Enrichment and characterization of human-associated mucin-degrading microbial consortia by sequential passage

Abstract

Mucin is a glycoprotein secreted throughout the mammalian gastrointestinal tract that can support endogenous microorganisms in the absence of complex polysaccharides. While several mucin-degrading bacteria have been identified, the interindividual differences in microbial communities capable of metabolizing this complex polymer are not well described. To determine whether community assembly on mucin is deterministic across individuals or whether taxonomically distinct but functionally similar mucin-degrading communities are selected across fecal inocula, we used a 10-day in vitro sequential batch culture fermentation from three human donors with mucin as the sole carbon source. For each donor, 16S rRNA gene amplicon sequencing was used to characterize microbial community succession, and the short-chain fatty acid profile was determined from the final community. All three communities reached a steady-state by day 7 in which the community composition stabilized. Taxonomic comparisons amongst communities revealed that one of the final communities had Desulfovibrio, another had Akkermansia, and all three shared other members, such as Bacteroides. Metabolic output differences were most notable for one of the donor's communities, with significantly less production of acetate and propionate than the other two communities. These findings demonstrate the feasibility of developing stable mucin-degrading communities with shared and unique taxa. Furthermore, the mechanisms and efficiencies of mucin degradation across individuals are important for understanding how this community-level process impacts human health.

Keywords: fermentation; gastrointestinal microbiome; microbial ecology; mucin.

Figure 1.

Final (day 10) optical density and pH measurements of microbial consortia from three human donors established with mucin as the sole carbon source supplemented with different amino acid sources. Each community grew to different final optical densities (A) and produced different levels of acid (B) after 10 days of incubation. Averages of three lineages from each final donor community ((Donor 1, D1; Donor 2, D2; and Donor 3, D3) are displayed with error bars representing standard deviation as calculated in R. Statistically significant differences are calculated by Tukey’s multiple comparisons test with P < .05. Symbol style: nonsignificant (ns), 0.05 (*), 0.01 (**), 0.001(***), and <0.0001(^****).

Figure 2.

SCFA profiles of microbial consortia from three human donors established with mucin as the sole carbon source after 10 days of sequential transfer. Mucin-degrading consortia from donors one and three produced the highest total concentration of SCFAs driven by acetate and propionate, whereas the donor two consortium produced lower concentrations, irrespective of amino acid source. Mean, first, and third quartiles of total SCFAs (A), acetate (B), propionate (C), and butyrate (D) for each final donor community (Donor 1, D1; Donor 2, D2; and Donor 3, D3) are represented. Statistically significant differences are calculated by Tukey’s multiple comparisons test. Symbol style: nonsignificant (ns), 0.05 (*), 0.01 (**), 0.001(***), and <0.0001(^****).

Figure 3.

Principal coordinates of analysis plots of Bray–Curtis dissimilarity between communities for each donor over 10 days with supplementation of either amino acids or tryptone. Donors are represented by fill color, days by hue of the dominant color, amino acid supplementation by border color, and lineage by shape. Principle coordinates 1 and 2 (A) and 1 and 3 (B) indicate that the community from each donor follows a distinct trajectory.

Figure 4.

Bacterial diversity at each passage for all three donors (Donor 1, D1; Donor 2, D2; and Donor 3, D3) when supplemented with amino acids provided as an equimolar mix ("AminoAcids") or peptides ("Tryptone"). Species richness as measured by the number of ASVs (A) and evenness as measured by Shannon entropy (B) plateaued for all three communities. Mean, first, and third quartiles are represented with fill color indicating amino acid source. Statistically significant differences at day 10 are calculated by Tukey’s multiple comparisons test. Symbol style: nonsignificant (ns), 0.05 (*), 0.01 (**), 0.001(***), and <0.0001(^****).

Figure 5.

Relative abundance of bacterial taxa for each donor over 10 days with supplementation of either amino acids or tryptone. Averaged abundance of three replicate lineages for each donor indicates mucin sustains diverse fermenting consortia over sequential dilutions that is dependent on the initial community. Day 0 (n = 1) is the initial fecal inoculum prior to incubation, whereas each subsequent day reflects the average of three lineages after incubation. Each shade represents a distinct ASV classified down to the level of genus, with the color representing a phylum: Bacteroidota (purple), Desulfobacterota (yellow), Firmicutes (green), Proteobacteria (grey), and Verrucomicrobiota (red). Numbers following genus names represent different ASVs. The top 40 ASVs across all donors (Donor 1, D1; Donor 2, D2; and Donor 3, D3) are shown, where remaining low-abundance ASVs are grouped in Other (light grey).