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Evaluation of Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity
journal of dentistry 42 (2014) 726–734
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Evaluation of Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity Ngoc Phuong Thanh Chau, Santosh Pandit, Ji-Eun Jung, Jae-Gyu Jeon * Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience and BK 21 Plus Program, Chonbuk National University, Jeonju 561-756, Republic of Korea
article info
abstract
Article history:
Objectives: The aim of this study was to evaluate Streptococcus mutans adhesion to fluoride
Received 6 January 2014
varnishes and subsequent change in biofilm accumulation and acidogenicity.
Received in revised form
Methods: After producing fluoride varnish-coated hydroxyapatite discs using Fluor Protector
17 March 2014
(FP), Bifluorid 12 (BIF), Cavity Shield (CASH), or Flor-Opal Varnish White (FO), S. mutans
Accepted 21 March 2014
biofilms were formed on the discs. To assess S. mutans adhesion to the discs, 4-h-old biofilms were analysed. To investigate the change in biofilm accumulation during subsequent biofilm formation, the biomass, colony forming units (CFU), and water-insoluble extracel-
Keywords:
lular polysaccharides (EP) of 46-, 70-, and 94-h-old biofilms were analysed. To investigate the
Acidogenicity
change in acidogenicity, pH values of the culture medium were determined during the
Adhesion
experimental period. The amount of fluoride in the culture medium was also determined
Biofilm accumulation
during the experimental period.
Fluoride varnishes
Results: BIF, CASH, and FO affected S. mutans adhesion (67–98% reduction) and subsequent
Streptococcus mutans
biofilm accumulation in 46-, 70-, and 94-h-old biofilms. However, the reducing effect of the fluoride varnishes on the biomass, CFU count, water-insoluble EP amount, and acid production rate of the biofilms decreased as the biofilm age increased. These results may be related to the fluoride-release pattern of the fluoride varnishes. Of the fluoride varnishes tested, FO showed the highest reducing effect against the bacterial adhesion and subsequent biofilm accumulation. Conclusions: Our findings suggest that if the results of these experiments are extrapolable to the in vivo situation, then reduced clinical benefit of using fluoride varnishes may occur with time. Clinical significance: Fluoride varnish application can affect cariogenic biofilm formation but the anti-biofilm activity may be reduced with time. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental caries is a biofilm-related oral disease that continues to afflict the majority of the world’s population.1 Although several studies have revealed that the level of mutans * Corresponding author. Tel.: +82 63 270 4036. E-mail address: [email protected] (J.-G. Jeon). http://dx.doi.org/10.1016/j.jdent.2014.03.009 0300-5712/# 2014 Elsevier Ltd. All rights reserved.
streptococci is not necessarily high during the development of dental caries,2,3 many studies have identified Streptococcus mutans as one of the main causative pathogens for dental caries.4,5 The ability to produce acids in biofilms is an important virulence trait of this bacterium.6,7 In addition, the bacterium can synthesise extracellular polysaccharides
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journal of dentistry 42 (2014) 726–734
(EP) from sucrose, which allow the bacterial cells to adhere to the tooth surfaces and contribute to the formation of cariogenic biofilms.8 The elevated levels of EP increase the bulk and structural stability of the biofilms. Thus, if the adhesion of S. mutans to tooth surfaces or the physiological ability (acid production and EP formation) of S. mutans in biofilms can be reduced, the potential for dental caries initiation will be decreased. It is well established that fluoride products play an important role in the prevention of dental caries. Fluoride varnishes have also been developed in an effort to improve upon the shortcomings of existing topical fluoride vehicles by prolonging the contact between the fluoride and the tooth enamel.9,10 Due to their effectiveness and safety, the use of fluoride varnishes has increased among the dental community in the United States since its introduction in the 1990s.11 Furthermore, previous studies have shown that fluoride varnishes are clinically effective in the prevention of dental caries.12,13 Recently, many studies have revealed that the anti-caries activity of fluoride varnishes can be attributed to the fluoride released from them and the mechanisms involved in the promotion of enamel remineralization and the inhibition of demineralization processes.14–17 However, study of the anti-biofilm activity of fluoride varnishes has received little attention, although there is much evidence showing that fluoride can affect caries-related bacterial adherence to the tooth surface and the virulence of cariogenic biofilms.18–20 According to previous studies of topical fluoride applications, decreases in the fluoride concentration in the saliva occur in a biphasic pattern, with an initial rapid decrease and a slower second phase.21,22 Fluoride varnishes also reportedly follow a similar pattern of fluoride decrease, although greater levels of fluoride in the saliva are evident for longer periods of time.21 It was also evident that salivary fluoride concentration of fluoride varnishes returned to <2 ppm after 2 h post application.23 Based on the information about the
anti-cariogenic biofilm activity of fluoride and the fluoriderelease pattern of fluoride varnishes, fluoride varnishes may affect caries-related bacterial adhesion and subsequent biofilm formation but cannot sustain their activity over time. However, little has been reported on the effect of fluoride varnishes on caries-related bacterial adhesion. Furthermore, there is no information about changes in the subsequent biofilm formation. Considering the continued widespread use of fluoride varnishes for the prevention of dental caries, it would be meaningful to test the hypothesis that, although fluoride varnishes can affect caries-related bacterial adhesion, they cannot sustain their activity during subsequent biofilm formation over time. Therefore, the aim of this study was to evaluate bacterial adhesion to fluoride varnishes and the change in biofilm accumulation and acidogenicity during subsequent biofilm formation using an S. mutans biofilm model.
2.
Materials and methods
2.1.
Fluoride varnishes and topical application
Table 1 lists fluoride varnishes used in this study. Four fluoride varnishes were selected: Fluor Protector (FP), Bifluorid 12 (BIF), Cavity Shield (CASH), and Flor-Opal Varnish White (FO). The varnishes were commercially available, and these were topically applied to hydroxyapatite discs (HA discs, 2.93 cm2, Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) according to the manufacturer’s recommendations. For the application of the varnishes, HA discs were autoclaved and were subsequently kept dry. One thin layer of each varnish was applied to the HA discs and dried at 59 8C for 24 h. The fluoride varnish-coated hydroxyapatite (fvHA) discs were used for S. mutans biofilm formation. Uncoated HA discs were also included as a control in this study.
Table 1 – Characteristics of fluoride varnishes used in this study. Product name
Fluoride form (wt.%)
Fluor protector
0.9% Difluorosilane
- Ethyl acetate (50–100%) - Isopentyl propionate (10–25%) - Polyisocyanate (10–25%)
- Fruit-like - Clear - Fluid
Ivoclar Vivadent AG, Liechtenstein
Bifluorid 12
5–10% Sodium fluoride
- Ethyl acetate (50–100%) - Cellulose nitrate with alcohol (10–25%) - Isopentyl propionate (10–25%)
- Fruit-like - Whitish - Fluid
VOCO GmbH, Germany
Cavity shielda
1–10% Sodium fluoride
-
- Colophony odour - Orange - Thick liquid
3 M ESPE Dental Products, USA
Flor-opal varnish whitea
4–6% Sodium fluoride
- Ethyl alcohol (18.9–28.9%) - Methyl Salicylate (<0.7%) - Hydrogenated rosin (<60%)
- Mint - White - Viscous
Ultradent, Products Inc., USA
Other ingredients (wt.%)
Colophony (20–70%) Polyamide resin (20–70%) Ethyl alcohol (4–30%) Flavour (1–5%)
This is based on information provided by safety data sheet of the products. Containing xylitol (based on instruction guide).
a
Odour, colour, physical state
Manufacturer
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2.2.
journal of dentistry 42 (2014) 726–734
Biofilm formation on fvHA discs
S. mutans UA159 was used for this study. For biofilm formation, 3 colonies of S. mutans UA159 were inoculated into 1% glucose (w/v) ultrafiltered (10 kDa molecular weight cut-off) tryptone yeast-extract (UTE) broth and incubated overnight (37 8C, 5% CO2) until an optical density of 1.4 at 600 nm (7.7 108–2 109 colony forming units (CFU)/ml were obtained. A 10-fold diluted S. mutans UA159 preparation in 1% sucrose (w/v) UTE broth was then prepared and used for biofilm formation. S. mutans UA159 biofilms were formed on fvHA discs placed in a vertical position in 24-well plates, as detailed elsewhere.24 Briefly, the fvHA discs were transferred to a 24-well plate containing 1% sucrose UTE broth, and subsequently inoculated with the 10-fold diluted S. mutans solution. The biofilms were grown undisturbed for 22 h to allow for initial biofilm growth. From this point time (22-hold biofilms), the culture medium was changed twice daily until it was 94 h old. The culture medium (2.7 ml/well) was changed a total of six times. The 4-h-old biofilms were used to evaluate the effect of fluoride varnishes on S. mutans adhesion. The 46-, 70-, and 94-h-old biofilms were analysed to investigate changes in biofilm accumulation during subsequent biofilm formation. The pH value and fluoride concentration in the culture medium were also determined during the experimental period (until biofilms reached 94 h of age). In this study, each assay was performed in duplicate in at least four different experiments (n = 8).
amount of water-insoluble EP was measured by a phenolsulfuric acid assay.
2.4.2.
Confocal laser scanning microscopy study
In addition to microbiological and biochemical analyses, confocal laser scanning microscopy (CLSM) study was performed to confirm the effect of fluoride varnishes on the bacterial cells and EP of S. mutans biofilms, using the 94-h-old biofilms, as described by Jeon et al.26 Briefly, 1 mM of Alexa Fluor1 647-labelled dextran conjugate (10,000 MW; absorbance/ fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) was added to the culture medium during biofilm formation. The fluorescence-labelled dextran serves as a primer for glucosyltransferases (GTFs) and can be incorporated into newly formed EP by GTFs.27 After 94 h, the bacterial cells in the biofilms were labelled by means of 25 mM of SYTO1 9 greenfluorescent nucleic acid stain (480/500 nm; Molecular Probes Inc., Eugene, OR, USA) for 30 min.27 CLSM imaging of the biofilms was performed using the LSM 510 META (Carl Zeiss, Jena, Germany) microscope equipped with argon-ion and helium– neon lasers. One experiment was performed and five image stacks (512 512 pixel tagged image file format) were collected.
2.5. Determination of pH and fluoride concentration in the culture medium
2.4. Evaluation of S. mutans biofilm accumulation on fvHA discs
To evaluate acid production during S. mutans adhesion to and subsequent biofilm formation on fvHA discs, the pH values of the culture medium of the 4-h-old biofilms and the old culture medium (until biofilms reached 94 h of age) were determined during the experimental period using a pH electrode. The concentration of fluoride in the culture medium of the 4-h-old biofilms and the old culture medium (until biofilms reached 94 h of age) were also determined to reveal the fluoride-release pattern of fvHA discs. For the determination of fluoride concentration, a total of 2.7 ml of each medium was mixed with a 270 ml of total ionic strength adjustment buffer (TISAB III). The fluorometer (Thermo Fisher Scientific Orion, MA, USA) was calibrated using three standard solutions (1, 10, and 100 ppm F ). To calculate acid production or fluoride-release rates, the concentrations of H+ or fluoride in the respective culture media were divided by the incubation time.
2.4.1.
2.6.
2.3.
Evaluation of S. mutans adhesion to fvHA discs
To evaluate S. mutans adhesion to fvHA discs, the 4-h-old biofilms were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath for 10 min to disperse the biofilms. The dispersed solution was re-sonicated at 7 W for 30 s after adding 3 ml of 0.89% NaCl (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA). An aliquot (0.1 ml) of the dispersed solution was serially diluted and plated onto Brain Heart Infusion (BHI) agar plates to count CFU.
Microbiological and biochemical analyses
To evaluate S. mutans biofilm accumulation on fvHA discs, the 46-, 70-, and 94-h-old biofilms were transferred into 2 ml of 0.89% NaCl and then dispersed by sonication, as described above. The dispersed solution (total volume: 5 ml) was used to evaluate the biofilm accumulation, as described elsewhere.25 Briefly, for the determination of CFU count, an aliquot (0.1 ml) of the dispersed solution was serially diluted and plated on BHI agar plates. For the determination of the biofilm biomass (dry weight) and the water-insoluble EP amount, the remaining solution (4.9 ml) was centrifuged (3000 g) for 20 min at 4 8C. The biofilm pellet was resuspended and washed twice in the same volume of water. The washed biofilm pellet was lyophilised and weighed to determine the biofilm biomass. After weighing, the water-insoluble EP was extracted from the dry pellet using 1 N sodium hydroxide before determination of the polysaccharide amount, as detailed elsewhere.25 The
Statistical analysis
The data are presented as the means standard deviations (SD). The intergroup differences were estimated by one-way analysis of variance (ANOVA), followed by a post hoc multiple comparison (Tukey test) to compare the multiple means. Values were considered statistically significant when the p value was <0.05.
3.
Results
3.1.
S. mutans adhesion to fvHA discs
As shown in Fig. 1, BIF, CASH, and FO reduced S. mutans adhesion. Of the fluoride varnishes tested, CASH and FO showed a strong inhibitory effect on the bacterial adhesion.
journal of dentistry 42 (2014) 726–734
Fig. 1 – Adhesion of S. mutans to fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. Values followed by the same superscript text are not significantly different from each other ( p > 0.05).
CASH and FO reduced approximately 88 and 98% of the bacterial adhesion, respectively ( p < 0.05). BIF also diminished approximately 67% of the bacterial adhesion ( p < 0.05). However, interestingly, FP did not reduce the bacterial adhesion ( p > 0.05). In general, the reduction of the bacterial adhesion depended on the type of fluoride varnish used.
3.2.
S. mutans biofilm accumulation on fvHA discs
As shown in Fig. 2, BIF, CASH, and FO affected the biomass of S. mutans biofilms, regardless of the biofilm age. Of the fluoride varnishes tested, FO showed the highest effect on biomass reduction across biofilms of all ages tested (99% reduction) ( p < 0.05). Biomass on FO did not increase as the biofilm age
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Fig. 2 – Biomass of 46-, 70-, and 94-h-old S. mutans biofilms on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. Values followed by the same superscript text are not significantly different from each other ( p > 0.05). The numbers in parenthesis represent the percentage of the respective control.
increased. However, although BIF and CASH could affect the biomass at all of the biofilm ages tested, the reducing effect gradually decreased as the biofilm age increased; BIF showed 32, 28, and 26% reductions in the 46-, 70-, and 94-h-old biofilms, respectively, and CASH showed 99, 97, and 79% reductions in the 46-, 70-, and 94-h-old biofilms, respectively. However, FP did not reduce the biomass of the biofilms, regardless of the age ( p > 0.05). The change in the CFU of S. mutans biofilms is shown in Fig. 3A. Of the fluoride varnishes tested, FO showed the highest reducing effect on the CFU count at all of the biofilm ages tested ( p < 0.05). Nevertheless, the reducing effect of FO on the
Fig. 3 – CFU (A) and water-insoluble EP (B) of 46-, 70-, and 94-h-old S. mutans biofilms on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnishcoated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. Values followed by the same superscript text are not significantly different from each other ( p > 0.05). The numbers in parenthesis represent the percentage of the respective control.
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journal of dentistry 42 (2014) 726–734
Fig. 4 – Representative images of confocal laser scanning microscopy (CLSM) from 94-h-old S. mutans biofilms on fluoride varnish-coated hydroxyapatite discs. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. In the CLSM images, the green indicates bacteria and the red indicates extracellular polysaccharides. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
CFU count sharply decreased as the biofilm age increased. CASH also reduced the CFU count in the 46- and 70-h-old biofilms ( p < 0.05), and the reducing effect in the 46-h-old biofilms was higher than that in the 70-h-old biofilms. FP and BIF did not decrease the CFU count at any of the biofilm ages tested ( p > 0.05). BIF, CASH, and FO affected the water-insoluble EP amount in S. mutans biofilms, regardless of the biofilm age (Fig. 3B). Of the fluoride varnishes tested, FO showed the highest reducing
effect on the water-insoluble EP amount at all of the biofilm ages tested (99% reduction) ( p < 0.05). The water-insoluble EP amount on FO did not increase as biofilm age increased. Although BIF could reduce the water-insoluble EP amount at all of the biofilm ages tested, the reducing effect gradually decreased as the biofilm age increased; it showed 54, 41, and 39% reductions in the 46-, 70-, and 94-h-old biofilms, respectively ( p < 0.05). CASH also reduced the water-insoluble EP amount by up to 99% in the 46- and 70-h-old biofilms, but
Fig. 5 – pH values in culture medium (A) and H+ production rate (B) during S. mutans adhesion to and subsequent biofilm formation on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White.
journal of dentistry 42 (2014) 726–734
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Fig. 6 – Fluoride concentration in culture medium (A) and fluoride-release rate (B) during S. mutans adhesion to and subsequent biofilm formation on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White.
the reducing effect in the 94-h-old biofilms was decreased (65% reduction) ( p < 0.05). FP did not reduce the water-insoluble EP amount at any of the biofilm ages tested ( p > 0.05). The 94 h effect of the test fluoride varnishes on the bacterial cells and EP of S. mutans biofilms was confirmed by CLSM. As shown in Fig. 4, FO could diminish the total volume of S. mutans cells (green colour) compared to the control. BIF, CASH, and FO also reduced the total volume of EP (red colour) compared to the control.
3.3.
Acid production of S. mutans biofilms on fvHA discs
The acid production ability of S. mutans biofilms was affected by BIF, CASH, and FO. As shown in Fig. 5A, BIF, CASH, and FO could inhibit the acid production during biofilm formation. However, the inhibition pattern varied according to the biofilm formation period. Thus, the H+ production rate of S. mutans biofilms was calculated to compare the inhibition pattern. As shown in Fig. 5B, BIF, CASH, and FO almost completely inhibited the H+ production rate in the early stages of biofilm formation (0–31 h) ( p < 0.05). However, the H+ production rate increased over time. The H+ production rate on BIF started to sharply increase during the middle stages of biofilm formation (31–46 h), reaching the normal H+ production rate of the control during the late stages of biofilm formation (46–94 h) ( p > 0.05). The H+ production rate on CASH and FO started to increase later than that of BIF, and did not reach the normal H+ production rate of the control during the experimental period. In general, CASH showed the highest inhibitory effect on the
H+ production rate, while FP did not affect the acid production of the biofilms.
3.4.
Fluoride-release pattern of fvHA discs
The fluoride concentrations and fluoride-release rates from the test fluoride varnishes were shown in Fig. 6A and B. Of the fluoride varnishes tested, BIF and FO released higher levels of fluoride (189 and 39 ppm F , respectively) into the culture medium during the bacterial adhesion (0–4 h) (Fig. 6A). However, as shown in Fig. 6B, the fluoride-release rates of BIF and FO sharply decreased over time, at which point it was similar to that of the control from the middle stages of biofilm formation (31–46 h) ( p > 0.05). Interestingly, although CASH released a lower level of fluoride (5.4 ppm F ) during the bacterial adhesion, the fluoride-release rate of CASH was maintained at a level higher than the other varnishes during the middle and late stages of biofilm formation (46–94 h) ( p < 0.05). In this study, FP did not release fluoride during the experimental period ( p > 0.05). In general, BIF showed the highest level of fluoride release during the bacterial adhesion and stages of early biofilm formation, while CASH showed the highest level of fluoride release over the middle and late stages of biofilm formation.
4.
Discussion
Dental caries is one of the most common infectious oral diseases, and is a biofilm-mediated disease.1,28 Biofilms
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develop following initial bacterial adhesion to and further accumulation on the tooth surface, suggesting that the incidence of dental caries can be lowered by the control of biofilm formation and accumulation. Thus, we focused on the activity of fluoride varnishes against both the virulence and accumulation of cariogenic biofilms, since the use of fluoride varnishes has been increasing for the prevention of dental caries. Although the S. mutans biofilm model used in the present study does not simulate the complex microbial community in the oral cavity, the model is useful in examining the pathogenesis of dental caries. Adhesion of bacteria to the tooth surface is a prerequisite for biofilm formation. The initial adhesion contributes to biofilm development as well as biofilm maturation and is affected by many factors such as growth environment, bacterial vitality and material surface characteristics.24,29–31 Thus, adhesion of caries-related bacteria to a fluoride varnish-coated surface was evaluated first. In this study, all of the test fluoride varnishes, except for FP, reduced S. mutans adhesion. As shown in Figs. 1 and 6, BIF, CASH, and FO, which released between 5.4 and 189 ppm F into the culture medium during the bacterial adhesion, could reduce the bacterial adhesion by between 67 and 98%. However, FP, which did not release fluoride into the culture medium during the experimental period, did not affect bacterial adhesion. This result suggests that the reducing effect may be closely related to the fluoride concentrations released during the bacterial adhesion. Although the precise mechanisms of the varnishes against bacterial adhesion were not revealed in this study, the reduction of bacterial adhesion may be due to the reducing effect of fluoride on bacterial growth. According to a previous study, the growth of S. mutans was reduced by sub-minimum inhibitory concentration (MIC) levels of fluoride (MIC: 282 ppm F ),32 indicating that the total number of bacteria that can adhere to the fluoride varnish-coated surface can be reduced by the fluoride concentrations released during the bacterial adhesion. In this study, although all of the test fluoride varnishes, except for FP, reduced biomass accumulation at all of the biofilm ages tested, the reducing effect of BIF and CASH gradually decreased as the biofilm age increased (Fig. 2). This result may be related to the fluoride concentration released in the middle and late stages of biofilm formation. As shown in Fig. 6A, the fluoride concentrations released from BIF and CASH in the middle and late stages of biofilm formation were much lower than that released during the bacterial adhesion, suggesting a physiological ability for biofilm cells in the middle and late stages of biofilm formation to recover and thereby initiate their normal functions pertaining to biomass accumulation. Since biofilms are mainly composed of bacterial cells and polysaccharides,33,34 we also evaluated the CFU count and water-insoluble EP amount of the biofilms on fluoride varnishes. In this study, the test fluoride varnishes, except for FP, also reduced the CFU count or water-insoluble EP amount, although their reducing effect gradually decreased as the biofilm age increased (Fig. 3A and B), confirming the change in the biomass accumulation (Fig. 2). Furthermore, our data indicated that the effect of the test fluoride varnishes on water-insoluble EP formation may be higher than that on biofilm bacterial growth. As shown in Fig. 3, although the CFU
count on BIF at all of the biofilm ages tested was similar to that of the control, water-insoluble EP formation on BIF was continuously affected. In addition, although the CFU count on CASH sharply increased according to the biofilm age, waterinsoluble EP formation on CASH was almost completely inhibited at all of the biofilm ages tested. This result may also be related to the low concentration of fluoride released in the middle and late stages of biofilm formation (Fig. 6). According to previous studies, fluoride at low concentrations (up to 3.8 ppm F ) partially inhibited the secretion of GTFs by S. mutans but did not affect the growth of S. mutans biofilm cells.20,35 Acid production is an important virulence factor of S. mutans biofilms that deserves attention during the study of dental caries prevention. As shown in Fig. 5, all of the fluoride varnishes tested, except for FP, reduced the acid production of S. mutans biofilms during the experimental period. The reducing effect of the test fluoride varnishes on the acid production may be closely related to their reducing effect on CFU count or the physiological ability of the biofilm cells. The effect of reducing acid production in the early stages of biofilm formation may be due to the activity of fluoride against the growth of S. mutans. On the other hand, the reducing effect of CASH on the acid production in the late stages of biofilm formation may be due to the activity of fluoride against the physiological ability of the biofilm cells since there was no difference in the CFU counts between the control and CASH (Figs. 3A and 6). According to a previous study, even 3 ppm F could reduce the acid production by S. mutans biofilm cells.36 Generally, the reducing effect of the test fluoride varnishes on acid production decreased with biofilm age (Fig. 5), suggesting that the physiological abilities of the biofilm cells related to acid production started to recover during the middle or late stages of biofilm formation. However, since this study determined the pH values of the culture medium rather than the biofilms, a more sophisticated study will be needed to identify the reducing effect of the test fluoride varnishes on the acid production of the biofilms. Of the fluoride varnishes tested, FO showed the highest inhibitory effect against the bacterial adhesion and biomass accumulation (Figs. 1 and 2). The varnish diminished the biomass accumulation by up to 99% in 46-h-old biofilms and the amount of biomass did not increase during the incubation time (Fig. 2), suggesting that FO can maintain its activity against biomass accumulation for a longer time than the other fluoride varnishes. Furthermore, the CFU count of the 46-h-old biofilms was not significantly different to that of the result of bacterial adhesion assay (Figs. 1 and 3A), indicating that FO may possess and release anti-bacterial (bacteriostatic) components since the MIC of fluoride against S. mutans was approximately 282 ppm F ,32 which is much higher than the concentration released from the varnish during the incubation period (Fig. 6). However, the CFU count of the biofilms sharply increased after 46 h of incubation (from 0.004% of the control to 2.9% of the control), suggesting that the bacteriostatic activity of the varnish that appeared once the varnish was applied can decrease over time. It has been sufficiently reported that the fluoride release from fluoride varnishes occurs in a biphasic pattern, with initial rapid decrease and a slower second phase.21 In this
journal of dentistry 42 (2014) 726–734
study, our data confirmed the fluoride-release pattern of fluoride varnishes. As shown in Fig. 6, despite the variations in the amount of fluoride from the fluoride varnishes tested, the largest amount of fluoride release occurs during the first day with a decrease thereafter. However, interestingly, the fluoride amount from FP was not different from that of the control during the experimental periods. This result suggests that a more sophisticated and long-term study will be needed to reveal the fluoride-release pattern of FP and its effect on dental caries. Although many studies revealed that fluoride has inhibitory effects on the virulence and accumulation of cariogenic biofilms,18–20 it is unclear whether fluoride varnishes can also affect cariogenic biofilms and whether they can maintain their anti-biofilm activity over a long period of time. To the best of our knowledge, this is the first report describing how fluoride varnishes can affect bacterial adhesion and subsequent biofilm formation. Furthermore, our data indicated that the anti-biofilm activity of fluoride varnishes may depend on their fluoride-release patterns. However, additional studies are required to reveal the possible effects of viscosity and other components released from fluoride varnishes on the bacterial adhesion and subsequent biofilm formation. As shown in Figs. 1–3, the reducing effect of the test fluoride varnishes on bacterial adhesion and subsequent biofilm accumulation was not fluoride concentration-dependent, suggesting that the other ingredients can also influence the level of bacterial adhesion and subsequent biofilm formation in addition to fluoride.
5.
Conclusions
In this study, although BIF, CASH, and FO reduced S. mutans adhesion and biofilm accumulation during subsequent biofilm formation, the reducing effect of the varnishes decreased over time, which may be related to the fluoride-release pattern of the varnishes. This finding suggests that if the findings of these in vitro studies are extrapolable to the in vivo situation, then reduced clinical effect may occur with time.
Acknowledgements This research was supported by research funds of Chonbuk National University in 2013 and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2005048).
references
1. Marsh PD. Dental plaque as a microbial biofilm. Caries Research 2004;38:204–11. 2. van Houte J. Role of micro-organisms in caries etiology. Journal of Dental Research 1994;73:672–81. 3. Beighton D. The complex oral microflora of high-risk individuals and groups and its role in the caries process. Community Dentistry and Oral Epidemiology 2005;33:248–55.
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4. Huis in ’t Veld JH, van Palenstein Helderman WH, Dirks OB. Streptococcus mutans and dental caries in humans: a bacteriological and immunological study. Antonie Van Leeuwenhoek 1979;45:25–33. 5. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiological Reviews 1986;50:353–80. 6. Harper DS, Loesche WJ. Growth and acid tolerance of human dental plaque bacteria. Archives of Oral Biology 1984;29:843–8. 7. Kuramitsu HK. Virulence factors of mutans streptococci: role of molecular genetics. Critical Reviews in Oral Biology and Medicine 1993;4:159–76. 8. Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Research 2011;45:69–86. 9. Beltra´n-Aguilar ED, Goldstein JW, Lockwood SA. Fluoride varnishes. A review of their clinical use, cariostatic mechanism, efficacy and safety. Journal of the American Dental Association 2000;131:589–96. 10. Bawden JW. Fluoride varnish: a useful new tool for public health dentistry. Journal of Public Health Dentistry 1998;58:266–9. 11. Autio-Gold J. Fluoride varnishes for everyday practice. Practical Procedures and Aesthetic Dentistry 2005;17:398. 400. 12. Strohmenger L, Brambilla E. The use of fluoride varnishes in the prevention of dental caries: a short review. Oral Diseases 2001;7:71–80. 13. Weintraub JA, Ramos-Gomez F, Jue B, Shain S, Hoover CI, Featherstone JD, et al. Fluoride varnish efficacy in preventing early childhood caries. Journal of Dental Research 2006;85:172–6. 14. Vivaldi-Rodriques G, Demito CF, Bowman SJ, Ramos AL. The effectiveness of a fluoride varnish in preventing the development of white spot lesions. World Journal of Orthodontics 2006;7:138–44. 15. Lee YE, Baek HJ, Choi YH, Jeong SH, Park YD, Song KB. Comparison of remineralization effect of three topical fluoride regimens on enamel initial carious lesions. Journal of Dentistry 2010;38:166–71. 16. Magalha˜es AC, Comar LP, Rios D, Delbem ACB, Buzalaf MAR. Effect of a 4% titanium tetrafluoride (TiF4) varnish on demineralisation and remineralisation of bovine enamel in vitro. Journal of Dentistry 2008;36:158–62. 17. Delbem ACB, Bergamaschi M, Sassaki KT, Cunha RF. Effect of fluoridated varnish and silver diamine fluoride solution on enamel demineralization: pH-cycling study. Journal of Applied Oral Science 2006;14:88–92. 18. Meurman JH. Effect of sodium and amine fluoride treatment on adsorption and ultrastructure of S. mutans and S. sanguis. Scandinavian Journal of Dental Research 1987;95:389–96. 19. Hayacibara MF, Rosa OP, Koo H, Torres SA, Costa B, Cury JA. Effects of fluoride and aluminum from ionomeric materials on S. mutans biofilm. Journal of Dental Research 2003;82:267– 71. 20. Pandit S, Kim JE, Jung KH, Chang KW, Jeon JG. Effect of sodium fluoride on the virulence factors and composition of Streptococcus mutans biofilms. Archives of Oral Biology 2011;56:643–9. 21. Castioni NV, Baehni PC, Gurny R. Current status in oral fluoride pharmacokinetics and implications the prophylaxis against dental caries. European Journal of Pharmaceutics and Biopharmaceutics 1998;45:101–11. 22. Mason SC, Shirodaria S, Sufi F, Rees GD, Birkhed D. Evaluation of salivary retention from a new high fluoride mouthrinse. Journal of Dentistry 2010;38(Suppl. 3):S30–6. 23. Eakle WS, Featherstone JD, Weintraub JA, Shain SG, Gansky SA. Salivary fluoride levels following application of fluoride varnish or fluoride rinse. Community Dentistry and Oral Epidemiology 2004;32:462–9.
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24. Pandit S, Kim GR, Lee MH, Jeon JG. Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites. Journal of Dentistry 2011;39:780–7. 25. Lemos JA, Abranches J, Koo H, Marquis RE, Burne RA. Protocols to study the physiology of oral biofilms. Methods in Molecular Biology 2010;666:87–102. 26. Jeon JG, Klein MI, Xiao J, Gregoire S, Rosalen PL, Koo H. Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms. BMC Microbiology 2009;9:228. 27. Koo H, Xiao J, Klein MI, Jeon JG. Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms. Journal of Bacteriology 2010;192:3024–32. 28. Bowen WH. Do we need to be concerned about dental caries in the coming millennium? Critical Reviews in Oral Biology and Medicine 2002;13:126–31. 29. Marsh PD. Dental plaque as a biofilm and a microbial community-implications for health and disease. Biomedical Central Oral Health 2006;6:14. 30. ElSalhy M, Sayed Zahid I, Honkala E. Effects of xylitol mouthrinse on Streptococcus mutans. Journal of Dentistry 2012;40:1151–4.
31. Fu D, Pei D, Huang C, Liu Y, Du X, Sun H. Effect of desensitising paste containing 8% arginine and calcium carbonate on biofilm formation of Streptococcus mutans in vitro. Journal of Dentistry 2013;41:619–27. 32. Dong L, Tong Z, Linghu D, Lin Y, Tao R, Liu J, et al. Effects of sub-minimum inhibitory concentrations of antimicrobial agents on Streptococcus mutans biofilm formation. International Journal of Antimicrobial Agents 2012;39:390–5. 33. Kokare CR, Chakraborty S, Khopade AN, Mahadik KR. Biofilm: importance and applications. Indian Journal of Biotechnology 2009;8:159–68. 34. Donlan RM. Biofilm: microbial life on surfaces. Emerging Infectious Diseases 2002;8:881–90. 35. Koo H, Sheng J, Nguyen PTM, Marquis RE. Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspension cultures and biofilms. FEMS Microbiology Letters 2006;254:134–40. 36. Pandit S, Kim HJ, Song KY, Jeon JG. Relationship between fluoride concentration and activity against virulence factors and viability of a cariogenic biofilm: in vitro study. Caries Research 2013;47:539–47.
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Evaluation of Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity Ngoc Phuong Thanh Chau, Santosh Pandit, Ji-Eun Jung, Jae-Gyu Jeon * Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience and BK 21 Plus Program, Chonbuk National University, Jeonju 561-756, Republic of Korea
article info
abstract
Article history:
Objectives: The aim of this study was to evaluate Streptococcus mutans adhesion to fluoride
Received 6 January 2014
varnishes and subsequent change in biofilm accumulation and acidogenicity.
Received in revised form
Methods: After producing fluoride varnish-coated hydroxyapatite discs using Fluor Protector
17 March 2014
(FP), Bifluorid 12 (BIF), Cavity Shield (CASH), or Flor-Opal Varnish White (FO), S. mutans
Accepted 21 March 2014
biofilms were formed on the discs. To assess S. mutans adhesion to the discs, 4-h-old biofilms were analysed. To investigate the change in biofilm accumulation during subsequent biofilm formation, the biomass, colony forming units (CFU), and water-insoluble extracel-
Keywords:
lular polysaccharides (EP) of 46-, 70-, and 94-h-old biofilms were analysed. To investigate the
Acidogenicity
change in acidogenicity, pH values of the culture medium were determined during the
Adhesion
experimental period. The amount of fluoride in the culture medium was also determined
Biofilm accumulation
during the experimental period.
Fluoride varnishes
Results: BIF, CASH, and FO affected S. mutans adhesion (67–98% reduction) and subsequent
Streptococcus mutans
biofilm accumulation in 46-, 70-, and 94-h-old biofilms. However, the reducing effect of the fluoride varnishes on the biomass, CFU count, water-insoluble EP amount, and acid production rate of the biofilms decreased as the biofilm age increased. These results may be related to the fluoride-release pattern of the fluoride varnishes. Of the fluoride varnishes tested, FO showed the highest reducing effect against the bacterial adhesion and subsequent biofilm accumulation. Conclusions: Our findings suggest that if the results of these experiments are extrapolable to the in vivo situation, then reduced clinical benefit of using fluoride varnishes may occur with time. Clinical significance: Fluoride varnish application can affect cariogenic biofilm formation but the anti-biofilm activity may be reduced with time. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental caries is a biofilm-related oral disease that continues to afflict the majority of the world’s population.1 Although several studies have revealed that the level of mutans * Corresponding author. Tel.: +82 63 270 4036. E-mail address: [email protected] (J.-G. Jeon). http://dx.doi.org/10.1016/j.jdent.2014.03.009 0300-5712/# 2014 Elsevier Ltd. All rights reserved.
streptococci is not necessarily high during the development of dental caries,2,3 many studies have identified Streptococcus mutans as one of the main causative pathogens for dental caries.4,5 The ability to produce acids in biofilms is an important virulence trait of this bacterium.6,7 In addition, the bacterium can synthesise extracellular polysaccharides
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journal of dentistry 42 (2014) 726–734
(EP) from sucrose, which allow the bacterial cells to adhere to the tooth surfaces and contribute to the formation of cariogenic biofilms.8 The elevated levels of EP increase the bulk and structural stability of the biofilms. Thus, if the adhesion of S. mutans to tooth surfaces or the physiological ability (acid production and EP formation) of S. mutans in biofilms can be reduced, the potential for dental caries initiation will be decreased. It is well established that fluoride products play an important role in the prevention of dental caries. Fluoride varnishes have also been developed in an effort to improve upon the shortcomings of existing topical fluoride vehicles by prolonging the contact between the fluoride and the tooth enamel.9,10 Due to their effectiveness and safety, the use of fluoride varnishes has increased among the dental community in the United States since its introduction in the 1990s.11 Furthermore, previous studies have shown that fluoride varnishes are clinically effective in the prevention of dental caries.12,13 Recently, many studies have revealed that the anti-caries activity of fluoride varnishes can be attributed to the fluoride released from them and the mechanisms involved in the promotion of enamel remineralization and the inhibition of demineralization processes.14–17 However, study of the anti-biofilm activity of fluoride varnishes has received little attention, although there is much evidence showing that fluoride can affect caries-related bacterial adherence to the tooth surface and the virulence of cariogenic biofilms.18–20 According to previous studies of topical fluoride applications, decreases in the fluoride concentration in the saliva occur in a biphasic pattern, with an initial rapid decrease and a slower second phase.21,22 Fluoride varnishes also reportedly follow a similar pattern of fluoride decrease, although greater levels of fluoride in the saliva are evident for longer periods of time.21 It was also evident that salivary fluoride concentration of fluoride varnishes returned to <2 ppm after 2 h post application.23 Based on the information about the
anti-cariogenic biofilm activity of fluoride and the fluoriderelease pattern of fluoride varnishes, fluoride varnishes may affect caries-related bacterial adhesion and subsequent biofilm formation but cannot sustain their activity over time. However, little has been reported on the effect of fluoride varnishes on caries-related bacterial adhesion. Furthermore, there is no information about changes in the subsequent biofilm formation. Considering the continued widespread use of fluoride varnishes for the prevention of dental caries, it would be meaningful to test the hypothesis that, although fluoride varnishes can affect caries-related bacterial adhesion, they cannot sustain their activity during subsequent biofilm formation over time. Therefore, the aim of this study was to evaluate bacterial adhesion to fluoride varnishes and the change in biofilm accumulation and acidogenicity during subsequent biofilm formation using an S. mutans biofilm model.
2.
Materials and methods
2.1.
Fluoride varnishes and topical application
Table 1 lists fluoride varnishes used in this study. Four fluoride varnishes were selected: Fluor Protector (FP), Bifluorid 12 (BIF), Cavity Shield (CASH), and Flor-Opal Varnish White (FO). The varnishes were commercially available, and these were topically applied to hydroxyapatite discs (HA discs, 2.93 cm2, Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) according to the manufacturer’s recommendations. For the application of the varnishes, HA discs were autoclaved and were subsequently kept dry. One thin layer of each varnish was applied to the HA discs and dried at 59 8C for 24 h. The fluoride varnish-coated hydroxyapatite (fvHA) discs were used for S. mutans biofilm formation. Uncoated HA discs were also included as a control in this study.
Table 1 – Characteristics of fluoride varnishes used in this study. Product name
Fluoride form (wt.%)
Fluor protector
0.9% Difluorosilane
- Ethyl acetate (50–100%) - Isopentyl propionate (10–25%) - Polyisocyanate (10–25%)
- Fruit-like - Clear - Fluid
Ivoclar Vivadent AG, Liechtenstein
Bifluorid 12
5–10% Sodium fluoride
- Ethyl acetate (50–100%) - Cellulose nitrate with alcohol (10–25%) - Isopentyl propionate (10–25%)
- Fruit-like - Whitish - Fluid
VOCO GmbH, Germany
Cavity shielda
1–10% Sodium fluoride
-
- Colophony odour - Orange - Thick liquid
3 M ESPE Dental Products, USA
Flor-opal varnish whitea
4–6% Sodium fluoride
- Ethyl alcohol (18.9–28.9%) - Methyl Salicylate (<0.7%) - Hydrogenated rosin (<60%)
- Mint - White - Viscous
Ultradent, Products Inc., USA
Other ingredients (wt.%)
Colophony (20–70%) Polyamide resin (20–70%) Ethyl alcohol (4–30%) Flavour (1–5%)
This is based on information provided by safety data sheet of the products. Containing xylitol (based on instruction guide).
a
Odour, colour, physical state
Manufacturer
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2.2.
journal of dentistry 42 (2014) 726–734
Biofilm formation on fvHA discs
S. mutans UA159 was used for this study. For biofilm formation, 3 colonies of S. mutans UA159 were inoculated into 1% glucose (w/v) ultrafiltered (10 kDa molecular weight cut-off) tryptone yeast-extract (UTE) broth and incubated overnight (37 8C, 5% CO2) until an optical density of 1.4 at 600 nm (7.7 108–2 109 colony forming units (CFU)/ml were obtained. A 10-fold diluted S. mutans UA159 preparation in 1% sucrose (w/v) UTE broth was then prepared and used for biofilm formation. S. mutans UA159 biofilms were formed on fvHA discs placed in a vertical position in 24-well plates, as detailed elsewhere.24 Briefly, the fvHA discs were transferred to a 24-well plate containing 1% sucrose UTE broth, and subsequently inoculated with the 10-fold diluted S. mutans solution. The biofilms were grown undisturbed for 22 h to allow for initial biofilm growth. From this point time (22-hold biofilms), the culture medium was changed twice daily until it was 94 h old. The culture medium (2.7 ml/well) was changed a total of six times. The 4-h-old biofilms were used to evaluate the effect of fluoride varnishes on S. mutans adhesion. The 46-, 70-, and 94-h-old biofilms were analysed to investigate changes in biofilm accumulation during subsequent biofilm formation. The pH value and fluoride concentration in the culture medium were also determined during the experimental period (until biofilms reached 94 h of age). In this study, each assay was performed in duplicate in at least four different experiments (n = 8).
amount of water-insoluble EP was measured by a phenolsulfuric acid assay.
2.4.2.
Confocal laser scanning microscopy study
In addition to microbiological and biochemical analyses, confocal laser scanning microscopy (CLSM) study was performed to confirm the effect of fluoride varnishes on the bacterial cells and EP of S. mutans biofilms, using the 94-h-old biofilms, as described by Jeon et al.26 Briefly, 1 mM of Alexa Fluor1 647-labelled dextran conjugate (10,000 MW; absorbance/ fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) was added to the culture medium during biofilm formation. The fluorescence-labelled dextran serves as a primer for glucosyltransferases (GTFs) and can be incorporated into newly formed EP by GTFs.27 After 94 h, the bacterial cells in the biofilms were labelled by means of 25 mM of SYTO1 9 greenfluorescent nucleic acid stain (480/500 nm; Molecular Probes Inc., Eugene, OR, USA) for 30 min.27 CLSM imaging of the biofilms was performed using the LSM 510 META (Carl Zeiss, Jena, Germany) microscope equipped with argon-ion and helium– neon lasers. One experiment was performed and five image stacks (512 512 pixel tagged image file format) were collected.
2.5. Determination of pH and fluoride concentration in the culture medium
2.4. Evaluation of S. mutans biofilm accumulation on fvHA discs
To evaluate acid production during S. mutans adhesion to and subsequent biofilm formation on fvHA discs, the pH values of the culture medium of the 4-h-old biofilms and the old culture medium (until biofilms reached 94 h of age) were determined during the experimental period using a pH electrode. The concentration of fluoride in the culture medium of the 4-h-old biofilms and the old culture medium (until biofilms reached 94 h of age) were also determined to reveal the fluoride-release pattern of fvHA discs. For the determination of fluoride concentration, a total of 2.7 ml of each medium was mixed with a 270 ml of total ionic strength adjustment buffer (TISAB III). The fluorometer (Thermo Fisher Scientific Orion, MA, USA) was calibrated using three standard solutions (1, 10, and 100 ppm F ). To calculate acid production or fluoride-release rates, the concentrations of H+ or fluoride in the respective culture media were divided by the incubation time.
2.4.1.
2.6.
2.3.
Evaluation of S. mutans adhesion to fvHA discs
To evaluate S. mutans adhesion to fvHA discs, the 4-h-old biofilms were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath for 10 min to disperse the biofilms. The dispersed solution was re-sonicated at 7 W for 30 s after adding 3 ml of 0.89% NaCl (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA). An aliquot (0.1 ml) of the dispersed solution was serially diluted and plated onto Brain Heart Infusion (BHI) agar plates to count CFU.
Microbiological and biochemical analyses
To evaluate S. mutans biofilm accumulation on fvHA discs, the 46-, 70-, and 94-h-old biofilms were transferred into 2 ml of 0.89% NaCl and then dispersed by sonication, as described above. The dispersed solution (total volume: 5 ml) was used to evaluate the biofilm accumulation, as described elsewhere.25 Briefly, for the determination of CFU count, an aliquot (0.1 ml) of the dispersed solution was serially diluted and plated on BHI agar plates. For the determination of the biofilm biomass (dry weight) and the water-insoluble EP amount, the remaining solution (4.9 ml) was centrifuged (3000 g) for 20 min at 4 8C. The biofilm pellet was resuspended and washed twice in the same volume of water. The washed biofilm pellet was lyophilised and weighed to determine the biofilm biomass. After weighing, the water-insoluble EP was extracted from the dry pellet using 1 N sodium hydroxide before determination of the polysaccharide amount, as detailed elsewhere.25 The
Statistical analysis
The data are presented as the means standard deviations (SD). The intergroup differences were estimated by one-way analysis of variance (ANOVA), followed by a post hoc multiple comparison (Tukey test) to compare the multiple means. Values were considered statistically significant when the p value was <0.05.
3.
Results
3.1.
S. mutans adhesion to fvHA discs
As shown in Fig. 1, BIF, CASH, and FO reduced S. mutans adhesion. Of the fluoride varnishes tested, CASH and FO showed a strong inhibitory effect on the bacterial adhesion.
journal of dentistry 42 (2014) 726–734
Fig. 1 – Adhesion of S. mutans to fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. Values followed by the same superscript text are not significantly different from each other ( p > 0.05).
CASH and FO reduced approximately 88 and 98% of the bacterial adhesion, respectively ( p < 0.05). BIF also diminished approximately 67% of the bacterial adhesion ( p < 0.05). However, interestingly, FP did not reduce the bacterial adhesion ( p > 0.05). In general, the reduction of the bacterial adhesion depended on the type of fluoride varnish used.
3.2.
S. mutans biofilm accumulation on fvHA discs
As shown in Fig. 2, BIF, CASH, and FO affected the biomass of S. mutans biofilms, regardless of the biofilm age. Of the fluoride varnishes tested, FO showed the highest effect on biomass reduction across biofilms of all ages tested (99% reduction) ( p < 0.05). Biomass on FO did not increase as the biofilm age
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Fig. 2 – Biomass of 46-, 70-, and 94-h-old S. mutans biofilms on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. Values followed by the same superscript text are not significantly different from each other ( p > 0.05). The numbers in parenthesis represent the percentage of the respective control.
increased. However, although BIF and CASH could affect the biomass at all of the biofilm ages tested, the reducing effect gradually decreased as the biofilm age increased; BIF showed 32, 28, and 26% reductions in the 46-, 70-, and 94-h-old biofilms, respectively, and CASH showed 99, 97, and 79% reductions in the 46-, 70-, and 94-h-old biofilms, respectively. However, FP did not reduce the biomass of the biofilms, regardless of the age ( p > 0.05). The change in the CFU of S. mutans biofilms is shown in Fig. 3A. Of the fluoride varnishes tested, FO showed the highest reducing effect on the CFU count at all of the biofilm ages tested ( p < 0.05). Nevertheless, the reducing effect of FO on the
Fig. 3 – CFU (A) and water-insoluble EP (B) of 46-, 70-, and 94-h-old S. mutans biofilms on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnishcoated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. Values followed by the same superscript text are not significantly different from each other ( p > 0.05). The numbers in parenthesis represent the percentage of the respective control.
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journal of dentistry 42 (2014) 726–734
Fig. 4 – Representative images of confocal laser scanning microscopy (CLSM) from 94-h-old S. mutans biofilms on fluoride varnish-coated hydroxyapatite discs. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White. In the CLSM images, the green indicates bacteria and the red indicates extracellular polysaccharides. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
CFU count sharply decreased as the biofilm age increased. CASH also reduced the CFU count in the 46- and 70-h-old biofilms ( p < 0.05), and the reducing effect in the 46-h-old biofilms was higher than that in the 70-h-old biofilms. FP and BIF did not decrease the CFU count at any of the biofilm ages tested ( p > 0.05). BIF, CASH, and FO affected the water-insoluble EP amount in S. mutans biofilms, regardless of the biofilm age (Fig. 3B). Of the fluoride varnishes tested, FO showed the highest reducing
effect on the water-insoluble EP amount at all of the biofilm ages tested (99% reduction) ( p < 0.05). The water-insoluble EP amount on FO did not increase as biofilm age increased. Although BIF could reduce the water-insoluble EP amount at all of the biofilm ages tested, the reducing effect gradually decreased as the biofilm age increased; it showed 54, 41, and 39% reductions in the 46-, 70-, and 94-h-old biofilms, respectively ( p < 0.05). CASH also reduced the water-insoluble EP amount by up to 99% in the 46- and 70-h-old biofilms, but
Fig. 5 – pH values in culture medium (A) and H+ production rate (B) during S. mutans adhesion to and subsequent biofilm formation on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White.
journal of dentistry 42 (2014) 726–734
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Fig. 6 – Fluoride concentration in culture medium (A) and fluoride-release rate (B) during S. mutans adhesion to and subsequent biofilm formation on fluoride varnish-coated hydroxyapatite discs. The data represent the means W standard deviations. The control (Control) was nonfluoride varnish-coated hydroxyapatite discs. FP, Fluor Protector; BIF, Bifluorid 12; CASH, Cavity Shield; FO, Flor-Opal Varnish White.
the reducing effect in the 94-h-old biofilms was decreased (65% reduction) ( p < 0.05). FP did not reduce the water-insoluble EP amount at any of the biofilm ages tested ( p > 0.05). The 94 h effect of the test fluoride varnishes on the bacterial cells and EP of S. mutans biofilms was confirmed by CLSM. As shown in Fig. 4, FO could diminish the total volume of S. mutans cells (green colour) compared to the control. BIF, CASH, and FO also reduced the total volume of EP (red colour) compared to the control.
3.3.
Acid production of S. mutans biofilms on fvHA discs
The acid production ability of S. mutans biofilms was affected by BIF, CASH, and FO. As shown in Fig. 5A, BIF, CASH, and FO could inhibit the acid production during biofilm formation. However, the inhibition pattern varied according to the biofilm formation period. Thus, the H+ production rate of S. mutans biofilms was calculated to compare the inhibition pattern. As shown in Fig. 5B, BIF, CASH, and FO almost completely inhibited the H+ production rate in the early stages of biofilm formation (0–31 h) ( p < 0.05). However, the H+ production rate increased over time. The H+ production rate on BIF started to sharply increase during the middle stages of biofilm formation (31–46 h), reaching the normal H+ production rate of the control during the late stages of biofilm formation (46–94 h) ( p > 0.05). The H+ production rate on CASH and FO started to increase later than that of BIF, and did not reach the normal H+ production rate of the control during the experimental period. In general, CASH showed the highest inhibitory effect on the
H+ production rate, while FP did not affect the acid production of the biofilms.
3.4.
Fluoride-release pattern of fvHA discs
The fluoride concentrations and fluoride-release rates from the test fluoride varnishes were shown in Fig. 6A and B. Of the fluoride varnishes tested, BIF and FO released higher levels of fluoride (189 and 39 ppm F , respectively) into the culture medium during the bacterial adhesion (0–4 h) (Fig. 6A). However, as shown in Fig. 6B, the fluoride-release rates of BIF and FO sharply decreased over time, at which point it was similar to that of the control from the middle stages of biofilm formation (31–46 h) ( p > 0.05). Interestingly, although CASH released a lower level of fluoride (5.4 ppm F ) during the bacterial adhesion, the fluoride-release rate of CASH was maintained at a level higher than the other varnishes during the middle and late stages of biofilm formation (46–94 h) ( p < 0.05). In this study, FP did not release fluoride during the experimental period ( p > 0.05). In general, BIF showed the highest level of fluoride release during the bacterial adhesion and stages of early biofilm formation, while CASH showed the highest level of fluoride release over the middle and late stages of biofilm formation.
4.
Discussion
Dental caries is one of the most common infectious oral diseases, and is a biofilm-mediated disease.1,28 Biofilms
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develop following initial bacterial adhesion to and further accumulation on the tooth surface, suggesting that the incidence of dental caries can be lowered by the control of biofilm formation and accumulation. Thus, we focused on the activity of fluoride varnishes against both the virulence and accumulation of cariogenic biofilms, since the use of fluoride varnishes has been increasing for the prevention of dental caries. Although the S. mutans biofilm model used in the present study does not simulate the complex microbial community in the oral cavity, the model is useful in examining the pathogenesis of dental caries. Adhesion of bacteria to the tooth surface is a prerequisite for biofilm formation. The initial adhesion contributes to biofilm development as well as biofilm maturation and is affected by many factors such as growth environment, bacterial vitality and material surface characteristics.24,29–31 Thus, adhesion of caries-related bacteria to a fluoride varnish-coated surface was evaluated first. In this study, all of the test fluoride varnishes, except for FP, reduced S. mutans adhesion. As shown in Figs. 1 and 6, BIF, CASH, and FO, which released between 5.4 and 189 ppm F into the culture medium during the bacterial adhesion, could reduce the bacterial adhesion by between 67 and 98%. However, FP, which did not release fluoride into the culture medium during the experimental period, did not affect bacterial adhesion. This result suggests that the reducing effect may be closely related to the fluoride concentrations released during the bacterial adhesion. Although the precise mechanisms of the varnishes against bacterial adhesion were not revealed in this study, the reduction of bacterial adhesion may be due to the reducing effect of fluoride on bacterial growth. According to a previous study, the growth of S. mutans was reduced by sub-minimum inhibitory concentration (MIC) levels of fluoride (MIC: 282 ppm F ),32 indicating that the total number of bacteria that can adhere to the fluoride varnish-coated surface can be reduced by the fluoride concentrations released during the bacterial adhesion. In this study, although all of the test fluoride varnishes, except for FP, reduced biomass accumulation at all of the biofilm ages tested, the reducing effect of BIF and CASH gradually decreased as the biofilm age increased (Fig. 2). This result may be related to the fluoride concentration released in the middle and late stages of biofilm formation. As shown in Fig. 6A, the fluoride concentrations released from BIF and CASH in the middle and late stages of biofilm formation were much lower than that released during the bacterial adhesion, suggesting a physiological ability for biofilm cells in the middle and late stages of biofilm formation to recover and thereby initiate their normal functions pertaining to biomass accumulation. Since biofilms are mainly composed of bacterial cells and polysaccharides,33,34 we also evaluated the CFU count and water-insoluble EP amount of the biofilms on fluoride varnishes. In this study, the test fluoride varnishes, except for FP, also reduced the CFU count or water-insoluble EP amount, although their reducing effect gradually decreased as the biofilm age increased (Fig. 3A and B), confirming the change in the biomass accumulation (Fig. 2). Furthermore, our data indicated that the effect of the test fluoride varnishes on water-insoluble EP formation may be higher than that on biofilm bacterial growth. As shown in Fig. 3, although the CFU
count on BIF at all of the biofilm ages tested was similar to that of the control, water-insoluble EP formation on BIF was continuously affected. In addition, although the CFU count on CASH sharply increased according to the biofilm age, waterinsoluble EP formation on CASH was almost completely inhibited at all of the biofilm ages tested. This result may also be related to the low concentration of fluoride released in the middle and late stages of biofilm formation (Fig. 6). According to previous studies, fluoride at low concentrations (up to 3.8 ppm F ) partially inhibited the secretion of GTFs by S. mutans but did not affect the growth of S. mutans biofilm cells.20,35 Acid production is an important virulence factor of S. mutans biofilms that deserves attention during the study of dental caries prevention. As shown in Fig. 5, all of the fluoride varnishes tested, except for FP, reduced the acid production of S. mutans biofilms during the experimental period. The reducing effect of the test fluoride varnishes on the acid production may be closely related to their reducing effect on CFU count or the physiological ability of the biofilm cells. The effect of reducing acid production in the early stages of biofilm formation may be due to the activity of fluoride against the growth of S. mutans. On the other hand, the reducing effect of CASH on the acid production in the late stages of biofilm formation may be due to the activity of fluoride against the physiological ability of the biofilm cells since there was no difference in the CFU counts between the control and CASH (Figs. 3A and 6). According to a previous study, even 3 ppm F could reduce the acid production by S. mutans biofilm cells.36 Generally, the reducing effect of the test fluoride varnishes on acid production decreased with biofilm age (Fig. 5), suggesting that the physiological abilities of the biofilm cells related to acid production started to recover during the middle or late stages of biofilm formation. However, since this study determined the pH values of the culture medium rather than the biofilms, a more sophisticated study will be needed to identify the reducing effect of the test fluoride varnishes on the acid production of the biofilms. Of the fluoride varnishes tested, FO showed the highest inhibitory effect against the bacterial adhesion and biomass accumulation (Figs. 1 and 2). The varnish diminished the biomass accumulation by up to 99% in 46-h-old biofilms and the amount of biomass did not increase during the incubation time (Fig. 2), suggesting that FO can maintain its activity against biomass accumulation for a longer time than the other fluoride varnishes. Furthermore, the CFU count of the 46-h-old biofilms was not significantly different to that of the result of bacterial adhesion assay (Figs. 1 and 3A), indicating that FO may possess and release anti-bacterial (bacteriostatic) components since the MIC of fluoride against S. mutans was approximately 282 ppm F ,32 which is much higher than the concentration released from the varnish during the incubation period (Fig. 6). However, the CFU count of the biofilms sharply increased after 46 h of incubation (from 0.004% of the control to 2.9% of the control), suggesting that the bacteriostatic activity of the varnish that appeared once the varnish was applied can decrease over time. It has been sufficiently reported that the fluoride release from fluoride varnishes occurs in a biphasic pattern, with initial rapid decrease and a slower second phase.21 In this
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study, our data confirmed the fluoride-release pattern of fluoride varnishes. As shown in Fig. 6, despite the variations in the amount of fluoride from the fluoride varnishes tested, the largest amount of fluoride release occurs during the first day with a decrease thereafter. However, interestingly, the fluoride amount from FP was not different from that of the control during the experimental periods. This result suggests that a more sophisticated and long-term study will be needed to reveal the fluoride-release pattern of FP and its effect on dental caries. Although many studies revealed that fluoride has inhibitory effects on the virulence and accumulation of cariogenic biofilms,18–20 it is unclear whether fluoride varnishes can also affect cariogenic biofilms and whether they can maintain their anti-biofilm activity over a long period of time. To the best of our knowledge, this is the first report describing how fluoride varnishes can affect bacterial adhesion and subsequent biofilm formation. Furthermore, our data indicated that the anti-biofilm activity of fluoride varnishes may depend on their fluoride-release patterns. However, additional studies are required to reveal the possible effects of viscosity and other components released from fluoride varnishes on the bacterial adhesion and subsequent biofilm formation. As shown in Figs. 1–3, the reducing effect of the test fluoride varnishes on bacterial adhesion and subsequent biofilm accumulation was not fluoride concentration-dependent, suggesting that the other ingredients can also influence the level of bacterial adhesion and subsequent biofilm formation in addition to fluoride.
5.
Conclusions
In this study, although BIF, CASH, and FO reduced S. mutans adhesion and biofilm accumulation during subsequent biofilm formation, the reducing effect of the varnishes decreased over time, which may be related to the fluoride-release pattern of the varnishes. This finding suggests that if the findings of these in vitro studies are extrapolable to the in vivo situation, then reduced clinical effect may occur with time.
Acknowledgements This research was supported by research funds of Chonbuk National University in 2013 and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2005048).
references
1. Marsh PD. Dental plaque as a microbial biofilm. Caries Research 2004;38:204–11. 2. van Houte J. Role of micro-organisms in caries etiology. Journal of Dental Research 1994;73:672–81. 3. Beighton D. The complex oral microflora of high-risk individuals and groups and its role in the caries process. Community Dentistry and Oral Epidemiology 2005;33:248–55.
733
4. Huis in ’t Veld JH, van Palenstein Helderman WH, Dirks OB. Streptococcus mutans and dental caries in humans: a bacteriological and immunological study. Antonie Van Leeuwenhoek 1979;45:25–33. 5. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiological Reviews 1986;50:353–80. 6. Harper DS, Loesche WJ. Growth and acid tolerance of human dental plaque bacteria. Archives of Oral Biology 1984;29:843–8. 7. Kuramitsu HK. Virulence factors of mutans streptococci: role of molecular genetics. Critical Reviews in Oral Biology and Medicine 1993;4:159–76. 8. Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Research 2011;45:69–86. 9. Beltra´n-Aguilar ED, Goldstein JW, Lockwood SA. Fluoride varnishes. A review of their clinical use, cariostatic mechanism, efficacy and safety. Journal of the American Dental Association 2000;131:589–96. 10. Bawden JW. Fluoride varnish: a useful new tool for public health dentistry. Journal of Public Health Dentistry 1998;58:266–9. 11. Autio-Gold J. Fluoride varnishes for everyday practice. Practical Procedures and Aesthetic Dentistry 2005;17:398. 400. 12. Strohmenger L, Brambilla E. The use of fluoride varnishes in the prevention of dental caries: a short review. Oral Diseases 2001;7:71–80. 13. Weintraub JA, Ramos-Gomez F, Jue B, Shain S, Hoover CI, Featherstone JD, et al. Fluoride varnish efficacy in preventing early childhood caries. Journal of Dental Research 2006;85:172–6. 14. Vivaldi-Rodriques G, Demito CF, Bowman SJ, Ramos AL. The effectiveness of a fluoride varnish in preventing the development of white spot lesions. World Journal of Orthodontics 2006;7:138–44. 15. Lee YE, Baek HJ, Choi YH, Jeong SH, Park YD, Song KB. Comparison of remineralization effect of three topical fluoride regimens on enamel initial carious lesions. Journal of Dentistry 2010;38:166–71. 16. Magalha˜es AC, Comar LP, Rios D, Delbem ACB, Buzalaf MAR. Effect of a 4% titanium tetrafluoride (TiF4) varnish on demineralisation and remineralisation of bovine enamel in vitro. Journal of Dentistry 2008;36:158–62. 17. Delbem ACB, Bergamaschi M, Sassaki KT, Cunha RF. Effect of fluoridated varnish and silver diamine fluoride solution on enamel demineralization: pH-cycling study. Journal of Applied Oral Science 2006;14:88–92. 18. Meurman JH. Effect of sodium and amine fluoride treatment on adsorption and ultrastructure of S. mutans and S. sanguis. Scandinavian Journal of Dental Research 1987;95:389–96. 19. Hayacibara MF, Rosa OP, Koo H, Torres SA, Costa B, Cury JA. Effects of fluoride and aluminum from ionomeric materials on S. mutans biofilm. Journal of Dental Research 2003;82:267– 71. 20. Pandit S, Kim JE, Jung KH, Chang KW, Jeon JG. Effect of sodium fluoride on the virulence factors and composition of Streptococcus mutans biofilms. Archives of Oral Biology 2011;56:643–9. 21. Castioni NV, Baehni PC, Gurny R. Current status in oral fluoride pharmacokinetics and implications the prophylaxis against dental caries. European Journal of Pharmaceutics and Biopharmaceutics 1998;45:101–11. 22. Mason SC, Shirodaria S, Sufi F, Rees GD, Birkhed D. Evaluation of salivary retention from a new high fluoride mouthrinse. Journal of Dentistry 2010;38(Suppl. 3):S30–6. 23. Eakle WS, Featherstone JD, Weintraub JA, Shain SG, Gansky SA. Salivary fluoride levels following application of fluoride varnish or fluoride rinse. Community Dentistry and Oral Epidemiology 2004;32:462–9.
734
journal of dentistry 42 (2014) 726–734
24. Pandit S, Kim GR, Lee MH, Jeon JG. Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites. Journal of Dentistry 2011;39:780–7. 25. Lemos JA, Abranches J, Koo H, Marquis RE, Burne RA. Protocols to study the physiology of oral biofilms. Methods in Molecular Biology 2010;666:87–102. 26. Jeon JG, Klein MI, Xiao J, Gregoire S, Rosalen PL, Koo H. Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms. BMC Microbiology 2009;9:228. 27. Koo H, Xiao J, Klein MI, Jeon JG. Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms. Journal of Bacteriology 2010;192:3024–32. 28. Bowen WH. Do we need to be concerned about dental caries in the coming millennium? Critical Reviews in Oral Biology and Medicine 2002;13:126–31. 29. Marsh PD. Dental plaque as a biofilm and a microbial community-implications for health and disease. Biomedical Central Oral Health 2006;6:14. 30. ElSalhy M, Sayed Zahid I, Honkala E. Effects of xylitol mouthrinse on Streptococcus mutans. Journal of Dentistry 2012;40:1151–4.
31. Fu D, Pei D, Huang C, Liu Y, Du X, Sun H. Effect of desensitising paste containing 8% arginine and calcium carbonate on biofilm formation of Streptococcus mutans in vitro. Journal of Dentistry 2013;41:619–27. 32. Dong L, Tong Z, Linghu D, Lin Y, Tao R, Liu J, et al. Effects of sub-minimum inhibitory concentrations of antimicrobial agents on Streptococcus mutans biofilm formation. International Journal of Antimicrobial Agents 2012;39:390–5. 33. Kokare CR, Chakraborty S, Khopade AN, Mahadik KR. Biofilm: importance and applications. Indian Journal of Biotechnology 2009;8:159–68. 34. Donlan RM. Biofilm: microbial life on surfaces. Emerging Infectious Diseases 2002;8:881–90. 35. Koo H, Sheng J, Nguyen PTM, Marquis RE. Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspension cultures and biofilms. FEMS Microbiology Letters 2006;254:134–40. 36. Pandit S, Kim HJ, Song KY, Jeon JG. Relationship between fluoride concentration and activity against virulence factors and viability of a cariogenic biofilm: in vitro study. Caries Research 2013;47:539–47.