Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon

Abstract

The release of energy from particulate substrates such as dietary fiber and resistant starch (RS) in the human colon may depend on the presence of specialist primary degraders (or 'keystone species') within the microbial community. We have explored the roles of four dominant amylolytic bacteria found in the human colon in the degradation and utilization of resistant starches. Eubacterium rectale and Bacteroides thetaiotaomicron showed limited ability to utilize RS2- and RS3-resistant starches by comparison with Bifidobacterium adolescentis and Ruminococcus bromii. In co-culture, however, R. bromii proved unique in stimulating RS2 and RS3 utilization by the other three bacterial species, even in a medium that does not permit growth of R. bromii itself. Having previously demonstrated low RS3 fermentation in vivo in two individuals with undetectable populations of R. bromii-related bacteria, we show here that supplementation of mixed fecal bacteria from one of these volunteers with R. bromii, but not with the other three species, greatly enhanced the extent of RS3 fermentation in vitro. This argues strongly that R. bromii has a pivotal role in fermentation of RS3 in the human large intestine, and that variation in the occurrence of this species and its close relatives may be a primary cause of variable energy recovery from this important component of the diet. This work also indicates that R. bromii possesses an exceptional ability to colonize and degrade starch particles when compared with previously studied amylolytic bacteria from the human colon.

Figure 1

Utilization of different starches by four species of amylolytic bacteria that are abundant in the human colon. The % decrease in total sugar in cultures is shown after 72 h incubation with Ruminococcus bromii L2-63 (R.bromii), Bifidobacterium adolescentis L2-32 (B.adol), Eubacterium rectale A1-86 (E.rectale) or Bacteroides thetaiotaomicron 5482 (B.theta). Strains were inoculated into YCFAS medium or M2S medium in the case of R.bromii that contained 0.2% starch. Starches shown are: high amylopectin corn starch (S9679 - S1); high amylose corn starch (S4180 - S2); and RS2- (HM-958 - S3) and RS3 (NL-330 - S4) -resistant starches. Results are shown for three different pretreatment regimes (autoclaved, boiled and raw). Data are given for three further starches in Supplementary Table S1.

Figure 2

Growth of human colonic amylolytic bacteria on starch-derived sugars. (a) Growth (monitored by OD₆₅₀) is shown for R. bromii in M2 medium containing 0.2% of the carbohydrate indicated. Fructose was included as a reference substrate as R. bromii was unable to utilize glucose. Growth of B. adolescentis L2-32, E. rectale A1-86 and B. thetaiotaomicron 5482 was on YCFA medium containing 0.2% carbohydrates. (b) Summary of the abilities of B. adolescentis (Bif), B. thetaiotaomicron (Bac), E. rectale (Erec) and R. bromii (Rum) to utilize different sugars. The darkest shading indicates the substrate supporting the most rapid growth rate for each strain, whereas lighter shadings indicate less rapid growth (see also Supplementary Figure S1). Blanks indicate no growth.

Figure 3

Stimulation of starch utilization in co-cultures containing R. bromii. Total sugar utilized and the concentration of free soluble reducing sugar are shown after 48 h incubation in YCFA medium containing RS2 (High-maize 958) or RS3 starch. Monocultures and co-cultures of the four bacterial strains are indicated as follows: Rum, R. bromii; Bif, B. adolescentis; Erec, E. rectale and Bac, B. thetaiotaomicron. Initial (t0) total sugar concentrations were 2039±39 and 2043±81 μg ml⁻¹ and reducing sugar concentrations were 91±39 and 63±25 μg ml⁻¹ for RS3 and RS2, respectively.

Figure 4

Interaction between R. bromii and E. rectale in co-culture. (a) Time course of total sugar utilization and reducing sugar release on YCFAS and M2S medium containing 0.2% boiled RS3 by monocultures and co-cultures of R. bromii L2-63 and E. rectale A1-86. (b) Comparison by qPCR, using primer pairs specific to each bacterium, of the relative growth of the two bacteria for the experiment shown in (a) in M2S medium. The signal obtained with each specific primer pair is expressed relative to the signal obtained with the ‘universal' primer pair. Equal volumes of the two monocultures were mixed immediately after inoculation (t0) or after 48 h growth (t48) confirming the greater growth of R. bromii relative to E. rectale on this substrate. The same analysis on the co-culture, however, showed that E. rectale had outgrown R. bromii after 48 h.

Figure 5

Fermentation of RS3 in vitro by mixed human fecal bacteria. (a) Utilization of RS3 is shown for fecal samples from three healthy control volunteers (v2, v6 and v7) and from one volunteer (v25) previously found to show low starch fermentation in vivo. (b) Representation of different bacterial groups estimated by 16S-rRNA-targeted qPCR in the fecal samples employed in (a). (Bif, bifidobacteria, Fprau, F. prausnitzii; Rum, cluster IV ruminococci; Bac, Bacteroidetes; Ros, relatives of E. rectale/Roseburia). Abundance for each group is expressed as a percentage of the 16S-rRNA signal obtained using universal primers. (c–f) Effect on RS3 utilization of adding each of the four isolated amylolytic bacteria to fecal inocula (F) from v25, v7, v2 and v6.

Figure 6

Effect of bacterial inoculation on relative abundance in incubations with fecal samples from v25. Changes in four groups of bacteria (Rum, cluster IV ruminococci, Bif, bifidobacteria, Ros, E. rectale and Roseburia spp., Bac, Bacteroidetes) were monitored by 16S rRNA-targeted qPCR for the experiment shown in Figure 5c. Abundance for each group is expressed as a percentage of the 16S rRNA signal obtained using universal primers. The impact of inoculation is easily seen for B. adolescentis and for R. bromii, as these groups were almost undetectable in the initial fecal samples, but is less evident for relatives of E. rectale and B. thetaiotaomicron because of the large populations in the fecal sample. Interestingly, a major increase in E. rectale-related bacteria was evident following inoculation with R. bromii. Data for one control sample (v7) are shown for comparison in Supplementary Figure S5.