Antagonim of Clostridium difficile by Bifidobacterium bifidum CIDCA 5310: in vitro and in vivo studies

TREJO, F. M.; PÉREZ, P. F. AND DE ANTONI, G. L.

Córdoba

Congreso; VI Congreso Argentino de Microbiología General (SAMIGE); 2009

SAMIGE

Clostridium difficile is responsible for antibiotic-associated colitis and diarrhea. Two protein toxins (TcdA and TcdB) are related to the virulence of this microorganism. Probiotic bacteria (i.e.bifidobacteria and lactobacilli) constitute an alternative approach to prevent/treat C. difficile associated diarrhea (CDAD). However, mechanisms involved in the protective effect remain unknown. The present work aims to gain insight on the underlying mechanisms of the protective effect by using both in vitro and in vivo models. Clostridium difficile is responsible for antibiotic-associated colitis and diarrhea. Two protein toxins (TcdA and TcdB) are related to the virulence of this microorganism. Probiotic bacteria (i.e.bifidobacteria and lactobacilli) constitute an alternative approach to prevent/treat C. difficile associated diarrhea (CDAD). However, mechanisms involved in the protective effect remain unknown. The present work aims to gain insight on the underlying mechanisms of the protective effect by using both in vitro and in vivo models. Bifidobacterium bifidum CIDCA 5310 and C. difficile strain 117 were grown in pure or mixed cultures (co-cultures) in BHI at 37 °C for 20 h. Biological activity of the culture supernatants (SN) was evaluated on Vero cells. Cells were incubated with SN from C. difficile pure cultures or co-cultures for 16 hours at 37°C. Fraction of detached cells was evaluated by staining remaining cells with crystal violet, further dye extraction and colorimetric determination. Biological activity was defined as the SN concentration that detaches 50 % of cells (DD50). DD50 is inversely related to the biological activity. Intra and extracellular toxins concentrations were evaluated through immunobloting by using monoclonal antibobies. For in vivo assays, female hamsters (4-7 weeks-old) were administered with bifidobacteria (108 CFU/ml) or placebo in drinking water starting at day 0. At day 7, a single dose of clindamycin (3 mg/animal) was administered by intragastric gavage. At day 10, all the animals were infected intragastrically with  108 CFU of C. difficile . Diarrhea, enterocolitis and survival of the animals were assessed. Cecal content was tested for biological activity on Vero cells and samples of cecum were processed for histology. In vitro, concentrations of both toxins were reduced around 50 % in co-cultures as compared with pure cultures of strain 117. Besides, intracellular toxin concentration was increased 3 times, compared with pure cultures. There was a significantly (p=0.05) higher ratio of survivors in the probiotic-treated group (0/13) as compared with the placebo-treated group (4/13). Ratio of animals showing enterocolitis was also significantly lower in the probiotic group (3/13) than in the placebo group (11/13, p=0,002). Histological analysis of cecum showed significant inflammation only in the placebo group and DD50 in cecal content was significantly higher in the animals administered with bifidobacteria (2,9 ± 1,2) as compared with those that received only placebo (0,6 ± 0,5). Our findings are compatible with 3 mechanisms: decrease of toxin production/secretion, blockade of the interaction between toxins and receptors or antagonism of signals triggered by toxins. The present work shows for the first time, the correlation between in vitro and in vivo results thus providing a rationale basis for the use of bifidobacteria-based probiotics for the prevention/treatment of intestinal pathologies associated to C. difficile.