Sucrose challenges to Streptococcus mutans biofilms and the curve fitting for the biofilm changes

Sucrose challenges to Streptococcus mutans biofilms and the curve fitting for the biofilm changes ABSTRACT The relationship between sugar level and development of dental caries has long been a main topic in dentistry. However, as a ubiquitous component of the modern diet, sucrose is mainly derived from three meals a day, rather than a long time exposure. In this study, various concentrations of sucrose were provided to Streptococcus mutans biofilms for 1 h per exposure (three times per day) to imitate a human meal pattern. And then the relationship between sucrose concentration and changes in the treated biofilms was determined. The results indicated that the components and acid production of the treated biofilms changed in a second-order polynomial curve pattern with sucrose concentration increase, which were confirmed by CLSM and SEM analyses. However, gene expression related to extracellular polysaccharides (EPS) formation, acid production and tolerance was up-regulated with sucrose concentration increase, which might have been due to compensation for the decrease in EPS formation and acid production by the biofilms at higher concentrations of sucrose. These findings suggest that sucrose in the range of 1%–5% can support the highest acid production and accumulation of S. mutans biofilms, which may further increase its cariogenic potential. However, additional studies are required to confirm the relationships in human cariogenic biofilms. sucrose, cariogenic biofilms, relationship, virulence INTRODUCTION Dental caries is a dental biofilm-related disease. If dental biofilms undergo significant environmental changes, such as an increase in sugar availability, aciduric and acidogenic bacteria become the dominant members of dental biofilms (Marsh 2003). The shift in microbial composition to aciduric and acidogenic bacteria (i.e. cariogenic biofilm formation) facilitates pH decrease and acidification of biofilms, which results in the development of cariogenic biofilms (Marsh 2003; 2010). Of the aciduric and acidogenic bacteria in dental biofilms, Streptococcus mutans is generally considered as an important cariogenic bacteria (Loesche 1986). This species can produce large amounts of acid by utilizing carbohydrates and survive in a low pH environment, which enhances the demineralization of dental hard tissue. Furthermore, S. mutans can utilize sucrose to synthesize extracellular polysaccharides (EPSs) by glucosyltransferases (GTFs) (Bowen and Koo 2011; Sendamangalam et al.2011). The intake of sugars including sucrose, glucose, fructose, lactose and maltose is also a contributory factor in the development of dental caries. Of dietary sugars, sucrose shows the highest cariogenic potential in dental caries development. Several in vitro and in vivo studies have demonstrated that sucrose can change the dental biofilm properties and increase enamel demineralization in a concentration or frequency dependent pattern (Aires et al.2006; Paes Leme et al.2006; Ccahuana-Vasquez et al.2007; Vale et al.2007; Sissons et al.2007; Diaz-Garrido, Lozano, Giacaman 2016). Sucrose is not only fermented by oral bacteria, but also enhances colonization and growth of mutans streptococci such as S. mutans in dental biofilms more than other sugars (Gupta et al.2013; Salli et al.2016). Furthermore, sucrose serves as a substrate for the synthesis of EPSs in dental biofilms, especially cariogenic biofilms (Newbrun 1967; Bowen 2002). It has been reported that EPSs contribute to the bulk, physical integrity and stability of biofilm matrixes, indicating that EPSs can be an essential virulence factor related to cariogenic biofilm formation (Koo et al.2010). Many studies have shown that the prevalence of dental caries is associated with the patterns of sugar intake in relation to frequency, stickiness and position within a meal sequence or a snack (Gupta et al.2013; Diaz-Garrido, Lozano and Giacaman 2016). Furthermore, a positive response relationship between free sugar intake and dental caries has been observed in many studies (Cury et al.2000; Sheiham and James 2015; Bernabe et al.2016). The WHO, therefore, recommends that both adults and children reduce their intake of free sugars to below 5% of total energy intake for the prevention of dental caries (Sheiham and James 2015). However, despite prolonged studies on both dental caries and sugars, the functional relationship between sugar concentration, especially sucrose, and virulence of cariogenic biofilms is not well documented. Cai et al. (2016) reported that the influence of sucrose on the components and acid production of a cariogenic biofilms follows a second-order polynomial curve with concentration dependence. However, the functional curve, which was derived from a 46 h experiment, could not explain the relationship in reality since dental biofilms are exposed to sucrose during meals or snack times rather than consistently over a 46 h time period. In this study, we designed a new experimental plan to mimic the sucrose exposure of three meals per day and then investigated change in a cariogenic biofilm according to sucrose level using an S. mutans biofilm model. This study also determined the mathematical relationship between sucrose level and accumulation and virulence of S. mutans biofilms. MATERIALS AND METHODS Streptococcus mutans biofilm formation and experimental scheme The S. mutans biofilm preparation and experimental schemes for this study are shown in Appendix Fig. 1. Streptococcusmutans UA159 (ATCC 700610; serotype c) biofilms were formed on saliva-coated hydroxyapatite (sHA) discs (2.93 cm2; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) placed in a vertical position in 24-well plates as described previously (Cai et al.2016). Briefly, HA discs were incubated in filter-sterilized (0.22-µm low protein-binding filter) human saliva for 1 h at 37°C. For biofilm formation, the sHA discs were transferred to a 24-well plate containing brain heart infusion (BHI; Difco, Detroit, MI, USA) broth with 1% (w/v) sucrose and S. mutans UA159 (2–5×106 colony-forming unit (CFU)/ml). The biofilms were grown at 37°C with 5% CO2 for 22 h to allow initial biofilm growth. After 22 h, the biofilms were grown in 20 mM potassium phosphate buffer (PPB, pH = 7.2) to the end of the experimental period (74 h). The biofilms in PPB were treated three times a day (at 22, 26, 31, 46, 50, 55 and 70 h) with 0%, 1%, 5%, 10%, 20% or 40% sucrose BHI broth. Specifically, the biofilms were exposed to the treatments for 1 h, dip-washed with water, and then transferred to PPB. Finally, the 74 h-old treated biofilms were analyzed in this study. Figure 1. View largeDownload slide Changes in composition and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A–C) Changes in dry weight, water-insoluble EPSs, and bacterial counts. (D-1) Change in acid production. (D-2) Initial rate and normalized initial rate of H+ production. (D-3) Total produced concentration and normalized total produced concentration of H+. In A–C, since x coordinate are plotted on a logarithmic scale, and since the log of 0 is undefined, we approximated 0 with an x coordinate of 0.1. *P < 0.05, significantly different from 0% sucrose. Figure 1. View largeDownload slide Changes in composition and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A–C) Changes in dry weight, water-insoluble EPSs, and bacterial counts. (D-1) Change in acid production. (D-2) Initial rate and normalized initial rate of H+ production. (D-3) Total produced concentration and normalized total produced concentration of H+. In A–C, since x coordinate are plotted on a logarithmic scale, and since the log of 0 is undefined, we approximated 0 with an x coordinate of 0.1. *P < 0.05, significantly different from 0% sucrose. Microbiological and biochemical analyses Dry weight (all biofilm components except water), the level of water-insoluble EPSs, and the number of CFUs of the 74 h-treated biofilms were determined as described elsewhere (Koo et al.2005; Pandit et al.2015). Briefly, the treated biofilms were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath (Power sonic 410; Hwashin Technology Co., Seoul, Korea) to disperse the biofilms. The suspension was homogenized by sonication (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA) for 30 s after adding 3 ml of 0.89% NaCl. An aliquot (100%µl) of the homogenized suspension was serially diluted and plated to determine the number of CFUs. To determine dry weight and amount of water-insoluble EPSs, the remaining solution (4.9 ml) was centrifuged (3000 g) for 20 min at 4°C. The biofilm pellet was then lyophilized and weighed to determine the dry weight. The water-insoluble EPSs were extracted from the dry pellet using 1 N sodium hydroxide before determination of the polysaccharide amount using a phenol-sulfuric acid assay. To evaluate differences in acid production of the 74 h-treated biofilms, a biofilm pH drop assay was performed as described elsewhere (Belli, Buckley and Marquis 1995). Briefly, the treated biofilms were incubated in 20 mM PPB/1 mM MgCl2 + 50 mM KCl (pH = 7.2) for 1 h to deplete endogenous catabolites and then transferred to a 6-well plate containing a salt solution (50 mM KCl + 1 mM MgCl2, pH = 7.0). The pH was adjusted to 7.2 with 0.2 M KOH solution. Glucose was then added to the mixture to produce a final concentration of 1% (w/v). The pH change was assessed using a glass electrode over a period of 120 min. The initial rate of H+ production (y) was derived from the equation: \begin{eqnarray*}{\rm y} = {\left( {{{\rm H}^ + }\,{\rm concentration}\,{\rm at}\ 30\,{\rm min} - {{\rm H}^ + }\,{\rm concentration}\ {\rm at}\,0\,{\rm min}} \right)} / 30\end{eqnarray*} The total produced concentration of H+ (y) was derived from the equation: \begin{eqnarray*}{\rm y} = {{\rm H}^ + }\ {\rm concentration}\ {\rm at}\,120\,{\rm min}\, - \,{{\rm H}^ + }\ {\rm concentration}\ {\rm at}\,0\,{\rm min}.\end{eqnarray*} Furthermore, the normalized initial rate of H+ production (or normalized total produced concentration of H+) was derived from the equation: y = initial rate of H+ production (total produced concentration of H+)/CFUs. Confocal laser scanning microscopy analysis Confocal laser scanning microscopy (CLSM) analysis was performed to confirm the results of microbiological and biochemical studies. The concentrations of sucrose tested in the CLSM study were 0%, 1%, 5% and 40% (w/v). To investigate difference in bacterial cells, the 74 h-treated biofilms were stained at room temperature in the dark for 30 min using the FilmTracer LIVE/DEAD Biofilm viability kit L10316 (Invitrogen, Molecular Probes Inc., Eugene, OR, USA). The final concentrations of SYTO9 and propidium iodide (PI) were 6.0 and 30 µM, respectively. This viability kit is based on plasma membrane integrity to determine live and dead cells. In this study, we regarded the cells with intact membranes (green) as live cells, whereas cells with damaged membranes (red) were regarded as the dead cells. To determine differences in EPSs, 1 µM of Alexa Fluor® 647-labeled dextran conjugate (10000MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) was added to 0%, 1%, 5% or 40% sucrose BHI broth during the 1 h sucrose treatments (total seven times). The stained live and dead bacterial cells and EPSs were observed using an LSM 510 META (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium–neon lasers. Three independent experiments were performed, and five image stacks per experiment were collected (n = 15). The biovolume (µm3/µm2), thickness (µm), or roughness coefficient and the coverage (%) of live and dead cells and EPSs were quantified from the entire stack using COMSTAT image-processing software (Heydorn et al.2000). The bio-volume is defined as the volume of the biomass (µm3) divided by the surface area of the substratum (HA discs) (µm2). The roughness coefficient, which provides a measure of how much the thickness of the biofilm varies, is calculated from the thickness distribution of the biofilm. The coverage (%), which is the fraction of the area occupied by biomass in each image of a stack, reflects how efficiently each stack is colonized by bacteria or EPSs (Heydorn et al.2000; Jeon et al.2009). The three-dimensional architecture of the biofilms was visualized using Imaris 8.0.2 (Bitplane, Zurich, Switzerland). Scanning electron microscopy analysis Scanning electron microscopy (SEM) was performed as detailed elsewhere (Takeuchi et al.2007). Briefly, 74 h-treated (0%, 5% or 40%) biofilms were rinsed three times in 0.1 M cacodylate buffer and prefixed with 3% glutaraldehyde solution for 1 h followed by post-fixation with a 1% osmium tetroxide solution for 1 h. The biofilms were then dehydrated in a graded series of ethanol (30%–100%) and infiltrated with nitrogen gas immediately before sputter coating with gold–palladium. The biofilm samples were analyzed by SEM (JSM-5900, Jeol, Japan). Gene expression analysis RNA from sucrose (0%, 1%, 5% or 40%) exposed biofilms was extracted and purified using standard protocols optimized for biofilms (Cury and Koo 2007). Briefly, the 74 h biofilms were removed and homogenized by sonication. RNA was extracted and purified using a Trizol® Max bacterial RNA isolation kit (Life Technologies, Carlsbad, CA, USA). DNA was digested with DNase I, amplification grade (Life Technologies, Carlsbad, CA, USA). Reverse transcription and real-time PCR were performed using a Top script® cDNA synthesis kit (Enzynomics, Daejeon, Korea) and a Power SYBR® Green PCR Master Mix (Life Technologies, Warrington, UK), respectively, according to the manufacturers’ instructions. Specific genes related to EPSs formation (gtfB, gtfC, gtfD), acid production (eno, ldh), and acid tolerance (atpD) were evaluated. The gene-specific primers used in this study were described previously (Lu, Liu and Yang 2008; Jeon et al.2009; Xu, Zhou and Wu 2011; Dong et al.2012). Relative expression was calculated by normalizing each test gene of the treated biofilms to the 16sRNA gene, which served as the reference gene (Shemesh et al.2008). Data analysis was performed using StepOne software v2.0 (Applied Biosystems, Foster City, CA, USA) according to the 2−ΔΔCT method. Statistical analysis To determine the relationship between sucrose concentration and S. mutans biofilm formation, a second-order polynomial fitting for sucrose concentration versus CFU count, dry weight, water-insoluble EPSs was performed. The determination coefficients (R2) of each fitted line were also calculated. All experiments were performed in duplicate, and at least three different experiments were conducted. The data are presented as mean ± standard deviation. Intergroup differences were estimated using one-way analysis of variance, followed by a post hoc multiple comparison (Tukey) test to compare multiple means. Values were considered statistically significant when the P value was < 0.05. RESULTS Changes in microbiological and biochemical studies The dry weight (all biofilm components except water), EPS amount and CFU counts of 74 h-treated biofilms were initially increased and then gradually decreased with sucrose concentration increase, which followed a second-order polynomial curves. The R2 values were 0.944, 0.914 and 0.997 (P < 0.05), respectively ( Fig. 1A, B and C). The maximum influence concentrations of sucrose for dry weight, EPS amount, and CFU counts were 2.1%, 2.4% and 0.6%, respectively. The change in acid production of the treated biofilms is shown in Fig. 1D-1. However, since only the general acid production tendency is shown, the initial rate (or normalized initial rate) of H+ production and total acid produced (or normalized total produced) were calculated to clearly describe acid production ability of the biofilms. The initial rate and normalized initial rate of H+ production all increased and then decreased as sucrose concentration increased, with 5% sucrose showing the highest acid production ability (Fig. 1D-2). A similar trend was shown in total produced H+ concentration; after normalization to total produced concentration of H+, 5% sucrose showed the highest values ( Fig. 1D-3). Changes in CLSM and SEM studies To further evaluate the effect of sucrose on biofilm components and structure, the CLSM analysis was performed. As shown in Fig. 2A and B, the bio-volume and mean thickness of live and dead cells of the 74 h treated biofilms initially increased and then decreased with sucrose concentration increase, with 1% or 5% sucrose showing the highest bacterial volume and mean thickness. Interestingly, the mean bio-volume and thickness of live cells at 0%, 1% and 5% sucrose were higher than those of dead cells, while the mean bio-volume and thickness of live cells at 40% sucrose were similar to those of dead cells. However, an opposite pattern was shown for the roughness coefficient (Fig. 2C), where 1% or 5% sucrose showed the lowest roughness coefficient. The coverage of live and dead cells of the treated biofilms was also calculated. The coverages of live cells at 0%, 1% and 5% sucrose were higher than those of dead cells, while the coverage of live cells at 40% sucrose was similar to that of dead cells (Fig. 2E). Representative three-dimensional images of live and dead cells are shown in Fig. 2D; biofilms at 5% sucrose showed the highest biofilm volume. The mean bio-volume and mean thickness of EPSs also gradually increased and then decreased as sucrose concentration increases ( Fig. 3A and B); the EPS was almost non-existent at 0% and 40% sucrose (Fig. 3D). The representative EPSs images showed that 1% and 5% sucrose exhibited large concentrations of EPSs with a homogeneous structure that covered and surrounded the bacteria micro-colonies to form a complex, structured biofilm (Fig. 3C). In this study, an SEM study was also performed to confirm the CLSM result. As shown in Fig. 4, biofilms treated with 5% sucrose showed micro-colonies surrounded with a large amount of EPSs. However, biofilms treated with 0% and 40% sucrose did not show EPSs (Fig. 4) Figure 2. View largeDownload slide Changes in CLSM images of bacterial cells in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) roughness coefficient, (D) representative confocal images and (E) coverage of live and dead cells. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 2. View largeDownload slide Changes in CLSM images of bacterial cells in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) roughness coefficient, (D) representative confocal images and (E) coverage of live and dead cells. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 3. View largeDownload slide Changes in CLSM images of EPSs in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) representative confocal images and (D) coverage of EPSs. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 3. View largeDownload slide Changes in CLSM images of EPSs in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) representative confocal images and (D) coverage of EPSs. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 4. View largeDownload slide Representative SEM images of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). Figure 4. View largeDownload slide Representative SEM images of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). Changes in gene expression In the 74 h-treated biofilms, the expressions of genes related to EPS formation (gtfB, gtfCandgtfD), glycolysis (ldh), and acid tolerance (atpD) were significantly upregulated at 40% sucrose (P < 0.05) (Fig. 5). However, the expression of eno was not upregulated at 40% sucrose (P > 0.05). Figure 5. View largeDownload slide Changes in gene expression related to EPS formation and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A-1) gtfB. (A-2) gtfC. (A-3) gtfD. (B-1) eno. (B-2) ldh. (B-3) atpD. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 5. View largeDownload slide Changes in gene expression related to EPS formation and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A-1) gtfB. (A-2) gtfC. (A-3) gtfD. (B-1) eno. (B-2) ldh. (B-3) atpD. Values followed by the same superscripts are not significantly different from each other (P > 0.05). DISCUSSION According to the ecological plaque hypothesis, environmental changes contribute to the formation of cariogenic biofilms, and these biofilms are mainly composed of aciduric and acidogenic bacteria (such as mutans streptococci and lactobacilli) (Marsh and Zaura 2017). Although the role of sucrose as a fermentable sugar in the development of carious lesions is vastly acknowledged, limited studies have been performed to reveal the functional relationship between sucrose concentration and cariogenic biofilm properties in reality. To define the relationships, we exposed S. mutans biofilms, a representative cariogenic biofilm, to different concentrations of sucrose for three times per day to mimic three meals per day, which may simulate the feast and famine episodes present in the oral cavity. However, although the biofilm model used in this study simulated the clinical situation to some extent, additional in vivo studies are required to confirm the relationships since the biofilm model does not precisely mimic the complex microbial community found in cariogenic biofilms on tooth surfaces. In this study, our data show that the influence of sucrose exposure on S. mutans biofilm components followed a second-order polynomial curve with concentration dependence, and the determination coefficients (R2) values ranged from 0.914 to 0.997 ( Fig. 1A, B and C). This suggests that the functional curve is appropriate to describe changes in the biofilm components by sucrose as 91.4%–99.7% of the variation in the functional curves can be explained by variation in sucrose concentration. In general, the functional curves indicate that increasing sucrose level in S. mutans biofilm formation increases biofilm components such as dry weight (all biofilm components except water), EPS amount, and CFU count up to a certain concentration (turning concentration), after which these biofilm components decrease as sucrose concentration increases. In the functional curves, the turning concentration for the biofilm formation ranged from 0.6% to 2.4% ( Fig. 1A, B and C), suggesting that sucrose level can have a strong influence on cariogenic biofilm accumulation and subsequent dental caries development. The relationships derived from microbiological and biochemical analyses were confirmed by CLSM and SEM studies. As shown in Figs. 2–4, bio-volume, thickness and coverage of bacterial cells (live or dead) and EPSs gradually increased and then decreased as sucrose concentration increased (turning concentration = 1% or 5%). Interestingly, the roughness coefficient of bacterial cells (live or dead) exhibited an opposite pattern with sucrose concentration increase (Fig. 2C). This finding suggests that bacteria cells at 1% or 5% sucrose showed a more homogenous structure than those at 0% and 40% sucrose since the roughness coefficient, which provides a measure of how much the thickness of the biofilm varies, is an indicator of biofilm heterogeneity (Heydorn et al.2000). Furthermore, the homogeneity of biofilms at 1% or 5% can be higher since the bio-volume and thickness of EPSs at 1% and 5% sucrose were higher than those at 0% and 40%. It has been reported that EPSs contribute to the bulk, physical integrity and stability of the biofilm matrix (Koo et al.2010). In this study, however, gene expression related to EPS formation of S. mutans was strongly upregulated at 40% sucrose (Fig. 5A), suggesting that high concentrations of sucrose could stimulate gtfB, gtfC and gtfD gene expression to compensate for the EPS reduction. Furthermore, our result was consistent with previous studies (Hudson and Curtiss 1990; Zhao et al.2014), in which the expression of gtfB and gtfC genes was stimulated in the presence of high level of sucrose. In addition to biofilm components, the acid production of S. mutans biofilms increased and then decreased as sucrose concentration increased, and 5% sucrose showed the highest acid production (Fig. 1D). This result confirmed a previous study, which showed the threshold concentration of sucrose related to acid production at 5% (Aires et al.2006). In this study, the reduction of the acid production by the biofilms at higher sucrose level (>5%) might be closely related to the decrease in CFU count (Fig. 1C) or to the decrease in physiological activity of the biofilm cells. As shown in Fig. 1D-2 and D-3, the normalized initial rate of H+ production and total produced concentration of H+ also decreased at >5% sucrose, suggesting that physiological activity related to acid production per bacterial cell was inhibited at high sucrose concentrations. However, gene expression related to acid production (eno and ldh) and acid tolerance (atpD) of S. mutans was upregulated at ≥5% sucrose and especially at 40% sucrose (Fig. 5B). The upregulation of eno, ldh and atpD genes might be due to compensation for the decrease in the glycolysis in response to a decrease in proton motive force and a drop in intracellular pH. In addition, the high expression of EPS (Fig. 5A) and acid production related genes (Fig. 5B) at 40% sucrose may be related to the experiment model used in this study, in which S. mutans biofilm were exposed to sucrose for 1 h and then transferred to PPB buffer for 4 or 5 h. These feast and famine episodes might influence the genes expression. In general, since RNA is not always translated to proteins (Vogel and Marcotte 2012), further studies in the enzymatic levels are needed to perfectly understand the inconsistencies. In this study, 10%, 20% and 40% sucrose significantly affected the components and acid production of S. mutans biofilms compared with 1% or 5% sucrose (Fig. 1). A possible explanation of this result might be related to the increase in osmotic pressure generated by the higher sucrose concentration (Chirife et al.1983), which can affect biofilm bacterial growth and physiological activity such as acid production. According to a previous study, the growth of Listeria monocytogenes, a gram-positive bacterium, was inhibited at 20%–60% sucrose (Meldrum et al.2003). However, the effect of osmotic pressure changes by sugars including sucrose on biofilm bacteria, especially cariogenic bacteria, has not been well-defined, even though the nutrient components play important roles in development of dental caries. In summary, our results showed that the effect of sucrose on S. mutans biofilm formation follows a second-order polynomial curve with concentration dependence; the turning concentration ranged from 0.6% to 2.4%. Acid production by the biofilms also significantly increased and gradually decreased with sucrose concentration increase (turning concentration = 5%). However, gene expression related to EPS formation, acid production and tolerance was up-regulated with sucrose concentration increase, which might have been due to compensation for the decrease in EPS formation and acid production by the biofilms at higher concentrations of sucrose. These results may provide fundamental information on the changes in virulence and formation of cariogenic biofilms in relation to sucrose concentration and subsequent dental caries development. However, additional studies are also required to confirm the exact relationship between pattern of sucrose consumption and dental caries formation in real-life conditions. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. FUNDING This work was supported by the National Research Foundation of Korea, funded by the Korea government (Ministry of Science, ICT and Future Planning; grants 2016R1A2B4006378 and 2014R1A 4A1005309). Conflict of interest. None declared. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Ecology Oxford University Press

Sucrose challenges to Streptococcus mutans biofilms and the curve fitting for the biofilm changes

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© FEMS 2018.
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0168-6496
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Abstract

ABSTRACT The relationship between sugar level and development of dental caries has long been a main topic in dentistry. However, as a ubiquitous component of the modern diet, sucrose is mainly derived from three meals a day, rather than a long time exposure. In this study, various concentrations of sucrose were provided to Streptococcus mutans biofilms for 1 h per exposure (three times per day) to imitate a human meal pattern. And then the relationship between sucrose concentration and changes in the treated biofilms was determined. The results indicated that the components and acid production of the treated biofilms changed in a second-order polynomial curve pattern with sucrose concentration increase, which were confirmed by CLSM and SEM analyses. However, gene expression related to extracellular polysaccharides (EPS) formation, acid production and tolerance was up-regulated with sucrose concentration increase, which might have been due to compensation for the decrease in EPS formation and acid production by the biofilms at higher concentrations of sucrose. These findings suggest that sucrose in the range of 1%–5% can support the highest acid production and accumulation of S. mutans biofilms, which may further increase its cariogenic potential. However, additional studies are required to confirm the relationships in human cariogenic biofilms. sucrose, cariogenic biofilms, relationship, virulence INTRODUCTION Dental caries is a dental biofilm-related disease. If dental biofilms undergo significant environmental changes, such as an increase in sugar availability, aciduric and acidogenic bacteria become the dominant members of dental biofilms (Marsh 2003). The shift in microbial composition to aciduric and acidogenic bacteria (i.e. cariogenic biofilm formation) facilitates pH decrease and acidification of biofilms, which results in the development of cariogenic biofilms (Marsh 2003; 2010). Of the aciduric and acidogenic bacteria in dental biofilms, Streptococcus mutans is generally considered as an important cariogenic bacteria (Loesche 1986). This species can produce large amounts of acid by utilizing carbohydrates and survive in a low pH environment, which enhances the demineralization of dental hard tissue. Furthermore, S. mutans can utilize sucrose to synthesize extracellular polysaccharides (EPSs) by glucosyltransferases (GTFs) (Bowen and Koo 2011; Sendamangalam et al.2011). The intake of sugars including sucrose, glucose, fructose, lactose and maltose is also a contributory factor in the development of dental caries. Of dietary sugars, sucrose shows the highest cariogenic potential in dental caries development. Several in vitro and in vivo studies have demonstrated that sucrose can change the dental biofilm properties and increase enamel demineralization in a concentration or frequency dependent pattern (Aires et al.2006; Paes Leme et al.2006; Ccahuana-Vasquez et al.2007; Vale et al.2007; Sissons et al.2007; Diaz-Garrido, Lozano, Giacaman 2016). Sucrose is not only fermented by oral bacteria, but also enhances colonization and growth of mutans streptococci such as S. mutans in dental biofilms more than other sugars (Gupta et al.2013; Salli et al.2016). Furthermore, sucrose serves as a substrate for the synthesis of EPSs in dental biofilms, especially cariogenic biofilms (Newbrun 1967; Bowen 2002). It has been reported that EPSs contribute to the bulk, physical integrity and stability of biofilm matrixes, indicating that EPSs can be an essential virulence factor related to cariogenic biofilm formation (Koo et al.2010). Many studies have shown that the prevalence of dental caries is associated with the patterns of sugar intake in relation to frequency, stickiness and position within a meal sequence or a snack (Gupta et al.2013; Diaz-Garrido, Lozano and Giacaman 2016). Furthermore, a positive response relationship between free sugar intake and dental caries has been observed in many studies (Cury et al.2000; Sheiham and James 2015; Bernabe et al.2016). The WHO, therefore, recommends that both adults and children reduce their intake of free sugars to below 5% of total energy intake for the prevention of dental caries (Sheiham and James 2015). However, despite prolonged studies on both dental caries and sugars, the functional relationship between sugar concentration, especially sucrose, and virulence of cariogenic biofilms is not well documented. Cai et al. (2016) reported that the influence of sucrose on the components and acid production of a cariogenic biofilms follows a second-order polynomial curve with concentration dependence. However, the functional curve, which was derived from a 46 h experiment, could not explain the relationship in reality since dental biofilms are exposed to sucrose during meals or snack times rather than consistently over a 46 h time period. In this study, we designed a new experimental plan to mimic the sucrose exposure of three meals per day and then investigated change in a cariogenic biofilm according to sucrose level using an S. mutans biofilm model. This study also determined the mathematical relationship between sucrose level and accumulation and virulence of S. mutans biofilms. MATERIALS AND METHODS Streptococcus mutans biofilm formation and experimental scheme The S. mutans biofilm preparation and experimental schemes for this study are shown in Appendix Fig. 1. Streptococcusmutans UA159 (ATCC 700610; serotype c) biofilms were formed on saliva-coated hydroxyapatite (sHA) discs (2.93 cm2; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) placed in a vertical position in 24-well plates as described previously (Cai et al.2016). Briefly, HA discs were incubated in filter-sterilized (0.22-µm low protein-binding filter) human saliva for 1 h at 37°C. For biofilm formation, the sHA discs were transferred to a 24-well plate containing brain heart infusion (BHI; Difco, Detroit, MI, USA) broth with 1% (w/v) sucrose and S. mutans UA159 (2–5×106 colony-forming unit (CFU)/ml). The biofilms were grown at 37°C with 5% CO2 for 22 h to allow initial biofilm growth. After 22 h, the biofilms were grown in 20 mM potassium phosphate buffer (PPB, pH = 7.2) to the end of the experimental period (74 h). The biofilms in PPB were treated three times a day (at 22, 26, 31, 46, 50, 55 and 70 h) with 0%, 1%, 5%, 10%, 20% or 40% sucrose BHI broth. Specifically, the biofilms were exposed to the treatments for 1 h, dip-washed with water, and then transferred to PPB. Finally, the 74 h-old treated biofilms were analyzed in this study. Figure 1. View largeDownload slide Changes in composition and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A–C) Changes in dry weight, water-insoluble EPSs, and bacterial counts. (D-1) Change in acid production. (D-2) Initial rate and normalized initial rate of H+ production. (D-3) Total produced concentration and normalized total produced concentration of H+. In A–C, since x coordinate are plotted on a logarithmic scale, and since the log of 0 is undefined, we approximated 0 with an x coordinate of 0.1. *P < 0.05, significantly different from 0% sucrose. Figure 1. View largeDownload slide Changes in composition and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A–C) Changes in dry weight, water-insoluble EPSs, and bacterial counts. (D-1) Change in acid production. (D-2) Initial rate and normalized initial rate of H+ production. (D-3) Total produced concentration and normalized total produced concentration of H+. In A–C, since x coordinate are plotted on a logarithmic scale, and since the log of 0 is undefined, we approximated 0 with an x coordinate of 0.1. *P < 0.05, significantly different from 0% sucrose. Microbiological and biochemical analyses Dry weight (all biofilm components except water), the level of water-insoluble EPSs, and the number of CFUs of the 74 h-treated biofilms were determined as described elsewhere (Koo et al.2005; Pandit et al.2015). Briefly, the treated biofilms were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath (Power sonic 410; Hwashin Technology Co., Seoul, Korea) to disperse the biofilms. The suspension was homogenized by sonication (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA) for 30 s after adding 3 ml of 0.89% NaCl. An aliquot (100%µl) of the homogenized suspension was serially diluted and plated to determine the number of CFUs. To determine dry weight and amount of water-insoluble EPSs, the remaining solution (4.9 ml) was centrifuged (3000 g) for 20 min at 4°C. The biofilm pellet was then lyophilized and weighed to determine the dry weight. The water-insoluble EPSs were extracted from the dry pellet using 1 N sodium hydroxide before determination of the polysaccharide amount using a phenol-sulfuric acid assay. To evaluate differences in acid production of the 74 h-treated biofilms, a biofilm pH drop assay was performed as described elsewhere (Belli, Buckley and Marquis 1995). Briefly, the treated biofilms were incubated in 20 mM PPB/1 mM MgCl2 + 50 mM KCl (pH = 7.2) for 1 h to deplete endogenous catabolites and then transferred to a 6-well plate containing a salt solution (50 mM KCl + 1 mM MgCl2, pH = 7.0). The pH was adjusted to 7.2 with 0.2 M KOH solution. Glucose was then added to the mixture to produce a final concentration of 1% (w/v). The pH change was assessed using a glass electrode over a period of 120 min. The initial rate of H+ production (y) was derived from the equation: \begin{eqnarray*}{\rm y} = {\left( {{{\rm H}^ + }\,{\rm concentration}\,{\rm at}\ 30\,{\rm min} - {{\rm H}^ + }\,{\rm concentration}\ {\rm at}\,0\,{\rm min}} \right)} / 30\end{eqnarray*} The total produced concentration of H+ (y) was derived from the equation: \begin{eqnarray*}{\rm y} = {{\rm H}^ + }\ {\rm concentration}\ {\rm at}\,120\,{\rm min}\, - \,{{\rm H}^ + }\ {\rm concentration}\ {\rm at}\,0\,{\rm min}.\end{eqnarray*} Furthermore, the normalized initial rate of H+ production (or normalized total produced concentration of H+) was derived from the equation: y = initial rate of H+ production (total produced concentration of H+)/CFUs. Confocal laser scanning microscopy analysis Confocal laser scanning microscopy (CLSM) analysis was performed to confirm the results of microbiological and biochemical studies. The concentrations of sucrose tested in the CLSM study were 0%, 1%, 5% and 40% (w/v). To investigate difference in bacterial cells, the 74 h-treated biofilms were stained at room temperature in the dark for 30 min using the FilmTracer LIVE/DEAD Biofilm viability kit L10316 (Invitrogen, Molecular Probes Inc., Eugene, OR, USA). The final concentrations of SYTO9 and propidium iodide (PI) were 6.0 and 30 µM, respectively. This viability kit is based on plasma membrane integrity to determine live and dead cells. In this study, we regarded the cells with intact membranes (green) as live cells, whereas cells with damaged membranes (red) were regarded as the dead cells. To determine differences in EPSs, 1 µM of Alexa Fluor® 647-labeled dextran conjugate (10000MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) was added to 0%, 1%, 5% or 40% sucrose BHI broth during the 1 h sucrose treatments (total seven times). The stained live and dead bacterial cells and EPSs were observed using an LSM 510 META (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium–neon lasers. Three independent experiments were performed, and five image stacks per experiment were collected (n = 15). The biovolume (µm3/µm2), thickness (µm), or roughness coefficient and the coverage (%) of live and dead cells and EPSs were quantified from the entire stack using COMSTAT image-processing software (Heydorn et al.2000). The bio-volume is defined as the volume of the biomass (µm3) divided by the surface area of the substratum (HA discs) (µm2). The roughness coefficient, which provides a measure of how much the thickness of the biofilm varies, is calculated from the thickness distribution of the biofilm. The coverage (%), which is the fraction of the area occupied by biomass in each image of a stack, reflects how efficiently each stack is colonized by bacteria or EPSs (Heydorn et al.2000; Jeon et al.2009). The three-dimensional architecture of the biofilms was visualized using Imaris 8.0.2 (Bitplane, Zurich, Switzerland). Scanning electron microscopy analysis Scanning electron microscopy (SEM) was performed as detailed elsewhere (Takeuchi et al.2007). Briefly, 74 h-treated (0%, 5% or 40%) biofilms were rinsed three times in 0.1 M cacodylate buffer and prefixed with 3% glutaraldehyde solution for 1 h followed by post-fixation with a 1% osmium tetroxide solution for 1 h. The biofilms were then dehydrated in a graded series of ethanol (30%–100%) and infiltrated with nitrogen gas immediately before sputter coating with gold–palladium. The biofilm samples were analyzed by SEM (JSM-5900, Jeol, Japan). Gene expression analysis RNA from sucrose (0%, 1%, 5% or 40%) exposed biofilms was extracted and purified using standard protocols optimized for biofilms (Cury and Koo 2007). Briefly, the 74 h biofilms were removed and homogenized by sonication. RNA was extracted and purified using a Trizol® Max bacterial RNA isolation kit (Life Technologies, Carlsbad, CA, USA). DNA was digested with DNase I, amplification grade (Life Technologies, Carlsbad, CA, USA). Reverse transcription and real-time PCR were performed using a Top script® cDNA synthesis kit (Enzynomics, Daejeon, Korea) and a Power SYBR® Green PCR Master Mix (Life Technologies, Warrington, UK), respectively, according to the manufacturers’ instructions. Specific genes related to EPSs formation (gtfB, gtfC, gtfD), acid production (eno, ldh), and acid tolerance (atpD) were evaluated. The gene-specific primers used in this study were described previously (Lu, Liu and Yang 2008; Jeon et al.2009; Xu, Zhou and Wu 2011; Dong et al.2012). Relative expression was calculated by normalizing each test gene of the treated biofilms to the 16sRNA gene, which served as the reference gene (Shemesh et al.2008). Data analysis was performed using StepOne software v2.0 (Applied Biosystems, Foster City, CA, USA) according to the 2−ΔΔCT method. Statistical analysis To determine the relationship between sucrose concentration and S. mutans biofilm formation, a second-order polynomial fitting for sucrose concentration versus CFU count, dry weight, water-insoluble EPSs was performed. The determination coefficients (R2) of each fitted line were also calculated. All experiments were performed in duplicate, and at least three different experiments were conducted. The data are presented as mean ± standard deviation. Intergroup differences were estimated using one-way analysis of variance, followed by a post hoc multiple comparison (Tukey) test to compare multiple means. Values were considered statistically significant when the P value was < 0.05. RESULTS Changes in microbiological and biochemical studies The dry weight (all biofilm components except water), EPS amount and CFU counts of 74 h-treated biofilms were initially increased and then gradually decreased with sucrose concentration increase, which followed a second-order polynomial curves. The R2 values were 0.944, 0.914 and 0.997 (P < 0.05), respectively ( Fig. 1A, B and C). The maximum influence concentrations of sucrose for dry weight, EPS amount, and CFU counts were 2.1%, 2.4% and 0.6%, respectively. The change in acid production of the treated biofilms is shown in Fig. 1D-1. However, since only the general acid production tendency is shown, the initial rate (or normalized initial rate) of H+ production and total acid produced (or normalized total produced) were calculated to clearly describe acid production ability of the biofilms. The initial rate and normalized initial rate of H+ production all increased and then decreased as sucrose concentration increased, with 5% sucrose showing the highest acid production ability (Fig. 1D-2). A similar trend was shown in total produced H+ concentration; after normalization to total produced concentration of H+, 5% sucrose showed the highest values ( Fig. 1D-3). Changes in CLSM and SEM studies To further evaluate the effect of sucrose on biofilm components and structure, the CLSM analysis was performed. As shown in Fig. 2A and B, the bio-volume and mean thickness of live and dead cells of the 74 h treated biofilms initially increased and then decreased with sucrose concentration increase, with 1% or 5% sucrose showing the highest bacterial volume and mean thickness. Interestingly, the mean bio-volume and thickness of live cells at 0%, 1% and 5% sucrose were higher than those of dead cells, while the mean bio-volume and thickness of live cells at 40% sucrose were similar to those of dead cells. However, an opposite pattern was shown for the roughness coefficient (Fig. 2C), where 1% or 5% sucrose showed the lowest roughness coefficient. The coverage of live and dead cells of the treated biofilms was also calculated. The coverages of live cells at 0%, 1% and 5% sucrose were higher than those of dead cells, while the coverage of live cells at 40% sucrose was similar to that of dead cells (Fig. 2E). Representative three-dimensional images of live and dead cells are shown in Fig. 2D; biofilms at 5% sucrose showed the highest biofilm volume. The mean bio-volume and mean thickness of EPSs also gradually increased and then decreased as sucrose concentration increases ( Fig. 3A and B); the EPS was almost non-existent at 0% and 40% sucrose (Fig. 3D). The representative EPSs images showed that 1% and 5% sucrose exhibited large concentrations of EPSs with a homogeneous structure that covered and surrounded the bacteria micro-colonies to form a complex, structured biofilm (Fig. 3C). In this study, an SEM study was also performed to confirm the CLSM result. As shown in Fig. 4, biofilms treated with 5% sucrose showed micro-colonies surrounded with a large amount of EPSs. However, biofilms treated with 0% and 40% sucrose did not show EPSs (Fig. 4) Figure 2. View largeDownload slide Changes in CLSM images of bacterial cells in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) roughness coefficient, (D) representative confocal images and (E) coverage of live and dead cells. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 2. View largeDownload slide Changes in CLSM images of bacterial cells in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) roughness coefficient, (D) representative confocal images and (E) coverage of live and dead cells. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 3. View largeDownload slide Changes in CLSM images of EPSs in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) representative confocal images and (D) coverage of EPSs. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 3. View largeDownload slide Changes in CLSM images of EPSs in the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A) Mean bio-volume, (B) mean thickness, (C) representative confocal images and (D) coverage of EPSs. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 4. View largeDownload slide Representative SEM images of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). Figure 4. View largeDownload slide Representative SEM images of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). Changes in gene expression In the 74 h-treated biofilms, the expressions of genes related to EPS formation (gtfB, gtfCandgtfD), glycolysis (ldh), and acid tolerance (atpD) were significantly upregulated at 40% sucrose (P < 0.05) (Fig. 5). However, the expression of eno was not upregulated at 40% sucrose (P > 0.05). Figure 5. View largeDownload slide Changes in gene expression related to EPS formation and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A-1) gtfB. (A-2) gtfC. (A-3) gtfD. (B-1) eno. (B-2) ldh. (B-3) atpD. Values followed by the same superscripts are not significantly different from each other (P > 0.05). Figure 5. View largeDownload slide Changes in gene expression related to EPS formation and acid production of the 74-h old Streptococcus mutans biofilms treated with sucrose (0%–40%) for 1 h (a total of seven times). (A-1) gtfB. (A-2) gtfC. (A-3) gtfD. (B-1) eno. (B-2) ldh. (B-3) atpD. Values followed by the same superscripts are not significantly different from each other (P > 0.05). DISCUSSION According to the ecological plaque hypothesis, environmental changes contribute to the formation of cariogenic biofilms, and these biofilms are mainly composed of aciduric and acidogenic bacteria (such as mutans streptococci and lactobacilli) (Marsh and Zaura 2017). Although the role of sucrose as a fermentable sugar in the development of carious lesions is vastly acknowledged, limited studies have been performed to reveal the functional relationship between sucrose concentration and cariogenic biofilm properties in reality. To define the relationships, we exposed S. mutans biofilms, a representative cariogenic biofilm, to different concentrations of sucrose for three times per day to mimic three meals per day, which may simulate the feast and famine episodes present in the oral cavity. However, although the biofilm model used in this study simulated the clinical situation to some extent, additional in vivo studies are required to confirm the relationships since the biofilm model does not precisely mimic the complex microbial community found in cariogenic biofilms on tooth surfaces. In this study, our data show that the influence of sucrose exposure on S. mutans biofilm components followed a second-order polynomial curve with concentration dependence, and the determination coefficients (R2) values ranged from 0.914 to 0.997 ( Fig. 1A, B and C). This suggests that the functional curve is appropriate to describe changes in the biofilm components by sucrose as 91.4%–99.7% of the variation in the functional curves can be explained by variation in sucrose concentration. In general, the functional curves indicate that increasing sucrose level in S. mutans biofilm formation increases biofilm components such as dry weight (all biofilm components except water), EPS amount, and CFU count up to a certain concentration (turning concentration), after which these biofilm components decrease as sucrose concentration increases. In the functional curves, the turning concentration for the biofilm formation ranged from 0.6% to 2.4% ( Fig. 1A, B and C), suggesting that sucrose level can have a strong influence on cariogenic biofilm accumulation and subsequent dental caries development. The relationships derived from microbiological and biochemical analyses were confirmed by CLSM and SEM studies. As shown in Figs. 2–4, bio-volume, thickness and coverage of bacterial cells (live or dead) and EPSs gradually increased and then decreased as sucrose concentration increased (turning concentration = 1% or 5%). Interestingly, the roughness coefficient of bacterial cells (live or dead) exhibited an opposite pattern with sucrose concentration increase (Fig. 2C). This finding suggests that bacteria cells at 1% or 5% sucrose showed a more homogenous structure than those at 0% and 40% sucrose since the roughness coefficient, which provides a measure of how much the thickness of the biofilm varies, is an indicator of biofilm heterogeneity (Heydorn et al.2000). Furthermore, the homogeneity of biofilms at 1% or 5% can be higher since the bio-volume and thickness of EPSs at 1% and 5% sucrose were higher than those at 0% and 40%. It has been reported that EPSs contribute to the bulk, physical integrity and stability of the biofilm matrix (Koo et al.2010). In this study, however, gene expression related to EPS formation of S. mutans was strongly upregulated at 40% sucrose (Fig. 5A), suggesting that high concentrations of sucrose could stimulate gtfB, gtfC and gtfD gene expression to compensate for the EPS reduction. Furthermore, our result was consistent with previous studies (Hudson and Curtiss 1990; Zhao et al.2014), in which the expression of gtfB and gtfC genes was stimulated in the presence of high level of sucrose. In addition to biofilm components, the acid production of S. mutans biofilms increased and then decreased as sucrose concentration increased, and 5% sucrose showed the highest acid production (Fig. 1D). This result confirmed a previous study, which showed the threshold concentration of sucrose related to acid production at 5% (Aires et al.2006). In this study, the reduction of the acid production by the biofilms at higher sucrose level (>5%) might be closely related to the decrease in CFU count (Fig. 1C) or to the decrease in physiological activity of the biofilm cells. As shown in Fig. 1D-2 and D-3, the normalized initial rate of H+ production and total produced concentration of H+ also decreased at >5% sucrose, suggesting that physiological activity related to acid production per bacterial cell was inhibited at high sucrose concentrations. However, gene expression related to acid production (eno and ldh) and acid tolerance (atpD) of S. mutans was upregulated at ≥5% sucrose and especially at 40% sucrose (Fig. 5B). The upregulation of eno, ldh and atpD genes might be due to compensation for the decrease in the glycolysis in response to a decrease in proton motive force and a drop in intracellular pH. In addition, the high expression of EPS (Fig. 5A) and acid production related genes (Fig. 5B) at 40% sucrose may be related to the experiment model used in this study, in which S. mutans biofilm were exposed to sucrose for 1 h and then transferred to PPB buffer for 4 or 5 h. These feast and famine episodes might influence the genes expression. In general, since RNA is not always translated to proteins (Vogel and Marcotte 2012), further studies in the enzymatic levels are needed to perfectly understand the inconsistencies. In this study, 10%, 20% and 40% sucrose significantly affected the components and acid production of S. mutans biofilms compared with 1% or 5% sucrose (Fig. 1). A possible explanation of this result might be related to the increase in osmotic pressure generated by the higher sucrose concentration (Chirife et al.1983), which can affect biofilm bacterial growth and physiological activity such as acid production. According to a previous study, the growth of Listeria monocytogenes, a gram-positive bacterium, was inhibited at 20%–60% sucrose (Meldrum et al.2003). However, the effect of osmotic pressure changes by sugars including sucrose on biofilm bacteria, especially cariogenic bacteria, has not been well-defined, even though the nutrient components play important roles in development of dental caries. In summary, our results showed that the effect of sucrose on S. mutans biofilm formation follows a second-order polynomial curve with concentration dependence; the turning concentration ranged from 0.6% to 2.4%. Acid production by the biofilms also significantly increased and gradually decreased with sucrose concentration increase (turning concentration = 5%). However, gene expression related to EPS formation, acid production and tolerance was up-regulated with sucrose concentration increase, which might have been due to compensation for the decrease in EPS formation and acid production by the biofilms at higher concentrations of sucrose. These results may provide fundamental information on the changes in virulence and formation of cariogenic biofilms in relation to sucrose concentration and subsequent dental caries development. However, additional studies are also required to confirm the exact relationship between pattern of sucrose consumption and dental caries formation in real-life conditions. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. FUNDING This work was supported by the National Research Foundation of Korea, funded by the Korea government (Ministry of Science, ICT and Future Planning; grants 2016R1A2B4006378 and 2014R1A 4A1005309). Conflict of interest. None declared. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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FEMS Microbiology EcologyOxford University Press

Published: May 17, 2018

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