# Inhibitory Effect of Cold Atmospheric Plasma on Chronic Wound-Related Multispecies Biofilms
## 4 Discussion  
The outcomes of this study showed that He-cold atmospheric plasma significantly reduced the viability of wound-related mono and multispecies biofilms. _S. aureus, P. aeruginosa_ and _E. faecalis_ are opportunistic pathogenic bacteria which cause chronic infections due a number of virulence factors and can interfere with wound healing process, in particular when strains resistant to antimicrobials are involved [55]. To the best of our knowledge, this is the first report on the activity of CAP on wound related multispecies biofilms, formed by MRSA, _P. aeruginosa and E. faecalis_, by using a model of wound-related biofilm. Although the main goal of this study was to evaluate the effect of cold atmospheric plasma in multispecies biofilms, experiments with monospecies biofilms were carried out in order to establish the operation parameters of the plasma device that generated antimicrobial activity against the tested microbial species. For this purpose and taking in account the cost of membrane used in the multispecies experiments, monospecies biofilms were grown in 96-well plates. We observed that the exposure to CAP for 5 min reduced in 3 log the viable cells of MRSA monospecieis biofilms. This result corroborates the findings reported by [PERSON] et al. [56], in which a plasma jet operating with 20 kV of voltage, frequency of 38 kHz, and He flow rate of 6.7 slm reduced in 3.6 log _S. aureus_ biofilms after 10 min exposure. CAPs generated using other working gases also showed similar inhibitory effects. [PERSON] et al. [41] observed a reduction of 1.7 log in MRSA biofilms after repeated 20 s exposure to Ar plasma during 6 h. Besides, [PERSON] et al. [57] observed a decrease of 4.67 \(\pm\) 0.29 log in the viability of _S. aureus_ biofilms after 10 min exposure to N\({}_{2}\) CAP. We also detected a significant reduction in the viability of _E. faecalis_ monospecies biofilm after 1 min exposure in the present study. The reduction varied from 1.2 to 1.9 log when compared to the untreated control group. This result is interesting, since _E. faecalis_ monospecies biofilms appear to be resistant to other therapies. In the case of _E. faecalis_ monospecies biofilm, it seems that other CAP generation setups lead to higher inhibitory effects when compared to He-CAP used in this study. [PERSON] et al. [37] observed 3 log reduction in _E. faecalis_ (ATCC 29212) biofilm viability after 5 min exposure to CAP generated by a surface micro-discharge reactor in open air operating with voltage of 3.5 kVp-p and a frequency of 4.0 kHz. It is important to highlight that in this study, differently than in ours, the treatment was applied indirectly (without direct contact between target and plasma) and the distribution of reactive species occurred by means of thermal convection and diffusion. Another study using CAP of Ar (98%) and O\({}_{2}\) (2%) observed 90% reduction in the viability of the 7-day biofilm of _E. faecalis_ formed within the dental root canals of human teeth after 8 min of exposure [58]. For _P. aeruginosa_ monospecies biofilms, we detected that He-CAP reduced in 2.4 log/CFU after 7 min of exposure. [PERSON] et al. [59] used a plasma jet with gas mixture (He/O\({}_{2}\)) with voltage of 6 kV, frequency of 40 kHz and gas flow rate of 2 slm for treatment of _P. aeruginosa_. Figure 5: Histogram of distribution of DNA indices (ID) obtained \(\times\) number of corresponding cells 4. _inosa_ and obtained a reduction of 4 log after treatment for 4 min. Besides, [PERSON] et al. [36] reported a reduction of 5 log in _P. aeruginosa_ counts after exposition for 90 s to microwave plasma jet (2.45 GHz) in air with flow rate of 5 slm, while we observed a reduction of 2.4 log after 7 min of exposure. However, it is important to mention that in both aforementioned studies the temperatures of CAP were above 40 \({}^{\circ}\)C, which could explain the higher reduction of the viable cells. After determining the optimal parameters for monospecies biofilms, the effect of CAP on multispecies biofilms was studied.