archives of oral biology 59 (2014) 318–323

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The effect of delmopinol and fluoride on acid adaptation and acid production in dental plaque biofilms Jessica Neilands *, Ulrika Troedsson, Torgny Sjo¨din, Julia R. Davies Department of Oral Biology, Faculty of Odontology, Malmo¨ University, Malmo¨, Sweden

article info

abstract

Article history:

Objective: To investigate the effect of delmopinol and fluoride alone or in combination on

Accepted 20 December 2013

acid adaptation and acid production in plaque biofilm bacteria in vitro. Design: The effect of delmopinol and fluoride on acid adaptation was tested by exposing the

Keywords:

biofilm bacteria, grown in a mini-flow cell system under static conditions, to pH 5.5 over-

Caries

night in the presence of 0.16 mM delmopinol, 1 mF NaF or a combination of both. The

Acid tolerance

following day, acid adaptation was evaluated by exposing the cells to an acid challenge for

Biofilm

2 h at a pH known to kill non-adapted cells (pH 2.5). The cells were stained using LIVE/

Delmopinol

DEAD1 BacLightTM Viability stain and the number of viable (acid tolerant) cells was

Fluoride

determined using confocal scanning laser microscopy. Control cells were treated in the same manner but without the exposure to delmopinol or fluoride. How delmopinol and fluoride affected acid production was assessed by measuring the pH-drop after glucose pulsing in the presence of delmopinol and/or different concentrations of fluoride. Results: Fluoride alone or in combination with delmopinol affected the acid adaptation and significantly reduced the acid tolerance of the plaque biofilm. This effect was more pronounced when the two compounds were combined. Delmopinol alone did not affect acid adaptation. A combination of delmopinol and fluoride also reduced acid production at concentrations where neither of the compounds in isolation had an effect. Conclusion: Fluoride and delmopinol can work synergistically to affect acid adaptation and acid production in plaque biofilm bacteria. # 2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Bacteria in the oral cavity are subjected to many environmental stresses, including recurrent cycles of low pH. This results from the production of organic acid by the bacteria themselves following exposure to dietary carbohydrates.1 Frequent intake of easily fermentable carbohydrates will

result in prolonged periods of low pH in plaque and this will favour the growth of bacteria that are acid-tolerant. While some bacteria such as lactobacilli are constitutively acid tolerant, others can, in response sub-lethal pH values, initiate an acid tolerance response (ATR) and thereby adapt to an acidic environment. The ATR is a phenotypic change of the bacteria and involves changes in protein expression, increased ATPase activity and shifts to lower pH optimum for glucose

* Corresponding author at: Department of Oral Biology, Faculty of Odontology, Malmo¨ University, SE-205 06 Malmo¨, Sweden. Tel.: +46 40 665 8494. E-mail address: [email protected] (J. Neilands). 0003–9969/$ – see front matter # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.008

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transport and glycolysis.2–4 The ATR thereby allows the bacteria to survive and continue to produce acids at low pH values.3,5,6 Streptococcus mutans biofilm grown cells have been shown to express a more acid tolerant phenotype. This characteristic disappears if the biofilms cells are dispersed indicating that the acid tolerance are of phenotypic and not genotypic nature.7 An acid tolerant microflora can cause prolonged periods of low pH in plaque and this will cause demineralization of the enamel and the development of dental caries.6,8 Fluoride is well known for its ability to reduce the incidence of caries. Its primary anti-caries effect is thought to be the conversion of hydroxyapatite to fluorapatite, which resists acid dissolution to a greater extent than hydroxyapatite.9,10 However, another important property of fluoride is its effect on bacterial physiology. Fluoride inhibits bacterial carbohydrate metabolism by affecting the enzyme enolase in the glycolytic pathway.11,12 Fluoride has also been shown to affect a range of other enzymes such as catalase, urease and the F-ATPases.13–15 The proton-translocating ATPases play a major role in the acid-base physiology of oral streptococci and lower ATPase activity results in a reduced bacterial acid tolerance.16 A more recently discovered property of fluoride is that it can inhibit the induction of an ATR in S. mutans and it has been shown to reduce acid tolerance in plaque biofilms in vivo.17,18 Delmopinol is a surface-active agent used in dental products. In comparison with other products on the market, delmopinol has a low antimicrobial profile and promotes a microbial flora compatible with dental health.19,20 The compound binds to hard and soft oral tissues as well as to bacterial surfaces and affects several of the steps in the formation and establishment of dental biofilms. These include displacement of components from the pellicle,21 and interference with the build-up and cohesion of plaque by reducing glucan synthesis and glucan viscosity.22–25 Delmopinol may also reduce cell to cell adhesion by changing the colloidal stability of bacterial suspensions26 and by detaching or solubilising surface structures of oral bacteria,27 and has been demonstrated to affect acid production in oral bacteria.19 The purpose of the present study was therefore to investigate whether delmopinol and/or fluoride can inhibit acid adaptation and/or acid production in plaque biofilms in vitro, and if the combination of delmopinol and fluoride would enhance the effect.

No. RB997 (Sinclair Pharma AB, Sweden) and sodium fluoride, (Sigma–Aldrich, USA).

2.2.

Plaque biofilm formation

After refraining from brushing the teeth for 12 hours, plaque was sampled in the morning from the buccal and lingual tooth surfaces of a healthy individual and pooled. The same donor was used throughout the entire study. The plaque sample was suspended in 50 ml 0.1 M potassium phosphate buffer (PBS) followed by the addition of 1.5 ml Todd–Hewitt broth pH 7.0 and mixed by vortexing. To each channel of a mini flow-cell, mSlide VI for Live Cell Analysis (Ibidi1, Ibidi GmbH, Martinried, Germany) with a growth area of 0.6 cm2, 120 ml of plaque suspension was added and incubated under static conditions for 24 h at 37 8C in air with 5% CO2 in a humid chamber. These biofilms were then used for either acid adaptation or acid production experiments (Fig. 1). All experiments were run under static conditions.

2.3.

Acid adaptation in plaque biofilms

To test if delmopinol and fluoride could affect acid adaptation in plaque biofilm cells, a modified version of a method developed previously for testing the effect of fluoride on acid adaptation was employed.17 The 24-h plaque biofilms, prepared as described above, were exposed to MM4 pH 5.5 with the addition of 1 mM fluoride or 0.16 mM delmopinol alone or in

Plaque suspended and added to Ibidi mini-flow cell Incubation 24 h

24 h plaque biofilm

Acid adaptation MM4 pH 5.5 +/- delmopinol and/or +/- fluoride Incubation 24h

MM4 pH 2.5

2.

Materials and methods

2.1.

Media and test compounds

For the establishment of a plaque biofilm, a semi-defined growth medium BactoTM Todd Hewitt Broth (Becton, Dickinson and Company, USA) was used. For the acid adaptation and acid killing experiment a fully defined minimal medium (MM4) was used. MM4 contains six amino acids: glutamate, serine, cysteine, valine, leucine, asparagine, 20 mM glucose and 40 mM phosphate/citrate buffer adjusted to pH 7.5, 5.5 or 2.5 and has been used previously in acid adaptation experiments.28 Test compounds used were delmopinol hydrochloride, batch

- delmopinol and fluoride Incubation 2h

LIVE/DEAD BacLight

Acid production MM4 pH 7.5 Incubation 24h

MM4 pH 7.5 +/- delmopinol and/or fluoride Incubation 30 min

MM4 pH 7.5 +/- delmopinol and/or fluoride + 100 mM glucose Incubation 3h

pH measurement Fig. 1 – Schematic overview plaque biofilm formation, acid adaptation and acid production experiments.

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combination and incubated for 24 h at 37 8C in air containing 5% CO2 in a humid chamber. Control cells were treated in the same way but without delmopinol and fluoride. After 24 h the MM4 medium was removed and the flow-cell rinsed twice with PBS to remove any medium containing fluoride and/or delmopinol. The biofilm was then exposed to MM4 pH 2.5, a pH that kills nonadapted cells, for two hours. To determine if the biofilm cells had undergone acid adaptation, after the two-hour incubation period the MM4 pH 2.5 media was removed from the flow cell and rinsed twice with PBS after which the biofilms were stained with the LIVE/DEAD1 BacLightTM Bacterial Viability kit for microscopy (Molecular Probes, Eugene, USA).17,29 All experiments were run three times on separate occasions with new plaque samples taken prior to each experiment.

2.4. Confocal scanning laser microscopy and image analysis The fluorescence from LIVE/DEAD1 BacLightTM stained cells was viewed using an Eclipse TE2000 inverted confocal scanning laser microscope (CSLM) (Nikon). A total of 10 CSLM images, per sample, were acquired from different parts of the flow cell using the software EZ-C1 v.3.40 build 691 (Nikon) at a resolution of 512  512 pixels and with a zoom factor of 1.0, giving a final pixel resolution of 0.42 mm/pixel. CSLM images were analyzed using a modification of the method described previously.18,20 The LIVE/ DEAD1 BacLightTM stains cells with an intact membrane (viable) green and cells with a damaged membrane (dead) red. Briefly, three independent evaluators examined the images without knowing the conditions for each image. Each image was then given a score of 1 to 4 depending on the proportion of red and green cells. The following criteria were used for each score: score 1, mostly red cells; score 2, more red than green cells; score 3, more green than red cells and score 4, mostly green cells. The median score value for each condition was calculated.

2.5.

Effect of delmopinol and fluoride on acid production

In 24-h plaque biofilms prepared as described above, the medium was carefully removed, 120 ml MM4 pH 7.5 was added and the biofilm incubated for a further 24 h during static conditions. On the following day the MM4 medium was removed and replaced with MM4 pH 7.5 with concentrations of fluoride between 0.01 and 10 mM alone or in combination with 0.32 mM delmopinol. The biofilms were incubated for 30 min at 37 8C during static conditions. After incubation, the medium was replaced with MM4 pH 7.5 containing delmopinol and/or fluoride as well as 100 mM glucose. Starting pH was recorded before the biofilm was incubated for a further 3 h at 37 8C, after which pH in the supernatant was measured with a pH-meter (Methrom 827, Herisau, Switzerland). Control cells were treated in the same way with MM4 pH 7.5 devoid of delmopinol and fluoride. The experiment was repeated three times on different occasions.

2.6.

Statistical analysis

Data were analyzed using InStat (version 3.0, Graph Pad, San Diego CA, USA) using the Mann–Whitney test for both the image analysis and the pH-experiments.

3.

Results

3.1. The effect of delmopinol and fluoride on acid adaptation in plaque biofilms Plaque biofilms were grown in the mini flow-cell system Ibidi m-Slide VI. Visual examination of the images taken in the CSLM indicated no distinct differences in biofilm coverage between the different groups, suggesting that delmopinol and fluoride alone or combined did not affect biofilm formation per se in this study. To induce acid adaptation, plaque biofilms were exposed to a sub-lethal pH (5.5) overnight. Acid adaptation was then assessed as the proportion of viable cells (acid tolerant cells) after 2 h exposure to pH 2.5, a pH that kills non-adapted cells.17 Each image was given a score 1–4 depending on the proportion of viable cells in the image. Where score 1 was mostly red cells; score 2, more red than green cells; score 3, more green than red cells and score 4, mostly green cells. Three evaluators examined the images and in 86% of cases the score value given was the same for all three evaluators. For the remaining images except two, the score given by two of the evaluators was the same while the third evaluator had given a score that was 1 unit higher or lower. In the control cells (C) exposed to pH 5.5 the median score value was 3, reflecting good viability of the cells. In cells maintained at pH 5.5 in the presence of delmopinol and/or fluoride the median score value for all three test groups was also 3, indicating that the viability of the cells had not been affected by the treatment (Fig. 1). After the acid challenge (pH 2.5) the score value for the C-cells was 3, demonstrating that a large proportion of the cells were acid tolerant. This was also the case for the delmopinol (D) exposed cells in which the score value also was 3 after acid challenge. However, the exposure to fluoride (F) alone or delmopinol and fluoride combined (D+F) significantly impaired acid adaptation as seen by the reduced number of viable cells after acid challenge. In both cases the number of acid tolerant cells was reduced compared to control ( p < 0.01). The median score value for Fexposed cells was 2 and for D+F-exposed cells 1 (Fig. 2). The effect of D+F was significantly greater than that of F alone ( p < 0.05).

3.2. Effect of delmopinol and fluoride on acid production in plaque biofilms The effect of D+F on acid production in plaque biofilms was tested after exposure to 100 mM glucose for 3 h. Initially 0.16 mM delmopinol combined with fluoride (0.1, 1 and 10 mM) was tested. A statistically significant reduction ( p < 0.05) in pH-drop compared to control was seen for D + 10 mM F and D + 1 mM F but not for D + 0.1 mM F. There was no additive effect when D was combined with F compared to F alone (data not shown) suggesting that the effect seen was due to fluoride. Therefore the delmopinol concentration was increased to 0.32 mM. In Fig. 3 the effect of 0.32 mM delmopinol in combination with various concentrations of fluoride is shown. At all four fluoride concentrations (0.01–10 mM) there was a statistically significant difference between C and D+F cells ( p < 0.01). At the higher concentrations of F (10 mM and 1 mM)

archives of oral biology 59 (2014) 318–323

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Fig. 3 – The effect of different concentrations of fluoride and 0.32 mM delmopinol on final pH in plaque biofilms in vitro, 3 h after glucose pulsing. There was a statistically significant difference ( p < 0.01) at all four fluoride concentrations between cells exposed to fluoride and delmopinol and the control cells (0 mM D/0 mM NaF). At the lower concentrations of fluoride (0.01 and 0.1 mM) there was a statistical difference ( p < 0.05) between cells exposed to delmopinol and fluoride compared to fluoride alone.

Fig. 2 – LIVE/DEADW BacLightTM stained plaque biofilms kept at pH 5.5 or exposed to pH 2.5. Control (A), 0.16 mM delmopinol (B), 1 mM fluoride (C) and 0.16 mM delmopinol + 1 mM fluoride (D). Green cells are viable (acid tolerant) while red cells are dead (not acid tolerant).

no additive effect of delmopinol was seen but at 0.1 and 0.01 mM F the final pH was higher in the D+F exposed cells compared to the cells exposed to F alone a difference that was statistically significant ( p < 0.05). Concentrations of fluoride alone at 0.1 mM and below did not affect acid production and the pH-drop did not differ from the control cells. Delmopinol alone did not affect acid production in plaque biofilms and the pH-drop after glucose pulsing was similar to the control.

4.

Discussion

In recent years there has been an increase in dental caries and there is a need for new preventive strategies.30 One such strategy could be the prevention of acid tolerant oral bacteria. Fluoride is an important factor in caries prevention, however

high doses of fluoride can result in the development of fluorosis.31 Identification of compounds that could be combined with fluoride, thereby allowing the use of lower concentrations of fluoride, would therefore be desirable. In this study we have shown that low concentrations of delmopinol in combination with low concentrations of fluoride inhibit acid adaptation in plaque biofilms in vitro and that the combination of the two also prevents pH decreases in plaque biofilms after glucose pulsing at levels where fluoride or delmopinol alone had no effect. Delmopinol is a surface-active agent and has previously been shown to affect plaque formation as well as acid production after glucose pulsing in planktonic cells of oral streptococci.19 How delmopinol affects these cellular processes is however not yet known. In this study delmopinol alone did not affect acid adaptation or acid production in plaque biofilms possibly due to the low concentrations used and the fact that biofilm cells are more resistant to most compounds than planktonic cells.32 However in combination with fluoride, an effect on acid production was seen at levels where fluoride alone had no effect. The data from the acid production experiments showed that when delmopinol was present, the inhibitory levels of fluoride were 100 times lower than for fluoride alone. Fluoride has previously been shown to inhibit the glycolytic enzyme, enolase, which leads to a diminished production of ATP and the acid end-products of bacterial metabolism.11,12,33 This was reflected in the reduced pH-decrease seen in this study. Fluoride crosses the cell membrane as HF and needs to be inside the cell in order to exert its action.11 Delmopinol has been shown to affect the cell wall and possibly the cell membrane of oral bacteria.27 One mode of action could therefore be that delmopinol by affecting the cell wall and cell membrane, facilitates the diffusion of fluoride across the bacterial cell membrane and thereby enhances the concentration of fluoride inside the cell. This effect would be more pronounced at sub-inhibitory levels of fluoride. Enhanced intracellular levels of fluoride could also explain the combined effect of fluoride and delmopinol on acid

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adaptation seen in this study. Although fluoride alone affected acid adaptation, the inhibitory effect was greater in the combination with delmopinol. Fluoride has previously been shown to affect acid adaptation in S. mutans as well as reducing the acid tolerance of plaque biofilms and thus the findings of this study are well in line with previous ones.17,18 Acid adaptation involves several different mechanisms including the synthesis of stress-response proteins, increased ATPase activity and reduced proton permeability which in turn makes the bacteria more acid tolerant.2,7,28,34–36 Fluoride inhibits the membrane bound ATPases that regulate intracellular pH by pumping protons out of the cell.37 An increased intracellular level of fluoride would therefore result in the accumulation of protons inside the cell which then would acidify the cytoplasm and affect cellular functions connected to acid adaptation. In S. mutans it has been shown that acid adaptation also involves changes in membrane fatty acid composition from short-chained to long-chained fatty acids.38,39 In a study by Zhu et al. it was shown that fluoride resistant strains are more acid-tolerant and these strains have a higher level of long-chained membrane fatty acids.40 The LIVE/DEAD BacLight stain used here consists of two different nucleic acid stains, SYTO1 9 and propidium iodine, with SYTO1 9 capable of penetrating all cells and staining them green. Propidium iodine, on the other hand, only penetrates cells with a damaged membrane and stains the cells red. The higher number of cells with a damaged membrane in the biofilms treated with fluoride alone or in the combination with delmopinol indicates that the cells might not have undergone such changes in membrane fatty acid composition. Overall, the data from this study show that fluoride and delmopinol can work together and that effect on acid adaptation and acid production can be achieved even at sub-inhibitory concentrations of fluoride when combined with delmopinol. That an additive effect can be achieved when combining two compounds is not a new phenomenon. Xylitol has been shown to work with fluoride to reduce acid production more effectively than either fluoride or xylitol alone.41 Apigenin and tt-farnesol, two compounds derived from the beehive product propolis, have also been shown to work more efficiently to prevent caries in combination with fluoride than each of the three compounds alone. However, further investigations are needed to understand how delmopinol can enhance the effect of fluoride on acid adaptation and acid production in plaque biofilms.

Conflict of interest There are no conflicts of interest.

Funding No funding, research supported by Malmo¨ University.

Ethical approval Ethical approval not needed for this type of study.

references

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The effect of delmopinol and fluoride on acid adaptation and acid production in dental plaque biofilms.

To investigate the effect of delmopinol and fluoride alone or in combination on acid adaptation and acid production in plaque biofilm bacteria in vitr...
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