Exploring the efficacy of in-vitro low-temperature plasma treatment on single and multispecies dental cariogenic biofilms

Abstract

The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log₁₀ in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log₁₀ at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms.

Keywords: Antibacterial; Dental caries; Low-temperature plasma; Oral biofilms.

Fig. 1

Mean values and standard deviations log₁₀ (CFU/mL) of single-species biofilms composed by C. albicans treated with LTP-argon 0.12% chlorhexidine digluconate (positive control) or 0.89% saline solution (negative control). *Values equal to zero. Different letters indicate significant statistical differences (n = 9; P ≤ 0.05; ANOVA, Tukey's Test). F30, F60, and F120 (argon gas–flow control treatment for 30, 60, and 120 s); P30, P60, and P120 (LTP-argon treatment for 30, 60, and 120 s), PC (positive control) and NC (negative control).

Fig. 2

Mean values and standard deviations log10 (CFU/mL) of single-species biofilms composed by L. casei treated with LTP-argon, 0.12% chlorhexidine digluconate (positive control) or 0.89% saline solution (negative control). *Values equal to zero. Different letters indicate significant statistical differences (n = 9; P ≤ 0.05; ANOVA, Tukey's Test). F30, F60, and F120 (argon gas–flow control treatment for 30, 60, and 120 s); P30, P60, and P120 (LTP-argon treatment for 30,60 and 120 s), PC (positive control) and NC (negative control).

Fig. 3

Mean values and standard deviations log10 (CFU/mL) of polymicrobial biofilms composed by Candida albicans, Lactobacillus casei, and Streptococcus mutans treated with LTP-argon, 0.12% chlorhexidine digluconate (positive control) or 0.89% saline solution (negative control). *Values equal to zero. Different letters indicate significant statistical differences (n = 9; P ≤ 0.05; ANOVA, Tukey's Test). F30, F60, and F120 (argon gas–flow control treatment for 30. 60, and 120 s); P30, P60, and P120 (LTP-argon treatment for 30, 60, and 120 s); PC (positive control) and NC (negative control).

Fig. 4

Confocal laser scanning microscopy of multispecies biofilm formed by Streptococcus mutans, Streptococcus gordonii, and Streptococcus sanguinis in the negative control group and LTP-argon (120 s) group; 25 × zoom. Biofilm stained with Live/Dead BacLight Viability kit. Live cells are in fluorescent green, and dead cells are in red. Live and Dead (live/dead) cells mixed in fluorescent green and red.