Figure 2. E. timonensis is sufficient to provide vegan fecal polymicrobial communities with the anaerobic metabolic transformation capability to produce TMA from L-carnitine.

(a) LC-MS/MS quantification of d₉-γBB consumption and d₉-TMA production by fifty different human fecal communities 24 h after exposure to d₉-γBB under either aerobic or anaerobic conditions. Results are presented as box-and-whisker plots with each point the mean metabolite value from three biological replicates of the same fecal bacterial community. The center line in the box shows the median for the data. The whiskers represent the expected variation of the data. The whiskers extend 1.5 times the IQR from the top and bottom of the box. If the data do not extend to the end of the whiskers, then the whiskers extend to the minimum and maximum data values. Fecal samples were collected from 50 healthy human subjects self-reported as omnivores. Significance was determined using two-way t-test.

(b) LC-MS/MS quantification of d₉-L-carnitine consumption and d₉-TMA production by fifty different human fecal communities 24 h after exposure to d₉-L -carnitine under either aerobic or anaerobic conditions. Results are presented as box-and-whisker plots with each point the mean metabolite value from three biological replicates of the same fecal bacterial community. The center line in the box shows the median for the data. The whiskers represent the expected variation of the data. The whiskers extend 1.5 times the IQR from the top and bottom of the box. If the data do not extend to the end of the whiskers, then the whiskers extend to the minimum and maximum data values. Significance was determined using two-way t-test.

(c) Schematic of anaerobic (red) vs aerobic (blue) conversion of L-carnitine → TMA transformation. Arrow widths approximate measured contribution.

(d-e). Production of d₉-TMA from exogenously added d₉-L-carnitine (E) or d₉-γBB (D) by fecal polymicrobial communities sourced from either healthy human omnivore (Omnivore FM [fecal microbiome]) or vegan (Vegan FM) donors. Where indicated, vegan polymicrobial communities were supplemented ex vivo with E. timonensis (Vegan FM + E. tim). E. timonensis alone (Ctrl) and media with no polymicrobial communities (No Addition) were used as additional controls. Samples were incubated for 24 h under anaerobic conditions. All biological replicates (n=3) are shown. Bar height represents the mean with error bars are ± one standard deviation from the mean.