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The aim of the present study was to investigate the colonization status of Clostridium difficile in healthy individuals. In total, 139 healthy adults from two study groups were examined at intervals of 3 months. Among the 18 positive subjects, the number of subjects from whom C. difficile was isolated once, twice, three times or four times was 10 (55.6 %), three (16.7 %), two (11.1 %) and three (16.7 %), respectively. In the student group, different subjects were colonized by different PCR ribotype/PFGE types. However, the same PCR ribotype/PFGE types of C. difficile were isolated from different subjects in the employee group, indicating that cross-transmission may have occurred in this group. Continuous colonization by the same PCR ribotype/PFGE type was only observed in three subjects. C. difficile-positive subjects were significantly more densely colonized by enterococci (P < 0.05) than C. difficile-negative subjects: subjects that were found to be C. difficile-positive three or four times appeared to have higher concentrations of enterococci. The present results demonstrate that, although colonization by a C. difficile strain is transient in many cases, there are healthy individuals that are colonized persistently by C. difficile. They also suggest that dense colonization of the intestine by enterococci may be associated with C. difficile colonization.

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