ObjectiveThe gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance.DesignA pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation.ResultsThe OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, p<0.0001) and improved the feasibility of OCCT.ConclusionThe OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment.Trial registration numberNCT02838732; Results.

The capability of gut microbiota in degrading foods and drugs administered orally can result in diversified efficacies and toxicity interpersonally and cause significant impact on human health. Production of atherogenic trimethylamine N-oxide (TMAO) from carnitine is a gut microbiota-directed pathway and varies widely among individuals. Here, we demonstrated a personalized TMAO formation and carnitine bioavailability from carnitine supplements by differentiating individual TMAO productivities with a recently developed oral carnitine challenge test (OCCT). By exploring gut microbiome in subjects characterized by TMAO producer phenotypes, we identified 39 operational taxonomy units that were highly correlated to TMAO productivity, including Emergencia timonensis, which has been recently discovered to convert γ-butyrobetaine to TMA in vitro. A microbiome-based random forest classifier was therefore constructed to predict the TMAO producer phenotype (AUROC = 0.81) which was then validated with an external cohort (AUROC = 0.80). A novel bacterium called Ihubacter massiliensis was also discovered to be a key microbe for TMA/TMAO production by using an OCCT-based humanized gnotobiotic mice model. Simply combining the presence of E. timonensis and I. massiliensis could account for 43% of high TMAO producers with 97% specificity. Collectively, this human gut microbiota phenotype-directed approach offers potential for developing precision medicine and provides insights into translational research.

Cardiovascular disease (CVD) is strongly associated with the gut microbiota and its metabolites, including trimethylamine-N-oxide (TMAO), formed from metaorganismal metabolism of ʟ-carnitine. Raw garlic juice, with allicin as its primary compound, exhibits considerable effects on the gut microbiota. This study validated the benefits of raw garlic juice against CVD risk via modulation of the gut microbiota and its metabolites. Allicin supplementation significantly decreased serum TMAO in ʟ-carnitine-fed C57BL/6 J mice, reduced aortic lesions, and altered the fecal microbiota in carnitine-induced, atherosclerosis-prone, apolipoprotein E-deficient (ApoE−/−) mice. In human subjects exhibiting high-TMAO production, raw garlic juice intake for a week reduced TMAO formation, improved gut microbial diversity, and increased the relative abundances of beneficial bacteria. In in vitro and ex vivo studies, raw garlic juice and allicin inhibited γ-butyrobetaine (γBB) and trimethylamine production by the gut microbiota. Thus, raw garlic juice and allicin can potentially prevent cardiovascular disease by decreasing TMAO production via gut microbiota modulation.

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Anti-acid drugs, proton pump inhibitor (PPI) and histamine-2 blocker (H2-blocker), are commonly prescribed to treat gastrointestinal disorders. These anti-acid drugs alter gut microbiota in the general population, but their effects are not known in hemodialysis patients. Hence, we investigated the microbiota composition in hemodialysis patients treated with PPIs or H2-blocker. Among 193 hemodialysis patients, we identified 32 H2-blocker users, 23 PPI users, and 138 no anti-acid drug subjects. Fecal samples were obtained to analyze the gut microbiome using 16S RNA amplicon sequencing. Differences in the microbial composition of the H2-blocker users, PPI users, and controls were assessed using linear discriminant analysis effect size and the random forest algorithm. The species richness or evenness (α-diversity) was similar among the three groups, whereas the inter-individual diversity (β-diversity) was different between H2-blocker users, PPI users, and controls. Hemodialysis patients treated with H2-blocker and PPIs had a higher microbial dysbiosis index than the controls, with a significant increase in the genera Provetella 2, Phascolarctobacterium, Christensenellaceae R-7 group, and Eubacterium oxidoreducens group in H2-blocker users, and Streptococcus and Veillonella in PPI users. In addition, compared to the H2-blocker users, there was a significant enrichment of the genera Streptococcus in PPI users, as confirmed by the random forest analysis and the confounder-adjusted regression model. In conclusion, PPIs significantly changed the gut microbiota composition in hemodialysis patients compared to H2-blocker users or controls. Importantly, the Streptococcus genus was significantly increased in PPI treatment. These findings caution against the overuse of PPIs.

This study demonstrates that even with limited exposure in early life, a residual dose of tylosin might cause long-lasting metabolic disturbances by altering the gut microbiota and its metabolites. Our findings reveal that the gut microbiota is susceptible to previously ignored environmental factors.

Gut dysbiosis in patients with chronic kidney disease (CKD) may induce chronic inflammation and increase morbidity. Phosphate-binding agents, generally used in patients with CKD, may potentially change the composition of the gut microbiota. This study aimed to compare the microbiota composition in hemodialysis patients treated with ferric citrate or calcium carbonate. The stool microbiota was investigated in hemodialysis patients treated with ferric citrate (n = 8) and calcium carbonate (n = 46) using 16S rRNA gene amplicon sequencing profiling using linear discriminant analysis of effect size. Further predictive functional profiling of microbial communities was obtained with Tax4Fun in R. Hemodialysis patients treated with calcium carbonate had a significantly reduced microbial species diversity (Shannon index and Simpson index) and an increased microbial alteration ratio compared with patients treated with ferric citrate. A distinct microbial community structure was found in patients treated with ferric citrate, with an increased abundance of the Bacteroidetes phylum and a decreased abundance of the phylum Firmicutes. Members of the order Lactobacillales were enriched in patients treated with calcium carbonate, whereas taxa of the genera Ruminococcaceae UCG-004, Flavonifractor, and Cronobacter were enriched in patients treated with ferric citrate phosphate binder. In conclusion, Ferric citrate therapy results in a more diverse microbiome community compared to calcium carbonate therapy in hemodialysis patients with phosphate binder treatment. The gut microbiome reflects the phosphate binder choice in hemodialysis patients, further affecting the physiological environment in the gastrointestinal tract.