Impact of early probiotic supplementation on the gut microbiota and urinary and fecal metabolome of formula-fed infants delivered via elective cesarean section

Abstract

Early postnatal life is a critical period for gut microbiota establishment and metabolic programming, particularly in infants born by cesarean section and fed with infant formula, two factors known to disrupt physiological microbial colonization. This exploratory pilot study aimed to provide a longitudinal characterization of microbiota and metabolome dynamics in infants born by elective cesarean section and formula-fed receiving Lactobif® (Buona S.p.a. Società Benefit, Sesto Fiorentino, Italy), a commercial probiotic formulation containing Lacticaseibacillus rhamnosus, LGG® (DSM33156) and Bifidobacterium longum subsp. infantis, Bifin02™ (DSM33361). A multi-omics approach was applied, integrating 16S rRNA gene-based microbiota profiles with ¹H NMR-based metabolomics of fecal and urinary samples collected before and during supplementation. Longitudinal analyses revealed dynamic shifts in gut microbial composition, notably a post-treatment increase in Bifidobacteriaceae and a reduction in Enterobacteriaceae, accompanied by changes in fecal metabolites associated with fermentation, including lactate, acetate, and propionate. An expansion of Veillonellaceae was also observed, suggesting the progressive emergence of a more functionally interconnected microbial ecosystem. Urinary metabolomic profiles showed time-dependent modulation of metabolites involved in energy me­tabolism, amino acid turnover, and microbial co-metabolism, reflecting systemic metabolic adaptation during early infancy. The longitudinal, multi-compartment design offers descriptive evidence of temporal positive changes in microbial composition and metabolic outputs, revealing both shared patterns and marked interindividual variability. The integrated analysis of microbiota and metabolomics data highlights the value of multi-biofluid, multi-omics strategies for capturing functional maturation of the gut ecosystem and supports the need for personalized approaches to early-life microbiome modulation.