Longitudinal profiling of one infant fecal microbiota over the first three years: effects of probiotics and prebiotics

Probiotics and prebiotics are increasingly added to infant formulas and supplements, potentially shaping early gut colonization. Their effects depend heavily on the existing microbiota, underscoring the need for individual, longitudinal, and polyphasic approaches. This study comprehensively analyzed microbial changes in an infant's stool over the first three years of life, capturing dietary transitions and interventions. The vaginally born, partially breastfed male infant received continuously formula with Limosilactobacillus reuteri DSM 17938 and 2´FL, and a two-week probiotic drop intervention with Bifidobacterium breve BR03 and Lactiplantibacillus plantarum LP01 to ease colic discomfort associated with clostridial dominance. Eighty-six fecal samples were analyzed using amplicon sequencing, complemented by qPCR and extensive culture-based throughput quantification of selected taxa identified by MALDI-TOF MS (>2000 isolates). Clostridia, enterobacteria and enterococci dominated the first month alongside supplemented Li. reuteri . Probiotic drops reduced clostridia and increased bifidobacteria, when B. breve persisted, while Lp. plantarum did not. During the liquid food period, supplemented B. breve and naturally occurring Lacticaseibacillus rhamnosus increased together, while Escherichia coli decreased in favor of Enterobacter cloacae . Later shifts reflected dietary transitions, including solid-food introduction and breastfeeding cessation. This longitudinal polyphasic study lies in its high-resolution design, which captures transient microecological successions and strain persistence that are typically masked in larger, less-frequently sampled cohorts. By combining molecular methods with extensive cultivation, we successfully bridge the gap between community-level sequencing and viable, strain-level absolute quantification. Ultimately, this work provides a rare micro-ecological view into real-time microbiota resilience and contributes an extensive repository of physiologically contextualized infant-derived strains.