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 resist C. difficile colonization after challenge, as measured by >100-fold reduction in levels of C. difficile in challenged communities. We screened 76 simplified communities diluted from cultures of six fecal donors and identified 24 simplified communities that inhibited C. difficile colonization in vitro. Sequencing revealed that simplified communities were composed of 19 to 67 operational taxonomic units (OTUs) and could be partitioned into four distinct community types. One simplified community could be further simplified from 56 to 28 OTUs through dilution and retain the ability to inhibit C. difficile. We tested the efficacy of seven simplified communities in a humanized microbiota mouse model. We 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 indigenous bacteria and led to stable engraftment of 19 to 22 OTUs 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. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and a significant health care burden. Fecal microbiota transplantation is highly effective at treating recurrent C. difficile disease; however, uncertainties about the undefined composition of fecal material and potential long-term unintended health consequences remain. These concerns have motivated studies to identify new communities of microbes with a simpler composition that will be effective at treating disease. This work describes a platform for rapidly identifying and screening new simplified communities for efficacy in treating C. difficile infection. Four new simplified communities of microbes with potential for development of new therapies to treat C. difficile disease are identified. 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.