Bowel preparation is routinely performed before colonoscopy, yet its immediate effects on the spatial organization of the colonic microbiota at the mucosal interface remain poorly resolved. Here, we introduce a high-resolution endoscopic mucus-harvesting approach, combined with luminal aspirates and mucosal biopsies, to generate a high-resolution, within-subject trajectory of microbiota alterations across distinct colonic niches in healthy adults over the first 24 hours after purging. While luminal bacterial communities remained remarkably stable, with no significant changes in alpha or beta diversity and proportional washout of taxa. In contrast, mucus-associated and mucosal communities underwent a rapid but reversible ecological restructuring, characterized by immediate post-cleansing shifts in composition and transient blooms of Proteobacteria, particularly Enterobacteriaceae. These perturbations were strongest in the 0-12-hour window and varied by individual, consistent with the dominance of personalized baseline microbial signatures. Critically, spatially resolved sampling revealed a key refinement: the Enterobacteriaceae expansion was confined almost exclusively to the superficial mucus layer, a glycan-rich, dynamically oxygenated compartment that is particularly susceptible to mechanical disturbance during lavage, whereas deeper mucus and mucosa-associated communities remained comparatively stable. By 24 hours, both mucosal and mucus-associated microbiota had largely returned to their individualized pre-cleansing configurations, indicating rapid ecosystem resilience and suggesting that the deeper mucus layer functions as a protected microbial reservoir that reseeds the epithelium and lumen once normal physiology is restored. This compartment-specific recovery trajectory contrasts with the prolonged dysbiosis typically observed after antibiotics or infection, underscoring the need for spatially precise sampling to interpret microbiome data collected during clinical endoscopy. Together, these findings establish an endoscopic strategy for probing microbe-mucus interactions in humans and provide a conceptual and methodological framework for interpreting microbiome data obtained during clinical endoscopy.
