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.

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.

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.

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.

Population-level analysis of gut microbiome variation.

Fecal microbiome variation in the average, healthy population has remained under-investigated. Here, we analyzed two independent, extensively phenotyped cohorts: the Belgian Flemish Gut Flora Project (FGFP; discovery cohort; N = 1106) and the Dutch LifeLines-DEEP study (LLDeep; replication; N = 1135). Integration with global data sets (N combined = 3948) revealed a 14-genera core microbiota, but the 664 identified genera still underexplore total gut diversity. Sixty-nine clinical and questionnaire-based covariates were found associated to microbiota compositional variation with a 92% replication rate. Stool consistency showed the largest effect size, whereas medication explained largest total variance and interacted with other covariate-microbiota associations. Early-life events such as birth mode were not reflected in adult microbiota composition. Finally, we found that proposed disease marker genera associated to host covariates, urging inclusion of the latter in study design.