Multi-kingdom metagenomic characterization of the gut bacteriome, mycobiome, and virome in chronic functional constipation

Background Chronic functional constipation (CFC) is a common gastrointestinal disorder increasingly linked to gut microbiome dysbiosis. However, multi-kingdom metagenomic characterization of bacterial, fungal, and viral communities in CFC remains limited. Methods Fecal samples from 53 CFC patients and 48 healthy controls were analyzed using whole-metagenome shotgun sequencing. Microbial composition, function, cross-kingdom interactions, and diagnostic potential were evaluated using diversity analyses, KEGG annotation, network analysis, and random forest modeling. Results Compared with healthy controls, CFC patients exhibited marked alterations across multiple microbial kingdoms. The gut bacteriome showed significant community-structure shifts despite comparable α-diversity, characterized by depletion of health-associated Firmicutes (e. g. , Faecalibacterium and Roseburia) and enrichment of Proteobacteria (e. g. , Klebsiella). The mycobiome displayed selective changes in diversity and composition, with several potentially pathogenic fungal taxa enriched in CFC (e. g. , Fusarium sp. c181). In the virome, community composition differed significantly between groups, with higher viral richness in CFC and widespread depletion of diverse bacteriophages in CFC patients. Functional profiling suggested feature-level functional differences without a clear global shift, including reduced carbohydrate transport and utilization pathways and relatively higher abundance of stress-response and metabolic adaptation modules in CFC. Cross-kingdom network analysis demonstrated substantially denser microbial interactions in CFC, dominated by viral associations, with Faecalibacterium prausnitzii and Faecalibacterium SGB15346 acting as central hubs. Machine-learning models showed strong discriminatory power for CFC classification based on bacterial and viral features, whereas fungal features contributed less. Conclusions CFC is associated with coordinated multi-kingdom gut microbiome dysbiosis involving bacteria, fungi, and viruses, accompanied by functional shifts and intensified cross-kingdom interactions. Bacterial and viral signatures show strong potential as microbiome-based biomarkers for CFC, highlighting the importance of integrating multi-kingdom analyses to better understand disease-associated gut ecosystem alterations.