Resveratrol, Bacterial strain, and growth conditions
Resveratrol (Sigma-Aldrich, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) to a stock concentration of 80 mg/mL and diluted in medium to the appropriate concentrations for each experiment. The bacterial strain S. mutans UA159 was grown in brain-heart infusion broth (BHI; Oxoid) anaerobically (85% N2, 10% H2 and 5% CO2) at 37°C in this study. The concentration of DMSO used was up to 1% (800 μg/mL in the resveratrol group), BHI broth or saline solution with 1% DMSO but without resveratrol acted as vehicle control.
Growth curve assay and Minimum inhibitory concentration (MIC)
The effect of various concentrations of resveratrol on the growth of S. mutans was assessed by growth curve assay. Briefly, aliquots of overnight culture of S. mutans were diluted in BHI broth to the final concentration of 1 × 107 CFU/mL. Various concentrations of resveratrol (0, 50, 100, 200, 400, 800 μg/mL) was added into BHI broth and anaerobically inoculated at 37°C for 24 h. The bacterial growth was measured using spectrophotometer (UV-1750, Shimadzu, Japan) at OD600 nm every 3 hour throughout 24 h of incubation. The growth curve assays were repeated three times independently.
Glycolysis pH drop assay
The effect of resveratrol on S. mutans glycolysis pH drop was measured according to earlier methodology . Briefly, S. mutans was harvested at mid-logarithmic phase by centrifugation, washed with a salt solution (50 mM KCl + 1 mM MgCl2), and resuspended in the same salt solution containing different concentrations of resveratrol (0, 50, 100, 200, 400 μg/mL), The pH of the mixture was adjusted to 7.2 with 0.1 M KOH solution and glucose was added in the mixture to a final concentration of 1% (w/v). The decrease in pH by glycolytic activity of S. mutans UA159 was assessed at 10 min intervals over a period of 120 min. The experiments were repeated for three times independently.
Lactate dehydrogenase (LDH) assay
S. mutans cells were collected at late exponential phase and incubated at 37 °C in Tris-HCl buffer (pH 7.0) containing 0.5 mg/mL of lysozyme for 1 h . The lysate was then sonicated on ice for 2 cycles of 60 s each, and the cell-free supernatant was collected by centrifugation for 10min at 4 °C. The crude extract was further dialyzed at 4 °C overnight against 10 mM phosphate buffer (pH 6.9). The dialyzed preparation was defined as crude LDH, and its total protein concentration was measured by the Bradford method to normalize the enzyme activity.
For the LDH assay, crude LDH was pretreated with concentrations of resveratrol (0, 50, 100, 200, 400 μg/mL) at room temperature for 30 min. The reaction mixture (200 μl) contained 180 μl of 50 mM phosphate-buffered saline (pH 6.9) with 0.167 mM NADH and 10 mM sodium pyruvate;
10 μl of fructose 1,6-diphos- phate (final concentration of 1 mM); and 10 μl of pretreated LDH.
Results were expressed as enzymatic activity relative to that of the untreated control. The experiments were performed in triplicates independently.
Acid tolerance assay
The effect of resveratrol on the acid tolerance of S. mutans was evaluated by measurement of the viability of bacteria after 120 min of exposure at pH 5.0 . S. mutans was grown in BHI medium until reaching the mid-logarithmic phase. The cells were collected by centrifugation and resuspended (1×107 CFU/mL) in TYEG (10% tryptone, 5% yeast extract, 3% K2HPO4, and 1% glucose medium buffered with 40 mM phosphate-citrate buffer (pH 5.0) containing different concentration of resveratrol（0, 50, 100, 200, 400 μg/mL）. After incubation at 37 °C for 2 h, cells were serially diluted and plated on BHI agar plates for viable counts. The experiments were repeated for three times independently.
S. mutans cells was permeabilized by subjecting the cells to 10% toluene (v/v) followed by two freezing and thawing cycles according to the method described by Belli et al . The F-ATPase activity was evaluated in terms of inorganic phosphate release in the following reaction mixture: 75 mM of Tris-maleate buffer (pH 7.0) containing 5 mM ATP, 10 mM of MgCl2, permeabilized cells, and different concentrations of resveratrol (0, 50, 100, 200, 400 μg/mL). After 30 min of reaction, the released phosphate was determined using the method of Bencini et al . The experiments were repeated for three times independently.
The extracellular polysaccharide of S. mutans was extracted as previously described with minor modifications . S. mutans was incubated with different concentration of resveratrol (0, 50, 100, 200, 400 μg/mL) for 24 h at 37 °C. An equivalent reaction mixture without resveratrol was set as control. The reaction mixture was centrifugated at 10000 rpm for 10 min to separate water-soluble polysaccharide (part 1, supernatant) and water-insoluble polysaccharide (part 2, precipitate). All the supernatant (Part 1) was pooled and added three volumes of cold ethanol. After centrifugation at 4 °C, the supernatant was discarded, and the precipitate (water-soluble polysaccharide) was collected and washed by cold 75% ethanol. The water-soluble polysaccharides were measured using the phenol-sulfuric acid method (0.1% glucose was used for the standard curve). The precipitate (part 2) was dried for 3 h in a Speed Vac concentrator, and used for determination of water-insoluble polysaccharides. The water-insoluble polysaccharides were extracted using 1 M NaOH with agitation at room temperature for 2 h. The water-insoluble polysaccharides were also centrifuged, precipitated, washed and quantified as described above. The experiments were repeated for three times independently.
Crystal violet assay
Crystal violet assays was used to determine the effect of resveratrol on S. mutans biofilm formation in a 96-well microtiter plate . Briefly, overnight culture of S. mutans was added into BHI broth with different concentrations of resveratrol (0, 50, 100, 200, 400 μg/mL). After incubation at 37°C for 6 h, 24 h, the supernatants were removed and washed by sterile PBS three times. Biofilm was stained with 0.1 % (w/v) crystal violet for 5 min at room temperature. After washed by sterile PBS three times, 200 μl of 95 % ethanol was added to each crystal violet-stained well. Plates were shaken for 10 min, and biofilm formation was quantified by measuring optical density at 595 nm. The experiments were repeated for three times independently.
The S. mutans biofilms with different concentrations of resveratrol (0, 50, 100, 200, 400 μg/mL) were inoculated on glass slides in 6-well plates at 37 °C to observe its structure by confocal laser scanning microscopy (CLSM). After incubation for 24 h, the supernatants were removed, washed by sterile PBS three times and stained by the LIVE/DEAD BacLight™ Bacterial Viability Kit for 15 min in the dark according to the manufacture recommendation. This Kit contains SYTO 9 which dyed live cells with intact membranes green fluorescent and propidium iodide (PI) which dyed dead cells with damaged cell membrane red fluorescence. Three random fields of each sample were imaged on a Leica SP5 confocal laser scanning microscopy.
RNA isolation and real time PCR
To analyse the effect of resveratrol on virulence genes (ldh, relA, gtfC, comDE) expression, total RNA of S. mutans with different concentrations of resveratrol (0, 50, 100, 200, 400 μg/mL) was extracted by TRIzol reagent (Sigma-Aldrich). cDNA conversion of isolated RNA was done by a cDNA synthesis kit (Takara, Dalian, China) according to the manufacturer’s instructions. The real-time PCR was performed in Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems). All primers used are listed in Table 1. The reaction mixture contained SYBR Green PCR Master Mix (Takara), template cDNA and forward and reverse primers. The PCR conditions included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s. Relative mRNA expression were using the ΔΔCt method. Each experiment was performed with three independent RNA samples in triplicate.
Statistical analyses were performed using SPSS Statistics 20.0 (IBM, USA). The results for groups with or without resveratrol were statistically analyzed by one-way analysis of variance (ANOVA) with post hoc test. A P-values of < 0.05 were considered statistically significant.