# Inhibitory Effect of Cold Atmospheric Plasma on Chronic Wound-Related Multispecies Biofilms
## 1 Introduction  
Chronic wounds, such as venous ulcers, diabetic foot ulcers, and pressure ulcers, are considered one of the most challenging problems in the medical area and affect millions of patients worldwide [1, 2]. The prevalence of chronic wounds is estimated in 2.21 per 1000 in the population [3] and these rates tend to rise due to the ageing population and increasing prevalence of obesity and associated conditions, such as diabetes and cardiovascular diseases [4]. This condition has significant economic impact on health systems [2, 5], with \(2\) to \(3\%\) of the budget in health applied in the treatment of chronic wounds in developed countries [1]. Microbial biofilms significantly affect wound healing and can contribute to therapeutic failures [6]. For these reasons, they have emerged as an important topic of discussion in the treatment of wounds [7]. The infections of wounds are usually polymicrobial, as their microenvironment is favorable for microbial growth [8; 9]. Despite this, previous evidence showed that species diversity in infected wounds is relatively low. It has been reported that _Staphylococcus aureus_, _Enterococcus faecalis_ and _Pseudomonas aeruginosa_ are the most prevalent species [4; 10], with occurrence of 93.5%, 71.7% and 52.2%, respectively [11]. Other bacterial species, such as _Proteus mirabilis_, _Klebsiella pneumoniae_ and _Escherichia coli_ were also isolated from wounds [12; 13]. The increasing rates of antimicrobial resistance also contributes to the unfavorable scenario [14; 15]. Thus, the control of the microbial biofilms is considered critical to the success of wound healing [7; 16]. The conventional therapeutic alternatives are focused on the reduction or eradication of biofilms by using debridement techniques and treatments with negative pressure or ultrasound [17; 18]. Other methods include the use of wound dressing impregnated with antimicrobial substances or compounds, such as gentian violet and methylene blue [15], honey [19], _Aloe vera_[20], nitric oxide releasing polymer [21], polyhexamethylenbiguanides [22] and ethylenediamine tetra acetic acid (EDTA) [23]. However, there is still need for alternatives to wound care, in particular, focusing on biofilm control [17; 21; 24]. Cold atmospheric plasma (CAP) has emerged as an attractive alternative approach to the biofilm control and is considered a rapid, environmental-friendly, energy saving, and versatile technology [25; 26]. Plasma is described as the fourth state of matter and is usually generated from a gaseous precursor in which a high electric field is applied, producing an electric discharge [27]. Plasma is a complex mixture composed of ions, reactive radicals, neutral particles, molecules and electrons, as well as excited species and energetic photons [28; 29]. These plasma components can damage cell structures, such as lipids, protein and DNA, and increase the levels of intracellular reactive species [30]. Thus, the biological activity of plasma is a product of synergetic action of several factors, such as neutral reactive species (oxygen reactive species--ROS, reactive nitrogen species--RNS) and UV radiation [31]. The anti-inflammatory and tissue repair inducing [32; 33] effects have been considered highly promising for the treatment of several diseases. The efficiency of CAP against the bacterial biofilms has been reported in the literature [34]. CAP was able to reduce the viability of monospecies biofilms of methicillin resistant _S. aureus_ (MRSA) [35], _P. aeruginosa_[36] and _E. faecalis_[37]. Monospecies biofilms formed by _E. faecalis_[38; 39] or _Escherichia coli_[40] were also inhibited by CAP. One of the main advantages of CAP when compared to conventional antibiotics is that the development of treatment resistance was not detected so far. Repeated exposure to CAP did not induce resistance in _S. aureus_[41].