In patients with Clostridioides difficile infection (CDI) treated with the most commonly used antibiotic, vancomycin (VAN), sustained clinical cure (SCC) rates can be as low as 50-60%. Methods to identify C. difficile strains likely to fail VAN therapy are needed. Our long-term goal is to develop clinically relevant CDI susceptibility measures predictive of patient outcomes. The parent grant explores and identifies antimicrobial resistance contributing to these poor failure rates. This project will expand these investigations into a newly identified resistance mechanism, biofilms. Infections caused by biofilm-producing bacteria are notoriously difficult to treat due to biofilms’ inherent increase in antibiotic resistance. Recently discovered by our group and others, C. difficile can be successfully grown in multispecies biofilms using representative gut microbiome species leading to a higher likelihood of in vitro CDI recurrence. Sessile C. difficile cells embedded in biofilm can have VAN MICs that are 100-times or higher than planktonic (non-biofilm) C. difficile. However, no studies have investigated a large number of clinical strains to assess the variance in C. difficile biofilm quantity among clinical strains or an effect on patient outcomes. Our hypothesis is that higher biofilm production decreases SCC rates. The central hypothesis will be supported by two specific aims: Aim 1 will determine the variance in biofilm formation among clinical C. difficile strains stratified by ribotype. This aim tests the hypothesis that biofilm development will be variable amongst C. difficile strains within ribotype groupings. Quantification of biofilms using the traditional crystal violet (CV) technique is ongoing with an innovative ATP bioluminescent biofilm technique developed as part of this proposal. Aim 2 will determine if biofilm quantity predicts VAN minimum biofilm eradication concentrations (MBEC) or sustained clinical cure (SCC) in hospitalized CDI patients treated with VAN. This aim tests the critical hypothesis that C. difficile biofilm formation is relevant in determining SCC. Biofilm quantitation will be evaluated in multivariate models to determine whether biofilm quantitation is predictive of MBEC values, SCC or both. We expect these findings will provide, for the first time, a biofilm quantity and VAN MBEC susceptibility measure that is predictive of poor patient outcomes. The proposed research will benefit the parent grant research by providing a tool to assess whether VAN resistance caused by biofilm-producing C. difficile strains impacts patient outcomes. These studies lay the foundation for the candidate to further expand into clinical microbiology translational research.