Contamination-controlled upper gastrointestinal microbiota profiling reveals salivary-duodenal community types linked to opportunistic pathogen carriage and inflammation

The upper gastrointestinal (uGI) microbiota has been implicated in infectious, metabolic, and immunological conditions, yet remains poorly characterized due to invasive sampling and low microbial biomass. We developed and validated a contamination-controlled 16S rRNA gene and transcript-based protocol to profile the murine and human uGI microbiota from low-biomass samples. We applied this protocol to murine esophageal, gastric, and duodenal tissues, and to human saliva, gastric, and duodenal aspirates from patients undergoing endoscopy for suspected food-related, mild GI symptoms. Our objective was to identify conserved compositional and structural uGI microbiota patterns and assess their clinical relevance in relation to pathogen burden and inflammation. In mice, we found evidence for transcriptionally inactive and active intestinal taxa along the uGI tract, supporting horizontal microbiota transfer. In humans, we identified two distinct, inversely correlated salivary microbiota types–one dominated by the Prevotella 7 genus–which were conserved in the duodenum. The Prevotella 7-dominated uGI microbiota type was associated with lower relative abundances of gastrointestinal and extraintestinal opportunistic pathogens. These patterns were reproducible in an independent cohort and associated with lower systemic TNF-α levels. Our findings suggest that noninvasive salivary microbiota profiling can stratify individuals based on uGI microbiota composition and inflammation-associated risk traits, offering new opportunities for clinical applications and translational studies.