The duodenal microbiota is compartmentalized and clinically stable yet rapidly responsive to nutrient exposure.

The duodenum is one of the most nutrient-exposed and immunologically active regions of the human gastrointestinal tract, yet its microbial ecology and short-term responses to dietary stimuli remain poorly defined. Most studies rely on fasting luminal aspirates, which fail to capture mucosa-associated communities and miss rapid ecological shifts during nutrient exposure, limiting insight into the spatial organization and dynamic behavior of the upper small intestinal microbiota (SIM). To address these limitations, we performed a compartment-resolved analysis of the duodenal microbiota in 94 individuals, including healthy controls, patients with obesity before and after bariatric surgery, individuals with IBS, and subjects with other SIBO-associated risk states. Paired luminal aspirates and mucosal biopsies were obtained during upper endoscopy; bacterial load was quantified by culture, and the community structure was assessed using 16S rRNA gene sequencing and PICRUSt-based pathway inference. In addition, healthy volunteers underwent a controlled intraluminal fat challenge with dense serial duodenal sampling over 180 min to resolve short-term nutrient-driven dynamics. Across all participants, the anatomical niche emerged as the dominant ecological determinant. Mucosal communities displayed higher species richness, broader phylogenetic representation, and distinct beta-diversity signatures compared with luminal aspirates, which were narrowly dominated by