Fructooligosaccharides and mannose affect Clostridium difficile adhesion and biofilm formation in a concentration-dependent manner

Abstract

The aim of this study was to investigate the effects that prebiotic and candidates for prebiotics on Clostridium difficile strains to adhere to various human epithelial cell lines and to compare the adhesive properties of specific C. difficile strains. We also sought to examine the effect of different concentrations of fructooligosaccharides and mannose on the formation of biofilms by C. difficile strains. The influence of cellobiose, fructooligosaccharides, inulin, mannose, and raffinose on the adherence properties of various C. difficile strains, including motile 630, non-motile M120, and 10 clinical motile ribotype 027 strains, to non-mucous secreting HT-29, mucous secreting HT-29 MXT, and CCD 841 CoN cells lines. The most effective prebiotics were used in biofilm formation assays. We demonstrated that all C. difficile strains adhered to all cell lines. However, the C. difficile M120 non-motile strain was statistically more likely to adhere to all three cell lines (CFU median, 40) compared to the motile strains (CFU median, 3; p < 0.001). Furthermore, among the carbohydrates examined, only fructooligosaccharides and mannose were found to significantly decrease adhesion (p < 0.001) of C. difficile strains. Alternatively, using a biofilm assay, we observed, via confocal laser scanning microscopy, that sub-inhibitory concentrations (1%) of fructooligosaccharides and mannose functioned to increase biofilm formation by C. difficile. We demonstrated that specific prebiotics and candidate prebiotics exhibit varying anti-adhesive properties towards C. difficile in vitro and that treatment with sub-inhibitory concentrations of prebiotics can cause an increase in biofilm formation by C. difficile.

Keywords: Bacterial adhesion; Biofilm formation; Clostridium difficile; Prebiotics, candidate prebiotics.

Fig. 1

Median adhesion of C. difficile strains to the three cell lines

Fig. 2

Adhesion of motile clinical C. difficile strains belonging to RT027 and two control strains non-motile M120 and motile 630

Fig. 3

Effect of examined prebiotics on adhesion of C. difficile to three cell lines

Fig. 4

Average biofilm formation by examined C. difficile strains with different concentrations FOS and MAN

Fig. 5

Effects of FOS and MAN on C. difficile biofilm formation. Representative confocal microscopy images of horizontal (xy) and vertical (xz and yz) projections of C. difficile biofilm structures. Vertical view was obtained using maximum intensity projection as a crosssection through marked line