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Background Clostridioides difficile infection (CDI) is the most common nosocomial infection in this country. The organism forms spores, which likely contribute to spread of infection in the hospital setting. Current guidelines call for special cleaning of rooms following discharge of patients who tested positive for CDI. There are currently no means to validate that cleaning has eradicated all of the spores. Canines (dogs) have been used to detect a variety of odors because of their superior olfactory abilities. A beagle was trained to detect the odor of C. difficile in the hospital setting. Harley the Clostridium difficile sniffing canine. Methods C. difficile was grown at the microbiology department at Vidant Medical Center (VMC). Culture plates containing the organism were used by a professional trainer to train a two-year old female beagle dog. The canine was taught to sit when she identified the odor. The canine was given positive reinforcement with oral treats each time she identified the odor. Once the dog was proficient in identifying the organism, the trainer used clinical stool specimens that tested either positive or negative for C. difficile. Prior to coming into the hospital setting, the canine was able to identify positive stool samples with near 100% accuracy. The dog was brought to search rooms occupied or previously occupied by patients who had CDI. The study was approved by the Animal Use and Care Committee at East Carolina University and took place between October 2019 and March 2020 at VMC. Results The canine detected the presence of C. difficile in 51% of 47 rooms inhabited by patients currently being treated for CDI and in 46% of 28 rooms previously inhabited by patients that tested positive for CDI. In all positive rooms, the organism was detected on the bedrails. She did not detect C. difficile in any positive areas after they had been cleaned with bleach wipes. Rooms found to be positive by canine. Conclusion Training a canine to detect C. difficile is an effective means of detecting the organism in the hospital environment. Use of a trained dog appears to be effective in validating the cleaning process of rooms that have been previously occupied by patients with CDI. Disclosures Paul P. Cook, MD, Contrafect (Grant/Research Support, Scientific Research Study Investigator)Gilead (Grant/Research Support, Scientific Research Study Investigator)Leonard-Meron (Grant/Research Support, Scientific Research Study Investigator)Lilly (Grant/Research Support, Scientific Research Study Investigator)