Bruno K Rodiño ^1,2 Olfat Khannous ^3,4 Marc Pigrau ^1,5 Jesse Willis ^3,4 Eloísa Salvo-Romero ^1,5,6 Adoración Nieto-Ruiz ^1,5 Elba Expósito ^1,5,6 Marina Fortea ^5,6 Cristina Pardo-Camacho ^1,5,6 Merce Albert-Bayo ^5,6 Ana María Gonzalez-Castro ^1,5,6,7 Danila Guagnozzi ^1,5,6,7 Cristina Martinez Martinez ^1,8,9 Beatriz Lobo ^1,5,7 María Vicario ^1,5 FRANCISCO JAVIER SANTOS VICENTE ^1,5,7 Toni Gabaldón ^10,11,3,4 Carmen Alonso-Cotoner ^1,12,5,7*

• ¹ Laboratory of Neuro-Immuno-Gastroenterology, Digestive System Research Unit, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Vall d'Hebron Research Institute (VHIR), Barcelona, Catalonia, Spain
• ² Department of Gastroenterology, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus,, Vall d'Hebron University Hospital, Barcelona, Catalonia, Spain
• ³ Barcelona Supercomputing Center, Barcelona, Catalonia, Spain
• ⁴ Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac, 10, 08028, Barcelona, Spain
• ⁵ Department of Gastroenterology, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Vall d'Hebron University Hospital, Barcelona, Catalonia, Spain
• ⁶ Laboratory of Translational Mucosal Immunology, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain
• ⁷ Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERHED), Instituto de Salud Carlos III, Madrid, Asturias, Spain
• ⁸ Department of Gastroenterology, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d'Hebron 119-129, 08035, Vall d'Hebron University Hospital, Barcelona, Catalonia, Spain
• ⁹ Vall d'Hebron Research Institute (VHIR), Barcelona, Balearic Islands, Spain
• ¹⁰ Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Catalonia, Spain
• ¹¹ Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
• ¹² Facultad de Medicina, Universitat Autònoma de Barcelona, Autonomous University of Barcelona, Barcelona, Catalonia, Spain

BACKGROUND/AIMS: Digestive disorders of gut-brain interaction (DGBI) are very common, predominant in females, and usually associated with intestinal barrier dysfunction, dysbiosis, and stress. We previously found that females have increased susceptibility to intestinal barrier dysfunction in response to acute stress. However, whether this is associated with changes in the small bowel microbiota remains unknown. We have evaluated changes in the small intestinal microbiota in response to acute stress to better understand stress-induced intestinal barrier dysfunction. METHODS: Jejunal biopsies were obtained at baseline and 90 min after cold pain or sham stress. Autonomic (blood pressure and heart rate), hormonal (plasma cortisol and adrenocorticotropic hormone) and psychological (Subjective Stress Rating Scale) responses to cold pain and sham stress were monitored. Microbial DNA of the biopsies was analyzed using a 16S metabarcoding approach before and after cold pain stress and sham stress. Differences in diversity and relative abundance of microbial taxa were examined. RESULTS: Cold pain stress was associated with a significant decrease in alpha diversity (p=0.015), which was more pronounced in females, along with significant sex differences in the abundance of specific taxa and the overall microbiota composition. Microbiota alterations significantly correlated with changes in psychological responses, hormones, and gene expression in the intestinal mucosal. Cold pain stress was also associated with activation of autonomic, hormonal and psychological response, with no differences between sexes. CONCLUSIONS: Acute stress elicits rapid alterations in bacterial composition in the jejunum of healthy subjects and these changes are more pronounced in females. Our results may contribute to the understanding of female predominance in DGBI.