Expanding the cultivated human archaeome by targeted isolation of novel Methanobrevibacter strains from fecal samples

Duller, S.; Vrbancic, S.; Szydlowski, L.; Mahnert, A.; Blohs, M.; Predl, M.; Kumpitsch, C.; Zrim, V.; Hoegenauer, C.; Kosciolek, T.; Schmitz, R. A.; Eberhard, A.; Dragovan, M.; Schmidberger, L.; Zurabischvili, T.; Weinberger, V.; Moser, A. M.; Kolb, D.; Pernitsch, D.; Mohammadzadeh, R.; Kuehnast, T.; Rattei, T.; Moissl-Eichinger, C.

doi:10.1101/2024.04.10.588852

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bioRxiv · 10.1101/2024.04.10.588852

Expanding the cultivated human archaeome by targeted isolation of novel Methanobrevibacter strains from fecal samples

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Abstract

Archaea are integral components of the human microbiome but persist as understudied entities within the gastrointestinal tract (GIT), primarily due to the lack of cultured representatives for comprehensive mechanistic investigations. With only four Methanobrevibacter smithii isolates from humans available according to the Global Catalogue of Microorganisms (GCM), the existing cultures fail to adequately represent the observed diversity, as underscored by recent findings. This study introduces a targeted cultivation method for enriching methanogenic archaea from human fecal samples. Applied to 16 stool samples from healthy and diseased donors, the method aimed to genomically characterize the archaeal cultures and establish correlations with gastrointestinal disorders. The procedure combines methane breath testing, in silico metabolic modelling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyses include co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which were absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from diverse participant cohorts revealed features potentially associated with gastrointestinal diseases. This work substantially broadens the scope of available archaeal representatives for functional and mechanistic studies in the human GIT. The established protocol facilitates the cultivation of methanogenic archaea from nearly every human fecal sample, offering insights into the adaptability of Candidatus Methanobrevibacter intestini genomes in critical microbiome situations.

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Duller, S., Vrbancic, S., Szydlowski, L., Mahnert, A., Blohs, M., Predl, M., Kumpitsch, C., Zrim, V., Hoegenauer, C., Kosciolek, T., Schmitz, R. A., Eberhard, A., Dragovan, M., Schmidberger, L., Zurabischvili, T., Weinberger, V., Moser, A. M., Kolb, D., Pernitsch, D., Mohammadzadeh, R., Kuehnast, T., Rattei, T., Moissl-Eichinger, C.. 2024-04-11. Expanding the cultivated human archaeome by targeted isolation of novel Methanobrevibacter strains from fecal samples. https://doi.org/10.1101/2024.04.10.588852

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