Summary To avoid detrimental interactions with intestinal microbes, the human epithelium is covered with a protective mucus layer that traps host defence molecules. Microbial properties such as adhesion to mucus further result in a unique mucosal microbiota with a great potential to interact with the host. As mucosal microbes are difficult to study in vivo , we incorporated mucin‐covered microcosms in a dynamic in vitro gut model, the simulator of the human intestinal microbial ecosystem (SHIME). We assessed the importance of the mucosal environment in this M‐SHIME (mucosal‐SHIME) for the colonization of lactobacilli, a group for which the mucus binding domain was recently discovered. Whereas the two dominant resident Lactobacilli, Lactobacillus mucosae and Pediococcus acidilactici, were both present in the lumen, L. mucosae was strongly enriched in mucus. As a possible explanation, the gene encoding a mucus binding ( mub ) protein was detected by PCR in L. mucosae . Also the strongly adherent Lactobacillus rhamnosus GG (LGG) specifically colonized mucus upon inoculation. Short‐term assays confirmed the strong mucin‐binding of both L. mucosae and LGG compared with P. acidilactici . The mucosal environment also increased long‐term colonization of L. mucosae and enhanced its stability upon antibiotic treatment (tetracycline, amoxicillin and ciprofloxacin). Incorporating a mucosal environment thus allowed colonization of specific microbes such as L. mucosae and LGG, in correspondence with the in vivo situation. This may lead to more in vivo ‐like microbial communities in such dynamic, long‐term in vitro simulations and allow the study of the unique mucosal microbiota in health and disease.
In this study, the prebiotic potential of arabinoxylan oligosaccharides (AXOS) was compared with inulin in two simulators of the human intestinal microbial ecosystem. Microbial breakdown of both oligosaccharides and short-chain fatty acid production was colon compartment specific, with ascending and transverse colon being the predominant site of inulin and AXOS degradation, respectively. Lactate levels (+5.5 mM) increased in the ascending colon during AXOS supplementation, while propionate levels (+5.1 mM) increased in the transverse colon. The concomitant decrease in lactate in the transverse colon suggests that propionate was partially formed over the acrylate pathway. Furthermore, AXOS supplementation strongly decreased butyrate in the ascending colon, this in parallel with a decrease in Roseburia spp. and Bacteroides/Prevotella/Porphyromonas (-1.4 and -2.0 log CFU) levels. Inulin treatment had moderate effects on lactate, propionate and butyrate levels. Denaturing gradient gel electrophoresis analysis revealed that inulin changed microbial metabolism by modulating the microbial community composition. In contrast, AXOS primarily affected microbial metabolism by 'switching on' AXOS-degrading enzymes (xylanase, arabinofuranosidase and xylosidase), without significantly affecting microbial community composition. Our results demonstrate that AXOS has a higher potency than inulin to shift part of the sugar fermentation toward the distal colon parts. Furthermore, due to its stronger propionate-stimulating effect, AXOS is a candidate prebiotic capable of lowering cholesterol and beneficially affecting fat metabolism of the host.
Akkermansia muciniphila is a champion of mucin degradation in the human gastrointestinal tract. Here, we report the isolation of six novel strains from healthy human donors and their genomic, proteomic and physiological characterization in comparison to the type-strains A. muciniphila MucT and A. glycaniphila PytT. Complete genome sequencing revealed that, despite their large genomic similarity (>97.6%), the novel isolates clustered into two distinct subspecies of A. muciniphila: Amuc1, which includes the type-strain MucT, and AmucU, a cluster of unassigned strains that have not yet been well characterized. CRISPR analysis showed all strains to be unique and confirmed that single healthy subjects can carry more than one A. muciniphila strain. Mucin degradation pathways were strongly conserved amongst all isolates, illustrating the exemplary niche adaptation of A. muciniphila to the mucin interface. This was confirmed by analysis of the predicted glycoside hydrolase profiles and supported by comparing the proteomes of A. muciniphila strain H2, belonging to the AmucU cluster, to MucT and A. glycaniphila PytT (including 610 and 727 proteins, respectively). While some intrinsic resistance was observed among the A. muciniphila straind, none of these seem to pose strain-specific risks in terms of their antibiotic resistance patterns nor a significant risk for the horizontal transfer of antibiotic resistance determinants, opening the way to apply the type-strain MucT or these new A. muciniphila strains as next generation beneficial microbes.
The human gut microbiome contributes crucial bioactive metabolites that support human health and is sensitive to perturbations from the ingestion of alcohol and antibiotics. We interrogated the response and recovery of human gut microbes after acute alcohol or broad-spectrum antibiotic administration in a gut model simulating the luminal and mucosal colonic environment with an inoculated human microbiome. Both alcohol and antibiotic treatments reduced the production of major short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate), which are established modulators of human health. Treatment with a microbial synbiotic restored and enhanced gut function. Butyrate and acetate production increased by up to 29.7% and 18.6%, respectively, relative to untreated, dysbiotic samples. In parallel, treatment led to increases in the relative abundances of beneficial commensal organisms not found in the synbiotic (e.g., Faecalibacterium prausnitzii and the urolithin-producing organism Gordonibacter pamelaeae) as well as species present in the synbiotic (e.g., Bifidobacterium infantis), suggesting synergistic interactions between supplemented and native microorganisms. These results lead us to conclude that functional shifts in the microbiome, evaluated by both metabolite production and specific taxonomic compositional changes, are an appropriate metric to assess microbiome "recovery" following a dysbiosis-inducing disruption. Overall, these findings support the execution of randomized clinical studies to determine whether a microbial synbiotic can help restore microbiome function after a disruption. IMPORTANCE The human gut microbiome is sensitive to disruptions by common stressors such as alcohol consumption and antibiotic treatment. In this study, we used an in vitro system modeling the gut microbiome to investigate whether treatment with a microbial synbiotic can help restore microbiome function after stress. We find that a complex gut community treated with alcohol or antibiotics showed reduced levels of production of short-chain fatty acids, which are critical beneficial molecules produced by a healthy gut microbiota. Treatment of stressed communities with a microbial synbiotic resulted in the recovery of SCFA production as well as an increase in the abundance of beneficial commensal organisms. Our results suggest that treatment with a microbial synbiotic has the potential to restore healthy gut microbiome function after stress and merits further investigation in clinical studies.
Along the human gastrointestinal tract, microorganisms are confronted with multiple barriers. Besides selective physical conditions, the epithelium is regularly replaced and covered with a protective mucus layer trapping immune molecules. Recent insights into host defense strategies show that the host selects the intestinal microbiota, particularly the mucosa-associated microbial community. In this context, humans coevolved with thousands of intestinal microbial species that have adapted to provide host benefits, while avoiding pathogenic behavior that might destabilize their host interaction. While mucosal microorganisms would be crucial for immunological priming, luminal microorganisms would be important for nutrient digestion. Further, we propose that the intestinal microorganisms also coevolved with each other, leading to coherently organized, resilient microbial associations. During disturbances, functionally redundant members become more abundant and are crucial for preserving community functionality. The outside of the mucus layer, where host defense molecules are more diluted, could serve as an environment where microorganisms are protected from disturbances in the lumen and from where they can recolonize the lumen after perturbations. This might explain the remarkable temporal stability of microbial communities. Finally, commensals that become renegade or a decreased exposure to essential coevolved microorganisms may cause particular health problems such as inflammatory bowel diseases, obesity or allergies.
The human gut microbiota has been linked to the health status of the host. Modulation of human gut microbiota through pro- and prebiotic interventions has yielded promising results; however, the effect of novel prebiotics, such as chitin–glucan, on gut microbiota–host interplay is still not fully characterized. We assessed the effect of chitin–glucan (CG) and chitin–glucan plus Bifidobacterium breve (CGB) on human gut microbiota from the luminal and mucosal environments in vitro. Further, we tested the effect of filter-sterilized fecal supernatants from CG and CGB fermentation for protective effects on inflammation-induced barrier disruption and cytokine production using a co-culture of enterocytes and macrophage-like cells. Overall, CG and CGB promote health-beneficial short-chain fatty acid production and shift human gut microbiota composition, with a consistent effect increasing Roseburia spp. and butyrate producing-bacteria. In two of three donors, CG and CGB also stimulated Faecalibacterium prausniitzi. Specific colonization of B. breve was observed in the lumen and mucosal compartment; however, no synergy was detected for different endpoints when comparing CGB and CG. Both treatments included a significant improvement of inflammation-disrupted epithelial barrier and shifts on cytokine production, especially by consistent increase in the immunomodulatory cytokines IL10 and IL6.
Modulation of the gut microbiome as a means to improve human health has recently gained increasing interest. In this study, it was investigated whether cRG-I, a carrot-derived pectic polysaccharide, enriched in rhamnogalacturonan-I (RG-I) classifies as a potential prebiotic ingredient using novel in vitro models. First, digestion methods involving α-amylase/brush border enzymes demonstrated the non-digestibility of cRG-I by host-derived enzymes versus digestible (starch/maltose) and non-digestible controls (inulin). Then, a recently developed short-term (48 h) colonic incubation strategy was applied and revealed that cRG-I fermentation increased levels of health-promoting short-chain fatty acids (SCFA; mainly acetate and propionate) and lactate comparable but not identical to the reference prebiotic inulin. Upon upgrading this fermentation model by inclusion of a simulated mucosal environment while applying quantitative 16S-targeted Illumina sequencing, cRG-I was additionally shown to specifically stimulate operational taxonomic units (OTUs) related to health-associated species such as Bifidobacterium longum, Bifidobacterium adolescentis, Bacteroides dorei, Bacteroides ovatus, Roseburia hominis, Faecalibacterium prausnitzii, and Eubacterium hallii. Finally, in a novel model to assess host–microbe interactions (Caco-2/peripheral blood mononuclear cells (PBMC) co-culture) fermented cRG-I increased barrier integrity while decreasing markers for inflammation. In conclusion, by using novel in vitro models, cRG-I was identified as a promising prebiotic candidate to proceed to clinical studies.
Abstract This chapter describes in vitro models of the gastrointestinal tract currently used in static and dynamic simulation studies of host-microbial interactions in the digestive tract.
A short-chain fructo-oligosaccharide (sc-FOS) was tested in a simulator of the human gut microbial ecosystem (SHIME) in vitro model to quantify its prebiotic effects according to Prebiotic Index (PI) and Measure of prebiotic effect (MPE) equations. FossenceTM, (sc-FOS, 0.5%) was fermented in a simulated human proximal colonic condition, using a fecal inoculum from a healthy individual. We analysed the pH reduction, substrate utilization, lactate and short-chain fatty acid (SCFA) production and microbial community modulation. Microbial fermentation of sc-FOS strongly reduced the media pH indicating the production of lactate and SCFA with accumulation of lactate and enhanced levels of acetate (34.38 ± 0.38 mM), propionate (20.93 ± 0.56 mM) and butyrate (4.93 ± 0.03 mM) compared to 18.46 ± 0.20 mM, 6.24 ± 0.10 mM and 3.3 ± 0.06 mM in the blank, respectively. Total SCFA production in test media was 61.91 ± 0.87 mM compared to 33.65 ± 0.36 mM in blank and the contribution of free-sugars present in sc-FOS to SCFAs was negligible. Modulation of the microbial community was analysed through 16S rRNA sequencing and we found that sc-FOS greatly stimulated the beneficial bacteria such as Bifidobacteria and Lactobacillus. We report the PI and MPE values for FossenceTM, as 14.9 and 0.01 respectively at the end of 24 h, which is an indicator of a strong prebiotic effect.
