Ecological Network Inference Reveals 737 Cross-Kingdom Associations Structuring Human Microbiomes

The human microbiome is a complex, multikingdom ecosystem where bacteria and fungi cohabit and interact. Despite their ecological and clinical significance, cross-kingdom dynamics remain poorly characterized due to dominant single-kingdom research approaches. To understand the principles structuring multi-kingdom microbial communities, we applied the sparse inference method SpiecEasi to 45 publicly available samples from the gastrointestinal tract, skin, and oral cavity. Bacterial (16S rRNA) and fungal (ITS) sequencing data were processed using QIIME2, managed in phyloseq, and co-occurrence networks were inferred via SpiecEasi with Meinshausen- Buhlmann estimation. To validate robustness, we employed SparCC as a secondary inference method and performed 100 bootstrap iterations. Body site stratification controlled for environmental confounders. Our analysis revealed a microbial network of 5,023 taxa (5,020 bacterial, 3 fungal) connected by 30,478 significant associations. Crucially, we identified 737 robust bacterial-fungal interkingdom interactions (689 positive, 48 negative) confirmed by both inference methods. The network exhibited sparse connectivity (density = 0.0024) and modular structure (modularity = 0.45). Hub analysis identified 15 keystone taxa, including Bacteroides uniformis and Faecalibacterium prausnitzii. Interaction patterns were body-site-specific (P < 0.001), with the gastrointestinal tract showing the highest interkingdom connectivity (385 edges). This study provides systematic evidence that bacterial-fungal interactions are abundant and integral to human microbiome architecture. The discovery of 737 cross-kingdom associations challenges the prevailing single-kingdom paradigm and advocates for an integrated multikingdom perspective. These interactions, particularly those mediated by keystone hubs, represent novel targets for microbiome-based therapeutics and diagnostics. ImportanceThis study challenges the prevailing single-kingdom paradigm in microbiome research by demonstrating that bacterial-fungal interactions are abundant and integral to human microbiome architecture. The discovery of 737 cross-kingdom associations across three body sites provides a foundational resource for understanding multikingdom microbial ecology. The identification of keystone bacterial hubs--particularly Bacteroides uniformis and Faecalibacterium prausnitzii--as central connectors in interkingdom networks opens new avenues for microbiome-based therapeutics and diagnostics. Our integrated analytical framework, combining SpiecEasi and SparCC with body site stratification, offers a robust methodological template for future cross-kingdom studies.