Reuterin disruptsClostridioides difficilemetabolism and pathogenicity through reactive oxygen species generation

Antibiotic resistance is one of the world's greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden.Clostridioides difficileinfection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derivedLactobacillus reuteriis a probiotic that produces the antimicrobial compound reuterin known to preventC. difficilecolonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibitsC. difficileoutgrowth from spores and vegetative cell growth, however, no effect onC. difficilegermination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) inC. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetativeC. difficileto vancomycin and metronidazole treatment and reduced toxin synthesis byC. difficile. We also demonstrate that reuterin is protective againstC. difficiletoxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production byL. reuteriis compatible as a therapeutic in a clinically relevant model.