Engineering probiotics to inhibit *Clostridioides difficile* infection by dynamic regulation of intestinal metabolism

Clostridioides difficile infection (CDI) results in significant morbidity and mortality in hospitalized patients. The pathogenesis of CDI is intrinsically related to the ability of C. difficile to shuffle between active vegetative cells and dormant endospores through the processes of germination and sporulation. Here, we hypothesise that dysregulation of microbiome-mediated bile salt metabolism contributes to CDI and that its alleviation can limit the pathogenesis of CDI. We engineer a genetic circuit harbouring a genetically encoded sensor, amplifier and actuator in probiotics to restore intestinal bile salt metabolism in response to antibiotic-induced microbiome dysbiosis. We demonstrate that the engineered probiotics limited the germination of endospores and the growth of vegetative cells of C. difficile in vitro and further significantly reduced CDI in model mice, as evidenced by a 100% survival rate and improved clinical outcomes. Our work presents an antimicrobial strategy that harnesses the host-pathogen microenvironment as the intervention target to limit the pathogenesis of infection.

Elvin Koh, In Young Hwang, Hui Ling Lee, Ryan De Sotto, Jonathan Wei Jie Lee, Yung Seng Lee, John C. March, Matthew Wook Chang