A Colombian strain of Clostridioides difficile ribotype 002 induces a highly inflammatory response in a mouse infection model

Abstract

Clostridioides difficile causes diarrhea associated with antibiotic use in hospitalized patients. Recent studies have identified that C. difficile ribotypes RT002, RT106, and RT591 as the most prevalent circulating strains in Colombia; thus, we aimed to assess the capability of these ribotypes to elicit an inflammatory response during in vivo infection. To achieve this, C57BL/6 mice were treated with cefoperazone (CPZ) for 5 d to develop C. difficile infection (CDI) model. Two days post-antibiotic treatment, the mice were orally inoculated with 1 × 10⁵ spores of C. difficile strains belonging to ribotypes RT002, RT106, RT591, and RT027 (ATCC strain, used as control). A group of animals was euthanized on day 7 post-infection to determine the bacterial load, total leukocyte number, and chemokines/cytokines levels in situ, and for histopathological analysis. RT002-infected groups showed significantly higher bacterial load, CD45+ leukocytes, and RANTES, eotaxin, MCP-1, G-CSF, and IL-2 levels compared to the other groups, suggesting a robust immune response. Furthermore, histopathological analysis of colonic tissue from the group infected with RT002 revealed the presence of an inflammatory response similar to the hypervirulent strain RT027. These results suggest that RT002 of C. difficile, one of the main circulating strains in Colombia, can induce a severe inflammatory response, potentially correlating with increased virulence and severity of these strains in CDI cases.

Keywords: CDI; Clostridioides difficile; RT002 ribotype; cytokines; inflammatory response.

Figure 1.

The Clostridioides difficile strains belonging to ribotypes 027 and 002 infection are related to an increased bacterial burden in the colon of mice. Bacterial load measurement was performed in the colon tissue of mice infected with different C. difficile strains belonging to the ribotypes RT027, RT591, RT106, and RT002 at 7 d post-infection. Data shown represent the median and IQR (n = 5–6 mice/group; representative of two independent experiments). A statistically significant increase in bacterial burden was observed in the colon of mice infected with the C. difficile strains belonging to the ribotypes RT027 and RT002 (*p < 0.05) compared to the infected mice with the other strains.

Figure 2.

Analysis of the accumulation of leukocytes in the colon of mice infected or uninfected with different C. difficile strains belonging to the ribotypes RT027, RT591, RT106, and RT002. The accumulation of leukocytes in the colon was analyzed by using flow cytometry on day 7 post-infection as described in M&M section. Data shown represent the median and IQR (n = 5–6 mice/group; representative of two independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3.

Levels of proinflammatory cytokines and chemokines in the colon of mice infected or uninfected with different C. difficile strains belonging to the ribotypes RT027, RT591, RT106, and RT002. Supernatants from colon homogenates of mice sacrificed 7 d after infection were analyzed using a commercial kit and the luminex system, as described in material and methods. Data shown represent the median and IQR (n = 5–6 mice/group; representative of two independent experiments). *p <0.05.

Figure 4.

Inflammatory response in colonic tissue of mice infected with the C. difficile strain belonging to the ribotype RT002. (a) Representative histology of caecum of healthy uninfected mice (control) with epithelium integrity; (b) colonic tissue of mice infected with a C. difficile strain belonging to the ribotype RT002 with edema (arrow) and loss of architecture of epithelium; (c) colonic tissue of mice infected with a C. difficile strain belonging to the ribotype RT002 with edema and leukocyte infiltration (arrow); (d) Inflammation histological scores for individual mice in each group. The horizontal lines represent the mean scores for each group of animals. (e) individual evaluation criteria of colonic inflammation. Data shown represent the median and IQR (n = 5–6 mice/group; representative of two independent experiments). *p < 0.05 compared to WT-infected mice. Tissue was stained with H&E; magnification 40X. Histopathological micrographs of tissues from animals infected with the other strains belonging to ribotypes RT027, RT591, and RT106 are not shown because after histopathological analyses, the slides and paraffin-embedded tissues were discarded without our consent before documenting the microphotographs.