Streptococcus mutans biofilms formation and cigarette smoking experimental scheme. Streptococcus mutans UA159 (ATCC 700610; serotype c) biofilms were formed on saliva-coated hydroxyapatite (sHA) discs (2.93 cm2; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) placed in a vertical position in 24-well plates. Briefly, an adult male (non-smoker) was selected for oral saliva collection. HA discs were incubated in filter-sterilized (0.22-µm low protein-binding filter) saliva (3 ml/disc) for 1 h at 37°C. For biofilms formation, the sHA discs were transferred to a 24-well plate containing brain heart infusion (BHI; D-ifco, Detroit, MI, USA) broth with 1% (w/v) sucrose and S. mutans UA159 (5–7×106 colony-forming unit (CFU)/ml) (3 ml/disc). The biofilms were grown at 37°C with 5% CO2 for 21 h to allow initial biofilms growth. After 21 h, S. mutans biofilms were divided into 4 groups. Experiment group 1 (control group) did not receive CS treatment and exposed to the air six times per day (at 8, 10, 12 a.m., 2, 4, 6 p.m.). Experiment group 2 was treated one time per day (at 10 a.m.) with CS and five times per day (at 8, 12 a.m., 2, 4, 6 p.m.) with air, a total of three times CS treatments in 74-h-old biofilms. Experiment group 3 was treated three times per day (at 10 a.m., 2, 6 p.m.) with CS and three times per day (at 8, 12 a.m., 4 p.m.) with air, a total of seven times CS treatments in 74-h-old biofilms. Experiment group 4 was treated six times per day (at 8, 10, 12 a.m., 2, 4, 6 p.m.) with CS, a total of fifteen times treatments in 74-h-old biofilms. Each treatment time was 5 minutes (simulate the real smoking time of smokers). The culture medium was changed twice daily (8 a.m. and 6 p.m.) (Oral sugar levels rise after 8 a.m. for breakfast and 6 p.m. for dinner). The hydroxyapatite disks were washed with distilled water three times a day (8 a.m., 1 p.m., 6 p.m.) (Simulate cleaning mouth after breakfast, lunch, and dinner) for the control group and all CS treatment groups. The incubated time of the S. mutans biofilms was 74 hours (Fig. 8). This study is approved by the ethics committee/institutional review board of the Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience, Jeonbuk National University. All experimental protocols were approved by the Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience, Jeonbuk National University. The author confirms that all methods were carried out in accordance with relevant guidelines and regulations. The author confirms that informed consent had been obtained from all subjects.
The microenvironment of plaque is easily affected by various bacteria and external factors. To minimize the influence of external factors and improve the internal validity of the results, an in vitro method was considered. Considering that S. mutans is a facultative anaerobe, a glass container was designed to allow air to enter while CS was inhaled to avoid the effect of complete hypoxia on the growth of S. mutans biofilm. At the time of treatment, the sHA discs were taken out from the culture medium, placed in a sterile glass container, and a cigarette was taken using a vacuum machine to simulate cigarette gas in the mouth during smoking, each treatment time was 5 minutes. After treatment, sHA discs were returned to the culture medium (Fig. 9).
In this experiment, a popular cigarette in Korean supermarkets was selected. Marlboro (tar: 8.0 mg; nicotine: 0.7 mg), it has the highest tar and nicotine content per cigarette of all cigarette brand. We used vacuum machines to provide smoking force.
Microbiological and biochemical biofilm analyses. The dry weight and colony-forming units (CFUs) in the homogenized suspension were analyzed. Briefly, the 74-h-old biofilms on the sHA disc 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 7W for 30 s after adding 3 ml of 0.89% NaCl (VCX 130PB; Sonics and Materials, Inc., Newtown, CT, USA). For the determination of CFUs count, an aliquot (0.1 ml) of the homogenized solution (5 ml) was serially diluted, plated onto brain heart infusion (BHI; Difco, Detroit, MI, USA) agar plates, and then incubated under aerobic conditions at 37°C to determine the CFUs count18, 19.
For the determination of the dry weight and amount of water-insoluble extracellular polysaccharides (water-insoluble EPSs) (ASP), water-soluble extracellular polysaccharides (water-soluble EPSs) (WSP), intracellular polysaccharides (IPS), the remaining solution (4.9 ml) was centrifuged (3000 ×g) for 20 min at 4°C. The biofilm pellet was resuspended and washed twice in the same volume of water. Mix the water washed the biofilms pellet with 95% alcohol and put it in a refrigerator at -20°C for at least 18 hours to precipitate the water-soluble EPS. Then calculate the content of water-soluble EPS in the biofilms. The washed biofilms pellet was evenly divided into two portions, lyophilized, and weighed to determine the dry weight. One part used 1 N sodium hydroxide to extract water-insoluble EPS from the dried precipitate. The other part was used to calculate the content of intracellular polysaccharides, as detailed elsewhere20.
The final pH values of the old culture media were also determined during the experimental period using a glass electrode (Beckman Coulter Inc., Brea, CA, USA) to investigate the change in acidogenicity of S. mutans biofilms by the treatments.
Glycolytic pH drop assay. To evaluate the activity of CS against acid production of S. mutans biofilms, a glycolytic pH drop assay was performed. Briefly, the 74-h-old S. mutans biofilms, which had been incubated in 20 mM potassium phosphate buffer (pH 7.2) for 1 h to deplete endogenous catabolites. And then transferred to a salt solution (50 mM KCl plus 1 mM MgCl2, pH 7.0). The pH was adjusted to 7.2 with a 0.2 M KOH solution. Glucose was then added to the mixture to give a final concentration of 1% (w/v). The decrease in pH was assessed using a glass electrode over 120 min (Futura Micro Combination pH electrode, 5 mm diameter; Beckman Coulter Inc., CA, USA). The effect of CS on the acid production of the biofilm was determined according to the acid production rate, calculated by the change in pH values over the linear portion (0–20, 30, 120 min) of the pH drop curves21.
Confocal laser scanning microscopy analysis
Live and dead bacterial cells staining. Confocal laser scanning microscopy (CLSM) analysis was performed to confirm the results of microbiological and biochemical studies. To investigate the difference in bacterial cells, the 74-h-treated biofilms were stained at room temperature in the dark for 30 min using the Film Tracer LIVE/DEAD Biofilm viability kit L10316 (Invitrogen, Molecular Probes Inc., Eugene, OR, USA). The final concentrations of SYTO®9 and propidium iodide (PI) were 6.0 and 30 μM, respectively. This viability kit was based on plasma membrane integrity to determine live and dead cells. In this study, we regarded the cells with intact membranes (green) as live cells, whereas cells with damaged membranes (red) were regarded as the dead cells. The excitation/emission wavelengths were 480/500nm for SYTO®9 and 490/635nm for PI for collecting the fluorescence. The stained live and dead bacterial cells were observed with an LSM 510 META microscope (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium–neon lasers. All confocal fluorescence images were taken with an EC Plan-Neofuar 10x/0.30 M27 objective lens. A stack of slices in 6.4 μm step sizes was captured from the top to the bottom of the biofilms. The biovolume and thickness of live and dead cells were quantified from the entire stack using COMSTAT image-processing software. The biovolume is defined as the volume of the biomass (μm3) divided by the substratum (hydroxyapatite surface) area (μm2). The three-dimensional architecture of the biofilms was visualized using ZEN 2.3 (blue edition) (Carl Zeiss Microscopy GmbH, Jena, Germany). The original confocal data was uploaded to ZEN 2.3 software and the intensity of green and red fluorescence in the full thickness of biofilms layers were captured automatically. The software reconstructed the 2-dimensional intensity of fluorescence in all the layers to a 3-dimensional volume stack22.
EPS staining. The EPSs of 74-h-old biofilms were also investigated by simultaneous in situ labeling as described elsewhere23. Briefly, Alexa Fluor® 647-labeled dextran conjugate (1 μM, 10,000 MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) was added to the culture medium during the formation of S. mutans biofilms (at 0, 21, 31, 45, 55, 69 h) to label the newly formed EPSs. As described above, the stained EPSs were observed with an LSM 510 META microscope (Carl Zeiss, Jena, Germany) (objective: EC Plan Neofuar 10x/0.30 M27) equipped with argon-ion and helium-neon lasers and visualized using ZEN 2.3. A stack of slices in 7.8 μm step sizes was captured from the top to the bottom of the biofilms. Four independent experiments were performed, and five image stacks per experiment were collected. The EPSs biovolume and thickness were quantified from the confocal stacks using COMSTAT.
Biofilm density. The density of the 74-h-old S. mutans biofilms was calculated using the dry weight, which was derived from the biochemical study above, and the total biovolume of the biofilms (live cells + dead cells + EPSs), which was derived from the CLSM study above. The biofilm density (μg/μm3) is defined as the dry weight (μg/μm2) divided by the total biovolume of the biofilms (μm3/μm2)24.
Statistical Analysis. All experiments (except CLSM and SEM) were performed in duplicate, and at least six different experiments were conducted. The data are presented as mean ± standard deviation. Inter-group differences were estimated using one-way analysis of variance, followed by a post hoc multiple comparison (Tukey) test to compare multiple means (SPSS® software, IBM). Values were considered statistically significant when the p-value was < 0.05.