Other investigators have described the recurrence of CDAD following Vanco withdrawal in mice[10, 19]. Chen et al. reported severe recurrent CDAD in mice following the removal of Vanco. CDAD was associated with severe diarrhea, prominent body weight loss, marked histological pathology, and a 58% mortality rate. In contrast, Sun and colleagues found only mild diarrhea, transient body weight loss, and no evidence of mortality following Vanco withdrawal in mice. It should be mentioned that different strains of C. difficile (VPI10463 or UK 101) were used in the two studies, as well as somewhat different Vanco treatment regimens[10, 11]. Despite the fact that we used the same strain of C. difficile (VPI10463) as Chen and colleagues, our mortality and stool consistency results (Figure3) are more similar to those reported by Sun et al.. Differences in these study results may also be related to alterations in endogenous bacterial flora populations within the colonies of mice. Certain types of bacteria that predominate in the colon (e.g., numbers of Firmicutes and Proteobacteria) have recently been shown by other investigators to critically influence the severity of C. difficile induced colitis in mice.
Interestingly, our results suggested that treatment of mice with BC30 slightly lowered the overall C. difficile infection rate (Figure2A), as well as the measured levels of associated toxins in the stool (Figure2B). However, statistically significant differences were not found compared to the corresponding cohort of vehicle treated animals. These results suggest the possibility that BC30 probiotic treatment may have lowered the actual numbers of C. difficile in the colonic lumen and/or mucosa. However, more detailed follow-up studies would be needed to critically test this possibility.
Previously, we found that pre-treatment of mice with B30 improved the stool consistency during the primary phase of C. difficile infection. In a similar fashion, our results show that BC30 treatment significantly improved both the stool consistency scores and percentage of mice with normal stools (Figure3) during the recurrence phase (days 11–17) following Vanco withdrawal in mice. Of note, mice treated with BC30 tended to have longer and firmer stools (increased stool size) than Vehicle/C. difficile treated mice. These results re-affirm the positive effects of this probiotic on stool consistency (Figure3).
Other laboratories have found that toxin A secreted by C. difficile can activate the NF-κB and AP-1 signal transduction system in monocytes and colonic epithelial cells[6, 8, 21]. This process leads to secretion of a key pro-inflammatory chemokine (IL-8) and subsequent neutrophil influx into the colonic tissue[6, 8, 21]. Interestingly, BC-30 can significantly inhibit IL-8 directed migration of human neutrophils in vitro. Based on these results, we measured the effects of BC30 on colonic MPO, as well the murine chemokine (KC) content in the colons of C. difficile infected mice. Probiotic treatment resulted in a significant reduction in colonic MPO (Figure4), as well as a diminution in the KC content. However, statistical significance was not achieved for reducing this chemokine, as compared to values in vehicle treated mice. Nevertheless, these positive effects of BC30 on parameters associated with neutrophil influx into the colon may also contribute to the observed improvement in stool consistency observed in the probiotic-treated mice.
Murine CDAD is associated with a specific colonic histopathology that includes crypt damage, submucosal edema and influx of inflammatory cells. These pathological changes were also evident during the recurrence phase in our Vehicle/C. difficile/Vanco treated mice (panel C, Figure5). Interestingly, histological pathology also persisted to some degree in the Vehicle/C. difficile/ No Vanco cohort of mice (panel B, Figure5), even at 8 to 11 days after the initial infection with C. difficile. In contrast, mice treated with BC30 showed evidence of improved colonic histopathology, including decreased leukocyte influx into the colon and diminished sub-mucosal edema (panel D, Figure5). Importantly, the comparisons of mean colonic histology scores showed a statistically significant reduction in B30 treated mice compared to the corresponding vehicle cohort of animals (Figure5E).
Other investigators have found evidence of in vitro and in vivo COX-2 induction in colonocytes or macrophages following exposure to C. difficile derived toxin A[9, 22]. Moreover, inducible COX-2 may contribute through prostaglandin formation to the alteration in stool consistency that is a prominent feature of CDAD[10, 18]. Therefore, it is interesting that colonic COX-2 immunostaining was dramatically diminished in the colon of BC30 treated mice (Figure6). It is possible that this probiotic may affect the CREB-COX-2-PGE2 pathway, which promotes fluid secretion and contributes to CDAD in mice[9, 10, 18]. Future studies could focus on more critically evaluating the effects of BC30, as well as other Bacillus coagulans probiotic strains, on this important pathway of CDAD.