Xylitol and erythritol inhibit real-time biofilm formation of Streptococcus mutans

Abstract

Background: Regular consumption of xylitol decreases the number of cariogenic streptococci in dental plaque. In vitro biofilm models to study the mechanism of xylitol action have been set-up, but the obtained results are contradictory. Biofilm growth is a dynamic process with time-specific characteristics that may remain undetected in conventional end-point biofilm tests. In this study we used an impedance spectroscopy instrument, xCELLigence Real Time Cell Analyzer (RTCA), that allows label-free, non-invasive real-time monitoring of biofilm formation, to explore effects of xylitol on biofilm formation by Streptococcus mutans. Based on the obtained information of biofilm dynamics, we assessed the number of viable bacteria, the polysaccharide content, and the expression levels of selected genes involved in glucan-mediated biofilm formation in different biofilm stages. Xylitol inhibition was compared with that of erythritol; another polyol suggested to have a positive impact on oral health.

Results: Our results showed that real-time monitoring provided new information of polyol-induced changes in S. mutans biofilm formation dynamics. The inhibitory effect of polyols was more pronounced in the early stages of biofilm formation but affected also the measured total amount of formed biofilm. Effects seen in the real-time biofilm assay were only partially explained by changes in CFU values and polysaccharide amounts in the biofilms. Both xylitol and erythritol inhibited real-time biofilm formation by all the nine tested S. mutans strains. Sensitivity of the strains to inhibition varied: some were more sensitive to xylitol and some to erythritol. Xylitol also modified the expression levels of gbpB, gtfB, gtfC and gtfD genes that are important in polysaccharide-mediated adherence of S. mutans.

Conclusion: The erythritol- and xylitol- induced inhibition of biofilm formation was only partly explained by decrease in the number of viable S. mutans cells or the amount of polysaccharides in the biofilm matrix, suggesting that in addition to reduced proliferation also the matrix composition and thereby the surface attachment quality of biofilm matrix may be altered by the polyols.

Keywords: Biofilm; Erythritol; Polysaccharides; Streptococcus mutans; Xylitol.

Fig. 1

Real-time biofilm formation of (a) S. mutans NCTC 10449 and (b) clinical isolate 2366 in BHI-sucrose medium or medium supplemented with 1, 2% or 5% xylitol and (c) S. mutans NCTC 10449 and (d) clinical isolate 2366 in BHI-sucrose medium or medium supplemented with 1, 2% or 5% erythritol. Bacteria were let to adhere on E-plate surface for 2 h, after which xylitol was added in the medium. dCI = cell index value change after addition of xylitol, see text for details. Mean ± SD, n = 4

Fig. 2

Real-time biofilm formation of different S. mutans strains in the BHI-sucrose medium (suc) or medium supplemented with 5% xylitol (xyl) or 5% erythritol (ery). aS. mutans NCTC10449 bS. mutans NG8 cS. mutans Ingbritt dS. mutans 2366 eS. mutans 195-s2 fS. mutans 199-s6 gS. mutans 124-s7 hS. mutans 113-s12 iS. mutans 117-s3. Bacteria were let to adhere on E-plate surface for 2 h, after which xylitol was added in the medium. dCI = cell index value change after addition of xylitol, see text for details. Mean ± SD, n = 2–4

Fig. 3

Formation rate and composition analysis of biofilms made by S. mutans NCTC 10449 (a, c, e) and clinical isolate 2366 (b, d, f) in BHI-sucrose medium (suc), and BHI-sucrose medium supplemented with eryhtritol (ery) or xylitol (xyl). a and b: Biofilm formation as measured in xCELLigence, the erythritol and xylitol curves significantly differed from sucrose in all time points, the line indicates the time period where there is a significant difference (p < 0.05) between erythritol and xylitol, c and d: Biofilm samples were collected at indicated time points and number of living bacteria were determined by plate counting, d and e: Amount of polysaccharides in the biofilm at different time points as measured by Anthrone method from collected biofilm samples. * significant difference (p < 0.05) between sucrose and xylitol, ^#significant difference (p < 0.05) between sucrose and erythritol, ^osignificant difference (p < 0.05) between erythritol and xylitol, **/^##/^oop < 0.01, ***/^###/^ooop < 0.001

Fig. 4

Expression of genes involved in glucose mediated adhesion and extra cellular polysaccharide formation in S. mutans 2366 biofilms at two different time points. a: 7 h, b: 10 h. Expression is depicted as fold change in gene expression in xylitol-biofilm in relation to biofilm formed in unsupplemented BHI-sucrose medium. * p < 0.0125