The human gastrointestinal tract hosts a diverse population of microorganisms that have a significant impact on host health. Among this population, Enterococcus faecalis (Ef ) represents a common member of intestinal microbiota, colonizing humans early in life but also capable of opportunistically infecting its host. Despite its importance in human health, investigations of its physiological adaptation to the mucosal environment remain limited. Building on recent advances in tissue engineering, we here leverage human colonic organoids (colonoids) to investigate the Ef 's mechanisms of mucosal surface colonization across space and time. Using high-resolution microscopy, we visualized Ef growth within the natively formed colonic mucus layer in colonoids. Leveraging a custom perfusion chamber, we tracked Ef growth within the mucus of live colonoids over time under flow, which revealed specific colonization strategies, including biofilm-like microcolony formation. To identify Ef fitness determinants in this niche, we implemented transposon insertion sequencing (Tn-seq) in the natively formed mucus of live colonoids. This approach revealed a large fitness rearrangement compared to typical liquid culture, mainly involving metabolic activity and regulatory response during mucosal colonization, as well as factors that may contribute to colony formation at the mucosal surface. Altogether, our results show important physiological and biophysical adaptation of Ef to the mucosal surface that are not captured by in vitro conditions and that cannot be revealed in vivo at high resolution.

Importance: Gut microbiota interactions with mucus during early intestinal colonization are critical for establishing stable communities and influencing host health. Using human colonic organoids combined with Tn-seq and live imaging, this study reveals how Enterococcus faecalis adapts to the mucosal surface by forming microcolonies and reprogramming its metabolism. This integrative approach provides a powerful platform to study other microbiota members in the native-like environment of the large intestine and evaluate potential therapeutic interventions.