Molecular Identification and Selection of Probiotic Strains Able to Reduce the Serum TMAO Level in Mice Challenged with Choline

Abstract

Trimethylamine oxide (TMAO) originates from trimethylamine (TMA), which is oxidized in the liver by hepatic flavin-containing monooxygenases (FMO3). TMA is produced by its dietary precursors such as choline, carnitine, and phosphatidylcholine by gut microbiota. TMAO attracts attention, identified as a novel and independent risk factor for promoting obesity, atherosclerosis and cardiovascular disease (CVD), chronic kidney disease (CKD), insulin tolerance, and colon cancer. Probiotics have been considered as live microorganisms, providing benefits to their host when they are given in sufficient quantities and administered continuously. The objective of this study is to suggest a method to select potential probiotic strains to reduce the serum concentration of TMAO in mice fed with choline. In this work, we chose three lactobacilli with strong adherence capability, and fed multistrain formula (MF) to the mice challenged with choline. On days 7, 14, and day 28, it was found that the MF-containing L. amylovorus LAM1345, Lpb. plantarum LP1145, and Lim. fermentum LF33 showed a significant reduction in serum TMAO and TMA levels. For the single strains, LP1145 reduced TMAO on days 14 and 28, and strain LAM1345 reduced TMAO significantly on days 7 and day 14. For strain LF1143 from strain LF33, it showed no significant effect on TMAO and TMA. Thus, MF showed the best effect, which may be due to the additive and synergetic effect and the contribution of strain LP1145 and LAM1345. Finally, for the LAM1345 and LP1145 strains, we used molecular identification and typing methods to assure that these two strains are unique strains. The methods used for LAM 1345 were leader peptidase A (lepA) gene analysis and phylogenetic analysis, while for strain LP 1145and other strains of Lpb. plantarum subsp. plantarum sequences were compared using the whole-genome multilocus sequence typing (wgMLST) method.

Keywords: L. amylovorus; Lpb. plantarum; TMAO; WgMLST; cardiovascular disease; choline-fed mice; lactobacilli.

Figure 1

Probiotic multistrain formula decreases the plasma TMAO and TMA levels in C57BL/6J mice. Experimental conditions were as described in Methods. Data are shown as mean values ± SD (n = 7). A p-value of <0.05 was considered to be statistically significant, * p <0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001.

Figure 2

Probiotic strain Lpb. plantarum decreases the plasma TMAO and TMA levels in C57BL/6J mice. Experimental conditions were as described in Methods. Data are expressed as mean values ± SD (n = 7). ** p < 0.01, *** p <0.001, **** p < 0.0001.

Figure 3

Effect of L. amylovorusLAM1345 and Lim. fermentum LF1145 on serum TMAO and TMA levels. Experimental conditions were as described in Methods. Values are represented as mean values ± SD (n = 7) ** p < 0.01, *** p <0.001, **** p < 0.0001.

Figure 4

Total lactobacilli counts in mice cecum. Experimental conditions were as described in Methods. Values are shown in mean value ± SD (n = 7).

Figure 5

Phylogenetic tree based on the 16S rRNA gene sequence showing the phylogenetic relationships between the LAM1345 strain and related lactobacilli species.

Figure 6

Phylogenetic tree of the lepA gene of strain LAM1345 strain and related lactobacilli species.

Figure 7

The allele-based minimum spanning tree constructed with cgMLST profiles for the 52 Lactiplantibacillus plantarum subsp. plantarum strains on the basis of a comparison of 2233 core genes. Each circle represents a different sequence type.

Figure 8

The allele-based UPGMA tree constructed with cgMLST profiles for the 52 Lactiplantibacillus plantarum subsp. plantarum strains on the basis of a comparison of 2233 differentiated core genes.