A comparison of the microbial community structure between inflamed and
non-inflamed sites in patients with ulcerative colitis
Running head: Dysbiosis in ulcerative colitis
Atsushi Hirano (1), Junji Umeno (1), Yasuharu Okamoto (1), Hiroki Shibata (2), Yoshitoshi
Ogura (3), Tomohiko Moriyama (1), Takehiro Torisu (1), Shin Fujioka (1), Yuta Fuyuno (1),
Yutaka Kawarabayasi (4), Takayuki Matsumoto (5), Takanari Kitazono (1), Motohiro Esaki
(1)
1. Department of Medicine and Clinical Science, Graduate School of Medical Sciences,
Kyushu University, Fukuoka, Japan
2. Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
3. Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University,
Fukuoka, Japan
4. National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan
5. Division of Gastroenterology, Department of Internal Medicine, Iwate Medical University,
Iwate, Japan
Correspondence to: Motohiro Esaki, M.D, PhD.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/jgh.14129
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Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu
University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
Phone: +81-92-642-5261; Fax: +81-92-642-5273; E-mail
mesaki@intmed2.med.kyushu-u.ac.jp
Financial support: This work was supported by grants awarded to AH by Mitsubishi Tanabe
Pharma Corporation [grant number MTPS20160411025]. The funder had no role in study
design, collection, or interpretation of data.
Declaration of conflict of interest: The authors declare that there is no conflict of interest.
Acknowledgments
We graciously thank the patients for providing samples.
Itemized list:
Word count for abstract: 245
Word count for text: 2661
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Tables: 1
Figure: 5
Supplementary figure: 4
References: 38
Abstract
Background and Aim: The gut microbiota is suggested to play an important role in the
pathogenesis of ulcerative colitis (UC). However, inter-individual and spatial variations
hamper the identification of UC-related changes. We thus investigated paired
mucosa-associated microbiota obtained from both inflamed and non-inflamed sites of UC
patients and corresponding sites of non-IBD controls.
Methods: Mucosal biopsies of both inflamed and non-inflamed sites were obtained from 14
patients with active UC of the left-sided or proctitis type. Paired mucosal biopsies of the
corresponding sites were obtained from 14 non-IBD controls. The microbial community
structure was investigated using 16S rRNA gene sequences, followed by data analysis using
Qiime and LEfSe software.
Results: Microbial alpha diversity in both inflamed and non-inflamed sites was significantly
lower in UC patients compared with non-IBD controls. There were more microbes of the
genus Cloacibacterium and the Tissierellaceae family, and there were less microbes of the
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genus Neisseria at the inflamed site when compared to the non-inflamed site in UC patients.
Decreased abundance of the genera Prevotella, Eubacterium, Neisseria, Leptotrichia,
Bilophila, Desulfovibrio, Butyricimonas was evident at the inflamed site of UC patients
compared to the corresponding site of non-IBD controls. Among these taxa, the genera
Prevotella and Butyricimonas were also less abundant at the non-inflamed site of UC patients
compared to the corresponding site in non-IBD controls.
Conclusions: Mucosal microbial dysbiosis occurs at both inflamed and non-inflamed sites in
UC patients. The taxa showing altered abundance in UC patients might mediate colonic
inflammation.
Keywords
ulcerative colitis; mucosal microbiota; dysbiosis; 16S rRNA gene sequence
Author contributions
AH, JU, TK and ME conceived and designed this study. AH, JU, YO, TM, TT, SF, YF and
ME collected and processed mucosal biopsies. AH and HS performed the experiments. AH,
JU, HS and YO analyzed and interpreted the data. AH, YK, TM and ME drafted the
manuscript. All authors approved the final version.
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Introduction
Ulcerative colitis (UC) and Crohn’s disease (CD) are the two principal forms of
inflammatory bowel disease (IBD). Although their etiology remains uncertain, complex
interactions between genetic susceptibility and environmental factors are considered to play a
role in the pathogenesis of IBD. An imbalance in the composition of the microbial population
has been proposed to be one of the most important environmental factors, and considerable
efforts have been focused on the identification of the microbial taxa that are associated with
gut inflammation.
Culture-independent techniques, which can identify bacteria on the basis of the
nucleic acid sequence of 16SrRNA molecules 1, have recently revolutionized the
understanding of the complex intestinal bacterial ecology associated with various diseases 2 3.
The dysbiosis characterized by a decreased abundance of Firmicutes, particularly of
Clostridium cluster IV or the enrichment of species belonging to the Enterobacteriaceae
family containing adherent invasive Escherichia coli (AIEC) occurs in CD4 5. In contrast,
associations between changes in the microbial population and UC are still inconsistent,
suggesting that dysbiosis might be less important for UC than CD 6. However, because the
genetics have been shown to contribute little to the etiology of UC when compared to CD in
twin studies 7, environmental triggers, including dysbiosis, are more likely to play important
roles in the pathogenesis of UC.
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Faecal samples have been used to investigate the microbial population in the
majority of previous studies. However, mounting evidence has indicated that the faecal
microbiota is distinct from the mucosa-associated microbiota 8 9. Because the
mucosa-associated microbiota shows intimate contact with the host, it is more likely to
induce direct immune responses than luminal microbiota does10. Therefore, mucosal samples
may be more appropriate for the identification of the microbiota associated with mucosal
inflammation. Moreover, paired mucosal sampling from inflamed and non-inflamed sites in
UC patients is a valid approach to minimize the effects of inter-individual variation in the
microbial population.
Studies of this type were carried out prior to the era of next-generation sequencing
technology, but those studies utilized relatively low-resolution methods, such as temporal
temperature-gradient gel electrophoresis (TTGE) 11, denaturing gradient gel electrophoresis
(DGGE) 12 or sequencing by capillary electrophoresis 13 14 for the determination of the
microbial population. Forbes et al. recently analysed differences in the bacterial population
between inflamed and non-inflamed sites using next-generation sequencer. However, the
results could have been influenced by inter-individual and spatial variations, because 3–4
mucosal samples were collected from various colonic sites in each UC patient 15.
In the present study, paired mucosal samples were collected unequivocally from
inflamed rectal mucosa and non-inflamed transverse colonic mucosa in UC patients. We
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believe that such samplings contribute to the characterization of the microbial population that
associates with colonic inflammation in UC. In addition, paired mucosal samples were also
collected from non-IBD controls, and microbial population at the corresponding sites were
compared between UC patients and non-IBD controls. With the analysis, site specific
difference of microbial population between UC patients and non-IBD controls could be
determined, while mucosa-associated microbiota has been reported to be mostly homogenous
throughout the colorectum9.
Using these specimens, we analysed the microbial community structure by 16S rRNA gene
sequencing with next-generation sequencer.
Methods
Subjects
Fourteen patients with active UC of the left-sided type or proctitis type, and 14
individuals without inflammatory bowel disease (non-IBD controls), who underwent
colonoscopy for polyp surveillance, were enrolled in the present study. If an adenoma larger
than 1 cm, cancer or inflammation were detected during surveillance colonoscopy of
non-IBD control patients, these individuals were excluded from the study. All the participants
were Japanese and none were related to each other. No individual had received antibiotics
during the preceding 2 months.
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Sample collection
Before colonoscopy, patients received standard bowel preparation using 2L of lavage
solution (NIFLEC®; EA Pharma Co., Tokyo，Japan). During colonoscopy, tissue samples
were obtained from the transverse colon and the rectum in each subject using disposable
biopsy forceps (Olympus, Tokyo, Japan) after washing the colorectal mucosa gently with tap
water containing dimethicone solution. Bioptic forceps were also rinsed with tap water before
taking each bioptic sample.
Based on the disease types of UC patients in the present study, the transverse colon
was regarded as the ‘non-inflamed’ site and the rectum was regarded as the ‘inflamed’ site in
UC patients (see Supplementary Figure 1). Mucosal biopsy samples were stored at -80°C
until extraction of DNA.
DNA extraction and 16S Ribosomal RNA Gene Sequencing
Mucosal bacterial DNA was extracted from mucosal samples using NucleoSpin®
Tissue XS (Macherey-Nagel, Düren, Germany) and 5 mm stainless steel beads in a Tissue
lyser (Qiagen Inc. CA, USA) vibrating at 25 times/sec for 1 min. Subsequently, 16S rRNA
gene sequencing was conducted as described previously with minor modifications 16. Briefly,
the extracted bacterial DNA was used as the template to amplify the V4 region of each 16S
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rRNA gene using the primer pair 515F/806R, which included the Illumina Flowcell adapter
sequences. The reverse primer also contained a 12 bases barcode sequence. Paired-end
sequencing of the PCR amplicons was performed on the Illumina MiSeq platform (Illumina
Inc. CA, USA) using custom primers.
Bioinformatics and Statistical analysis
Raw Illumina fastq files were demultiplexed, quality filtered, and analysed using
QIIME v 1.9.1 software 17. The 16S rRNA operational taxonomic units (OTUs) were
clustered using “open-reference OTU” of QIIME. In this open-reference OTU picking
process, reads were firstly clustered against a Greengenes 13_8 reference 18 using
closed-reference OTUs picking. Subsequently, 0.1% of the reads which failed to hit the
reference sequence collection were randomly subsampled and clustered de novo using
UCLUST 19, with an OTU cluster defined by a sequence similarity of 97%. ChimeraSlayer
was employed to remove chimeric sequences 20. Alpha diversity (microbial diversity within
samples) was calculated using Observed Species, Phylogenetic Diversity (PD) Whole Tree
and Chao1. OTUs were rarefied at a depth of 5,500 sequences, and subsampling was
performed 10 times. The measured alpha diversities were compared between each group
using a non-parametric two sample t-test and the default number of Monte Carlo
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permutations (999). Principal Coordinate Analysis (PCoA), based on the weighted UniFrac
distances, was used to evaluate beta diversity (community diversity between samples). The
significance of the distance between two groups was calculated by non-parametric
MANOVA.
We used linear discriminant analysis (LDA) effect size (LEfSe) 21, which is an
algorithm used to discover high-dimensional biomarkers characterizing the differences
between biological conditions, to identify taxa that differed consistently between sample
types. LEfSe employs the non-parametric factorial Kruskal-Wallis (KS) sum-rank test
(α=0.05) to identify taxa with significantly different abundances between categories,
followed by LDA to estimate the effect size of each feature of the differential abundance. We
regarded differences in abundance as statistically significant when the logarithmic LDA score
was > 2.0. Significant taxa were used to generate taxonomic cladograms illustrating
differences between sample classes in the Galaxy framework
(http://huttenhower.sph.harvard.edu/galaxy) 22. If multiple varieties with different ranks
showed significance in the same taxon, the lowest-ranked varieties were regarded as
responsible.
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Ethical considerations
The present study was conducted in accordance with the Declaration of Helsinki.
The protocol was approved by the Ethical Committee of Kyushu University (approval
number: 27-176), and written informed consent was obtained from all participants.
Results
Fifty-six mucosal samples were collected from 28 participants, including paired
samples from 14 patients with active UC and 14 non-IBD controls. The clinical
characteristics of the participants are listed in Table1. Most of the UC patients exhibited the
left-sided type, and their endoscopic findings were classified as mild to moderately active.
Mesalazine was administered to 13 UC patients and probiotics were administered to 10 UC
patients. Three UC patients and two non-IBD controls were taking proton pump inhibitor.
While no UC patient experienced abdominal surgery, two non-IBD controls experienced
appendectomy. A total of 2,732,338 quality-filtered sequences were obtained from the
samples, with a mean of 48,791±32,360 (standard deviation; SD) sequences per sample. The
filtered data were assigned to 11 bacterial phyla. The most diverse phylum was Firmicutes,
followed by Bacteroidetes and Proteobacteria, and the rank order of abundance of these three
phyla in the four sample groups (transverse colon and rectum of UC patients and non-IBD
controls) was similar (see Supplementary Figure 2).
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Comparison of alpha and beta diversity according to colonic site and disease status
The alpha diversities expressed as observed species index were significantly lower in
both the transverse colon (non-inflamed site) and the rectum (inflamed site) of UC patients
compared with those of non-IBD controls (Figure 1). Other indices of alpha diversity (PD
Whole Tree, Chao1) showed similar trends (see Supplementary Figure 3). These results
indicate that the mucosal microbial diversities of UC patients were lower in both
non-inflamed and inflamed sites.
No apparent clustering was observed in PCoA using the beta diversity metrics of
weighted UniFrac between the transverse colon and the rectum of both UC patients and
non-IBD controls (Figure 2 a, b). Conversely, the composition of the bacterial populations
tended to be different between UC and non-IBD controls in both the transverse colon
(p=0.18) and rectum (p=0.12) (non-parametric MANOVA), although the differences did not
reach statistical significance (Figure 2 c, d).
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Comparison of taxonomic composition according to biopsy site in UC patients and
non-IBD controls
When the taxonomic composition was compared between the rectum (inflamed site)
and the transverse colon (non-inflamed site) of UC patients using LEfSe, the abundances of
the genus Cloacibacterium and the Tissierellaceae family were significantly greater at the
inflamed site compared to the non-inflamed site. In contrast, the abundance of the genus
Neisseria was lower at the inflamed site than the non-inflamed site (Figure 3). However, no
significant difference was observed in taxonomic composition between the transverse colon
and the rectum among non-IBD controls.
Comparison of taxonomic composition between UC patients and non-IBD controls
When the taxonomic composition of microbiota in the rectum of UC patients and
non-IBD controls was compared, the abundance of the genera Prevotella, Eubacterium,
Neisseria, Leptotrichia, Bilophila, Desulfovibrio, and Butyricimonas was lower in UC
patients than non-IBD controls (Figure 4). Conversely, only the abundance of the
Bifidobacterium genus was greater in UC patients than non-IBD controls.
When the taxonomic composition of microbiota in the transverse colon was
compared between UC patients and non-IBD controls, the abundance of the genera Prevotella
and Butyricimonas was significantly lower in UC patients than non-IBD controls (Figure 5).
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Thus, the abundance of the genera Prevotella and Butyricimonas was lower in both the
non-inflamed and inflamed sites of UC patients.
Discussion
In this study, we have demonstrated decreased alpha diversity in both inflamed and
non-inflamed sites of UC patients compared to non-IBD controls, indicating that dysbiosis
was present not only at inflamed sites but also at non-inflamed sites in UC patients. The
strength of the present study is that we were able to compare the microbiota between
inflamed and non-inflamed sites by paired mucosal sampling in patients with UC. Moreover,
we compared the mucosal microbiota at each site between UC patients and non-IBD controls.
By using these sampling, we could minimize inter-individual and spatial variations in
mucosal microbiota.
Consistent with the finding of the previous study 9, the populations of mucosal
microbiota in the present study were roughly similar between the transverse colon and rectum.
However, there was a trend towards a larger alpha diversity in the rectum compared to the
transverse colon in both UC patients and non-IBD controls (see Supplementary Figure 4).
This result strongly suggests the necessity of a comparison of mucosal samples obtained from
the identical sites between UC patients and non-IBD controls to avoid the confounding effect
of spatial variation and to detect site-specific differences in the microbial populations of the
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large bowel.
With regard to specific taxa in UC, greater abundance of the Cloacibacterium and
Tissierellaceae were found at the inflamed site compared to the non-inflamed site.
Cloacibacteria are Gram-negative bacteria that were first identified in untreated human
wastewater 23. Furthermore, their greater abundance in the submucosal tissues was found in
patients with Crohn's disease 24. Tissierellaceae have recently been identified within the
phylum Firmicutes by reconstruction of high-rank 16S rRNA gene-based phylogenies 25, and
they have been shown to be more abundant in patients with Parkinson’s disease 26.
Considering the difference in their abundance among inflamed and non-inflamed sites in UC
and their presence in non-IBD controls, these taxa may act as pro-inflammatory property
when microbial diversity is low. Further investigation using animal models is necessary to
determine whether these bacteria can actually induce colonic inflammation under specific
conditions.
In the present study, the abundance of the genera Eubacterium, Butyricimonas,
Prevotella, Neisseria, Leptotrichia, Bilophila and Desulfovibrio was lower at the inflamed
site in UC compared to the corresponding site in non-IBD controls. In addition, the
abundance of the Prevotella and Butyricimonas was also lower at the non-inflamed site of
UC compared to the corresponding site in non-IBD controls. Among those species,
Eubacterium is categorized in cluster XIVa of the Clostridia, which have recently gained a lot
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of attentions because of their anti-inflammatory properties mediated by induction of
regulatory T-cells 27. Recently, the lower abundance of Eubacterium has also been shown in
UC patients 28 29. A lower abundance of Butyricimonas with a negative correlation with
pro-inflammatory cytokines, has recently been reported in patients with multiple sclerosis 30.
In addition, Prevotella, which showed the greatest difference at both the inflamed and
non-inflamed sites of UC from non-IBD controls in the present study, was reported to be less
abundant in untreated multiple sclerosis patients, while this difference was ameliorated after
the treatment 30. Although the potential influence of Prevotella on gut inflammation has not
been fully investigated, the “Prevotella enterotype” is known to be associated with
non-Western rural communities, where residents consume a plant-based diet rich in
polysaccharides and fibre known to be protective against inflammation 31 32. Therefore,
Prevotella provisionally plays a protective role against gut inflammation. Neisseria is
considered to an orally resident microbe, with the exception of the pathogenic N.
gonorrhoeae and N. meningitides species. In a previous study of the salivary microbiota in
IBD patients, Neisseria was less abundant in patients with CD and UC when compared to
controls 33. Given the association between the lower abundance of specific taxa and
proinflammatory conditions in the present study, such taxa could play an important role in
maintaining microbial homeostasis.
Bilophila and Desulfovibrio have been shown to be involved in inflammation,
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including UC 34 35. Leptotrichia are commensal oral and female genital bacteria, and are
considered not typical of pathognomonic property 36, while invasive Leptotrichia infections
have been reported in immunosuppressed patients. However, further investigation of the
association of these taxa with UC is necessary.
The present study has some limitations. First, most UC patients were taking
probiotics (Table 1), because a possible beneficial effect on UC has been suggested 37.
Consequently, the abundance of the Bifidobacteria was greater in UC patients than non-IBD
controls, which was the opposite trend to that observed in a previous study38. Thus, the
probiotics might have influenced the results of the present study to some degree. Second,
possible bacterial contamination through the endoscopy channel needs to be considered.
However, mucosal samples were collected using biopsy forceps. Therefore, biopsy samples
were considered to be less susceptible to contamination, because tissue samples were covered
by biopsy forceps during the procedure of sample collection. Third, because the present study
included a small sample size, it is necessary to validate our observations in a larger cohort
free from the use of probiotics.
In conclusion, the present study demonstrated a disturbed mucosa-associated
microbial population at the inflamed and the non-inflamed site of UC patients. Moreover, we
identified two taxa with greater abundance at the inflamed site of UC by comparing the
results with the non-inflamed site of UC patients, and also seven genera that were less
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abundant at the inflamed site when compared to non-IBD controls. In addition, the genera
Prevotella and Butyricimonas were significantly less abundant at both the inflamed and
non-inflamed sites of the UC patients, suggesting that they may play an important role in the
pathogenesis of UC.
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Figure 1 Comparison of alpha diversity index between ulcerative colitis (UC) patients
and individuals without inflammatory bowel disease.
An alpha diversity index expressed as the observed species was compared between UC
patients (n=14) and individuals without inflammatory bowel disease (non-IBD controls)
(n=14) in the transverse colon (a, b), and rectum (c, d). Rarefaction curves show the observed
species at various sequencing depths (a, c), and boxplots show these at higher sequencing
depth (5500 reads per sample). The alpha diversities of UC patients were significantly lower
in both the transverse colon and rectum.
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Figure 2 Comparison of microbial diversity clustering between anatomical sites and
patient groups.
Principal Coordinate Analysis (PCoA) using the beta diversity metrics of weighted UniFrac
within UC patients (a), non-IBD controls (b), transverse colon (c), and rectum (d),
respectively. The P value was calculated using non-parametric MANOVA. There were no
apparent differences in the composition of the microbial population between the transverse
colon and rectum in both UC patients and non-IBD controls (a, b). Conversely, the
composition of the microbial population tended to be phenotypically different between UC
and non-IBD controls in both the transverse colon and rectum, although these differences did
not reach statistical significance.
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Figure 3 Comparison of the taxonomic composition of the microbiome in the transverse
colon and rectum of ulcerative colitis patients.
The cladogram and bar graph indicate the taxa that discriminate between transverse colon
(non-inflamed site) and rectum (inflamed site) of UC patients, based on LEfSe method and
LDA analysis. If multiple varieties with different ranks showed significance in the same
taxon, the lowest-ranked varieties were regarded to be responsible. While the abundance of
the genus Cloacibacterium and the Tissierellaceae family was greater in the rectum, the
abundance of the genus Neisseria was lower.
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Figure 4 Comparison of the taxonomic composition of the microbiome in the rectum of
ulcerative colitis patients and controls.
The cladogram and bar graph indicate the taxa that discriminate between UC patients and
non-IBD controls in the rectum. If multiple varieties with different ranks showed significance
in the same taxon, the lowest-ranked varieties were regarded to be responsible. The
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abundance of the genera Prevotella, Eubacterium, Neisseria, Leptotrichia, Bilophila,
Desulfovibrio, and Butyricimonas was lower in UC patients compared to non-IBD controls.
Conversely, the abundance of the genus Bifidobacterium was greater in UC patients.
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Figure 5 Comparison of the taxonomic composition of the microbiome in the transverse
colon of ulcerative colitis patients and controls.
The cladogram and bar graph indicate the taxa that discriminate between UC patients and
non-IBD controls in the transverse colon. If multiple varieties with different ranks showed
significance in the same taxon, the lowest-ranked varieties were regarded to be responsible.
The abundance of the genera Prevotella and Butyricimonas was lower in UC patients
compared to non-IBD controls.
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Table1. The clinical characteristics of participants
UC patients (n=14)
Non-IBD controls (n=14)
6/8
44.5 (17-67)
9 (1-30)
8/6
58.5 (41-73)
-
13
1
-
Mayo endoscopic subscore
1
2
3
8
5
1
-
Concomitant drugs (n)
Mesalazine
Corticosteroids(oral/local)
Probiotics
Thiopurines
Tacrolimus
13
4/3
10
3
1
-
3
1
3
0
2
2
Gender (male/female)
Age (years)
Disease duration (years)
Ulcerative colitis extension
Left-sided
Proctitis
Infliximab
Adalimumab
Proton pump inhibitor
History of abdominal surgery
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