pH-Dependent Effects of Short-chain Carboxylic Acids and Buffer Systems On Clostridioides difficile in Vitro and in Vivo

The spore-forming anaerobe Clostridioides difficile colonizes the highly dynamic gut environment early after birth, frequently without causing disease. In this study, we aimed to determine how environmental conditions indicative of the infant gut impacted prevalence and physiology of C. difficile. We examined the effect of pH, fermentation derived short-chain carboxylic acids (SCCA) and buffering systems combining in vitro and in vivo analysis, and experimental and modelling approaches. In vivo, the prevalence of Clostridioides significantly increased between 3 months (30.2%) and 12 months (56.2%) after birth. At 12 months, the occurrence of Clostridioides was the highest in feces with near neutral pH (6.7 (IQR 6.5‒7.3). In vitro, C. difficile showed pH-dependent growth and metabolic activity with an optimum around pH 5.8–6.3. Most SCCA conferred antimicrobial activity at pH 5.2 and 6.1 while at pH 6.5, high concentrations of SCCA promoted growth. The presence of phosphate buffer enhanced antimicrobial activity of SCCA, particularly at lower pH values (5.2–5.8). Two multilinear regression models indicated that ionic strength was inversely related to optical density in vitro, while in vivo, the abundance of Clostridioides was inversely linked to the presence of undissociated SCCA. Together, this study highlights that the that occurrence and performance of the opportunistic pathogen C. difficile was affected by chemical systems such as pH, the presence of buffer systems and concentration and chemical state of SCCA. Our results suggest novel targets that could be modulated to impact C. difficile colonization.