Isolation and metabolomic analysis of the culturable human gut mycobiota during dysbiosis

The human gut mycobiome, though less diverse than the bacterial microbiome, plays a significant role in health and disease. This study investigates the culturable fungal communities in fecal samples from hospitalized patients with diarrhea in Mexico City. We isolated and characterized 26 fungal strains using culture-dependent methods, including 20 yeasts and six filamentous fungi. The most prevalent organisms were Candida albicans, Rhodotorula mucilaginosa, Penicillium spp., and Paecilomyces spp. Fungal isolates were tested for their ability to withstand gut-like conditions, including temperature, pH, oxidative stress, and bile salts. Notably, Paecilomyces variotii demonstrated thermotolerance, surviving at 42{degrees}C, and exhibited competitive growth against other fungi. Co-occurrence analysis revealed associations between fungal isolates and bacterial pathogens such as Salmonella and Clostridioides difficile, suggesting potential interkingdom interactions. Cytotoxicity assays on Caco-2 cells showed that cell-free supernatants from Candida inospicua and filamentous fungi reduced cell viability by up to 40%. Finally, dereplication and untargeted metabolomic analyses of P. variotii, Penicillium crustosum, and Penicillium chrysogenum revealed the presence of several bioactive metabolites, including mycotoxins and antimicrobial compounds, highlighting their potential roles in gut ecology and disease. Overall, this study underscores the importance of the gut mycobiome in dysbiosis and its interactions with bacterial pathogens. The findings suggest that fungi, particularly thermotolerant species such as P. variotii, may contribute to gut dysbiosis and disease progression, particularly in immunocompromised patients. Further research is needed to elucidate the functional roles of these fungi and their metabolites in gut health and disease.