Clostridium difficile from Healthy Food Animals: Optimized Isolation and Prevalence

Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by alcohol shock and isolation on tryptic soy agar supplemented with 5% sheep blood, and cycloserine-cefoxitin fructose agar. The double alcohol shock method (DS) used alcohol shock prior to and after selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by isolation on tryptic soy agar supplemented with 5% sheep blood and cycloserine-cefoxitin fructose agar. A total of 55 (15.9%, n = 345) swine fecal samples, 32 (2.4%, n = 1,325) dairy cattle fecal samples, and 188 (6.3%, n = 2,965) beef cattle fecal samples were positive for C. difficile by either method. However, the DS was significantly better than the SS for the recovery of C. difficile from swine feces, while the SS was significantly better than the DS for the recovery of C. difficile from beef cattle feces. There was no significant difference between methods for the recovery of C. difficile from dairy cattle feces. This study suggests that food animals might harbor C. difficile and it provides critical information that isolation methods might not have universal application across animal species.