Isolation and Preservation of C. difficile

In total, 76 C. difficile clinical isolates were obtained from a University-affiliated tertiary hospital and the National Institute of Health of Thailand. The isolation of C. difficile from stool samples of diarrheal patients was performed in previous studies [39 (link),40 (link)]. These isolates were separated into 2 groups based on collection periods. The THA group was composed of 50 isolates collected from 2006 to 2009, and the THB group contained 26 isolates collected from 2010 to 2012. Each isolate was cultured on cycloserine–cefoxitin fructose agar (CCFA) for 24 h at 37 °C under anaerobic conditions (Coy Laboratory Products, Glass Lake, MI, USA) supplemented with 0.1% taurocholate to recover and enrich C. difficile cells. A single colony was cultured in fresh brain heart infusion (BHI) broth and incubated in an anaerobic chamber at 37 °C for 24–48 h. The culture was preserved with 10% (v/v) glycerol at −80 °C for further use.