Identification of simplified microbial communities that inhibit Clostridioides difficile infection through dilution/extinction

The gastrointestinal microbiome plays an important role in limiting susceptibility to infection with Clostridioides difficile. To better understand the ecology of bacteria important for C. difficile colonization resistance, we developed an experimental platform to simplify complex communities of fecal bacteria through dilution and rapidly screen for their ability to inhibit C. difficile in vitro. We simplified complex communities from six fecal donors and found that 17% of simplified communities inhibited C. difficile growth when initially isolated and when re-cultured from frozen stocks. Composition varied between simplified communities based upon fecal donor used for dilution; complexity ranged from 19-67 OTUs. One simplified community could be further simplified through dilution and retain the ability to inhibit C. difficile. We tested efficacy of seven simplified communities in a humanized microbiota mouse model and found that four communities were able to significantly reduce the severity of the initial C. difficile infection and limit susceptibility to disease relapse. Analysis of fecal microbiomes from treated mice demonstrated that simplified communities accelerated recovery of endogenous bacteria and led to stable engraftment of at least 20% of bacteria from simplified communities. Overall, the insights gained through the identification and characterization of these simplified communities increase our understanding of the microbial dynamics of C. difficile infection and recovery. ImportanceClostridioides difficile is the leading cause of antibiotic-associated diarrhea and a significant healthcare burden. While fecal microbiota transplantation is highly effective at treating recurrent C. difficile disease, uncertainties about the undefined composition of fecal material and potential long-term unintended health consequences have motivated studies to identify new communities of simple microbes that will be effective at treating disease. This work describes a platform for rapidly identifying and screening new simplified communities of microbes for efficacy in treating C. difficile infection and identifies four new simplified communities of microbes with potential for development of new therapies to treat C. difficile disease in humans. While this platform was developed and validated to model infection with C. difficile, the underlying principles described in the paper could be easily modified to develop therapeutics to treat other gastrointestinal diseases.