Bifidobacterium antagonizes key virulence factors of Clostridium difficile.

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

Boise-Idaho-USA

Congreso; The 8th Bienal Congreso of the Anaerobe Society oh the Ameritas.; 2006

The pathology associated to Clostridium difficile overgrowth ranges from slight diarrhea to fatal pseudomembranous colitis. Two toxins: TcdA and TcdB, are the main virulence factors. Since both Clostridium difficile and bifidobacteria localize in the colon, bifidobacteria are candidates for the formulation of probiotic preparations against this pathogen. The present work sought to determine, in vitro, the effect of Bifidobacterium on key virulence factors of Clostridium difficile (i. e. adhesion to cultured human enterocytes and biological activity). Two Clostridium difficile strains (ATCC 9689, TcdA+/TcdB+ and ATCC 43593, TcdA-/TcdB-) and 21 Bifidobacterium strains were studied. Clostridia were grown in BHI broth and bifidobacteria were grown in MRS broth. Adhesion assays: Clostridial suspensions were pre-incubated with bifidobacterial supernatants (1h, 37°C) and then co-incubated (108 CFU/ml) with Caco-2/TC7 cells (3 h at 37°C). Adhesion was assessed by microscopical counts. Different treatments of the bifidobacterial supernatants were done, i. e. heating (100°C for 10 min) and protease treatment (pepsin, trypsin and proteinase K). Culture medium supplemented with lactic and acetic acids was also included. Co-culture assays: bifidobacteria were incubated with Clostridium difficile ATCC 9689 (37°C for 20 h), centrifuged, filtered and the supernatants were tested for toxicity. Toxin adsorption: clostridial supernatants were incubated with concentrated bifidobacterial suspensions. Toxicity was assessed on Vero cells. Supernatants from 10 out of 21 bifidobacteria (e. g. CIDCA 5323 and 5325) inhibited the adhesion of Clostridium difficile to enterocytes. Thermal and pepsin treatments partially abrogated this effect. Filtered supernatants of Clostridium-Bifidobacteria co-cultures diminished the cytopathic activity as compared with pure cultures of clostridia. No effect was observed in the adsorption experiments. Conclusions: bifidobacteria produce extracellular factors (proteinaceous and thermolabile) that prevent the adhesion of Clostridium difficile to enterocytes. Co-culture with bifidobacteria affect the cytopathic ability of clostridia. This effect is not related to toxin adsorption. Our findings support the suitability of a Bifidobacterium-based probiotic approach for the treatment of Clostridium difficile associated diarrhoea.