Human-origin probiotic cocktail increases short-chain fatty acid production via modulation of mice and human gut microbiome

Abstract

The gut bacteria producing metabolites like short-chain fatty acids (SCFAs; e.g., acetate, propionate and butyrate), are frequently reduced in Patients with diabetes, obesity, autoimmune disorders, and cancers. Hence, microbiome modulators such as probiotics may be helpful in maintaining or even restoring normal gut microbiome composition to benefit host health. Herein, we developed a human-origin probiotic cocktail with the ability to modulate gut microbiota to increase native SCFA production. Following a robust protocol of isolation, characterization and safety validation of infant gut-origin Lactobacillus and Enterococcus strains with probiotic attributes (tolerance to simulated gastric and intestinal conditions, adherence to intestinal epithelial cells, absence of potential virulence genes, cell-surface hydrophobicity, and susceptibility to common antibiotics), we select 10 strains (5 from each genera) out of total 321 isolates. A single dose (oral gavage) as well as 5 consecutive doses of this 10-strain probiotic cocktail in mice modulates gut microbiome and increases SCFA production (particularly propionate and butyrate). Inoculation of these probiotics in human feces also increases SCFA production along with microbiome modulation. Results indicate that human-origin probiotic lactobacilli and enterococci could ameliorate gut microbiome dysbiosis and hence may prove to be a potential therapy for diseases involving reduced SCFAs production in the gut.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Flowchart depicting the process of isolation, characterization, and screening of human-origin probiotic lactobacilli and enterococci strains used in these experiments.

Figure 2

Human-origin probiotics inhibit uropathogenic bacterial growth. (a,b) Probiotic culture supernatant significantly reduced growth of clinical isolate of the human uropathogen E. coli (a) and Klebsiella pneumoniae (b) based on well diffusion assays. (c,d) Anti-microbial activity of probiotics supernatant against E. coli after heat (c) and proteinase K (d) treatments. (e) Neutralizing pH to 6.5 of probiotic supernatant abolished antimicrobial activity against E. coli. (f) Lactate inhibits E. coli in a dose-dependent fashion. (g–i) Probiotic produced significant amount of organic acid like lactate (g), acetate (h) and butyrate (i) in the supernatant. All the values plotted are means of 3–6 replicates or independent experiments and standard error of means. All the values are significantly different compared to media only (no-zone) control.

Figure 3

Single-dose feeding of human-origin probiotics causes mild changes in gut microbiome and increases SCFA production in the mouse gut. (a) PCA analysis showing the β-diversity clustering of gut microbiome from mice (n = 6 each group) treated with single-dose of five lactobacilli, five enterococci, and the cocktail containing all 10 strains, before treatment (0 h) and after 8 h, 1d, 3d and 10d of feeding. (b) Shannon index representing the alpha-diversity of gut microbiome at 0 h and after 8 h, 1d, 3d and 10d of lactobacilli, enterococci and cocktail treatments compared to non-treated control groups. (c) Major changes in bacteria phyla after a single dose of probiotics for 10 days. (d) Cladograms of linear discrimination analysis (LDA) showing clustering of gut microbiome after 1d of single-dose treatment of lactobacilli (upper panel), enterococci (middle panel) and cocktail (lower panel) compared to control group. (e) Fecal levels of SCFAs, lactate (upper panel), acetate (upper middle panel), propionate (lower middle panel) and butyrate (lower panel) increase after lactobacilli, enterococci and cocktail treatments. Values are mean ± SEM of n = 6 animals per group. Each dot in PCA analysis represents 2 samples pooled from 2 independent mice from same group, hence gut microbiome data will have this factor in all the data analyses at different time-points. SCFAs data represents duplicates of 5–6 and standard error of means. Values with *<0.05, **0.01 and ***0.001 are significantly different within the same group compared to 0 h time point. Values with ● < 0.05, ●● < 0.01 are significantly different compared to controls at the same time points.

Figure 4

Five-day feeding of selected probiotics significantly increases microbiome diversity and SCFAs production in the mouse gut. (a) PCA analysis showing the β-diversity clustering of gut microbiome in mice after 5 days of once-daily feeding of lactobacilli, enterococci and their combination (cocktail) and non-treated control mice on 0d (before treatment), 5d, 2wks, 3wks and 5wks (after 1, 2 and 4 weeks of treatment). (b) Alpha-diversity (Shannon index) in mice undergoing 5 days of once-daily feeding of lactobacilli, enterococci and cocktail compared to control mice. (c) Major phyla changes upon 5 days of once-daily feeding of probiotic treatments up to 5 weeks. (d) Cladogram showing microbial signatures upon 5 days of once-daily feeding of lactobacilli, enterococci and cocktail compared to control mice. (e) SCFAs including lactate, acetate, propionate and butyrate levels after 5 days of once-daily feeding of probiotics treatment over the time course up to 30 days. Each dot in PCA analysis represents 2 samples pooled from 2 independent mice from same group, hence gut microbiome data will have this factor in all the data analyses at different time-points. SCFA data are duplicates of 5–6 and standard error of means. Values with *<0.05, **0.01 and ***0.001 are significantly different within the same group compared to 0 h time point. Values with ● < 0.05, ●● < 0.01 are significantly different compared to controls at the same time points.

Figure 5

Probiotic inoculation differentially changes the human fecal microbiome ex-vivo with increased production of SCFAs. (a) PCA analysis of human fecal microbiome showing beta-diversity upon treatment with lactobacilli, enterococci and probiotics cocktail after 9 and 24 h of anaerobic incubation in conditioned medium following the probiotic inoculation. (b,c) Shannon index (b) and major phyla changes upon lactobacilli, enterococci and cocktail treatment over the time up to 24 h. (d) Microbial cladogram indicating microbial clustering of human fecal microbiome in lactobacilli, enterococci and cocktail treated specimen compared to control specimen. (e) SCFAs including lactate, acetate, propionate and butyrate levels in human fecal microbiome after lactobacilli, enterococci and cocktail treatment up to 24 h. Values with *< 0.05, **0.01 and ***0.001 are significantly different within the same group compared to 0 h time point. Values with ● < 0.05, ●● < 0.01 are significantly different compared to controls at the same time points.