2.1 Different biological properties of the clinical strains of S. mutans
As shown in Fig. 1A, the biomass of the clinical isolates was different between two groups. The amount of biomass in the SECC group was greater than that in the CF group, and the difference was statistically significant (t = 4.296, P < 0.001). As shown in Fig. 1B, the reduction of pH value was 1.868 ± 0.028 in SECC group and 1.772 ± 0.225 in CF group, there was no significant difference in the acid production ability between two groups(t = 1.272, P = 0.238). The aciduricity assay showed that no CFU can be counted on the BHI plates. It indicated that no bacteria survived in the SECC group and the CF group under the pH = 2.5 environment at 60 min.
2.2 The effect of curcumin on biofilm activity of S. mutans clinical strains
As shown in Fig. 2A, there was no significant effect of curcumin on the biofilm viability of the two groups after 5 minutes’ treatment. After long-term (24 hours) action of curcumin in Fig. 2B, the biofilm activity of the SECC group was reduced to 61.865 ± 7.108%, the difference was statistically significant (t = 10.731, P = 0.002). The biofilm viability of the CF group decreased to 84.059 ± 10.227%, the difference was statistically significant (t = 3.485, P = 0.025).
Figure 2C showed the net reduction of biofilm viability after 5 min and 24 h curcumin treatment. There was no significant difference between 5 min treatment. However, the biofilm viability (%) in the SECC group decreased by 38.135 ± 1.708%, and that in the CF group decreased by 15.941 ± 1.023% at 24 h. The difference between the two groups was statistically significant at 24 h. (t = 3.832, P = 0.007)
2.3 Effect of curcumin on the ration of live/dead bacteria in clinical strains of S. mutans
The images of CLSM were showed in Fig. 3. Green fluorescence represented live bacteria, red fluorescence represented dead bacteria, and yellow fluorescence was an overlap of live bacteria and dead bacteria. The image of one of the nine strains from each group was showed in Fig. 3A&3B. The green fluorescence density of the curcumin-treated group was lighter than that of the control group in both SECC group and CF group. The red fluorescence density of the curcumin-treated group was much darker in curcumin-treated group than that of the control group (Fig. 3A & 3B).
The results of all the strains were combined and showed in Fig. 3C&3D. At 5 minutes, the amount of live bacteria in the clinical strains of S. mutans decreased. The change was from (22.560 ± 1.736) µm3/µm2 to (14.999 ± 3.116) µm3/µm2 (t = 6.824, P < 0.001) in CF group while was from (25.460 ± 3.579) µm3/µm2 to (14.228 ± 3.237) µm3/µm2 (t = 7.726, P < 0.001) in SECC group. The total bacteria also showed the same trend. The CF group decreased from (42.841 ± 2.284) µm3/µm2 to (29.671 ± 4.197) µm3/µm2 (t = 7.509, P < 0.001) and the SECC group decreased from (48.007 ± 1.676) µm3/µm2 to (29.716 ± 4.101) µm3/µm2 (t = 10.304, P < 0.001). The net reduction of live bacteria and total bacteria in the SECC group was significantly higher than that of the CF group (live bacteria t = 3.017, P = 0.030; total bacteria t = 2.881, P = 0.045) (Fig. 3C).
After treated with curcumin for 24 h, the amount of live bacteria in the biofilm formed by the clinical strains of the CF group decreased from (18.906 ± 1.934) µm3/µm2 to (8.860 ± 1.192) µm3/µm2 (t = 17.129, P < 0.001). The amount of live bacteria in the biofilm formed by SECC group decreased from (20.684 ± 2.320) µm3/µm2 to (9.840 ± 2.274) µm3/µm2 (t = 12.927, P < 0.001). The amount of total bacteria also showed the descending trend. In CF group, it was decreased from (38.943 ± 2.615) µm3/µm2 to (21.005 ± 2.381) µm3/µm2 (t = 16.038, P < 0.001). In SECC group, it was decreased from (43.716 ± 3.812) µm3/µm2 to (20.839 ± 4.016) µm3/µm2 (t = 12.008, P < 0.001). The net reduction of live bacteria and total bacteria in the SECC group was significantly higher than that of the CF group (live bacteria t = 3.305, P = 0.016; total bacteria t = 2.378, P = 0.045) (Fig. 3D).
2.4 The effect of curcumin on the thickness of biofilm of S. mutans clinical strains
As shown in Fig. 3E, the short-term (5 minutes) effect of curcumin inhibited the biofilm thickness of the clinical strains from SECC group. The biofilm thickness formed by the SECC group decreased from (23.767 ± 2.656) µm to (15.844 ± 2.424) µm (t = 4.806, P = 0.001). There was no difference in the strains from CF group (t = 1.903, P = 0.084). But, the biofilm thickness formed by the CF group was reduced from (18.400 ± 1.229) µm to (11.500 ± 2.129) µm (t = 7.937, P < 0.001) after long-term (24 hours) effect of curcumin. SECC group decreased from (26.450 ± 3.984) to (12.075 ± 2.381) µm (t = 5.690, P < 0.001). The net reduction of biofilm thickness reduced significantly in short-term effect (t = 4.110, P = 0.015) and in long-term effect (t = 3.453, P = 0.014) (Fig. 3E).
2.5 Effect of curcumin on the formation of EPS of S. mutans biofilms formed by clinical strains
In Fig. 4A and 4B, the green color represented the total bacteria, the red color represented EPS, and the yellow color was an overlap of EPS and bacteria. It can be seen that after treating with curcumin for 5 minutes, no inhibitory effect on the biofilm EPS can be seen in both groups (Fig. 4C). However, long-term (24 h) curcumin treatment inhibited the amount of EPS in SECC group from (25.980 ± 1.156) µm3/µm2 to (20.136 ± 1.042) µm3/µm2, the difference was statistically significant (t = 7.510, P < 0.001). There was also significant inhibition on the biomass of EPS in the CF group (t = 4.082, P = 0.005) (Fig. 4D).
2.6 Effect of curcumin on biofilm formation related genes of clinical strains of S. mutans
As showed in Fig. 5, most gene expression of strains was downregulated compared to control groups. After treated by curcumin for 5 minutes, gtfC, gtfD, ftf, gbpB, fruA and srtA in CF group showed a decreasing trend, which down-regulated to 0.365-,0.501-,0.541-,0.82-,0.587-,0.408-fold. In SECC group, the gtfB, gtfC, gtfD, ftf, gbpB, srtA were respectively reduced by 0.840, 0.905, 0.641, 0.813, 0.816, 0.787 times. All the down-regulation in both group were statistically significant (Fig. 5A).
Curcumin treatment for 24 hours significantly inhibited the expression of genes related to the clinical strains of the SECC group and the CF group. Among them, gtfB, gtfC, gtfD, ftf, gbpB, fruA and srtA in the CF group showed a downward trend, which decreased by 0.526, 0.553, 0.549, 0.486, 0.507, 0.482, 0.530 times, all the differences were statistically significant. In the SECC group, the gtfB, gtfC, gtfD, ftf, gbpB, fruA, srtA down-regulated 0.530, 0.522, 0.671, 0.648, 0.674, 0.664, 0.570 times, the differences were statistically significant (Fig. 5B).