The inhibitory effects of polypyrrole on the biofilm formation of Streptococcus mutans

Abstract

Streptococcus mutans primary thrives on the biofilm formation on the tooth surface in sticky biofilms and under certain conditions can lead to carious lesions on the tooth surface. To search for a new preventive material for oral biofilm-associated diseases, including dental caries, we investigated the effects of polypyrrole, which contains an electrochemical polymer and causes protonation and incorporation of anion under low pH condition, on the biofilm formation of S. mutans and other streptococci. In this study, polypyrrole was applied in biofilm formation assays with the S. mutans strains UA159 and its gtfB and gtfC double mutant (gtfBC mutant), S. sanguinis, S. mitis and S. gordonii on human saliva and bovine serum albumin-coated 96-well microtiter plates in tryptic soy broth supplemented with 0.25% sucrose. The effects of polypyrrole on biofilm formation were quantitatively and qualitatively observed. High concentrations of polypyrrole significantly inhibited the biofilm formation of S. mutans UA159 and S. sanguinis. As an inhibition mechanism, polypyrrole attached to the surface of bacterial cells, increased chains and aggregates, and incorporated proteins involving GTF-I and GTF-SI produced by S. mutans. In contrast, the biofilm formation of gtfBC mutant, S. sanguinis, S. mitis and S. gordonii was temporarily induced by the addition of low polypyrrole concentrations on human saliva-coated plate but not on the uncoated and bovine serum albumin-coated plates. Moreover, biofilm formation depended on live cells and, likewise, specific interaction between cells and binding components in saliva. However, these biofilms were easily removed by increased frequency of water washing. In this regard, the physical and electrochemical properties in polypyrrole worked effectively in the removal of streptococci biofilms. Polypyrrole may have the potential to alter the development of biofilms associated with dental diseases.

Fig 1. The inhibitory effects of polypyrrole on the biofilm formation of S. mutans.

Effects of various concentrations of polypyrrole on the biofilm formation of S. mutans UA159 were observed on human saliva–coated 96-well microtiter plates after incubation for 16 h in TSB supplemented with 0.25% sucrose. A: Biofilm formation in S. mutans was present at high concentrations of polypyrrole (63, 125, 250 and 500 x 10 μg/ml) on human saliva–coated 96-well microtiter plates. B: Biofilm formation in S. mutans UA159 was quantitatively analyzed at high concentrations of polypyrrole. C: Biofilm formation in S. mutans UA159 and MT8148 was quantitatively analyzed at various concentrations of polypyrrole. D: Biofilm formation in the S. mutans strains GS-5 and MT8148, clinical isolates FSC-8, FSM-5, and FSC-7, and S. sobrinus AHT and OMZ176 were quantitatively analyzed at 2.5 mg/ml polypyrrole. The data indicated the mean ± SD of triplicate experiments. The independent experiments were performed 3 times, with similar results obtained in each. The asterisks indicated a significant difference between 2 groups (*: p < 0.05, no polypyrrole vs polypyrrole).

Fig 2. The effects of polypyrrole on the aggregation and production of insoluble glucan in S. mutans.

The effects of polypyrrole on the aggregation and production of insoluble glucan by S. mutans were observed after incubation for 16 h in TSB supplemented with 0.25% sucrose. A: Aggregations of S. mutans UA159 were observed at a high concentration of polypyrrole (2.5 mg/ml). B: Effects of polypyrrole (2.5 mg/ml) on the proteins produced by S. mutans UA159 in culture supernatant were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining, and western blotting. M: Molecular size marker, 1: culture supernatant experiment 1, 2: culture supernatant experiment 2, 3: culture supernatant experiment 1 + 2.5 mg/ ml polypyrrole, 4: culture supernatant experiment 2 + 2.5 mg/ ml polypyrrole, 5: 2.5 mg/ ml polypyrrole/PBS. C: The production of insoluble glucan by S. mutans UA159 was quantitatively analyzed at a high concentration of polypyrrole (2.5 mg/ml). The data indicated the mean ± SD of triplicate experiments. The independent experiments were performed 3 times, with similar results obtained in each. The asterisks indicated a statistically significant difference between 2 groups (Student’s t-test; p < 0.05, vs no treatment control).

Fig 3. Biofilm formation in S. mutans induced by polypyrrole.

Effects of polypyrrole on the biofilm formation of S. mutans were observed in the condition without glucan. Biofilm formation in gtfBC mutant was quantitatively assessed in conditions with various concentrations of polypyrrole on human saliva-coated (A), uncoated (B), BSA-coated (C), anti-PAc antiserum-coated (D), K4A (anti-PAc monoclonal antibody)-coated (E) and anti-GbpC antiserum-coated (F) 96-well microtiter plates in TSB supplemented with 0.25% sucrose. The data indicated the mean ± SD of triplicate experiments. The independent experiments were performed 3 times, with similar results obtained in each. The asterisks indicated a significant difference among multiple groups (ANOVA with Bonferroni correction; p-values < 0.05, various concentrations of polypyrrole vs no polypyrrole control).

Fig 4. Microscopic observation of biofilms treated with high concentrations of polypyrrole.

GtfBC mutant was incubated with 0 (control), 8, 16, 32 and 63 x 10 μg/ml polypyrrole and stained without (A) or with (B) a Gram staining kit. The aggregates were observed by microscopy. Squares were presented as an enlarged view. Representative data from more than three independent experiments were presented in the images.

Fig 5. Biofilm formation by S. sanguinis induced by polypyrrole.

The effects of polypyrrole on the biofilm formation of S. sanguinis were observed. Biofilm formation by S. sanguinis ATCC 10556 was quantitatively assessed with various concentrations of polypyrrole on human saliva-coated (A)-, uncoated (B), and BSA-coated (C) 96-well microtiter plates in TSB supplemented with 0.25% sucrose, including various concentrations of polypyrrole. (D) The growth of S. sanguinis was assessed by measuring the cell colonization numbers on BHI agar plates after 16 h of incubation in TSB supplemented with 0.25% sucrose, including various concentrations of polypyrrole. Small C indicated no polypyrrole as a control. The data indicated the mean ± SD of triplicate experiments. The independent experiments were performed 3 times, with similar results obtained in each. The asterisks indicated a statistically significant difference among multiple groups (ANOVA with Bonferroni correction; p-values < 0.05, various concentrations of polypyrrole vs no polypyrrole control).

Fig 6. Biofilm formation by S. gordonii and S. mitis induced by polypyrrole.

The effects of polypyrrole on the biofilm formation by S. gordonii and S. mitis were observed. Biofilm formation by S. gordonii ATCC 10558 (A and C) and S. mitis ATCC 6249 (B and D) was quantitatively assessed in various concentrations of polypyrrole on uncoated (A and B) and human saliva-coated (C and D) 96-well microtiter plates in TSB supplemented with 0.25% sucrose in various concentrations of polypyrrole. The data indicated the mean ± SD of triplicate experiments. The independent experiments were performed 3 times, with similar results obtained in each. The asterisks indicated a statistically significant difference among multiple groups (ANOVA with Bonferroni correction; p-values < 0.05, various concentrations of polypyrrole vs no polypyrrole control).

Fig 7. The effects of hyaluronic acid on polypyrrole-dependent biofilm formation.

The effects of hyaluronic acid on the polypyrrole-dependent biofilm formation of S. mutans were observed. A: Biofilm formation by gtfBC mutant was quantitatively assessed in various concentrations of polypyrrole on human saliva-coated 96-well microtiter plates in TSB supplemented with 0.25% sucrose and various concentrations of polypyrrole. B: Polypyrrole was preincubated with hyaluronic acid and applied in the biofilm formation assay with gtfBC mutant. The data indicated the mean ± SD of triplicate experiments. The independent experiments were performed 3 times, with similar results obtained in each. The asterisks indicated a statistically significant difference among multiple groups (ANOVA with Bonferroni correction; * p-values < 0.05, various concentrations of polypyrrole vs no polypyrrole control: Student’s t-test; **p-values < 0.05, only polypyrrole vs polypyrrole + hyaluronic acid at 1, 2 and 4 x 10, μg/ml). Biofilms at some concentrations of polypyrrole were stained by the LIVE/DEAD BacLight Viability Kit, observed by confocal microscopy and analyzed (Zen). Confocal images for the biofilm formation are indicated by arrows and presented in the top left image (live cells), top right image (dead cells) and bottom left image (merged live and dead cells). Representative data from more than 3 independent experiments were present in the images.