Eukaryotic and bacterial gut communities vary along a lifestyle-associated urbanization gradient: comparative analysis of Germany and Madagascar.

Intestinal eukaryotes, often neglected in gut microbiome studies, play crucial roles in human health and cause life-threatening diseases affecting millions worldwide. This neglect has also been driven by the underrepresentation of samples from regions with a high prevalence of (parasitic) intestinal eukaryotes. As a result, the overall understanding of how intestinal eukaryotes vary among broad lifestyle and socioeconomic differences, remains limited. Addressing this gap is increasingly urgent given the global rise of urbanization and industrialization and their profound effects on lifestyle, pathogen exposure, and environmental factors. We characterized the diversity and composition of eukaryotic and bacterial microbiota in 1387 fecal samples from Madagascar (Andina, Ankazomborona, Tsiroanomandidy) and Germany (Kiel), spanning a composite gradient of urbanization-associated lifestyles. Using a parallel approach of 18S V4-V5 rRNA and 16S V3-V4 rRNA amplicon gene sequencing, we identified distinct regional patterns in eukaryotic and bacterial community composition. Malagasy cohorts showed higher prevalence of helminths (e.g. Schistosoma, Necator) and protozoa (e.g. Entamoeba, Dientamoeba). Notably, the diversity of particularly food-associated fungi increased along the composite urbanization-associated lifestyle gradient, peaking in samples from Germany. Bacterial 16S amplicon sequencing confirmed and extended known geographical differences, showing a dominance of Bacteroides in Germany versus Prevotella and Firmicutes in Madagascar. This work highlights the importance of integrating eukaryotic and prokaryotic data, as well as considering different lifestyle-associated factors in microbiome research. We further highlight the need for deeper investigation into the role of dietary and environmental fungi in the human gut ecosystem.