Feeding-mode-defined microbial communities modulate prebiotic responses and alters colonic motility in early life

Prebiotic supplementation a well-established impact on early-life gut microbiota, yet how substrate type and baseline community structure interact to shape microbial and metabolic responses remains incompletely understood. Using donor-derived infant faecal fermentations, a defined synthetic microbial community (SynCom), and multi-layered metabolomics, we demonstrate that prebiotic responses are strongly dependent on feeding-mode and mediated by species-specific metabolic interactions. While both human milk oligosaccharides (HMOs) and galacto-oligosaccharides (GOS) robustly promoted bifidobacterial expansion, modulation of Bacteroides/Phocaeicola species varied according to feeding background and substrate identity with 2'-fucosyllactose (2'FL) selectively alleviating growth inhibition in breast milk-derived communities. Mechanistic dissection using SynCom revealed extensive cross-feeding networks and substrate-dependent interaction rewiring, identifying Phocaeicola vulgatus as a context-dependent ecological hub that disproportionately shaped succinate and propionate production and altered Escherichia coli metabolic activity without affecting its abundance. Competitive outcomes were determined not only by environmental acidification, but also by the chemical identity of fermentation products, with lactate exerting species-specific inhibitory effects independent of bulk pH changes. Using an in-house ex vivo IntestiFlow gut organ platform, we further show that metabolites produced by the defined community induced a strong but reversible suppression of colonic peristaltic activity, an effect not recapitulated by SCFAs alone. Biogenic amine metabolites, including phenethylamine, partially reproduced key aspects of this response, suggesting a role for specific microbial fermentation products in modulating intestinal motility. Together, these findings demonstrate that early-life prebiotic responses extend beyond uniform bifidogenic expansion and instead emerge from interaction-driven metabolic specialization within feeding-mode-specific community contexts, with direct functional consequences for infant intestinal physiology.