Understanding how commensal bacteria achieve persistent colonization in the human gut is essential for microbiome-based treatments. This project targets Bifidobacterium longum, a pioneer of early gut colinization and a hallmark for the development of the microbiome in healthy humans. B. longum adheres to the mucosa and supports microbiome maturation by metabolizing human milk oligosaccharides (HMO), producing short-chain fatty acids. We hypothesize that within-infant evolution – via de novo mutations, horizontal gene transfer, and selection on lineage-specific genes – drives transmission, transient dominance, and stable colonization during the first year of life when the composition of the microbiome is most dynamic. To resolve the genetic mechanisms underlying adaptation at genome-wide, single-mutation resolution, we will apply a high-throughput, culture-based whole-genome strategy to longitudinal, family-wide samples collected across multiple time points from birth until the first birthday. Evolutionary reconstruction, comparative genomics, and statistical and phylogenetic analyses will identify adaptive genes and genotypes linked to HMO use, metabolism, and host interaction. Functional consequences will be tested using isolate panels, targeted assays, and synthetic infant microbial communities, complemented by metabolomics and expert collaborations within the SPP. The study will deliver an extensive B. longum isolate resource and delineate gene functions that enable transmission and persistence. Outcomes will reveal fundamental rules of early-life colonization and inform precision prebiotic or probiotic design towards more targeted microbiome engineering.