RESUMEN
Recent advances in the human microbiome characterization have revealed significant oral microbial detection in stools of dysbiotic patients. However, little is known about the potential interactions of these invasive oral microorganisms with commensal intestinal microbiota and the host. In this proof-of-concept study, we proposed a new model of oral-to-gut invasion by the combined use of an in vitro model simulating both the physicochemical and microbial (lumen- and mucus-associated microbes) parameters of the human colon (M-ARCOL), a salivary enrichment protocol, and whole-metagenome shotgun sequencing. Oral invasion of the intestinal microbiota was simulated by injection of enriched saliva in the in vitro colon model inoculated with a fecal sample from the same healthy adult donor. The mucosal compartment of M-ARCOL was able to retain the highest species richness levels over time, while species richness levels decreased in the luminal compartment. This study also showed that oral microorganisms preferably colonized the mucosal microenvironment, suggesting potential oral-to-intestinal mucosal competitions. This new model of oral-to-gut invasion can provide useful mechanistic insights into the role of oral microbiome in various disease processes. IMPORTANCE Here, we propose a new model of oral-to-gut invasion by the combined use of an in vitro model simulating both the physicochemical and microbial (lumen- and mucus-associated microbes) parameters of the human colon (M-ARCOL), a salivary enrichment protocol, and whole-metagenome shotgun sequencing. Our study revealed the importance of integrating the mucus compartment, which retained higher microbial richness during fermentation, showed the preference of oral microbial invaders for the mucosal resources, and indicated potential oral-to-intestinal mucosal competitions. It also underlined promising opportunities to further understand mechanisms of oral invasion into the human gut microbiome, define microbe-microbe and mucus-microbe interactions in a compartmentalized fashion, and help to better characterize the potential of oral microbial invasion and their persistence in the gut.
RESUMEN
In vitro gut models, such as the mucosal artificial colon (M-ARCOL), provide timely and cost-efficient alternatives to in vivo assays allowing mechanistic studies to better understand the role of human microbiome in health and disease. Using such models inoculated with human fecal samples may require a critical step of stool storage. The effects of preservation methods on microbial structure and function in in vitro gut models have been poorly investigated. This study aimed to assess the impact of three commonly used preserving methods, compared with fresh fecal samples used as a control, on the kinetics of lumen and mucus-associated microbiota colonization in the M-ARCOL model. Feces from two healthy donors were frozen 48 h at - 80 °C with or without cryoprotectant (10% glycerol) or lyophilized with maltodextrin and trehalose prior to inoculation of four parallel bioreactors (e.g., fresh stool, raw stool stored at - 80 °C, stool stored at - 80 °C with glycerol and lyophilized stool). Microbiota composition and diversity (qPCR and 16S metabarcoding) as well as metabolic activity (gases and short chain fatty acids) were monitored throughout the fermentation process (9 days). All the preservative treatments allowed the maintaining inside the M-ARCOL of a complex and functional microbiota, but considering stabilization time of microbial profiles and activities (and not technical constraints associated with the supply of frozen material), our results highlighted 48 h freezing at - 80 °C without cryoprotectant as the most efficient method. These results will help scientists to determine the most accurate method for fecal storage prior to inoculation of in vitro gut microbiome models. KEY POINTS: ⢠In vitro ARCOL model reproduces luminal and mucosal human microbiome. ⢠Short-term storage of fecal sample influences microbial stabilization and activity. ⢠48 h freezing at - 80°C: most efficient method to preserve microbial ecosystem. ⢠Scientific and technical requirements: influencers of preservation method.