Characteristics of intestinal flora of ileocecal region in pediatric patients with Crohn disease

doi:10.21203/rs.3.rs-2332398/v1

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Characteristics of intestinal flora of ileocecal region in pediatric patients with Crohn disease

https://doi.org/10.21203/rs.3.rs-2332398/v1

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Purpose

Intestinal microbiota was considered to be one of the main factors leading to changes in the immune response and had a significant correlation with Crohn disease (CD).This study aimed to reveal the intestinal microbiota profiles of Crohn's disease patients.

Methods

We analyzed the alterations of the gut mucosal microbiota using a 16S rRNA sequencing approach in CD patients in active stage (CD), CD patients in the remission stage (CDR) and the control group (Col).

Results

The results showed that there was a higher abundance of the genus Enterococcus, Lachnoclostridium and Prevotella in CD group than that in CDR and Col. Also, we found a lower abundance of the genus Ruminococcus gnavus group in CD and CDR patients, but higher in Col, indicating that Ruminococcus gnavus group was closely related to CD.

Conclusions

The decreased abundance of Ruminococcus gnavus may result in the reduction of mucin and subsequently lead to the degradation of the host’s ability to maintain a bacteria-free zone within the mucin layer. The increasing of Prevotella may be one of the inducing factors of Crohn's disease. Also, high level Enterococcus faecalis colonization had a significant, positive relationship with Pediatric Crohn disease activity index (PCDAI).

intestinal microbiota

Crohn disease

16S rRNA

mucous membrane

Intestinal microbiota was identified as the main factor responsible for the immune response alternation, including Veillonella, Streptococcus, Enterococcus, etc., and has a significant correlation with Crohn disease.
Reduction in the abundance of OTUs of the genus Ruminococcus gnavus group had been proved to be closely related to Crohn disease.

What is new

The increasing of Prevotella may be one of the inducing factors of Crohn's disease.
High-level Enterococcus faecalis colonization had a significant, positive relationship with Crohn disease activity index (CDA) as well as with fecal calprotectin (FC) levels.

Crohn disease is a type of inflammatory bowel disease (IBD) that may affect any segment of the gastrointestinal tract from the mouth to the anus [1]. Symptoms often include abdominal pain, diarrhea (which may be bloody if inflammation is severe), fever, abdominal distension, and weight loss [2]. Microorganisms are considered to play a key role in the occurrence and development of CD, so it is of considerable interest in targeting the commensal microbiome for therapeutic purpose. Intestinal microbiota is a complex microbial ecosystem, and its balance and homeostasis are maintained by the continuous influx of symbiotic microorganisms [3]. Microbial communities support intestinal homeostasis and the immune system function and it is widely accepted that disruptions in host–microbe interactions are the driving force behind tissue-damaging inflammation in CD [4–5]. As we know, intestinal microbiota was identified as the main factor responsible for the immune response alternation, including Veillonella, Streptococcus, Enterococcus, etc., and has a significant correlation with Crohn's disease. For example, Klebsiella pneumoniae strains derived from gnotobiotic mice transplanted with PSC-IBD microbiota were found to induce pore formation in human intestinal epithelial cells and enhanced T helper cell 17 responses [6]. However, etiology of CD is still poorly understood and treatment of CD patients generally consists of maintaining long-term remission [7–8]. Therefore, it is important to understand the intestinal microbiota of CD patients and CD remission patients to reveal the relationship between specific microorganisms and CD.

In this study, we collected intestinal mucosal samples of ileocecal region from CD patients (CD), CD remission patients (CDR), and the control group (Col) for 16S RNA sequencing. We hypothesized that specific gut microbes are closely related to Crohn disease, especially in the ileocecal region. We intend to screen for specific bacteria by comparing the differences in bacterial abundance among the three groups. It is hoped that the results of this study can provide data support for the diagnosis and treatment of Crohn disease.

Diagnosis and treatment of Crohn disease

Diagnosis and treatment of all patients followed the European Crohn’s and Colitis Organization (ECCO) guidelines and the Chinese expert consensus on the diagnosis and management of pediatric inflammatory bowel disease [9–11]. The diagnosis of CD should be based on clinical manifestations, endoscopy, histopathology, and imaging results. Follow up observation was made to improve the accuracy of diagnosis. Disease activity was assessed by pediatric Crohn disease activity index (PCDAI) and patients with PCDAI < 10.0 were considered to be in remission. The PCDAI between 10.0-27.5 was defined as mild activity, 30.0-37.5 as moderate activity, and 40.0-100.0 as severe activity.

Study Cohort And Recruitment Of Subjects

To investigate the gut microbiota in CD patients, we first recruited a total of 163 children with chronic abdominal pain from the department of Pediatrics of the First Affiliated Hospital of Fujian Medical University between January 2018 and December 2021.The inclusion criteria for patients with chronic abdominal pain were as follows: 4 to 14 years of age, the course of abdominal pain is more than 3 months, at least once a month abdominal pain attacks, affecting children's daily life and learning. The after-exclusion criteria were applied to initial recruitment.

The exclusion criteria include: using antibiotics, probiotics, prebiotics, symbiosis, hormonal drugs, laxatives, proton pump inhibitors, insulin sensitizers, or herbal medicine in the recent 3 months. In addition, volunteers with a history of other autoimmune diseases, such as autoimmune thyroid disease, multiple sclerosis, rheumatoid arthritis, malignant tumors or history of gastrointestinal surgery, etc. are also excluded.

All the volunteers were divided into 3 groups according to the results of examination items: the control group (Col, n = 9), CD patient group (CD, n = 7). CD patients in remission group (CDR, n = 5) were obtained from the treatment and follow-up of the CD group. The inclusion criteria of the control group were as follows: children with chronic abdominal pain, normal C-reactive protein (CRP), normal procalcitonin (PCT), normal erythrocyte sedimentation rate (ESR), normal gastroenteroscopy, no abnormality of small intestine observed in intestinal magnetic resonance, and pathological results of mucosal biopsy not consistent with Crohn's disease. The experimental design is illustrated in Fig. 1.

Sample Collection

For bowel preparation for colonoscopy, each subject ingested 2 liters of polyethylene glycol electrolyte (PEG) solution. Then, the specimen was obtained from the ileocecal junction with biopsy forceps. The diameter of the specimen is about 2 mm. Fresh samples were then immediately placed in liquid nitrogen, and transferred to -80 ℃ refrigerator until use. Whole blood was collected via venipuncture from healthy donors after obtaining written informed consent. Blood was drawn into citrate (3.2% w/v) vacutainers using a 21-gauge needle. Whole blood samples were kept at low temperature until use.

Blood tests were performed at various stages of the disease for hemoglobin (Hb), hematocrit (Hct), white blood cell count (WBC), serum protein (Alb), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP).

Dna Isolation And Sequencing

DNA isolation, 16S rRNA gene amplification and sequencing were done as described previously [12]. Briefly, DNA were isolated by QIAamp UCP Pathogen Mini Kit (Qigen, Hilden, Germany). Distinct regions of V3-V4 were amplified by specific primers (341F: 5’-CCTACGGGRSGCAGCAG-3’; 806R: 5’-GGACTACVVGGGTATCTAATC-3’) with the barcode. Then, PCR products PCR product was purified with GeneJET™ Gel Extraction Kit (Thermo Scientific). Library preparation and sequencing were carried out by the Lybaybio library preparation kit (Lybaybio, Tianjin, China), and the library quality was assessed on the Qubit 2.0 Fluorometer (Thermo Scientific, CA, USA). At last, the library was sequenced on an Illumina Miseq platform (Illumina, CA, USA) and 300bp pare-end reads were generated.

Sequencing Data Analysis

The quality of the raw sequence data was initially evaluated with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Then the sequences were split into groups corresponding to their taxonomy at the level of order, and then were assigned to operational taxonomic units (OTUs) at 97% similarity level [13]. All effective tags were clustered by Uparse software (V7.0.1001) [14]. A representative sequence of each OTU was assigned to a taxonomic level in the SILVA database (http://www.arb-silva.de). According to the results of OTU taxonomy, the abundance distribution and composition of each sample at five classification levels (phylum, class, order, family and genus) were obtained using QIIME software. The alpha diversity and beta diversity analyses were performed based on OTUs. Alpha diversity indices, including chao1, good-coverage, observed-species, PD_whole_tree, and Shannon, were calculated using QIIME (V1.8, http://qiime.org/scripts/alpha_rarefaction.html). Beta diversity on unweighted unifrac was also calculated by the QIIME software. Principal coordinate analysis (PCoA) was performed to visualize principal coordinates from complex multidimensional data. According to the composition and sequence distribution of each sample at each taxonomic level, the abundance differences of each taxon between two or more samples (groups) could be compared one by one. In the present study, the difference of abundance between groups at each taxonomic level was analyzed using Wilcoxon test. Hierarchical cluatering analysis (Hcluster) was generated using the UPGMA (Unweighted pair group method with arithmetic mean) sequential clustering method through the DendroUPGMA web server (URL: genomes.urv.cat/UPGMA). In addition, LDA Effect Size (LEfSe) analysis was conducted to search biomarkers with statistical differences between groups using LEfSe software with LDA Score of 3.

Diagnosis and treatment of patients

After diagnosis, a total of 7 patients were included, of which 5 met the expectation of remission after treatment (Table 1). The PCDAI of all patients were significantly reduced after treatment.

Table 1 Blood tests before and after treatment

Hb, hemoglobin; Hct, hematocritg; WBC, white blood cell count; Alb, serum protein; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; FC, fecal calprotectin; PCDAI, pediatric Crohn′ s disease activity index.

Intestinal bacterial profiles among the three groups

A total of 890,213 valid tags were generated from 21 samples in three groups by Illumina sequencing, and then clustered into 2,017 OTUs at the 97% similarity level. The flower diagram of OTUs distribution in 37 samples of the five groups is shown in Fig. 2A, which showed only 6 common OTUs were commonly identified in all samples. Rarefaction curve of each sample tends to flatten, suggesting that sequencing depths meet the need of analysis (Fig.2B). And, rank abundance curves that reflected the evenness and abundance of species in samples horizontally and vertically were demonstrated in Fig.2C. PCoA analysis showed that the samples of the same group were not clustered together obviously (Fig.2D), indicating that the individual differences of samples cannot be ignored. Additionally, according to the results of OTUs annotation, the top 20 maximum abundance of species in each group in five taxonomic levels (phylum, class, order, family, and genus) were selected to generate the column accumulation graph of species relative abundance. The Hcluster tree results revealed that samples in CD group had similar clustering characteristics; The samples in CDR group had similar clustering characteristics to that in Col group (Fig.2E).

Differential Analysis Of Taxonomic Composition Among The Three Groups

Top 30 in genus level in different groups showed that the relative abundance of many bacteria is more than 1% in the CD group, which was higher than those in CDR and Col groups (Fig. 3A). However, the bacterial abundance profile in the remission stage of CD patients was closer to that of Col. The relative abundance of dominating bacteria was plotted (Fig. 3B). The relative abundance of Ruminococcus torques group and Faecalibacterium in CD and CDR was significantly lower than that in Col. Fusobacterium and Bifidobacterium in CD were significantly lower than that in Col and CDR. In addition, some genera with low relative abundance (< 1%) showed significant differences among the three group. The abundance of Abiotrophia and Mycobacterium was higher in CDR group than that in CD and Col group (Fig. 3C). In addition, the abundance of Enterococcus and Prevotella in CD was higher than that in Col. The results indicated that these significantly changed bacteria might be indicators and play a key role in Crohn's disease.

Redundancy Analysis

Redundancy analysis (RDA) was used to explore the correlations between gut bacteria at the genus level and environmental factors (Fig. 4). The RDA results showed that gut bacteria at CD, Col, and CDR clustered in groups. RDA1 and RDA2 explained 8.43% and 4.54% of the total variance, respectively. PCDAI, CRP and ESR were strongly negatively correlated with the relative abundance of Ruminococcus torques group, Bifidobacterium, Lactobacillus, Blautia, whereas WBC was negatively correlated with the Ruminococcus gnavus group. The other blood indicators showed no statistically significant correlation with the bacterial abundance.

In the current study, we analyzed the alterations of the gut mucosal microbiota using a 16S rRNA sequencing approach in CD patients, CD patients in the remission stage and the control group. We found that the dominant bacteria in each group were different. A significant increase in the abundance of OTUs of the genus Enterococcus, Lachnoclostridium and Prevotella in our CD patients was consistent with the findings of other authors [15–17]. We also found a significant reduction in the abundance of OTUs of the genus Ruminococcus gnavus group, which had been proved to be closely related to Crohn's disease [18]. Ruminococcus gnavus group is an anaerobic, Gram-positive member of the human gut microbiome. Several studies link an increased relative abundance of Ruminococcus gnavus to increased symptoms of CD, a major form of inflammatory bowel disease [18–22]. Matthew has identified and described a potent, TLR4-dependent, inflammatory glucorhamnan polysaccharide made by Ruminococcus gnavus. Ruminococcus gnavus may contribute to breakdown in gut barrier function via regulating mucin levels (Ruminococcus gnavus can utilize mucin as a carbon source) [18, 23–25]. The decreased abundance of Ruminococcus gnavus may result in the reduction of mucin and subsequently lead to the degradation of the host’s ability to maintain a bacteria-free zone within the mucin layer. In this way, mucin destruction and glucorhamnan production by Ruminococcus gnavus could have profound effects on human inflammatory responses. It has been demonstrated that Ruminococcus torques was decreased in patients with CD when compared to healthy subjects [26]. Min et al reported that Ruminococcus torques may induce excessive mucus degradation of the small intestine in CD patients [27]. Ruminococcus torques might lead to the mucus barrier dysfunction at a young age, which might influence the microbial community on the intestinal mucosa and be instrumental in the development of CD [27]. In our RDA results, the abundance of Ruminococcus torques was negatively related to PCDAI, CRP and ESR. Hence, the abundance of Ruminococcus torques might be an important indicator of the CD.

In the gut, Prevotella was detected in Crohn’s disease, which was similar to the previous study [28]. Elinav et al reported that Prevotella induced spontaneous colitis in mice by intestinal induction of CC-chemokine ligand 5 (CCL5) [29]. The increasing of Prevotella may be one of the inducing factors of Crohn's disease.

In addition, Enterococcus is a common opportunistic pathogen found in the alimentary tract of both humans and animals that can trigger inflammatory bowel disease (IBD) [30]. Our study reveals that increased Enterococcus are prominent features in patients with CD. Zhou et al found that high-level Enterococcus faecalis colonization had a significant, positive relationship with Pediatric Crohn disease activity index (PCDA) as well as with fecal calprotectin (FC) levels[30]. Elevated FC is a predictor of relapse and clinically active disease in patients with IBD.

Qiu et al reported that Lachnoclostridium faecalis was increased in ulcerative colitis (UC) patients, not in CD patients [16]. However, in our study, the abundance of Lachnoclostridium faecalis increased significantly in CD patients. Lachnoclostridium marker had been identified for the noninvasive diagnosis of colorectal adenoma and cancer [31]. More experiments are needed to study and verify the relationship between the abundance of Lachnoclostridium and CD. Lachnoclostridium may not be a key factor, but there may still have a more or less connection with the CD.

We found that a significant increase in the abundance of OTUs of the genus Enterococcus, Lachnoclostridium and Prevotella in CD patients. Also, there was a significant reduction in the abundance of OTUs of the genus Ruminococcus gnavus group, which had been proved to be closely related to Crohn's disease. The decreased abundance of Ruminococcus gnavus may result in the reduction of mucin and subsequently lead to the degradation of the host’s ability to maintain a bacteria-free zone within the mucin layer. Similarly, the abundance of Ruminococcus torques might be an important indicator of CD. The increasing of Prevotella may be one of the inducing factors of Crohn's disease. Our study reveals that increased Enterococcus are prominent features in patients with CD. High-level Enterococcus faecalis colonization had a significant, positive relationship with Crohn disease activity index (CDA) as well as with fecal calprotectin (FC) levels. Elevated FC is a predictor of relapse and clinically active disease in patients with IBD. For Lachnoclostridium, it may not be a key factor, but there may still have a more or less connection with CD.

CD Crohn disease

CDA Crohn disease activity index

CRP C-reactive protein

FC Fecal calprotectin

IBD Inflammatory bowel disease

OTUs Operational taxonomic units

PCT Procalcitonin

PCDAI Pediatric Crohn disease activity index

RDA Redundancy analysis

Funding

This work was supported by the First Affiliated Hospital of Fujian Medical University.

Competing Interests

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Peilin Wu, Junhong Liu, Linliang Hong and Junxi Wang. The first draft of the manuscript was written by Peilin Wu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of The First Affiliated Hospital of Fujian Medical University for Medical Research (Approval NO.MARCTA, ECFAH of FMU[2019]219).

Consent to participate

For all research involving human subjects, freely-given, informed consent to participate in the study must be obtained from participants (or their parent or legal guardian in the case of children under 16) and a statement to this effect should appear in the manuscript.

A written informed consent must be obtained from all patients and volunteers before the trial. This will also be documented in the clinical file. Informed consent includes general characteristics of the study, alternative treatment options, potential risks and benefits from the study, as well as the right to withdraw consent at any given time.

Consent to publish

Not applicable.

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No competing interests reported.

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Version 1

posted

You are reading this latest preprint version

Characteristics of intestinal flora of ileocecal region in pediatric patients with Crohn disease

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusions

Figures

What Is Known

Introduction

Materials And Methods

Diagnosis and treatment of Crohn disease

Study Cohort And Recruitment Of Subjects

Sample Collection

Dna Isolation And Sequencing

Sequencing Data Analysis

Results

Diagnosis and treatment of patients

Differential Analysis Of Taxonomic Composition Among The Three Groups

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1