AbstractClostridioides difficile can cause life-threatening diarrhea and C. difficile Infections (CDIs) typically occur inpatients who have been administered antibiotics. CDI is dominant in both healthcare and community settingswith >500000 cases annually in the USA. The CDC designated C. difficile as one of five `Urgent Threats' to UShealthcare in 2013 and again in 2019.Currently antibiotics are the only fully FDA-approved treatment for CDI. The persistent dysbiosis induced byantibiotics however can contribute to recurrent infections in a significant proportion of patients. Microbiotarestoration via fecal transplants (FMT) can be highly effective against recurrent CDI but have also beenassociated 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 avaccine based on the C. difficile toxins met the criteria for futility and that the study was terminated. To date allC. 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. Thenew anti-infective agent will prevent CDI in a strain-agnostic and multi-pronged manner highlighted by nicheoccupancy (colonization resistance) as well as adaptive immunity (oral vaccine) against the pathogen and itstoxins. In Aim 1 we will build key metabolic and new antigen-display features into the existing bacterial platformand characterize it in vitro. Aim 2 studies will employ two rodent models to assess the ability of the anti-infectiveagent to prevent both primary and recurrent CDI. Given that biocontrol and immunity features will requirevalidation before other in-depth pre-clinical testing we propose the high-risk/high-payoff R21 mechanism forthis 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 extensivedysbiosis attendant with antibiotic use. The long-term impact of this effort may therefore be realized viadeployment of this novel anti-infective for all at-risk patients (not just those with recurrent CDI).