Because of its antimicrobial properties, nonthermal plasma could serve as an alternative to chemical antisepsis in wound treatment. Therefore, this study investigated the inactivation of biofilm-embedded Pseudomonas aeruginosa SG81 by a surface barrier-discharged (SBD) plasma for 30, 60, 150 and 300 s. In order to optimize the efficacy of the plasma, different carrier gases (argon, argon admixed with 1% oxygen, and argon with increased humidity up to approx. 80%) were tested and compared against 0.1% chlorhexidine digluconate (CHG) exposure for 600 s. The antimicrobial efficacy was determined by calculating the difference between the numbers of colony-forming units (CFU) of treated and untreated biofilms. Living bacteria were distinguished from dead by fluorescent staining and confocal laser scanning microscopy. Both SBD plasmas and CHG showed significant antimicrobial effects compared to the untreated control. However, plasma treatment led to a higher antimicrobial reduction (argon plasma 4.9 log10 CFU/cm2, argon with admixed oxygen 3 log10 CFU/cm2, and with increased gas humidity 2.7 log10 CFU/cm2 after 300 s) compared to CHG. In conclusion, SBD plasma is suitable as an alternative to CHG for inactivation of Pseudomonas aeruginosa embedded in biofilm. Further development of SBD plasma sources and research on the role of carrier gases and humidity may allow their clinical application for wound management in the future.

1.
Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318-1322.
2.
Burmølle M, Thomsen TR, Fazli M, Dige I, Christensen L, Homøe P, Tvede M, Nyvad B, Tolker-Nielsen T, Givskov M, Moser C, Kirketerp-Moller K, Johansen HK, Høiby N, Jensen PØ, Sørensen SJ, Bjarnsholt T: Biofilms in chronic infections - a matter of opportunity - monospecies biofilms in multispecies infections. FEMS Immunol Med Microbiol 2010;59:324-336.
3.
Nakagami G, Morohoshi T, Ikeda T, Ohta Y, Sagara H, Huang L, Nagase T, Sugama J, Sanada H: Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in pressure ulcer infection in rats. Wound Repair Regen 2011;19:214-222.
4.
Davis SC, Ricotti C, Cazzaniga A, Welsh E, Eaglstein WH, Mertz PM: Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo. Wound Repair Regen 2008;16:23-29.
5.
Percival SL, Bowler P, Woods EJ: Assessing the effect of an antimicrobial wound dressing on biofilms. Wound Repair Regen 2008;16:52-57.
6.
Bjarnsholt T, Kirketerp-Møller K, Jensen PØ, Madsen KG, Phipps R, Krogfelt K, Høiby N, Givskov M: Why chronic wounds will not heal: a novel hypothesis. Wound Repair Regen 2008;16:2-10.
7.
Bowler PG, Duerden BI, Armstrong DG: Wound microbiology and associated approaches to wound management. Clin Microbiol Rev 2001;14:244-269.
8.
McCarty SM, Cochrane CA, Clegg PD, Percival SL: The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing. Wound Repair Regen 2012;20:125-136.
9.
Jimenez PN, Koch G, Thompson JA, Xavier KB, Cool RH, Quax WJ: The multiple signaling systems regulating virulence in Pseudomonas aeruginosa. Microbiol Mol Biol Rev 2012;76:46-65.
10.
Eberlein T, Assadian O: Clinical use of polihexanide on acute and chronic wounds for antisepsis and decontamination. Skin Pharmacol Physiol 2010;23:45-51.
11.
Hübner NO, Kramer A: Review on the efficacy, safety and clinical applications of polyhexanide, a modern wound antiseptic. Skin Pharmacol Physiol 2010;23:17-27.
12.
Ammons CB: Anti-biofilm strategies and the need for innovations in wound care. Recent Pat Anti-Infect Drug Discovery 2010;5:10-17.
13.
Ermolaeva SA, Varfolomeev AF, Chernukha MY, Yurov DS, Vasiliev MM, Kaminskaya AA, Moisenovich MM, Romanova JM, Murashev AN, Selezneva II, Shimizu T, Sysolyatina EV, Shaginyan IA, Petrov OF, Mayevsky EI, Fortov VE, Morfill GE, Naroditsky BS, Gintsburg AL: Bactericidal effects of non-thermal argon plasma in vitro, in biofilms and in the animal model of infected wounds. J Med Microbiol 2011;60:75-83.
14.
Kong MG, Kroesen G, Morfill G, Nosenko T, Shimizu T, Dijk JV, Zimmermann JL: Plasma medicine: an introductory review. New J Phys 2009;11:115012.
15.
Laroussi M: Low temperature plasma-based sterilization: overview and state-of-the-art. Plasma Process Polym 2005;2:391-400.
16.
Nosenko T, Shimizu T, Morfill GE: Designing plasmas for chronic wound disinfection. New J Phys 2009;11:115013.
17.
Moshrefi A: Chlorhexidine. J West Soc Periodontol Periodontal Abstr 2002;50:5-9.
18.
Muangman P, Nitimonton S, Aramwit P: Comparative clinical study of Bactigras and Telfa AMD for skin graft donor-site dressing. Int J Mol Sci 2011;12:5031-5038.
19.
Hübner NO, Matthes R, Koban I, Rändler C, Müller G, Bender C, Kindel E, Kocher T, Kramer A: Efficacy of chlorhexidine, polihexanide and tissue-tolerable plasma against Pseudomonas aeruginosa biofilms grown on polystyrene and silicone materials. Skin Pharmacol Physiol 2010;23:28-34.
20.
Afilalo M, Dankoff J, Guttman A, Lloyd J: Duoderm hydroactive dressing versus silver sulphadiazine/Bactigras in the emergency treatment of partial skin thickness burns. Burns 1992;18:313-316.
21.
Wagner HE, Brandenburg R, Kozlov KV, Sonnenfeld A, Michel P, Behnke JF: The barrier discharge: basic properties and applications to surface treatment. Vacuum 2003;71:417-436.
22.
Kogelschatz U: Dielectric-barrier discharges: their history, discharge physics, and industrial applications. Plasma Chem Plasma Process 2003;23:1-46.
23.
Weltmann K-D, Fricke K, Stieber M, Brandenburg R, Woedtke TV, Schnabel U: New nonthermal atmospheric-pressure plasma sources for decontamination of human extremities. IEEE Trans Plasma Sci 2012;40:2963-2969.
24.
Weltmann KD, Kindel E, von Woedtke T, Hähnel M, Stieber M, Brandenburg R: Atmospheric-pressure plasma sources: prospective tools for plasma medicine. Pure Appl Chem 2010;82:1223-1237.
25.
ICNIRP: Guidelines on limits of exposure to ultraviolet radiation of wavelengths between 180 and 400 nm (incoherent optical radiation). Health Phys 2004;87:117-186.
26.
European Commission: Biological effects of ultraviolet radiation relevant to health with particular reference to sunbeds for cosmetic purposes. Brussels, Scientific Committee on Consumer Products Report, 2006, No 0949/05.
27.
Grobe S, Wingender J, Trüper HG: Characterization of mucoid Pseudomonas aeruginosa strains isolated from technical water systems. J Appl Microbiol 1995;79:94-102.
28.
Matthes R, Hübner N, Bender C, Koban I, Weltmann K, Kramer A: Screening test for quality control of surface barrier discharged plasma sources with the microorganism-agar test (MAT). GMS Krankenhhyg Interdiszip 2010;5:DOC02.
29.
Müller G, Kramer A: Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. J Antimicrob Chemother 2008;61:1281-1287.
30.
DIN EN 1040: Chemical disinfectants and antiseptics - quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics - test method and requirements (phase 1), German version EN 1040, 2005. Berlin, Beuth, 2006.
31.
Boulos L, Prevost M, Barbeau B, Coallier J, Desjardins R: Live/dead® Baclight™: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Methods 1999;37:77-86.
32.
Hseu YC, Chou CW, Senthil Kumar KJ, Fu KT, Wang HM, Hsu LS, Kuo YH, Wu CR, Chen SC, Yang HL: Ellagic acid protects human keratinocyte (HaCaT) cells against UVA-induced oxidative stress and apoptosis through the upregulation of the HO-1 and Nrf-2 antioxidant genes. Food Chem Toxicol 2012;50:1245-1255.
33.
Nasti TH, Timares L: Inflammasome activation of IL-1 family mediators in response to cutaneous photodamage. Photochem Photobiol 2012;88:1111-1125.
34.
Lin F, Xu W, Guan C, Zhou M, Hong W, Fu L, Liu D, Xu A: Niacin protects against UVB radiation-induced apoptosis in cultured human skin keratinocytes. Int J Mol Med 2012;29:593-600.
35.
Haywood R, Wardman P, Sanders R, Linge C: Sunscreens inadequately protect against ultraviolet-A-induced free radicals in skin: implications for skin aging and melanoma? J Invest Dermatol 2003;121:862-868.
36.
Isbary G, Heinlin J, Shimizu T, Zimmermann JL, Morfill G, Schmidt HU, Monetti R, Steffes B, Bunk W, Li Y, Klaempfl T, Karrer S, Landthaler M, Stolz W: Successful and safe use of 2 min cold atmospheric argon plasma in chronic wounds: results of a randomized controlled trial. Br J Dermatol 2012;167:404-410.
37.
Rändler C, Matthes R, McBain AJ, Giese B, Fraunholz M, Sietmann R, Kohlmann T, Hübner N-O, Kramer A: A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses. BMC Microbiol 2010;10:282-212.
38.
Becker K: Exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions. Microb Ecol 1996;32:23-33.
39.
Koban I, Holtfreter B, Hübner N-O, Matthes R, Sietmann R, Kindel E, Weltmann K-D, Welk A, Kramer A, Kocher T: Antimicrobial efficacy of non-thermal plasma in comparison to chlorhexidine against dental biofilms on titanium discs in vitro - proof of principle experiment. J Clin Periodontol 2011;38:956-965.
40.
Koban I, Matthes R, Hübner NO, Welk A, Meisel P, Holtfreter B, Sietmann R, Kindel E, Weltmann KD, Kramer A, Kocher T: Treatment of Candida albicans biofilms with low-temperature plasma induced by dielectric barrier discharge and atmospheric pressure plasma jet. New J Phys 2010;12:073039.
41.
Alkawareek MY, Algwari QT, Laverty G, Gorman SP, Graham WG, O'Connell D, Gilmore BF: Eradication of Pseudomonas aeruginosa biofilms by atmospheric pressure non-thermal plasma. PLoS One 2012;7:e44289.
42.
Matthes R, Koban I, Bender C, Masur K, Kindel E, Weltmann K-D, Kocher T, Kramer A, Hübner N-O: Antimicrobial efficacy of an atmospheric pressure plasma jet against biofilms of Pseudomonas aeruginosa and Staphylococcus epidermidis. Plasma Process Polym 2013;10:161-166.
43.
Lim JP, Uhm HS, Li SZ: Influence of oxygen in atmospheric-pressure argon plasma jet on sterilization of Bacillus atrophaeus spores. Phys Plasmas 2007;14:093504.
44.
McKay K, Liu DX, Rong MZ, Iza F, Kong MG: Generation and loss of reactive oxygen species in low-temperature atmospheric-pressure RF He + O2 + H2O plasmas. J Phys D Appl Phys 2012;45:172001.
45.
Lee HW, Park GY, Seo YS, Im YH, Shim SB, Lee HJ: Modelling of atmospheric pressure plasmas for biomedical applications. J Phys D Appl Phys 2011;44:053001.
46.
Ehlbeck J, Schnabel U, Polak M, Winter J, von Woedtke T, Brandenburg R, von dem Hagen T, Weltmann KD: Low temperature atmospheric pressure plasma sources for microbial decontamination. J Phys D Appl Phys 2011;44:013002.
47.
Hähnel M, von Woedtke T, Weltmann K-D: Influence of the air humidity on the reduction of bacillus spores in a defined environment at atmospheric pressure using a dielectric barrier surface discharge. Plasma Process Polym 2010;7:244-249.
48.
Dobrynin D, Friedman G, Fridman A, Starikovskiy A: Inactivation of bacteria using DC corona discharge: role of ions and humidity. New J Phys 2011;13:103033.
49.
Srivastava N, Wang CJ: Effects of water addition on OH radical generation and plasma properties in an atmospheric argon microwave plasma jet. J Appl Phys 2011;110:053304.
50.
Moisan M, Barbeau J, Moreau S, Pelletier J, Tabrizian M, Yahia LH: Low-temperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms. Int J Pharm 2001;226:1-21.
51.
Shimizu T, Zimmermann JL, Morfill GE: The bactericidal effect of surface micro-discharge plasma under different ambient conditions. New J Phys 2011;13:023026.
52.
Gaunt LF, Beggs CB, Georghiou GE: Bactericidal action of the reactive species produced by gas-discharge nonthermal plasma at atmospheric pressure: a review. IEEE Trans Plasma Sci 2006;34:1257-1269.
53.
Shin DH, Choi YS, Cho YH: Unusual properties of catalase A (KatA) of Pseudomonas aeruginosa PA14 are associated with its biofilm peroxide resistance. J Bacteriol 2008;190:2663-2670.
54.
Bjarnsholt T, Jensen PØ, Burmølle M, Hentzer M, Haagensen JA, Hougen HP, Calum H, Madsen KG, Moser C, Molin S, Høiby N, Givskov M: Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology 2005;151:373-383.
55.
Wagatsuma K, Hirokawa K: Effect of oxygen addition to an argon glow-discharge plasma source in atomic-emission spectrometry. Anal Chim Acta 1995;306:193-200.
56.
Sharma A, Pruden A, Yu ZQ, Collins GJ: Bacterial inactivation in open air by the afterglow plume emitted from a grounded hollow slot electrode. Environ Sci Technol 2005;39:339-344.
57.
Kostov KG, Rocha V, Koga-Ito CY, Matos BM, Algatti MA, Honda RY, Kayama ME, Mota RP: Bacterial sterilization by a dielectric barrier discharge (DBD) in air. Surf Coat Technol 2010;204:2954-2959.
58.
Xu KD, Stewart PS, Xia F, Huang CT, McFeters GA: Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability. Appl Environ Microbiol 1998;64:4035-4039.
59.
De Beer D, Stoodley P, Roe F, Lewandowski Z: Effects of biofilm structures on oxygen distribution and mass transport. Biotechnol Bioeng 1994;43:1131-1138.
60.
Pei X, Lu X, Liu J, Liu D, Yang Y, Ostrikov K, Chu PK, Pan Y: Inactivation of a 25.5 µm Enterococcus faecalis biofilm by a room-temperature, battery-operated, handheld air plasma jet. J Phys D Appl Phys 2012;45:165205.
61.
James GA, Swogger E, Wolcott R, Pulcini E, Secor P, Sestrich J, Costerton JW, Stewart PS: Biofilms in chronic wounds. Wound Repair Regen 2008;16:37-44.
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