Impact of maternal, infant, and household factors on early-life gut microbiome development in a rural setting

Early-life gut microbiome development is influenced by host, microbial, environmental, and social factors. Rural infants typically exhibit greater microbial diversity than their urban counterparts, yet microbiome maturation patterns in less industrialized settings remain underexplored. Additionally, though microbial eukaryotes are integral to gut ecology, most studies to date have focused predominantly on bacterial communities. Using shallow shotgun metagenomics and 18S ribosomal RNA gene sequencing, we characterized bacterial and eukaryotic gut microbiomes in an intensively sampled longitudinal cohort of 10 infants from a rural community in Morelos, Mexico, each followed monthly from the first to the 18th month, providing a detailed view of early-life microbiome development in a low-resource setting. Although both bacterial and eukaryotic alpha diversity increased over time, they showed distinct colonization trajectories. Age, delivery mode, and environmental exposures, such as animal contact and household factors, influenced bacterial and eukaryotic community compositions, and bacterial metabolic composition. Inter-kingdom microbial networks varied with age, with a reduction in taxonomic diversity after the first year of life. Age and birth mode also influenced changes in the overall community structure and connectivity of microbial co-occurrence patterns, but did not impact the associations among specific microbial taxa. Functional profiling revealed that bacterial metabolic potential diversified with age, whereas the mode of birth had a minimal impact on functional variation. These findings highlight the dynamic nature of bacterial and eukaryotic microbiota in early life and underscore the need to explore how rural environmental exposures shape microbial maturation, with potential implications for immune development and long-term health.