From Multi-Omics to Molecular Causality: A Five-Tier Evidence Framework for the Single-Bacterium-Metabolite Axis in...

Gut microbiota research in inflammatory bowel disease (IBD) is transitioning from holistic community-level associations toward causal validation of individual bacteria and their metabolites. However, existing causal evidence is dispersed across diverse pathways and experimental models, lacking systematic integration and comparative assessment of evidence strength. Building on the concept of grading causal evidence in microbiome studies, we extend this approach into a five-tier progressive validation framework that covers the entire causal chain-from initial association discovery to the identification of specific effector metabolites. Using this framework, we systematically collate the single-bacterium-metabolite-host target axes that have been validated by high-level experimental methods. Focusing on four major pathways-AhR/tryptophan metabolism, bile acid/nuclear receptor signaling, short-chain fatty acid/vitamin/energy metabolism, and cell death-related pathogenic mechanisms-we integrate the molecular mechanisms through which specific bacteria and their causally linked metabolites regulate intestinal barrier integrity, immune responses, and inflammation resolution. We further discuss the modulatory influences of host genetics, diet, and medications on these axes, as well as therapeutic strategies such as live bacterial preparations, engineered probiotics, and postbiotics. Causal studies on individual bacteria and metabolites in IBD have progressed from isolated mechanistic discoveries towards systematic consolidation. It should be acknowledged, however, that most of the high-level evidence summarized in this review derives from animal models and preclinical studies, whereas robust human validation remains limited. In addition, protective mechanisms appear to considerably outnumber pathogenic ones, suggesting a marked imbalance in the current evidence base. To bridge the gap between mechanistic validation and clinical application, future research should focus on validating causal chains from animal models in well-designed human cohorts, exploring pathogenic mechanisms that drive disease onset, and clarifying interactions among different metabolites. By synthesizing available evidence, identifying key research gaps, and acknowledging translational limitations, this review aims to provide a foundational reference for researchers, while recognizing that further translational efforts are needed to move these preclinical findings toward clinical application.