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Despite recent advances in sequencing and culturing, a deep knowledge of the wiring and functioning of the human gut ecosystem and its microbiota as a community is still missing. A holistic mechanistic understanding will require study of the gut microbiota as an interactive and spatially organized biological system, which is difficult to do in complex natural communities. Synthetic gut microbial ecosystems can function as model systems to further current understanding of the composition, stability and functional activities of the microbiota. In this Review, we provide an overview of the current synthetic ecology strategies that can be used towards a more comprehensive understanding of the human gut ecosystem. Such approaches that integrate in vitro experiments using cultured isolates with mathematical modelling will enable the ultimate goal: translating mechanistic and ecological knowledge into novel and effective therapies.

Interest in gut microbiome dysbiosis and its potential association with the development and progression of chronic kidney disease (CKD) has increased substantially in the past 6 years. In parallel, the microbiome field has matured considerably as the importance of host-related and environmental factors is increasingly recognized. Past research output in the context of CKD insufficiently considered the myriad confounding factors that are characteristic of the disease. Gut microbiota-derived metabolites remain an interesting therapeutic target to decrease uraemic (cardio)toxicity. However, future studies on the effect of dietary and biotic interventions will require harmonization of relevant readouts to enable an in-depth understanding of the underlying beneficial mechanisms. High-quality standards throughout the entire microbiome analysis workflow are also of utmost importance to obtain reliable and reproducible results. Importantly, investigating the relative composition and abundance of gut bacteria, and their potential association with plasma uraemic toxins levels is not sufficient. As in other fields, the time has come to move towards in-depth quantitative and functional exploration of the patient’s gut microbiome by relying on confounder-controlled quantitative microbial profiling, shotgun metagenomics and in vitro simulations of microorganism–microorganism and host–microorganism interactions. This step is crucial to enable the rational selection and monitoring of dietary and biotic intervention strategies that can be deployed as a personalized intervention in CKD.Gut microbiome studies have potential to provide novel therapeutic targets in chronic kidney disease. Here, the authors not only examine the current state of the field and discuss potential gut-related therapies for targeting uraemic metabolites, but also provide guidelines for improving microbiome study design, and data collection and analysis.

BackgroundThe gut microbiota has been linked to non-communicable diseases, including chronic kidney disease (CKD). However, the relationships between gut microbiome composition changes, uraemic toxins (UTs) accumulation, and diet on CKD severity and progression remain underexplored.ObjectiveTo characterise relationships between gut microbiome composition and functionality, UTs diet, and CKD severity and progression, as well as assess microbial contributions to UTs accumulation through mice faecal microbiota transplantation (FMT).DesignThis study profiled the gut microbiome of 240 non-dialysis patients with CKD (CKD-REIN cohort) using shotgun metagenomics, with follow-up in 103 patients after 3 years, with comparisons with healthy volunteers from the Milieu Intérieur cohort. A multiomics approach identifies features associated with CKD severity (and progression), with validation in an independent Belgian cohort. Experimental models used FMT to test CKD gut microbiome effects on UTs and kidney fibrosis. Changes in gut microbiome over time were evaluated, and the impact of diet on these changes was assessed.ResultsCompared with matched healthy controls, patients with CKD exhibited gut microbiota alteration, with enrichment of UT precursor-producing species. Patients with severe CKD exhibited higher UT levels and greater enrichment of UT (precursor)-producing species in the microbiota than patients with moderate CKD. Over time, UT (precursor)-producing species increased, and a plant-based low protein diet appeared to mitigate these changes. FMT from patients with CKD to antibiotic-treated CKD model mice increased serum UT levels and exacerbated kidney fibrosis.ConclusionsThis study highlights the role of the microbiome and UTs in CKD, suggesting a potential therapeutic target to slow disease progression.

Background Novel strategies for anaerobic bacterial isolations from human faecal samples and various initiatives to generate culture collections of gut-derived bacteria have instigated considerable interest for the development of novel microbiota-based treatments. Early in the process of building a culture collection, optimal faecal sample preservation is essential to safeguard the viability of the broadest taxonomic diversity range possible. In contrast to the much more established faecal storage conditions for meta-omics applications, the impact of stool sample preservation conditions on bacterial growth recovery and isolation remains largely unexplored. In this study, aliquoted faecal samples from eleven healthy human volunteers selected based on a range of physicochemical and microbiological gradients were cryopreserved at – 80 °C either without the addition of any medium (dry condition) or in different Cary-Blair medium conditions with or without a cryoprotectant, i.e. 20% (v/v) glycerol or 5% (v/v) DMSO. Faecal aliquots were subjected to bulk 16S rRNA gene sequencing as well as dilution plating on modified Gifu Anaerobic Medium after preservation for culturable fraction profiling and generation of bacterial culture collections. Results Analyses of compositional variation showed that cryopreservation medium conditions affected quantitative recovery but not the overall community composition of cultured fractions. Post-preservation sample dilution and richness of the uncultured source samples were the major drivers of the cultured fraction richness at genus level. However, preservation conditions differentially affected recovery of specific genera. Presence-absence analysis indicated that twenty-two of the 45 most abundant common genera (>0.01% abundance, dilution 10 −4 ) were recovered in cultured fractions from all preservation conditions, while nine genera were only detected in fractions from a single preservation condition. Overall, the highest number of common genera (i.e. 35/45) in cultured fractions were recovered from sample aliquots preserved without medium and in the presence of Cary-Blair medium containing 5% (v/v) DMSO. Also, in the culture collection generated from the cultured fractions, these two preservation conditions yielded the highest species richness (72 and 66, respectively). Conclusion Our results demonstrate that preservation methods partly determine richness and taxonomic diversity of gut anaerobes recovered from faecal samples. Complementing the current standard practice of cryopreserving stool samples in dry conditions with other preservation conditions, such as Cary-Blair medium with DMSO, could increase the species diversity of gut-associated culture collections. 5AZP4afEVR163YDfjLx79n Video abstract

In chronic kidney disease (CKD), impaired kidney function results in accumulation of uremic toxins, which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality. Protein-bound uremic toxins (PBUTs), such as p-cresyl sulfate, indoxyl sulfate and indole-3-acetic acid, originate from phenolic and indolic compounds, which are end products of gut bacterial metabolization of aromatic amino acids (AAA). This study investigates gut microbial composition at different CKD stages by isolating, identifying and quantifying PBUT precursor-generating bacteria. Fecal DNA extracts from 14 controls and 138 CKD patients were used to quantify total bacterial number and 11 bacterial taxa with qPCR. Moreover, isolated bacteria from CKD 1 and CKD 5 fecal samples were cultured in broth medium supplemented with AAA under aerobic and anaerobic conditions, and classified as PBUT precursor-generators based on their generation capacity of phenolic and indolic compounds, measured with U(H)PLC. In total, 148 different fecal bacterial species were isolated, of which 92 were PBUT precursor-generators. These bacterial species can be a potential target for reducing PBUT plasma levels in CKD. qPCR indicated lower abundance of short chain fatty acid-generating bacteria, Bifidobacterium spp. and Streptococcus spp., and higher Enterobacteriaceae and E. coli with impaired kidney function, confirming an altered gut microbial composition in CKD.

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

A bottleneck for microbial community experiments with many samples and/or replicates is the fast quantification of individual taxon abundances, which is commonly achieved through sequencing marker genes such as the 16S rRNA gene. Here, we propose a new approach for high-throughput and high-quality enumeration of human gut bacteria in a defined community, combining flow cytometry and supervised classification to identify and quantify species mixed in silico and in defined communities in vitro. We identified species in a 5-species in silico community with an F1 score of 71%. In addition, we demonstrate in vitro that our method performs equally well or better than 16S rRNA gene sequencing in two-species cocultures and agrees with 16S rRNA gene sequencing data on the most abundant species in a four-species community. We found that shape and size differences alone are insufficient to distinguish species, and that it is thus necessary to exploit the multivariate nature of flow cytometry data. Finally, we observed that variability of flow cytometry data across replicates differs between gut bacterial species. In conclusion, the performance of supervised classification of gut species in flow cytometry data is species-dependent, but is for some combinations accurate enough to serve as a faster alternative to 16S rRNA gene sequencing.

Acetate-producing Saccharomyces cerevisiae var. boulardii strains could exert improved effects on ulcerative colitis, which here, was preclinically evaluated in an acute dextran sodium sulphate induced model of colitis. Nine-week-old female mice were divided into 12 groups, receiving either drinking water or 2.75% dextran sodium sulphate for 7 days, combined with a daily gavage of various treatments with different levels of acetate accumulation: sham control (phosphate buffered saline, no acetate), non-probiotic control (Baker’s yeast, no acetate), probiotic control (Enterol®, transient acetate), and additionally several Saccharomyces cerevisiae var. boulardii strains with respectively no, high, and extra-high acetate accumulation. Disease activity was monitored daily, and feces samples were collected at different timepoints. On day 14, the mice were sacrificed, upon which blood and colonic tissue were collected for analysis. Disease activity in inflamed mice was lower when treated with the high-acetate-producing strain compared to sham and non-probiotic controls. The non-acetate-producing strain showed higher disease activity compared to the acetate-producing strains. Accordingly, higher histologic inflammation was observed in non- or transient-acetate-producing strains compared to the sham control, whereas this increase was not observed for high- and extra-high-acetate-producing strains upon induction of inflammation. These anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes. Moreover, only the strain with the highest acetate production was superior in maintaining a stable gut microbial alpha-diversity upon inflammation. These findings support new possibilities for acetate-mediated management of inflammation in inflammatory bowel disease by administrating high-acetate-producing Saccharomyces cerevisae var. boulardii strains.

Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins which exert deleterious effects on various organ systems. Several of these uremic toxins originate from the bacterial metabolization of aromatic amino acids in the colon. This study assessed whether the gut microbial composition varies among patients in different stages of CKD. Uremic metabolites were quantified by UPLC/fluorescence detection and microbial profiling by 16S rRNA amplicon sequencing. Gut microbial profiles of CKD patients were compared among stages 1–2, stage 3 and stages 4–5. Although a substantial inter-individual difference in abundance of the top 15 genera was observed, no significant difference was observed between groups. Bristol stool scale (BSS) correlated negatively with p-cresyl sulfate and hippuric acid levels, irrespective of the intake of laxatives. Butyricicoccus, a genus with butyrate-generating properties, was decreased in abundance in advanced stages of CKD compared to the earlier stages (p = 0.043). In conclusion, in this cross-sectional study no gradual differences in the gut microbial profile over the different stages of CKD were observed. However, the decrease in the abundance of Butyricicoccus genus with loss of kidney function stresses the need for more in-depth functional exploration of the gut microbiome in CKD patients not on dialysis.