Gut microbes with the <i>gbu</i> genes determine TMAO production from L-carnitine intake and serve as a biomarker for precision nutrition

Gut microbial metabolism of L-carnitine, which leads to the production of detrimental trimethylamine N-oxide (TMAO), offers a plausible link between red meat consumption and cardiovascular risks. Several microbial genes, including <i>cntA/B</i>, the <i>cai</i> operon, and the recently identified <i>gbu</i> gene cluster, have been implicated in the conversion of dietary L-carnitine into TMA(O). However, the key microbial genes and associated gut microbes involved in this pathway have not been fully explored. Utilizing the oral carnitine challenge test (OCCT), which specifically measures TMAO production from L-carnitine intake and identifies TMAO producer phenotypes, we compared the abundance of microbial genes between low- and high-TMAO producers across three independent cohorts. Our findings consistently revealed that the <i>gbu</i> gene cluster, rather than <i>cntA/B</i> or the <i>cai</i> operon, was significantly enriched in high-TMAO producers. We further analyzed 292 paired multi-omic datasets from OCCT and shotgun metagenomic sequencing, which demonstrated a significant positive correlation between the abundance of fecal <i>gbu</i> genes and L-carnitine-induced TMAO production, with <i>gbuB</i> showing the strongest correlation. Interestingly, these fecal <i>gbu</i> genes were found to increase with L-carnitine supplementation and decrease with a plant-based diet. Notably, we verified a previously uncultured <i>gbu</i>-containing bacterium, JAGTTR01 sp018223385, as the major contributor to TMA formation in the human gut. We isolated these <i>gbu</i>-containing gut microbes and confirmed their role in TMA/TMAO production using anaerobic incubation and a gnotobiotic mouse model. Using an in-house collection of <i>gbu</i>-containing isolates, we developed a qPCR-based method to quantify fecal <i>gbuB</i> and validated its correlation with L-carnitine-mediated TMAO production as measured by OCCT. Overall, these findings suggest that <i>gbu</i>-containing gut microbes are crucial for TMAO increases following L-carnitine intake and may serve as biomarkers or targets for personalized nutrition.