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Background  Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD).  Clostridium difficile  is the most common cause of AAD and the resulting  C. difficile  – mediated infection (CDI), is potentially deadly.  C. difficile  associated diarrhea (CDAD) is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by  C. difficile  causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria  Lactobacillus delbrueckii  ssp.  bulgaricus  B-30892 (LDB B-30892) on  C. difficile -mediated cytotoxicity using Caco-2 cells as a model.    Methods  Experiments were carried out to test if the cytotoxicity induced by  C. difficile- conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS) from LDB B-30892 in different dilutions (1:2 to 1:2048). In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect  C. difficile  induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of  C. difficile  to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on  C. difficile .    Results and discussion  Co-culturing of LDB B-30892 with  C. difficile  inhibited the  C. difficile- mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892- C. difficile  co-culture was administered (up to a dilution of 1:16) on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin preparation (CFT) of separately cultured  C. difficile , the LDB B-30892 CFS was inhibitory to  C. difficile  CFT-mediated cytotoxicity at a ratio of 1:8 (LDB B-30892 CFS: C. difficile  CFT). We failed to find any similar inhibition of  C. difficile- mediated cytotoxicity when other probiotic organisms were tested in parallel to LDB B-30892. Our data of cytotoxicity experiments suggest that LDB B-30892 releases one or more bioactive component(s) into the CFS, which neutralizes the cytotoxicity induced by  C. difficile , probably by inactivating its toxin(s). Our data also indicate that CFS from LDB B-30892 reduced the adhesion of  C. difficile  by 81%, which is significantly ( P  <0.01) higher than all other probiotic organisms tested in this study.    Conclusion  This study reveals the very first findings that  Lactobacillus delbrueckii  ssp.  bulgaricus  B-30892 (LDB B-30892) can eliminate  C. difficile -mediated cytotoxicity, using Caco-2 cells as a model. The study also demonstrates that LDB B-30892 can reduce the colonization of  C. difficile  cells in colorectal cells. More study is warranted to elucidate the specific mechanism of action of such reduction of cytotoxicity and colonization.

Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells