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

Abstract

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.

Figure 1

Role of LDB B-30892 on CD-9689 mediatedcytotoxicity. Confluent Caco-2 cultures were incubated 24 h in DMEM containing 20% FBS. Cell free supernatants (CFS or CFT) from different treatments were added to the cell monolayer. (A) Caco-2 control cells (200× original magnification); (B) Normal-appearance after 24 h of incubation with fresh RCM diluted 1:4 into DMEM containing 20% FBS; (C) Appearance of Caco-2 in LDB B-30892 CFS, diluted 1:4 into DMEM, 20% FBS. (D) Caco-2 cultures incubated 24 h in cell-free RCM after growing CD-9689 24 h in it. Note the cytopathic effect of the CD-9689 cytotoxin. (E) Caco-2 cultures incubated 24 h in cell-free RCM after growing CD-9689 and LDB B-30892 24 hours together in it. (F) Caco-2 cultures incubated 24 h in cell-free RCM after growing CD-9689 for 24 h.

Figure 2

Live and dead status of Caco-2 cells after exposures to CFT from CD-9689 with or without treatments with different lactobacillus CFS. After the treatments with CFS or CFTs, Caco-2 cells were trypsinized and then florescence dye, PI and AO were added. A green fluorescence (AO) indicates live cell, while a red fluorescence (PI) indicates dead cell.

Figure 3

Quantitative assessment of live and dead status of Caco-2 cells after exposures to CFT from CD-9689 with or without treatments with different lactobacilli CFS. Cont, control without any treatment, CFS from LDB B-30892 was added to CD-9689 CFT so that the resulting ratio of CFT from CD and CFS from LDB are 1:1, 4:1 and 8:1. The ratio of CFS from other lactobacilli (LB-1 through LB-6) to CFT from CD-9689 were 1:1. Values are presented as Mean ± Standard error of mean (SEM) of three experiments done in duplicate.

Figure 4

Inhibition of CD-9689 to Caco-2 monolayer by cell free supernatants from different lactobacilli. CD-9689 cells were suspended in 1:1 volume/volume RCM- lactobacillus conditioning MRS at a MOI of 100: 1 and incubated at 37°C in 7% CO₂ for 2 h. After incubation, Caco-2 cells were washed, stained with Giemsa and counted. The above formula was used to calculate the percent inhibition of adhesion CD-9689 to Caco-2 monolayer. Values are presented as Mean ± Standard error of mean (SEM) of three experiments done in duplicate. ** P < 0.01.

Figure 5

Failure of LDB B-30892 (Lactobacillus) to inhibit the growth of CD-9689 (C. diff). LDB B-30892 and CD-9689 were co-cultured on RCM agar anaerobically at 37°C for 48 h. LDB B-30892 and CD-9689 were co-cultured on TSA anaerobically at 37°C for 48 h.