Characterization of the gut mycobiome in patients with non-alcoholic fatty liver disease and correlations with serum metabolome.

BACKGROUND: Emerging evidence suggests that the gut microbiome plays a key role in metabolic diseases such as non-alcoholic fatty liver disease, yet the contribution of the gut mycobiome remains largely overlooked. METHODS: We performed a comprehensive analysis of publicly available fecal metagenomic sequencing data and matched serum metabolomic profiles from 90 non-alcoholic fatty liver disease patients and 90 healthy controls. A curated fungal genome database was constructed for taxonomic profiling. We integrated fungal, bacterial, and metabolomic data to assess taxon-specific associations, cross-kingdom interactions, and predictive potential. RESULTS: Although overall fungal diversity showed no significant differences between groups, four fungal species-Pseudopithomyces sp. c174, Mucor sp. c176, Aspergillus sp. c25, and Ascochyta c213-were significantly enriched in non-alcoholic fatty liver disease patients. The gut mycobiome explained 38.2% of the variance in serum metabolomic profiles, with several species displaying strong correlations with non-alcoholic fatty liver disease relevant metabolites. For instance, Pseudopithomyces sp. c174 was positively associated with protective metabolites such as glycoursodeoxycholic acid and alpha-linolenic acid, while Aureobasidium c170 and Basipetospora c193 were linked to phenylacetic acid, a metabolite implicated in hepatic lipid accumulation. Network analysis revealed altered fungal-bacterial co-abundance patterns in non-alcoholic fatty liver disease, with fungal taxa such as Alternaria alternata c42 and Malassezia c303 emerging as key hubs. A random forest classifier integrating 42 bacterial and fungal features achieved an AUC of 0.772 for distinguishing non-alcoholic fatty liver disease from controls, highlighting the predictive value of the mycobiome. CONCLUSIONS: Our findings reveal that gut fungal communities are functionally and ecologically altered in non-alcoholic fatty liver disease and contribute to shaping the host metabolic environment. These results underscore the need to incorporate the gut mycobiome into future microbiome-based strategies for non-alcoholic fatty liver disease diagnosis and treatment.