Probiotic Bifidobacterium longum alters gut luminal metabolism through modification of the gut microbial community

Hirosuke Sugahara, Toshitaka Odamaki, Shinji Fukuda, Tamotsu Kato, Jin-zhong Xiao, Fumiaki Abe, Jun Kikuchi, Hiroshi Ohno, Hirosuke Sugahara, Toshitaka Odamaki, Shinji Fukuda, Tamotsu Kato, Jin-zhong Xiao, Fumiaki Abe, Jun Kikuchi, Hiroshi Ohno

Abstract

Probiotics are well known as health-promoting agents that modulate intestinal microbiota. However, the molecular mechanisms underlying this effect remain unclear. Using gnotobiotic mice harboring 15 strains of predominant human gut-derived microbiota (HGM), we investigated the effects of Bifidobacterium longum BB536 (BB536-HGM) supplementation on the gut luminal metabolism. Nuclear magnetic resonance (NMR)-based metabolomics showed significantly increased fecal levels of pimelate, a precursor of biotin, and butyrate in the BB536-HGM group. In addition, the bioassay revealed significantly elevated fecal levels of biotin in the BB536-HGM group. Metatranscriptomic analysis of fecal microbiota followed by an in vitro bioassay indicated that the elevated biotin level was due to an alteration in metabolism related to biotin synthesis by Bacteroides caccae in this mouse model. Furthermore, the proportion of Eubacterium rectale, a butyrate producer, was significantly higher in the BB536-HGM group than in the group without B. longum BB536 supplementation. Our findings help to elucidate the molecular basis underlying the effect of B. longum BB536 on the gut luminal metabolism through its interactions with the microbial community.

Conflict of interest statement

H.S., T.O., J.-z.X. and F.A. are employees of Morinaga Milk Industry Co., Ltd.

Figures

Figure 1. Experimental design of the mouse…
Figure 1. Experimental design of the mouse study.
Germ-free mice were inoculated with a single gavage of 15 species of a predominant human gut-derived microbiota cocktail at day -14 and were administered PBS containing Bifidobacterium longum BB536 or nothing (n = 6) every day for 14 days. Fecal samples were collected on days 0 and 13.
Figure 2. Water-soluble metabolic profiling in fecal…
Figure 2. Water-soluble metabolic profiling in fecal samples of the human gut-derived microbiota (HGM) mice.
(a) Principal component analysis (PCA) of fecal 1D 1H-NMR spectroscopy profiles. The contributions of PC1, PC2 and PC3 were 63.7%, 20.1% and 6.3%, respectively. (b) Fecal pimelate, butyrate and acetate levels at day 13. All compounds were calculated and identified using 1H-13C HSQC 2D-NMR measurements (n = 6). Boxes denote the interquartile range between the first and third quartiles, and the line within denotes the median. P-values were calculated using Mann–Whitney U test. *P < 0.05; **P < 0.01. (c) Biotin concentrations in fecal samples at day 13. Data are shown as the mean ± SD (n = 6). P-values were calculated using Student’s t-test. *P < 0.05. (d) Correlation analysis based on Spearman’s rank correlation coefficient between fecal pimelate and biotin levels. Fecal pimelate and biotin levels are shown as open symbols, and the line indicates a simple regression model.
Figure 3. Metabolite levels in germ-free and…
Figure 3. Metabolite levels in germ-free and Bifidobacterium longum mono-associated mice.
(a) Fecal pimelate, butyrate and acetate levels measured by 2D-NMR. The levels were based on the normalized intensities calculated from 1H-13C HSQC 2D-NMR measurements (n = 5). Boxes denote the interquartile range between the first and third quartiles, and lines within the boxes denote the median. P-values were calculated using the Mann–Whitney U test. (b) Acetate concentration in fecal samples measured by enzyme method. Data are shown as the mean ± SD (n = 5). P-values were calculated using Student’s t-test. **P < 0.01. (c) Biotin concentration in fecal samples after treatment. Data are shown as the mean ± SD (n = 5). P-values were calculated using Student’s t-test. *P < 0.05.
Figure 4. Distribution of bacterial transcripts in…
Figure 4. Distribution of bacterial transcripts in fecal samples of the human gut-derived microbiota (HGM) mice.
Boxes denote the interquartile range between the first and third quartiles, and the line within denotes the median. P-values were calculated using the Mann–Whitney U test. *P 

Figure 5. Predicted contribution to biotin production…

Figure 5. Predicted contribution to biotin production in gut microbiota.

( a ) Components of…

Figure 5. Predicted contribution to biotin production in gut microbiota.
(a) Components of pathways from pimelate to biotin. The black arrow indicates the step with a significant difference between the two groups. (b) Gene expression with a significant difference between the two groups. RPKM-normalized reads are shown as boxes that denote the interquartile range between the first and third quartiles, and the line within denotes the median (n = 6). P-values were calculated using the Mann–Whitney U test. *P < 0.05. (c) In vitro assay of biotin synthesis. Each strain was cultivated for 16 h in GAM broth with or without pimelate (10 mg/ml). Data are shown as the mean ± SD (n = 3). P-values were calculated using Student’s t-test. **P < 0.01.

Figure 6. Proposed effect of Bifidobacterium longum…

Figure 6. Proposed effect of Bifidobacterium longum BB536 on the gut luminal metabolism mediated by…

Figure 6. Proposed effect of Bifidobacterium longum BB536 on the gut luminal metabolism mediated by interaction with the human gut-derived microbiota (HGM) community.
Figure 5. Predicted contribution to biotin production…
Figure 5. Predicted contribution to biotin production in gut microbiota.
(a) Components of pathways from pimelate to biotin. The black arrow indicates the step with a significant difference between the two groups. (b) Gene expression with a significant difference between the two groups. RPKM-normalized reads are shown as boxes that denote the interquartile range between the first and third quartiles, and the line within denotes the median (n = 6). P-values were calculated using the Mann–Whitney U test. *P < 0.05. (c) In vitro assay of biotin synthesis. Each strain was cultivated for 16 h in GAM broth with or without pimelate (10 mg/ml). Data are shown as the mean ± SD (n = 3). P-values were calculated using Student’s t-test. **P < 0.01.
Figure 6. Proposed effect of Bifidobacterium longum…
Figure 6. Proposed effect of Bifidobacterium longum BB536 on the gut luminal metabolism mediated by interaction with the human gut-derived microbiota (HGM) community.

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