The human digestive tract is a key player at the boundary between the external environment and the host. At the interface between the digestive lumen and the intestinal epithelium, the mucus layer, a complex viscoelastic adherent secretion, acts as a major modulator of human health. In order to reach the intestinal cells and/or colonize, several enteric pathogens have to interact with and get through this physical, chemical and biological line of defense. Enterotoxigenic Escherichia coli (ETEC), the main pathogenic agent of travelers’ diarrhea, does not escape this rule. To fulfil its infection cycle, ETEC is equipped with an arsenal of adhesins and mucinases allowing cellular adhesion and mucus degradation, respectively. These colonization mechanisms facilitate the production and release of heat labile (LT) and/or heat-stable (ST) enterotoxins, ultimately responsible for cholera-like watery diarrhea. To date the treatment of ETEC infection remains mainly symptomatic with a frequent use of antibiotics. Given the global burden of antibiotic resistance and its negative impact on human health, it is urgent to find new preventive strategies against these infections. Among the candidates, dietary fibers have been recently investigated for their antagonistic properties against enteric pathogens. A low number of studies has suggested that they may act through various means: (i) direct antagonism (bacteriostatic effect, inhibition of cell adhesion and toxin production) or (ii) indirect antagonism via modulation of gut microbiota composition/activity or decoy of resident gut microbes from mucus layer consumption. In this context, this joint doctoral research work between Ghent University (Belgium) and Université Clermont Auvergne (France) aimed to (1) unravel how the mucus compartment can modulate the prototypical ETEC strain H10407 survival and virulence, and (2) decipher if dietary fiber-containing products could present ETEC anti-infectious properties, notably by preventing ETEC-mucus interactions. In the first axis, we confirmed the prototypical ETEC strain H10407 adhesion propensity for the intestinal mucus by using different simple in vitro approaches. The introduction of mucin secretion and physical surface in the dynamic TIM-1 digestive model showed that mucus could favor ETEC survival during gastrointestinal passage without significantly affecting its virulence. However, when reaching the host intestinal cells simulated by mucus-secreting Caco-2/HT29-MTX co-culture, ETEC virulence gene expression was significantly induced confirming that the host is a key driver of pathogen’s virulence. When simulating the complex microbial background of the human gut, mucin addition did not impact significantly ETEC survival, but we showed that the mucosal compartment was colonized by a specific microbiota particularly affected by ETEC. In the second axis, a screening program was first performed to select among 8 fiber candidates from cereals, legumes or microbes the two most relevant based on their anti-infectious properties against ETEC strain H10407, namely a lentil fiber extract and specific yeast cell walls from Saccharomyces cerevisiae. In-depth investigations indicated that the lentil extract reduced LT toxin concentration while the yeast product decreased ETEC adhesion to the mucus secreting co-culture model. Also, in cell assays, both lentils and yeast fiber products were able to modulate ETEC virulence gene expression and innate immune response induction. Mainly yeast cell walls were able to strengthen intestinal barrier function. Finally, in batch experiments with fecal microbiota, we reported that the yeast product supported the prevalence of some phylogroups as Parabacteroides or commensal E. coli, which could be of interest in traveler’s diarrhea prevention. (...)