Contact-free inactivation of Candida albicans biofilms by cold atmospheric air plasma.

Abstract

Candida albicans is one of the main species able to form a biofilm on almost any surface, causing both skin and superficial mucosal infections. The worldwide increase in antifungal resistance has led to a decrease in the efficacy of standard therapies, prolonging treatment time and increasing health care costs. Therefore, the aim of this work was to demonstrate the applicability of atmospheric plasma at room temperature for inactivating C. albicans growing in biofilms without thermally damaging heat-sensitive materials. This so-called cold atmospheric plasma is produced by applying high voltage to accelerate electrons, which ionize the surrounding air, leading to the production of charged particles, reactive species, and photons. A newly developed plasma device was used, which exhibits a large plasma-generating surface area of 9 by 13 cm (117 cm(2)). Different time points were selected to achieve an optimum inactivation efficacy range of ≥3 log(10) to 5 log(10) reduction in CFU per milliliter, and the results were compared with those of 70% ethanol. The results obtained show that contact-free antifungal inactivation of Candida biofilms by cold atmospheric plasma is a promising tool for disinfection of surfaces (and items) in both health care settings and the food industry, where ethanol disinfection should be avoided.

Full text

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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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1 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 Department of Dermatology, Allergology, and Environmental Medicine, Hospital
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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: tim.maisch@klinik.uni-regensburg.de
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.
Appl. Environ. Microbiol. doi:10.1128/AEM.07235-11
AEM Accepts, published online ahead of print on 30 March 2012

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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
26
increase of antifungal resistance led to a decrease in the efficacy of standard
27
therapies, prolonging treatment time and increasing healthcare costs. Therefore the
28
aim of this work is to demonstrate the applicability of atmospheric plasma at room
29
temperature for inactivating biofilm growing Candida albicans without thermally
30
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
35
generating surface area of 9 x 13 cm2 (117 cm2). Different time points were selected
36
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
40
(and items) in both health care settings and foodstuff industry where ethanol
41
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
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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
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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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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
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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
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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
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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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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
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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
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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.
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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.
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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.
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