Research Article: Biofilm extracellular polysaccharides degradation during starvation and enamel demineralization

Date Published: July 17, 2017

Publisher: Public Library of Science

Author(s): Bárbara Emanoele Costa Oliveira, Jaime Aparecido Cury, Antônio Pedro Ricomini Filho, Marcelle Nascimento.

http://doi.org/10.1371/journal.pone.0181168

Abstract

This study was conducted to evaluate if extracellular polysaccharides (EPS) are used by Streptococcus mutans (Sm) biofilm during night starvation, contributing to enamel demineralization increasing occurred during daily sugar exposure. Sm biofilms were formed during 5 days on bovine enamel slabs of known surface hardness (SH). The biofilms were exposed to sucrose 10% or glucose + fructose 10.5% (carbohydrates that differ on EPS formation), 8x/day but were maintained in starvation during the night. Biofilm samples were harvested during two moments, on the end of the 4th day and in the morning of the 5th day, conditions of sugar abundance and starvation, respectively. The slabs were also collected to evaluate the percentage of surface hardness loss (%SHL). The biofilms were analyzed for EPS soluble and insoluble and intracellular polysaccharides (IPS), viable bacteria (CFU), biofilm architecture and biomass. pH, calcium and acid concentration were determined in the culture medium. The data were analyzed by two-way ANOVA followed by Tukey’s test or Student’s t-test. The effect of the factor carbohydrate treatment for polysaccharide analysis was significant (p < 0.05) but not the harvest moment (p > 0.05). Larger amounts of soluble and insoluble EPS and IPS were formed in the sucrose group when compared to glucose + fructose group (p < 0.05), but they were not metabolized during starvation time (S-EPS, p = 0.93; I-EPS, p = 0.11; and IPS = 0.96). Greater enamel %SHL was also found for the sucrose group (p < 0.05) but the demineralization did not increase during starvation (p = 0.09). In conclusion, the findings suggest that EPS metabolization by S. mutans during night starvation do not contribute to increase enamel demineralization occurred during the daily abundance of sugar.

Partial Text

Dental caries is a biofilm-sugar related disease that depends on biofilm accumulation on tooth surface and its frequent exposure to dietary carbohydrates [1]. The cariogenic biofilm forms and grows on dental surfaces in a dynamic condition in which the exposure to dietary carbohydrates occurs intermittently [2]. During the day, the biofilm is frequently exposed to short periods of great amount of carbohydrates, followed by long periods of non-exposure between the meals and overnight. These episodes are known as “feast” and “famine” periods and they are determinant for bacterial metabolism and biofilm growth [3, 4, 5]. In “feast” periods, bacterium such as S. mutans is able to store the excess of available carbohydrate as intracellular polysaccharides (IPS), which act as an energy reserve source in “famine” periods [6, 7]. Besides the IPS storage, glucosyl- and fructosyltransferases enzymes produced by S. mutans synthesize extracellular polysaccharides (EPS) when sucrose is the available carbohydrate [8, 9, 10].

For the amounts of polysaccharides, the statistical analyses showed a significant difference for the factor carbohydrate (p < 0.05), however no differences were observed for the harvest moment (S-EPS, p = 0.93; I-EPS, p = 0.11; and IPS = 0.96) and for the interaction effect between the two factors (carbohydrate and harvest moment) (S-EPS, p = 0.82; I-EPS, p = 0.08; and IPS = 0.70). The same statistical pattern was observed for the variables: biofilm dry weight, CFU per biofilm dry weight, %SHL and biomass. The amounts of intracellular (IPS), and soluble (S-EPS) and insoluble (I-EPS) extracellular polysaccharides in biofilms formed under daily exposure to sucrose were higher, when compared to those found in biofilms exposed to glucose + fructose (Fig 1) (p < 0.05). In this study, we used a validated S. mutans biofilm model for caries study [5] to evaluate the degradation of extracellular polysaccharides (EPS) as an energy source during overnight starvation of carbohydrate and the consequent effect on enamel demineralization. Once this approach has not been performed using biofilms, this study seems to be a pioneer in the field. The experimental design using different carbohydrates allowed us to develop biofilms with EPS (sucrose) or without them (glucose + fructose), however, for both groups, bacterial cells could also storage intracellular polysaccharides (IPS), which could also be used as an energy source. Our findings showed no reduction in the amounts of EPS and IPS (Fig 1), and no increase in enamel demineralization after the overnight period of starvation (Table 1). The non-reduction of EPS and IPS quantities may indicate that they were not metabolized during the starvation period, or that they were metabolized in a small amount, just to maintain the bacterial basal metabolism, causing no additional damage to the enamel.   Source: http://doi.org/10.1371/journal.pone.0181168

 

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