ObjectiveHuman gut microbiome studies are mainly bacteria- and archaea-oriented, overlooking the presence of single-cell eukaryotes such as Blastocystis, an enteric stramenopiles with worldwide distribution. Here, we surveyed the prevalence and subtype variation of Blastocystis in faecal samples collected as part of the Flemish Gut Flora Project (FGFP), a Western population cohort. We assessed potential links between Blastocystis subtypes and identified microbiota–host covariates and quantified microbiota differentiation relative to subtype abundances.DesignWe profiled stool samples from 616 healthy individuals from the FGFP cohort as well as 107 patients with IBD using amplicon sequencing targeting the V4 variable region of the 16S rRNA and 18S rRNA genes. We evaluated associations of Blastocystis, and their subtypes, with host parameters, diversity and composition of bacterial and archaeal communities.ResultsBlastocystis prevalence in the non-clinical population cohort was 30% compared with 4% among Flemish patients with IBD. Within the FGFP cohort, out of 69 previously identified gut microbiota covariates, only age was associated with Blastocystis subtype carrier status. In contrast, a strong association between microbiota community composition and Blastocystis subtypes was observed, with effect sizes larger than that of host covariates. Microbial richness and diversity were linked to both Blastocystis prevalence and subtype variation. All Blastocystis subtypes detected in this cohort were found to be less prevalent in Bacteroides enterotyped samples. Interestingly, Blastocystis subtypes 3 and 4 were inversely correlated with Akkermansia, suggesting differential associations of subtypes with host health.ConclusionsThese results emphasise the role of Blastocystis as a common constituent of the healthy gut microbiota. We show its prevalence is reduced in patients with active IBD and demonstrate that subtype characterisation is essential for assessing the relationship between Blastocystis, microbiota profile and host health. These findings have direct clinical applications, especially in donor selection for faecal transplantation.