In vitro biofilms of Clostridioides difficile undomesticated strains: Morphology and properties according to strain diversity

Clostridioides difficile is a Gram-positive, spore-forming, obligate anaerobe and an entero-pathogen representing a One Health problem. C. difficile infections are difficult to treat and recurrences frequent. C. difficile biofilms may play a role in persistence. We studied the biofilms formed in vitro by a collection of C. difficile undomesticated strains (n=27, 20 toxigenic and 7 non-toxigenic) of 11 PCR-ribotypes (WRT) and isolated from equids. After growth in rich BHIS medium supplemented with glucose for 48h, the biomass of biofilms stained by crystal violet, i. e. adhesive to the surface, was measured, while intact biofilms were observed in situ, by confocal laser scanning microscopy, to determine their total biovolume and 3D morphology. All biofilms were relatively similar, even though adhesive, stained biofilms of non-toxigenic strains of ribotype 009 significantly displayed highest biomass. These results suggested that strains of ribotype 009 were able to form a highly cohesive and polystyrene-adhesive biofilm than any other strains. 8 common, representative ribotypes in equids, including 6 clinically important ribotypes in humans, were then selected and one strain chosen for each of them. Their biofilms and planktonic cultures grown under the same conditions were compared for sporulation, toxin production and susceptibility to vancomycin at a highly inhibitory concentration (8 MIC). In both the biofilm and planktonic culture of all selected strains, sporulation was low and toxin production undetectable. The addition of vancomycin at T0=24h followed by a 24 hours exposure barely reduced the viability of neither planktonic cultures nor biofilms of any strains. On the contrary, for all selected strains, addition of vancomycin at T0=6h followed by a 24 hours exposure significantly and more efficiently reduced the viability of planktonic cultures than that of biofilms, which was not decreased at all in the case of strains of ribotype WRT 005, 009 and 035. The biofilms of these selected strains of eight ribotypes, compared to their planktonic cultures, therefore showed a higher tolerance to vancomycin, suggesting that they could play a role in persistence.