Non-targeted metabolomics reveals metabolic signatures associated with Clostridioides difficile virulence

Huixin Pan¹Miao Zhang²Dongxiao Zhao³Qinglu Wang⁴Hua Shang^1*Ying Luo^1*

• ¹Zibo Central Hospital, Shandong, China
• ²Zibo Center for Disease Control and Prevention, Zibo, China
• ³Ningxia integrated Chinese and western medicine hospital, Yinchuan, China
• ⁴Shandong Sport University, Jinan, China

Background: Clostridioides difficile(C. difficile ) is a major pathogen causing antibiotic-associated diarrhea and pseudomembranous colitis, with Clostridioides difficile infection (CDI) showing a global upward trend. Significant differences exist in clinical manifestations and pathogenic potential among strains of varying virulence, yet their underlying metabolic basis and molecular mechanisms remain poorly understood. Systematic investigation of metabolic characteristics across strains with differing virulence levels is crucial for elucidating pathogenic mechanisms and identifying potential metabolic targets. Methods: Four C. difficile strains with varying virulence gradients (RT027/ST1, RT046/ST35, RT017/ST37, RT012/ST54) were selected. Liquid chromatography-mass spectrometry (LC–MS)-based non-targeted metabolomics was employed, combined with principal component analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and pathway enrichment analysis to compare metabolic differences among strains. Results: A total of 3,255 metabolites were identified (1,735 in positive ion mode and 1,520 in negative ion mode). Multivariate statistical models revealed significant metabolic profile separation among the four strains. The highly virulent strain (ST1) exhibited significantly enhanced activation in lipid metabolism, bile acid metabolism, nicotinic acid/nicotinamide energy metabolism, and branched-chain amino acid fermentation pathways compared to the low-virulence strain (ST54). Analysis of virulence gradient-related metabolites identified differentially expressed metabolites with potential biological significance, including upregulated isomangiferin, ginsenoside ro, glycocholic acid, lactic acid, isovalerate, and downregulated inosine, n-acetylmuramate, n-acetylglucosamine, cholesterol. These metabolites were primarily enriched in pathways involving bile acid synthesis, pyruvate metabolism, amino sugar and nucleotide sugar metabolism, and sterol biosynthesis. Conclusion: This study systematically characterized the metabolomic profiles of C. difficile strains of different ST types, revealing that their enhanced virulence is closely associated with the reprogramming of energy metabolism, membrane lipid structural remodeling, and bile acid metabolism. Metabolic differences suggest that highly virulent strains may enhance fermentation and lipid synthesis pathways to gain stronger survival and infection capabilities. The 13 candidate metabolites identified hold promise as potential biomarkers for distinguishing strain virulence levels, providing new theoretical basis for subsequent targeted metabolic regulation and anti-C. difficile therapies.