# Low-Temperature Plasma as an Approach for Inhibiting a Multi-Species Carigenic Biofilm
## 4 Discussion  
Dental caries can cause partial or total destruction of dental hard tissues by (acidic) by-products of biochemical processes in the fermentation of carbohydrates from the diet [15; 47]. It also relies on microorganisms and other host factors, such as dental surface [47]. Dental caries is considered a biofilm-dependent disease [48]. Dental biofilm is a highly organized polymicrobial structure embedded in an extracellular matrix formed by extracellular eposysaccharides (EPS) [49; 50]. In this study, different species that are commonly related to the formation of the cariogenic biofilm on the tooth surface were used. The results of this study show that multi-species cariogenic biofilm treated with LTP had a significant \(\log_{10}\) CFU/mL reduction in all plasma-treated samples compared to when exposed to the negative and positive controls. A previous study that treated _S. mutans_ and _S. sanguitiis_ dual-species biofilms formed on the bottom of polystyrene plates with atmospheric non-thermal argon/oxygen plasma for 120 s observed a reduction of 99% of viable cells when compared to their exposure to the negative control [51]. Our results corroborate this previous report because a significant reduction in the viability of a multi-species biofilm formed by _S. mutans_, _S. gordonii_, and _S. sanguitiis_ was detected after exposure to LTP for as low as 30 s.  
Argon gas was the choice for the plasma-generating device; the distance from the plasma outlet to the sample surface was 10 mm; and the biofilms were treated for 30 s, 60 s, and 120 s. [PERSON] et al. [52] analyzed the effect of a plasma-generating laboratory-developed device, which was different from the one used in our work, on free-floating planktonic cells. Bacterial cell suspensions containing \(10^{7}\) CFU/mL of either _S. mutans_, _Porhyromonas gingialis_, or _E. faecalis_ were treated. The treatment with LTP caused a significant reduction of more than 4 logs in the number of _S. mutans_ after 3 min of treatment, and almost all the bacteria were killed after 5 min. The gas used in their study was compressed air with a pulsed energy generator. The distances of 1 mm, 3 mm, and 5 mm were set between the plasma outlet and the single-species _S. mutans_ biofilm surface, and the samples were treated for 1, 3, 5, and 7 min [52]. Remarkably, in our study, in addition to using a greater distance from the output of the plasma device to the surface of the sample and less time of exposure, we treated a multi-species biofilm, which is known to be more resistant to anti-biofilm therapies than single-species biofilms [10]. Furthermore, there was no recovery for _S. gordonii_\(\log_{10}\) CFU/mL after treatment with LTP in all exposure time tested in this work for both single- and multi-species biofilms. _S. gordonii_ is one of the early stage colonizers of the oral cavity that can bind directly to salivary proteins on the tooth surface, establishing a basis for the construction of microbial communities of the cariogenic biofilm [53; 54]. The reduction of these microorganisms in the oral cavity can impair the formation of the cariogenic biofilm. Argon plasma produced by kINPen09 TM was also reported as a treatment option for biofilms formed by _S. sanguitiis_, where the biofilms were grown in a European biofilm reactor (EUREBI) as well as in 24-well plates [55]. For both biofilms formed in the different model systems, substrate for biofilm formation was the surface of titanium plates and the number of CFU were determined at 0, 24, 48, and 72 h (per exposure time). Using this model, the authors defined that, after 72 h, a mature biofilm was established. They concluded based on this study that EUREBI was the most effective system. Subsequently, biofilms were treated with a plasma jet for 180 s at 10 mm of distance between the top of the disc and the plasma source.