Interactions of Clostridium difficile and Escherichia coli with microfloras in continuous-flow cultures and gnotobiotic mice

Abstract

We studied the interactions between the entire cecal flora of hamsters and the pathogens Clostridium difficile and Escherichia coli in gnotobiotic mice and in a continuous-flow (CF) culture system in which the growth medium consisted of an extract of fecal pellets from germfree mice. CF cultures and germfree mice were colonized first with C. difficile and E. coli and then with the cecal flora of hamsters. Both in vivo and in vitro hamster flora markedly suppressed the potential pathogens. Contents of CF cultures inoculated with hamster flora were introduced into gnotobiotic mice previously colonized with C. difficile and E. coli. These mice were compared with mice given homogenates of hamster ceca. In both groups, the C. difficile population decreased by a factor of more than 106 and the E. coli population decreased by a factor of 104 to 105. CF culture contents also reduced the size of the dilated germfree mouse cecum to normal. When veal infusion broth was used as a medium, contents of CF cultures colonized with hamster flora failed to eliminate the C. difficile from mice. Thus, the extract of fecal pellets appeared to contain a substance important for sustained colonization by important components of the cecal flora. We also studied the ability of collections of isolates to suppress the potential pathogens in both gnotobiotic mice and CF cultures. A total of 150 isolates obtained from predominant hamster flora at the ecologic climax stage (C flora) suppressed C. difficile and E. coli to 10 and 1 to 3%, respectively, of the population sizes attained in monoassociated mice. A total of 67 isolates obtained during ecologic succession combined with a C flora consisting of 100 isolates suppessed the potential pathogens to 0.3 and 0.03% of their original levels, respectively. Similar degrees of suppression occurred in CF cultures, further indicating that anaerobic CF cultures are promising models for investigation of the microbial ecology of C. difficile.