Real-time acid production and extracellular matrix formation in mature biofilms of three Streptococcus mutans strains with special reference to xylitol

Abstract

Background: Acidogenicity and production of an extracellular matrix (ECM) are important virulence factors for the dental caries-associated bacteria, such as Streptococcus mutans, that live in biofilms on tooth surface. The ECM protects the bacteria from the flushing and buffering effects of saliva resulting in highly acidic microenvironments inside the biofilm.

Materials and methods: In this in vitro study, we applied real-time assays to follow biofilm formation and pH decrease in a growth medium and saliva by three S. mutans strains, as well as acid neutralization inside the mature biofilm. Results were compared with the biofilm composition. Effects of a non-fermentable polyol, xylitol, on acid production and acid neutralization in mature biofilms were evaluated by real-time pH measurements and confocal microscopy.

Results: Combination of real-time pH measurements with biofilm accumulation assays revealed growth media dependent differences in the pH decrease and biofilm accumulation, as well as strain differences in acid production and biofilm formation but not in the buffer diffusion through ECM. The presence of xylitol reduced the pH drop during biofilm formation of all strains. In addition, with strain Ingbritt xylitol reduced the amount of ECM in biofilm, which increased the rate of acid neutralization inside the biofilm after buffer exposure.

Conclusion: Our results stress the importance of biofilm matrix in creating the acidic environment inside a S. mutans biofilm, especially in the presence of saliva. In addition, our results suggest a novel mechanism of xylitol action. The observed increase in the permeability of the S. mutans ECM after xylitol exposure may allow acid-neutralizing saliva to reach deeper layer of the biofilms and thus, in part, explain previous clinical observations of reduced plaque acidogenicity after frequent xylitol use.

Keywords: Biofilm; Real-time assay; Streptococcus mutans; Xylitol; pH.

Fig. 1

Biofilm accumulation and pH changes in Brain Heart Infusion (BHI) media and in saliva of S. mutans strains Ingbritt, NCTC 10449 and CI2366. Overnight grown bacteria were washed and supended in NaCl solution and added into indicated growth media. Biofilm accumulation was followed by impedance based measurement (a,b) and pH changes measured by integrated fiberoptic pH sensors (c,d). CI: cell index. Mean ± SD.

Fig. 2

S. mutans strains were grown in BHI -sucrose medium with and without xylitol and pH changes monitored by real-time with fiberoptic pH sensors intergated in the growth plate. * indicates the first time point for significant difference Mean ± SD.

Fig. 3

Changes of the pH after exposing the biofilm to PBS buffer. The mature biofilms of three S. mutans strains Ingbritt, NCTC10449 and CI2366 were washed and exposed to neutral buffer. The pH changes inside the biofilm were measured by integrated pH sensors. At indicated times aliquots of the buffer in the wells were withdrawn and pH measured with a pH electrode. Inside: the pH inside the biofilm, top: the pH of the buffer on the top of the biofilm.

Fig. 4

The pH neutralization inside the mature biofilm formed by S. mutans Ingbritt in the presence (suc-xyl) or absence (suc) of xylitol after buffer exposure. The biofilms were allowed to accumulate in the presence and absence of the xylitol. After 48 h the biofilms were washed and exposed to neutral buffer and the pH was followed by integrated pH sensors. Mean ± SD.

Fig. 5

The pH neutralization inside the mature biofilm formed in growth medium with and without xylitol supplemented with pH sensitive fluorophore. The biofilms were exposed to neutral buffer and the pH changes were measured by confocal microscopy as changes in fluorescence ratio of the fluorophore in different levels of the biofilm. Relative Fl: fluorescence 452/521, higher ratio reflects higher pH.

Fig. 6

The effect of xylitol on biofilm composition. S. mutans Ingbritt biofilms were allowed to accumulate in BHI-sucrose growth medium with and without xylitol. The amount of Non-soluble and water -soluble exctracellular carbohydrates as well as extracellular DNA (eDNA) and the amount of bacteria (colony forming units, CFU) were measured.