The composition and activity of the microbiota in the human gastrointestinal tract are primarily shaped by nutrients derived from either food or the host. Bacteria colonizing the mucus layer have evolved to use mucin as a carbon and energy source. One of the members of the mucosa-associated microbiota is , which is capable of producing an extensive repertoire of mucin-degrading enzymes. To further study the substrate utilization abilities of, we constructed a genome-scale metabolic model to test amino acid auxotrophy, vitamin biosynthesis, and sugar-degrading capacities. The model-supported predictions were validated by experiments, which showed to be able to utilize the mucin-derived monosaccharides fucose, galactose, and -acetylglucosamine. Growth was also observed on-acetylgalactosamine, even though the metabolic model did not predict this. The uptake of these sugars, as well as the nonmucin sugar glucose, was enhanced in the presence of mucin, indicating that additional mucin-derived components are needed for optimal growth. An analysis of whole-transcriptome sequencing (RNA-Seq) comparing the gene expression of grown on mucin with that of the same bacterium grown on glucose confirmed the activity of the genes involved in mucin degradation and revealed most of these to be upregulated in the presence of mucin. The transcriptional response was confirmed by a proteome analysis, altogether revealing a hierarchy in the use of sugars and reflecting the adaptation of to the mucosal environment. In conclusion, these findings provide molecular insights into the lifestyle of and further confirm its role as a mucin specialist in the gut. is among the most abundant mucosal bacteria in humans and in a wide range of other animals. Recently, has attracted considerable attention because of its capacity to protect against diet-induced obesity in mouse models. However, the physiology of has not been studied in detail. Hence, we constructed a genome-scale model and describe its validation by transcriptomic and proteomic approaches on bacterial cells grown on mucus and glucose, a nonmucus sugar. The results provide detailed molecular insight into the mucus-degrading lifestyle of and further confirm the role of this mucin specialist in producing propionate and acetate under conditions of the intestinal tract.

Background: Next generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy. Results: The metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single-or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems. Conclusions: A reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche.

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...

Disruption of the intestinal microbiota occurs frequently in allogeneic hematopoietic cell transplantation (allo-HCT) recipients and predisposes them to development of graft-versus-host disease (GvHD). In a prospective, single-center, single-arm study, we investigated the effect of donor fecal microbiota transplantation (FMT) on symptoms of steroid-refractory or steroid-dependent, acute or late-onset acute intestinal GvHD in 15 individuals who had undergone allo-HCT. Study participants received a fecal suspension from an unrelated healthy donor via nasoduodenal infusion. Donor FMT was well tolerated, and infection-related adverse events did not seem to be related to the FMT procedure. In 10 of 15 study participants, a complete clinical response was observed within 1 month after FMT, without additional interventions to alleviate GvHD symptoms. This response was accompanied by an increase in gut microbial α-diversity, a partial engraftment of donor bacterial species, and increased abundance of butyrate-producing bacteria, including Clostridiales and Blautia species. In 6 of the 10 responding donor FMT recipients, immunosuppressant drug therapy was successfully tapered. Durable remission of steroid-refractory or steroid-dependent GvHD after donor FMT was associated with improved survival at 24 weeks after donor FMT. This study highlights the potential of donor FMT as a treatment for steroid-refractory or steroid-dependent GvHD, but larger clinical trials are needed to confirm the safety and efficacy of this procedure.

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Background Commercial pre-weaning diets are formulated to be highly digestible and nutrient-dense and contain low levels of dietary fibre. In contrast, pigs in a natural setting are manipulating fibre-rich plant material from a young age. Moreover, dietary fibre affects gastrointestinal tract (GIT) development and health in older pigs. We hypothesised that supplemental diets that contain vegetal fibres are accelerating GIT development in suckling piglets in terms of size and functionality. From d 2 of life, sow-suckled piglets had access to a low fibre diet (CON), a diet with a fermentable long-chain arabinoxylan (lc-AXOS), a diet with a largely non-fermentable purified cellulose (CELL), or a diet containing both fibres. During the initial 2 weeks, the control diet was a high-density milk replacer, followed by a dry and highly digestible creep meal. Upon weaning at 25 d, 15 piglets from each treatment group, identified as eaters and originating from six or seven litters, were sacrificed for post-mortem examination of GIT morphology, small intestinal permeability and metabolic profile of the digesta. The microbiota composition of the mid-colon was evaluated in a sub-set of ten piglets. Results No major statistical interactions between the fibre sources were observed. Piglets consumed the fibre-containing milk supplements and creep diets well. Stomach size and small intestinal permeability was not affected. Large intestinal fill was increased with lc-AXOS only, while relative large intestinal weight was increased with both fibre sources (P < 0.050). Also, CELL decreased ileal pH and tended to increase ileal DM content compared to CON (P < 0.050). Moreover, the concentration of volatile fatty acids was increased in the caecum (P < 0.100) and mid-colon (P < 0.050) by addition of CELL. lc-AXOS only stimulated caecal propionate (P < 0.050). The microbiota composition showed a high individual variation and limited dietary impact. Nonetheless, CELL induced minor shifts in specific genera, with notable reductions of Escherichia-Shigella. Conclusions Adding dietary fibres to the supplemental diet of suckling piglets altered large intestinal morphology but not small intestinal permeability. Moreover, dietary fibre showed effects on fermentation and modest changes of microbial populations in the hindgut, with more prominent effects from the low-fermentable cellulose.

Lactobacillus rhamnosus is a diverse Gram-positive species with strains isolated from different ecological niches. Here, we report the genome sequence analysis of 40 diverse strains of L. rhamnosus and their genomic comparison, with a focus on the variable genome. Genomic comparison of 40 L. rhamnosus strains discriminated the conserved genes (core genome) and regions of plasticity involving frequent rearrangements and horizontal transfer (variome). The L. rhamnosus core genome encompasses 2,164 genes, out of 4,711 genes in total (the pan-genome). The accessory genome is dominated by genes encoding carbohydrate transport and metabolism, extracellular polysaccharides (EPS) biosynthesis, bacteriocin production, pili production, the cas system, and the associated clustered regularly interspaced short palindromic repeat (CRISPR) loci, and more than 100 transporter functions and mobile genetic elements like phages, plasmid genes, and transposons. A clade distribution based on amino acid differences between core (shared) proteins matched with the clade distribution obtained from the presence–absence of variable genes. The phylogenetic and variome tree overlap indicated that frequent events of gene acquisition and loss dominated the evolutionary segregation of the strains within this species, which is paralleled by evolutionary diversification of core gene functions. The CRISPR-Cas system could have contributed to this evolutionary segregation. Lactobacillus rhamnosus strains contain the genetic and metabolic machinery with strain-specific gene functions required to adapt to a large range of environments. A remarkable congruency of the evolutionary relatedness of the strains’ core and variome functions, possibly favoring interspecies genetic exchanges, underlines the importance of gene-acquisition and loss within the L. rhamnosus strain diversification.