Synthetic Communities of Gut Microbes for Basic Research and Translational Approaches in Animal Health and Nutrition.

Microbes and animals have a symbiotic relationship that greatly influences nutrient uptake and animal health. This relationship can be studied using selections of microbes termed synthetic communities, or SynComs. SynComs are used in many different animal hosts, including agricultural animals, to investigate microbial interactions with nutrients and how these affect animal health. The most common host focuses for SynComs are currently mouse and human, from basic mechanistic research through to translational disease models and live biotherapeutic products (LBPs) as treatments. We discuss SynComs used in basic research models and findings that relate to human and animal health and nutrition. Translational use cases of SynComs are discussed, followed by LBPs, especially within the context of agriculture. SynComs still face challenges, such as standardization for reproducibility and contamination risks. However, the future of SynComs is hopeful, especially in the areas of genome-guided SynCom design and custom SynCom-based treatments.

Extension of the Segatella copri complex to 13 species with distinct large extrachromosomal elements and associations with host conditions.

The Segatella copri (formerly Prevotella copri) complex (ScC) comprises taxa that are key members of the human gut microbiome. It was previously described to contain four distinct phylogenetic clades. Combining targeted isolation with large-scale metagenomic analysis, we defined 13 distinct Segatella copri-related species, expanding the ScC complex beyond four clades. Complete genome reconstruction of thirteen strains from seven species unveiled the presence of genetically diverse large circular extrachromosomal elements. These elements are consistently present in most ScC species, contributing to intra- and inter-species diversities. The nine species-level clades present in humans display striking differences in prevalence and intra-species genetic makeup across human populations. Based on a meta-analysis, we found reproducible associations between members of ScC and the male sex and positive correlations with lower visceral fat and favorable markers of cardiometabolic health. Our work uncovers genomic diversity within ScC, facilitating a better characterization of the human microbiome.

A physiologically based model of bile acid metabolism in mice.

Bile acid (BA) metabolism is a complex system that includes a wide variety of primary and secondary, as well as conjugated and unconjugated BAs that undergo continuous enterohepatic circulation (EHC). Alterations in both composition and dynamics of BAs have been associated with various diseases. However, a mechanistic understanding of the relationship between altered BA metabolism and related diseases is lacking. Computational modeling may support functional analyses of the physiological processes involved in the EHC of BAs along the gut-liver axis. In this study, we developed a physiologically based model of murine BA metabolism describing synthesis, hepatic and microbial transformations, systemic distribution, excretion, and EHC of BAs at the whole-body level. For model development, BA metabolism of specific pathogen-free (SPF) mice was characterized in vivo by measuring BA levels and composition in various organs, expression of transporters along the gut, and cecal microbiota composition. We found significantly different BA levels between male and female mice that could only be explained by adjusted expression of the hepatic enzymes and transporters in the model. Of note, this finding was in agreement with experimental observations. The model for SPF mice could also describe equivalent experimental data in germ-free mice by specifically switching off microbial activity in the intestine. The here presented model can therefore facilitate and guide functional analyses of BA metabolism in mice, e.g., the effect of pathophysiological alterations on BA metabolism and translation of results from mouse studies to a clinically relevant context through cross-species extrapolation.

Bacteriophages targeting protective commensals impair resistance against Salmonella Typhimurium infection in gnotobiotic mice.

Gut microbial communities protect the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages) are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, they are an attractive tool for microbiome engineering due to the lack of known serious adverse effects on the host. However, the functional role of phages within the gastrointestinal microbiome remain poorly understood. Here, we investigated the effects of microbiota-directed phages on infection with the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm), using a gnotobiotic mouse model (OMM14) for colonization resistance (CR). We show, that phage cocktails targeting Escherichia coli and Enterococcus faecalis acted in a strain-specific manner. They transiently reduced the population density of their respective target before establishing coexistence for up to 9 days. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails. Surprisingly, OMM14 mice were also susceptible 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Concluding, our work shows that phages that dynamically modulate the density of protective members of the gut microbiota can provide opportunities for invasion of bacterial pathogens, in particular at early time points after phage application. This suggests, that phages targeting protective members of the microbiota may increase the risk for Salmonella infection.

Corrigendum: A MALDI-TOF MS library for rapid identification of human commensal gut bacteria from the class Clostridia.

[This corrects the article DOI: 10.3389/fmicb.2023.1104707.].

M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer's patch.

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ T cell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

Gut-liver axis: barriers and functional circuits.

The gut and the liver are characterized by mutual interactions between both organs, the microbiome, diet and other environmental factors. The sum of these interactions is conceptualized as the gut-liver axis. In this Review we discuss the gut-liver axis, concentrating on the barriers formed by the enterohepatic tissues to restrict gut-derived microorganisms, microbial stimuli and dietary constituents. In addition, we discuss the establishment of barriers in the gut and liver during development and their cooperative function in the adult host. We detail the interplay between microbial and dietary metabolites, the intestinal epithelium, vascular endothelium, the immune system and the various host soluble factors, and how this interplay establishes a homeostatic balance in the healthy gut and liver. Finally, we highlight how this balance is disrupted in diseases of the gut and liver, outline the existing therapeutics and describe the cutting-edge discoveries that could lead to the development of novel treatment approaches.

A MALDI-TOF MS library for rapid identification of human commensal gut bacteria from the class Clostridia.

Introduction: Microbial isolates from culture can be identified using 16S or whole-genome sequencing which generates substantial costs and requires time and expertise. Protein fingerprinting via Matrix-assisted Laser Desorption Ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid bacterial identification in routine diagnostics but shows a poor performance and resolution on commensal bacteria due to currently limited database entries. The aim of this study was to develop a MALDI-TOF MS plugin database (CLOSTRI-TOF) allowing for rapid identification of non-pathogenic human commensal gastrointestinal bacteria. Methods: We constructed a database containing mass spectral profiles (MSP) from 142 bacterial strains representing 47 species and 21 genera within the class Clostridia. Each strain-specific MSP was constructed using >20 raw spectra measured on a microflex Biotyper system (Bruker-Daltonics) from two independent cultures. Results: For validation, we used 58 sequence-confirmed strains and the CLOSTRI-TOF database successfully identified 98 and 93% of the strains, respectively, in two independent laboratories. Next, we applied the database to 326 isolates from stool of healthy Swiss volunteers and identified 264 (82%) of all isolates (compared to 170 (52.1%) with the Bruker-Daltonics library alone), thus classifying 60% of the formerly unknown isolates. Discussion: We describe a new open-source MSP database for fast and accurate identification of the Clostridia class from the human gut microbiota. CLOSTRI-TOF expands the number of species which can be rapidly identified by MALDI-TOF MS.

Early life gut microbiota profiles linked to synbiotic formula effects: a randomized clinical trial in European infants.

BACKGROUND: Microbial colonization of the gastrointestinal tract after birth is an essential event that influences infant health with life-long consequences. Therefore, it is important to investigate strategies to positively modulate colonization in early life. OBJECTIVES: This randomized, controlled intervention study included 540 infants to investigate the effects of a synbiotic intervention formula (IF) containing Limosilactobacillus fermentum CECT5716 and galacto-oligosaccharides on the fecal microbiome. METHODS: The fecal microbiota from infants was analyzed by 16S rRNA amplicon sequencing at 4, 12, and 24 months of age. Metabolites (e.g., short-chain fatty acids) and other milieu parameters (e.g., pH, humidity, and IgA) were also measured in stool samples. RESULTS: Microbiota profiles changed with age, with major differences in diversity and composition. Significant effects of the synbiotic IF compared with control formula (CF) were visible at month 4, including higher occurrence of Bifidobacterium spp. and Lactobacillaceae and lower occurrence of Blautia spp., as well as Ruminoccocus gnavus and relatives. This was accompanied by lower fecal pH and concentrations of butyrate. After de novo clustering at 4 months of age, overall phylogenetic profiles of the infants receiving IF were closer to reference profiles of those fed with human milk than infants fed CF. The changes owing to IF were associated with fecal microbiota states characterized by lower occurrence of Bacteroides compared with higher levels of Firmicutes (valid name Bacillota), Proteobacteria (valid name Pseudomonadota), and Bifidobacterium at 4 months of age. These microbiota states were linked to higher prevalence of infants born by Cesarean section. CONCLUSIONS: The synbiotic intervention influenced fecal microbiota and milieu parameters at an early age depending on the overall microbiota profiles of the infants, sharing a few similarities with breastfed infants. This trial was registered at clinicaltrials.gov as NCT02221687.

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities.

Microbiome research needs comprehensive repositories of cultured bacteria from the intestine of mammalian hosts. We expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes strain-level diversity, small-sized bacteria, and previously undescribed taxa (one family, 10 genera, and 39 species). This collection enabled metagenome-educated prediction of synthetic communities (SYNs) that capture key functional differences between microbiomes, notably identifying communities associated with either resistance or susceptibility to DSS-induced colitis. Additionally, nine species were used to amend the Oligo-Mouse Microbiota (OMM)12 model, yielding the OMM19.1 model. The added strains compensated for phenotype differences between OMM12 and specific pathogen-free mice, including body composition and immune cells in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available for future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

Eggerthella lenta DSM 2243 Alleviates Bile Acid Stress Response in Clostridium ramosum and Anaerostipes caccae by Transformation of Bile Acids.

Bile acids are crucial for the uptake of dietary lipids and can shape the gut-microbiome composition. This latter function is associated with the toxicity of bile acids and can be modulated by bile acid modifying bacteria such as Eggerthella lenta, but the molecular details of the interaction of bacteria depending on bile acid modifications are not well understood. In order to unravel the molecular response to bile acids and their metabolites, we cultivated eight strains from a human intestinal microbiome model alone and in co-culture with Eggerthella lenta in the presence of cholic acid (CA) and deoxycholic acid (DCA). We observed growth inhibition of particularly gram-positive strains such as Clostridium ramosum and the gram-variable Anaerostipes cacae by CA and DCA stress. C. ramosum was alleviated through co-culturing with Eggerthella lenta. We approached effects on the membrane by zeta potential and genotoxic and metabolic effects by (meta)proteomic and metabolomic analyses. Co-culturing with Eggerthella lenta decreased both CA and DCA by the formation of oxidized and epimerized bile acids. Eggerthella lenta also produces microbial bile salt conjugates in a co-cultured species-specific manner. This study highlights how the interaction with other bacteria can influence the functionality of bacteria.

A taxonomic note on the genus Prevotella: Description of four novel genera and emended description of the genera Hallella and Xylanibacter.

The genus Prevotella comprises 55 species with validly published, and correct, names (at June 2021) that are phenotypically, ecologically and functionally diverse. This study used a range of comparative genome approaches (marker gene-based genome phylogeny, core genome phylogeny, average amino acid identity, percentage of conserved proteins and clade-specific marker genes) to identify large differences between the 53 species for which genomes are available, as well as two effectively published yet not validly named species and four novel species. These differences were consistent between the various analysis methods and justify the separation of Prevotella into multiple genera. While the distribution across 19 ecosystem types was unique for each species and inconsistent within clades, the functional repertoire based on the presence/absence of both PFAMs and CAZy families revealed distinct clustering based on the proposed genera. Based on the integration of all results, we propose the reclassification of species previously assigned to the genus Prevotella into seven genera, including four novel genera for which the names Segatella, Hoylesella, Leyella and Palleniella are proposed. In addition to the reclassification of Prevotella, this work describes four novel species, Hallella faecis, Xylanibacter rodentium, Xylanibacter muris, and Palleniella intestinalis.

Microbiome-based interventions to modulate gut ecology and the immune system.

The gut microbiome lies at the intersection between the environment and the host, with the ability to modify host responses to disease-relevant exposures and stimuli. This is evident in how enteric microbes interact with the immune system, e.g., supporting immune maturation in early life, affecting drug efficacy via modulation of immune responses, or influencing development of immune cell populations and their mediators. Many factors modulate gut ecosystem dynamics during daily life and we are just beginning to realise the therapeutic and prophylactic potential of microbiome-based interventions. These approaches vary in application, goal, and mechanisms of action. Some modify the entire community, such as nutritional approaches or faecal microbiota transplantation, while others, such as phage therapy, probiotics, and prebiotics, target specific taxa or strains. In this review, we assessed the experimental evidence for microbiome-based interventions, with a particular focus on their clinical relevance, ecological effects, and modulation of the immune system.

Ring Trial on Quantitative Assessment of Bile Acids Reveals a Method- and Analyte-Specific Accuracy and Reproducibility.

Bile acids are a key mediator of the molecular microbiome-host interaction, and various mass spectrometry-based assays have been developed in the recent decade to quantify a wide range of bile acids. We compare existing methodologies to harmonize them. Methodology for absolute quantification of bile acids from six laboratories in Europe were compared for the quantification of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) and conjugated products glycocholic acid (GCA) and taurocholic acid (TCA). For the bacterially modified secondary bile acids, the quantification of deoxycholic acid (DCA) and lithocholic acid (LCA) was compared. For the murine bile acids, we used the primary muricholic acids (α-MCA and, ß-MCA) and the intestinally produced secondary bile acid muricholic (ω-MCA). The standards were spiked into methanol:water (1:1) mix as well as in human and murine serum at either low concentration range (150-3000 nM) or high concentration range (1500-40,000 nM). The precision was better for higher concentrations. Measurements for the hydrophobic unconjugated bile acids LCA and ω-MCA were the most challenging. The quality assessments were generally very similar, and the comprehensive analyses demonstrated that data from chosen locations can be used for comparisons between studies.

Metadata harmonization-Standards are the key for a better usage of omics data for integrative microbiome analysis.

BACKGROUND: Tremendous amounts of data generated from microbiome research studies during the last decades require not only standards for sampling and preparation of omics data but also clear concepts of how the metadata is prepared to ensure re-use for integrative and interdisciplinary microbiome analysis. RESULTS: In this Commentary, we present our views on the key issues related to the current system for metadata submission in omics research, and propose the development of a global metadata system. Such a system should be easy to use, clearly structured in a hierarchical way, and should be compatible with all existing microbiome data repositories, following common standards for minimal required information and common ontology. Although minimum metadata requirements are essential for microbiome datasets, the immense technological progress requires a flexible system, which will have to be constantly improved and re-thought. While FAIR principles (Findable, Accessible, Interoperable, and Reusable) are already considered, international legal issues on genetic resource and sequence sharing provided by the Convention on Biological Diversity need more awareness and engagement of the scientific community. CONCLUSIONS: The suggested approach for metadata entries would strongly improve retrieving and re-using data as demonstrated in several representative use cases. These integrative analyses, in turn, would further advance the potential of microbiome research for novel scientific discoveries and the development of microbiome-derived products.

Anaerobic single-cell dispensing facilitates the cultivation of human gut bacteria.

Cultivation via classical agar plate (CAP) approaches is widely used to study microbial communities, but they are time-consuming. An alternative approach is the application of single-cell dispensing (SCD), which allows high-throughput, label-free sorting of microscopic particles. We aimed to develop a new anaerobic SCD workflow to cultivate human gut bacteria and compared it with CAP using faecal communities on three rich culture media. We found that the SCD approach significantly decreased the experimental time to obtain pure cultures from 17 ± 4 to 5 ± 0 days, while the isolate diversity and relative abundance coverage were comparable for both approaches. We further tested the total captured fraction by sequencing the sorted bacteria directly after growth as bulk biomass from 2400 dispensed single cells without downstream identification of individual strains. In this approach, the cultured fraction increased from 35.2% to 52.2% for SCD, highlighting the potential for deeper cultivation projects from single samples. SCD-based cultivation also captured species not detected by sequencing (16 ± 5 per sample, including seven novel taxa). From this work, 82 human gut bacterial species across five phyla (Actinobacteriota, Bacteroidota, Desulfobacterota, Firmicutes and Proteobacteria) and 24 families were obtained, including the first cultured member of 11 novel genera and 10 novel species that were fully characterized taxonomically.

Species-targeted sorting and cultivation of commensal bacteria from the gut microbiome using flow cytometry under anaerobic conditions.

BACKGROUND: There is a growing interest in using gut commensal bacteria as "next generation" probiotics. However, this approach is still hampered by the fact that there are few or no strains available for specific species that are difficult to cultivate. Our objective was to adapt flow cytometry and cell sorting to be able to detect, separate, isolate, and cultivate new strains of commensal species from fecal material. We focused on the extremely oxygen sensitive (EOS) species Faecalibacterium prausnitzii and the under-represented, health-associated keystone species Christensenella minuta as proof-of-concept. RESULTS: A BD Influx® cell sorter was equipped with a glovebox that covered the sorting area. This box was flushed with nitrogen to deplete oxygen in the enclosure. Anaerobic conditions were maintained during the whole process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii strains ATCC 27766, ATCC 27768, and DSM 17677. We then generated polyclonal antibodies against target species by immunizing rabbits with heat-inactivated bacteria. Two polyclonal antibodies were directed against F. prausnitzii type strains that belong to different phylogroups, whereas one was directed against C. minuta strain DSM 22607. The specificity of the antibodies was demonstrated by sorting and sequencing the stained bacterial fractions from fecal material. In addition, staining solutions including LIVE/DEAD™ BacLight™ Bacterial Viability staining and polyclonal antibodies did not severely impact bacterial viability while allowing discrimination between groups of strains. Finally, we combined these staining strategies as well as additional criteria based on bacterial shape for C. minuta and were able to detect, isolate, and cultivate new F. prausnitzii and C. minuta strains from healthy volunteer's fecal samples. CONCLUSIONS: Targeted cell-sorting under anaerobic conditions is a promising tool for the study of fecal microbiota. It gives the opportunity to quickly analyze microbial populations, and can be used to sort EOS and/or under-represented strains of interest using specific antibodies, thus opening new avenues for culture experiments. Video abstract.

NLRP6 Inflammasome Modulates Disease Progression in a Chronic-Plus-Binge Mouse Model of Alcoholic Liver Disease.

A considerable percentage of the population is affected by alcoholic liver disease (ALD). It is characterized by inflammatory signals from the liver and other organs, such as the intestine. The NLR family pyrin domain containing 6 (NLRP6) inflammasome complex is one of the most important inflammatory mediators. The aim of this study was to evaluate a novel mouse model for ALD characterized by 8-week chronic-plus-binge ethanol administration and to investigate the role of NLRP6 inflammasome for intestinal homeostasis and ALD progression using Nlrp6-/- mice. We showed that chronic-plus-binge ethanol administration triggers hepatic steatosis, injury, and neutrophil infiltration. Furthermore, we discovered significant changes of intestinal microbial communities, including increased relative abundances of bacteria within the phyla Bacteroidota and Campilobacterota, as well as reduced Firmicutes. In this ALD model, inhibiting NLRP6 signaling had no effect on liver steatosis or damage, but had a minor impact on intestinal homeostasis via affecting intestinal epithelium function and gut microbiota. Surprisingly, Nlrp6 loss resulted in significantly decreased hepatic immune cell infiltration. As a result, our novel mouse model encompasses several aspects of human ALD, such as intestinal dysbiosis. Interfering with NLRP6 inflammasome activity reduced hepatic immune cell recruitment, indicating a disease-aggravating role of NLRP6 during ALD.

Allulose in human diet: the knowns and the unknowns.

D-Allulose, also referred to as psicose, is a C3-epimer of D-fructose used as a sugar substitute in low energy products. It can be formed naturally during processing of food and drinks containing sucrose and fructose or is prepared by chemical synthesis or via enzymatic treatment with epimerases from fructose. Estimated intakes via Western style diets including sweetened beverages are below 500 mg per d but, when used as a sugar replacement, intake may reach 10 to 30 g per d depending on the food consumed. Due to its structural similarity with fructose, allulose uses the same transport and distribution pathways. But in contrast to fructose, the human genome does not encode for enzymes that are able to metabolise allulose leading to an almost complete renal excretion of the absorbed dose and near-to-zero energetic yield. However, in vitro studies have shown that certain bacteria such as Klebsiella pneumonia are able to utilise allulose as a substrate. This finding has been a subject of concern, since Klebsiella pneumoniae represents an opportunistic human pathogen. It therefore raised the question of whether a high dietary intake of allulose may cause an undesirable growth advantage for potentially harmful bacteria at mucosal sites such as the intestine or at systemic sites following invasive infection. In this brief review, we discuss the current state of science on these issues and define the research needs to better understand the fate of allulose and its metabolic and microbiological effects when ingested as a sugar substitute.

Next steps after 15 stimulating years of human gut microbiome research.

Gut microbiome research has bloomed over the past 15 years. We have learnt a lot about the complex microbial communities that colonize our intestine. Promising avenues of research and microbiome-based applications are being implemented, with the goal of sustaining host health and applying personalized disease management strategies. Despite this exciting outlook, many fundamental questions about enteric microbial ecosystems remain to be answered. Organizational measures will also need to be taken to optimize the outcome of discoveries happening at an extremely rapid pace. This article highlights our own view of the field and perspectives for the next 15 years.