Background: Recent studies suggest links between Clostridioides difficile infection (CDI) and liver disorders, with non-alcoholic fatty liver disease (NAFLD) increasing CDI risk and CDI exacerbating the progression and prognosis of liver cirrhosis. Moreover, gut dysbiosis, often leading to C. difficile overgrowth, is implicated in chronic liver conditions. We approach this association from a different angle, exploring CDI as a potential direct contributor to metabolic liver diseases. Our study explores CDI-associated liver changes at gene expression and metabolomic levels.
Methods: Fifty-four C57BL/6 mice aged 3-4 weeks were divided into Control, Antibiotic control (Abx), and C. difficile infection (C. diff) groups. The Abx and C. diff groups received an antibiotic mixture in drinking water for four days to induce gut dysbiosis and facilitate C. difficile colonization. The C. diff group was then inoculated orally with 1.4 X 10⁴ C. difficile spores. Control animals received sterile PBS at each corresponding step. Animals were euthanized 48 hours post-inoculation to collect liver, intestine, and cecal contents for metabolomic, transcriptomic, and metagenomic analysis and histopathology.
Results: Gut dysbiosis and C. difficile colitis were confirmed in respective treatment groups by histopathology and shotgun metagenomic analysis. The RNA sequencing analysis of liver tissue showed a marked increase in the expression of genes associated with insulin resistance, lipid accumulation, oxidative stress, endoplasmic reticulum stress response, inflammation, and liver fibrosis in C. diff group compared to control. Conversely, genes involved in antioxidant defense, triglyceride and cholesterol hydrolysis, lipogenesis regulation, xenobiotic detoxification, and anti-inflammatory pathways were significantly downregulated. Untargeted metabolomic analysis indicated a significant alteration in fatty acid metabolism, highlighted by increased levels of palmitic acid; amino acid metabolism, with elevated levels of free valine, glycine, tyrosine, and aspartate; and Coenzyme A synthesis, showing reduced dephospho-CoA and pantothenic acid levels. Additionally, energy metabolism was altered, evidenced by reduced citrate levels. Increased levels of inflammatory mediators, such as prostaglandins and leukotrienes, along with reduced levels of free and oxidized glutathione and glucuronate, were observed in the C. diff group compared to controls.
Conclusion: Our results indicate that CDI triggers gene expression and metabolic shifts that promote inflammation, lipid accumulation, oxidative damage, and insulin resistance, which are critical in NAFLD development and progression. These insights suggest that recurrent CDI may contribute to NAFLD onset and progression in infected individuals. However, further studies are necessary to validate these observations.

Farnaz Yousefi

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