Modeling the effects of prebiotic interventions on luminal and mucosa-associated gut microbiota without and with Clostridium difficile challenge in vitro

Prebiotics can modulate the gut microbial community composition and function for improved (gut) health and increase resilience against infections. <i>In vitro</i> models of the gut facilitate the study of intervention effects on the gut microbial community relevant to health. The mucosa-associated gut microbiota, which thrives in close contact with the host plays a pivotal role in colonization resistance and health. Therefore, we here introduce the Mi-screen, an experimental approach implementing a 96-well plate equipped with a mucus agar layer for the additional culturing of mucosa-associated microbiota <i>in vitro</i>. In this study, we screened the effects of 2'-Fucosyllactose (2'-FL), fructooligosaccharides (FOS), and inulin within a complex microbiota without and with infection with the <i>C. difficile</i> strains ATCC 43599 (Ribotype 001) or ATCC BAA-1870 (Ribotype 027). We analyzed the microbial community composition and short-chain fatty acid levels after 48 h of incubation. The inclusion of an additional substrate and surface in the form of the mucus agar layer allowed us to culture a microbial richness ranging between 100-160 in Chao index, with Shannon indices of 5-6 across culture conditions, indicative of a microbial diversity of physiological relevance. The mucus agar layer stimulated the growth of characteristic mucosa-associated bacteria such as <i>Roseburia inulinovorans</i>. The prebiotic interventions affected luminal and mucosal microbial communities cultured <i>in vitro</i> and stimulated short-chain fatty acid production. FOS, inulin and 2'-FL promoted the growth of <i>Bifidobacterium adolescentis</i> within the mucosa-associated microbiota cultured <i>in vitro</i>. When spiking the untreated conditions with pathogenic <i>C. difficile</i>, the strains thrived within the luminal and the mucosal sample types, whereas prebiotic treatments exhibited inhibitory effects on <i>C. difficile</i> growth and prevented colonization. In conclusion, the Mi-screen facilitates the screening of luminal and mucosa-associated gut microbial community dynamics <i>in vitro</i> and therefore fills an important gap in the field of <i>in vitro</i> modeling.