Methanobrevibacter smithii Is Associated with a Constipation-Like Phenotype in Rats

To determine the effects of increased M. smithii levels on stool phenotypes, adult male Sprague Dawley rats (N = 30) were placed on a HFD. Absolute levels of stool M. smithii were increased significantly after 3 days on HFD when compared to baseline (1.69 × 105 ± 1.57 × 105 copies/gram vs. 0.91 × 105 ± 0.71 × 105 copies/gram, P = 0.0127) and were further increased after 52 days on HFD (3.06 × 105 ± 2.89 × 105 copies/gram, P < 0.001, Fig. 2A). Stool % wet weights were significantly decreased after 12 days on HFD compared to baseline (P < 0.0001) and remained significantly decreased after 59 days on HFD (P < 0.0001, Fig. 2B). Further, the decrease in % wet weight after 59 days on HFD was associated with the increase in M. smithii levels (R = − 0.38, P = 0.037), indicating that higher M. smithii levels were associated with drier stool.

Fig. 2

Changes in absolute levels of M. smithii in stool (A) and stool % wet weights (B) of rats after 59 days on HFD. Changes in absolute levels of M. smithii in stool (C) and stool wet weights (D) after 10 days of treatment with water (controls), lovastatin hydroxyacid, or lovastatin lactone. E Changes in absolute levels of M. smithii in the small bowel (ileum) after 10 days of treatment with water (controls), lovastatin hydroxyacid, or lovastatin lactone. Horizontal bars denote mean ± SD

To confirm that these effects were due to increased M. smithii, rather than the effects of the HFD alone, rats were randomized into 3 groups and treated for 10 days with lovastatin lactone (N = 10), lovastatin hydroxyacid (N = 10), or water (controls, N = 10) while being maintained on the HFD. Levels of M. smithii in stool samples were not significantly different between controls and rats treated with either form of lovastatin (Fig. 2C). However, rats treated with lovastatin hydroxyacid exhibited a significant increase in % wet weight when compared to controls (P = 0.0246, Fig. 2D), indicating a partial reversal of the constipation-like phenotype.

In contrast to stool, rats treated with lovastatin lactone exhibited significant reductions in absolute M. smithii levels in the distal small bowel (ileum) (29.24 × 105 ± 19.91 × 105 copies/gram) when compared both to controls (144.80 × 105 ± 82.15 × 105 copies/gram, P < 0.0006) and to rats treated with lovastatin hydroxyacid (P = 0.0003, Fig. 2E). Levels of M. smithii in the small bowel of rats treated with lovastatin hydroxyacid were not significantly different from those in controls (Fig. 2E).

H2S-Producing Bacteria Induce H2S Production and a Diarrhea-Like Phenotype in Rats

To determine the effects of increased levels of H2S producers on stool phenotypes, adult male Sprague Dawley rats were gavaged with D. piger (N = 16), F. varium (N = 16) ,or PBS (controls, N = 8). Rats gavaged with D. piger had increased % wet weights on day 10 compared to controls (P < 0.0001, Fig. 3A). Rats gavaged with F. varium had significantly increased % wet weights at all time points when compared to controls [day 5 (P = 0.019), day 7 (P = 0.005), day 10 (P < 0.0001), day 12 (P = 0.027), and day 20 (P = 0.032)], with the peak occurring on day 10 (Fig. 3B). In addition, H2S production in stool samples from rats gavaged with D. piger (P = 0.0005) or F. varium (P = 0.006) was significantly greater than H2S production in stool samples from control rats (Fig. 3C). Using a cutoff of 0.48 ppm of H2S (95% Cl 0.65–1.0), stool H2S levels in rats gavaged with F. varium were positively associated with stool % wet weight on day 20 (r = 0.447, P = 0.042).

Fig. 3

Changes in stool wet weight in rats gavaged with A D. piger and B F. varium. P-values denote comparisons between SRB-gavaged rats and controls. C H2S production in stool collected from D. piger- or F. varium-gavaged rats and controls. Horizontal bars denote mean ± SD. Statistical analyses by Mann–Whitney U-test

H2S Producers Induce Changes in Stool Microbial Profiles

16S rRNA gene sequencing was performed using stool samples obtained at euthanasia, as rats gavaged with H2S producers exhibited increased stool H2S levels when compared to controls at this timepoint. Alpha diversity analysis (Shannon index) showed no differences between rats gavaged with H2S producers and controls, D. piger vs. controls, P = 0.5; F. varium vs. controls, P = 0.2). Although D. piger and F. varium were not detected in stool samples at euthanasia (17 days after the last gavage) and alpha diversity was not different between groups, there were significant differences in the stool microbial profiles of rats gavaged with H2S producers when compared to controls. Beta diversity (PERMANOVA) was different between control rats and rats gavaged with D. piger (P = 0.0001) and between control rats and rats gavaged with F. varium (P = 0.001) (Fig. 4). Moreover, beta diversity was positively associated with stool H2S levels (PCo3: r = − 0.401, P = 0.014).

Fig. 4

Principal coordinates analysis (PCoA) plot of the unweighted UniFrac distances between control rats (green), rats gavaged with D. piger (purple), and rats gavaged with F. varium (red)

Taxonomic differences were identified even at the phylum level between rats gavaged with H2S producers and controls, including increases in relative abundance (RA) of Deferribacterota (D. piger vs. controls, fold change (FC) = 8.93, P = 0.0006, Adj. P-value = 0.006; F. varium vs. controls, FC = 7.70, P = 0.002, Adj. P-value = 0.007), Bacteroidota (D. piger vs. controls, FC = 1.58, P = 0.002, Adj. P-value = 0.01; F. varium vs. controls, FC = 1.72, P = 0.0003, Adj. P-value = 0.004), Desulfobacterota (D. piger vs. controls, FC = 2.13, P = 0.017, Adj. P-value = 0.06; F. varium vs. controls, FC = 2.61, P = 0.003, Adj. P-value = 0.008). F. varium-gavaged rats also exhibited increased RA of phylum Firmicutes compared to controls (FC = 1.78, P = 0.002, Adj. P-value = 0.007).

At the family level, rats gavaged with D. piger exhibited increased RA of Bacteroidaceae (FC = 1.83, P < 0.0001, Adj. P-value = 0.002), Deferribacteraceae (FC = 10.15, P = 0.0005, Adj. P-value = 0.015), Lachnospiraceae (FC = 2.21, P = 0.0007, Adj. P-value = 0.015), Enterobacteriaceae (FC = 7.12, P = 0.001, Adj. P-value = 0.016), Staphylococcaceae (FC = 4.54, P = 0.003, Adj. P-value = 0.032), and Rikenellaceae (FC = 1.60, P = 0.01, Adj. P-value = 0.074), and decreased RA of Eggerthellaceae (FC = − 1.52, P = 0.006, Adj. P-value = 0.055), when compared to controls. Rats gavaged with F. varium exhibited increased RA of family Butyricicoccaceae (FC = 2.24, P = 0.0004, Adj. P-value = 0.023) when compared to controls. However, at the genus level, the RA of several genera were found to be different in F. varium-gavaged rats compared to controls, including increased RA of an unknown genus from family Lachnospiraceae (FC = 19.94, P = 0.0008, Adj. P-value = 0.039) and of Bilophila (FC = 4.77, P = 0.001, Adj. P-value = 0.048), and decreased RA of Akkermansia (FC = − 19.03, P = 0.004, Adj. P-value = 0.076). D. piger-gavaged rats also exhibited increased RA of the unknown genus from family Lachnospiraceae (FC = 18.96, P = 0.0009, Adj. P-value = 0.052), as well as Frisingicoccus (FC = 106.70, P = 0.0005, Adj. P-value = 0.052) and Mucispirillum (FC = 6.86, P-value = 0.002, Adj. P-value = 0.069), and a trend toward increased RA of Bilophila (FC = 3.66, P = 0.007, Adj. P-value = 0.116) and Sutterella (FC = 31.47, P = 0.006, Adj. P-value = FDR P = 0.116) (Supplemental Table 1).

A Spearman correlation was performed, including all animals, between stool H2S levels and the RA of known H2S-producing bacteria, as well as the most abundant bacteria found in the stool of these animals. There were positive associations between stool H2S levels and the RA of genera Bilophila (r = 0.282, P = 0.091), Escherichia-Shigella (r = 0.315, P = 0.058), the unknown genus from family Lachnospiraceae (r = 0.28, P = 0.093), and Sutterella (r = 0.331, P = 0.046). The positive association between H2S levels and the unknown genus from family Lachnospiraceae (r = 0.49, P = 0.024) was stronger in rats gavaged with F. varium, whereas the association with Sutterella was stronger in rats gavaged with D. piger (r = 0.592, P-value = 0.003).

Predicted microbial metabolic pathways were also analyzed and compared to those in controls, and several pathways were found to be different between groups. Importantly, the L-homocysteine biosynthesis pathway, which is used by specific bacteria to consume and produce H2S, was positively associated with stool % wet weight on day 20 (r = 0.30, P = 0.06). This pathway was increased in rats gavaged with F. varium compared to controls (P < 0.001) and was positively associated with H2S levels in these animals (r = 0.5, P = 0.021), but this was not observed when D. piger-gavaged animals were analyzed (r = 0.24, P = 0.264).