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AEM Accepts, published online ahead of print on 30 March 2012 Appl. Environ. Microbiol. doi:10.1128/AEM.07235-11 Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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Contact-free inactivation of Candida albicans biofilm by Cold-Atmospheric Air

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Plasma

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Tim Maisch*1, Tetsuji Shimizu2, Georg Isbary3, Julia Heinlin1, Sigrid Karrer1, Tobias

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G. Klämpfl², Yang-Fang Li2, Gregor Morfill2 and Julia L. Zimmermann2

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Department of Dermatology, Regensburg University Hospital, Germany

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2

Max Planck Institute for Extraterrestrial Physics, Garching, Germany

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3

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Department of Dermatology, Allergology, and Environmental Medicine, Hospital Munich-Schwabing, Germany

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Running title: Candida biofilm inactivation by plasma

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Key words:

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Candida albicans, disinfection, cold atmospheric plasma

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* Correspondent footnote:

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Tim Maisch, Ph.D.

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Department of Dermatology, Regensburg University Hospital

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Franz-Josef-Strauss-Allee 11

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93053 Regensburg

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Germany

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e-mail: [email protected]

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1

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Abstract

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Candida albicans is one of the main species which is able to form a biofilm on almost

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every surface, causing both skin and superficial mucosal infections. Worldwide

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increase of antifungal resistance led to a decrease in the efficacy of standard

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therapies, prolonging treatment time and increasing healthcare costs. Therefore the

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aim of this work is to demonstrate the applicability of atmospheric plasma at room

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temperature for inactivating biofilm growing Candida albicans without thermally

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damaging heat-sensitive materials. This so-called cold atmospheric plasma is

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produced by applying high voltage to accelerate electrons which ionize the

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surrounding air - leading to the production of charged particles, reactive species and

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photons.

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A newly developed plasma device was used, which exhibits a large plasma

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generating surface area of 9 x 13 cm2 (117 cm2). Different time points were selected

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to achieve an optimum inactivation efficacy range of ≥ 3log10 to 5log10 reduction of

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CFU/ml and compared with 70% ethanol.

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The obtained results show that contact-free antifungal inactivation of Candida

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biofilms by cold atmospheric plasma is a promising tool for disinfection of surfaces

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(and items) in both health care settings and foodstuff industry where ethanol

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disinfection should be avoided.

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Introduction

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Candida albicans is one of the main fungal species which has the ability to grow as a

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biofilm on almost all surfaces, such as medical devices as well as human epithelial

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surfaces (31, 32). In the last decade, biofilm growing Candida showed increased

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levels of resistance to a wide spectrum of conventional antifungal drugs used in

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clinical practice, such as amphotericin B and fluconazole (15). Chandra and

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colleagues demonstrated that C. albicans biofilms can be up to 20-times more

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resistant to amphotericin B and more than 100-times more resistant to fluconazole

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compared with their planktonic counterparts (4, 5). Environmental contamination by

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microbes which originate in dust and soil is a major problem where the maintenance

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of high level hygiene is indispensable such as in clinics or in foodstuff facilities.

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Several studies demonstrated that conventional methods to inactivate free-floating

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microorganisms through antimicrobial agents or disinfection solutions are often less

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effective against pathogens within a biofilm (6, 9, 13). Furthermore Vazquez et al.

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demonstrated the possibility of exogenous nosocomial colonisation of Candida,

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including the possible acquisition from the hospital environment (33). Transmission

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may be by indirect contact since identical strains of Candida were recovered from

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patients who were not in direct contact but temporally associated via a transmission

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route of indirect contact between the patients. Adherence of Candida species to host

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tissues and non-biological materials is not a problem (28). Mucosal cells, fibrin-

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platelet matrices, vascular endothelial cells or plastic materials can be colonized by

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Candida. Radford et al. showed that rough surfaces on e.g. denture-base materials

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promote the adhesion of Candida albicans (26).

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Therefore research efforts have led to the development of new antimicrobial

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strategies, especially for killing of biofilm growing microorganisms (10, 11, 16).

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Another new approach in combating biofilm growing organisms is cold atmospheric 3

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plasma which has demonstrated its bactericidal, virucidal and fungicidal properties

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due to the generation of reactive species, charged molecules and photons (20, 29,

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34). Numerous studies so far have demonstrated the effectiveness of gas-discharges

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or cold atmospheric plasmas in killing planktonic microorganisms only (14, 17, 18,

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29). The killing efficacies were dependent on the plasma exposure-time (10 sec up to

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10 min), the material the microorganisms were located on and the cell density. Only

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recently the bactericidal effect of a non-thermal argon plasma was shown in vitro and

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also in biofilms (8). Furthermore Koban et al. showed inactivation of Candida biofilm

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using different plasma devices (19). An atmospheric plasma jet pen showed only

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minimal antifungal effects on Candida biofilms, whereas an argon dielectric barrier

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discharge plasma device showed a reduction of CFU/ml of up to 5 log10. The

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disadvantage of these dielectric barrier discharge plasmas is the fact that the surface

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of the growing Candida biofilm acts as the counter electrode for the plasma

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generation. This means that the electric current flows through the biofilm, which

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increases the electric field strength of the plasma, which in turn increases the

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bacteria killing efficacy of the biofilm. This is critical for in vivo applications. In the

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study presented here we used the Surface Micro-Discharge (SMD) plasma

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technology to generate plasma in ambient air (25) to inactivate Candida albicans

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biofilm via a contact-free disinfection procedure. The plasma is produced indirectly

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and transported to the biofilm on the surface via diffusion. The surface does not serve

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as a counter electrode and complications with electric currents are avoided and

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"safe" applications in vivo are possible. The SMD plasma device operates at

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approximately room temperature and produces so-called electrical micro-discharges.

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This plasma discharge is generated by applying high voltage to accelerate electrons

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to ionize the surrounding air molecules. Due to the high electric field (voltage), the

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electrons have high energies and therefore do not only produce ions but also 4

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chemically reactive atoms/molecules (O3, OH, O, NO, etc.). In addition to these

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particles, light - including UV - is emitted.

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Our study on contact-free inactivation of Candida albicans biofilm with cold

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atmospheric pressure plasma indicates efficient disinfection of inanimate surfaces

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where liquid disinfectants fail, because protection of corrosive material is mandatory.

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5

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Materials and Methods

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SMD Plasma Device

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The SMD (Surface Micro-Discharge) plasma device is incorporated in a box made

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out of plastic (Teflon and polyoxymethylene). The electrode for producing the plasma

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is located inside the box which is shown in figure 1. On one side of the box a door is

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installed so that the produced plasma gas is confined inside (Fig. 1). The plasma

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device is designed for the efficient treatment of a 96-well plate – therefore, the

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maximum area to treat is large and equals 9 x 13 cm2. The electrode, which

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produces the plasma, is located above the respective samples to treat and the

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distance between the electrode and the sample is adjustable. In this study, the

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distance was set to approximately 6 mm. The electrode for plasma production

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consists of a 0.5 mm thick Teflon plate sandwiched by a brass planar plate and a

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stainless steel mesh grid (line width 2 mm, opening 10 mm, height 1.5 mm). By

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applying a high sinusoidal voltage of 9 kVpp with a frequency of 1 kHz between the

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brass plate and the mesh grid the plasma is produced homogenously in ambient air

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by many micro-discharges (25). The plasma discharge is sustained through

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ionization processes with electrons accelerated by applying the high voltage. These

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electrons produce electron/ion pairs. In addition to these pairs, chemically reactive

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species (O3, O, NO, etc.) are produced by approximately 600 chemical reactions

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driven by the electrons (dissociation of molecules, recombination, etc.). Furthermore,

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due to activated molecules/atoms, light emission can also be detected. The UV light

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emitted by plasma is mainly observed from the N2 positive system between 280 and

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420 nm in wavelength (see spectrum in fig. 2). Furthermore peaks in the UVC region

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of the spectrum resulting from the NO γ system can be detected. The UV power

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density measured with a power meter (HAMAMATSU UV-Power Meter C8026,

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Japan) equalled 25 nW/cm2. This value is far below ICNIRP (International 6

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Commission Non-Ionizing Radiation Protection) safety levels. The measurement of

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ozone was performed by using an UV absorption spectroscopy, NO2 concentration

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was measured using a gas detector (Dräger AG, Multiwarn II, Germany) (table 1).

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The power consumption for the plasma discharge was approximately 0.02 W/cm2

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measured with the Lissajous method using a 1 μF capacitance.

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Microorganism

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In order to obtain well isolated discrete colonies, Candida albicans were streaked on

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a Sabouraud dextrose agar plate and cultured at 37°C. A single colony of Candida

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albicans (ATCC-MYA-273) was picked up using a sterile inoculation loop and

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suspended in 5 ml of Sabouraud dextrose broth (SDB) (Sigma Chemical Co., St.

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Louis, Mo, USA). The suspension was cultured overnight at 37°C on a shaker

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platform (200 rpm). When the cultures reached the stationary phase of growth, the

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cells were harvested by centrifugation (1800 rcf, 5 min) and washed once with PBS

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(Dulbecco's phosphate-buffered saline without Ca2+ and Mg2+, PAA Laboratories

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GmbH, Austria).

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Biofilm formation

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The Candida cell density was determined using a microscope counting chamber. For

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biofilm formation, C. albicans were diluted to 106 cells ml-1 in 25% of fetal bovine

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serum (FBS) and 2.5 ml were added to sterile flat-bottomed 6-well polystyrene plates

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for cell culture and incubated at 37°C for 24 h without shaking.

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Cold atmospheric plasma treatment

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In a first step a proof of concept study was adopted from Shimizu et al. (29).

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Suspensions of free-floating Candida cells with a density of 2 x 106/ml were prepared 7

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in PBS. 3 x 20 µl of serially diluted samples were dropped out on Sabouraud

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dextrose agar plates and kept at RT under laminar flow for 30 min to dry the surface.

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After treatment with plasma, the agar plates were incubated at 37°C for 24 h until

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surviving Candida colonies were counted.

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Second, 24 h after biofilm generation, the samples were washed twice with PBS to

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remove free-floating Candida cells. The 6-well plates containing the biofilm were

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placed inside the device and treated for different time intervals as follows: 0 min, 2, 5,

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6, 7, 8, 9, 10 and 15 min. As controls, non plasma-treated samples were placed as

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well in the SMD device for the respective time points.

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Furthermore the disinfectant efficacy of 70% Ethanol was tested on the Candida

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biofilm as recently published by Théraud et al. (30).

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Quantification of inactivation efficacy

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Post plasma treatment the biofilm was scraped out of the 6-well plate using a sterile

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cell scraper (Sarstedt, Newtonk, NC, USA). Each sample was sonicated for 3 min in

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an ultrasonic bath sonicator (Merck Eurolab, USR 30H, Germany) at a frequency of

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35kHz to disrupt the biofilm, because the colony forming unit assay was used to

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determine the survivors by the Miles and Misra technique for viable counts (24).

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Serially diluted aliquots (20 µl) of treated and untreated samples were plated on

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Sabouraud dextrose agar, and the number of CFU per millilitre was counted after 24

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h of incubation at 37°C.

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Statistical methods

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All results are shown as medians, including the 25% and 75% quartiles, which were

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calculated from the values of at least three independent experiments. Each

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experiment was conducted in triplicates (three wells of a 6-well-plate corresponding 8

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to a biofilm area of 28.9 cm2), with Prism 4 for Windows (GraphPad Software Inc.,

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San Diego, CA, U.S.A). The calculation (reduction of CFU/ml) was then compared

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with the untreated controls (non plasma treated). In figures 3-4, medians on or below

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the dotted horizontal line in red or green represent ≥ 99.9% efficacy or ≥ 99.999% of

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Candida cell killing corresponding to at least more than three magnitudes or five

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magnitudes of log10 reduction compared with the matching untreated controls (non

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plasma treated). A reduction of at least three magnitudes of log10 of viable median

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numbers of Candida cells was stated as biologically relevant with regard to the

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guidelines of hand hygiene (3).

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9

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Results

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SMD Plasma device

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A SMD plasma device was used for all experiments. During the experiments, the

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door (as shown in figure 1) of the plasma device was closed, i.e. almost no gas

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exchange was able to take place. Thermal effects on the Candida biofilm can be

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ruled out in this study because the increase of the gas temperature during 10 min

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application did not exceed 4°C (data not shown). As mentioned in Materials and

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Methods the UV light emission was mainly observed from the N2 positive system

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between 280 and 420 nm (Fig. 2). Furthermore, emission in the UVC region resulting

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from the NO γ system was detected. Nevertheless the main content of the measured

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UV radiation refers to the UV-A wavelength range between 320 to 400 nm. The UV

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power density of the plasma equalled 25 nW/cm2. The main components produced

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by the SMD plasma device are shown in table 1 which are relevant for biomedical

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applications. The ozone concentration in the device (inside the box, door closed)

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after 60 seconds of plasma production was approximately 500 ppm. The NO2

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concentration was approximately 3 ppm.

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Plasma rapidly kills planktonic Candida cells: A proof of concept

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First the susceptibility of the SMD plasma device was determined by applying plasma

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to planktonic Candida cells plated on Sabouraud Dextrose agar plates. The fungicidal

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effect of plasma is shown in figure 3. A killing efficacy of 99.9% of viable Candida

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cells was achieved upon a plasma treatment time of 40 sec. The reduction of viable

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Candida cells was increased further up to 5 log10 steps when the plasma treatment

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time was 5 min. No fungicidal effect was observed for the untreated controls.

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Killing efficacy of Plasma against Candida biofilms 10

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The Candida biofilms formed on inanimate surfaces were exposed to different

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plasma treatment times (Fig. 4). A biofilm surface area of 28.9 cm2 was treated with

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plasma corresponding to three single wells of a 6-well plate. The plasma application

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of Candida biofilms resulted in a successful inactivation of Candida cells within the

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biofilm, which increased with increasing treatment time (Fig. 4). After 7 min, more

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than 99.9% of Candida cells were inactivated. 8 min results in a reduction of 6 log10

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steps which corresponds to a killing efficacy of 99.9999%. Again no fungicidal effect

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was observed for the untreated controls. Here we also want to mention, that the

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inactivation efficacy also depends on the distance between the electrode and the

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biofilm (data not shown). The tested distances ranged from 6 to 10 mm. All the

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results in this study refer to the most effective distance of 6 mm. In a second set of

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experiments, 70% ethanol was tested on biofilm inactivation. A reduction efficacy of

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only 1.5 log10 , 2.8 log10 and >3 log10 was achieved within a treatment time of 5 min,

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7min and 10 min, respectively (data not shown).

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Discussion

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Candida growing as a biofilm produces a broad range of infections, ranging from non

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life-threatening mucocutaneous illnesses to severe invasive diseases (4, 27). This

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study demonstrates the efficacy of SMD plasma for killing biofilm growing C.

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albicans. The results of this study clearly show, that cold atmospheric plasma

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treatment is an attractive potential approach for Candida biofilm inactivation growing

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on inanimate surfaces. A reduction of up to 99.9999% was achieved in a time-

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dependent manner (8 min) without any direct contact to the biofilm. A biofilm surface

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area of 28.9 cm2 was efficiently inactivated. This area is 72-times greater than the

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biofilm surface area of a 96-well plate, which was used in other studies (19). The

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possibility of removing large areas of biofilm in one step, without screening the

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surface, clearly exhibits advantages for industrial applications. Koban et al. measured

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a reduction factor of 5.2 log10 steps of a Candida biofilm grown in a 96-microtitre

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plate upon 10 min plasma treatment using the dielectric barrier discharge (DBD)

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plasma device (19). As already stated in the introduction, the great advantage of

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SMD plasma compared to DBD is that there is no direct contact to the surface

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necessary and no current passes through the plate and the biofilm.

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The main species which contribute to the inactivation of the biofilm using SMD

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plasma in ambient air are reactive species and charged particles. As indicated

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earlier, the density of the charged particles which interact with the biofilm is low due

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to excitation, dissociation, attachment and recombination processes on their way

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from the electrode to the sample (25). Therefore the main impact on the biofilm

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results from the produced reactive species: Measurements of the produced ozone in

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the device after a treatment time of 60s showed a value of approximately 500 ppm.

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The NO2 concentration was approximately 3 ppm. Concerning the ozone

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concentration, Kowalski et al. reported that 15 s with 1500 ppm of ozone were 12

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necessary for a 3 log10 reduction of E. coli plated on agar (21). In our study, the

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reduction of even Candida albicans (and E.coli (data not shown)) is faster with a

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smaller amount of ozone. This leads to the conclusion that the produced plasma

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agents have synergetic effects and therefore result in a faster inactivation of

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pathogens. Various inactivation experiments with bacteria showed that the UV

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photons (gained by treating a planktonic bacteria sample with plasma filtered by a

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quartz glass) do not have any bactericidal property up to 120 s (data not shown).

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Therefore we conclude that the UV radiation alone does not contribute to the

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fungicidal property as well. The electric field produced by the plasma can cause

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stress on the cell wall of fungi. However, we could not observe any inactivation of

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fungi due to the electric field produced by the plasma (data not shown). As stated

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earlier the electrical current through the sample is negligibly small.

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In recent years opportunistic pathogens from the genera Candida are the main cause

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of fungal infections, especially as biofilm-mediated infections (4, 23). Therefore

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contamination of inanimate surfaces by Candida may lead to the formation of

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Candida biofilm and if left untreated or not disinfected it may become a risk in health

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care services. Candida biofilms are more resistant to standard antifungal agents than

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free-floating Candida cells (7). This study supports this statement as we clearly

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showed that planktonic Candida cells can be easier killed using shorter plasma

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treatment times than biofilm growing cells. Therefore we conclude that not only the

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amount of cells per ml limits the inactivation but also the growing conditions:

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planktonic or biofilm. Single Candida cells can survive for a longer time embedded in

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a biofilm and grow with slower rates, and their metabolic and antifungal responses

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are often different from their planktonic counterpart. Furthermore we were able to

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show that the disinfectant ethanol (70%) was less effective in killing the Candida

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biofilm which is in agreement with already published data (30). Théraud et al. 13

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demonstrated that the overall efficacy of antiseptics and disinfectants on yeast

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isolates is different when the cells are grown in planktonic or biofilm conditions (30).

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Eight out of nine agents investigated (10% iodine polyvinylpyrrolidone, 2% sodium

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lauryl sufate, 0.5% and 0.05% chlorhexidine digluconate, 3% hydrogen peroxide,

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70% ethanol, 0.5% alkylamine, 0.5% alkylamine + sodium hypochlorite and UV

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radiation at 365 nm were ineffective against biofilm growing Candida (30).

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Chlorhexidine digluconate at a concentration of 0.5% was the only antifungal agent

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which was active both on planktonic Candida cells suspension and biofilm growing

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Candida albicans. Today chlorhexidine digluconate is used as a “gold standard”

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antiseptic in the oral cavity, but some risks must be considered (12). Chlorhexidine

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has a mutagenic potential, possesses a neurotoxic side effect and can induce

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hypersensitiveness (22). The Japanes Ministry of Health recommended in 1984 to

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avoid the use of chlorhexidine. Therefore the long-term use of chlorhexidine has to

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be questioned (1).

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As stated in the introduction, the high antifungal resistance of biofilms is a multifactor

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process where an antifungal drug with just one single mechanism of action is unlikely

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to be effective (2). In contrast, the mechanism of action of cold atmospheric plasma is

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different from that of an antifungal agent. Typically an antifungal drug acts using the

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lock-and-key principle. This means that a specific antifungal agent has to fit in a

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certain way inside or outside of a Candida cell to induce an antifungal effect. In

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contrast cold atmospheric plasma produced by this device results in the generation of

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reactive oxygen and nitrogen species as well as atomic O and N and hydrogen

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peroxide interacting with water vapour. All these species are capable to induce

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oxidative and radical damage of the biofilm immediately during the plasma treatment.

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Therefore a rapid fungicidal disinfection is possible. In this case no specific

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interaction is necessary to induce the antifungal effect. 14

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Overall the use of cold atmospheric plasma for disinfection of inanimate surfaces

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could lead to a major development preventing biofilm-associated Candida infections

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in the future. Therefore the advantage of a concept of a contact-free application of

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plasma to sterilize Candida biofilm contaminated surfaces (and items) in health care

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settings might have a positive impact on preventing community-acquired and

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nosocomial infections in the medical field, but also in foodstuff industry where time

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saving is a critical point to achieve efficient disinfection in the future.

15

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Acknowledgement

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The excellent technical assistant of Judith Heider is gratefully acknowledged. Dr. Tim

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Maisch was supported by a grant (M.TT.A.EXT00002) of the Max Planck Institute for

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Extraterrestrial Physics, Garching, Germany.

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16

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Table 1 Plasma components relevant to biomedical applications Charged particles

electrons, ions

at the surface of the electrode: ~1011 cm-3

Reactive species

O3

~ 500 ppm

NO

< 1 ppm

NO2

~ 3 ppm

O, OH

presence

according

to

the

literature Heat

Max. 4 deg. above the ambient temperature

Photons

UV, Visible

UV power ~25 nW/cm2 mainly UVA

Static electric field

Max. 106 V/m

Electrical current

Negligibly small, below 100 μA

through samples 326 327

17

328

Figure legend

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Figure 1. A sketch of the plasma device.

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a) The plasma device contains one SMD electrode and the sample to treat is placed

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below the electrode. In this study the distance between the electrode and the sample

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was fixed to 6 mm.

333 334

Figure 2. Spectra of the plasma produced by the SMD device

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The spectrum of the produced SMD plasma was measured in front of the electrode at

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a distance of 6 mm. The main UV components are in the wavelength range between

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280 and 400 nm and are produced from nitrogen molecules excited by electron

338

impact.

339 340

Figure 3. Proof of concept inactivation of C. albicans by cold atmospheric

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plasma

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20 µl of serial diluted free-floating C. albicans suspensions according to the Miles and

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Misra technique were applied to agar-plates and dried for 45 min. Cold atmospheric

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plasma treatment was performed using different time intervals. Surviving colonies

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were counted 24 h later. Black dotted line: baseline of viable C. albicans per ml; red

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dotted line: reduction of three log10 steps of viable C. albicans (99.9%); green dotted

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line: reduction of five log10 steps of viable C. albicans (99.999%). (n = 3, median ±

348

interquartile range). Arrows pointing downwards mark the appropriate upper limits.

349 350

Figure 4. Cold atmospheric plasma treatment of C. albicans biofilm

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C. albicans biofilm was grown on the surface of 6-well-plates for 24 h. After washing

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the biofilm to remove free floating cells, the biofilm was dried for 30 min. Cold

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atmospheric plasma treatment was done using different time intervals [20sec to 18

354

10min].

A CFU assay was performed immediately after the plasma treatment.

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Colonies were counted 24h later. Black dotted line: baseline of viable C. albicans per

356

ml; red dotted line: reduction of three log10 step of viable C. albicans (99.9%); green

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dotted line: reduction of five log10 steps of viable C. albicans (99.999%). (n = 3,

358

median ± interquartile range). Arrows pointing downwards mark the appropriate

359

upper limits.

360 361

19

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