Urinary TMAO Levels Are Associated with the Taxonomic Composition of the Gut Microbiota and with the Choline TMA-Lyase Gene (cutC) Harbored by Enterobacteriaceae

Abstract

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

Keywords: 16S rRNA gene profiling; choline; linear mixed models; qPCR; trimethylamine; trimethylamine n-oxide.

Figure 1

Detection of the choline-utilization activity in pure bacterial cultures. Panels (A,B) represent agarose gel resulting from end-point PCR with primers cut-Dd (A) and cut-Kp (B). Panel (C) summarizes the detection of TMA in cell-free broth by mass spectrometry (MS) and nuclear magnetic resonance (NMR); +, TMA detected; -, TMA not detected. Lanes: 1, Escherichia coli 3.1; 2, Lactococcus garvieae FMBgr; 3, Enterococcus gilvus MD160; 4, Enterococcus hirae MD179; 5, Klebsiella oxytoca MIMgr; 6, Klebsiella sp. A1.2; 7, Streptococcus dysgalactiae 485; 8, Streptococcus dysgalactiae 486; 9, Streptococcus dysgalactiae A 1.2; NC, negative control (i.e., M17 broth incubated without bacteria).

Figure 2

Fecal levels of the cutC gene and daily urinary excretion of trimethylamine-N-oxide (TMAO). The relative abundance of cutC was determined by quantitative real-time polymerase chain reaction (qPCR) with the primer pair cut-Dd-F/R (panel A) and cut-Kp-F/R (B). The TMAO concentration was determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) in urine collected over 24 h (C). Green bars represent the mean ± standard deviation of three measurements per subject.

Figure 3

Analysis of the associations among fecal cutC gene abundances, fecal bacterial operational taxonomic units (OTUs), and urinary excreted TMAO carried out through a linear mixed model (LMM). Only OTUs that showed a significant association with cutC or TMAO are reported. The heatmap on the right represents TMAO levels, and cutC gene and OTU relative abundances. White boxes in the blue-yellow-red heatmap indicate that the OTU was not detected in that specific sample. The taxonomic lineage of each taxon is shown: p, phylum; c, class; o, order; f, family; g, genus; s, species. The black-yellow heatmap represents the Akaike’s information criterion (AIC) values of the LMM analysis. Asterisks indicate significant associations: * p < 0.05; ** p < 0.01; *** p < 0.001; ⁺, p < 0.1. syn. cut-Kp/Dd = synergy between cut-Kp and cut-Dd in LMM analysis.