer ELISA EK-CAL method by including 23 healthy controls and 54 IBD

patients (Experiment 2). Of 54 patients included in second part of the study, 28 were diagnosed with

UCA, 6 with UCI, 6 with CDA, 7 with CDI, 2 with PI and 5 with PA. In order to evaluate

correlation between faecal calprotectin values and clinical activity indices using CALPRO method,

additionally 21 IBD patients were included in the study, 11 with UCI, 2 with CDA, 3 with CDI, 2

with PI and 3 with PA. These samples were obtained in a previous study.

3.4.3 Statistical approach

The software "GraphPad Prism 5" was used for statistical analysis. The differences between the

faecal calprotectin levels among patients with active or inactive disease were analyzed using the

Mann-Whitney test. MS Excel tools were used to produce Receiver Operating Characteristic (ROC)

curves showing the distribution between specificity and sensitivity and to perform regression

analysis between clinical activity indices and faecal calprotectin values in CD, UC and pouch

patients. A p<0.05 is considered significant.

32

3.5 AIM IV: Extra intestinal pathogenic Escherichia coli are

associated with intestinal inflammation in patients with

Ulcerative Colitis

3.5.1 Study material

One hundred patients with a flare of UC were randomized to Ciprofloxacin or placebo for 1 week

followed by EcN or placebo for 7 weeks. All 4 treatments were given as add-on treatments. Stool

samples were collected; at week 0, 1, 8 and 12 and cultured for E coli. Phylogenetic E. coli groups

(A, B1, B2 and D) were determined by a simple PCR. Faecal calprotectin was measured with

CALPROLABTM Calprotectin ELISA (ALP) assay.

3.5.2 Statistical Analysis

Kaplan-Meier curves were used to compare groups. Test of equality of survival distributions for the

different clinical treatment groups was performed using the Mantel-Cox (log-rank) test. The

software "SAS 9.4" and "GraphPad Prism 5" was used for statistical analysis. The differences

between the faecal calprotectin levels in the four patient groups among patients treated/ not treated

with EcN, were analyzed using SAS- Two-way ANOVA test. The differences between faecal

calprotectin levels at week 0 among patients colonized/ not colonized with B2 E. coli, were

analysed using t-test.

33

4 Results and Conclusion

4.1 Aim I: Microbial diversity in fecal samples depends on DNA

extraction method: easyMag DNA extraction compared to QIAamp

DNA stool mini kit extraction

4.1.1 Aim and Hypothesis

There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics,

since faecal samples also contain DNA from human cells and many different substances derived

from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study

was to investigate the DNA extraction quality of two systems, a semi-automatic and a manual DNA

extraction system in order to choose the most efficient method for studying IBD patients’ intestinal

bacterial diversity using DGGE.

4.1.2 Results and Conclusion

The automated easyMag® protocol recommends the use of 140 μL silica161 to extract faecal DNA.

In this study, both 140 and 35 μL silica were tested and the amount of extracted faecal DNA was

compared to the amount of extracted faecal DNA using the QIAamp DNA Stool Mini Kit (Qiagen).

Faecal DNA extraction using easyMag® with 140 and 35 μL silica yielded an average of 0.8 μg/mL

(Standard Deviation (SD) 0.5) and 1.8 μg/mL (SD 2.0) DNA, respectively. Faecal samples purified

using QIAamp DNA Stool Mini Kit (Qiagen) yielded an average of 6.3 μg/mL (SD 9.4) DNA. 16S

rDNA PCR products were visualized using electrophoresis on 2% agarose gel and analysed by

DGGE. Lanes exhibiting 16S rDNA PCR products obtained from easyMag® DNA were blank,

while PCR products obtained from DNA extracted by Qiagen were clearly visible (Figure 4). It was

noted that using less silica than suggested by the easyMag® manufacturer resulted in a higher DNA

yield. An explanation could be that when using the semi-automatic easyMag® with 140 μL silica

relatively more non-DNA compounds such as protein will be extracted from the sample compared

to 35 μL silica. These non-DNA compounds probably interfere with the DNA extraction.

34

Figure 4 DGGE gel pictures show, 16S rDNA PCR products on faecal DNA extracts obtained using easyMag®

with140 μL silica and Qiagen methods. Lanes 1, 3, 5, 7, 9, 11, 13, and 15 are 16S rDNA PCR products using DNA

extracted by easyMag®. Lanes 2, 4, 6, 8, 10, 12, 14, and 16 are 16S rDNA PCR products using DNA extracted by

Qiagen.

In order to investigate whether impurities and/or inhibitory compounds had any effect on the

visualization of the DNA, DNA extractions from HC-1, HC-2, HC-3, IBD1, IBD2 and IBD3 were

diluted 5, 10, 15, and 20 times. PCR was performed and the PCR-products were analysed by

DGGE. 16S rDNA PCR products obtained using easyMag® revealed more bands and showed

bands with higher densities in the lanes where the DNA was diluted 10 and 15 times. Additionally,

almost identical bands appear for each faecal sample in DGGE gel lane in both DNA extracts;

diluted easyMag® DNA extracts and Qiagen DNA extracts. However, the lanes in DGGE gels

representing DNA extracted by Qiagen show brightest bands in comparison to DNA extracted by

easyMag®.

Interestingly, CD patients frequently have a reduced intestinal bacterial diversity (bacterial DNA)

with predominant prevalence of uncultured conventional intestinal bacteria in comparison to

healthy controls162–164. This was confirmed, when analysing IBD patients’ faecal DNA extraction by

DGGE, using 16S rDNA PCR products. When DGGE band patterns were analysed for similarities

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by BioNumerics 7.0, the results show a reduced number of DGGE gel bands/reduced bacterial

diversity presented in IBD patients in comparison to control group (figure 5). Multidimensional

scaling (MDS) analysis of control group versus CD and UC patients shows, 72% of IBD patients

are clustered together in the blue circle, while only 28% of control group are clustered among

IBD patients (Figure 5A) in the blue circle. MDS image of control group versus CDA and CDI

patients shows, 77% of CD patients are clustered in the blue circle, while only 22% of control

group is clustered among CD patients (Figure 5B) in the blue circle. These results indicate that

bacterial colonisation in the GI of the IBD patients differs from the control group.

Figure 5 Multidimensional scaling (MDS) analysis performed using BioNumerics 7.0 A) MDS image of control

group versus CD and UC patients shows IBD patients are mostly clustered together in the blue circle. B) MDS image

of control group versus CDA and CDI patients shows CD patients mostly clustered together in the blue circle.

Additionally, when analysing GI bacterial diversity in the Pouch patients compared to control groups,

using MDS, 89% of pouch patients are clustered together in the white circle, while none of control

group are clustered among pouch patients in the white circle (Figure 6). Interestingly, DGGE gel

picture shows an identical very bright band (equal to high bacteria concentration) among pouch

patients (marked with red line around). Furthermore, the diversity is seen to be diminished in patients

with active vs. inactive pouchitis. Studies reports up to 50% of pouch patients develop at least one

episode of pouchitis165. Additionally, within 12 months of ileostomy surgery, 40% of the pouch

26 Control A

42 CD

38 UC

23 Control B

18 CDA

20 CDI

36

patients develop pouchitis165. Evidence suggests that an abnormal mucosal immune response to altered

microbiota in the pouch leads to acute and/ or chronic inflammation166.

Figure 6 Multidimensional scaling (MDS) analysis performed using BioNumerics 7.0 A) 89% of pouch patients are

clustered together in the white circle, while none of the control groups are clustered among pouch patients. B) one

identical brighter band appears in DGGE gel of all pouch patients (marked with red line around).

17 Control 7 PA

11 PI A B

37

4.2 Aim II: Secretion of alpha-hemolysin by Escherichia coli

disrupts tight junctions in inflammatory bowel disease patients


An increased GI prevalence of E. coli has been associated with IBD. Many of the IBD-associated E.

coli belong to the phylogenetic group B2 and harbour virulence genes present in extra intestinal

pathogenic E. coli including alpha-hemolysin (hly) and cnf1. In this study, the goal was to

investigate the potential of E. coli, isolated from IBD patients with active and inactive disease, to

damage the integrity of the intestinal epithelium and the potential contribution of hly and cnf1in

vitro.


Three of five E. coli strains from patients with active IBD (strains p7, p19A and p22) completely

disrupted epithelial cell TJ after only 3-6 hours of co-culture at an MOI of 10. This was not

observed with EcN, LF82, the four E. coli isolated from patients with inactive UC and three E. coli

strains from healthy controls. The three strains disrupting TJ in epithelial cells, all belonged to the

phylogenetic group B2 and they were all alpha-hemolysin positive. IBD associated E. coli p19A

was chosen for further experiments to elucidate the role of alpha-hemolysin and cnf1. P19A was

found to possess two copies of the hly gene cluster, one of which was located upstream of the cnf1

gene. Deletion mutants of the individual hemolysin genes as well as a cnf1 retained the ability to

damage Caco-2 cell TJ after less than 6 hrs of co-culture. However, this phenotypic characteristic

was lost in a double knockout mutant of both hly genes (Figure 7). Interestingly, there were no

differences in cytotoxicity between p19A WT and mutants.

38

Figure 7 p19A WT and mutants effect on Caco-2 cell TJ, using the TER method. p19A WT followed by ∆ hlyI, ∆ hlyII

and ∆ cnf1 mutant all disrupted the epithelial barrier in less than 6 hrs of co- culture with Caco-2 cells. Whereas p19A ∆

hlyI, II mutant did not disrupt epithelial barrier until after 12 hrs of co-incubation. As control TER was also performed

on Caco-2 cells without any bacteria added.

Hemolytic titration assays were performed with bacteria and bacterial culture supernatants in order

to investigate the hemolytic activity of clinical isolate E. coli p19A WT and the hly and cnf1 mutants.

The hemolytic activity was only completely abolished in the double mutant lacking hly clusters I and

II (p<0.05). Deletion of hly cluster II only partially decreased hemolytic activity, compared to the WT

suggesting hly cluster I does contribute to the overall hemolytic activity of the WT strain, despite the

fact that no reduction in hemolysis was observed in the hly cluster I mutant. RT-PCR was used to

quantify the relative amounts of the hly transcript in E. coli p19A WT and the different hly deletion

mutants. Relative expression of hly was two-fold higher in the WT p19A than in the ∆hlyI, mutant

and four-fold higher than in the ∆hlyII mutant. As expected only the double ∆hlyI, II mutant of p19A

lacked hly gene expression. A significant reduction in immunofluorescent staining of occludin was

evident, from 2 to 3 hours of co-culture with wild type p19A (P<0.001), as confirmed by Western

blotting with antibodies to occludin (Figure 8A). Similar results were obtained with all three Hly-

producing E. coli strains p7, p19A or p22, but not with the p19A ∆hlyI, II double mutant

demonstrating a link between hly expression and loss of TJ occludin (Figure 8B).

39

Figure 8 A) Western blot analysis of occludin expression in Caco-2 cell monolayers infected with p19A, p19A ∆ clbA,

p19A ∆ cnf1 and p19A WT. The occludin expression is high in p19A mutant ∆ hlyI, II, single p19A mutant ∆hlyI and

∆hlyII. The beta actin controls are equal for all. B) Confocal images of Caco-2 cell-monolayers immunofluorescence

stained for occludin (red) and nuclei (blue) for 3 hrs (MOI: 50) during apical incubation with E. coli p 19A from a

patient with active Ulcerative colitis. P-values for differences were, after 2 and 3 hrs, P <0.01 and P<0.001,

respectively. Notice, there appears a non-specific band on top of occluding band in p19A ∆hlyI, II.

IBD associated E. coli producing alpha-hemolysin can cause rapid loss of TJ integrity in differentiated

Caco-2 cell monolayers. This effect is abolished in a mutant unable to express alpha-hemolysin.

These results indicate that high alpha-hemolysin expression may be a mechanism by which strains of

E. coli isolated from IBD patients can contribute to epithelial barrier dysfunction and pathophysiology

of IBD.

A

B

40

4.3 Aim III: Comparison of three commercial fecal calprotectin

ELISA test kits used in patients with inflammatory Bowel Disease


Faecal calprotectin is a non-invasive marker of intestinal inflammation used to distinguish between

functional and organic bowel diseases and to evaluate disease activity among patients with IBD.

The goal of this study was to compare three different ELISA tests measuring calprotectin in their

accuracy to detect IBD and to distinguish between IBD patients with active or inactive disease.


Comparison of HK325 VS CALPRO ELISA kit: When measuring faecal calprotectin using

CALPRO, 92% of the samples from healthy controls and 9% of the samples from IBD patients had

a faecal calprotectin result below the cutoff value of 50 mg/kg. With HK325, only 7% of the

samples from healthy controls and none of the samples from IBD patients had a faecal calprotectin

result below the cutoff value (50 mg/kg). Based on a ROC curve, HK325 calprotectin

measurements (cutoff value of 50 mg/kg) had 95% sensitivity and 28% specificity. CALPRO

calprotectin measurements (cutoff value of 50 mg/kg) had 90% sensitivity and 93% specificity.

Comparison of EK-CAL VS CALPRO ELISA kit: Measuring faecal calprotectin using EK-

CAL, 70% of the samples from healthy controls and 9% of samples from IBD patients had a faecal

calprotectin result below the cutoff value of 50 mg/kg. Using the CALPRO calprotectin ELISA test,

96% of the samples from healthy controls and 25% of the samples from IBD patients had faecal

calprotectin value below the cutoff value of 50 mg/kg (Figure 9). Based on a ROC curve, CALPRO

calprotectin measurements (calprotectin cutoff value of 50 mg/kg) had 72% sensitivity and 96%

specificity (Figure 10). The stated EK-CAL calprotectin cutoff value of 50 mg/kg, gave 92%

sensitivity and 74% specificity. The CALPRO ELISA test performed best in measuring calprotectin

in faeces.

41

Figure 9 Frequency ratio for the EK-CAL and CALPRO ELISA: calprotectin results for healthy controls and IBD

patients. As it is shown, the faecal calprotectin values of 16 of 23 healthy controls are seen below the cutoff value

(log10≈ 1.7) compared to only 5 of 54 IBD patients for EK-CAL. For CALPRO ELISA method, the faecal calprotectin

values of 22 of 23 healthy controls and 7 of 54 IBD patients are seen below the cutoff value (log10≈ 1.7).

42

Figure 10 ROC curve of false-positive ratio for the HK325 and CALPRO ELISA: False-positive calprotectin results

of the HK325 versus the CALPRO calprotectin ELISA methods. As it is shown, EK-CAL has 26% false-positive

results, while CALPRO has 4% false-positive calprotectin results.

When comparing active vs inactive disease based on clinical activity indices, a statistically

significant difference in the CALPRO levels was found in patients with UC with median

(interquartile range [IQR]) faecal calprotectin levels of 150 mg/kg (39-1843) in UCA and of 39

mg/kg (38-47) in UCI, p<0.05. In patients with an ileoanal pouch, the median (interquartile range

[IQR]) faecal calprotectin levels were higher in active disease, 296 mg/kg (103-1783) in PA, and

117 mg/kg (60-187) in PI. The median (interquartile range [IQR]) level of faecal calprotectin was

222 mg/kg (149-789) in CDA and 125 mg/kg (57-850) in CDI, NS.

A significant correlation between clinical activity indices and faecal calprotectin in patients with

UC (p<0.05***) and patients with an ileoanal pouch (p<0.05) was shown (Figure 11). However,

regression analysis shows no correlation between faecal calprotectin and disease activity indices, in

patients with CD (p=0.078). In healthy controls, the median (interquartile range [IQR]) level of

faecal calprotectin of 8 mg/kg (8-17) was significantly lower than all active or inactive IBD patient

groups, p<0.05 (Figure 11).

43

Figure 11 Faecal calprotectin in IBD-patients: Faecal calprotectin results using the CALPRO calprotectin ELISA

method in different IBD-patient groups during active and inactive disease compared to controls. Error bars show the

mean and standard division (SD). As it is shown, faecal calprotectin concentrations are associated with clinical disease

activity indices in patients with UC and pouchitis. Controls are significantly below the cutoff value. Faecal calprotectin

is not a suitable marker for differentiating between disease activities in Crohn’s disease patients.

44

4.4 Aim IV: Extra intestinal pathogenetic Escherichia coli are

associated with intestinal inflammation in patients with Ulcerative

Colitis


E. coli of the phylogenetic group B2 frequently colonize the intestine of patients with IBD. We have

previously tested the effect of Ciprofloxacin (Cipro) and/or orally administered E. coli EcN as add-

on treatment, to conventional therapies in patients with UCA. Studies show that IBD patients

between relapses have a calprotectin value between 123-213 mg/kg167. Therefore, in this follow-up

study a calprotectin value of >200 mg/kg was used as criteria for active UC. In this study, the goal

was to investigate the influence of EcN treatment and initial colonization with phylogenetic group

B2 E. coli on intestinal inflammation evaluated by measurement of faecal calprotectin.


As described previously129 100 patients with UC were included in this study, and randomized to

four treatment groups: 1 week of Cipro or placebo followed by 7 weeks of EcN or placebo, with 25

patients in each group. Activity was primarily evaluated by a CAI score>/= 6.

When faecal calprotectin of patients with an initial faecal calprotectin level >200 mg/kg and a CAI

score≥ 6 were analysed (UCA), we confirmed that in the UCA patient group receiving placebo/EcN

fewer patients, 17 %, reached remission defined by a faecal calprotectin <200 mg/kg compared to

the UCA patient group receiving placebo/placebo, 61 %, p<0.05 (Figure 12). When comparing

UCA patient groups receiving EcN (Cipro/EcN and placebo/EcN), with UCA patient groups not

receiving EcN, (Cipro/placebo and placebo/placebo), it was demonstrated that groups receiving

EcN reached remission (faecal calprotectin < 200 mg/kg) less frequent (31.4% ) than groups not

receiving EcN (59% ), p≤0.05 (figure 12).

However, in order to see whether EcN has an overall effect on the level of calprotectin, groups

treated with EcN were compared to groups not treated with EcN. Faecal calprotectin values from

weeks 0, 1, 8 and 12 of 100 UC patients with CAI score≥ 6 (regardless of the calprotectin value of

the initial stool sample) was evaluated using the Two-way ANOVA test. When comparing the

experimental groups receiving EcN (Cipro/EcN and placebo/EcN), with groups not receiving EcN,

45

(Cipro/placebo and placebo/placebo), higher faecal calprotectin values were found in patients

treated with EcN, however this was not statistically significant, p=0.053.

Figure 12 Comparison of the UC patient groups reaching faecal calprotectin values <200 mg/kg during 12 weeks

follow-up (intention to treat- analysis) (A) Treated with: placebo-placebo (D), Cipro/EcN (A), Cipro/placebo (B) or

placebo/EcN (C). (B) 31% of the UC patients treated with EcN (AC) as add-on treatment reach calprotectin value <200

mg/kg, while 59% of the patients not treated with EcN (BD) reach a calprotectin value <200 mg/kg, p<0.05*.

When analysing all 100 UC patients with CAI score≥ 6, in the patient group treated with

Cipro/placebo, 48% (12/25) were initially colonized with B2 E. coli at week 0, prior to

ciprofloxacin treatment. After antibiotic treatment, the number of the patients colonized with B2 E.

coli was reduced to 18% (4/22), 33% (7/21) and 36% (5/14) at week 1, 8 and 12, respectively

(figure 13). These results demonstrates, that even though one week of Cipro treatment did reduce

the number of UCA patients colonized with B2 E. coli immediately after the treatment, no long

term effect on B2 E. coli colonization could be demonstrated. In the group treated with

placebo/placebo, 52 % (13/25) of the patients were colonized with B2 E. coli at week 0. In this

group, the number of patients colonized with B2 E. coli were, 42% (10/24), 45% (9/20) and 38%

(6/16) at week 1, 8 and 12, respectively (figure 13).

46

Figure 13 Project flow of all patient groups, with number of the submitted stool samples, Median faecal calprotectin

value, the number of the B2 E. coli isolates and number of the patients with a faecal calprotectin <200 mg/kg, at week

0, 1, 8 and 12.

Interestingly, UC patients, initially colonized with B2 E. coli (before treatment with EcN) had an

increased faecal calprotectin value in comparison to UC patients not colonized with B2 E. coli,

median (interquartile range [IQR]) 1336 mg/kg (672-1818) and 386 (126-787), respectively,

p<0.05*** (figure 14).

Our results demonstrate that there are no beneficial effect of using EcN as add-on treatment in UCA

patients. On the contrary, an increased number of patients treated with EcN as add-on treatment

were unable to reach remission. UC patients included in this study, initially colonized with B2 E.

coli at week 0, have increased burden of inflammation measured using CAI score and faecal

calprotectin compared to UC patient not colonized with B2 E. coli.

47

Figure 14 week 0 faecal calprotectin value in mg/kg in experimental groups (25 patients in each group) included in the

study. There is significant difference between faecal calprotectin values of group initially colonized with B2 E. coli

(46/87) in comparison to group not colonized with B2 E. coli (41/87), p<0.05*(Median with rang) (there was no

bacterial growth in 13 patients faecal samples).

48

5. Discussion and Perspectives

The colon and the distal ileum are colonized with a complex mixture of microorganisms that are

highly metabolically active. Enteric bacteria and fungi provide a constant antigenic stimulus that

drives a pathogenic immune response seen in genetically susceptible individuals such as IBD

patients. A variety of genetic defects in either mucosal barrier function or in the innate bacterial

killing lead to an abnormally aggressive T-cell activation by bacterial antigens and/or the loss of

normal immunologic tolerance by the mucosal immune system. Recently developed concepts of

IBD pathogenesis include defective innate immune function resulting in diminished bacterial killing

as well as functional alterations in the composition of the commensal bacteria such as E .coli, which

in turn leads to enhanced epithelial adherence and invasion, bacterial persistence within intestinal

epithelial and phagocytic cells, and metabolic derangements that negatively influence the epithelial

function. However, this might explain reduced intestinal bacterial diversity in IBD patients in

comparison to healthy controls162–164(manuscript I), which might be caused by intestinal overgrowth

of aggressive bacteria in IBD patients, which are normally in symbiotic balance with other intestinal

bacteria in healthy people.

Studies show increased numbers of E. coli strains from the B2 phylogenetic group harbouring

ExPEC genes within the intestines of IBD patients. Patients colonized with E. coli of the B2

phylogenetic group display increased burdens of inflammation as measured using the CAI score and

faecal calprotectin levels, in comparison to patients not colonized with B2 E. coli (manuscript IV).

DAEC have been associated with UC, while AIEC has been linked to CD. Both AIEC and DAEC

are known as enteropathogenic96 E. coli and often belongs to the B2 phylogenetic group168. The B2

phylogenetic E. coli is frequently isolated from the intestines of IBD patients169. Martinez-Medina93

et al show in a collection of 22 AIEC isolates, 14 from CD and 6 from healthy controls, 63% of the

strains were from the B2 phylogenetic group and here of 79% of the stains was isolated from CD

patients93. However, Escobar-Paramo170 et al showed that of 16 DAEC strains isolated from patients

with diarrhoea, 31.3% was from B2 and 31.3% was from D phylogenetic group. Petersen et al

200924 reported the prevalence of E. coli isolated from a UC patient harbouring afa/Dr gene, which

is related to DAEC, while other studies showed the high prevalence of E. coli isolates from CD

patients sharing ExPEC genes and the strains belonged to the AIEC pathovar93. The question

remains whether B2 E. coli strains isolated from faecal samples of IBD patients generally, are

related to the AIEC and DAEC pathovars. The role of E. coli pathobionts in the pathophysiology in

49

IBD has been attributed to their capability to adhere and invade epithelial cells and replicate in

macrophages and the most well-studied prototype strain is LF82. Previous studies have shown that

UC associated E. coli (p19A) from the B2 phylogenetic group, harbouring two alpha-hemolysin

genes, induce cell death in dendritic cells, stimulate the release of TNF-α, IL-6, IL-23 cytokines98

(poster) and causes rapid loss of TJ integrity in differentiated Caco2-cell monolayer (manuscript II).

In contrast to p19A, strain LF82 does not cause rapid dissolution of epithelial TJ, clearly indicating

that B2 phylotype of IBD-associated strains differ markedly in pathogenic mechanisms.

The IBD-associated E. coli strains included in our study causing loss of TJ in epithelial cell

monolayers were hemolytic (manuscript II). Four types of hemolysin have been demonstrated in E.

coli: alpha-hemolysin (hly A), plasmid- and phage- carried enterohemolysin (EhxA, a-hlyA) and

silent hemolysin (SheA); EhxA and HlyA belong to the RTX (repeat in toxin) related family, which

lyse erythrocytes from different mammalian species171–173. It is known that a number of E. coli

pathotypes, i.e. urinary tract pathogenic (UPEC), Verocytotoxin-producing E. coli (VTEC), EPEC

and Enterotoxigenic E. coli (ETEC) all harbor alpha-hemolysin174. The E. coli alpha-hemolysin is

known to be able to lyse erythrocytes through binding to the surface protein glycoporin175–177 but

also other cell types including leucocytic cells, bladder and renal tubular cells in a dose dependent

manner153,171,172,178,179.

The role of hlyA in TJ disruption was further investigated in E. coli strain p19A, which possessed

two hlyA clusters as previously reported for isolates of uropathogenic E. coli belonging to phylotype

B2180. We showed that both hyl A gene clusters in p19A contributed to the damaging effects on

epithelial integrity, suggesting that intestinal E. coli strains expressing possessing more than one

hlyA locus can have increased pathological consequences in intestinal inflammation. Although our

IBD-associated strains did not induce epithelial cell apoptosis, a hly-expressing uropathogenic E.

coli was previously shown to cause localized regions of apoptosis in HT29/B6 cell monolayers. The

difference between these findings and our results may be due to use of a higher MOI than in our

study, the use of different strains or the amount of hlyA expressed181 (manuscript II).

These findings suggest that IBD associated E. coli might play a role in pathogenesis of IBD and

might play a role in disease relapses in IBD patients. Additional studies of gut microbiota and IBD

associated E. coli species are needed to confirm whether IBD reflects an abnormal host response to

commensal bacteria or if the acquisition of pathogenic features by specific E. coli strains drives the

onset of the IBD. However, clinical experience with treatment effects (although varying) of both

50

immune-suppressants and antibiotics (and probiotics) would suggest that the truth about IBD

pathogenesis lies in between the interaction between the predisposing host genetic factors and the

host immune response to intestinal bacteria.

51

6 Conclusion

There is a reduced diversity of intestinal microbial species in IBD patients in comparison to healthy

controls (Manuscript I). Patients colonized with E. coli from the B2 phylogenetic group display

increased burdens of inflammation, as measured by CAI scores and faecal calprotectin levels, in

comparison to patients not colonized with B2 E. coli (Manuscript IV). UC associated E. coli (p19A)

from the B2 phylogenetic group, harbouring two alpha-hemolysin genes, induce cell death in

dendritic cells, stimulate the release of TNF-α, IL-6, IL-23 cytokines98 (poster) and causes rapid

loss of TJ integrity in differentiated Caco2-cell monolayer (manuscript II).

52

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Manuscript I:

Microbial diversity in fecal samples depends on DNA extraction method:

easyMag DNA extraction compared to QIAamp DNA stool mini kit

extraction

Hengameh Mirsepasi, Søren Persson, Carsten Struve, Lee O B Andersen, Andreas M

Petersen and Karen A Krogfelt. BMC Research Notes 2014, 7:50.

67

Manuscript II:

Secretion of alpha-hemolysin by Escherichia coli disrupts tight

junctions in inflammatory bowel disease patients

Hengameh Mirsepasi, Zhengyu Du, Carsten Struve, Godefroid Charbon, Jurgen Karczewski,

Karen Angeliki Krogfelt, Andreas Munk Petersen, Jerry M. Wells. Submitted to American

Journal of Gastroenterology.

68

Manuscript III:

Comparison of three commercial fecal calprotectin ELISA test kits in

patients with Inflammatory Bowel Disease

Hengameh Chloé Mirsepasi-Lauridsen, Ulla Bachmann Holmetoft, Sofie Ingdam Halkjær,

Karen Angeliki Krogfelt, Andreas Munk Petersen. Revision submitted for

publication in Scandinavian Journal of Gastroenterology.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Krogfelt%20KA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Petersen%20AM%22%5BAuthor%5D

69

Manuscript IV:

Extra intestinal pathogenic Escherichia coli are associated with

intestinal inflammation in patients with Ulcerative Colitis

Hengameh Chloé Mirsepasi-Lauridsen, Sofie Ingdam Halkjaer, Esben Munk Mortensen,

Magnus Christian Lydolph, Inge Nordgaard-Lassen, Karen Angeliki Krogfelt and Andreas

Munk Petersen. (Manuscript for submission).

70

Appendices

Conferences and Poster Presentations

Documents

PhD Thesis20Chlo%E9%20Mirsepasi... · Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, traditionally divided into Crohn’s disease