Short-term supplementation with ω-3 polyunsaturated fatty acids modulates primarily mucolytic species from the gut luminal mucin niche in a human fermentation system

Abstract

Consumption of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) provides multifaceted health benefits. Recent studies suggest that ω-3 PUFAs modulate the gut microbiota by enhancing health-promoting bacteria, such as the mucin specialist Akkermansia muciniphila. However, these prebiotic properties have been poorly investigated and direct effects on the gut microbiome have never been explored dynamically across gut regions and niches (lumen vs. mucus-associated microbiota). Thus, we studied the effects of 1 week EPA- and DHA-enriched ω-3 fish-oil supplementation on the composition and functionality of the human microbiome in a Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME®). Gut microbial communities derived from one individual harvested in two different seasons were tested in duplicate. Luminal and outer mucus-associated microbiota of the ileum, ascending, transverse and descending colons were cultivated over 28 d from fecal inoculates and supplemented with ω-3 PUFAs for the last 7 d. We show that ω-3 PUFA supplementation modulates the microbiota in a gut region- and niche-dependent fashion. The outer mucus-associated microbiota displayed a higher resilience than the luminal mucin habitat to ω-3 PUFAs, with a remarkable blooming of Akkermansia muciniphila in opposition to a decrease of Firmicutes-mucolytic bacteria. The ω-3 PUFAs also induced a gradual and significant depletion of non-mucolytic Clostridia members in luminal habitats. Finally, increased concentrations of the short chain fatty acids (SCFA) propionate in colon regions at the end of the supplementation was associated positively with the bloom of Akkermansia muciniphila and members of the Desulfovibrionia class.

Keywords: Akkermansia muciniphila; DHA; EPA; M-SHIME® fermentation system; SCFA; fish oil; gut microbiota; mucolytic bacteria; prebiotics; Ω-3 PUFAs.

Figure 1.

Gut habitats, niches, and ω-3 supplementation as the main explanatory variables to the microbiota community structure at the genus level, in the M-SHIME®. (a) Schematic representation of the different gut habitats and niches recreated in the M-SHIME®. (b-d) Type II scaling triplots obtained using partial distance-based redundancy analysis (db-RDA) of the microbial community composition detected using 16S rRNA gene amplicon sequencing. Treatment condition (b), gut habitat (c) and lumen (l)/mucus (m) niche (d) were set as explanatory variables (in blue) and abundances of genera as response variables (purple arrows). Only the top ten genera were displayed for adequate visibility. Axes are annotated with their contribution to the total variance. “Vexplained” indicates the variability in the gut microbiota composition explained by the variables condition, gut habitat, and lumen/mucus niches. ***indicate the p < .001 significance of the observed group separation, as assessed with a Permutational Multivariate Analysis of Variance (PERMANOVA) using distance matrixes. (e) Genus level relative abundance of the luminal (on top) and mucosa-associated (bottom) microbiota composition following a 1-week control (C; blue lines) versus 1-week ω-3 treatment (T; Orange lines) at indicated days across the successive gut habitats/niches. For technical reasons (see Methods), mucin beads containing mucosa-associated microbiota were sampled 2 times under each condition. The barplots represent the mean composition of four replicates. The 20 most abundant genera are represented.

Figure 2.

Contrasting response to ω-3 supplementation between luminal/mucus-associated microbiota in the M-SHIME®. (a) Evolution of Shannon diversity index following a 1-week control versus 1-week ω-3 supplementation across the successive gut habitats/niches (n = 4 replicates). * represent the p < .05 significant differences with control based on Wilcoxon Rank Sum tests with Holm’s correction (α = .05). (b-e) Volcano plots indicating the genera significantly enriched by the ω-3 supplementation in the transverse (b and d) and descending (c and e) gut habitats of the luminal and mucosa-associated (m) niches of the M-SHIME®. A positive log2 fold-change indicates a stimulation of the genus under the ω-3 supplementation period (in Orange) while a negative log2 fold-change indicates a decrease of the genera compared to the control period (in blue), as determined by Deseq2 analysis. Statistical differences between the control and ω-3 PUFA supplementation across gut habitats were determined using a Wald Test. The log transformed adjusted p-value is displayed on the y-axis and the α = .05 significance level is indicated by a dashed line.

Figure 3.

Mucolytic bacteria of luminal floating mucin habitats dynamically shifted following the ω-3 supplementation. (a-c) Selection of mucolytic bacteria that displayed significant differences in ASVs level abundance between control and ω-3 supplementation, as assessed by DESeq2 analysis in the different gut habitats and over time (gradient of colors). Statistical differences between the control and ω-3 PUFA supplementation across gut habitats were determined using a Wald Test. (d) Phylum level relative abundance of the luminal microbiota composition following a 1-week control (C) versus 1-week treatment (T) across the successive gut habitats. Control and treatment conditions are demarcated in blue and Orange lines, respectively. The barplots represent the mean composition of four replicates.

Figure 4.

Temporal depletion of genera in luminal floating mucin habitats following the ω-3 supplementation. Bacteria displaying significant differences in genus level abundance between control and ω-3 supplementation, as assessed by DESeq2 analysis in the different gut habitats and over time (gradient of colors). Statistical differences between the control and ω-3 PUFA supplementation across gut habitats were determined using a Wald Test. Colored labels indicate the class of the respective genera. Scissors indicate the genus known for their mucolytic functions.

Figure 5.

Temporal stimulation of genera in luminal floating mucin habitats following the ω-3 supplementation. Bacteria displaying significant differences in genus level abundance between control and ω-3 supplementation, as assessed by DESeq2 analysis in the different gut habitats and over time (gradient of colors). Statistical differences between the control and ω-3 PUFA supplementation across gut habitats were determined using a wald test. colored labels indicate the class of the respective genera.

Figure 6.

SCFA modulation under ω-3 supplementation. Mean concentrations of major (a) and minor (b) SCFAs in the 4 replicates ± SD across each luminal gut habitat in control (C; blue lines) and treated (T; Orange lines) at different days. Statistically significant differences between control period and ω-3 supplementation are denoted for p < .05 (*), p < .01 (**), and p < .001 (***) as determined by Pairwise Wilcoxon rank sum tests with holm correction.

Figure 7.

Associations between class taxa and SCFA concentrations in habitats and treatment-dependent fashion. Correlation networks illustrating the Spearman correlations between the SCFA produced under control (c) vs. ω-3 supplementation (t) and their corresponding class enrichment in opposed environments displayed in the luminal ileum (a) and descending colon (b). Gradient color, distance, and thickness of the lines were applied to nodes depending on coefficients of correlation. Only significant nodes that are upper to a coefficient of correlation of 0.5 or −0.5 are shown. Negative and positive correlations are denoted in shades of red and blue, respectively. SCFA nodes are highlighted in yellow.