Combinatorial effects of arginine and fluoride on oral bacteria

Abstract

Dental caries is closely associated with the microbial disequilibrium between acidogenic/aciduric pathogens and alkali-generating commensal residents within the dental plaque. Fluoride is a widely used anticaries agent, which promotes tooth hard-tissue remineralization and suppresses bacterial activities. Recent clinical trials have shown that oral hygiene products containing both fluoride and arginine possess a greater anticaries effect compared with those containing fluoride alone, indicating synergy between fluoride and arginine in caries management. Here, we hypothesize that arginine may augment the ecological benefit of fluoride by enriching alkali-generating bacteria in the plaque biofilm and thus synergizes with fluoride in controlling dental caries. Specifically, we assessed the combinatory effects of NaF/arginine on planktonic and biofilm cultures of Streptococcus mutans, Streptococcus sanguinis, and Porphyromonas gingivalis with checkerboard microdilution assays. The optimal NaF/arginine combinations were selected, and their combinatory effects on microbial composition were further examined in single-, dual-, and 3-species biofilm using bacterial species-specific fluorescence in situ hybridization and quantitative polymerase chain reaction. We found that arginine synergized with fluoride in suppressing acidogenic S. mutans in both planktonic and biofilm cultures. In addition, the NaF/arginine combination synergistically reduced S. mutans but enriched S. sanguinis within the multispecies biofilms. More importantly, the optimal combination of NaF/arginine maintained a "streptococcal pressure" against the potential growth of oral anaerobe P. gingivalis within the alkalized biofilm. Taken together, we conclude that the combinatory application of fluoride and arginine has a potential synergistic effect in maintaining a healthy oral microbial equilibrium and thus represents a promising ecological approach to caries management.

Keywords: Porphyromonas gingivalis; Streptococcus mutans; Streptococcus sanguis; biofilms; dental caries; drug synergism.

Figure 1.

The antimicrobial effects of NaF and arginine alone or in combination against the planktonic and biofilm cultures of S. mutans/S. sanguinis. (A, B) Checkerboard microdilution assays on S. mutans planktonic (A) and biofilm cultures (B), respectively. (C, D) Checkerboard microdilution assays on S. sanguinis planktonic (C) and biofilm cultures (D), respectively. The data (normalized by the mean of nonagent control) are reported as the mean of at least 3 separate tests. Black arrows indicate the minimum inhibitory concentrations (MICs) or minimum biofilm inhibitory concentrations (MBICs) of NaF and arginine alone, and the blue arrows indicate the MICs or MBICs in combination. The red dashed box indicates the NaF/arginine combinations that showed no significant inhibitory effect on the growth of S. sanguinis.

Figure 2.

Effects of NaF/arginine combinations on the S. mutans and S. sanguinis single-species biofilms. (A, B) Representative images of S. mutans biofilms treated with either high concentration (HC, A) or low concentration (LC, B) of NaF/arginine combinations. (C, D) Representative images of S. sanguinis biofilms treated with either HC (C) or LC (D) of NaF/arginine combinations. Green, bacteria (SYTO 9); red, extracellular polysaccharides (EPS; Alexa Fluor 647 [Life Technologies, Grand Island, NY, USA]). Confocal images were taken at 63× magnification. (E, F) The quantitative data of bacterial/EPS amount and pH values of spent media of either S. mutans (E) or S. sanguinis (F). Results were averaged from 3 separate experiments and are presented as mean ± standard deviation. Different lowercase letters indicate a significant intergroup difference of bacterial (green letters)/EPS (red letters) amount or pH values (black letters).

Figure 3.

Effects of NaF/arginine combinations on the S. mutans/S. sanguinis dual-species biofilms. (A, B) Representative confocal microscopic images of S. mutans/S. sanguinis dual-species biofilms treated with high concentration (HC, A) or low concentration (LC, B) of NaF/arginine combinations or each agent alone. S. mutans (S. m, green) and S. sanguinis (S. s, red) were labeled with species-specific fluorescent in situ hybridization probes. Image stacks were captured by confocal laser scanning microscope at 63× magnification and reconstructed using Imaris 7.0.0. (C, D) Quantitative data of bacterial composition of S. mutans/S. sanguinis dual-species biofilms treated with either HC (C) or LC (D) of NaF/arginine combinations. The microbial composition was quantified by species-specific quantitative polymerase chain reaction. Results were averaged from 3 separate experiments and are presented as mean ± standard deviation. Different lowercase letters indicate significant intergroup difference of S. mutans (blue letters) or S. sanguinis (red letters), respectively. ***Significant difference compared with nontreated controls (P < 0.001).

Figure 4.

pH values of spent media of the dual-species (A) or 3-species (B) biofilms treated with either high concentrations (HC) or low concentrations (LC) of NaF/arginine combinations. Results were averaged from 3 separate experiments and are presented as mean ± standard deviation. Different lowercase letters indicate significant intergroup differences of pH values.

Figure 5.

Effects NaF/arginine combinations on the S. mutans/S. sanguinis/P. gingivalis 3-species biofilms. (A–C) Representative images of 3-species biofilms treated with phosphate-buffered saline (A), high concentration (HC, B) or low concentration (LC, C) of NaF/arginine combinations. S. sanguinis (S. s, red), S. mutans (S. m, green), and P. gingivalis (P. g, blue) were labeled with species-specific fluorescent in situ hybridization (FISH) probes. Images were captured by fluorescence microscope at 60× magnification. (D) Quantitative data of bacterial composition based on the integral optical density of FISH-labeled biofilms. Results were averaged from 3 separate experiments and are presented as mean ± standard deviation. Different lowercase letters indicate significant intergroup differences of S. mutans (green letters), S. sanguinis (red letters), or P. gingivalis (blue letters), respectively.