Characteristics of Faecal Microbiota in Korean Patients with Clostridioides difficile-associated Diarrhea

1. Study population and sample collection

This study was conducted at a 2,500 beds tertiary hospital of Korea. Stool samples were collected from hospitalized patients with CDI (CD+), hospitalized patients with loose stool negative for C. difficile (CD−), hospitalized patients with VRE colonisation, and healthy controls between December 2014 and March 2015. CD+ and VRE groups included only patients who were first diagnosed as CDI or VRE colonization. CDI was diagnosed in subjects with diarrhea (passage of three or more unformed or loose stools per day for more than 2 days) and who satisfied at least one of the following criteria: a toxin-producing C. difficile strain was successfully cultured, or a toxin gene of C. difficile was detected by PCR [21, 22]. It was confirmed that subjects in the CD+ and CD− groups did not have VRE colonisation within their stool samples. Likewise, subjects in the VRE group were confirmed to be without CDI. Subjects with both VRE and CDI were excluded. Healthy controls were recruited from individuals visiting the Severance Hospital for routine health check-ups who had no malignancy or gastrointestinal disease and no history of antibiotics or chemotherapy for 3 months prior to investigation. Healthy controls were confirmed to be without CDI or VRE colonisation. All of the participants were over 20 years old. The Institutional Review Board (IRB) of the Severance Hospital approved the study (IRB #4-2014-0792). The participants for this study provided informed signed consent. All methods in this study were performed in accordance with the Declaration of Helsinki.

2. Microbiological tests

Stool specimens were cultured to confirm the presence of toxigenic C. difficile. Before inoculation, each specimen was pre-treated using the alcohol-shock method, mixed with an equivalent volume of alcohol, and incubated for 30 min. Alcohol-shocked specimens were inoculated into C. difficile selective agar (BD Co., Franklin Lakes, NJ, USA) and incubated at 35°C for 48 h under anaerobic conditions. Colonies suspected of being C. difficile were identified using a VITEK 2 automated microbiology system (bioMérieux, Marcy l'Etoile, France). After isolating C. difficile, PCR was performed to determine the presence of C. difficile toxin genes (tcdA, tcdB, cdtA and cdtB) as previously described [23, 24]. The presence of the C. difficile toxin B gene in patient samples was examined by real-time PCR using the GeneXpert DX system (Cepheid, Sunnyvale, CA, USA).

The identification of enterococcal growth was performed using the VITEK 2 automated system (bioMérieux), and antibiotic susceptibility was determined using a VITEK 2 antibiotic susceptibility test card (bioMérieux).

3. DNA extraction

DNA extraction was performed using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Stool samples stored at −70°C since the day of sample collection were used for DNA extraction. First, 500 μg (wet weight) stool sample was collected, 10 ml PBS was added, and the solution was vortexed vigorously until the stool sample was thoroughly homogenized. Then, the diluted sample was filtered through a cell strainer (Falcon, Corning Inc, Corning, NY, USA) and centrifuged at 1,300 rpm for 10 min. The sample was resuspended in AL buffer (Qiagen) and disrupted the bacterial cell walls by bead-beating according to the manufacturer's recommendations. The quantity and quality of DNA was confirmed by measuring the absorbance at 260 and 280 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Rockland, DE, USA).

4. PCR amplification and pyrosequencing

PCR amplification was performed using primers targeting the V1 to V3 regions of the bacterial 16S rRNA gene. For bacterial amplification, barcoded primers of 9F (5′-CCTATCCCCTGTGTGCCTTGGCAGTC-TCAG-AC-AGAGTTTGATCMTGGCTCAG-3′; underlined sequence indicates the target region primer) and 541R (5′-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-AC-ATTACCGCGGCTGCTGG-3′; ‘X’ indicates the unique barcode for each subject). Amplification was carried out under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30s, and 72°C for 30s, with a final elongation at 72°C for 5 min. The PCR product was confirmed by 2% agarose gel electrophoresis and visualized under a Gel Doc system (BioRad Laboratories, Hercules, CA, USA). The amplified products were purified with the QIAquick PCR purification kit (Qiagen). Equal concentrations of purified products were pooled, and short fragments (non-target products) were removed with the Ampure beads kit (Agencourt Bioscience, Beverley, MA, USA). The quality and size of the products was assessed on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using a DNA 7500 chip. Mixed amplicons were analysed by emulsion PCR, and then deposited on Picotiter plates. Sequencing was carried out using the GS Junior Sequencing system (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions.

5. Pyrosequencing data analysis

Basic analysis was conducted as described previously [25, 26, 27]. The obtained reads from the different samples were sorted based on the unique barcodes of each PCR product. The sequences of the barcode, linker, and primers were removed from the original sequencing reads. Any reads containing two or more ambiguous nucleotides, low-quality reads (average score < 25), or reads shorter than 300 bp, were discarded. Potential chimeric sequences were detected using the Bellerophon method, which compares the BLASTN search results between forward half and reverse half sequences [28]. After removing chimeric sequences, the taxonomic classification of each read was assigned against the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net) [29], which contains the 16S rRNA gene sequences of type strains with valid published names and representative species level phylotypes of either cultured or uncultured entries in the GenBank database with complete hierarchical taxonomic classification from the phylum to the species level. The richness and diversity of samples were determined by Chao1 estimation and Shannon diversity index at the 3% distance. Random subsampling was conducted to equalize the read size of samples to compare different read sizes among samples. The overall phylogenetic distance between communities was estimated using Fast UniFrac [30] and visualized using PCoA. To compare operational taxonomic units (OTUs) between samples, shared OTUs were obtained by XOR analysis in the CLcommunity software (Chunlab Inc., Seoul, Korea).

6. Statistical Analysis

Statistical tests were performed using SPSS version 19 (SPSS Inc., Chicago, IL, USA) and CLcommunity (ChunLab Inc.). Data are shown as the mean ± standard deviation (SD). The statistical significance of continuous variables was assessed using Student's t-test or the Mann-Whitney U test between two groups. One-way analysis of variance (ANOVA) and the Kruskal-Wallis test were used to evaluate the differences between more than two groups. A value of P <0.05 was considered statistically significant.

1. Characteristics of study subjects

We collected stool samples from hospitalized patients with CD+, hospitalized patients with CD−, hospitalized patients with VRE colonization, and healthy controls. A total of sixty-six stool samples were collected from twenty-four patients with CD+, eighteen patients with CD−, eleven patients with VRE colonization, and thirteen healthy controls. The baseline characteristics of each group are shown in Table 1. The mean age was 59.6 years and there were 35 male patients (53.0%). The Charlson comorbidity index was the lowest in the healthy controls (CD+: 4.2; CD−: 2.1; VRE: 4.1; Healthy: 0.5). The rate of hospitalization in the 3 months prior to the investigation was 79.2% in CD+, 44.4% in CD−, 90.9% in VRE, and 0% in healthy controls. The mean white blood cell count was highest in the CD+ group (CD+: 11232.6 (/μL); CD−: 7680.6; VRE: 7147.3; Healthy: 5701.5) and the mean albumin was the lowest in the CD+ group (CD+: 3.0 (g/dL); CD−: 3.1; VRE: 3.6; Healthy: 4.2). The clinical symptoms of the patients are also shown in Table 1.

Table 1 Demographic and clinical characteristics of subjects Click for larger imageClick for full tableDownload as Excel file

The proportion of patients who had used antibiotics in the 3 months prior to this study was 95.2% in CD+, 83.3% in CD−, and 100% in VRE (Table 2). The total duration of antibiotic use was the longest in VRE patients (CD+: 26.2 (days); CD−: 20.9; VRE: 45.2). The duration of treatment with each antibiotic class is also shown in Table 2.

Table 2 History of antibiotic use in subjects within 3 months before the start of experiments Click for larger imageClick for full tableDownload as Excel file

2. Species richness and diversity of intestinal microbiota

The alpha diversity of the bacteria in the analysed samples is shown in Table 3 and Fig. 1. The species richness, as estimated by the Chao1 index, was significantly lower in the CD+ group compared to that in the healthy control (P <0.001), but did not differ significantly between the CD+ group and any other group. Microbial diversity, as calculated by the Shannon index, was significantly lower in the CD+ group compared to that in the healthy control group (P = 0.001), but did not differ significantly between the CD+ group and any other group.

Table 3 Alpha diversity and relative abundance within each group at the phylum and genus level Click for larger imageClick for full tableDownload as Excel file

Figure 1 Alpha diversity of analysed bacterial samples. (A) Chao1 index to determine species richness. (B) Shannon index to determine microbial diversity. CD+, hospitalized patients with Clostridioides difficile infection; CD−, hospitalized patients with colitis not due to C. difficile; VRE, hospitalized patients with vancomycin-resistant enterococci colonisation. Click for larger imageDownload as PowerPoint slide

Principal coordinate analysis (PCoA) was used to evaluate the beta diversity within each group (Fig. 2). Healthy controls were clustered separately from the CD+, CD−, and VRE group, while the CD+, CD−, and VRE groups could not be separated.

Figure 2 Principal coordinate analysis (PCoA) was used to evaluate the beta diversity of each group of bacteria. CD+, hospitalized patients with Clostridioides difficile infection; CD−, hospitalized patients with colitis not due to C. difficile; VRE, hospitalized patients with vancomycin-resistant enterococci colonisation. Click for larger imageDownload as PowerPoint slide

3. Relative abundance of intestinal microbes

A comparison of the mean relative abundance of each major phylum is shown in Table 3. Phylum Firmicutes was predominant in all groups. In the healthy group, Actinobacteria was the second-most abundant. The abundance of Actinobacteria was significantly lower in the CD+ group (P = 0.001) compared to that in healthy control, but there was no difference in its abundance between the CD+ group and any other group. Proteobacteria was the second-most predominant phylum in the CD+, CD−, and VRE groups. The proportion of Proteobacteria was slightly higher in the CD+, CD−, and VRE groups than in healthy controls, but not significantly (12.8% in CD+; 23.6% in CD−; 17.5% in VRE; 3.5% in healthy control). Compared to that in the VRE group, the proportion of Bacteroidetes was higher in the CD+ and CD− groups, but not significantly (7.1% in CD+; 5.9% in CD−; 2.7% in VRE; 4.3% in healthy control, P = 0.673). Verrucomicrobia was detected at a proportion of 2.5% in the CD+ group, but was below the detection threshold in the CD−, VRE, and healthy groups.

At the genus level, genera with a mean relative abundance of >1.0% (based on healthy controls) were analysed, as described in Table 3. Compared to those in the healthy group, the CD+ group showed significantly lower proportions of Blautia, Bifidobacterium, Faecalibacterium, Anaerostipes, Dorea, Collinsella, Eubacterium_g5, Fusicatenibacter, Prevotella, Roseburia, Subdoligranulum, GQ871709_g, Catenibacterium, Clostridium, Ruminococcus_g2, Dialister, and Ruminococcus_g5. However, the proportions of Streptococcus, Escherichia, and Bacteroides in the CD+ group were higher than those in the healthy control, but not significantly. Compared to those in the CD− group, subjects in the CD+ group showed no significant difference in the abundance of any bacteria at the genus level. Compared to those in the VRE group, subjects in the CD+ group showed a significantly higher proportion of Anaerostipes.