Gut microbiome, an avenue towards understanding colitis-complex diarrhea

Farhad M. Panah

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling

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Colitis-complex diarrhea (CCD) has a blurred etiology and can often happen with the involvement of different factors such as pathogens, dietary factors, and management. We hypothesized that colonic microbial changes had underlying roles in the incidence of CCD. The initial aim of this PhD project was to understand the changes in the colonic microbial composition and their fermentation patterns in growing pigs with CCD. Paper I was a literature review to establish a clear definition for CCD. Paper II was to characterize the microbial changes in the colon of pigs with CCD, which was an observational study on 21 pigs, five without diarrhea (NoDiar) and 16 with diarrhea. The group with diarrhea was further classified as with diarrhea and no inflammation in the colon (DiarNoInf; n = 4), and a group with diarrhea and inflammation in the colon (DiarInfl; n = 12). 16S rRNA gene sequencing revealed that microbial composition of mucus (MAB) and digesta (DAB) of pigs differed; therefore, we analyzed them separately. Compositional diversity was significantly different between diarrheal groups in both DAB and MAB. Compared to NoDiar, DiarInfl showed increased abundance of various taxa, incl. certain pathogens, in both digesta and mucus, as well as decreased digesta butyrate concentration. However, DiarNoInfl showed reduced number of different genera compared to NoDiar, but still lower butyrate concentration, highlighting the importance of butyrate. With these results, we suggested that diarrhea with inflammation can happen due to a dysbiosis of gut microbiota, associated with an increase in facultative anaerobic taxa, e.g., Escherichia-Shigella (Proteobacteria), Helicobacter (Campylobacterota), and Bifidobacterium (Actinobacteriota), which may tolerate or utilize oxygen and cause epithelial hypoxia and inflammation. The increased consumption of oxygen in epithelial mucosal layer by infiltrated neutrophils may also have added up to this hypoxia. Furthermore, for community-based analysis we suggest that fecal samples can well-represent digesta.
In Paper III we tested the hypothesis that a westernized diet high in fat and protein, could be a risk factor or an exacerbating agent in a pig model for human Ulcerative Colitis (UC). Colitis was induced in 24 six-week-old pigs using Dextran Sodium Sulfate (DexSS) and digesta from proximal and distal colon, and fecal samples were collected for microbial and chemical analysis. A group of pigs was on a standard diet (CT), or the standard diet substituted with 15% ground beef to simulate a typical westernized diet (WD). Colitis was induced in half of the pigs on each dietary treatment by oral administration of DexSS (DSS and WD+DSS, respectively). WD+DSS group showed the lowest alpha diversity compared to the other treatment groups. The westernized diet and DexSS resulted in three and seven differentially abundant phyla, 21 and 65 species, respectively, mainly associated with the Firmicutes and Bacteroidota phyla followed by Spirochaetota, Desulfobacterota, and Proteobacteria. Treatment had a slight effect on the estimates for microbial metabolites that might have valuable biological relevance for future studies. The concentration of putrescine in the colon and feces and that of total biogenic amines was highest in the WD+DSS group. We concluded that the westernized diet could be a potential risk factor and an exacerbating agent for UC by reducing the abundance of SCFA-producing bacteria, increasing the abundance of pathogens such as Helicobacter trogontum, and by increasing the concentration of microbial proteolytic-derived metabolites in the colon. Furthermore, we did not observe differences in microbial composition and concentrations of fermentation products among sample types, indicating that fecal samples could suffice for analysis of the microbiale changes in the colon. Moreover, there were indications of anaerobic dysbiosis in the colon of animals treated with DexSS.

Paper IV reports the results of an experimental study designed based on the results from paper I-III, in which we wished to establish an in vivo pig model for CCD in growing pigs. The experiment was done in three complete blocks with 24 six-week-old pigs from six litters, eight pigs from two litters per block. Pigs were fed a basal diet (CT), challenged with DSS (DSS), challenged with B. pilosicoli (BP) or a combination of chemical and pathogen (DSSBP). All pigs were sacrificed eight days after the last oral administration of B. pilosicoli on day 37 for postmortem sampling. Blood samples, fecal samples, digesta and tissue samples were taken for different analyses. Histopathological analysis showed mild lesions for the BP group, characterized with edema in lamina propria, whereas the DSS-treated groups showed severe hemorrhagic diarrhea with symptoms of necrotizing ulcerative colitis. The number of total and individual leucocytes in the peripheral blood significantly increased in DSS-treated animals. Metagenomics analysis of colonic microbiome did not show any changes in BP-groups except with reduced Shannon diversity. However, DSS showed to be associated with reduced alpha diversity and differentially abundance of 38 species correspondent with eight metabolic pathways being reduced and increase of one metabolic pathway related to biosynthesis of spermidine from putrescine. Concentrations of SCFA did not differ by treatment, while total biogenic amines and putrescine showed a significant increase in the DSSBP group compared to the other groups. Indoles such as 4-methylphenol and 1-benzopyrrol were lowest in the DSSBP groups, while skatole was higher in the DSS group compared to CT and BP in both digesta and fecal samples. These results confirmed that we successfully established an in vivo pig model for CCD based on DSS, albeit with relatively more severe lesions. The results of this study are suggested to be considered for research on human UC as well.
All in all, the results of these papers indicate that there are associations between the incidence of colitis-complex diarrhea with colonic microbial dysbiosis. The results further suggest that in case of mild inflammation in the colon the microbial dysbiosis may be linked to mucosal hypoxia due to increased oxygen consumption, while in case of ulcerative colitis there might be oxygen leakage from seral space into the lumen due to disrupted epithelial integrity, hence anaerobic dysbiosis rather than anoxia might be present.
ForlagAarhus University
Antal sider188
StatusUdgivet - jul. 2023


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