# Low-Temperature Plasma as an Approach for Inhibiting a Multi-Species Carigenic Biofilm
## 4 Discussion
The work showed a significant reduction (0.6 \(\log_{10}\) CFU/mL or 0.5 \(\log_{10}\) CFU/mL) in biofilms compared to the control in either the EUREBI produced biofilm model or in 24-well plates [55]. The methodology we used uses hydroxyapatite discs as the substrate for biofilm formation, which closely represents the tooth mineral. Our results indicate that, for the treatment of multi-species biofilms formed on a hydroxyapatite disc surface, and treated with LTP for 30 s, 60 s, and 120 s at a 10 mm distance from the tip of the plasma to the sample, _S. sanguitiis_ was not recovered. For single-species biofilms formed by the same microorganism, there was CFU recovery of _S. sanguitiis_ afterLTP treatment with significant CFU reduction observed in all tested exposure times when compared to their exposure to the positive and negative controls. _S. sanguinis_ is typically associated with the formation of the cariogenic biofilm in the oral cavity [56; 57]. Like _S. gordonii_, _S. sanguinis_ is a pioneer colonizer that is closely related to the further binding of other microorganisms on the tooth surface and subsequent development of a mature cariogenic biofilm [25; 58]. Chlorexidine is a gold-standard antiseptic [59] that was chosen as a positive control for the present study. However, a single treatment with 0.12% chlorexidine, as expected, is not able to reduce the CFU amount when compared to the negative control on both single- and multi-species biofilms. However, as a mouthwash, 0.12% chlorexidine is used twice daily as an anti-plaque agent for oral infections due to its effectiveness in reducing bacteria present in the saliva and on the tongue [60; 61]. The low efficacy of a single treatment of chlorexidine on mature biofilms [62; 63] is associated with the resistance that a well-established and mature biofilm presents, which protects the microorganisms against desiccation, oxidizing, or charged biocides, some antibiotics, and metallic cations, ultraviolet radiation, some protozoan grazers, and host immune defenses [64]. Plasmas produce electromagnetic radiation, such as ultra-violet radiation and light in the visible spectrum, and comprise excited gas particles, charged ions, free electrons, free radicals, neutral reactive oxygen (ROS) and nitrogen species (RNS), and molecule fragments. LTP-reactive species are rapidly produced and are good sources of reactive oxygen and nitrogen species including singlet oxygen, ozone, hydroxyl radicals, nitrous oxide, and nitrogen dioxide [65]. The antibacterial effects of LTP mediated by ROS have not been demonstrated to increase bacterial resistance when bacteria are continuously exposed to it. Plasmas can reach good gas-phase chemistry deprived of high gas temperatures. This happens because plasmas display much higher electron energies than that of the ions and the neutral species. The energetic electrons collide with the background gas, producing higher dissociation, excitation, and ionization levels. Since ions and neutrals continue to be quite cold, as there is no contact thermal damage caused by the plasma [65]. The unique characteristics of LTP have opened a new era in dental care. Even though this study has limitations, such as the lack of analysis of the action of LTP in biofilm formed in situ and the absence of more complex cytotoxicity tests, it has shown promising results in oral biofilm-related disease. Dental treatments with LTP are painless and drill-less, thus making them patient-friendly. These findings indicate that LTP is potentially a good candidate for future clinical treatments [65].