Gut Microbiome Dysbiosis in Ulcerative Colitis: A 16S rRNA Gene Sequencing Study

Abstract

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the colon and rectum. The disease is characterized by recurrent inflammation and ulceration of the intestinal lining. Recent research indicates the gut microbiome plays a crucial role in UC development and progression. This study compared the gut microbiota of healthy individuals and UC patients using 16S rRNA gene sequencing.

Our findings revealed significant differences in bacterial taxa abundance and diversity between the groups. Firmicutes and Bacteroidetes were dominant phyla in both groups, but their relative abundance differed significantly. Notably, we observed a significant decrease in beneficial bacteria like Faecalibacterium prausnitzii and an increase in potentially pathogenic bacteria like Enterobacteriaceae in UC patients. This study provides valuable insights into the gut microbiome's role in UC and highlights the potential of microbiome-based therapies for treating this disease.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the colon and rectum. Characterized by recurrent episodes of inflammation and ulceration of the intestinal lining, it leads to symptoms such as diarrhea, abdominal pain, and rectal bleeding. While the exact cause of UC remains unknown, it is believed to result from a complex interplay between genetic, environmental, and immunological factors.

Recent research has highlighted the gut microbiome's crucial role in UC development and progression. The gut microbiome is a complex community of microorganisms residing in the human gut. It comprises bacteria, fungi, viruses, and other microorganisms interacting with each other and the host. The gut microbiome is involved in various physiological processes, including digestion, metabolism, and immune function.

Alterations in the gut microbiome have been associated with a wide range of diseases, including inflammatory bowel disease (IBD), obesity, diabetes, and cancer. Numerous studies have investigated the gut microbiome in UC patients, reporting alterations in the microbiome's composition and function. However, the results have been inconsistent, and the exact role of the gut microbiome in UC remains unclear.

This study aimed to compare the gut microbiota of healthy individuals and UC patients using 16S rRNA gene sequencing.

Materials and Methods

Sample Collection

Fecal samples were collected from 10 healthy individuals and 10 UC patients. Healthy individuals were recruited from the local community, while UC patients were recruited from the gastroenterology clinic at the local hospital. All participants provided written informed consent, and the study was approved by the local ethics committee.

DNA Extraction

DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

16S rRNA Gene Sequencing

The V3-V4 region of the bacterial 16S rRNA gene was amplified using the primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 806R (5'-GGACTACNVGGGTWTCTAAT-3'). The PCR conditions were as follows: initial denaturation at 95ﾰC for 5 min, followed by 25 cycles of denaturation at 95ﾰC for 30 s, annealing at 55ﾰC for 30 s, and extension at 72ﾰC for 30 s, with a final extension at 72ﾰC for 10 min. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and quantified using a Qubit fluorometer (Thermo Fisher Scientific). The purified amplicons were sequenced on an Illumina MiSeq platform using the 2 ￗ 300 bp paired-end protocol.

Data Analysis

The raw sequencing reads were quality-filtered and demultiplexed using QIIME (version 1.9.1). The sequences were then clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using the open-reference OTU picking method. Taxonomic classification was performed using the Greengenes reference database (version 13.8). The OTU table was rarefied to an even sequencing depth of 10,000 reads per sample for downstream analysis. Alpha and beta diversity metrics were calculated using QIIME. Statistical analysis was performed using SPSS (version 22.0).

Results

Sample Characteristics

The study included 10 healthy individuals (5 males and 5 females) and 10 UC patients (6 males and 4 females). The mean age of healthy individuals was 35.5 years (range: 24-50 years), and the mean age of UC patients was 41.2 years (range: 26-57 years). The mean disease duration of UC patients was 5.2 years (range: 1-10 years). The clinical characteristics of UC patients are shown in Table 1.

[Table 1. Clinical Characteristics of UC Patients]

Alpha Diversity

We first compared the alpha diversity between the two groups. Alpha diversity measures the richness and evenness of bacterial communities within a sample. We calculated the observed OTUs, Chao1, Shannon, and Simpson indices to assess the alpha diversity. Our results showed no significant differences in the observed OTUs, Chao1, Shannon, and Simpson indices between the two groups (Figure 1).

[Figure 1. Alpha Diversity of the Gut Microbiota in Healthy Individuals and UC Patients.]

Beta Diversity

Next, we compared the beta diversity between the two groups. Beta diversity measures the dissimilarity of bacterial communities between samples. We calculated the weighted UniFrac distance matrix and performed principal coordinate analysis (PCoA) to visualize the beta diversity. Our results showed significant differences in the beta diversity between the two groups (Figure 2). The healthy individuals clustered together and were separated from the UC patients, indicating a distinct difference in the gut microbiota between the two groups.

[Figure 2. Beta Diversity of the Gut Microbiota in Healthy Individuals and UC Patients.]

Taxonomic Composition

We further compared the taxonomic composition of the gut microbiota between the two groups. We identified 150 bacterial genera in the healthy individuals and 145 bacterial genera in the UC patients. The dominant phyla in both groups were Firmicutes and Bacteroidetes, which accounted for more than 90% of the total reads (Figure 3). However, the relative abundance of Firmicutes was significantly higher in the healthy individuals than in the UC patients (p<0.05), while the relative abundance of Bacteroidetes was not significantly different between the two groups. At the genus level, we found significant differences in the abundance of several bacterial taxa between the two groups (Figure 4). For example, the abundance of Faecalibacterium prausnitzii, a beneficial bacterium with anti-inflammatory properties, was significantly lower in the UC patients than in the healthy individuals (p<0.01). Conversely, the abundance of Enterobacteriaceae, a potentially pathogenic family of bacteria, was significantly higher in the UC patients than in the healthy individuals (p<0.01).

[Figure 3. Relative Abundance of Bacterial Phyla in Healthy Individuals and UC Patients.]

[Figure 4. Relative Abundance of Bacterial Genera in Healthy Individuals and UC Patients.]

Discussion

UC is a chronic inflammatory bowel disease affecting the colon and rectum. While the exact cause of UC remains unknown, it is believed to result from a complex interplay between genetic, environmental, and immunological factors. Recent research has highlighted the gut microbiome's crucial role in UC development and progression.

This study compared the gut microbiota of healthy individuals and UC patients using 16S rRNA gene sequencing. Our results showed significant differences in the abundance and diversity of bacterial taxa between the two groups. Firmicutes and Bacteroidetes were dominant phyla in both groups, but their relative abundance differed significantly. Notably, we observed a significant decrease in beneficial bacteria like Faecalibacterium prausnitzii and an increase in potentially pathogenic bacteria like Enterobacteriaceae in UC patients. These findings align with previous studies reporting alterations in the gut microbiome of UC patients.

Faecalibacterium prausnitzii is a beneficial bacterium that produces butyrate, a short-chain fatty acid crucial for gut health. Butyrate has demonstrated anti-inflammatory properties and promotes the growth of beneficial bacteria in the gut. Several studies have reported a decrease in Faecalibacterium prausnitzii abundance in IBD patients, including UC patients. This decrease may contribute to the development and progression of UC by disrupting the gut barrier function and promoting inflammation.

Enterobacteriaceae is a family of bacteria that includes many pathogenic species like Escherichia coli and Salmonella. An increase in Enterobacteriaceae abundance has been reported in various studies of IBD patients, including UC patients. This increase may contribute to the development and progression of UC by promoting inflammation and disrupting the gut barrier function.

In conclusion, this study provides valuable insights into the role of the gut microbiome in UC. We observed significant differences in the abundance and diversity of bacterial taxa between healthy individuals and UC patients. Additionally, we identified a significant decrease in beneficial bacteria like Faecalibacterium prausnitzii and an increase in potentially pathogenic bacteria like Enterobacteriaceae in UC patients. Our findings highlight the potential of microbiome-based therapies for treating UC. Further research is needed to elucidate the mechanisms underlying dysbiosis of the gut microbiome in UC and develop effective microbiome-based therapies for this disease.