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Serine proteases are involved in many biological processes and are associated with irritable bowel syndrome (IBS) pathology. An increase in serine protease activity has been widely reported in IBS patients. While most of the studies focused on host proteases, the contribution of microbial proteases are poorly studied. In the present study, we report the analysis of proteolytic activities in fecal samples from the first Tunisian cohort of IBS-M patients and healthy individuals. We demonstrated, for the first time, that metalloproteases activities were fourfold higher in fecal samples of IBS patients compared to controls. Of interest, the functional characterization of serine protease activities revealed a 50-fold increase in trypsin-like activities and a threefold in both elastase- and cathepsin G-like activities. Remarkably, we also showed a fourfold increase in proteinase 3-like activity in the case of IBS. This study also provides insight into the alteration of gut microbiota and its potential role in proteolytic modulation in IBS. Our results stressed the impact of the disequilibrium of serine proteases, metalloproteases and gut microbiota in IBS and the need of the further characterization of these targets to set out new therapeutic approaches. [ABSTRACT FROM AUTHOR]