# Inhibitory Effect of Cold Atmospheric Plasma on Chronic Wound-Related Multispecies Biofilms
## 4 Discussion
The study of mixed-species biofilms is relevant as, clinically, they can cause persistent infections and usually lead to worse infections [60, 61, 62]. The model of polymicrobial biofilm obtained on a porcine collagen membrane gave strong evidences that CAP can reduce the bacterial biofilm viability in complex models. The model described by [PERSON] [8] for wound-related biofilms was selected because it mimics the conditions observed in wounds, by using a culture medium with a constitution close to the exudate and a nutrient flow diffusion from the bottom up. Besides, the model also mimics the air-wound-liquid interface that occurs in vivo [8]. The selection of the microbial species used to form the multispecies biofilms were based on their relevance in the context of chronic wounds infection. Previous studies reported that _S. aureus_ and _P. aeruginosa_ were the most prevalent species in wounds [4, 11]. Besides, the presence of certain species, such as _E. faecalis_, seems to be related to worse clinical course of the wound [4, 10, 63]. In this study, _P. aeruginosa_ was more susceptible to CAP in a multispecies biofilm, with reduction of 1.48 log. It is an unexpected result since previous report suggested that _P. aeruginosa_ is one of the most benefited species in a mixed species culture, probably because it is present in a more protected niche of the biofilm [64]. It is known that CAP generates large variety of reactive species that can cause decontamination. Among them, ROS (e.g., O\({}_{3}\), O\({}_{2}\)-, OH, O) are indicated as the major agent responsible for bactericidal effects of plasmas [65]. In plasma jets, the generated plasma plume excites and ionizes air molecules present in the surrounding ambient leading to formation of RONS [66]. The concentration of generated reactive species can be enhanced by adding molecular gases (e.g., air, O\({}_{2}\) or N\({}_{2}\)) to the main carrier gas or applying a shielding gas curtain around the plasma plume [67]. In plasma jets, the produced reactive species are driven by the gas flow to the sample allowing short and long-living species to act on the treated surface, which can increase the treatment efficiency when compared to indirect methods. The plasma jet device used in the present study was operated with commercial low-grade purity helium that contains few impurities (mostly air) and ROS formation in the plasma plume was detected in previous studies [47, 48]. Thus, the MRSA biofilm was significantly more susceptible than _P. aeruginosa_ and _E. faecalis_ under ROS-dominated conditions. Interestingly, the increased susceptibility of MRSA in a polymicrobial biofilm with _P. aeruginosa_, _E. faecalis and Klebsiella pneumoniae_, to RNS-dominated conditions were previously reported [PERSON] et al. [31]. Our results showed that exposure to CAP for 1, 3, 5 and 7 min was not cytotoxic for fibroblasts 3T3 cells analyzed immediately after the exposure. Similarly, [PERSON] et al. [45] found the cell viability to be higher than 80% immediately and 24 h after amplitude modulated cold atmospheric pressure plasma jet (AM-CAPP) exposure of Vero cells for 3 and 5 min. On the other hand, [PERSON] et al. [45] showed that cell viability was maintained at around 80%, 48 h after AM-CAPPJ exposure, which was different from the present work where the cell viability has decreased to around 60% after 48 h. This could be explained by the internal cell signaling cascade that some reactive species may trigger as previously described [30, 68, 69]. Therefore, a potential cytotoxicity may be related to this lower cell viability after 48 h (late effects) in relation to the immediate time or 24 h, which could have side effects on cells. However, further chronic toxicity studies of CAP should be carried out. These late effects on fibroblasts could also exist for microorganisms and should be investigated in future studies. Aiming the clinical application of CAP for wound care, it was extremally important to investigate whether plasma conditions would lead to DNA damage or not.