Figures & data
Table 1. List of bacterial strains used in this study
Figure 1. Growth-promoting effects of existing prebiotics and Gal-β1,4-Rha on beneficial bacteria and the most prominent 27 bacterial species in human gut microbiota
(a-f) Bacterial species were cultured in GAM-wos with or without 0.5% prebiotic supplements for 24 h. Bacterial growth was measured by determining the OD600. To compare the growth promotion ability by prebiotics for each bacterium, the growth-promoting value (Ratio of OD600) was obtained by dividing the average of growth (OD600) in the medium supplemented with prebiotics (n = 3) by that without prebiotics (n = 3). White bars, the most prominent 27 bacterial species in the human gut microbiota; gray bars, pathogenic bacterium; black bars, lactic acid bacteria; diagonal bars, bifidobacteria. GAM-wos was supplemented with raffinose (a), 1-kestose (b), lactulose (c), GOS (d), FOS (e), and Gal-β1,4-Rha (f). Data represent means ± standard deviation.
Figure 2. The importance of the BL105A_0502 gene for the utilization of Gal-β1,4-Rha in B. longum 105-A
(a-b) Growth curve of B. longum 105-A wild-type and mutants with a disruption of the gene encoding ABC transporter solute-binding protein in mMRS-wos containing 0.5% Gal-β1,4-Rha (a) or 0.5% galactose (b) as a carbon source (n = 1). The disrupted genes were indicated by the locus tag. Black triangle, BL105A_0201 mutant; black rhombus, BL105A_0500 mutant; black circle, BL105A_0501 mutant; black square, BL105A_0502 mutant; gray triangle, BL105A_1223 mutant; gray rhombus, BL105A_1604 mutant; gray circle, BL105A_1817 mutant; gray square, BL105A_1867 mutant; white triangle, BL105A_1888 mutant; white rhombus, BL105A_1890 mutant; white circle, BL105A_1896 mutant; white square, wild-type. (c-d) Growth of B. longum wild-type (white squares) and the BL105A_0502 mutant (black squares) in mMRS-wos containing 0.5% Gal-β1,4-Rha (c), or 0.5% galactose (d) as a carbon source (n = 3). Data represent means ± standard deviation. (e) Presence of BL105A_0502 homolog in bifidobacteria and 56 prominent bacteria in the human gut microbiota. The results shown are based on the Protein BLAST analysis of genomes in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The color of each column indicates the score from the Protein BLAST analysis: (black) >500 bits and (white) ≤500 bits. If no genome information was available in the NCBI database, it was indicated by N/A.
Figure 3. Growth inhibition of C. difficile by B. infantis in the presence of Gal-β1,4-Rha and the importance of BLIJ_2090 gene for the growth inhibition
(a, c, e, and g) Circles indicated the viable cell number of C. difficile. Monocultures, co-cultures with B. infantis wild-type, and co-culture with B. infantis BLIJ_2090 mutant are indicated by white, black, and gray symbols, respectively. (b, d, f, and h) The viable cell number of wild-type B. infantis and the BLIJ_2090 mutant is indicated by squares and rhombuses, respectively. Mono-cultures, co-cultures with B. infantis wild-type, and co-culture with the B. infantis BLIJ_2090 mutant are indicated by white, black, and gray symbols, respectively. GAM-wos was supplemented with 0.5% of Gal-β1,4-Rha (a-b, g-f and i), 0.5% of glucose (e-f), or without sugar (c-d). In i, viable cell numbers of C. difficile co-cultured with wild-type B. infantis (deduced from panel a) was compared to that with B. infantis BLIJ_2090 mutant (deduced from panel g). Data represent means ± standard deviation from biological replicates (n = 3).
Figure 4. Inhibition of the production of CD toxins A and B by the combination of Gal-β1,4-Rha and B. infantis in fecal culture
C. difficile, B. infantis, and/or human fecal suspension from 5 donors were cultured in GAM-wos supplemented with or without 0.5% Gal-β1,4-Rha for 48 h, and CD toxins A and B in the fecal cultures were quantified by ELISA. Black bar, fecal suspension, and C. difficile were cultured in GAM-wos; gray bar, C. difficile, B. infantis, and fecal suspension were co-cultured in GAM-wos; checkered bar, C. difficile B. infantis, and fecal suspension were co-cultured in GAM-wos supplemented with 0.5% Gal-β1,4-Rha; diagonal bar, C. difficile and fecal suspension were cultured in GAM-wos supplemented with Gal-β1,4-Rha. These culture conditions are summarized in the table below the Figure (+, when the components were added to the medium; -, when not added). For all fecal suspensions, CD toxin was not detected when C. difficile was not inoculated into the culture and is not shown in the figure. Data represent means ± standard deviation. Paired one-way ANOVA followed by multiple comparison with Bonferroni correction was performed. Individual data points are represented by circles.
Figure 5. Effects of Gal-β1,4-Rha and B. infantis on weight loss in mice owing to C. difficile infection
(a) Experimental outline. The experimental groups are shown in the table (+, administered; −, not administered). The experimental schedule is shown below the table. The arrows indicate the timing of Gal-β1,4-Rha or bacterial administration. (b-c) Changes in body weights. b: Asterisks indicate statistical significance versus the same group on the day of the previous measurement after C. difficile infection. c: Asterisks indicate statistical significance versus groups A and D on day 7. (*p <.05 and **p <.01, two-way ANOVA with repeated measurement followed by Tukey multiple comparison test) Data represent means ± standard deviation. Individual data points are represented by circles. (d-e) Copy number of beta-galactosidase 42B gene of B. infantis (d) and 16S rRNA gene of C. difficile (e) in feces on day 7 quantified by qPCR. B. infantis has 1 copy of beta-galactosidase 42B gene per cell, and C. difficile has 12 copies of the 16S rRNA gene per cell. Asterisks indicate statistical significance (*p <.05, one-way ANOVA with Tukey-Kramer multiple comparison test). Data represent means ± standard deviation. Individual data points are represented by circles.
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