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 Streptococcaceae. Under fasting conditions, SIM remained remarkably stable across obesity, IBS, and culture-defined SIBO, with only minor taxonomic differences in SIBO-positive individuals. In contrast, acute nutrient exposure triggered rapid microbial blooming, increased culture positivity, and a transient rise in species richness within 30-60 min, revealing a highly responsive ecosystem not captured by fasting samples. Together, these findings show that the defining feature of the human duodenal microbiota is not disease-associated dysbiosis under fasting conditions, but rather a conserved spatial architecture coupled with rapid, nutrient-driven ecological plasticity, highlighting the dynamic and compartmentalized nature of the upper small intestinal microbiome.
