Treatment of Candida albicans biofilms with low-temperature plasma induced by dielectric barrier discharge and atmospheric pressure plasma jet

Because of some disadvantages of chemical disinfection in dental practice (especially denture cleaning), we investigated the effects of physical methods on Candida albicans biofilms. For this purpose, the antifungal efficacy of three different low-temperature plasma devices (an atmospheric pressure plasma jet and two different dielectric barrier discharges (DBDs)) on Candida albicans biofilms grown on titanium discs in vitro was investigated. As positive treatment controls, we used 0.1% chlorhexidine digluconate (CHX) and 0.6% sodium hypochlorite (NaOCl). The corresponding gas streams without plasma ignition served as negative treatment controls. The efficacy of the plasma treatment was determined evaluating the number of colony-forming units (CFU) recovered from titanium discs. The plasma treatment reduced the CFU significantly compared to chemical disinfectants. While 10 min CHX or NaOCl exposure led to a CFU log10 reduction factor of 1.5, the log10 reduction factor of DBD plasma was up to 5. In conclusion, the use of low-temperature plasma is a promising physical alternative to chemical antiseptics for dental practice.


Introduction
Oral candidosis is an opportunistic infection (caused by pathogens that usually do not cause disease in a healthy host) of the oral cavity with a broad spectrum of clinical signs and symptoms (e.g. denture stomatitis, pseudomembranous candidosis, erythematous candidosis and angular cheilitis) [5]. Oral yeast infections are predominantly caused by Candida albicans (C. albicans) [6]. In approximately 40% of the total human population and 75% of persons with dentures, the oral cavity is colonized with C. albicans forming biofilms.
Biofilm formation on denture surfaces is crucial to the pathogenesis of most oral yeast infections. C. albicans is able to colonize and penetrate into pits and fissures of rough denture surfaces. Thus the initial colonization of commensal (part of the human flora) Candida could be regarded as the origin of infections. Extracellular polysaccharides (EPS) secreted by C. albicans after adhesion on dentures are the basis for the biofilm matrix, which protects the cells from different physical and chemical environmental influences. It has been repeatedly confirmed that C. albicans biofilms are more resistant to antifungal agents than planktonic (free-floating cells, non-adherent, without EPS) C. albicans cells [13].
Approximately 10-75% of denture wearers present a denture-related stomatitis (inflammation of the mucous membrane of any of the structures in the mouth) caused by C. albicans biofilms [2]. Denture stomatitis is an erythematous (redness of the mucous membrane) pathogenic condition of the denture-bearing mucosa [22]. The main reservoir of C. albicans is the fitting surface of the upper partial and complete dentures [26]. Denture cleaning should therefore include the removal of Candida species. Chemical cleansing baths with peroxides, hypochlorites or chlorhexidine (CHX) digluconates in which dentures are immersed are suggested as the first choice of plaque control. The disadvantages of these agents 3 comprise a discolouring of dentures by oxygenating agents and black staining in the case of CHX. Also, ultrasonic cleansing baths are not effective [15].
The use of low-temperature plasma as a physical alternative to chemical antiseptics appears to be an interesting alternative due to the dose-dependent microbicidal effectiveness [32]. Furthermore, neither decolouration nor staining of dentures has been observed.
Physical plasma is an electrically neutral, highly ionized gas composed of ions, electrons, excited atoms and molecules, vacuum ultraviolet and ultraviolet (VUV/UV) irradiation, free radicals and chemically reactive neutral particles. There are many possibilities of producing nonthermal plasma at atmospheric pressure [27].
In the present study, we tested the antifungal potential of different plasma devices (atmospheric pressure plasma jet (APPJ); kINPen ® 09), hollow electrode dielectric barrier discharge (DBD) and volume DBD to reduce C. albicans biofilms in vitro.

Biofilm formation
Candida albicans ATCC 10231 (ATCC is American Type Culture Collection, Rockville, MD, USA), a strongly biofilm-forming strain, was used for the tests [14]. Biofilms were cultured on titanium discs with a diameter of 5 mm and a thickness of 1 mm (Straumann, Basel, Switzerland) using standard conditions, as published before [16]. Briefly, C. albicans was cultured overnight at 37 • C on Columbia sheep blood agar (BBL™, BD, Heidelberg, Germany). One inoculation loop of this culture was resuspended into 30 ml of YPD broth (Yeast Extract Peptone Dextrose; a complete medium for yeast growth, Sigma, Steinheim, Germany). The sterile test objects were positioned in 96-well microtitre plates (Techno Plastic Products AG, Trasadingen, Switzerland), covered with 100 µl of microorganism suspension and incubated aerobically at 37 • C. Every 24 h the medium was changed. After 48 h the medium was drawn off, and the discs were washed with 0.9% NaCl solution and transferred into a new, sterile microtitre plate.
After plasma treatment, the titanium discs were placed into wells with 200 µl of 0.9% NaCl solution and the biofilm was removed by ultrasonic scaling (20 min). Serial dilutions of this resuspended biofilm solution were made by transferring 0.1 ml of the resultant suspension to 0.9 ml of freshly prepared 0.9% NaCl solution. Afterwards an aliquot portion of 0.1 ml from each dilution was plated on sabouraud glucose (4%) agar plates (Carl Roth, Karlsruhe, Germany) and incubated at 37 • C for 48 h. The colonies were counted and expressed as colonyforming units (CFU). The log 10 reduction factor (RF) for each contact time was calculated according to the formula [25]: RF = log 10 n c − log 10 n u , where n c is the number of viable cells (CFU) in the resuspended biofilm solution of NaCl control (in the presence of 0.9% NaCl solution); and n u is the number of viable cells (CFU) in the resuspended biofilm solution after contact with CHX, sodium hypochlorite (NaOCl) or plasma.

Antiseptic treatment
CHX digluconate (an extensively used antiseptic in dentistry) was used as a 0.1% aqueous solution. Firstly, the discs with the C. albicans biofilm were covered with 100 µl of the antiseptic and incubated for 1, 2, 5 or 10 min. Secondly, CHX was drawn off and the antiseptic effect was halted by adding 1 µl of an inactivator consisting of 40 g l −1 Tween 80, 30 g l −1 saponin, Additionally, a 0.6% sodium hypochlorite solution (ApplyChem GmbH, Darmstadt, Germany) was used in the same way. An inactivator consisting of 30 g l −1 Tween 80, 3 g l −1 lecithin, 1 g l −1 histidin and 5 g l −1 sodium thiosulfate was used to halt the antifungal effect.

Plasma treatment
For plasma application, an APPJ 'kINPen09', a hollow electrode DBD and a volume DBD (figure 1), all developed by Leibniz Institute for Plasma Science and Technology (INP), Greifswald, Germany, were used.  [8]. The device has got the CE mark, which certifies that the product has met EU consumer safety, health or environmental requirements. The device consists of a hand-held unit (dimensions: length = 170 mm, diameter = 20 mm and weight = 170 g) for the generation of a plasma jet at atmospheric pressure, a dc power supply (system power: 8 W at 220 V, 50/60 Hz) and a gas supply unit. The principal scheme of the plasma source is shown in figure 1(B). At the centre of a quartz or ceramic capillary (inner diameter 1.6 mm), a pin-type electrode (1 mm diameter) is mounted. In the continuous working mode, a high-frequency (HF) voltage (1.82 MHz, 2-6 kV pp ) is coupled to the pin-type electrode. The plasma is generated from the top of the centred electrode and expands to the surrounding air outside the nozzle.
The whole system works with all rare gases (especially argon) with gas flow rates between 5 and 10 slm (standard litres per minute). Small admixtures ( 1%) of molecular gases to the feed gas are possible. With these gas flow rates and a maximal input dc power of 3.5 W to the hand-held unit, the ignited plasma jet has a length of up to 12 mm. For our experiments the argon (Ar) gas flow was set to 5 slm. The flow rate was controlled by a flow controller (MKS Instruments, Munich, Germany). Temperature measurement resulted in 42 • C at the tip of the plasma jet, which is connected with a thermal output of 150 mW (input power 3 W). We chose a pin-to-sample distance of 7 mm, held constant during the application.
Generally, argon is the working gas for maintaining the discharge, while small amounts of oxygen can be admixed [8]. The plasma treatment is named 'Ar plasma' if no oxygen is admixed to the discharge and 'Ar + 1% O 2 plasma' if 1.0% (0.05 slm) oxygen is used as admixture, respectively.
Temperature measurement. Axial temperature profiles of the plasma jet were obtained by fibre optic temperature measurement (Luxtron, model 755, Santa Clara, USA). A temperaturedependent fluorescent signal of luminescent magnesium fluorogermanate was monitored, which was excited with an Xe flash lamp.

Dielectric barrier discharge.
With the dielectric barrier discharge (DBD), at least one of the two metal electrodes is covered with a dielectric layer. Due to an inherent wall charge mechanism, the transition to an arc discharge is prevented. As a result, the discharge creates a non-thermal plasma without substantial heating of the gas.

Hollow electrode DBD.
Here, another novel approach for the direct DBD treatment of microbiological samples is presented, the so-called hollow electrode DBD (HDBD; see figure 1(D) for a schematic view of the device). The system has been developed for the treatment of samples placed in wells of microtitre plates. Since microtitre plates are made of dielectric materials, they can serve as the barrier in a DBD arrangement. Therefore, microtitre plates were placed on the grounded electrode, which was cooled by a Peltier element in order to control the temperature of the objects to be treated. Six hollow and thin metal tubes served as high-voltage electrodes and gas injection pipes in one function. The gas flowed through these electrodes, while a high rf-voltage (37.6 kHz, 9 W, 9 kV) was coupled. Argon flow was set to 1 slm per well, which is equivalent to a total flow rate of 6 slm (1 slm through each of the six metal tubes). If oxygen was admixed, the total oxygen flow was set to 0.06 slm, which was equivalent to 0.01 slm per well. Two columns of the well plate remain for control samples. Figure 1(C) shows a photograph of the discharges in the microtitre plates. 6 The kINPen09 and the HDBD were clamped onto a computer-driven three-axis (x, y, z) motorized stage, under which a microtitre plate with the titanium discs was positioned. The discs were approached in succession and treated punctually according to the respective treatment period.

Volume DBD.
Volume DBD (VDBD; see figure 1(E) for a photograph of the discharges and figure 1(F) for a schematic view of the device) consisted of two flat, round, metal electrodes, with one of them being electrically grounded. The high-voltage electrode was perforated to get a better insight into the discharge gap. A petri dish (Techno Plastic Products AG, Trasadingen, Switzerland) with the titanium discs was located between these electrodes. The bottom of the petri dish acted as the dielectric for the DBD. For cooling, a Peltier element was used. The gap (the distance between the disc and electrode) in this system was 15 mm and sealed air-tight. Argon gas (0.05 slm) flowed into the system. The high sinusoidal voltage (40 kHz, 16 W, 10 kV) applied between both electrodes generated the plasma. All eight samples were treated simultaneously.
There were two adjustable parameters for our plasma treatment: (i) time intervals of 1, 2, 5 and 10 min for each titanium sample and (ii) plasma gas composition.
Thus, two different gas compositions of plasma (in the case of VDBD, only argon was available) were applied for 1, 2, 5 or 10 min to the discs with the C. albicans biofilm. We repeated the treatment procedure eight times. Altogether, 160 discs were treated with three plasma devices (two gas admixtures × four treatment intervals and eight repetitions in the case of kINPen09 and HDBD; one gas admixture × four treatment intervals and eight repetitions in the case of VDBD).
To assess the effects of biofilm dehydration by gas flow (negative control), eight discs were treated with gas (argon or argon + 1% oxygen) for each treatment time (altogether again 160 discs). As a negative control for CHX and NaOCl treatment, we used 100 µl 0.9% NaCl solution (called NaCl control).

Scanning electron microscopy
For the electron microscopy, the biofilms were prepared as follows. After a fixation step (1 h in 1% glutaraldehyde, 2% paraformaldehyde, 0.2% picric acid, 5 mM HEPES (pH 7.4) and 50 mM NaN 3 ), the samples were treated with 2% tannic acid for 1 h, 1% osmium tetroxide for 1 h, 1% thiocarbohydrazide for 30 min, 1% osmium tetroxide at 4 • C overnight and 2% uranyl acetate for 2 h with washing steps in between. The samples were dehydrated in a graded series of acetone solutions (10-100%) and then critical point dried. Finally, they were mounted on aluminium stubs, sputtered with gold-palladium and examined in an EVO LS10 (Zeiss, Oberkochen, Germany).
The preparation described is a typical protocol for the preparation of yeast cells for scanning electron microscopy. The first fixation step is necessary to fix the proteins in the cell. The following treatment-especially with osmium tetroxide-is used to stabilize the membranes. After fixation, the samples need to be dehydrated and dried because the examination occurs in the scanning electron microscope under high vacuum. Critical point drying with CO 2 is a very gentle method for sample drying because the critical point of CO 2 is in a range in which biological samples are not damaged.

7
From the authors' point of view, the preparation offsets the antiseptic and plasma-treated samples equally, because the damage occurred prior to processing the samples for electron microscopy. This preparation is intended to maintain the condition of the cells they had right before fixation.

Statistics
Continuous data are presented as means ± standard deviation.
Firstly, CFU were compared for each procedure and exposure time versus the NaCl control (treatment time 0 min) using the Mann-Whitney-U tests. Secondly, CFU values were compared for each procedure (kINPen09, HDBD and VDBD combined with gas/plasma compositions) versus the positive controls (CHX and NaOCl) within varying exposure times using one-sided Mann-Whitney-U tests. For each step, P values were corrected for multiple testing according to Benjamini and Hochberg [4]. Procedures (kINPen09, HDBD or VDBD with gas/plasma compositions) that did not reduce CFU values significantly compared to the control or the positive controls were excluded from further analyses.
Then, analysis of variance (ANOVA) and linear regression analyses were applied to evaluate differences in CFU values for different values of plasma sources (HDBD and VDBD combined with Ar gas, Ar plasma, Ar + 1% O 2 gas or Ar + 1% O 2 plasma) and treatment time (1, 2, 5 or 10 min). The twofold interaction term between both factors was considered and confirmed to be statistically significant (P < 0.001). Post hoc analyses of linear combinations of coefficients were carried out to examine differences in CFU reduction between different procedures and gas/plasma compositions.
Finally, for each exposure time, the best performing procedure was compared against the second best performing procedure applying post hoc Wald tests for linear hypotheses. Accordant P values for post hoc tests were adjusted for multiple testing [4]. Statistical differences were considered as significant at P < 0.05. Statistical analyses were performed with STATA/SE 10.0 (Stata Corp. LP, College Station, TX, USA).

Positive controls and kINPen09
Firstly, l g (CFU ml −1 ) values were compared for each procedure and exposure time versus controls. Except for three procedures (CHX and NaOCl, 1 min, and kINPen09 with Ar gas, 2 min), all procedures revealed a better antifungal effect (P < 0.05).
Secondly, the different plasma sources were compared against CHX and NaOCl within exposure times. CHX and NaOCl treatment of 1 min did not lead to a significant reduction in CFU. Between CHX, NaOCl and kINPen09 plasma (both gas compositions), a significant difference was not observed. At no time was treatment with CHX significantly superior to treatment with kINPen09. The results of NaOCl treatment were similar to those of CHX (CHX: RF 1 min = 0.3, RF 2 min = 0.8, RF 5 min = 0.9, RF 10 min = 1.4; NaOCl: RF 1 min = 0.2, RF 2 min = 0.5, RF 5 min = 0.7, RF 10 min = 1.5).
NaOCl-treated cells analyzed by scanning electron microscopy looked rugose ( figure 4(B)). In contrast, CHX-treated cells were as undamaged as the NaCl controls. They seemed to be covered by a layer ( figure 4(A)).

Dielectric barrier discharges (DBDs)
All DBD procedures revealed better antifungal effects (P < 0.05) than the NaCl controls. Overall, for HDBD and VDBD, reduction in CFU was significantly higher compared to positive controls (CHX and NaOCl; P < 0.05).
After 10 min, the three HDBD groups (Ar plasma, Ar + 1% O 2 gas and Ar + 1% O 2 plasma) reduced the CFU ml −1 by a log 10 RF of 2.9, 3.2 and 3.3, respectively, while CHX only achieved a reduction factor of 1.5 (table 1). In the case of VDBD, the CFUs were reduced by a log 10 RF of 2.3 for 1 min, 2.2 for 2 min, 3.5 for 5 min and 5.2 for 10 min of plasma treatment. To evaluate the impact of method and exposure time on CFU, ANOVAs with accordant linear regression models were computed (table 2). According to the ANOVA, 34.7 and 22.8% of the variation in CFU values could be explained by the method and exposure time, respectively, whereas 19.1% of the variation was explained by the interaction between both factors (P < 0.001). Overall, the model explains 76.6% of the total variance. After 1 min, CFUs were significantly reduced for HDBD Ar plasma, VDBD Ar gas and VDBD Ar plasma compared to HDBD Ar gas (P = 0.007, P = 0.003 and P < 0.001, respectively). Increased exposure time (i.e. 5 or 10 min) further reduced CFU values significantly for HDBD Ar + 1% O 2 gas, HDBD Ar + 1% O 2 plasma, VDBD Ar gas (only for 5 min) and VDBD Ar plasma.
Post hoc analyses of linear combinations of coefficients (table 3 and figure 2) revealed that, compared to HDBD Ar gas 1 min, CFU values were significantly reduced for VDBD Ar gas (all times, except 10 min), VDBD Ar plasma (all times), HDBD combined with Ar plasma with   treatment times of 2, 5 or 10 min (P < 0.05), and HDBD combined with Ar + 1% O 2 gas or Ar + 1% O 2 plasma with treatment times of 5 or 10 min (P < 0.05).
To compare CFU values for VDBD plasma with the procedure with the second best performance within exposure times, we applied post hoc Wald tests for linear hypotheses (adjusted for multiple testing). They revealed that VDBD combined with plasma was the significantly best performing procedure for an exposure time of 10 min among all procedures (B = −4.29 (−4.87; −3.70), P < 0.05).
SEM micrographs of 10 min HDBD-treated cells revealed massive perforations to cell walls. Most cells seemed to be evenly split (figure 4(C)). SEM micrographs of 10 min VDBDtreated cells were similar to those of HDBD (figure 4(D)). In contrast to kINPen09-treated cells, only small areas of intact cells could be examined under the scanning electron microscope.

Discussion
The presence of C. albicans is crucial in the etiology of prothesis stomatitis and is reported to be found often on surfaces of acrylic resin materials in vivo. It has been, furthermore, repeatedly isolated from infected peri-implant sites (inflammatory tissue surrounding dental implants) [20]. Those kinds of yeast infections are difficult to treat with topical measures, so new methods need to be developed for effective treatment [17].
Since denture brushing is seldom performed as a daily routine by elderly people with acceptable efficacy, and treatment with ultrasonic baths is not effective, chemical cleansing with immersion baths is the first choice for denture plaque control [12]. Many cleansers have been commercialized. Among alkaline peroxides, CHX gluconate and NaOCl, NaOCl is considered as the most effective and CHX as the least effective against adhered C. albicans cells [11]. New approaches with enhanced efficacy are therefore highly appreciated [31]. Here, we have demonstrated that DBD plasma from different sources was even more effective in the treatment of C. albicans biofilms when compared to standard procedures.
Our study had several limitations. We used an in vitro model to compare the efficacy of different plasma devices with chemical cleansing. To ensure reproducibility of the results, a standard method of biofilm research was used. For a better understanding of the antifungal effects of plasma treatment, we performed our experiments on titanium discs to exclude material hydrophobicity and retention niches, such as cavities and porosities into which the biofilm could adhere. Furthermore, we wanted to exclude putative chemical interactions of residual monomers or of surface properties with adhesion of C. albicans. Future experimental series will be performed on denture resin discs to better simulate the oral environment. Since other groups showed that C. albicans is a common colonizer of infected dental implants [20], our results may also be applicable to implant dentistry and may help to develop future treatment protocols against the peri-implant biofilms. The plasma effects against C. albicans biofilms in vitro were compared with those of CHX and NaOCl, representing chemical cleansers [11,38]. CHX is a frequently used antiseptic [18,37]. But, depending on the test system, CHX was proven to be ineffective against biofilms [35]. The efficacy of CHX depends on its concentration and particularly on its exposure time. It also has a fungistatic effect on yeasts [1]. Our results are in agreement with Gedik and Özkan [11], who showed only a moderate effect of CHX despite 90 min immersion in a cleansing bath. NaOCl has been used for over 200 years for disinfection purposes and is one of the most effective substances currently used in dentistry for biofilm treatment [33].
The antimicrobial mechanism of plasma is more complex than that of CHX. CHX attacks all cell membranes, which are dissolved and the cytoplasm leaks out. Plasma affects many cell components (especially proteins, lipids and DNA/RNA) [7]. Disinfection with plasma is based on short-lived active species, radicals (reactive oxygen species, ROS) and UV radiation. ROS can cause lipid peroxidation. As a result, the membrane is damaged and the cell lyses [7]. Electrostatic effects of the electric field have an additional antimicrobial effect [23]. Inactivation, bio-decontamination and sterilization have received much attention in recent years [9]. Plasma is known to inactivate planktonic bacteria, yeasts and spores [10,17,28,34]. Morfill et al [24] reduced 4 log 10 of C. albicans on agar plates after 5 s plasma treatment. Using a plasma pen, a 3-4 log 10 reduction in adherent bacteria and in C. albicans (non-biofilm) has been demonstrated [29]. In contrast, a log 10 reduction factor of only 1.5 of a 24-h-old Pantoea agglomerans biofilm was achieved (plasma pen with 5 slm He and 0.01% O 2 ) [36]. Our results are in agreement with these findings. We also observed a minor log 10 reduction factor of only 1 with kINPen09 with similar settings on mature biofilms. Regarding our SEM images (figures 3 and 4), plasma could remove 48-h-old C. albicans biofilms. Lee et al [19] showed similar effects, but only on 24-h-old bacterial biofilms. Comparing the different plasmas used in our study, the antifungal efficacy of DBD plasma (VDBD and HDBD) was significantly better compared to kINPen09 (P < 0.05). One reason for these minimal antifungal effects of kINPen09 could be that the spot treatments with the narrow plasma afterglow damage only yeast cells at the actual touch point and that the reactive plasma species, etc do not spread over the entire disc in a sufficient dose. Analyses by scanning electron microscopy showed clear boundaries of damaged cells underlining this hypothesis ( figure 3(B)). Therefore, in further experiments, the pen should be moved all over the disc, instead of treating only one area. Increased exposure time of plasma treatment showed no effects for kINPen09. Compared to kINPen09, the DBD plasma achieved a log 10 reduction factor between 3.1 (HDBD) and 5.2 (VDBD). After DBD plasma treatment, single cells looked exactly like those treated with kINPen09 ( figure 4C). We also observed boundaries between treated and untreated or not sufficiently treated areas. Thus, most cells were severely damaged by DBD plasma treatment. Only in small areas did cells look intact.
In a plasma process, a high gas stream is necessary to keep the plasma cool [3]. In comparison with the NaCl control discs, a gas stream has a certain antifungal effect. Antimicrobial effects of irrigation with non-inert gases were shown in vivo [30]. DBD plasma processes allow the use of a low gas flow. We have admixed oxygen to provide the necessary oxygen species. Treatment with flowing argon + 1% O 2 gas resulted in higher reductions than treatment with pure argon gas. The reduction factor increased with increasing exposure time, probably because of oxidative stress. DBD plasma consists of many microstreamers that last only a few nanoseconds [21]. The disadvantage of DBD plasma is that it is not homogeneous throughout the volume. However, the whole disc was treated in contrast to kINPen09. Thus, some regions of an intact biofilm stayed undamaged, probably due to the inhomogeneities of the plasma. The DBD plasma was subdivided into HDBD and VDBD. VDBD plasma was significantly the best procedure for an exposure time of 10 min among all procedures. While the VDBD worked with 16 W, 40 kHz and 10 kV, the HDBD worked with 9 W, 37.6 kHz and 9 kV. Hence, at first sight, the higher input power of the VDBD seems to be the reason for the increased reduction factor of VDBD (5.2 after 10 min) in contrast to HDBD (3.1) after 10 min. On the other hand, both measured powers are system powers and did not equal the real power injected into the plasma. Therefore, a direct comparison of both plasmas is difficult. For this purpose, further diagnostics are necessary. After the first 2 min, the log 10 reduction factor was the same for both devices. After 5 min treatment with HDBD, Ar + 1% O 2 plasma was the second best procedure.
In contrast to kINPen09 plasma, biofilms treated with DBD plasma were located between electrodes in the middle of an electric field. The disc was the counterelectrode, so that the electric current went through the biofilm. The increased electric field strength could be another reason for better inactivation of C. albicans by both DBDs in comparison with kINPen09. This property, and the possibility of treating a whole denture with plasma, are ideal for denture cleaning. Therefore, atmospheric pressure plasma may become an alternative to chemical cleansing.

Conclusions
C. albicans biofilms play a key role in the pathogenesis of denture stomatitis. CHX and NaOCl were shown to be only moderately effective against yeast cells in biofilms. In this study, the effect of three different plasma devices on C. albicans biofilms in comparison with CHX and NaOCl was investigated. We found that antifungal therapy with nonthermal plasma is effective against biofilms. DBD plasmas exceed the antifungal effects of CHX and NaOCl. Further research is going on to evaluate the cause of the different efficacies and the possible role of physical therapies for biofilm-derived infections in dental practice.