# Low-Temperature Plasma as an Approach for Inhibiting a Multi-Species Carigenic Biofilm
## 1 Introduction
mutans_ is not considered an early colonizer, it has unique mechanisms to metabolize carbohydrates, which results in acids and generate an adherent extracellular polysaccharide matrix involved in the progress of dental caries [29]. Another important fact to question is how we can improve the current protocols existing today in the treatment of dental caries. In this sense, minimally invasive dentistry (MID) has been presented as an alternative treatment for dental caries disease. MID aims to control or paralyze disease activity using non-operative (non-invasive) techniques that prevent unnecessary tissue loss and cause less damage to the dentin pulp complex [30; 31; 32]. Recently, plasma therapy has been suggested as a potential therapeutic tool in the treatment of dental caries due to its antimicrobial efficacy against cariogenic bacteria [33; 34]. Plasma is considered the fourth state of matter, consisting of a mixture of atoms, molecules, and ions at different densities and temperatures. It is conductive and responds to electric and magnetic fields [35]. Plasma is produced by applying an electric field to noble or molecular gases, such as argon, helium, oxygen, and nitrogen [36; 37; 38]. Cold plasmas that reach temperatures below 40 \({}^{\circ}\)C are known as biologically viable sources of energy [36; 38]. In addition, plasma can be generated under different pressures. Biologically applied cold plasmas are produced at atmospheric pressure and are named cold atmospheric pressure plasmas [39]. Among different types of plasmas, low-temperature plasma (LTP) is the most promising for biomedical applications. [PERSON] et al. [40] determined the effects of LTP on mono- and dual-species biofilms composed by _Candida albicans_ and _Staphylococcus aureus_. Biofilms were exposed to LTPfor 60 s, and all plasma-treated biofilms showed a significant reduction in comparison to the negative control. Furthermore, LTP showed low cytotoxicity based on a test with reconstituted oral epithelium [40]. [PERSON] et al. [41] reported that LTP reduced the viability of _C. albicans_ biofilms after 5 min of LTP exposure, with low cytotoxicity to Vero cells. In the same study, LTP was used to treat experimentally induced oral candidiasis and the histological analysis showed a remarkable decrease in candidial tissue invasion [41]. The effect of LTP on _Porphyromonas gingivalis_ biofilm grown on titanium discs was also reported [42]. After 1 min and 3 min of treatment, a significant reduction in _P. gingivalis_ viability was observed when compared to the negative control. Moreover, low toxicity to cells of the gingival epithelium was observed in the same study [42]. Another study demonstrated that _S. mutans_ sown on the agar plates can be inactivated by needle LTP [43]. Culture of _S. mutans_ that was sown on the agar surface was treated with plasma. After plasma application and incubation, a spatially resolved biological diagnosis was performed, and the pattern of the bacterial colonies present in the sample was photographed. The images of this biological diagnosis revealed that _S. mutans_ was killed in a solid circle of 5 mm diameter, showing that a precise treatment of the site is possible [43]. Experiments were conducted to test the sterilizing capability of LTP against oral microorganisms (_Streptococcus mutans_, _Candida albicans_, and _Enterococcus faecalis_) to translate its potential for clinical application by a direct exposure test on a solid surface, indirect exposure test in a liquid phase, and inhibitory test of reactive oxygen species (ROS) [44]. This study showed that the LTP jet inactivates oral microorganisms present in both solid and liquid phases and that ROS influenced this sterilization effect [44]. Thus, considering the effect of LTP on a different range of oral biofilms, and the potential therapeutic effect on oral diseases, the goal of this present study was to determine how LTP treatment at different exposure times affects single and multi-species cariogenic biofilms formed by _S. mutans_, _S. gordonii_, and _S.