Establishment of an Endogenous Clostridium difficile Rat Infection Model and Evaluation of the Effects of Clostridium butyricum MIYAIRI 588 Probiotic Strain

Abstract

<i>Clostridium difficile</i> is well known as an agent responsible for pseudomembranous colitis and antibiotic-associated diarrhea. The hamster model utilizing an oral route for infection of <i>C. difficile</i> has been considered to be the standard model for analysis of <i>C. difficile</i> infection (CDI) but this model exhibits differences to human CDI, most notably as most hamsters die without exhibiting diarrhea. Therefore, we attempted to develop a new non-lethal and diarrheal rat CDI model caused by endogenous <i>C. difficile</i> using metronidazole (MNZ) and egg white. In addition, the effects of probiotic strain <i>Clostridium butyricum</i> MIYAIRI 588 (CBM) on CDI were examined using this model. Syrian Golden hamsters received clindamycin phosphate orally at 30 mg/kg on 5 days before challenge with either <i>C. difficile</i> VPI10463 (hypertoxigenic strain) or KY34 (low toxigenic clinical isolate). Mortality and the presence of diarrhea were observed twice a day for the duration of the experiment. Wistar rats received 10% egg white dissolved in drinking water for 1 week <i>ad libitum</i> following intramuscular administration of 200 mg/kg MNZ twice a day for 3 days. Diarrhea score was determined for each day and fecal water content, biotin concentration, and cytotoxin titer in feces were examined. More than 70% of hamsters orally infected with <i>C. difficile</i> died without exhibiting diarrhea regardless of toxigenicity of strain. The rats receiving egg white after MNZ administration developed diarrhea due to overgrowth of endogenous <i>C. difficile</i>. This CDI model is non-lethal and diarrheal, and some rats in this model were spontaneously cured. The incidence of diarrhea was significantly decreased in <i>C. butyricum</i> treated rats. These results indicate that the CDI model using egg white and MNZ has potentially better similarity to human CDI, and implies that treatment with <i>C. butyricum</i> may reduce the risk of CDI.