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Analysis by confocal laser scanning microscopy of the MDPB bactericidal effect on S. mutans biofilm CLSM analysis of MDPB bactericidal effect on biofilm

Since bacteria remain in the dentin following caries removal, restorative materials with antibacterial properties are desirable to help maintaining the residual microorganisms inactive. The adhesive system Clearfil Protect Bond (PB) contains the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) in its primer, which has shown antimicrobial activity. However, its bactericidal effect against biofilm on the dentin has been little investigated. Objective The aim of this study was to analyze by confocal laser scanning microscopy (CLSM) and viable bacteria counting (CFU) the MDPB bactericidal effect against S. mutans biofilm on the dentin surface. Material and Methods Bovine dentin surfaces were obtained and subjected to S. mutans biofilm formation in BHI broth supplemented with 1% (w/v) sucrose for 18 h. Samples were divided into three groups, according to the primer application (n=3): Clearfil Protect Bond (PB), Clearfil SE Bond, which does not contain MDPB, (SE) and saline (control group). After the biofilm formation, Live/Dead stain was applied directly to the surface of each sample. Next, 10 µL of each primer were applied on the samples during 590 s for the real-time CLSM analysis. The experiment was conducted in triplicate. The primers and saline were also applied on the other dentin samples during 20, 90, 300 and 590 s (n=9 for each group and period evaluated) and the CFU were assessed by colonies counting. Results The results of the CLSM showed that with the SE application, although non-viable bacteria were detected at 20 s, there was no increase in their count during 590 s. In contrast, after the PB application there was a gradual increase of non-viable bacteria over 590 s. Conclusions The quantitative analysis demonstrated a significant decrease of S. mutans CFU at 90 s PB exposure and only after 300 s of SE application. Protect Bond showed an earlier antibacterial effect than SE Bond.


INTRODUCTION
The use of cavity disinfectants before restoration is an important clinical step because bacteria can remain in the smear layer or in the dentinal tubules, and can potentially multiply 16 . The ability of restorative materials to control bacteria by antimicrobial components is also desirable to help maintain residual microorganisms inactive 14 . The self-etching adhesive system, Clearfil Protect Bond, contains the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) in its primer. This monomer was incorporated to inactivate residual microorganisms on the dentin surface and to prevent bacterial invasion through the gap tooth/restoration 7 . MDPB monomer is a compound of the antibacterial agent quaternary ammonium and a methacryloyl group, which is covalently bound to the polymer matrix by its copolymerization with other monomers 7 . MDPB has shown antimicrobial activity against oral streptococci, lactobacilli and microorganisms clinically isolated from root caries 23 . However, studies that evaluated the MDPB antibacterial effect were usually conducted in bacterial suspension and by agar diffusion testing 7,9,11 .
Since in the clinical practice adhesive systems are directly applied to the dentin with some level of contamination 12,14,21 , it would be crucial to evaluate the MDPB antibacterial effect against bacteria colonized on the dentin surface. This fact is also relevant because the bacteria adhered on the surfaces, forming biofilms, are less susceptible to antimicrobials than planktonic microorganisms (bacteria in suspension) 4 . However, few studies evaluated the antibacterial effect of adhesive systems containing MDPB against the biofilm 10, 15 . Roland, et al. 15 (2006) investigated the antibacterial effect of MDPB and fluoride on biofilm formation of dental adhesive surfaces to establish whether these components had the potential to reduce biofilm formation on adhesive system surfaces. It was found that the antibacterial agents within the resins have a minimal effect on biofilm formation 15 . Izutani, et al. 10 (2011) evaluated the antibacterial effect of MDPB on S. mutans biofilm and showed that high concentrations of MDPB in solution are necessary to effectively kill biofilm S. mutans cells within an acceptable clinical time. Based on these results, it becomes necessary to evaluate if the application time of MDPB-containing a primer recommended by the manufacturer (20 s) is sufficient to decrease or eliminate microorganisms colonized on the dentin surface or for how long the bactericidal effect of MDPB-containing primer acts against these microorganisms.
Confocal laser scanning microscopy (CLSM) has been used to evaluate bacterial viability on dentin biofilms 15 . Fluorescence dyes are applied on the biofilm to differentiate live and dead bacteria, allowing bacteria to be distinguished according to cytoplasmic membrane permeability 9 . This holds particular relevance for studying the antimicrobial action of adhesive systems containing MDPB, because MDPB causes cell death through disruption of the cytoplasmic membrane [7][8][9]15 . Furthermore, CLSM can capture a series of image-scans showing changes in the viability of the bacterial cell over time, making the visualization of real-time death of microorganisms possible.
The aim of this study was to evaluate the bactericidal effect against S. mutans biofilm between self-etching priming solutions with and without MDPB (Clearfil Protect Bond and Clearfil SE Bond, respectively) over 590 s by real time CLSM analysis and count of viable bacteria test. The hypothesis tested in this study was that there is a difference in the bactericidal effect against S. mutans biofilm between primers with or without MDPB during 590 s by CLSM analysis and count of viable bacteria test.

Adhesive systems
Two self-etching/priming solutions were tested, an antibacterial primer containing a M D P B m o n o m e r -C l e a r f i l P r o t e c t B o n d (Kuraray, Tokyo, Japan) -containing MDPB, MDP (10-methacryloxydecyldihydrogen phosphate), HeMA (2-hydroxyethylmethacrylate) and water, and a primer without an antibacterial monomer -Clearfil SE Bond (Kuraray, Tokyo, Japan) -containing MDP, HeMA, dl-camphorquinone, N,N-diethanol-ptoluidine and water.

Samples preparation
One hundred fourteen bovine incisors were obtained and stored in 0.1% thymol solution. The buccal portion of the enamel was wet ground (Arotec, São Paulo, SP, Brazil) using 400 and 600 grit silicon carbide paper (Saint-Gobain, São Paulo, SP, Brazil). Dentin samples with 4x4 mm were obtained of each tooth using a low-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) and ground with 240 grit silicon carbide paper to reach 1.5 mm thickness. All samples were sterilized by steam autoclave (Phoenix, Araraquara, SP, Brazil) at 121°C for 15 min.

Bacterial colonization on the dentin
S. mutans UA159 was used in this study. To prepare the inoculums, this microorganism strain was grown on Mitis salivarius agar plates at 37ºC for 48 h in a 5% supplemented CO 2 environment. Subsequently, single colonies were inoculated into 5 mL of BHI broth and incubated at 37ºC for 24 hrs. An overnight culture was adjusted to 1x10 6 [colony forming units (CFU)/mL] with an optical density of 0.6 at 600 nm. The dentin surface of each sample was exposed to 20 µL aliquot of S. mutans suspension in brain heart infusion broth (BHI) (Becton Dickinson, Sparks, MD, USA) without any disturbance, to allow for bacterial adherence. After 2 hrs at room temperature, the non-adhering cells were removed by washing three times with saline solution 5 . A sample was placed in each well of a 24-well multi-dish polystyrene plate (Nunclon, Thomas Scientific, Swedesboro, NJ, USA). Two mL of BHI broth supplemented with 1% (w/v) sucrose (LabSynth, Diadema, SP, Brazil) were then added to the wells to simulate bacterial colonization 5 . The plates were incubated for 18 h at 37ºC in a 5% CO 2 environment. The lack of contamination in the media of each well was verified using Gram staining and by plating the samples. The 18 h incubation period was established by a previous study, which verified that it was sufficient to develop a thin S. mutans biofilm (containing 10 8 colony unit formation) without producing an intense demineralization of the dentin specimens, since the demineralization process could increase the dentin autofluorescence 3 and harm the biofilm visualization by CLSM.

Assessment of bactericidal effects by CLSM
Live/Dead Baclight bacterial viability stain (L13152) (Molecular Probes, eugene, OR, USA) was used in this study. It consists of a two nucleic acid-binding stains mixture: Syto 9 and propidium iodide. Syto 9 stains all viable bacteria in green, while propidium iodide stains in red the bacteria whose membranes were damaged (non-viable bacteria). Three samples were used for each solution application: Clearfil Protect Bond primer, Clearfil Se Bond primer and saline (control group). After 18 hrs of bacterial colonization, the non-adhering cells were removed by washing three times with saline solution 5 . Live/Dead was mixed according to the manufacturer's instructions and one drop was applied directly to the surface of each sample 15 . After 15 min in dark incubation 5 , the stain surplus was removed by absorbent paper and 10 µL of each solution was applied on the samples' surface, according to each group. Immediately after, samples were analyzed by CLSM for 590 s (LSM510 MeTA, Zeiss, Jena, Germany). This period was used based on the Imazato, et al. 9 (2008) study, which found rapid killing of planktonic bacteria after contact with MDPB for 590 s. An excitation wavelength of 488 nm was used, and all light emitted between 500 -550 nm and over 560 nm was collected by different filters. The scan mode time series was used to take a series of time-lapse scans (xyt) at intervals of 10 s during 590 s using continuous scanning with 10x objective lens. Scans were taken in 8 bits at a resolution of 512 by 512 pixels. To standardize the gain and offset values to take a series scan, a preliminary sample prepared as described before was used. The experiment was conducted in triplicate.

Assessment of bactericidal effects by viable bacteria count
The primer application time was tested based on the CLSM results (20 s, 90 s, 300 s, 590 s). One-hundred eight dentin samples were used and twelve groups were obtained according to the time application of each primer or saline (n=3): saline application for 20 s, 90 s, 300 s and 590 s; Protect Bond application for 20 s, 90 s, 300 s and 590 s and Se Bond application for 20 s, 90 s, 300 s and 590 s. The groups with the saline application were considered the negative control groups (no primer application). The experiment was repeated three times (triplicate). After 18 h of bacterial colonization as described before, 10 µL of each primer or 0.9% NaCl solution was applied on the sample surface. The solutions were kept in contact with the S. mutans biofilm, without any disturbance for each group. The time was recorded by a chronometer. After each experimental time, the excess solutions were removed by sterile absorbent paper. Samples were immersed in individual eppendorfs with 1 mL 0.9% NaCl solution and sonicated for 30 s at 30% amplitude. The suspension was diluted in decimal series from 10 −1 to 10 −6 in 0.9% NaCl solution and inoculated on BHI agar plates containing bacitracin (2 U/mL) 13 . The plates were incubated for 48 h at 37ºC in a 5% supplemented CO 2 environment and the number of viable bacteria (CFU) were assessed by counting the colonies formed. The data of CFU were submitted to the Kruskal-Wallis and Dunn's Multiple Comparisons tests at a significance level of p<0.05.

RESULTS
CLSM images showed S. mutans biofilm on the dentin surface with viable and non-viable colonies (green and yellow/red, respectively) and non-stained (black) bubble-like structures within the biofilm architecture (Figures 1, 2 and 3). The control group demonstrated viable bacteria during 590 s, all colonies remained green, indicating that S. mutans were viable during the period evaluated ( Figure 1). Non-viable bacteria (red colonies) were seen after the Clearfil SE Bond primer application (Figures 2A and 2B), nonetheless, there was no increase of these bacteria during 590 s and viable bacteria were observed at this time ( Figure 2C). After the Clearfil Protect Bond primer application some non-viable bacteria were visualized in 0 s ( Figures 3A and 3B). However, at 20 s (application time recommended by the manufacturer) viable bacteria was still visualized, and no difference was found between the viable and non-viable bacteria in 0 s and 20 s time-lapse scans ( Figure 3A). The color change of S. mutans colonies to a yellowish or orange indicated the death of bacteria after the Clearfil Protect Bond primer application, ( Figure 3A) showing a gradual increase of non-viable bacteria over 590 s. At 590 s, yellowish and red colonies were predominant ( Figure 3C).   (Figures A and B), nonetheless, there was no increase of these bacteria during 590 s and viable bacteria were observed at this time ( Figure C). *Low-intensity noise that can be attributed to the fluorescence of the primer Table 1 shows a quantitative analysis of the number of viable bacteria (CFU) of S. mutans biofilm on the dentin after the Clearfil Protect Bond, Clearfil SE Bond and saline application. There was no statistical difference among CFU after 20, 90, 300 and 590 s for the control and Clearfil SE Bond groups. In the contrary, the Clearfil Protect Bond group at 300 s and 590 s primer exposures showed a significant decreasing CFU compared to 20 s.
When the comparisons were made among the primers and saline in each experimental time, at the initial time (20 s), no difference was found among the two primers and saline (Table 1). At 90 s, the Clearfil Protect Bond group showed a significant decreasing CFU compared to the control group and Clearfil SE Bond showed intermediate CFU between the control and Clearfil Protect Bond groups (Table  1). For the Clearfil SE Bond group the CFU decreased significantly after 300 s and 590 s of application, consequently the Clearfil Protect Bond showed an earlier antibacterial effect than the Se Bond (Table  1). At 300 s and 590 s, both primers showed decreasing CFU compared to the control group, but with no difference between them (Table 1).

DISCUSSION
The null hypothesis was rejected because there was a difference in the bactericidal effect against S. mutans biofilm between self-etching priming solutions with and without MDPB by CLSM analysis and count of viable bacteria test. MDPB monomer can be considered a bactericidal agent against S. mutans biofilm, since it affected the bacteria membrane that could be detectable by fluorescent indicators.
Antimicrobial compounds that do not directly affect the cell membrane, such as those that interfere with protein or nucleic acid synthesis, are normally considered bacteriostatic and are not suitable agents to be studied by a viability staining method 1 . Live/Dead Baclight has been shown to be an effective stain to distinguish viable and nonviable bacteria according to cytoplasmic membrane permeability 2 . As MDPB is a derivate of quaternary ammonium, it has a high affinity for negatively charged bacterial cells by a nitrogen atom on the pyridinium ring, which binds to the cell surface. The cell membrane loses its electrical balance, creating a disruption in the cytoplasmic membrane and the cell dies 7 .
Few studies evaluated the antibacterial effect of adhesive systems containing MDPB against the biofilm by CLSM analysis 10,15 . However, these earlier studies evaluated the effect of the MDPB monomer to reduce biofilm formation on the Clearfil Protect Bond adhesive system surface 15 and the effect of high concentrations of unpolymerized MDPB solutions to kill S. mutans in planktonic or biofilm forms after 20, 40 or 60 s of contact 10 . The primer containing-MDPB real-time effect against S. mutans biofilm by CLSM evaluation over 590 s had never been tested.
There was a good relation among the CFU results and the elapsed times of the CLSM analysis. The CLSM time-lapse scans and CFU results of the control group showed viable bacteria during 590 s ( Figure 1 and Table 1). After the Clearfil SE primer application some non-viable bacteria were seen, however, the green staining was maintained in all periods tested (Figure 2). No statistical difference was found among the CFU during 590 s for the Clearfil SE Bond group ( Table 1). The acidic nature of the primer of self-etching adhesive systems has been considered as one of the key factors related to bacterial inhibition 6 . The MDP monomer of Clearfil SE and Clearfil Protect Bond can have antibacterial activity because of its low pH level (1.9) 6,18 . However, the dentin can act as a buffer, because the acidic dissolution of the dentin apatite may be neutralized by the bonding between the dentin ions and H+ ions of the monomer 6 . Consequently, the pH level of the MDP may have been neutralized by the buffering action of the dentin, leading to the visualization of viable bacteria by CLSM after the Clearfil SE primer application. Likewise, the MDP of the Clearfil SE Protect primer may also have been neutralized; however, the MDPB monomer showed a gradual bactericidal effect over 590 s and nonviable bacteria were predominant at 590 s. The CFU results also showed a decrease after the 90, 300 and 590 s Clearfil Protect Bond application. These results confirmed previous studies that a MDPBcontaining primer has an antibacterial effect against S. mutans colonized in the dentin 8,20 .
Bacterial biofilms associated with surfaces are complex three-dimensional structures in which bacteria are embedded in a matrix mainly made of exopolysaccharides (ePS) 19 . ePS functions as an ion-exchange matrix and hinders the penetration of positively-charged antimicrobial agents 17 . The activation of an adaptive stress response and changes in gene and protein expression of bacteria are also factors that make biofilms 10 to 1000 times less sensitive to antimicrobial agents than planktonic bacteria 1 . Three main factors can be considered to the difference in susceptibility of planktonic and biofilm bacteria to antimicrobials 10 . One is the hindrance of penetration of antimicrobials into the biofilm, owing to the presence of a copious amount of an extracellular matrix, which acts as a molecular sieve. A second factor is that the chemical micro-environment, such as nutrientdepletion or waste products within the biofilm, may act as antagonists of antimicrobials. A third, and still speculative hypothesis is that some of the bacteria may differentiate into a protected phenotypic state 10 .
Some studies reported declining S. mutans counts in dentin chips and bacterial suspension after 20 s of the Clearfil Protect Bond primer application (application time recommended by the manufacturer) 7,8,11,20 . However, the present study showed that there were viable bacteria at 20 s similar to 0 s time-lapse scan ( Figure 1). Furthermore, the CFU results showed a decreasing number of bacteria after the 90 s Clearfil Protect Bond primer application (Table 1). Probably, the EPS among the biofilm channels may have impaired the penetration of MDPB and consequently its action, needing more time to kill bacteria since the mechanism of action for MDPB is based on the contact of immobilized antibacterial molecules with bacteria 7 . Thus, a gradual bactericidal effect was evidenced during the period evaluated. The results of the present study were contradictory to the Imazato, et al. 9 (2008) results, which showed rapid killing of all bacteria (S. mutans) after 590 s contact with MDPB solution using Live/Dead Baclight bacterial viability stain and fluorescence microscopy, but the MDPB solution was tested against planktonic bacteria. Another study 10 showed that high concentrations of MDPB are necessary to effectively kill planktonic and biofilm S. mutans cells within 60 s of contact, however, the biofilm produced under the experimental conditions of the Izutani, et al. 10 (2011) study had a sparse structure (i.e. without a dense extracellular matrix) and this fact may have helped the penetration of MDPB and its antimicrobial effect 10 . The CLSM analysis showed a dense biofilm with some characteristics of in vivo biofilm (Figures 1, 2 and 3), such as the bubble-like structures and dead bacteria that were initially present. This dense structure of biofilm may also have impaired the antimicrobial effects of the Clearfil Protect Bond primer. The bubblelike structures were voids that were possibly filled with biological substances, such as EPS and glycoproteins, which are not stainable by the stain used 22 . Besides, the dead bacteria might have been located in deeper regions of the biofilm and had reduced access to nutrients from the exposed medium 15 .
The viability staining method provided a rapid and sensitive way to test the MDPB bactericidal effect against S. mutans biofilm. As the evidence of in vitro study must not be extrapolated to in vivo situations, clinical studies must be conducted to evaluate the application time of the Clearfil Protect Bond to disinfect the enamel/dentin cavity to obtain the antimicrobial activity. The collection of softened carious dentin or the use of swabs rubbed on to the base of the cavity before and after the primer application could be performed to clinically measure the antimicrobial activity 14 . Further investigations also need to be conducted against other cariogenic microorganisms in the biofilm or multispecies biofilm. Some attempts were made in this present study to test the MDPB effect after the bond application and cure, but the polymerized adhesive (bond) autofluorescence interfered with the bacteria visualization using CLSM.

CONCLUSIONS
Based on the findings of this study, and within the limitation of the in vitro investigation, it can be concluded that the Clearfil SE Bond primer presented a bactericidal effect only after 300 s against S. mutans biofilm and the Clearfil Protect Bond primer had a bactericidal effect against S. mutans biofilm after 90 s and showed a gradual bactericidal effect over 590 s by real-time CLSM evaluation and viable bacteria counting.