Antimicrobial Activity of Cinnamaldehyde on Streptococcus mutans Biofilms

Abstract

Streptococcus mutans is considered the most relevant bacteria in the transition of non-pathogenic commensal oral microbiota to biofilms which contribute to the dental caries process. The present study aimed to evaluate the antimicrobial activity of a natural plant product, cinnamaldehyde against S. mutans biofilms. Minimum inhibitory concentrations (MIC), minimal bactericidal concentration (MBC), and growth curves were determined to assess its antimicrobial effect against planktonic S. mutans. The biofilm biomass and metabolism with different concentrations of cinnamaldehyde and different incubation time points were assessed using the crystal violet and MTT assays. The biofilms were visualized using confocal laser scanning microscopy (CLSM). Bacterial cell surface hydrophobicity, aggregation, acid production, and acid tolerance were evaluated after cinnamaldehyde treatment. The gene expression of virulence-related factors (gtfB, gtfC, gtfD, gbpB, comDE, vicR, ciaH, ldh and relA) was investigated by real-time PCR. The MIC and MBC of cinnamaldehyde against planktonic S. mutans were 1000 and 2000 μg/mL, respectively. The results showed that cinnamaldehyde can decrease biofilm biomass and metabolism at sub-MIC concentrations. CLSM images revealed that the biofilm-covered surface areas decreased with increasing concentrations of cinnamaldehyde. Cinnamaldehyde increased cell surface hydrophobicity, reduced S. mutans aggregation, inhibited acid production, and acid tolerance. Genes expressions in the biofilms were down-regulated in the presence of cinnamaldehyde. Therefore, our data demonstrated that cinnamaldehyde at sub-MIC level suppressed the microbial activity on S. mutans biofilm by modulating hydrophobicity, aggregation, acid production, acid tolerance, and virulence gene expression.

Keywords: Streptococcus mutans; antimicrobial activity; biofilm; cinnamaldehyde; dental caries; virulence.

FIGURE 1

Antibacterial Activity of Cinnamaldehyde. (A) MIC and MBC values of cinnamaldehyde against planktonic S. mutans. (B) The number of colonies on BHI agar. (C) Growth curve of planktonic S. mutans with different concentrations of cinnamaldehyde. ^∗P < 0.05, ^∗∗P < 0.01, significantly different from the control group.

FIGURE 2

Effect of cinnamaldehyde on overall biomass and metabolic activity of single-species and dual-species biofilm formation at 4 and 24 h time points. (A) overall biomass of S. mutans biofilm (B) metabolic activity of S. mutans biofilm (C) overall biomass of S. mutans and S. sanguinis biofilm (D) metabolic activity of S. mutans and S. sanguinis biofilm. ^∗P < 0.05, ^∗∗P < 0.01, significantly different from the control group at 4 and 24 h time points.

FIGURE 3

Confocal laser scanning micrographs of biofilms with different concentrations of cinnamaldehyde. (A) Control; (B) 125 μg/mL; (C) 250 μg/mL; (D) 500 μg/mL. Red, non-viable cells; green, viable cells; yellow, overlap of non-viable and viable cells. Bar = 50 μm.

FIGURE 4

Effect of cinnamaldehyde on hydrophobicity (A) and aggregation (B) of S. mutans.*P < 0.05, significantly different from the control group.

FIGURE 5

Effect of cinnamaldehyde on acid production (A) and acid tolerance (B) of S. mutans.*P < 0.05, significantly different from the control group.

FIGURE 6

Effect of cinnamaldehyde on gene expression of S. mutans biofilms. The results represent the means and SD of three independent experiments performed in triplicate. ^∗P < 0.05, ^∗∗P < 0.01, significantly different from the control group.