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

## TL;DR

This study shows how pH and short-chain carboxylic acids affect the growth and prevalence of Clostridioides difficile in infants' guts.

## Contribution

The study reveals novel pH- and buffer-dependent interactions between SCCA and C. difficile that influence colonization.

## Key findings

- C. difficile prevalence in infants increased from 30.2% at 3 months to 56.2% at 12 months.
- C. difficile growth was optimal at pH 5.8–6.3, with SCCA showing antimicrobial activity at lower pH and promoting growth at higher pH.
- Phosphate buffer enhanced SCCA antimicrobial effects at lower pH, and undissociated SCCA inversely correlated with C. difficile abundance in vivo.

## Abstract

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.

The online version contains supplementary material available at 10.1007/s00248-026-02694-6.