In this study, we worked with three different species of microorganisms commonly related to the composition of cariogenic biofilm on the tooth surface. Here, we were able to show that the cariogenic biofilm formed by the association of these microorganisms was significantly reduced after treatment in all plasma-treated samples in comparison to both negative and positive controls.
Our results indicate that LTP treatment reduced the viability of C. albicans by 1.95 log10 CFU/mL on the multispecies biofilm after 120 s of treatment compared to negative control. This is corroborated by a previous study, where the authors used LTP-helium at a 1.5 cm distance from the plasma tip to the sample. They observed significant viable C. albicans cell reduction. Approximately 2-log reduction was observed after 7.5 min of exposure. [37]. Another study that also used an LTP-helium analyzed the capacity of the LTP-helium to disrupt the biofilm matrix, the cellular structure, and the C. albicans viability after exposure by applying 60 s of treatment and 10 mm of distance from the plasma tip to the biofilm surface. The results indicated that after treatment, significant log10 CFU/mL reductions were observed by changing the microorganism morphology compared to controls [38].
Ebrahimi-Shaghaghi et al. (2021) evaluated the effects of LTP-helium/O2 (2%) on the growth of C. albicans, submitted to 90, 120, 150, 180, and 210 s of treatment. The percentage of biofilm inhibition was 31.43% after 90 s of exposure and reached 41.15% after 120 s of treatment. [39]. Similar trends were observed in the present work both for monospecies and multispecies biofilms; as the dosage of LTP increased, a more significant reduction of the biofilm was observed.
This study also significantly reduced L. casei in multispecies cariogenic biofilms. Another study analyzed the action of the LTP-argon brush on monospecies biofilms formed by Lactobacillus and Streptococcus on hydroxyapatite discs. Three, 9, 13, 15 and 18 s of treatment were applied. Initially, the biofilm was created by a lower concentration of inoculum (between 2.1×108 and 2.4×108 CFU/mL). After treatments, the authors observed that applying the plasma brush for just 13 s was necessary to reduce the viability of the biofilms to the point that they could not be recovered. However, when biofilms were formed at a higher concentration (between 1.7×1010 and 3.5×1010 CFU/mL), the biofilm showed greater resistance to LTP-argon and the reductions were between 1.5 and 2. 5 logs for both biofilms, respectively [40]. This study may help to understand better how LTP and its generated reactive oxygen species can act to reduce the viability of biofilms of Lactobacillus and S. mutans, two sugar metabolizers that are important in the process of caries evolution, and also to understand better how single jet that can only treat a specific point can act in comparison to LTP-brush that produces more quantity reactive species.
In a previous study carried out by our team, we analyzed the action of LTP-argon using the same plasma device commercially obtained, as well as the same parameters [29]; the argon gas flow was also used, and the effect of LTP-argon jet was analyzed on single and multispecies biofilms formed by S. mutans, Streptococcus sanguinis, and Streptococcus gordonii on top the same hydroxyapatite disk. After treatment with LTP-argon per 30 s, 60 s, and 120 s, we observed a significant reduction in viability (log10 CFU/mL) for S. mutans on single and muti-species biofilm [29]. This study obtained similar results for S. mutans on multi-species biofilm formed by C.albicans, L. casei and S. mutans. We observed a significant reduction in viability (log10 CFU/mL) for all plasma-treated samples. This allowed us to understand better the action of LTP-argon on different associations of cariogenic microorganisms present in the biofilm, especially the activity of LTP-argon on S. mutans.
Qing H et al. (2016) investigated the effect of LTP-argon brush treatment on the biofilm of S. mutans nomo-species [41] by applying a 6 mm distance from the plasma tip to the sample surface. Biofilms were treated for 1, 2, and 5 min. After just 1 minute of treatment, results show 90% biofilm reduction. Our results were important for better understanding the LTP jet since the plasma brush can treat a larger surface, producing more species of oxygen, nitrogen, and other agents. The plasma jet can only treat a specific point, which leads to less production of these agents that cause the effect [42]. Nima G et al. (2021) evaluated the action of LTP against S. mutans biofilms formed on sterile resin discs under anaerobic conditions. The plasma jet was applied for 30, 90, 120, and 150 s. After CFU analysis, a significant reduction was proven at all times of treatment, using jets of LTP formed by a device similar to a brush [43]. Yang et al. (2011) observed the effectiveness of the LTP device in reducing the viability of biofilms formed by S. mutans and Lactobacillus acidophilus. The authors conclude in this study that the complete elimination of S. mutans took only 15 seconds and 5 minutes to eradicate L. acidophilus [44].
Using 0.12% chlorhexidine as a positive control was based on knowing that chlorhexidine is a gold-standard chemical substance with an antibacterial action against gram-positive and gram-negative bacteria [45]. In this study, treatment with 0.12% chlorhexidine reduced C. albicans, L. casei, and S. mutans log10 CFU/mL amount according to the exposure.
There is an urgency to develop alternative methods to contain the exponential growth of the microbial population comprised in biofilm without increasing the burden of bacterial resistance. Because it is safe, effective, non-toxic, and resistance-free [46], LTP is an optimal alternative for the microbial resistance response.
Plasmas are produced at various pressures, generally close to atmospheric pressure by high electric field intensities, which have electromagnetic radiation, ultraviolet radiation, and light in the visible spectrum, free radicals, free electrons, neutral reactive oxygen species (ROS – O, O2●-, O3, OH), and nitrogen (RNS – N, N2*, NO, NO2) that play a synergistic role in antimicrobial action [47]. LTPs are produced at the temperature of heavy species (neutral ions), which is much lower than the electron temperature and can provide energetic fluxes of ions to the substrates. This gaseous reaction at low temperatures is the main reason for using LTPs for biological interests [47]. Due to these fundamental characteristics, scientists began to innovate to update existing dentistry protocols.
This study showed significant and promising results in areas related to biofilm, specifically cariogenic biofilms. Studies also demonstrate the effect of LTP in keratinocytes, gingival fibroblasts, and reconstituted tissue [48, 38]. Future studies will be necessary to analyze the action of LTP on biofilms formed in situ. The data generated in the present work may contribute to developing LTP equipment with specific parameters for treating dental caries and developing new techniques based on minimal intervention dentistry and other forms of treatment.
Low-temperature plasma showed a significant antibiofilm effect against single and multispecies biofilms formed by C. albicans, L. casei, and S. mutans. The association of microorganisms to perform the biofilm analyzed in this work was based on studies that indicated the microorganisms that metabolize acids and are critical for colonizing the dental surface. C. albicans and L. casei were inactivated by LTP treatment in short exposure times. CFU/mL of all microorganisms studied were significantly reduced in single and multispecies biofilms. In conclusion, all tested exposure times with LTP-argon greatly affected cariogenic biofilms of single and multiple species in short exposure times. LTP-argon may be a promising therapy, capable of reducing the viability of microorganisms present in a cariogenic biofilm, thus contributing to protocols for treating and controlling dental caries in children and adults.