Improvement of Clostridium difficile isolation by heat-shock and typing of the isolated strains by SDS-PAGE

Clostridium difficile plays an essential role in causing pseudomembranous colitis. We looked for the presence of these bacteria in the stools of 169 hospitalized patients and 38 nurses from wards with cases of diarrhea (207 subjects). The study was divided into three parts. In the first part, we compared three methods for isolating Clostridium difficile from stool samples: pre-selection with heat-shock, direct plating on Cycloserine-Cefotaxime-Fructose Agar (CCFA) and culturing in a selective broth medium. Final identification of Clostridium difficile was achieved by gas-chromatography and ApiZym. From the 207 consecutively obtained stool specimens, Clostridium difficile was isolated in 108 (52%) when pre-treated by heat-shock compared to only 26 (13%) when plated on modified CCFA and 23 (11%) when cultured in selective broth medium. Pre-selection significantly increases the isolation rate for Clostridium difficile and should be used in further epidemiological research. In the second part of our study, a retrospective review of subjects' records showed that the heat-shock method detected Clostridium difficile in all age groups at a higher rate than the other methods. In the third part of our study, we typed the 157 isolates of Clostridium difficile strains by protein patterns using SDS-PAGE, and 16 distinct groups were identified. In 19 cases different Clostridium difficile strains were found in the same subject by SDS-PAGE. Finally, the isolated strains were compared with strains from Brussels and Freiburg. Matching patterns were noted in only three cases.