bacteria in biofilms can be up to a thousand times more resistant to antimicrobial challenge than the planktonic cells of the same species [16,17]. This recalcitrance to antimicrobial challenge can explain why biofilm-mediated infections often fail to respond to conventional antibiotic treatment regimens [14]. Many mecha- nisms of biofilm resistance to antimicrobial agents have been proposed; a first mechanism is the failure of antimicrobial agents to penetrate into the whole structure of the biofilm as a result of the barrier properties offered by the polymeric matrix [2,5,13]. Another mechanism explaining biofilm resistance is that a large number of microorganisms within a biofilm experience nutrient depletion which renders them slow-growing or metabolically inactive [2,13]. Most antimicrobials require at least some degree of cellular activity to be effective; since their mechanism of action usually relies on disrupting different microbial metabolic processes [5]. The third mechanism, of biofilm resistance, is related to the presence of subpopulations that exhibit distinct resistant pheno- types [2,5]. These subpopulations are frequently referred to as ‘‘persister cells’’ [18,19]. Pseudomonas aeruginosa is an opportunistic Gram negative pathogen. The ability of this pathogen to survive in multiple niches and to utilize many naturally occurring compounds as energy sources makes it one of the most ubiquitous bacteria in both the environment and the clinical setting, where it contam- inates the floor, bed rails, and sinks, and the skin of patients and healthcare personnel [20]. Unsurprisingly, in the European Prevalence of Infection in Intensive Care Study, this bacterium was found to be responsible for up to 28% of nosocomial infections in intensive care units [21]. The majority of these infections were found to affect immunocompromised patients or those having severe underlying diseases like cystic fibrosis and severe burns in addition to those who are in contact with contaminated medical devices [21]. Unfortunately, such nosocomial infections are frequently life threatening and often challenging to treat; especially with the frequent emergence of resistance to multiple drugs in this pathogen [22]. This resistance can emerge gradually during exposure to antipseudomonal antibiotics [23], this emergence was reported in 27–72% of patients with initially susceptible P. aeruginosa isolates and usually results in higher morbidity, mortality and economic burden [22]. As an antimicrobial strategy, atmospheric pressure non-thermal plasmas benefit from simple design, relatively low capital and operational cost [24], utilisation of non-toxic gases [25], operating at gas temperatures at or near room temperature [24–26], and the absence of harmful residues [24]. Importantly, these plasmas produce large quantities of microbicidal active agents [27]; which include a mixture of charged particles (positive and negative ion and electrons), chemically reactive species, UV radiation and electromagnetic fields [28]. The diversity and the small size of these active agents are believed to target multiple cellular components and metabolic processes in microorganisms and therefore make the emergence of resistance mechanisms less likely. The exact mechanisms driving plasma-mediated bacterial inactivation are not yet well understood [29,30]. However, several plasma products are believed to play a role in this process, these products include reactive oxygen species (ROS) [29–31], reactive nitrogen species (RNS) [31], ultraviolet radiation (UV) [30] and charged particles [30]. Among the ROS believed to be involved in this process are ozone, atomic oxygen, single delta oxygen, superoxide, peroxide, and hydroxyl radicals [30,32]. Although many of the aforementioned ROS have documented antibacterial activities through their interactions with different cellular compo- nents [30,33,34], it is highly important to consider the additive and synergistic effects of these species with each other’s and with other P. aeruginosa Biofilms Removal by APNTP plasma products like UV radiation and charged particles, in such a physically and chemically complex environment, before any successful conclusions can be drawn about the responsible cellular inactivation processes. The use of atmospheric pressure non-thermal plasmas has been evaluated for a number of biomedical applications, including: wound healing [26,35], blood coagulation [35,36], skin regener- ation [35], tooth bleaching [36], and apoptosis of cancer cells [35,36]. In addition, many atmospheric pressure plasma systems have been studied and proven to be effective in terms of microbial inactivation and even surface decontamination and sterilization [24,26,35–38]. However, most of these studies have been carried out using planktonic bacteria, which do not represent the entire spectrum of bacterial growth and survival in the natural as well as clinical settings. Whilst a number of studies have examined the effect of atmospheric pressure non-thermal plasmas on microbial biofilms [39–47], this study presents a comprehensive investigation of the activity of an in-house designed kHz-driven atmospheric pressure non-thermal plasma jet for the in vitro eradication of the clinically significant P. aeruginosa biofilms grown on inanimate surfaces. Multiple approaches were adopted to evaluate the bacterial cell viability prior to and after plasma treatment. These approaches are based on colony count method, XTT assay and LIVE/DEAD differential staining followed by confocal laser scanning microscopy. Furthermore, the effect of changing the plasma operating frequency on the anti-biofilm activity of the plasma jet was also investigated. Although the exact explanation for the difference in plasma activity upon changing the frequency was not provided in this manuscript since it needs further investigations, to our knowledge, this is the first research paper to date to report the effect of changing the frequency, within the same jet configuration, on biofilm eradication by plasma; which adds useful information about tailoring this approach to suit different biofilm-related applications. Materials and Methods Bacterial Strain(s) & Growth Conditions Pseudomonas aeruginosa PA01 (ATCC BAA-47, obtained from the American Type Culture Collection) was stored at 270uC in Microbank vials (Pro-Lab Diagnostics, Cheshire, UK) and was subcultured in Mu¨ ller-Hinton Broth (MHB) several times prior to conducting the microbiological assessments. Plasma Source A schematic diagram and a photograph of the plasma jet used in this study, as previously described in [43,48], are presented in Figure 1. It consists of a quartz tube with inner and outer diameters of 4 mm and 6 mm, respectively. Two copper electrodes (2 mm wide) encircle the tube, separated by 25 mm. For the experiments presented here the output of a high voltage pulse source (Haiden PHK-2k), operating at variable repetition frequencies, of between 20 and 40 kHz, and voltage amplitude 6 kV, is applied to the downstream electrode, which is 5 mm from the end of the plasma tube. The upstream electrode is grounded. The plasma jet was operated with a gas mixture of 0.5% oxygen and 99.5% helium, at a total flow rate of 2 Standard Litres per Minute (SLM) into ambient air. This plasma jet can be observed to generate an intense core plasma between the two electrodes and a luminous plume, which under the operating conditions discussed here, extends up to several centimetres beyond the exit of the tube. Spatially and temporally resolved images in the main plasma production region and plume regions confirm the presence of PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e44289