Butyrate enhances Clostridioides difficile sporulation in vitro

Abstract

Short-chain fatty acids (SCFAs) are products of bacterial fermentation that help maintain important gut functions such as maintenance of the intestinal barrier, cell signaling, and immune homeostasis. The main SCFAs acetate, propionate, and butyrate have demonstrated beneficial effects for the host, including its importance in alleviating infections caused by pathogens such as Clostridioides difficile. Despite the potential role of SCFAs in mitigating C. difficile infection, their direct effect on C. difficile remains unclear. Through a set of in vitro experiments, we investigated how SCFAs influence C. difficile growth, sporulation, and toxin production. Similar to previous studies, we observed that butyrate decreased growth of C. difficile strain 630 in a dose-dependent manner. The presence of butyrate also increased C. difficile sporulation, with minimal increases in toxin production. RNA-Seq analysis validated our experimental results, demonstrating increased expression of sporulation-related genes in conjunction with changes in metabolic and regulatory genes, such as a putative carbon starvation protein, CstA. Collectively, these data suggest that butyrate may induce alternative C. difficile survival pathways, modifying its growth ability and virulence to persist in the gut environment. IMPORTANCE Several studies suggest that butyrate may modulate gut infections, such as reducing inflammation caused by the healthcare-associated Clostridioides difficile. While studies in both animal models and human studies correlate high levels of butyrate with reduced C. difficile burden, the direct impact of butyrate on C. difficile remains unclear. Our study demonstrates that butyrate directly influences C. difficile by increasing its sporulation and modifying its metabolism, potentially using butyrate as a biomarker to shift survival strategies in a changing gut environment. These data point to additional therapeutic approaches to combat C. difficile in a butyrate-directed manner.

Keywords: Clostridioides difficile; butyrate; growth assay; metabolism; sporulation.

Fig 1

Butyrate inhibits the growth of C. difficile strain 630. (A) Growth curves [log₁₀(OD₆₀₀)] over 24 h (n = 7 per condition). (B) CFUs after 24 h (n = 21 per condition) and (C) at 6, 12, 18, and 24 h of growth in BHI supplemented with 5 and 25 mM acetate, propionate, or butyrate (n > 15 per condition). (D) Growth curves [log₁₀(OD₆₀₀)] over 24 h in BHI supplemented with increasing concentrations of butyrate (0, 5, 10, 25, 50 mM; n = 3 per condition). Statistical significance calculated using Dunnett’s test: *P-value <0.05; **P-value <0.01; ***P-value <0.001.

Fig 2

Butyrate-induced inhibition of C. difficile growth is dependent on the metabolic environment. Growth curves of C. difficile strain 630 [log₁₀(OD₆₀₀)] over 24 h in minimal media (CDMM) in the presence of a single sugar supplemented with (red) and without (black) 25 mM butyrate (n = 3 per condition). Statistical significance calculated using Dunnett’s test: *P-value <0.05; **P-value <0.01; ***P-value <0.001.

Fig 3

Short-chain fatty acids increase C. difficile toxin production. Toxin activity (log₁₀ of toxin mg/mL) of C. difficile strain 630 normalized to the average C. difficile load [log₁₀(CFU/mL)] per condition, as measured by an in vitro cell assay (A) after 24 h of growth in BHI supplemented with 5 and 25 mM acetate, propionate, and butyrate (n = 21 per condition), and (B) at 6, 12, 18, and 24 h in BHI supplemented with 5 and 25 mM acetate, propionate, or butyrate; n > 6 per condition. (C) Toxin activity (log₁₀ of toxin mg/mL) of C. difficile strain 630 after 24 h in BHI with increasing concentrations of butyrate (0, 5, 10, 25, 50 mM; n > 3 per condition). (D) Log₂ fold change of tcdC and tcdR expressions in C. difficile strain 630 growing in BHI with 25 mM butyrate over without butyrate (measured at early and late log growth using RT-qPCR, n = 5 per condition). Statistical significance calculated using Dunnett’s test, *P-value <0.05; **P-value <0.01; ***P-value <0.001.

Fig 4

Butyrate increases C. difficile spore production. (A) Growth (log₁₀ of colony-forming units) of C. difficile strain 630 spores (A) after 24 h of growth (n = 9 per condition) and (B) throughout growth at 6, 12, 18, and 24 h in BHI supplemented with 5 and 25 mM acetate, propionate, and butyrate (n = 9 per condition per timepoint), or (C) after 24 h of growth in BHI supplemented with increasing concentrations of butyrate (5, 10, 25, and 50 mM; n = 9 per condition). Cultures were collected at indicated timepoints and heated at 65°C for 20 min to kill off vegetative cells, reflecting spore CFUs. Representative phase contrast images (100×) of C. difficile strain 630 cells grown for 24 h in (D) 70:30 media alone (E) supplemented with 25 mM butyrate. (F) Sporulation efficiency calculated over 1,000 cells in 70:30 media with or without 25 mM butyrate (n = 3 experiments; >5 frames per experiment per condition). Welch’s two-sample test, *P-value <0.05; **P-value <0.01; ***P-value <0.001.

Fig 5

Butyrate modulates C. difficile gene expression. (A) NMDS of C. difficile strain 630 transcriptomic sequences (n = 3 per condition; total n = 12) using Bray-Curtis dissimilarity and normalized enrichment scores (NES) for KEGG assignments significantly upregulated and downregulated genes at (B) early log (~0.2 OD₆₀₀), and (C) late log (~0.5 OD₆₀₀) for C. difficile strain 630 grown in BHI with or without 25 mM butyrate. NES was calculated using clusterprofiler (gseKEGG) in R with P-values adjusted post hoc using false discovery rate.

Fig 6

Butyrate upregulates genes related to spore formation and metabolism. Volcano plots of significantly upregulated and downregulated genes (Wald’s test, adjusted P < 10⁻⁶, log₂ fold change > 1) at (A) early log and (B) late log growth of C. difficile strain 630 grown in BHI with or without 25 mM butyrate. Heatmap depicting normalized transcript counts of the top 50 significantly upregulated (top panels) and downregulated (bottom panels) genes in the presence of butyrate at (C) early log and (D) late log growth.

Fig 7

Model for potential mechanism of butyrate effectiveness against C. difficile strain 630. Increasing butyrate may alleviate host inflammation during recovery of the microbiota (such as via fecal microbiota transplantation) but also signals C. difficile to change metabolic strategies to increase survival. This may involve increased expression of PTS transporters for mannose, fructose, and mannitol and decreasing expression of butyrate-producing genes (4hbD, abfD), inducing alternate metabolic pathways for carbohydrate utilization in metabolically active cells. While growth of vegetative cells may be inhibited, sporulation and toxin genes are upregulated to optimize colonization. Figure illustrated in part with Biorender.