Objectives: Dysbiosis of the intestinal microbiota is associated with Crohn's disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNF ^deltaARE mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement. Design: Heterozygous TNF ^deltaARE mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis. Results: GF-TNF ^deltaARE mice were free of inflammation in the gut and antibiotic treatment of CONV-TNF ^deltaARE mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNF ^deltaARE mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNF ^deltaARE recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNF ^deltaARE mice with the human CD-related Escherichia coli LF82 did not induce ileitis. Conclusions: We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.

Breastfeeding provides the ideal nutrition for infants. Human milk contains a plethora of functional ingredients which foster the development of the immune system. The human milk microbiota predominantly contributes to this protective effect. This is mediated by various mechanisms, such as an antimicrobial effect, pathogen exclusion and barrier integrity, beneficial effects on the gastrointestinal microbiota, vitamin synthesis, immunity enhancement, secreted probiotic factors, and postbiotic mechanisms. Therefore, human milk is a good source for isolating probiotics for infants who cannot be exclusively breastfed. One such probiotic which was isolated from human milk is Limosilactobacillus fermentum CECT5716. In this review, we give an overview of available interventional studies using Limosilactobacillus fermentum CECT5716 and summarise preclinical trials in several animal models of different pathologies, which have given first insights into its mechanisms of action. We present several randomised clinical studies, which have been conducted to investigate the clinical efficacy of the Limosilactobacillus fermentum CECT5716 strain in supporting the host’s health.

Antibiotic (ABx) therapy is associated with increased risk for Crohn's disease but underlying mechanisms are unknown. We observed high fecal serine protease activity (PA) to be a frequent side effect of ABx therapy. The aim of the present study was to unravel whether this rise in large intestinal PA may promote colitis development via detrimental effects on the large intestinal barrier. Transwell experiments were used to assess the impact of high PA in ABx-treated patients or vancomycin/metronidazole-treated mice on the epithelial barrier. Serine protease profiling was performed using liquid chromatography-mass spectrometry/mass spectrometry analysis. The impact of high large intestinal PA on the intestinal barrier in wild-type and interleukin (IL)10^-/- mice and on colitis development in IL10^-/- mice was investigated using vancomycin/metronidazole with or without oral serine protease inhibitor (AEBSF) treatment. The ABx-induced, high large intestinal PA was caused by significantly increased levels of pancreatic proteases and impaired epithelial barrier integrity. In wild-type mice, the rise in PA caused a transient increase in intestinal permeability but did not affect susceptibility to chemically induced acute colitis. In IL10^-/- mice, increased PA caused a consistent impairment of the intestinal barrier associated with inflammatory activation in the large intestinal tissue. In the long term, the vancomycin/metronidazole-induced lasting increase in PA aggravated colitis development in IL10^-/- mice. High large intestinal PA is a frequent adverse effect of ABx therapy, which is detrimental to the large intestinal barrier and may contribute to the development of chronic intestinal inflammation in susceptible individuals.

Diet is an emerging but poorly defined disease modulator in inflammatory bowel diseases (IBDs). Dietary factors exert direct effects on epithelial and immune cells and indirectly modulate immune homeostasis by shaping the intestinal microbiota. The increase in IBD prevalence in industrialized countries is associated with lifestyle changes including diets rich in energy, saturated fats, meat and sugar. Despite the fact that the intestinal ecosystem shows high stability and resilience to short-term perturbations, long-term dietary habits have profound effects on composition and function eventually leading to dysbiosis, that is changes in microbial composition associated with deleterious effects to the host. High-throughput sequencing data generated deeper insights of the intestinal ecosystems related to health and disease. However, the available cohort-studies establish associative relationships between microbiota changes and disease, rather than causality. New mouse models of intestinal inflammation and the possibility to transfer disease-associated microbial consortia state an essential tool to unravel the potential of diet-induced microbial shifts. This review will discuss new insights of how nutrition or single dietary factors shape the intestinal ecosystem. Furthermore, we want to provide perspectives for clinical translation of this knowledge to treat or prevent IBD.

Human milk is the best nutrition for infants, providing optimal support for the developing immune system and gut microbiota. Hence, it has been used as source for probiotic strain isolation, including members of the genus Bifidobacterium, in an effort to provide beneficial effects to infants who cannot be exclusively breastfed. However, not all supplemented bifidobacteria can effectively colonise the infant gut, nor confer health benefits to the individual infant host; therefore, new isolates are needed to develop a range of dietary products for this specific age group. Here, we investigated the beneficial potential of Bifidobacterium breve DSM 32583 isolated from human milk. We show that in vitro B. breve DSM 32583 exhibited several characteristics considered fundamental for beneficial bacteria, including survival in conditions simulating those present in the digestive tract, adherence to human epithelial cell lines, and inhibition of growth of potentially pathogenic microorganisms. Its antibiotic resistance patterns were comparable to those of known beneficial bifidobacterial strains, and its genome did not contain plasmids nor virulence-associated genes. These results suggest that B. breve DSM 32583 is a potential probiotic candidate. Graphical Abstract

Aim: Microbial colonization of the neonatal gut is pivotal in priming the infant’s immune system. Human milk (HM) is the best nutrition for infants and supports the development of the microbiota due to prebiotic compounds and probiotic microorganisms. When exclusive breastfeeding is not possible, infant formula (IF) with probiotics is a strategy to support the infant’s microbiome development. However, knowledge about the effects of the infant gut microbiota and different compounds in IF on individual probiotic strains is limited, as strain-level detection in a complex ecosystem is challenging. The aim of the present study was to show the effects of IF with different protein forms on the metabolic activity of two probiotic strains isolated from HM in a complex ecosystem. Methods: By using an ex-vivo infant gut model containing infant donor-microbiota, the effects of IF with either intact or extensively hydrolyzed protein on the metabolic activity of the donor microbiota, as well as two probiotic strains [Limosilactobacillus fermentum (L. fermentum ) CECT 5716 (Lf) and Bifidobacterium breve (B. breve ) DSM 32583 (Bb)], were analyzed. A new bioinformatic pipeline combined with a specific infant microbiome database was used to explore shotgun metagenome datasets (1200 Megabases) for taxonomic identification and strain-level tracking. Results: Both protein forms (i.e., intact or extensively hydrolyzed protein) in IF supported infant gut microbial metabolic activity equally, as evidenced by similar levels of short-chain fatty acids (SCFAs). Interestingly, gut microbial metabolic activity was found to be differently activated in a strain-dependent manner. Taxonomic profiling of the microbiome at the strain level enabled monitoring of the prevalence and abundance of both probiotic strains, even in a complex ecosystem. Conclusion: Food matrix and host microbiota interactions should be considered when evaluating strain-specific probiotic effects in the future.