Mucosa-associated biohydrogenating microbes protect the simulated colon microbiome from stress associated with high concentrations of poly-unsaturated fat.

Polyunsaturated fatty acids (PUFAs) may affect colon microbiome homeostasis by exerting (specific) antimicrobial effects and/or interfering with mucosal biofilm formation at the gut mucosal interface. We used standardized batch incubations and the Mucosal-Simulator of the Human Microbial Intestinal Ecosystem (M-SHIME) to show the in vitro luminal and mucosal effects of the main PUFA in the Western diet, linoleic acid (LA). High concentrations of LA were found to decrease butyrate production and Faecalibacterium prausnitzii numbers dependent on LA biohydrogenation to vaccenic acid (VA) and stearic acid (SA). In faecal batch incubations, LA biohydrogenation and butyrate production were positively correlated and SA did not inhibit butyrate production. In the M-SHIME, addition of a mucosal environment stimulated biohydrogenation to SA and protected F. prausnitzii from inhibition by LA. This was probably due to the preference of two biohydrogenating genera Roseburia and Pseudobutyrivibrio for the mucosal niche. Co-culture batch incubations using Roseburia hominis and F. prausnitzii validated these observations. Correlations networks further uncovered the central role of Roseburia and Pseudobutyrivibrio in protecting luminal and mucosal SHIME microbiota from LA-induced stress. Our results confirm how cross-shielding interactions provide resilience to the microbiome and demonstrate the importance of biohydrogenating, mucosal bacteria for recovery from LA stress.

Arabinoxylans, inulin and Lactobacillus reuteri 1063 repress the adherent-invasive Escherichia coli from mucus in a mucosa-comprising gut model.

The microbiota that colonises the intestinal mucus may particularly affect human health given its proximity to the epithelium. For instance, the presence of the adherent-invasive Escherichia coli (AIEC) in this mucosal microbiota has been correlated with Crohn's disease. Using short-term screening assays and a novel long-term dynamic gut model, which comprises a simulated mucosal environment (M-SHIME), we investigated how (potential) pro- and prebiotics may repress colonisation of AIEC from mucus. Despite that during the short-term screening assays, some of the investigated Lactobacillus strains adhered strongly to mucins, none of them competed with AIEC for mucin-adhesion. In contrast, AIEC survival and growth during co-culture batch incubations was decreased by Lactobacillus rhamnosus GG and L. reuteri 1063, which correlated with (undissociated) lactic acid and reuterin levels. Regarding the prebiotics, long-chain arabinoxylans (LC-AX) lowered the initial mucin-adhesion of AIEC, while both inulin (IN) and galacto-oligosaccharides (GOS) limited AIEC survival and growth during batch incubations. L. reuteri 1063, LC-AX and IN were thus retained for a long-term study with the M-SHIME. All treatments repressed AIEC from mucus without affecting AIEC numbers in the luminal content. As a possible explanation, L. reuteri 1063 treatment increased lactobacilli levels in mucus, while LC-AX and IN additionally increased mucosal bifidobacteria levels, thus leading to antimicrobial effects against AIEC in mucus. Overall, this study shows that pro- and prebiotics can beneficially modulate the in vitro mucosal microbiota, thus limiting occurrence of opportunistic pathogens among those mucosal microbes which may directly interact with the host given their proximity to the epithelium.

Micromanagement in the gut: microenvironmental factors govern colon mucosal biofilm structure and functionality.

The human gut microbiome provides us with functional features that we did not have to evolve ourselves and can be viewed as a structured microbial community that operates like a microbial organ within the human host. A minor but important part of this microbiome is the ability to colonise and thrive within the mucous layer that covers the colon epithelium. These mucosal microbes intimately interact with the intestinal tissue and seem to be important modulators of human health. Embedded in the host-secreted mucous matrix, they form a 'mucosal biofilm' with a distinct composition and functionality. In this review, we provide evidence that six specific (micro)environmental factors near the colon mucosa shape and determine mucosal biofilm formation and stability, that is, (1) mucous rigidity, (2) gradients of fluid shear, (3) radial oxygen gradients, (4) secretions of host defense molecules, (5) the presence of a rich but challenging nutrient platform and (6) the presence of niches at the colon epithelial surface. In addition, it appears that microbes actively participate in shaping their mucosal environment. Current insights into the interaction between mucosal microbes and their environment are rather limited, and many questions regarding the contribution of mucosal biofilm functionality and stability to human health remain to be answered. Yet, given the higher potency of mucosal microbes than their luminal counterparts to interact with the host, new insights can accelerate the development of novel disease-preventive or therapeutic strategies.

Biofilm models for the food industry: hot spots for plasmid transfer?

Biofilms represent a substantial problem in the food industry, with food spoilage, equipment failure, and public health aspects to consider. Besides, biofilms may be a hot spot for plasmid transfer, by which antibiotic resistance can be disseminated to potential foodborne pathogens. This study investigated biomass and plasmid transfer in dual-species (Pseudomonas putida and Escherichia coli) biofilm models relevant to the food industry. Two different configurations (flow-through and drip-flow) and two different inoculation procedures (donor-recipient and recipient-donor) were tested. The drip-flow configuration integrated stainless steel coupons in the setup while the flow-through configuration included a glass flow cell and silicone tubing. The highest biomass density [10 log (cells cm-²)] was obtained in the silicone tubing when first the recipient strain was inoculated. High plasmid transfer ratios, up to 1/10 (transconjugants/total bacteria), were found. Depending on the order of inoculation, a difference in transfer efficiency between the biofilm models could be found. The ease by which the multiresistance plasmid was transferred highlights the importance of biofilms in the food industry as hot spots for the acquisition of multiresistance plasmids. This can impede the treatment of foodborne illnesses if pathogens acquire this multiresistance in or from the biofilm.

Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model.

The human gut is colonized by a complex microbiota with multiple benefits. Although the surface-attached, mucosal microbiota has a unique composition and potential to influence human health, it remains difficult to study in vivo. Therefore, we performed an in-depth microbial characterization (human intestinal tract chip (HITChip)) of a recently developed dynamic in vitro gut model, which simulates both luminal and mucosal gut microbes (mucosal-simulator of human intestinal microbial ecosystem (M-SHIME)). Inter-individual differences among human subjects were confirmed and microbial patterns unique for each individual were preserved in vitro. Furthermore, in correspondence with in vivo studies, Bacteroidetes and Proteobacteria were enriched in the luminal content while Firmicutes rather colonized the mucin layer, with Clostridium cluster XIVa accounting for almost 60% of the mucin-adhered microbiota. Of the many acetate and/or lactate-converting butyrate producers within this cluster, Roseburia intestinalis and Eubacterium rectale most specifically colonized mucins. These 16S rRNA gene-based results were confirmed at a functional level as butyryl-CoA:acetate-CoA transferase gene sequences belonged to different species in the luminal as opposed to the mucin-adhered microbiota, with Roseburia species governing the mucosal butyrate production. Correspondingly, the simulated mucosal environment induced a shift from acetate towards butyrate. As not only inter-individual differences were preserved but also because compared with conventional models, washout of relevant mucin-adhered microbes was avoided, simulating the mucosal gut microbiota represents a breakthrough in modeling and mechanistically studying the human intestinal microbiome in health and disease. Finally, as mucosal butyrate producers produce butyrate close to the epithelium, they may enhance butyrate bioavailability, which could be useful in treating diseases, such as inflammatory bowel disease.

Glycerol supplementation enhances L. reuteri's protective effect against S. Typhimurium colonization in a 3-D model of colonic epithelium.

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.

Human faecal microbiota display variable patterns of glycerol metabolism.

Significant amounts of glycerol reach the colon microbiota daily through the diet and/or by in situ microbial production or release from desquamated epithelial cells. Some gut microorganisms may anaerobically reduce glycerol to 1,3-propanediol (1,3-PDO), with 3-hydroxypropanal as an intermediate. Accumulation of the latter intermediate may result in the formation of reuterin, which is known for its biological activity (e.g. antimicrobial properties). To date, glycerol metabolism in mixed cultures from the human colon has received little attention. Using in vitro batch incubations of faeces from 10 human individuals, we demonstrated that glycerol addition (140 mM) significantly affects the metabolism and composition of the microbial community. About a third of the samples exhibited rapid glycerol conversion, yielding proportionally higher levels of acetate and 1,3-PDO. In contrast, a slower glycerol metabolism resulted in higher levels of propionate. Furthermore, rapid glycerol metabolism correlated with significant shifts in the Lactobacillus-Enterococcus community, which were not observed in slower glycerol-metabolizing samples. As the conversion of glycerol to 1,3-PDO is a highly reducing process, we infer that the glycerol metabolism may act as an effective hydrogen sink. Given the importance of hydrogen-consuming processes in the gut, this work suggests that glycerol may have potential as a tool for modulating fermentation kinetics and profiles in the gastrointestinal tract.