Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma Mahmoud Y. Alkawareek1, Qais Th. Algwari2,4, Garry Laverty1, Sean P. Gorman 1 , William G. Graham2, Deborah O’Connell2,3, Brendan F. Gilmore1* 1 School of Pharmacy, Queen’s University of Belfast, Belfast, United Kingdom, 2 Centre for Plasma Physics, Queen’s University of Belfast, Belfast, United Kingdom, 3 York Plasma Institute, Department of Physics, University of York, York, United Kingdom, 4 Department of Electronics, College of Electronic Engineering, University of Mosul, Mosul, Iraq Abstract Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self- produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (,109s s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity. Citation: Alkawareek MY, Algwari QT, Laverty G, Gorman SP, Graham WG, et al. (2012) Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma. PLoS ONE 7(8): e44289. doi:10.1371/journal.pone.0044289 Editor: Jamunarani Vadivelu, University of Malaya, Malaysia Received April 17, 2012; Accepted August 1, 2012; Published August 31, 2012 Copyright: 2012 Alkawareek et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors would like to thank The University of Jordan for supporting Mahmoud Alkawareek in his PhD study, as well as EPSRC for supporting Deborah O’Connell through a Career Acceleration Fellowship (Grant No. EP/H003797/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: b.gilmore@qub.ac.uk Introduction Microbial biofilms are organised, multicellular communities held together by a self-produced matrix forming architecturally complex structures [1–3]. Biofilms are ubiquitous in both natural and pathogenic ecosystems [4] and may be present on almost every biotic or abiotic surface or interface [2]. The process of biofilm formation begins with cellular attachment to a substratum, and formation of microcolonies on the surface, which become embedded within a self-produced extracellular, hydrated matrix and which subsequently differentiate into a mature biofilm, These exhibit architecturally defined, complex three-dimensional struc- tures and a functionally heterogeneous bacterial population [4,5]. Dispersal mechanisms facilitate colonisation of new niches [2], including repopulation of surfaces following sub-lethal antimicro- bial challenges. Biofilms are estimated to be implicated in around 80% of all chronic human infections [6] and are important mediators of healthcare-associated infections [7], around half of which are related to the use of an indwelling medical device [8]. In 2008, more than 4 million patients acquired healthcare-associated infections in European hospitals; which resulted in about 37000 deaths as a direct consequence [7]. In the US, the number of such infections was more than 1.7 million in 2002 with almost 100,000 associated deaths [9]. Infections related to medical devices were the first clinical infections to be identified as having biofilm aetiology [5,10]. With many millions of medical devices being used each year [11], biofilms constitute a significant public health risk for patients requiring such devices [12]. Among these devices are: intravenous catheters, prosthetic heart valves, joint prostheses, peritoneal dialysis catheters, cardiac pacemakers, cerebrospinal fluid shunts, urethral catheters, urinary stents and endotracheal tubes, which all have an intrinsic risk of surface-associated infections [5]. Biofilms have also been associated with many other conditions, on biotic surfaces, including dental plaque, upper respiratory infections, peritonitis, and urogenital infections [11]. Biofilms constitute a protected mode of bacterial growth [2] and bacteria within biofilms typically exhibit significantly enhanced tolerance to antimicrobial challenge and host defences, compared to planktonic bacteria of the same species [2,5,13–15]. Indeed, PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e44289