Gut microbiota profiles in diarrheic patients with co-occurrence of Clostridioides difficile and Blastocystis

Abstract

Blastocystis and Clostridioides difficile co-occurrence is considered a rare event since the colonization by Blastocystis is prevented under a decrease in beneficial bacteria in the microbiota when there is C. difficile infection (CDI). This scenario has been reported once, but no information on the gut microbiota profiling is available. The present study is motivated by knowing which members of the microbiota can be found in this rare scenario and how this co-occurrence may impact the abundance of other bacteria, eukaryotes or archaea present in the gut microbiota. This study aimed to describe the bacterial and eukaryotic communities using amplicon-based sequencing of the 16S- and 18S-rRNA regions of three patient groups: (1) Blastocystis and C. difficile infection (B+/C+, n = 31), (2) C. difficile infection only (B-/C+, n = 44), and (3) without Blastocystis or C. difficile (B-/C-, n = 40). Blastocystis was subtyped using amplicon-based sequencing of the 18S-rRNA gene, revealing circulation of subtypes ST1 (43.4%), ST3 (35.85%) and ST5 (20.75%) among the study population. We found that B+/C+ patients had a higher abundance of some beneficial bacteria (such as butyrate producers or bacteria with anti-inflammatory properties) compared with non-Blastocystis-colonized patients, which may suggest a shift towards an increase in beneficial bacteria when Blastocystis colonizes patients with CDI. Regarding eukaryotic communities, statistical differences in the abundance of some eukaryotic genera between the study groups were not observed. Thus, this study provides preliminary descriptive information of a potential microbiota profiling of differential presence by Blastocystis and C. difficile.

Fig 1. Frequency of Blastocystis subtypes in the 31 fecal samples (B+/C+).

The absolute frequency of each ST is displayed in the lateral bar plot (green). The panel with the dots represents the single or mixed infections, which frequency is displayed in the main bar plot (blue).

Fig 2. Alpha diversity measures and description of the bacterial phyla and eukaryotic divisions in the three groups.

(A) Richness and abundance measures for the ASVs corresponding to bacterial communities of the three study groups and (B) Richness and abundance measures for the ASVs corresponding to eukaryotic communities of the three study groups. No significant differences were found in the richness and abundance of the bacterial and eukaryotic communities of the study groups (p<0.05). (C) Relative abundance of bacterial phyla identified for each of the three study groups, where the predominant phyla within each group were Firmicutes, Bacteroidetes and Proteobacteria. (D) Relative abundance of the eukaryotic classes identified for each of the three study groups, where the predominant classes within each group were Ascomycota and Basidiomycota.

Fig 3. Description and evaluation of the statistical differences in the abundance of bacterial biomarkers among the three study groups.

(A) Description of the relative abundance of 21 genera of bacteria considered as possible biomarkers of the intestinal microbiota among the three study groups. (B) Three genera of bacterial biomarkers presented significant differences in their relative abundance among some of the study groups, where all of these genera display a higher abundance in the B+/C+ group. (C) Changes in the relative abundance of Faecalibacterium considering the colonization of a determined Blastocystis subtype, showing that this genus has a higher abundance in ST3 colonized patients. Significant differences between the study groups were evaluated using Kruskal-Wallis test and ANOVA (*, p<0.05; **, p<0.01).

Fig 4. Intra-domain and inter-domain correlations in the three study groups.

Correlation networks of the B+/C+ (top left), B-/C+ (top center), and B-/C- (top right) groups. The nodes represent the bacterial and eukaryotic families, and its color represents the phylum to which it belongs. Blue edges and red edges indicate positive and negative correlations, respectively. The networks only represent significant correlations (p<0.05, after FDR correction) with ρ above 0.6 and lower than -0.6, and that were computed with the Spearman correlation test. This figure also displays correlation plots of the genera belonging to the most abundant bacterial and eukaryotic families of the B+/C+ (bottom left), B-/C+ (bottom center), and B-/C- (bottom right) groups. Blue and red squares represent positive and negative correlations, respectively. Only significant correlations, computed with the Spearman correlation test, (p<0.05, after FDR correction) were represented in the mentioned plots.