Abstract Clostridioides difficile can cause life-threatening diarrhea, and C. difficile Infections (CDIs) typically occur in patients who have been administered antibiotics. CDI is dominant in both healthcare and community settings, with >500,000 cases annually in the USA. The CDC designated C. difficile as one of five `Urgent Threats' to US healthcare in 2013, and again in 2019. Currently, antibiotics are the only fully FDA-approved treatment for CDI. The persistent dysbiosis induced by antibiotics, however, can contribute to recurrent infections in a significant proportion of patients. Microbiota restoration via fecal transplants (FMT) can be highly effective against recurrent CDI, but have also been associated with adverse outcomes, including death. There are no licensed vaccines to prevent CDIs. In 2020, it was reported that a large Phase III Clinical Trial of a vaccine based on the C. difficile toxins met the criteria for futility and that the study was terminated. To date, all C. difficile vaccine candidates in late-stage clinical trials continue to be based solely on the toxins. Herein, we propose to systematically refine a synthetic bacterium that we recently developed and tested. The new anti-infective agent will prevent CDI in a strain-agnostic and multi-pronged manner highlighted by niche occupancy (colonization resistance) as well as adaptive immunity (oral vaccine) against the pathogen and its toxins. In Aim 1, we will build key metabolic and new antigen-display features into the existing bacterial platform, and characterize it in vitro. Aim 2 studies will employ two rodent models to assess the ability of the anti-infective agent to prevent both primary and recurrent CDI. Given that biocontrol and immunity features will require validation before other in-depth pre-clinical testing, we propose the “high-risk/high-payoff” R21 mechanism for this project. Our approach builds on prior NIH-funded research in our laboratory, and focuses on a safer alternative to FMT. Further, and via the built-in precision viability that we will engineer, our technology will not engender the extensive dysbiosis attendant with antibiotic use. The long-term impact of this effort may therefore be realized via deployment of this novel anti-infective for all at-risk patients (not just those with recurrent CDI).