Butyrate enhances <i>Clostridioides difficile</i> sporulation <i>in vitro</i>

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 <i>Clostridioides difficile</i>. Despite the potential role of SCFAs in mitigating <i>C. difficile</i> infection, their direct effect on <i>C. difficile</i> remains unclear. Through a set of <i>in vitro</i> experiments, we investigated how SCFAs influence <i>C. difficile</i> growth, sporulation, and toxin production. Similar to previous studies, we observed that butyrate decreased growth of <i>C. difficile</i> strain 630 in a dose-dependent manner. The presence of butyrate also increased <i>C. difficile</i> 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 <i>C. difficile</i> 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 <i>Clostridioides difficile</i>. While studies in both animal models and human studies correlate high levels of butyrate with reduced <i>C. difficile</i> burden, the direct impact of butyrate on <i>C. difficile</i> remains unclear. Our study demonstrates that butyrate directly influences <i>C. difficile</i> 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 <i>C. difficile</i> in a butyrate-directed manner.