PROJECT SUMMARY Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and death worldwide. As a result, the U.S. CDC has classified C. difficile (CD) as an urgent public health threat. Recent guidelines from Infectious Diseases Society of America and Society for Healthcare Epidemiology of America recommended oral vancomycin or fidaxomicin for both non-severe and severe CDI cases. Because of the high cost of fidaxomicin, vancomycin is now the drug of choice, making it the most important antibiotic for the treatment of CDI. The foundation for this work is based on our recent discovery of CD strains in patients from Texas and Kenya exhibiting reduced susceptibility to both metronidazole and vancomycin. We examined diarrhea CDI stools from 438 patients from Texas and 98 from Kenya for the presence of metronidazole- and vancomycin-non- susceptible CD isolates. Of the stools from Houston, 114/438 (26%) grew CD isolates that were not susceptible to vancomycin, 128/438 (29%) to metronidazole, and 97/438 (22%) to both metronidazole and vancomycin. Among the Kenyan patients, 66/98 (67%) were not susceptible to vancomycin, 83/98 (85%) to metronidazole, and 57/98 (58%) to both antibiotics. Alarmingly, many of the isolates from both locations showed levels of non-susceptibility to these antibiotics that far exceeded their known MICs. Whole-genome sequencing showed the presence of homologs of vanA and vanB gene clusters, common mediators of high-level vancomycin resistance in many hospital-associated pathogens. Until now, such high-level vancomycin non-susceptibility has not been reported in CD strains. The spread of CD strains resistant to vancomycin, a front-line antibiotic for this life-threatening pathogen, will have serious clinical and public health implications. This underscores an urgent need for a comprehensive analysis of the circulating strains, mechanisms of resistance, and how it impacts clinical outcomes to help inform clinical decisions. Our preliminary evidence strongly supports the hypothesis that vancomycin non-susceptible CD strains may be widespread in the CDI patient population and that strains circulating on the African continent may be genetically different from strains circulating in North America. To investigate this hypothesis, we will (i) assess the proportion of CDI patients from Texas and Kenya infected with vancomycin non-susceptible strains and compare the infecting strains; (ii) characterize the genetic elements associated with vancomycin non-susceptibility; and (iii) prospectively follow CDI patients infected with vancomycin non-susceptible strains to assess disease severity, clinical outcome, and rate of recurrence of the infection following treatment. Due to the current importance of vancomycin in CDI treatment, the proposed research will have a major impact on clinical decisions. Importantly, the genetic elements responsible for high-level vancomycin non- susceptibility in CD strains...