Vincent, E. C. | Search bioRxiv

bioRxiv2026

Bile acid dependent attenuation of toxin mediated disease is independent of colonization resistance against C. difficile

Clostridioides difficile infection (CDI) is a severe antibiotic associated disease and a major cause of morbidity and mortality worldwide. CDI is thought to arise from the loss of protective gut microbes that mediate functions such as secondary bile acid metabolism and nutrient competition, yet the relative contributions of these mechanisms remain unclear. To determine how these processes influence C. difficile growth, virulence, and disease, we performed in vitro and in vivo experiments using two Clostridia strains previously associated with colonization resistance against C. difficile. Neither organism prevented colonization or growth through nutrient competition alone. In contrast, secondary bile acid metabolism significantly reduced toxin-mediated disease in vivo in a strain dependent manner. These findings demonstrate that secondary bile acid modulation is an important component of CDI prevention independent of nutrient competition and suggest that attenuating virulence, in addition to limiting colonization, may represent a key strategy for next-generation CDI therapeutics.

microbiology↗

bioRxiv2026

Unlocking the Bile Acid Universe: Advanced Workflows and a Multidimensional Library of 280 Unique Species

Microbes and bile acids are tightly intertwined, especially in the gut. While the liver produces primary bile acids from cholesterol, gut bacteria transform these into diverse secondary forms which act as powerful signaling molecules, influencing host metabolism and immune function. Since bile acid changes are increasingly linked to health and disease, their accurate measurement in the gut and circulation is essential. Analytical evaluations, however, remain challenging as many bile acids co-elute in liquid chromatography (LC), share identical precursor masses in mass spectrometry (MS), and produce similar tandem mass spectrometry (MS/MS) spectra. As a result, conventional LC-MS/MS workflows struggle to differentiate bile acids, motivating the addition of orthogonal separations such as ion mobility spectrometry (IMS). Here, we assess optimal bile acid extraction parameters for stool, serum, and plasma; compare LC conditions; and assess electrospray ionization performance across polarities. Additionally, we created a multidimensional reference library containing LC retention times, IMS collision cross section values, and accurate precursor masses for 280 unique bile acids (264 endogenous and 16 deuterium-labeled species) including unconjugated, host-conjugated, and microbially conjugated bile acids. This multidimensional library empowers bile acid identification in complex samples and enables a more comprehensive exploration of their biological roles and disease associations.

microbiology↗