Study population

Sampled mice are representative of an underlying study population that recapitulates the previously observed sex-specific longevity effects of ACA. Across all three sites, ACA increased the median male survival of the underlying study population by 17% from 830 to 975 days (log-rank test P<0.001, see Fig. 1). Female median survival increased 5% from 889 to 931 days (P=0.003). These results are consistent with the increased longevity due to ACA previously reported [2]. Fecal samples from 48 mice at each of three study sites—The Jackson Laboratory (TJL), the University of Michigan (UM), and the University of Texas Health Science Center at San Antonio (UT)—were collected between 762 and 973 days of age with a balanced factorial design over sex and treatment group. While having samples collected at the same age for all mice might have reduced some of the observed variability, and this does represent one limitation of the study, the collection window is narrow relative to other biological variation of the mice. For example, the standard deviation in the longevity of mice sampled for this study was 112 days, versus a standard deviation of 50 days for age at sample collection. Visual inspection of the overall survival curves (Fig. 1) confirmed that the longevity of mice sampled for microbiome analyses at UM and UT was representative of the other, surviving, unsampled mice. The total number of mice from which microbiome or longevity data were obtained is summarized in Table 1. Samples from TJL were not matched to individual mice and longevity measures are therefore not available for the subset described here.

Fig. 1 Survival curves of sampled cohort. Survival curves for mice fed a control diet (blue lines) or mice fed the same diet containing ACA (gold) at each of three sites: TJL, UM, and UT. Median longevity for each group of mice is indicated by a dashed vertical line. Grey circles indicate the age at death for each of the sampled mice at UM and UT. The number of samples summarized by each line is described in Table. 1 Full size image

Table 1 Number of mice sampled Full size table

Differences in fecal community in ACA-treated mice

ACA-treated mice had a substantially different microbial community composition from control mice at all three study sites. In a multivariate analysis of variance on site, sex, and treatment using Bray-Curtis dissimilarities and including all two-way interactions, significant effects were found for treatment (partial r2=9.6%, PERMANOVA P<0.001) and site (partial r2=16.4%,P<0.001), as well as their interaction (partial r2=3.4%,P<0.001). These statistical results reflect the separation apparent in a principal coordinates ordination (see Fig. 2). A much smaller but still significant effect of sex (partial r2=1.0%,P=0.014) was also identified, but there was no interaction between sex and treatment (P=0.344). Despite the unbalanced design, significance of the PERMANOVA was not affected by changing the order of predictors. Based on a test of multivariate homogeneity of variances, dispersion differed between sites (PERMDISP P<0.001) and sexes (P=0.023), which may bias the PERMANOVA results, but did not differ between treatments (P=0.425). The small effect of sex and the lack of significant sex-by-treatment interaction effects suggest that community composition itself, at the level of overall diversity, is not directly responsible for differential effects of ACA on longevity in male and female mice. However, the substantial differences in community composition due to treatment, while not surprising, suggests that the effects of ACA on the microbiome have the potential to modulate host health.

Fig. 2 Microbial community composition. Fecal bacterial community composition in mice fed the control diet (circles) or the same diet supplemented with ACA (triangles). The two dominant principal coordinates, based on Bray-Curtis dissimilarities among community profiles, are plotted, and percent of variation explained by each is indicated in parentheses on the axes. The location of points in each panel is identical. Markers denote whether mice were treated (triangles) or controls (circles). In (a) points are colored by treatment: control mice (blue) and ACA-treated (gold), in (b) points are colored by site: TJL (bright pink), UM (blue), and UT (green), and in (c) points are colored by sex: male (light blue) and female (pink). The number of samples depicted in each panel is described in Table 1 Full size image

The fecal bacterial community in control mice was dominated by a handful of bacterial families (see Table 2 for details). Across control mice at all three sites, a median of 30% of sequences were classified as members of the largely uncultured family Muribaculaceae—historically called the S24-7—belonging to the phylum Bacteroidetes [22]. Other abundant families included the Lachnospiraceae (27%), Ruminococcaceae (14%), Lactobacillaceae (9%), and Erysipelotrichaceae (1%), all of which are classified in the phylum Firmicutes. More than 99.99% of sequences across all mice were classified at or below the family level. More detailed classification of sequences is provided in Additional file 1.

Table 2 Abundance of common bacterial families Full size table

At a 97% sequence similarity cutoff, 271 operational taxonomic units (OTUs) had a mean relative abundance across all samples of greater than 0.01% and an incidence of greater than 5%. Of these, the relative abundance of 113 OTUs differed between treated and control mice, correcting for a false discovery rate (FDR) of 5%. Together, these OTUs account for a median relative abundance of 54% across both control and treated mice. OTUs differing between sexes or reflecting an interaction between sex and treatment were a substantially less abundant. 5 OTUs were identified after FDR correction that differed significantly in relative abundance between male and female mice, accounting for a median, summed relative abundance of 6%. 7 OTUs were found to be subject to an interaction between treatment and sex, with a median relative abundances of 2%. Details of OTUs responsive to treatment, sex, and a treatment-by-sex interaction have been included in Additional file 1.

Differences between control and ACA mice at TJL and UM were dominated by the increased abundance of a single OTU, OTU-1, which had a median relative abundance of 7.7% in control mice compared to 28.8% in ACA mice at TJL (Mann-Whitney U test P<0.001), and 10.4% compared to 39.0% at UM (P<0.001) (see Fig. 3). At UT, OTU-1 was higher in ACA-treated mice—a median of 5.4% and 11.0% in control and treated mice, respectively—but this increase was not statistically significant (P=0.344). Instead, a different OTU, designated OTU-4, was strongly affected by ACA treatment at UT, with a median relative abundance of 6.3% in control mice that increased to 25.6% in ACA-treated mice (P=0.007). OTU-4 was nearly absent at TJL and UM, with only one mouse out of 95 having a relative abundance above 0.1%, compared to 39 out of 48 mice at UT. Differences in abundance between sexes were not observed for OTU-1 at TJL or UM, but at UT results were suggestive of an increased abundance of OTU-1 in females (P=0.076) and an increased abundance of OTU-4 in males (P=0.060). Interestingly, their combined abundance did not differ between males and females (P=0.344) at UT. Both OTU-1 and OTU-4 were classified as members of the Muribaculaceae, and subsequent phylogenetic analysis confirmed this placement (see Additional file 2). OTU-1 and OTU-4 are approximately 90% identical to each other and to the most closely related cultivar (DSM-28989) over the V4 hypervariable region of the 16S rRNA gene. These OTUs are notable both for their high abundance overall, as well as the large difference between control and ACA-treated mice. It is surprising that OTU-4 is common and differentially abundant at UT, while remaining rare at both of the other sites, suggesting that local community composition modulates the effects of ACA. While OTU-1 is made up of multiple unique sequences, the composition within the OTU does not differ substantially with ACA treatment (see Additional file 3).

Fig. 3 Abundance of dominant OTUs. Relative abundance of the 16S rRNA gene from two OTUs classified as belonging to the family Muribaculaceae, abundant in ACA-treated mice. Points in each panel correspond with samples collected from individual mice at each of three replicate study sites. Samples were obtained from mice fed either the control diet (blue, labeled ‘-’) or the same diet supplemented with ACA (gold, labeled ‘+’). Markers indicate the sex of the mouse: male (triangle) or female (circle). The number of samples illustrated in each panel is described in Table. 1. Boxes span the interquartile range and the internal line indicates the median. (*: P<0.05, **: P<0.001 by Mann-Whitney U test) Full size image

The increased relative abundance of OTU-1 and OTU-4 in mice treated with ACA appears to be due to greater abundance of these sequences, and is not explained solely by a decrease in other groups. The abundance of taxa in control and treated mice was compared based on the recovery of spiked-in standard relative to the sequence of interest. The median combined spike-adjusted abundance of 16S rRNA gene copies from OTU-1 and OTU-4 was 4.3 times greater per gram of feces in ACA-treated mice compared to controls (Mann-Whitney U test P<0.001), suggesting a corresponding increase in population density.

While OTU-1 and OTU-4 are classified to the same family and are similarly affected by ACA, other OTUs in the Muribaculaceae have decreased abundance in treated mice. The combined relative abundance of all other OTUs in the family—excluding OTU-1 and OTU-4—was 8.3% in treated mice versus 16.8% of sequences in control mice (Mann-Whitney U test P<0.001). The median combined spike-adjusted abundance of all other Muribaculaceae OTUs was 0.5 times the median in control mice (P=0.001), suggesting a decrease in the population density of these taxa. This is consistent with competition between OTUs in this family.

Three of the five most abundant families all exhibit decreased relative abundance in ACA treated mice (see Table 2). However, the large increase in abundance of OTU-1 and OTU-4 suggests that some changes in the relative abundance of other taxa may be the result of compositional effects, rather than decreased density. For instance, although the relative abundance of Ruminococcaceae was lower in ACA-treated mice, the spike-adjusted abundance was little changed (P=0.327), emphasizing the value of this complementary analysis. Conversely, decreased relative abundance was matched by decreased spike-adjusted abundance for both the Lactobacillaceae (P=0.014) and the Erysipelotrichaceae (P=0.063).

ACA-treated mice exhibited decreased fecal community diversity. The median Chao1 richness estimate was decreased from 229 in control mice to 199 in treated mice (Mann-Whitney U test P<0.001). The Simpson’s evenness index was also lower in ACA mice: 0.044 versus 0.075 in controls (P<0.001). This reduced richness and evenness is not surprising given the much greater abundance of a single OTU in treated mice at each site. To understand changes in diversity while controlling for compositional effects, we measured the effect of ACA ignoring counts for OTU-1 and OTU-4. While Simpson’s evenness was not decreased by treatment in this fraction of the community (P=0.26), the Chao1 richness—subsampling to equal counts after partitioning—was decreased (P=0.005), suggesting that the bloom of OTU-1 and OTU-4 may have, in fact, resulted in the local extinction of rare community members.

Changes in fecal metabolite concentrations

The effects of long-term ACA treatment on fecal metabolite profiles was variable across study sites, but with increased concentrations of propionate at all three (see Fig. 4). Consistent with the expected increase in starch in the lower gut, glucose concentrations were higher in ACA-treated mice at UM (from a median of 6.1 μmols/g in control mice to 12.9 in treated mice, Mann-Whitney U test P<0.001), and UT (2.8 to 9.1, P<0.001). At TJL, while the statistical test did not surpass the significance threshold, there was also an observed increase in glucose concentrations (6.6 to 9.1, P=0.069). However, this presumed increase in fermentable substrate did not result in increased total SCFA—here defined as the sum of acetate, butyrate, and propionate concentrations—at either TJL (22.4 to 18.8, P=0.443), or UM (19.1 to 24.8, P=0.092). Interestingly, total SCFA was sharply higher in ACA treated mice at UT alone (17.7 to 31.9, P<0.001). This same pattern of variation across sites was found in the effects of ACA on fecal acetate and butyrate concentrations, where statistically significant increases were only found at UT (acetate: P=0.003, butyrate: P<0.001), but not at either of the other two sites. Fecal propionate concentrations, on the other hand, were elevated in ACA mice at all three study sites, from a median of 0.93 to 1.9 at TJL (P=0.001), from 1.1 to 2.5 at UM (P<0.001), and from 1.9 to 2.9 at UT (P=0.008).

Fig. 4 Fecal metabolites. Concentrations of metabolites in feces obtained from mice fed either the control diet (open circles) or the same diet supplemented with ACA (solid circles), at each of three study sites: TJL (bright pink), UM (blue), and UT (green). Lines span the interquartile range and the circle indicates the median. The summed concentration of acetate, butyrate, and propionate is plotted as “total SCFA”. The number of samples summarized in each panel is described in Table. 1. Symbols indicate the significance of a Mann-Whitney U test comparing control to ACA-treated mice. (†:P<0.1,∗:P<0.05,∗∗:P<0.001) Full size image

The consistent increase in fecal propionate was matched by consistently lower concentrations of one of its metabolic precursors, succinate, at all three sites, from 2.8 in control to 1.5 in ACA-treated mice at TJL (P=0.009), from 3.0 to 1.2 at UM (P<0.001), and from 3.1 to 2.1 at UT (P=0.022). Median lactate concentrations were also lower in ACA treated mice, although this was only statistically significant at UT (P=0.002). It is surprising that these fermentation intermediates are reduced, given the expected increase in available polysaccharide. It is possible that their concentrations reflect greater consumption in downstream pathways, or perhaps ACA directly inhibits the metabolism and growth of relevant bacteria; such effects have been previously reported for in vitro fermentations of starch with human fecal slurries [8]. Concentrations of formate, valerate, isobutyrate, and isovalerate were generally below the detection limit. Fresh pellet weight was increased from a median of 36 to 74 mg (P<0.001).

Given the inconsistant observation of changes in metabolite concentrations with ACA across study sites, we tested for treatment-by-site interaction effect using an ANOVA framework, with log-transformed metabolite concentrations to better accommodate distributional assumptions. Using this approach, significant interactions were found for glucose (P=0.005), and acetate (P=0.044), and results were suggestive for succinate (P=0.069), butyrate (P=0.071), and total SCFA (P=0.053). Treatment-by-site interaction effects were not found for lactate (P=0.266) or propionate (P=0.542).

Differences in SCFAs between sexes are particularly interesting given the greater longevity effects of ACA in male mice. In a log-linear model that included terms for site, sex, and treatment, as well as all other two-way interactions, including a treatment-by-sex interaction significantly improved the fit of the model for propionate (LRT P=0.020), but not for butyrate or acetate. This interaction results in a larger difference in propionate concentrations for male mice (from an overall median of 1.4 μmols/g in control mice to 2.7 in ACA) than for female mice (from 1.0 to 1.9) with ACA treatment. The significance of the interaction term was not corrected for multiple testing, and therefore additional studies would greatly increase our confidence in this result.

Community predictors of fecal SCFA concentrations

As both community composition and the effects of ACA on metabolite concentrations in feces varied greatly between study sites, we explored the relationship between microbial abundance and metabolite concentration. Community composition was correlated with metabolite concentrations in both control and ACA-treated mice. Numerous strong correlations were detected between the spike-adjusted abundance of 16S rRNA copies from the most common bacterial families and the concentrations of SCFAs and lactate. Notably, Muribaculaceae abundance was particularly strongly correlated with propionate concentrations in both control (Spearman’s ρ=0.36,P=0.002; see Fig. 5) and ACA mice (ρ=0.64,P<0.001). Likewise, Lachnospiraceae were correlated with butyrate (ρ=0.61 in control and 0.77 in ACA, P<0.001 for both), and Lactobacillaceae with lactate concentrations (ρ=0.63 in control and 0.67 in ACA, P<0.001 for both). Strikingly, concentrations of acetate and butyrate were especially correlated with each other (ρ=0.67 in control and 0.80 in ACA, P<0.001 for both). What’s more, all of these correlations were found in treated mice even when independently tested at each of the three sites, suggesting that they cannot be explained by simple associations between metabolite concentrations and community composition at the site level.

Fig. 5 Microbiota/metabolite correlations. Correlations among metabolite concentrations in feces and family level, spike-adjusted 16S rRNA gene abundances. Points correspond with samples collected from individual mice and colors indicate whether they were obtained from mice fed the control diet (blue) or the same diet supplemented with ACA (gold). Markers indicate the site where the mouse was housed: TJL (diamond), UM (circle), or UT (triangle). Metabolite concentrations are reported normalized to feces wet weight, and abundances are in spike-equivalent units. Values are on a linear scale between 0 and the subsequent tick label, above which, points are plotted logarithmically. The number of samples illustrated in each panel is described in Table 1 Full size image

Although our study was not an unambiguous test, these results support the hypothesis that the fecal metabolite response to treatment is dependent on the population density of relevant microbes in the gut community. Similarly, environmental and host factors that promote or inhibit the growth of particular community members would be expected to modulate the response.

To identify key players in these associations, we examined the relationship between metabolite concentrations and the spike-adjusted abundances of OTUs. Based on a least absolute shrinkage and selection operator (LASSO) regression analysis, the abundances of a number of OTUs can be used to predict concentrations of propionate, butyrate, acetate, and lactate even after accounting for treatment, sex, and study site. Consistent with the correlations found between Muribaculaceae abundance and propionate, OTU-1 and OTU-4 were identified as predictors of increased propionate, along with a third taxon, OTU-5, also classified as a member of the family. For both butyrate and acetate, OTUs classified as members of both the Lachnospiraceae and Ruminococcaceae were most predictive of increased concentrations. Unsurprisingly, the most abundant OTU classified as a member of the Lactobacillaceae, OTU-2, was found to be highly predictive of increased lactate. However, 8 OTUs were also associated with decreased lactate concentrations, most of which were among those associated with increased butyrate and acetate. Among other explanations, this is consistent with these taxa either being inhibited by lactate or being lactate utilizers, which are likely to be producing SCFAs as secondary fermentation products. Regression coefficients for each metabolite are included in Additional file 1. Overall, results were both consistent with a priori expectations, and useful for generating hypotheses about which taxa might be associated with the generation of fermentation products.

Fecal SCFA concentrations as predictors of longevity

Given the documented health benefits of SCFAs in the gut (reviewed in [19]) and their increased levels in ACA-treated mice, we tested the relationship between the acetate, butyrate, and propionate concentrations in feces, and the lifespan of individual mice. Lifespans of fecal donors were not available for mice at TJL, so survival analyses were carried out only with UM and UT mice, and effect sizes are reported for SCFAs as standardized hazard ratios (HRs). Due to the reduced number of mice sampled for this study, data were pooled across sexes and sites. The shared effects of the design parameters—treatment, sex, and study site—on both SCFAs and longevity, were accounted for by including terms for these covariates as well as their two and three-way interactions. Analyses reinforcing our interpretations are discussed in Additional file 4. Tested individually against this null model, an association between longevity and propionate was found (standardized HR of 0.727, P=0.031), but no relationship was observed with butyrate (P=0.240) or acetate (P=0.742). However, when the model was fit with all three SCFAs simultaneously, each was found to be associated with longevity (P=0.012, 0.030, 0.042 for propionate, butyrate, and acetate, respectively). Coefficients for SCFA covariates in this full model suggest a positive association with longevity for both propionate and butyrate (standardized HR of 0.674 and 0.586, respectively). Interestingly, a negative association was found with acetate (standardized HR of 1.576) using this model. The discrepancy between this result and the lack of association when butyrate and acetate are each tested alone likely reflects the strong positive correlation between acetate and butyrate concentrations, masking their individual, opposing associations with longevity. The overall fit of the full model was improved compared to the null model with only design covariates (likelihood ratio test, P=0.023).