Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota

Abstract

Background: The dietary methylamines choline, carnitine, and phosphatidylcholine are used by the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However, little is known about the use of trimethylamine N-oxide (TMAO) by this consortium of microbes.

Results: A feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture fermentation systems inoculated with human faeces, growth of Enterobacteriaceae was stimulated in the presence of TMAO. Human-derived faecal and caecal bacteria (n = 66 isolates) were screened on solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by ¹H-NMR. As with the in vitro fermentation experiments, TMAO stimulated the growth of Enterobacteriaceae; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates of Escherichia coli produced more TMA from TMAO than their faecal counterparts. Lactic acid bacteria produced increased amounts of lactate when grown in the presence of TMAO but did not produce large amounts of TMA. Clostridia (sensu stricto), bifidobacteria, and coriobacteria were significantly correlated with TMA production in the mixed fermentation system but did not produce notable quantities of TMA from TMAO in pure culture.

Conclusions: Reduction of TMAO by the gut microbiota (predominantly Enterobacteriaceae) to TMA followed by host uptake of TMA into the bloodstream from the intestine and its conversion back to TMAO by host hepatic enzymes is an example of metabolic retroconversion. TMAO influences microbial metabolism depending on isolation source and taxon of gut bacterium. Correlation of metabolomic and abundance data from mixed microbiota fermentation systems did not give a true picture of which members of the gut microbiota were responsible for converting TMAO to TMA; only by supplementing the study with pure culture work and additional metabolomics was it possible to increase our understanding of TMAO bioconversions by the human gut microbiota.

Keywords: Co-metabolic axis; Enterobacteriaceae; Gut–liver axis; Lactic acid bacteria; Metabolomics.

Fig. 1

In vivo confirmation of metabolic retroconversion of TMAO. Reduction of d₉-TMAO to d₉-TMA was quantified by UPLC–MS/MS up to 6 h after d₉-TMAO gavage and antibiotic treatment, together with unlabelled TMA and TMAO levels. Plasma quantification of post-gavage a d₉-TMA and b d₉-TMAO. *Significantly (P < 0.05; t test and corrected for multiple comparison using the Holm–Sidak method) different from the respective groups not treated with antibiotics. c d₉-TMA bioavailability (AUC). d d₉-TMAO bioavailability (AUC). Plasma quantification of post-gavage unlabelled/endogenous e TMA and f TMAO. *Significant between d₉ and d₉ antibiotic treatment; ^$significant between TMAO and TMAO antibiotic treatment. g Unlabelled/endogenous TMA bioavailability (AUC). h Unlabelled/endogenous TMAO bioavailability (AUC). Data (n = 6 per group) are shown as mean ± SEM (a, b, e, f). Differences between the bioavailabilities (c, d, g, h) were assessed using one-way analysis of variance (ANOVA), followed by Holm–Sidak post hoc tests. Data with different superscript letters are significantly different (P < 0.05)

Fig. 2

Effect of TMAO on mixed faecal microbial population in vitro. a Enumeration of selected bacteria in fermentation vessels by FISH analysis. Red lines, TMAO-containing systems; blue lines, negative controls. Data are shown as mean + SD (n = 3). Eub338, total bacteria; Ent, Enterobacteriaceae; Bif164, Bifidobacterium spp.; Lab158, lactic acid bacteria. *Statistically significantly different (adjusted P < 0.05) from the control at the same time point. Full data are shown in Additional file 1: Figure S1. b ¹H-NMR data for batch culture samples. Data are shown as mean ± SD (n = 3). Red lines, TMAO-containing systems; blue lines, negative controls. *Statistically significantly different (P < 0.05) from the negative control at the same time point. c Bidirectional clustering of correlation matrix of FISH data and data for the six metabolites found in the highest amounts in the NMR spectra from the batch-culture samples. ⁺Adjusted P value (Benjamini–Hochberg) statistically significant (P < 0.05). FISH and metabolite data and a table of correlations and adjusted P values (Benjamini–Hochberg) for the batch-culture samples are available in Additional file 1: Table S3–S5

Fig. 3

Influence of TMAO on growth and metabolism of pure cultures of gut bacteria. a Representative growth curves for isolates grown in the presence and absence of TMAO. Red lines, TMAO-supplemented cultures; blue lines, negative controls. Data are shown as mean ± SD (n = 3). b Biplot showing production of various metabolites when isolates were grown in the presence of TMAO. Summary of data from Additional file 1: Table S2. The larger a circle, the more of the metabolite produced by an isolate. c Differences in metabolites produced when caecal and faecal isolates of Escherichia coli were grown in the presence (+) and absence (−) of 1% TMAO. Adjusted (Benjamini–Hochberg) P values indicate the caecal isolates were significantly different from the faecal isolates for a particular metabolite. d Lactate production by lactic acid bacteria was increased in the presence of TMAO. Enterobacteriaceae, n = 20; Bifidobacteriaceae, n = 17; Streptococcaceae, n = 7; Enterococcaceae, n = 5. Members of the Enterococcaceae and Streptococcaceae are homofermenters (produce only lactic acid from glucose fermentation), whereas the Bifidobacteriaceae are heterofermenters (produce ethanol, CO₂, and lactic acid from glucose fermentation), though it should be noted the bifidobacteria included in this study were grown on raffinose-containing media. Red, TMAO-containing medium; blue, negative control. *Statistically significantly different from its negative control (adjusted P value < 0.05)