Application of Mendelian randomization to explore the causal role of the human gut microbiome in colorectal cancer.

The role of the human gut microbiome in colorectal cancer (CRC) is unclear as most studies on the topic are unable to discern correlation from causation. We apply two-sample Mendelian randomization (MR) to estimate the causal relationship between the gut microbiome and CRC. We used summary-level data from independent genome-wide association studies to estimate the causal effect of 14 microbial traits (n = 3890 individuals) on overall CRC (55,168 cases, 65,160 controls) and site-specific CRC risk, conducting several sensitivity analyses to understand the nature of results. Initial MR analysis suggested that a higher abundance of Bifidobacterium and presence of an unclassified group of bacteria within the Bacteroidales order in the gut increased overall and site-specific CRC risk. However, sensitivity analyses suggested that instruments used to estimate relationships were likely complex and involved in many potential horizontal pleiotropic pathways, demonstrating that caution is needed when interpreting MR analyses with gut microbiome exposures. In assessing reverse causality, we did not find strong evidence that CRC causally affected these microbial traits. Whilst our study initially identified potential causal roles for two microbial traits in CRC, importantly, further exploration of these relationships highlighted that these were unlikely to reflect causality.

Alterations of the bile microbiome in primary sclerosing cholangitis.

BACKGROUND: Patients with primary sclerosing cholangitis (PSC) display an altered colonic microbiome compared with healthy controls. However, little is known on the bile duct microbiome and its interplay with bile acid metabolism in PSC. METHODS: Patients with PSC (n=43) and controls without sclerosing cholangitis (n=22) requiring endoscopic retrograde cholangiography were included prospectively. Leading indications in controls were sporadic choledocholithiasis and papillary adenoma. A total of 260 biospecimens were collected from the oral cavity, duodenal fluid and mucosa and ductal bile. Microbiomes of the upper alimentary tract and ductal bile were profiled by sequencing the 16S-rRNA-encoding gene (V1-V2). Bile fluid bile acid composition was measured by high-performance liquid chromatography mass spectrometry and validated in an external cohort (n=20). RESULTS: The bile fluid harboured a diverse microbiome that was distinct from the oral cavity, the duodenal fluid and duodenal mucosa communities. The upper alimentary tract microbiome differed between PSC patients and controls. However, the strongest differences between PSC patients and controls were observed in the ductal bile fluid, including reduced biodiversity (Shannon entropy, p=0.0127) and increase of pathogen Enterococcus faecalis (FDR=4.18×10-5) in PSC. Enterococcus abundance in ductal bile was strongly correlated with concentration of the noxious secondary bile acid taurolithocholic acid (r=0.60, p=0.0021). CONCLUSION: PSC is characterised by an altered microbiome of the upper alimentary tract and bile ducts. Biliary dysbiosis is linked with increased concentrations of the proinflammatory and potentially cancerogenic agent taurolithocholic acid.

Normal gut microbiome in NMDA receptor encephalitis.

OBJECTIVE: To determine whether the gut microbiota shows overabundance of commensal bacteria species in patients with anti-NMDA receptor (NMDAR) encephalitis, similar to patients with MS or neuromyelitis optica where they potentially balance pro- and anti-inflammatory immune responses or participate in disease pathogenesis by molecular mimicry. METHODS: Intestinal microbiota was characterized in patients with NMDAR encephalitis (n = 23, mean age: 34 ± 12.7 years; 21 females) and age/sex/environment-matched healthy controls (n = 24, 40 ± 14.2 years; 22 females) using stool bacteria 16S rDNA sequencing and classification in operational taxonomic units (OTUs). Statistical analyses focused on intraindividual and interindividual bacterial diversity and identification of differentially abundant taxa. RESULTS: Patients with NMDAR encephalitis and controls had similar microbiome profiles of the gut microbiota regarding intraindividual bacterial diversity, OTU distribution, ratio between regional and local species diversity when testing all OTUs, and genera with a relative abundance greater than 0.5%. Similarly, the subgroup of NMDAR encephalitis patients with an ovarian teratoma (n = 3) showed no differences in microbiome variation compared with controls. Patients in the acute encephalitis stage (n = 8) showed significant differences in the numbers of Clostridium XVIII, Clostridium IV, Oscillibacter, Prevotella, and Blautia; however, significance was lost after correction for multiple testing. CONCLUSION: Patients with NMDAR encephalitis and controls both had a normal gut microbiome. The lack of overabundance of certain bacterial species in patients suggests that microbiome changes are no major contributors to the pathogenesis, disease course, or prognosis in NMDAR encephalitis. Despite the small sample size and heterogeneous groups, findings indicate differences to other neuroimmunologic diseases.

Application of the distance-based F test in an mGWAS investigating ß diversity of intestinal microbiota identifies variants in SLC9A8 (NHE8) and 3 other loci.

Factors shaping the human intestinal microbiota range from environmental influences, like smoking and exercise, over dietary patterns and disease to the host's genetic variation. Recently, we could show in a microbiome genome-wide association study (mGWAS) targeting genetic variation influencing the ß diversity of gut microbial communities, that approximately 10% of the overall gut microbiome variation can be explained by host genetics. Here, we report on the application of a new method for genotype-ß-diversity association testing, the distance-based F (DBF) test. With this we identified 4 loci with genome-wide significant associations, harboring the genes CBEP4, SLC9A8, TNFSF4, and SP140, respectively. Our findings highlight the utility of the high-performance DBF test in ß diversity GWAS and emphasize the important role of host genetics and immunity in shaping the human intestinal microbiota.

Functional sequencing read annotation for high precision microbiome analysis.

The vast majority of microorganisms on Earth reside in often-inseparable environment-specific communities-microbiomes. Meta-genomic/-transcriptomic sequencing could reveal the otherwise inaccessible functionality of microbiomes. However, existing analytical approaches focus on attributing sequencing reads to known genes/genomes, often failing to make maximal use of available data. We created faser (functional annotation of sequencing reads), an algorithm that is optimized to map reads to molecular functions encoded by the read-correspondent genes. The mi-faser microbiome analysis pipeline, combining faser with our manually curated reference database of protein functions, accurately annotates microbiome molecular functionality. mi-faser's minutes-per-microbiome processing speed is significantly faster than that of other methods, allowing for large scale comparisons. Microbiome function vectors can be compared between different conditions to highlight environment-specific and/or time-dependent changes in functionality. Here, we identified previously unseen oil degradation-specific functions in BP oil-spill data, as well as functional signatures of individual-specific gut microbiome responses to a dietary intervention in children with Prader-Willi syndrome. Our method also revealed variability in Crohn's Disease patient microbiomes and clearly distinguished them from those of related healthy individuals. Our analysis highlighted the microbiome role in CD pathogenicity, demonstrating enrichment of patient microbiomes in functions that promote inflammation and that help bacteria survive it.

Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis.

Adaptive thermogenesis is an energy-demanding process that is mediated by cold-activated beige and brown adipocytes, and it entails increased uptake of carbohydrates, as well as lipoprotein-derived triglycerides and cholesterol, into these thermogenic cells. Here we report that cold exposure in mice triggers a metabolic program that orchestrates lipoprotein processing in brown adipose tissue (BAT) and hepatic conversion of cholesterol to bile acids via the alternative synthesis pathway. This process is dependent on hepatic induction of cytochrome P450, family 7, subfamily b, polypeptide 1 (CYP7B1) and results in increased plasma levels, as well as fecal excretion, of bile acids that is accompanied by distinct changes in gut microbiota and increased heat production. Genetic and pharmacological interventions that targeted the synthesis and biliary excretion of bile acids prevented the rise in fecal bile acid excretion, changed the bacterial composition of the gut and modulated thermogenic responses. These results identify bile acids as important metabolic effectors under conditions of sustained BAT activation and highlight the relevance of cholesterol metabolism by the host for diet-induced changes of the gut microbiota and energy metabolism.