Comparison between Symptomatic and Asymptomatic Mice after Clostridioides difficile Infection Reveals Novel Inflammatory Pathways and Contributing Microbiota

Abstract

Clostridioides difficile causes the highest number of nosocomial infections. Currently, treatment options for C. difficile infection (CDI) are very limited, resulting in poor treatment outcomes and high recurrence rates. Although the disease caused by CDI is inflammatory in nature, the role of inflammation in the development of CDI symptoms is contradictory and not completely understood. Hence, the use of anti-inflammatory medication is debatable in CDI. In the current study, we evaluated the genetic and microbiome profiles of mice after infection with C. difficile. These mice were categorized based on the severity of CDI and the results were viewed accordingly. Our results indicate that certain genes are upregulated in severe CDI more than in the moderate case. These include oncostatin-M (OSM), matrix metalloprotease 8 (MMP8), triggering receptor expressed on myeloid cells 1 (Trem-1), and dual oxidase 2 (Duox2). We also investigated the microbiome composition of CDI mice before and after infecting with C. difficile. The results show that C. difficile abundance is not indicative of diseases severity. Certain bacterial species (e.g., Citrobacter) were enriched while others (e.g., Turicibacter) were absent in severe CDI. This study identifies novel inflammatory pathways and bacterial species with a potential role in determining the severity of CDI.

Keywords: Citrobacter amalonaticus; Clostridioides difficile; Duox2; MMP; OSM; Trem-1; Turicibacter; inflammation; microbiome; mouse model.

Figure 1

Kaplan–Meier survival curve of mice infected with C. difficile ATCC 43255 and uninfected mice.

Figure 2

(A) Volcano plot (RNA-seq); symptomatic CDI mice had 1812 upregulated and 1257 downregulated genes compared with uninfected mice. (B) Overall cluster analysis of differential expression between C. difficile-infected symptomatic, asymptomatic mice, and uninfected mice.

Figure 3

Quantitative PCR (Q-PCR) confirmation of the upregulation of Trem-1, OSM, and Duox2 in infected symptomatic mice (2, 3, and 4). No upregulation is observed with asymptomatic (5 and 6) or uninfected (9 and 10) mice. (*) denotes significant difference from the control (uninfected) average using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test.

Figure 4

Histological examination of H&E-stained colons of (A) uninfected, (B) infected symptomatic, and (C) infected asymptomatic mice. (A,C) show normal microscopic morphology, (B) shows severe submucosal edema, marked mucosal and luminal neutrophilic infiltrates, and moderate epithelial necrosis and ulceration.

Figure 5

Microbiome analysis of asymptomatic (A), moderately symptomatic (M), and severely symptomatic (S) mice after infection with C. difficile spores. Samples were taken from each mouse at three timepoints: before antibiotic administration, after antibiotic administration and before infection, and right before euthanasia.