Bacterial–fungal shifts and functional reprogramming in CDI: insights from matched metagenomic and metatranscriptomic profiling

Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea, yet most microbiome studies have focused on bacterial composition, overlooking fungal contributions and transcriptionally active functions. Here, we applied matched metagenomic (MG) and metatranscriptomic (MT) sequencing to diarrheal stool from 100 adults (50 CDI+, 50 CDI−) to characterize taxonomic structure, functional potential and expression, predicted metabolites, and bacterial–fungal interactions. CDI− samples exhibited relatively greater taxonomic evenness, whereas CDI+ samples showed relatively higher MT KEGG Ortholog richness, indicating broader transcriptional activity. Beta diversity analyses demonstrated significant separation by CDI status, strongest in MT functional profiles. CDI+ microbiomes were enriched for opportunistic and transcriptionally active taxa, including C. difficile , Lactococcus cremoris , Nitratidesulfovibrio vulgaris , and fungi such as Candida albicans and Nakaseomyces glabratus , while CDI− communities were relatively enriched for obligate anaerobic commensals (e.g., Faecalibacterium , Roseburia , Subdoligranulum ). Functionally, CDI+ communities were relatively enriched in transport, stress-response, motility, quorum sensing, and amino acid metabolism pathways, whereas CDI− retained core metabolic functions. Network analyses identified significant enrichment of fungal-commensal co-exclusion relationships in the CDI+ metatranscriptomic network relative to CDI-, and predicted metabolites implicated bile acid disruption and lipid remodeling. Together, these findings define CDI as a trans-kingdom, functionally reprogrammed ecosystem and highlight the importance of integrating fungal ecology and transcriptional activity in CDI pathogenesis.