Recently, Chen et al. described a murine model of CDAD that could be used for testing the efficacy of applicable pharmacological agents (antibiotics, probiotics) . Infection of female C57BL/6 mice with 103 to 104 CFU of C. dififcile was associated with significant weight loss, diarrhea and mortality . In a similar fashion, we also showed that the infection of vehicle treated mice resulted in transient weight loss, altered stool consistency and some evidence of mortality (11.5%).
In contrast to vehicle treatment, mice treated with the probiotic (BC30) had a delayed onset in mortality (no deaths until day 14), as well as a slightly reduced overall mortality rate (8.7%), when compared to vehicle treated animals (Figure 2). In a similar fashion, mice treated with BC30 had a delayed onset in the appearance of altered stool consistency (Figure 3). Specifically, on day 12, 87% of vehicle treated mice had evidence of loose stools or diarrhea. In contrast, only 33% of BC30 treated animals had evidence of altered stool consistency (p < 0.05 vs. vehicle). Moreover, on the final study day (day 16), BC30 treated mice still had a higher incidence of normal stools (Figure 3), as well as a significantly lower stool consistency score (Figure 4). These results demonstrate evidence of improved stool consistency in C. difficile infected mice that were pre-treated with BC30.
Murine CDAD is associated with a specific colonic histopathology that includes crypt damage, submucosal edema and the influx of inflammatory cells . These pathological changes were also evident in our Vehicle/C. diificle treated cohort of mice (panel B, Figure 5). In contrast, mice treated with BC30 showed some evidence of attenuated colonic histopathology, including decreased leukocyte influx into the colon (panel C, Figure 5). However, the overall comparisons of mean colonic histology scores were not statistically different on day 16.
Data from other studies suggests that toxin A secreted by C. difficile can activate the NF-κB signal transduction system in monocytes and colonic epithelial cells [4, 5]. This activation of NF-κB leads to the secretion of a key pro-inflammatory chemokine (IL-8) and subsequently neutrophil influx into the colonic tissue [4, 5]. Interestingly, BC-30 can significantly inhibit the IL-8 directed migration of human neutrophils in vitro. Based on these results, we measured the effects of BC30 on the nuclear binding of NF-κB p65, as well the murine chemokine (MIP-2) content in the colons of C. difficile infected mice. Probiotic treatment resulted in reductions of both colonic p65, as well as the MIP-2 content (Figure 6). However, statistical significance was not achieved compared to values in vehicle treated mice.
Nevertheless, these effects of BC30 on NF-κB mediated pathological processes (Figure 6) may contribute to the observed improvement in stool consistency observed in the probiotic-treated mice. For example, NF-κB activation is involved in the up-regulation of Fas-ligand, which subsequently leads to colonocyte apoptosis . Colonocyte apoptosis could diminish the barrier function of the colonic mucosa, and contribute to the altered stool consistency associated with CDAD . By reducing the colonic activation of NF-κB (Figure 6), it is possible that BC-30 treatment improved the barrier function of the colonic mucosa (Figure 5C), thereby improving stool consistency (Figures 2 and 3). Possibly, the improvement in stool consistency was unrelated to direct effects on colonization of C. difficile, or an alteration in the production of toxins A and B, because all infected mice showed evidence of infection and exotoxin production by ELISA (data not shown). Nevertheless, the ELISA kit utilized in this study does not quantify either the numbers of C. difficile in the colon, or the actual amounts of toxin production. Therefore, it is also possible that BC30 attenuated the level of C. difficile colonization and/or production of toxins in the colon. Future studies are needed to better understand the mechanisms, by which BC30 favorably impact stool consistency, as we observed in this study.
Of importance to this study, it is probable that the use of antibiotics in this murine CDAD model (during study days 5 to 10) resulted in anti-microbial effects that altered the levels of BC30 in the colon (David Keller, personal communication). Therefore, future studies with this murine model of CDAD should focus on effects of BC30 on the recurrence of C. difficile following treatment with vancomycin . Using this recurrence paradigm, the unwanted anti-microbial effects of antibiotics will not negatively impact the presence of BC-30 in the mouse colon. Finally, it would also be interesting to test other Bacillus coagulans strains in this type of experimental paradigm.