Obesity and type 2 diabetes are associated with low-grade inflammation and specific 34 changes in gut microbiota composition [1][2][3][4][5][6][7] . We previously demonstrated that administration 35 of Akkermansia muciniphila prevents the development of obesity and associated 36 complications 8 . However, its mechanisms of action remain unclear, whilst the sensitivity of 37 A. muciniphila to oxygen and the presence of animal-derived compounds in its growth 38 medium currently limit the development of translational approaches for human medicine 9 . 39Here we addressed these issues by showing that A. muciniphila retains its efficacy when Akkermansia muciniphila is one of the most abundant members of the human gut 53 microbiota, representing between 1 and 5% of our intestinal microbes 10,11 to improve glucose intolerance and insulin resistance regardless of the growth medium used and 71 independently of food intake ( Fig. 1a-g). 72 We previously showed that autoclaving A. muciniphila abolished its beneficial effects 8 . (Fig. 1a-c and Supplemental Fig. 1a-c). In both sets of 81 experiments, we found that mice treated with pasteurized A. muciniphila displayed a much lower 82 glucose intolerance and insulin concentration when compared to the HFD group, resulting in a 83 lower insulin resistance (IR) index (Fig. 1d-g and Supplemental Fig. 1d-g). Treatment with 84 pasteurized A. muciniphila also led to greater goblet cell density in the ileum when compared to 85 ND-fed mice (Fig. 1h), suggesting a higher mucus production, while normalizing the mean 86 adipocyte diameter (Fig. 2a-b) and significantly lowering plasma leptin when compared to HFD-87 fed mice (Fig. 2c). These effects were not observed in mice treated with live A. muciniphila. A 88 similar trend could be observed for plasma resistin (Supplemental Fig. 1h), thereby suggesting 89 improved insulin sensitivity, while plasma adiponectin remained unaffected in all conditions 90 (Supplemental Fig. 1i). We found that mice treated with pasteurized A. muciniphila had a higher 91 fecal caloric content when compared to all other groups (Fig. 2d), suggesting a lower energy (Fig. 2e-g). This resulted in a normalization of the HFD-induced shift of 37% with the 104 pasteurized bacterium, and 17% with the live bacterium ( Fig. 2f). 105By comparing the metabolic profiles of the different groups, we found that the shift 106 induced by pasteurized A. muciniphila was mainly associated with trimethylamine (TMA) and TMA to TMAO, a metabolite associated with atherosclerosis 19,20 . While exposure to a HFD led 114 to a two-fold higher Fmo3 expression when compared to ND-fed mice, treatment with 115 pasteurized A. muciniphila reversed this effect (Fig. 2j) Fmo3 expression were not associated with a modification of plasma TMA and TMAO, as all 121 HFD-fed group displayed similar concentrations for both metabolites (Fig. 2k,l) (Fig. 3a), but not cells expressing TLR5, TLR9 or the NOD2 receptor (Fig. 3b-131 d). 132Genomic and proteomic analyses of A. muciniphila identified p...

Akkermansia muciniphila colonizes the mucus layer of the gastrointestinal tract, where the organism can be exposed to the oxygen that diffuses from epithelial cells. To understand how A. muciniphila is able to survive and grow at this oxic-anoxic interface, its oxygen tolerance and response and reduction capacities were studied. A. muciniphila was found to be oxygen tolerant. On top of this, under aerated conditions, A. muciniphila showed significant oxygen reduction capacities and its growth rate and yield were increased compared to those seen under strict anaerobic conditions. Transcriptome analysis revealed an initial oxygen stress response upon exposure to oxygen. Thereafter, genes related to respiration were expressed, including those coding for the cytochrome bd complex, which can function as a terminal oxidase. The functionality of A. muciniphila cytochrome bd genes was proven by successfully complementing cytochrome-deficient Escherichia coli strain ECOM4. We conclude that A. muciniphila can use oxygen when it is present at nanomolar concentrations. IMPORTANCEThis article explains how Akkermansia muciniphila, previously described as a strictly anaerobic bacterium, is able to tolerate and even benefit from low levels of oxygen. Interestingly, we measured growth enhancement of A. muciniphila and changes in metabolism as a result of the oxygen exposure. In this article, we discuss similarities and differences of this oxygen-responsive mechanism with respect to those of other intestinal anaerobic isolates. Taken together, we think that these are valuable data that indicate how anaerobic intestinal colonizing bacteria can exploit low levels of oxygen present in the mucus layer and that our results have direct relevance for applicability, as addition of low oxygen concentrations could benefit the in vitro growth of certain anaerobic organisms.T he gastrointestinal (GI) tract harbors a rich and diverse microbial community, which has proven to play a role in host health and physiology (1). This microbial community is not in direct contact with epithelial cells; a thin layer of host-derived mucus separates them. The outer layer of mucus is colonized with microbes that differ in composition from the luminal microbiota (2, 3). The mucin glycans are used by some bacteria as growth substrates, resulting in the production of short-chain fatty acids (SCFAs) (4). To the host, the SCFAs are important modulators of gut health (4). To the microbial community, SCFAs are a necessary waste product, and the process of SCFA production is required to maintain the redox balance in the cell, as it can restore the NAD ϩ /NADH ratio (5). One member of the mucosa-associated microbiota is Akkermansia muciniphila, a mucin-degrading specialist that can use mucin as a sole carbon and nitrogen source (6). A. muciniphila is associated with a healthy GI tract, as its abundance is inversely correlated with several GI tract-related disorders (7). Moreover, it has been shown that A. muciniphila has immune-stimulatory capacities, stimul...

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections, this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies, permeable membrane based monolayer approaches are needed. In this paper, we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures, which were characterized with respect to epithelial cell types, polarization, barrier function, and gene expression. In addition, viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated, with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research.

The human gut is colonized with a myriad of microbes, with substantial interpersonal variation. This complex ecosystem is an integral part of the gastrointestinal tract and plays a major role in the maintenance of homeostasis. Its dysfunction has been correlated to a wide array of diseases, but the understanding of causal mechanisms is hampered by the limited amount of cultured microbes, poor understanding of phenotypes, and the limited knowledge about interspecies interactions. Genome-scale metabolic models (GEMs) have been used in many different fields, ranging from metabolic engineering to the prediction of interspecies interactions. We provide showcase examples for the application of GEMs for gut microbes and focus on (i) the prediction of minimal, synthetic, or defined media; (ii) the prediction of possible functions and phenotypes; and (iii) the prediction of interspecies interactions. All three applications are key in understanding the role of individual species in the gut ecosystem as well as the role of the microbiota as a whole. Using GEMs in the described fashions has led to designs of minimal growth media, an increased understanding of microbial phenotypes and their influence on the host immune system, and dietary interventions to improve human health. Ultimately, an increased understanding of the gut ecosystem will enable targeted interventions in gut microbial composition to restore homeostasis and appropriate host-microbe crosstalk.

Streptococcus suis is an emerging zoonotic pathogen. Over 100 putative virulence factors have been described, but it is unclear to what extent these virulence factors could contribute to zoonotic potential of S. suis . We identified all S. suis virulence factors studied in experimental models of human origin in a systematic review and assessed their contribution to zoonotic potential in a subsequent genomic meta-analysis. PubMed and Scopus were searched for English-language articles that studied S. suis virulence published until 31 March 2021. Articles that analyzed a virulence factor by knockout mutation, purified protein, and/or recombinant protein in a model of human origin, were included. Data on virulence factor, strain characteristics, used human models and experimental outcomes were extracted. All publicly available S. suis genomes with available metadata on host, disease status and country of origin, were included in a genomic meta-analysis. We calculated the ratio of the prevalence of each virulence factor in human and pig isolates. We included 130 articles and 1703 S. suis genomes in the analysis. We identified 53 putative virulence factors that were encoded by genes which are part of the S. suis core genome and 26 factors that were at least twice as prevalent in human isolates as in pig isolates. Hhly3 and NisK/R were particularly enriched in human isolates, after stratification by genetic lineage and country of isolation. This systematic review and genomic meta-analysis have identified virulence factors that are likely to contribute to the zoonotic potential of S. suis .

SummaryThe abundance of the human intestinal symbiont Akkermansia muciniphila has found to be inversely correlated with several diseases, including metabolic syndrome and obesity. A. muciniphila is known to use mucin as sole carbon and nitrogen source. To study the physiology and the potential for therapeutic applications of this bacterium, we designed a defined minimal medium. The composition of the medium was based on the genome‐scale metabolic model of A. muciniphila and the composition of mucin. Our results indicate that A. muciniphila does not code for GlmS, the enzyme that mediates the conversion of fructose‐6‐phosphate (Fru6P) to glucosamine‐6‐phosphate (GlcN6P), which is essential in peptidoglycan formation. The only annotated enzyme that could mediate this conversion is Amuc‐NagB on locus Amuc_1822. We found that Amuc‐NagB was unable to form GlcN6P from Fru6P at physiological conditions, while it efficiently catalyzed the reverse reaction. To overcome this inability, N‐acetylglucosamine needs to be present in the medium for A. muciniphila growth. With these findings, the genome‐scale metabolic model was updated and used to accurately predict growth of A. muciniphila on synthetic media. The finding that A. muciniphila has a necessity for GlcNAc, which is present in mucin further prompts the adaptation to its mucosal niche.

There is considerable attention for developing Akkermansia muciniphila as a new therapeutic microbe since it has shown to prevent diet-induced obesity and type 2 diabetes in mice. However, A. muciniphila is sensitive to gastric conditions such as low pH and oxygen. Therefore, we explored the possibility of encapsulating A. muciniphila in a water-in-oil-in-water (W/O/W) double emulsion, to allow for protection during gastric passage and subsequent release in the small intestine. The bacteria were efficiently encapsulated in the inner emulsion droplets and remained entrapped during in vitro gastric digestion. The cells were then released in the simulated intestinal phase of the in vitro system. The viability of encapsulated cells was found to be higher when compared to cells dispersed in buffer, that had been subjected to similar mechanical process as the one conducted to prepare the emulsion systems. Surprisingly, the viability of the processed cells was even higher than that of the cells dispersed in buffer without processing, likely due to shear-induced stress tolerance. To conclude, encapsulation in a double emulsion seems to be a promising strategy to protect A. muciniphila during gastric passage in oral formulations.

Streptococcus suis is a porcine pathogen, causing severe invasive infections. S. suis serotype 9 is increasingly causing disease in Dutch and Chinese pig herds, but it is unknown whether all serotype 9 isolates are equally virulent and markers that can identify virulent strains are not available. Therefore, discrimination between virulent isolates and carriage isolates typically not associated with disease, is currently not possible. We collected tonsillar S. suis isolates from 6 herds not previously diagnosed with S. suis infections, and clinical S. suis isolates of previously diseased pigs. We confirmed the virulence of a virulent type strain and one representative clinical isolate, and the lack of virulence of two carriage isolates, in a pig infection model. Phylogenetic analysis of whole genome sequences of 124 isolates resulted in 10 groups, of which two were almost uniquely populated by clinical isolates. The population structure of S. suis serotype 9 appears highly diverse. However, analysis of the capsule loci sequences showed variation in a single region which fully correlated with a virulent genotype. Transmission electron microscopy suggested differences in capsule thickness between carriage and clinical genotypes. In conclusion, we found that that the S. suis serotype 9 population in the Netherlands is diverse. A distinct virulence-associated lineage was identified and could be discriminated based on the capsule locus sequence. Whilst the difference in virulence cannot be directly attributed to the DNA sequence, the correlation of capsule locus sequence with virulence could be used in the development of diagnostic tests to identify potential virulent S. suis serotype 9 in pigs.