Abstract
Blastocystis is the most prevalent intestinal protist in humans, yet its role in gut health remains poorly understood. Increasing evidence suggests subtype-specific interactions with the gut microbiome and metabolome may underlie its variable associations with health and disease. In this pilot study, we performed an integrated analysis of the microbiota and metabolite profiles of nine Blastocystis subtypes (ST1-ST9) grown in vitro using xenic cultures. Using 16S rRNA amplicon sequencing and proton nuclear magnetic resonance ((1)H-NMR) metabolomics, we characterised the microbial communities and extracellular metabolites across subtypes. ST3 exhibited the most distinct microbiome and metabolomic profile, characterised by a significant enrichment of short-chain fatty acids (SCFAs) and amino-acid derivatives. Benzoate, a known antimicrobial, was uniquely downregulated in ST3. Linear discriminant analysis identified several bacterial genera, such as Methanobrevibacter and Enterobacter, as biomarkers for ST3. Correlations between key metabolites and microbial taxa suggest potential syntrophic interactions. These findings suggest that individual Blastocystis subtypes establish distinct microenvironments in vitro, with implications for their ecological roles in vivo. Our study provides a foundational framework for understanding subtype-specific biology and offers a platform for improving culture conditions and investigating host-microbe interactions.