ABSTRACT
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. 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. Antibiotic-treated mice lacked microbial activity necessary to convert TMAO to TMA, 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. In summary, TMA can be produced by the gut microbiota (predominantly Enterobacteriaceae) from TMAO. This TMA is then taken up by the host and converted back to TMAO. That is, metabolic retroconversion occurs. In addition, TMAO influences microbial metabolism depending on isolation source and taxon of gut bacterium.
INTRODUCTION
Dietary methylamines such as choline, trimethylamine N-oxide (TMAO), phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish, nuts and eggs. It has long been known that gut bacteria are able to use choline in a fermentation-like process, with trimethylamine (TMA), ethanol, acetate and ATP among the known main end-products (Wünsche, 1940; Bradbeer, 1965; Zeisel et al., 1983). TMA can be used by members of the order Methanomassiliicoccales (Archaea) present in the human gut to produce methane (Borrel et al., 2017), or taken up by the host. Microbially produced TMA derived from PC is absorbed in the small intestine (Stremmel et al., 2017). TMA diffuses into the bloodstream from the intestine via the hepatic vein to hepatocytes, where it is converted to trimethylamine N-oxide (TMAO) by hepatic flavin-containing mono-oxygenases (FMOs; Bennett et al., 2013). The bulk of TMAO, and lesser amounts of TMA, derived from dietary methylamines can be detected in urine within 6 h of ingestion, and both compounds are excreted in urine but not faeces (de la Huerga and Popper, 1951; Taesuwan et al., 2017). TMAO can also be detected in human skeletal muscle within 6 h of an oral dose of TMAO (Taesuwan et al., 2017).
TMAO present in urine and plasma is considered a biomarker for non-alcoholic fatty liver disease (NAFLD), insulin resistance and cardiovascular disease (Dumas et al., 2006; Spencer et al., 2011; Tang et al., 2013; Dumas et al., 2017). Feeding TMAO to high-fat-fed C57BL/6 mice exacerbates impaired glucose tolerance, though the effect on the gut microbiota is unknown (Gao et al., 2014). Low plasma PC and high urinary methylamines (including TMAO) were observed in insulin-resistant mice on a high-fat diet, suggesting microbial methylamine metabolism directly influences choline bioavailability (Dumas et al., 2006), and choline bioavailability is known to contribute to hepatic steatosis in patients (Spencer et al., 2011). High levels of circulating TMAO are associated with cardiovascular disease (Tang et al., 2013). However, circulating TMAO has been suggested to play a role in protection from hyperammonemia, acting as an osmoprotectant, and from glutamate neurotoxicity (Kloiber et al., 1988; Miñana et al., 1996). Recently, chronic exposure to TMAO has been shown to attenuate diet-associated impaired glucose tolerance in mice, and reduce endoplasmic reticulum stress and adipogenesis in adipocytes (Dumas et al., 2017). There is a basal level of TMA and TMAO detected in human urine even in the absence of dietary supplementation (de la Huerga et al., 1953), suggesting use of (microbial and/or host) cell-derived choline or PC in the intestinal tract by the gut microbiota.
While it is well established that choline (Zeisel et al., 1983; Wang et al., 2011; Spencer et al., 2011), PC (Zeisel et al., 1983; Tang et al., 2013) and carnitine (Rebouche et al., 1984; Seim et al., 1985; Rebouche and Chenard, 1991; Koeth et al., 2013) are used by the human and rodent gut microbiotas to produce TMA, little is known about the reduction of TMAO (predominantly from fish) to TMA (or other compounds) by members of these consortia. TMAO is found at concentrations of 20–120 mg per 100 g fish fillet (Jebsen and Riaz, 1977). Ingestion of fish by humans leads to increased urinary excretion of dimethylamine (DMA) and TMA, from 5.6 to 24.1 and from 0.2 to 1.6 μmol/24 h/kg of body weight, respectively (Zeisel and Da Costa, 1986). Individuals with trimethylaminuria (fish odour syndrome), in which TMA is not detoxified to TMAO by hepatic FMOs, have been shown to reduce 40–60 % of an oral dose of TMAO to TMA (al-Waiz et al., 1987). It was suggested the gut microbiota was responsible for reducing TMAO in these individuals, and in those individuals without trimethylaminuria the TMA was re-oxidized in the liver before being excreted in the urine. The process was termed ‘metabolic retroversion’ “a(i.e., a cycle of reductive followed by oxidative reactions to regenerate TMAO)” (al-Waiz et al., 1987). The reduction of TMAO to TMA is most commonly associated with the gut microbiota of marine fish, with the TMA generated by these bacteria contributing to the characteristic odour of rotting fish (Barrett and Kwan, 1985). Members of the class Gammaproteobacteria, which includes a number of food spoilage organisms, are known to reduce TMAO to TMA quantitatively and are unable to reduce TMA further (Takagi and Ishimoto, 1983; Barrett and Kwan, 1985). The conversion of TMAO to TMA by bacteria represents a unique form of anaerobic respiration, in which TMAO reductase acts as a terminal electron acceptor (from NADH or formate) for respiratory electron flow (Takagi and Ishimoto, 1983).
Mining of metagenomic data (torA) suggests Proteobacteria (particularly Escherichia and Klebsiella spp.) are likely to contribute greatest to the production of TMA from TMAO in the human gut via the TMAO reductase pathway, with Actinobacteria (Eggerthellaceae) becoming more important under stress (Jameson et al., 2016). Other microbial genes are associated with production of TMA from choline (cutC), glycine-betaine (grdH), L-carnitine (cntA) and γ-butyrobetaine (cntA) (Borrel et al., 2017). A search of the NCBI nucleotide database with the phrase ‘TMAO reductase’ also suggests many other bacteria of human intestinal origin (namely, Salmonella, Helicobacter, Prevotella, Bacillus and Bacteroides spp.) should be able to reduce TMAO to TMA. However, this trait has not been examined in vitro for isolates of intestinal origin.
Consequently, we carried out an in vivo study in mice to confirm use of TMAO by the mouse gut microbiota and to allow us to examine metabolic retroconversion of TMAO in a murine model. We then used an in vitro fermentation system to highlight the effect of TMAO on the human faecal microbiota. Finally, we screened a panel of human faecal and caecal/small intestinal isolates to determine which members of the human gut microbiota were able to reduce TMAO to TMA, and whether their metabolism was affected by being grown in the presence of TMAO.
METHODS
Animal work
Groups of six-week-old C57BL6/J mice (Janvier Labs, Courtaboeuf, France) were received and acclimated in specific pathogen-free (SPF) maintenance conditions. They were fed with a standard chow diet (R04-40, Safe, Augy, France) and were either given free access to tap water or treated with the antibiotic cocktail (0.5 g/L vancomycin hydrochloride, 1 g/L neomycin trisulfate, 1 g/L metronidazole, 1 g/L ampicillin sodium; all antibiotics were purchased from Sigma-Aldrich) for 14 days. Mice were given in the morning by gavage either a solution of d9-TMAO at 1×10-4 M (Cambridge Isotope Laboratories Inc., DLM-4779-0, UK) or saline and euthanized 6 h later by cervical dislocation. Blood samples were collected by tail tipping every 2 h in Microvette® CB 300 Lithium Heparin (Sarstedt, Marnay, France). Plasma was separated by centrifugation (10 min, 5000 g, 4 °C) and stored at −80 °C until analysed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS).
All procedures were authorized following review by the institutional ethics committee (Sorbonne Universities) and carried out under national license conditions.
UPLC–MS/MS determination of plasma TMA, d9-TMA, TMAO and d9-TMAO
UPLC-MS/MS was employed for the determination of TMA, d9-TMA, TMAO and d9-TMAO. Samples (10 μL) were spiked with 10 μL Internal Standard solution (d9-choline and 13C3/15N-TMA in water; 1 mg/L, Sigma-Aldrich). Ethyl 2-bromoacetate solution (45 μL) (15 g/L ethyl 2-bromoacetate, 1 % NH4OH in acetonitrile; ChromaSolv grade, Sigma-Aldrich) was added and derivatization of TMAs (TMA, d9-TMA and 13C3/15N-TMA) to their ethoxy-analogues was completed after 30 min at room temperature. Protein/lipid precipitation solution (935 μL) (94 % acetonitrile/5 %water/1 % formic acid; ChromaSolv grade, Sigma-Aldrich) was added, samples were centrifuged (20 min, 20,000 g, 4 °C) and were transferred to UPLC-autosampler vials. Sample injections (5 μL loop) were performed with a Waters Acquity UPLC-Xevo TQ-S UPLC-MS/MS system equipped with an Acquity BEH HILIC (2.1×100 mm, 1.7 μm) chromatographic column. An isocratic elution was applied with 10 mM ammonium formate (Sigma-Aldrich) in 95:5 (v/v) acetronitrile:water for 7 min at 750 μL/min and 50 °C. Positive electrospray (ESI+) was used as ionization source and mass spectrometer parameters were set as follows: capillary, cone and sources voltages at −700, −18 and 50 V, respectively, desolvation temperature at 600 °C, desolvation/cone/nebuliser gases were high purity nitrogen (BOC) at 1000 L/h, 150 L/h and 7 bar, respectively. Collision gas was high-purity argon (BOC). Mass spectrometer was operated in multiple reaction monitoring mode. The monitored transitions were the following: for TMA, +146 → +118/59 m/z (23/27 V); for d9-TMA, +155 → +127/68 m/z (21/23 V); for 13C3/15N-TMA, +150 → +63/122 m/z (27/22 V); for TMAO, +76 → +59/58 m/z (12/13 V); for d9-TMAO, +85 → +68/66 m/z (18/20 V); and for d9-choline, +108 → +60/45 m/z (20/22 V). The system was controlled by MassLynx software, also used for data acquisition and analysis.
1H-NMR spectroscopy and data analysis
Medium samples were randomized and centrifuged (5 min, 16000 g). Aliquots (50 μL) of the supernatants were diluted in 550 μL D2O containing 1 mM trimethylsilyl-(2,2,3,3-2H4)-1-propionate. Samples were transferred to 5 mm NMR tubes and measured on a NMR spectrometer (Bruker) operating at 600.22 MHz 1H frequency as described previously (Dona et al., 2014). 1H-NMR spectra were pre-processed and analysed as described previously (Dumas et al., 2006) using the Statistical Recoupling of Variables-algorithm (Blaise et al., 2009). Structural assignment was performed as described in Dona et al. (2016), using in-house and publicly available databases.
In vitro fermentation systems
Freshly voided faecal samples were obtained from three healthy human volunteers (one male and two females; age range 29–31 years), none of whom had been prescribed antibiotics 6 months prior to the study, eaten fish 3 days prior to sample collection or had any history of gastrointestinal disease. The University of Reading’s Research Ethics Committee (UREC) does not require that specific ethical review and approval be given by UREC for the collection of faecal samples from healthy human volunteers to inoculate in vitro fermentation systems. Samples were processed immediately by diluting them 1 in 10 (w/w) in pre-reduced minimal broth and homogenizing them in a filter bag in a stomacher (Stomacher 400 Lab System; Seward) for 2 min at ‘high’ speed. For each donor, a batch culture vessel containing 175 mL of pre-reduced minimal broth was inoculated with 20 mL of the faecal homogenate and 5 mL of sterile H2O containing 2 g TMAO dihydrate (Sigma-Aldrich). Control vessels were inoculated with 20 mL of the faecal homogenate and 5 mL of sterile H2O. The final working volume of each batch culture vessel was 200 mL. The pH of each vessel (pH 6.5) was controlled automatically by the addition of 2 M HCl or 2 M NaOH. pH controllers were supplied by Electrolab. The contents of each vessel were stirred constantly. An anaerobic environment was maintained by constantly sparging the vessels with O2-free N2. The temperature of each vessel was maintained at 37 °C by use of a circulating waterbath connected to each fermentation vessel. The experiment was run for 9 h, with samples taken at 0, 1, 2, 3, 4, 5, 6 and 9 h.
Fluorescence in situ hybridization (FISH) analysis
Aliquots (2× 375 μL) of sample were fixed in ice-cold 4 % paraformaldehyde for 4 h, washed in sterile phosphate-buffered saline and stored for FISH analysis as described by Martín-Peláez et al. (2008). Supplementary Table 1 gives details for probes used in this study. Probes were synthesized commercially (MWG-Biotech) and labelled with the fluorescent dye cyanine 3 (Cy3; excitation λ, 514 nm; emission λ, 566 nm; fluorescence colour, orange–red). FISH was done as described by Martín-Peláez et al. (2008).
Slides were viewed under a Nikon E400 Eclipse microscope. DAPI slides were visualized with the aid of a DM400 filter; probe slides were visualized with the aid of a DM575 filter. Cells (between 15 and 50 per field of view) were counted for 15 fields of view, and the numbers of bacteria were determined by using the following equation:
Where the DF (dilution factor) was calculated by taking into account the concentration of the original sample (375 μL to 300 μL = 0.8×). ACC (average cell count) was determined by counting 15 fields of view and assumes that a normal distribution was observed for the counts. The figure 6,732.42 refers to the area of the well divided by the area of the field of view. DFsample refers to the dilution of sample used with a particular probe (e.g. 5× for t0 Lab158 counts). The detection limit of this method was 89,766 bacteria/mL of sample (= log10 4.95).
Screening of bacteria for ability to reduce TMAO
Part of an in-house collection (University of Reading) of bacteria isolated from human caecal/small intestinal and faecal samples (Table 1) was screened on minimal agar [g/L: glucose (Fisher Scientific), 4; bacteriological peptone (Oxoid), 20; NaCl (Fisher Scientific), 5; neutral red (Sigma-Aldrich), 0.03; agar technical no. 3 (Oxoid), 15; pH 7] with and without 1 % (w/v) TMAO dihydrate (Sigma-Aldrich). The composition of the agar was based on that of Takagi and Ishimoto (1983), who used the medium with crystal violet and bile salts to screen members of the Enterobacteriaceae for TMAO reductase activity. Colonies of isolates able to ferment glucose were red, whereas those able to reduce TMAO were white. Growth curves (OD600 measured hourly for 10–12 h, then at 24 h) were determined for selected isolates grown anaerobically at 37 °C in anaerobic minimal broth [g/L: glucose, 4; bacteriological peptone, 20; NaCl, 5; L-cysteine HCl (Sigma-Aldrich), 0.5; resazurin solution (0.25 mg/mL; Sigma-Aldrich), 4 mL; pH 7] with and without 1 % (w/v) TMAO dihydrate. Glucose was substituted by raffinose (Sigma-Aldrich) (Trojanová et al., 2006) in the minimal broth when working with bifidobacteria because of the poor growth of these bacteria on the glucose-based medium. pH and metabolite profiles of culture medium were examined at the end of the growth experiment (i.e. at t24).
Statistical analyses
Differences between metabolites produced by faecal and caecal/small intestinal isolates of Escherichia coli and lactic acid bacteria in the presence and absence of TMAO were analysed using Student’s t test. FISH data from the in vitro fermentation systems were analysed using the Kolmogorov–Smirnov test with statistical significance, after correction for multiple testing, taken at P < 0.05. Because of the presence of ties in the metabolite data from in vitro fermentation systems, the bootstrapped Kolmogorov–Smirnov test (10,000 replications) was used with these data. Data from in vitro fermentation systems (FISH and NMR) were correlated using Spearman’s rank correlation (corrected for ties) with results corrected for multiple testing using the method of Benjamini and Hochberg (1995).
RESULTS
In vivo confirmation of metabolic retroconversion
First, to confirm in vivo metabolic retroconversion (i.e. microbial conversion of TMAO to TMA, followed by host conversion of TMA to TMAO), we administered isotopically-labelled d9-TMAO or saline to mice that had or had not been treated with a broad-spectrum antibiotic cocktail for 14 days to suppress the gut microbiota (Tang et al., 2013). Reduction of d9-TMAO to d9-TMA was quantified in urine by UPLC–MS/MS up to 6 h after d9-TMAO gavage, together with unlabelled TMA and TMAO potentially produced from dietary sources (Fig. 1).
In the absence of antibiotics, d9-TMAO was converted to d9-TMA within 2 h of gavage. This conversion was dramatically reduced, leading to a significantly lower concentration of d9-TMA and a higher concentration of d9-TMAO, when the gut microbiota was suppressed by antibiotics (Fig. 1A-D). We noted that in the absence of antibiotics all animals excreted approximately three times higher levels of urinary unlabelled TMA than TMAO (Fig. 1E-H), corresponding to constitutively low FMO3 activity in mice (Zhang et al., 2007). Levels of unlabelled TMAO/TMA were not significantly different from one another in the control and d9-TMAO-fed animals, while antibiotic treatment significantly reduced the amount of both TMAO and TMA (Fig. 1E, F). Bioavailability of unlabelled TMAO/TMA, as assessed by area under the curve (AUC), was significantly reduced by antibiotic treatment in both saline and d9-TMAO-gavaged animals, with almost no TMA detected in either experimental group (Fig. 1G, H).
Effect of TMAO on human gut bacteria within a mixed system
After in vivo validation of the role of the gut microbiota in metabolic retroconversion, we analysed the effect of TMAO on the faecal microbiota in an anaerobic batch-culture fermentation system. Fermenter vessels filled with the glucose-containing medium supplemented or not with 1 % (w/v) TMAO were inoculated with faecal slurries from three healthy donors and monitored for 9 h. With the exception of enhanced growth of the Enterobacteriaceae (probe Ent), the presence of TMAO in the medium had no statistically significant effect on the growth of bacteria within the fermentation systems at 9 h (Fig. 2A, Supplementary Figure 1).
Huge variability, as measured by 1H-NMR, was observed in the amount of TMA and DMA produced by gut bacteria in the TMAO-containing fermentation systems, with the concentrations of both metabolites increasing steadily from 0 to 9 h and differing significantly (P < 0.05) from the control systems (Fig. 2B). The amount of TMAO in the systems was seen to decrease at 8 h.
Correlation of metabolite and FISH data demonstrated Clostridium clusters I and II (Chis150), Enterobacteriaceae (Ent), bifidobacteria (Bif164) and coriobacteriia (Ato291) were associated with TMA, acetate, ethanol and lactate (Fig. 2C). The Betaproteobacteria (Bet42a) were anti-correlated with TMA, acetate, ethanol and lactate, which is unsurprising given this was the only group of bacteria whose representation decreased in the fermentation systems over the course of the experiment (Supplementary Figure 1, Fig. 2C). The Enterobacteriaceae and clostridia were positively correlated with DMA production. The lactic acid bacteria (Lab158) were not significantly correlated with any of the metabolites in the mixed culture.
Growth of pure cultures of gut bacteria in the presence of TMAO
Using an in-house collection of bacteria, we initially screened isolates on a modified version of the agar of Takagi and Ishimoto (1983) as a rapid means of screening bacteria for TMAO reductase activity, and thereby their ability to reduce TMAO to TMA. While members of the Enterobacteriaceae produced expected results (i.e. white colonies when grown in the presence of TMAO and glucose, rather than red colonies on the glucose only control), we observed a number of unexpected outcomes depending on the species under study. For example, colonies of lactic acid bacteria were larger (almost twice their usual size) on TMAO-containing agar than on the glucose control but remained red in colour, suggesting they had not reduced TMAO to TMA at detectable levels but TMAO was influencing their growth. The clostridia examined produced mixed results on the control and TMAO-containing media (i.e. white colonies on both plates or on the control plate only, larger colonies on the TMAO-containing medium but without a colour change of the medium).
To determine whether these isolates were converting TMAO to TMA but a low level, we examined the growth of all isolates in liquid culture and metabolites in the spent medium using NMR. The growth of the Enterobacteriaceae was most greatly affected by the presence of TMAO in the medium, with a faster, longer-lasting exponential phase than for the same isolates grown in the control medium (Fig. 3A). pH of the spent medium (after 24 h) when Enterobacteriaceae were grown in the presence of TMAO increased from a mean of 4.7 ± 0.3 (for the control) to 7.6 ± 0.3 (n = 20) (the change in pH is what causes the colonies to appear white on TMAO-containing agar). The growth of lactic acid bacteria, including Enterococcus and Streptococcus (Fig. 3A) spp., was enhanced in the presence of TMAO, but not to the same extent as seen for the Enterobacteriaceae. There was no significant difference (P = 0.27, t test) in the pH of the spent medium for these bacteria after 24 h (mean 4.67 ± 0.9 compared with 4.33 ± 0.27 for the control). The growth of members of Clostridium cluster I (e.g. Clostridium perfringens, Fig. 3A) was not enhanced in the presence of TMAO, though some of these bacteria changed the colour of both the control and TMAO-containing plates yellow during their growth. The pH of the spent liquid medium confirmed this observation to be due to the alkalinity of the media in the control and TMAO-containing media after 24 h incubation: e.g. Clostridium sporogenes D1(9) (pH 6.25 compared with 6.76 in the control medium), Clostridium paraputrificum L16-FAA6 (pH 6.45 vs 5.38) and Clostridium perfringens L20-BSM1 (pH 5.56 vs 4.64).
1H-NMR analysis of spent medium from the TMAO-containing and control samples demonstrated that, as expected, the Enterobacteriaceae produced the greatest amount of TMA from TMAO (mean 38.79 ± 11.08 mM compared with 0.03 ± 0.01 mM, n = 20) (Fig. 3B; Supplementary Table 2). Members of the families Peptostreptococcaceae (3.72 mM, n = 1), Clostridiaceae (Cluster I) (2.62 ± 1.83 mM, n = 3), Porphyromonadaceae (1.42 mM, n = 1), Bacteroidaceae (1.40 ± 0.31 mM, n = 3), Enterococcaceae (1.19 ± 0.05 mM, n = 5), Erysipelotrichaceae (0.94 mM, n = 1; [Clostridium] ramosum), Staphylococcaceae (0.34 mM, n = 1), Streptococcaceae (0.30 ± 0.16 mM, n = 5), Lactobacillaceae (0.17 ± 0.07 mM, n = 2), Pseudomonadaceae (0.12 mM, n = 1) and Bifidobacteriaceae (0.13 ± 0.1 mM, n = 17) produced low levels of TMA from TMAO (Fig. 3B; Supplementary Table 2). There was great variability in the ability of the bifidobacteria to produce TMA from TMAO, with several isolates and [Clostridium] innocuum, Actinomyces odontolyticus, Fusobacterium ulcerans and Actinomyces viscosus not producing TMA from TMAO (Fig. 3B; Supplementary Table 2).
Differences in the metabolic capabilities of faecal and caecal Escherichia coli isolates
Comparison of the amounts of TMA, and co-metabolites, produced by the faecal (n = 7) and caecal (n = 9) isolates of Escherichia coli demonstrated significantly higher amounts of TMA were produced by the caecal isolates compared with the faecal isolates in the TMAO-containing medium (Fig. 3C). Escherichia coli of caecal origin produced more TMA than faecal isolates of the same bacterium or other enterobacteria (Hafnia, Citrobacter and Klebsiella spp.) (Supplementary Table 2). The faecal isolates produced more acetate and lactate than the caecal isolates when grown in the control medium. Taken together, these results demonstrate the different metabolic capabilities of isolates of Escherichia coli recovered from different regions of the human gut.
Lactic acid bacteria produce more lactate in the presence of TMAO
Differences were also seen in the amount of lactic acid produced by lactic acid bacteria in the presence and absence of TMAO (Fig. 3B, D). In raffinose-containing medium, bifidobacteria produced increased of amounts of lactate when grown in the presence of TMAO (the bifidobacteria grew poorly, if at all, in glucose-containing media). Unlike the bifidobacteria, the Streptococcaceae and Enterococcaceae grew well in the glucose-containing medium and produced over 25 mM lactic acid in the TMAO-containing samples compared with < 5 mM in the control samples (Fig. 3D). To the best of our knowledge, this is the first time TMAO has been shown to influence the metabolism of gut bacteria – specifically lactic acid bacteria – without producing appreciable amounts of TMA.
DISCUSSION
TMAO is a circulating metabolite produced as a direct result of microbial degradation of dietary methylamines in the intestinal tract, and can be readily detected along with its precursor TMA in human urine, blood and skeletal muscle (Wang et al., 2011; Koeth et al., 2013; Tang et al., 2013; Taesuwan et al., 2017). It worsens atherosclerosis in some mouse models of cardiovascular disease, and is positively correlated with cardiovascular disease severity in humans. Beneficial effects associated with TMAO include potential protection from hyperammonia and glutamate neurotoxicity, alleviation of endoplasmic reticulum stress and improved glucose homeostasis by stimulating insulin secretion by pancreatic cells (Kloiber et al., 1988; Minana et al., 1996; Dumas et al., 2017).
Over many decades it has been established that choline, PC and carnitine are dietary methylamines that contribute directly to microbiome-associated circulating levels of TMAO found in humans and other animals (e.g. Zeisel et al., 1983; Rebouche et al., 1984; Seim et al., 1985; Wang et al., 2011; Koeth et al., 2013; Tang et al., 2013). However, TMAO itself is a water-soluble osmolyte found in high abundance in fish. Based on their observations that individuals with trimethylaminuria could reduce an oral dose of TMAO to TMA could not detoxify it to TMAO, al-Waiz et al. (1987) suggested the gut microbiota could use TMAO as a substrate, and in those individuals without trimethylaminuria the TMA was re-oxidized in the liver before being excreted in the urine. This led these authors to propose the process of ‘metabolic retroversion’. Reference to the literature associated with fish spoilage and recent mining of metagenomic data have predicted members of the Enterobacteriaceae (particularly Escherichia coli and Klebsiella pneumoniae) have the potential to convert TMAO to TMA in the intestinal tract, but this has not been tested in vitro or in vivo to date (Takagi and Ishimoto, 1983; Barrett and Kwan, 1985; Jameson et al., 2016; Borrel et al., 2017; Taesuwan et al., 2017). Consequently, we instigated this study to demonstrate metabolic retroconversion of TMAO, and to determine the effect of TMAO on the growth and metabolism of human-derived intestinal bacteria in pure and mixed cultures.
Through in vivo administration of deuterated TMAO to mice via oral gavage in mice, we unambiguously demonstrated that TMAO is converted to TMA, with this TMA detectable in plasma within 2 h of administration. This conversion was highly dependent on the gut microbiota, as conversion of TMAO to TMA was dramatically reduced when the microbiota was suppressed by treatment of animals with broad-spectrum antibiotics. Even in the presence of antibiotics, there was low-level conversion of TMAO to TMA, suggesting a subpopulation of the microbiota was resistant to the antibiotics used in our experiment. However, administration of a broad-spectrum antibiotic cocktail for 14 days has been demonstrated to be an effective means of suppressing the gut microbiota in studies associated with gut microbial use of dietary methylamines (Tang et al., 2013).
Our in vivo experiment clearly demonstrates the gut microbiota converts TMAO to TMA, and that this TMA is re-oxidized to TMAO, in line with the process of ‘metabolic retroversion’ defined by al-Waiz et al. (1987). Gut-associated microbial conversion of the majority of TMAO to TMA is at odds with the findings of Taesuwan et al. (2017), who suggested TMAO is taken up intact and not metabolised by the gut microbiota of humans. The ratio of TMAO-to-TMA is around 10:1 in humans and 1:10 in mice, meaning TMAO was N-oxidized before Taesuwan et al. (2017) were able to observe it in circulating blood of their human subjects. In a human system with high FMO3 activity, O17-labelled TMAO would need to be used in any study evaluating O turnover to allow calculation of the true rate of retroconversion.
Having conducted our in vivo experiment in mice, we examined the ability of a range of human-derived gut bacteria to convert TMAO to TMA. In the mixed microbiota system and in pure cultures, the growth of the Enterobacteriaceae – the main TMA-producers – was quickly affected by the presence of TMAO. This is likely to happen in the human gut also. Consequently, we believe dietary TMAO undergoes metabolic retroconversion in mammals, with the TMA produced as a result of bacterial activity in the gut available to the host for conversion back to TMAO by FMOs in hepatocytes. It should be noted that Taesuwan et al. (2017) did not suppress or monitor the intestinal/faecal microbiota when they administered isotopically labeled TMAO to humans, nor did they measure d9-TMA and d9-TMAO in the portal vein, bypassing subsequent hepatic N-oxidation of d9-TMA, so it is not possible to interpret their results in the context of presence/absence of microbial activity.
Of note is the finding that caecal isolates of enterobacteria produce more TMA from TMAO than faecal isolates of the same bacterium. The speed with which TMAO was reduced to TMA by the Enterobacteriaceae in the present study suggests bacterial conversion of TMAO to TMA takes place in the small intestine/proximal colon of humans and small intestine/caecum of mice. It is, therefore, unsurprising that caecal bacteria – representing the microbiota present at the intersection of the small and large intestine – are metabolically more active than their faecal counterparts with respect to TMAO metabolism. This finding is relevant to functional studies of the gut microbiota where gene annotations are based largely on faecal isolates, whose functionalities may be greatly different from those of bacteria in other regions of the human intestinal tract. It has already been demonstrated that the microbiota of the small intestine is enriched for functions associated with rapid uptake and fermentation of simple carbohydrates compared with the faecal microbiota, and that streptococci isolated from this niche are functionally very different from the same bacteria isolated from different habitats (Zoetendal et al., 2012; van den Bogert et al., 2013). It is, therefore, important we characterize the functions and genomes of bacteria isolated from all regions of the intestinal tract, not just those of faecal bacteria, to gain a true picture of how microbial activity influences host health.
Enterobacteriaceae made the greatest contribution to the conversion of TMAO to TMA, both in pure culture and in a mixed microbiota. These Gram-negative bacteria are a source of the virulence factor lipopolysaccharide (LPS), which is associated with low-grade inflammation in high-fat-fed mice and elevated plasma levels that define metabolic endotoxaemia (Cani et al., 2007, 2008). High-fat feeding has been shown to increase the representation of Enterobacteriaceae in the caecal microbiota of obesity-prone Sprague–Dawley rats (de La Serre et al., 2010), though this mode of feeding is known to modulate the microbiota of mice independent of obesity (Hildebrandt et al., 2009). Non-LPS-associated virulence of Enterobacteriaceae, Vibrio cholerae and Helicobacter pylori is increased when these bacteria are grown anaerobically or microaerophilically in the presence of as little as 5–10 mM TMAO (Ando et al., 2007; Lee et al., 2012; Paiva et al., 2009; Wu et al., 2017), and may be an additional means by which the gut microbiota contributes to cardiovascular and other diseases in which increased representation of Gammaproteobacteria is observed. This warrants attention in future animal studies.
Lactic acid bacteria clearly grow better in the presence of TMAO. The relatively high concentration (1 %) of TMAO used in this study may have contributed to this improved growth, as TMAO is an osmolyte that stabilizes proteins. Future work will involve growing lactic acid bacteria in a range of TMAO concentrations to determine how this compound affects their growth and gene expression, and comparing faecal and caecal isolates. Similar to the Enterobacteriaceae, a large number of these bacteria are facultative anaerobes able to grow over a range of conditions, and whose representation is increased in obese, and cirrhotic patients (Le Chatelier et al., 2013; Qin et al., 2014). Streptococcus and Enterococcus spp. are commensal lactic acid bacteria of the gut microbiota known to modulate immune function; but little is known about their metabolic activities in mixed microbial populations (van den Bogert et al., 2014). Understanding how commensal lactic acid bacteria influence the host in dysbiosis in mixed microbial communities may allow the development of approaches to modulate their activity and influence host health.
With respect to the lactic acid bacteria, it is important to note that our mixed culture work did not highlight these as being relevant to TMAO metabolism. This is unsurprising given these bacteria do not produce large quantities of TMA from TMAO. However, we have shown their metabolism is affected by presence of TMAO in growth medium, and the increased lactate they produce in its presence may contribute to cross-feeding associated with short-chain fatty acid production (Hoyles and Wallace, 2010). It is difficult to determine relevance of correlations from mixed microbial ecosystems in the absence of isotope labelling or pure culture work: i.e. correlation does not equate with causation. As an example, three (Clostridium clusters I and II, bifidobacteria, coriobacteriia) of the four groups of bacteria correlated with TMA production in our fermentation study did not produce notable quantities of TMA from TMAO based on our pure culture work. Therefore, correlating microbiota and metabolite data derived from complex systems will not give a true picture of which members of microbiota contribute to specific metabolic processes, and work with pure cultures is required to supplement functional studies to increase our understanding of poorly understood microbially driven metabolic processes within the human gut.
In summary, we have demonstrated metabolic retroconversion – another example of host– microbial co-metabolism – occurs in the mammalian system with respect to TMAO, whereby TMAO is reduced by the gut microbiota to TMA and regenerated by host hepatic enzymes. We have also demonstrated that growth and metabolism of members of the gut microbiota are affected by TMAO in a source- and taxon-dependent manner, with the family Enterobacteriaceae making the greatest contribution to production of TMA in the gut.
ACKNOWLEDGEMENTS
M.L.J.-P., L.H. and A.L.M. performed the microbiological work, J.C. was responsible for NMR analysis and interpretation of the NMR data, T.A., F.B. and N.P. did the animal work, M.-E.D. supervised the NMR work, A.L.M. supervised the microbiological work and D.G. supervised the animal work. All authors contributed to the writing of the manuscript. M.L.J.-P. was sponsored by Fundación Alfonso Martin Escudero (Spain). Work was supported by a grant from the EU-FP7 (METACARDIS, HEALTH-F4-2012-305312) to D.G. and M.-E.D. L.H. is in receipt of an MRC Intermediate Research Fellowship in Data Science (grant number MR/L01632X/1, UK Med-Bio).
Footnotes
Abbreviations: AUC, area under the curve; DMA, dimethylamine; FISH, fluorescence in situ hybridization; FMO, flavin mono-oxygenase; MMA, monomethylamine; PC, phosphatidylcholine; TMA, trimethylamine; TMAO, trimethylamine N-oxide; UPLC–MS/MS, ultra-performance liquid chromatography–tandem mass spectrometry