Microbiome confounders and quantitative profiling challenge predicted microbial targets in colorectal cancer development.

Despite substantial progress in cancer microbiome research, recognized confounders and advances in absolute microbiome quantification remain underused; this raises concerns regarding potential spurious associations. Here we study the fecal microbiota of 589 patients at different colorectal cancer (CRC) stages and compare observations with up to 15 published studies (4,439 patients and controls total). Using quantitative microbiome profiling based on 16S ribosomal RNA amplicon sequencing, combined with rigorous confounder control, we identified transit time, fecal calprotectin (intestinal inflammation) and body mass index as primary microbial covariates, superseding variance explained by CRC diagnostic groups. Well-established microbiome CRC targets, such as Fusobacterium nucleatum, did not significantly associate with CRC diagnostic groups (healthy, adenoma and carcinoma) when controlling for these covariates. In contrast, the associations of Anaerococcus vaginalis, Dialister pneumosintes, Parvimonas micra, Peptostreptococcus anaerobius, Porphyromonas asaccharolytica and Prevotella intermedia remained robust, highlighting their future target potential. Finally, control individuals (age 22-80 years, mean 57.7 years, standard deviation 11.3) meeting criteria for colonoscopy (for example, through a positive fecal immunochemical test) but without colonic lesions are enriched for the dysbiotic Bacteroides2 enterotype, emphasizing uncertainties in defining healthy controls in cancer microbiome research. Together, these results indicate the importance of quantitative microbiome profiling and covariate control for biomarker identification in CRC microbiome studies.

Associations of HIV and iron status with gut microbiota composition, gut inflammation and gut integrity in South African school-age children: a two-way factorial case-control study.

BACKGROUND: Human immunodeficiency virus (HIV) and iron deficiency (ID) affect many African children. Both HIV and iron status interact with gut microbiota composition and related biomarkers. The study's aim was to determine the associations of HIV and iron status with gut microbiota composition, gut inflammation and gut integrity in South African school-age children. METHODS: In this two-way factorial case-control study, 8- to 13-year-old children were enrolled into four groups based on their HIV and iron status: (1) With HIV (HIV+) and ID (n = 43), (2) HIV+ and iron-sufficient nonanaemic (n = 41), (3) without HIV (HIV-) and ID (n = 44) and (4) HIV- and iron-sufficient nonanaemic (n = 38). HIV+ children were virally suppressed (<50 HIV RNA copies/ml) on antiretroviral therapy (ART). Microbial composition of faecal samples (16S rRNA sequencing) and markers of gut inflammation (faecal calprotectin) and gut integrity (plasma intestinal fatty acid-binding protein [I-FABP]) were assessed. RESULTS: Faecal calprotectin was higher in ID versus iron-sufficient nonanaemic children (p = 0.007). I-FABP did not significantly differ by HIV or iron status. ART-treated HIV (redundancy analysis [RDA] R2 = 0.009, p = 0.029) and age (RDA R2 = 0.013 p = 0.004) explained the variance in the gut microbiota across the four groups. Probabilistic models showed that the relative abundance of the butyrate-producing genera Anaerostipes and Anaerotruncus was lower in ID versus iron-sufficient children. Fusicatenibacter was lower in HIV+ and in ID children versus their respective counterparts. The prevalence of the inflammation-associated genus Megamonas was 42% higher in children with both HIV and ID versus HIV- and iron-sufficient nonanaemic counterparts. CONCLUSIONS: In our sample of 8- to 13-year-old virally suppressed HIV+ and HIV- children with or without ID, ID was associated with increased gut inflammation and changes in the relative abundance of specific microbiota. Moreover, in HIV+ children, ID had a cumulative effect that further shifted the gut microbiota to an unfavourable composition.

Untargeted Fecal Metabolomic Analyses across an Industrialization Gradient Reveal Shared Metabolites and Impact of Industrialization on Fecal Microbiome-Metabolome Interactions.

The metabolome is a central determinant of human phenotypes and includes the plethora of small molecules produced by host and microbiome or taken up from exogenous sources. However, studies of the metabolome have so far focused predominantly on urban, industrialized populations. Through an untargeted metabolomic analysis of 90 fecal samples from human individuals from Africa and the Americas-the birthplace and the last continental expansion of our species, respectively-we characterized a shared human fecal metabolome. The majority of detected metabolite features were ubiquitous across populations, despite any geographic, dietary, or behavioral differences. Such shared metabolite features included hyocholic acid and cholesterol. However, any characterization of the shared human fecal metabolome is insufficient without exploring the influence of industrialization. Here, we show chemical differences along an industrialization gradient, where the degree of industrialization correlates with metabolomic changes. We identified differential metabolite features such as amino acid-conjugated bile acids and urobilin as major metabolic correlates of these behavioral shifts. Additionally, coanalyses with over 5,000 publicly available human fecal samples and cooccurrence probability analyses with the gut microbiome highlight connections between the human fecal metabolome and gut microbiome. Our results indicate that industrialization significantly influences the human fecal metabolome, but diverse human lifestyles and behavior still maintain a shared human fecal metabolome. This study represents the first characterization of the shared human fecal metabolome through untargeted analyses of populations along an industrialization gradient. IMPORTANCE As the world becomes increasingly industrialized, understanding the biological consequences of these lifestyle shifts and what it means for past, present, and future human health is critical. Indeed, industrialization is associated with rises in allergic and autoimmune health conditions and reduced microbial diversity. Exploring these health effects on a chemical level requires consideration of human lifestyle diversity, but understanding the significance of any differences also requires knowledge of what molecular components are shared between human groups. Our study reveals the key chemistry of the human gut as defined by varied industrialization-based differences and ubiquitous shared features. Ultimately, these novel findings extend our knowledge of human molecular biology, especially as it is influenced by lifestyle and behavior, and provide steps toward understanding how human biology has changed over our species' history.

Effect of cryopreservation medium conditions on growth and isolation of gut anaerobes from human faecal samples.

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. Video abstract.

The effect of oral iron supplementation on the gut microbiota, gut inflammation, and iron status in iron-depleted South African school-age children with virally suppressed HIV and without HIV.

PURPOSE: Both HIV and oral iron interventions may alter gut microbiota composition and increase gut inflammation. We determined the effect of oral iron supplementation on gut microbiota composition, gut inflammation, and iron status in iron-depleted South Africa school-aged children living with HIV (HIV+) but virally suppressed on antiretroviral therapy and children without HIV (HIV-ve). METHODS: In this before-after intervention study with case-control comparisons, we provided 55 mg elemental iron from ferrous sulphate, once daily for 3 months, to 33 virally suppressed (< 50 HIV RNA copies/mL) HIV+ and 31 HIV-ve children. At baseline and endpoint, we assessed microbial composition of faecal samples (16S rRNA sequencing), and markers of gut inflammation (faecal calprotectin), anaemia (haemoglobin) and iron status (plasma ferritin, soluble transferrin receptor). This study was nested within a larger trial registered at clinicaltrials.gov as NCT03572010. RESULTS: HIV+ (11.3y SD ± 1.8, 46% male) and HIV-ve (11.1y SD ± 1.7, 52% male) groups did not significantly differ in age or sex ratio. Following iron supplementation, improvements were observed in haemoglobin (HIV+ : 118 to 124 g/L, P = 0.003; HIV-ve: 120 to 124 g/L, P = 0.003), plasma ferritin (HIV+ : 15 to 34 µg/L, P < 0.001; HIV-ve: 18 to 37 µg/L, P < 0.001), and soluble transferrin receptor (HIV+ : 7.1 to 5.9 mg/L, P < 0.001; HIV-ve: 6.6 to 5.7 mg/L, P < 0.001), with no significant change in the relative abundance of any genera, alpha diversity of the gut microbiota (HIV+ : P = 0.37; HIV-ve: P = 0.77), or faecal calprotectin (HIV+ : P = 0.42; HIV-ve: P = 0.80). CONCLUSION: Our findings suggest that oral iron supplementation can significantly improve haemoglobin and iron status without increasing pathogenic gut microbial taxa or gut inflammation in iron-depleted virally suppressed HIV+ and HIV-ve school-age children.

Variation and transmission of the human gut microbiota across multiple familial generations.

Although the composition and functional potential of the human gut microbiota evolve over the lifespan, kinship has been identified as a key covariate of microbial community diversification. However, to date, sharing of microbiota features within families has mostly been assessed between parents and their direct offspring. Here we investigate the potential transmission and persistence of familial microbiome patterns and microbial genotypes in a family cohort (n = 102) spanning 3 to 5 generations over the same female bloodline. We observe microbiome community composition associated with kinship, with seven low abundant genera displaying familial distribution patterns. While kinship and current cohabitation emerge as closely entangled variables, our explorative analyses of microbial genotype distribution and transmission estimates point at the latter as a key covariate of strain dissemination. Highest potential transmission rates are estimated between sisters and mother-daughter pairs, decreasing with increasing daughter's age and being higher among cohabiting pairs than those living apart. Although rare, we detect potential transmission events spanning three and four generations, primarily involving species of the genera Alistipes and Bacteroides. Overall, while our analyses confirm the existence of family-bound microbiome community profiles, transmission or co-acquisition of bacterial strains appears to be strongly linked to cohabitation.

Duodenal Dysbiosis and Relation to the Efficacy of Proton Pump Inhibitors in Functional Dyspepsia.

Proton pump inhibitors (PPI) may improve symptoms in functional dyspepsia (FD) through duodenal eosinophil-reducing effects. However, the contribution of the microbiome to FD symptoms and its interaction with PPI remains elusive. Aseptic duodenal brushings and biopsies were performed before and after PPI intake (4 weeks Pantoprazole 40 mg daily, FD-starters and controls) or withdrawal (2 months, FD-stoppers) for 16S-rRNA sequencing. Between- and within-group changes in genera or diversity and associations with symptoms or duodenal factors were analyzed. In total, 30 controls, 28 FD-starters and 19 FD-stoppers were followed. Mucus-associated Porphyromonas was lower in FD-starters vs. controls and correlated with symptoms in FD and duodenal eosinophils in both groups, while Streptococcus correlated with eosinophils in controls. Although clinical and eosinophil-reducing effects of PPI therapy were unrelated to microbiota changes in FD-starters, increased Streptococcus was associated with duodenal PPI effects in controls and remained higher despite withdrawal of long-term PPI therapy in FD-stoppers. Thus, duodenal microbiome analysis demonstrated differential mucus-associated genera, with a potential role of Porphyromonas in FD pathophysiology. While beneficial effects of short-term PPI therapy were not associated with microbial changes in FD-starters, increased Streptococcus and its association with PPIeffects in controls suggest a role for duodenal dysbiosis after long-term PPI therapy.

Temporal variability in quantitative human gut microbiome profiles and implications for clinical research.

While clinical gut microbiota research is ever-expanding, extending reference knowledge of healthy between- and within-subject gut microbiota variation and its drivers remains essential; in particular, temporal variability is under-explored, and a comparison with cross-sectional variation is missing. Here, we perform daily quantitative microbiome profiling on 713 fecal samples from 20 Belgian women over six weeks, combined with extensive anthropometric measurements, blood panels, dietary data, and stool characteristics. We show substantial temporal variation for most major gut genera; we find that for 78% of microbial genera, day-to-day absolute abundance variation is substantially larger within than between individuals, with up to 100-fold shifts over the study period. Diversity, and especially evenness indicators also fluctuate substantially. Relative abundance profiles show similar but less pronounced temporal variation. Stool moisture, and to a lesser extent diet, are the only significant host covariates of temporal microbiota variation, while menstrual cycle parameters did not show significant effects. We find that the dysbiotic Bact2 enterotype shows increased between- and within-subject compositional variability. Our results suggest that to increase diagnostic as well as target discovery power, studies could adopt a repeated measurement design and/or focus analysis on community-wide microbiome descriptors and indices.

Analysis of global human gut metagenomes shows that metabolic resilience potential for short-chain fatty acid production is strongly influenced by lifestyle.

High taxonomic diversity in non-industrial human gut microbiomes is often interpreted as beneficial; however, it is unclear if taxonomic diversity engenders ecological resilience (i.e. community stability and metabolic continuity). We estimate resilience through genus and species-level richness, phylogenetic diversity, and evenness in short-chain fatty acid (SCFA) production among a global gut metagenome panel of 12 populations (n = 451) representing industrial and non-industrial lifestyles, including novel metagenomic data from Burkina Faso (n = 90). We observe significantly higher genus-level resilience in non-industrial populations, while SCFA production in industrial populations is driven by a few phylogenetically closely related species (belonging to Bacteroides and Clostridium), meaning industrial microbiomes have low resilience potential. Additionally, database bias obfuscates resilience estimates, as we were 2-5 times more likely to identify SCFA-encoding species in industrial microbiomes compared to non-industrial. Overall, we find high phylogenetic diversity, richness, and evenness of bacteria encoding SCFAs in non-industrial gut microbiomes, signaling high potential for resilience in SCFA production, despite database biases that limit metagenomic analysis of non-industrial populations.

Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

Genome-wide associations of human gut microbiome variation and implications for causal inference analyses.

Recent population-based1-4 and clinical studies5 have identified a range of factors associated with human gut microbiome variation. Murine quantitative trait loci6, human twin studies7 and microbiome genome-wide association studies1,3,8-12 have provided evidence for genetic contributions to microbiome composition. Despite this, there is still poor overlap in genetic association across human studies. Using appropriate taxon-specific models, along with support from independent cohorts, we show an association between human host genotype and gut microbiome variation. We also suggest that interpretation of applied analyses using genetic associations is complicated by the probable overlap between genetic contributions and heritable components of host environment. Using faecal 16S ribosomal RNA gene sequences and host genotype data from the Flemish Gut Flora Project (n = 2,223) and two German cohorts (FoCus, n = 950; PopGen, n = 717), we identify genetic associations involving multiple microbial traits. Two of these associations achieved a study-level threshold of P = 1.57 × 10-10; an association between Ruminococcus and rs150018970 near RAPGEF1 on chromosome 9, and between Coprococcus and rs561177583 within LINC01787 on chromosome 1. Exploratory analyses were undertaken using 11 other genome-wide associations with strong evidence for association (P < 2.5 × 10-8) and a previously reported signal of association between rs4988235 (MCM6/LCT) and Bifidobacterium. Across these 14 single-nucleotide polymorphisms there was evidence of signal overlap with other genome-wide association studies, including those for age at menarche and cardiometabolic traits. Mendelian randomization analysis was able to estimate associations between microbial traits and disease (including Bifidobacterium and body composition); however, in the absence of clear microbiome-driven effects, caution is needed in interpretation. Overall, this work marks a growing catalogue of genetic associations that will provide insight into the contribution of host genotype to gut microbiome. Despite this, the uncertain origin of association signals will likely complicate future work looking to dissect function or use associations for causal inference analysis.

Microbial communities of the house fly Musca domestica vary with geographical location and habitat.

House flies (Musca domestica) are widespread, synanthropic filth flies commonly found on decaying matter, garbage, and feces as well as human food. They have been shown to vector microbes, including clinically relevant pathogens. Previous studies have demonstrated that house flies carry a complex and variable prokaryotic microbiota, but the main drivers underlying this variability and the influence of habitat on the microbiota remain understudied. Moreover, the differences between the external and internal microbiota and the eukaryotic components have not been examined. To obtain a comprehensive view of the fly microbiota and its environmental drivers, we sampled over 400 flies from two geographically distinct countries (Belgium and Rwanda) and three different environments-farms, homes, and hospitals. Both the internal as well as external microbiota of the house flies were studied, using amplicon sequencing targeting both bacteria and fungi. Results show that the house fly's internal bacterial community is very diverse yet relatively consistent across geographic location and habitat, dominated by genera Staphylococcus and Weissella. The external bacterial community, however, varies with geographic location and habitat. The fly fungal microbiota carries a distinct signature correlating with the country of sampling, with order Capnodiales and genus Wallemia dominating Belgian flies and genus Cladosporium dominating Rwandan fly samples. Together, our results reveal an intricate country-specific pattern for fungal communities, a relatively stable internal bacterial microbiota and a variable external bacterial microbiota that depends on geographical location and habitat. These findings suggest that vectoring of a wide spectrum of environmental microbes occurs principally through the external fly body surface, while the internal microbiome is likely more limited by fly physiology.

The neuroactive potential of the human gut microbiota in quality of life and depression.

The relationship between gut microbial metabolism and mental health is one of the most intriguing and controversial topics in microbiome research. Bidirectional microbiota-gut-brain communication has mostly been explored in animal models, with human research lagging behind. Large-scale metagenomics studies could facilitate the translational process, but their interpretation is hampered by a lack of dedicated reference databases and tools to study the microbial neuroactive potential. Surveying a large microbiome population cohort (Flemish Gut Flora Project, n = 1,054) with validation in independent data sets (ntotal = 1,070), we studied how microbiome features correlate with host quality of life and depression. Butyrate-producing Faecalibacterium and Coprococcus bacteria were consistently associated with higher quality of life indicators. Together with Dialister, Coprococcus spp. were also depleted in depression, even after correcting for the confounding effects of antidepressants. Using a module-based analytical framework, we assembled a catalogue of neuroactive potential of sequenced gut prokaryotes. Gut-brain module analysis of faecal metagenomes identified the microbial synthesis potential of the dopamine metabolite 3,4-dihydroxyphenylacetic acid as correlating positively with mental quality of life and indicated a potential role of microbial γ-aminobutyric acid production in depression. Our results provide population-scale evidence for microbiome links to mental health, while emphasizing confounder importance.

Citroniella saccharovorans gen. nov. sp. nov., a member of the family Peptoniphilaceae isolated from a human fecal sample from a coastal traditional community member.

A novel Gram-stain-positive, non-motile, non-spore-forming coccus-shaped obligately anaerobic bacterium was recovered from a fecal sample obtained from an individual from a traditional community located on the southern coast of Peru. The results of analysis based on 16S rRNA gene sequencing indicated the novel bacterium to be phylogenetically distinct from other genera of members of the Peptoniphilaceae family, sharing a loose affinity with the genera Ezakiella, Finegoldia, Gallicola and Parvimonas. The major cellular fatty acids of the novel isolate were determined to be C16:0, C17:1ω8c, and C18:1ω9c. The DNA G+C content was 29.9 mol%. End products of metabolism from peptone yeast glucose broth (PYG) were determined to be acetate and methyl succinate. The diagnostic diamino acid present in the cell wall was lysine. On the basis of the phenotypic, chemotaxonomic and phylogenetic results the organism is a member of a novel genus belonging to the family Peptoniphilaceae for which the name Citroniella saccharovorans gen nov. sp. nov., is proposed. The type strain is M6.X9T (DSM 29873T=CCUG 66799T).

Metformin induces weight loss associated with gut microbiota alteration in non-diabetic obese women: a randomized double-blind clinical trial.

OBJECTIVE: The increasing prevalence of obesity over the past few decades constitutes a global health challenge. Pharmacological therapy is recommended to accompany life-style modification for obesity management. Here, we perform a clinical trial to investigate the effects of metformin on anthropometric indices and gut microbiota composition in non-diabetic, treatment-naive obese women with a low-calorie diet (LCD). DESIGN: Randomized double-blind parallel-group clinical trial. METHODS: Forty-six obese women were randomly assigned to the metformin (500 mg/tab) or placebo groups using computer-generated random numbers. Subjects in both groups took two tablets per day for 2 months. Anthropometric measurements and collection of blood and fecal samples were done at the baseline and at the end of the trial. Gut microbiota composition was assessed using 16S rRNA amplicon sequencing. RESULTS: Twenty-four and twenty-two subjects were included in the metformin + LCD and placebo + LCD groups, respectively; at the end of trial, 20 and 16 subjects were analyzed. The metformin + LCD and placebo + LCD caused a 4.5 and 2.6% decrease in BMI from the baseline values, respectively (P < 0.01). Insulin concentration decreased in the metformin + LCD group (P = 0.046). The overall fecal microbiota composition and diversity were unaffected in the metformin + LCD group. However, a significant specific increase in Escherichia/Shigella abundance was observed after metformin + LCD intervention (P = 0.026). Fecal acetate concentration, but not producers, was significantly higher in the placebo + LCD group, adjusted for baseline values and BMI (P = 0.002). CONCLUSIONS: Despite the weight reduction after metformin intake, the overall fecal microbiota composition remained largely unchanged in obese women, with exception of changes in specific proteobacterial groups.

Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota.

OBJECTIVE: Human gut microbiome studies are mainly bacteria- and archaea-oriented, overlooking the presence of single-cell eukaryotes such as Blastocystis, an enteric stramenopiles with worldwide distribution. Here, we surveyed the prevalence and subtype variation of Blastocystis in faecal samples collected as part of the Flemish Gut Flora Project (FGFP), a Western population cohort. We assessed potential links between Blastocystis subtypes and identified microbiota-host covariates and quantified microbiota differentiation relative to subtype abundances. DESIGN: We profiled stool samples from 616 healthy individuals from the FGFP cohort as well as 107 patients with IBD using amplicon sequencing targeting the V4 variable region of the 16S rRNA and 18S rRNA genes. We evaluated associations of Blastocystis, and their subtypes, with host parameters, diversity and composition of bacterial and archaeal communities. RESULTS: Blastocystis prevalence in the non-clinical population cohort was 30% compared with 4% among Flemish patients with IBD. Within the FGFP cohort, out of 69 previously identified gut microbiota covariates, only age was associated with Blastocystis subtype carrier status. In contrast, a strong association between microbiota community composition and Blastocystis subtypes was observed, with effect sizes larger than that of host covariates. Microbial richness and diversity were linked to both Blastocystis prevalence and subtype variation. All Blastocystis subtypes detected in this cohort were found to be less prevalent in Bacteroides enterotyped samples. Interestingly, Blastocystis subtypes 3 and 4 were inversely correlated with Akkermansia, suggesting differential associations of subtypes with host health. CONCLUSIONS: These results emphasise the role of Blastocystis as a common constituent of the healthy gut microbiota. We show its prevalence is reduced in patients with active IBD and demonstrate that subtype characterisation is essential for assessing the relationship between Blastocystis, microbiota profile and host health. These findings have direct clinical applications, especially in donor selection for faecal transplantation.

Quantitative microbiome profiling links gut community variation to microbial load.

Current sequencing-based analyses of faecal microbiota quantify microbial taxa and metabolic pathways as fractions of the sample sequence library generated by each analysis. Although these relative approaches permit detection of disease-associated microbiome variation, they are limited in their ability to reveal the interplay between microbiota and host health. Comparative analyses of relative microbiome data cannot provide information about the extent or directionality of changes in taxa abundance or metabolic potential. If microbial load varies substantially between samples, relative profiling will hamper attempts to link microbiome features to quantitative data such as physiological parameters or metabolite concentrations. Saliently, relative approaches ignore the possibility that altered overall microbiota abundance itself could be a key identifier of a disease-associated ecosystem configuration. To enable genuine characterization of host-microbiota interactions, microbiome research must exchange ratios for counts. Here we build a workflow for the quantitative microbiome profiling of faecal material, through parallelization of amplicon sequencing and flow cytometric enumeration of microbial cells. We observe up to tenfold differences in the microbial loads of healthy individuals and relate this variation to enterotype differentiation. We show how microbial abundances underpin both microbiota variation between individuals and covariation with host phenotype. Quantitative profiling bypasses compositionality effects in the reconstruction of gut microbiota interaction networks and reveals that the taxonomic trade-off between Bacteroides and Prevotella is an artefact of relative microbiome analyses. Finally, we identify microbial load as a key driver of observed microbiota alterations in a cohort of patients with Crohn's disease, here associated with a low-cell-count Bacteroides enterotype (as defined through relative profiling).

Practical considerations for large-scale gut microbiome studies.

First insights on the human gut microbiome have been gained from medium-sized, cross-sectional studies. However, given the modest portion of explained variance of currently identified covariates and the small effect size of gut microbiota modulation strategies, upscaling seems essential for further discovery and characterisation of the multiple influencing factors and their relative contribution. In order to guide future research projects and standardisation efforts, we here review currently applied collection and preservation methods for gut microbiome research. We discuss aspects such as sample quality, applicable omics techniques, user experience and time and cost efficiency. In addition, we evaluate the protocols of a large-scale microbiome cohort initiative, the Flemish Gut Flora Project, to give an idea of perspectives, and pitfalls of large-scale faecal sampling studies. Although cryopreservation can be regarded as the gold standard, freezing protocols generally require more resources due to cold chain management. However, here we show that much can be gained from an optimised transport chain and sample aliquoting before freezing. Other protocols can be useful as long as they preserve the microbial signature of a sample such that relevant conclusions can be drawn regarding the research question, and the obtained data are stable and reproducible over time.