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.
1
Contact-free inactivation of Candida albicans biofilm by Cold-Atmospheric Air
2
Plasma
3
Tim Maisch*1, Tetsuji Shimizu2, Georg Isbary3, Julia Heinlin1, Sigrid Karrer1, Tobias
4
G. Klämpfl², Yang-Fang Li2, Gregor Morfill2 and Julia L. Zimmermann2
5 6
1
Department of Dermatology, Regensburg University Hospital, Germany
7
2
Max Planck Institute for Extraterrestrial Physics, Garching, Germany
8
3
9
Department of Dermatology, Allergology, and Environmental Medicine, Hospital Munich-Schwabing, Germany
10 11
Running title: Candida biofilm inactivation by plasma
12 13
Key words:
14
Candida albicans, disinfection, cold atmospheric plasma
15 16
* Correspondent footnote:
17
Tim Maisch, Ph.D.
18
Department of Dermatology, Regensburg University Hospital
19
Franz-Josef-Strauss-Allee 11
20
93053 Regensburg
21
Germany
22
e-mail:
[email protected]
23
1
24
Abstract
25
Candida albicans is one of the main species which is able to form a biofilm on almost
26
every surface, causing both skin and superficial mucosal infections. Worldwide
27
increase of antifungal resistance led to a decrease in the efficacy of standard
28
therapies, prolonging treatment time and increasing healthcare costs. Therefore the
29
aim of this work is to demonstrate the applicability of atmospheric plasma at room
30
temperature for inactivating biofilm growing Candida albicans without thermally
31
damaging heat-sensitive materials. This so-called cold atmospheric plasma is
32
produced by applying high voltage to accelerate electrons which ionize the
33
surrounding air - leading to the production of charged particles, reactive species and
34
photons.
35
A newly developed plasma device was used, which exhibits a large plasma
36
generating surface area of 9 x 13 cm2 (117 cm2). Different time points were selected
37
to achieve an optimum inactivation efficacy range of ≥ 3log10 to 5log10 reduction of
38
CFU/ml and compared with 70% ethanol.
39
The obtained results show that contact-free antifungal inactivation of Candida
40
biofilms by cold atmospheric plasma is a promising tool for disinfection of surfaces
41
(and items) in both health care settings and foodstuff industry where ethanol
42
disinfection should be avoided.
43
2
44
Introduction
45
Candida albicans is one of the main fungal species which has the ability to grow as a
46
biofilm on almost all surfaces, such as medical devices as well as human epithelial
47
surfaces (31, 32). In the last decade, biofilm growing Candida showed increased
48
levels of resistance to a wide spectrum of conventional antifungal drugs used in
49
clinical practice, such as amphotericin B and fluconazole (15). Chandra and
50
colleagues demonstrated that C. albicans biofilms can be up to 20-times more
51
resistant to amphotericin B and more than 100-times more resistant to fluconazole
52
compared with their planktonic counterparts (4, 5). Environmental contamination by
53
microbes which originate in dust and soil is a major problem where the maintenance
54
of high level hygiene is indispensable such as in clinics or in foodstuff facilities.
55
Several studies demonstrated that conventional methods to inactivate free-floating
56
microorganisms through antimicrobial agents or disinfection solutions are often less
57
effective against pathogens within a biofilm (6, 9, 13). Furthermore Vazquez et al.
58
demonstrated the possibility of exogenous nosocomial colonisation of Candida,
59
including the possible acquisition from the hospital environment (33). Transmission
60
may be by indirect contact since identical strains of Candida were recovered from
61
patients who were not in direct contact but temporally associated via a transmission
62
route of indirect contact between the patients. Adherence of Candida species to host
63
tissues and non-biological materials is not a problem (28). Mucosal cells, fibrin-
64
platelet matrices, vascular endothelial cells or plastic materials can be colonized by
65
Candida. Radford et al. showed that rough surfaces on e.g. denture-base materials
66
promote the adhesion of Candida albicans (26).
67
Therefore research efforts have led to the development of new antimicrobial
68
strategies, especially for killing of biofilm growing microorganisms (10, 11, 16).
69
Another new approach in combating biofilm growing organisms is cold atmospheric 3
70
plasma which has demonstrated its bactericidal, virucidal and fungicidal properties
71
due to the generation of reactive species, charged molecules and photons (20, 29,
72
34). Numerous studies so far have demonstrated the effectiveness of gas-discharges
73
or cold atmospheric plasmas in killing planktonic microorganisms only (14, 17, 18,
74
29). The killing efficacies were dependent on the plasma exposure-time (10 sec up to
75
10 min), the material the microorganisms were located on and the cell density. Only
76
recently the bactericidal effect of a non-thermal argon plasma was shown in vitro and
77
also in biofilms (8). Furthermore Koban et al. showed inactivation of Candida biofilm
78
using different plasma devices (19). An atmospheric plasma jet pen showed only
79
minimal antifungal effects on Candida biofilms, whereas an argon dielectric barrier
80
discharge plasma device showed a reduction of CFU/ml of up to 5 log10. The
81
disadvantage of these dielectric barrier discharge plasmas is the fact that the surface
82
of the growing Candida biofilm acts as the counter electrode for the plasma
83
generation. This means that the electric current flows through the biofilm, which
84
increases the electric field strength of the plasma, which in turn increases the
85
bacteria killing efficacy of the biofilm. This is critical for in vivo applications. In the
86
study presented here we used the Surface Micro-Discharge (SMD) plasma
87
technology to generate plasma in ambient air (25) to inactivate Candida albicans
88
biofilm via a contact-free disinfection procedure. The plasma is produced indirectly
89
and transported to the biofilm on the surface via diffusion. The surface does not serve
90
as a counter electrode and complications with electric currents are avoided and
91
"safe" applications in vivo are possible. The SMD plasma device operates at
92
approximately room temperature and produces so-called electrical micro-discharges.
93
This plasma discharge is generated by applying high voltage to accelerate electrons
94
to ionize the surrounding air molecules. Due to the high electric field (voltage), the
95
electrons have high energies and therefore do not only produce ions but also 4
96
chemically reactive atoms/molecules (O3, OH, O, NO, etc.). In addition to these
97
particles, light - including UV - is emitted.
98
Our study on contact-free inactivation of Candida albicans biofilm with cold
99
atmospheric pressure plasma indicates efficient disinfection of inanimate surfaces
100
where liquid disinfectants fail, because protection of corrosive material is mandatory.
101
5
102
Materials and Methods
103
SMD Plasma Device
104
The SMD (Surface Micro-Discharge) plasma device is incorporated in a box made
105
out of plastic (Teflon and polyoxymethylene). The electrode for producing the plasma
106
is located inside the box which is shown in figure 1. On one side of the box a door is
107
installed so that the produced plasma gas is confined inside (Fig. 1). The plasma
108
device is designed for the efficient treatment of a 96-well plate – therefore, the
109
maximum area to treat is large and equals 9 x 13 cm2. The electrode, which
110
produces the plasma, is located above the respective samples to treat and the
111
distance between the electrode and the sample is adjustable. In this study, the
112
distance was set to approximately 6 mm. The electrode for plasma production
113
consists of a 0.5 mm thick Teflon plate sandwiched by a brass planar plate and a
114
stainless steel mesh grid (line width 2 mm, opening 10 mm, height 1.5 mm). By
115
applying a high sinusoidal voltage of 9 kVpp with a frequency of 1 kHz between the
116
brass plate and the mesh grid the plasma is produced homogenously in ambient air
117
by many micro-discharges (25). The plasma discharge is sustained through
118
ionization processes with electrons accelerated by applying the high voltage. These
119
electrons produce electron/ion pairs. In addition to these pairs, chemically reactive
120
species (O3, O, NO, etc.) are produced by approximately 600 chemical reactions
121
driven by the electrons (dissociation of molecules, recombination, etc.). Furthermore,
122
due to activated molecules/atoms, light emission can also be detected. The UV light
123
emitted by plasma is mainly observed from the N2 positive system between 280 and
124
420 nm in wavelength (see spectrum in fig. 2). Furthermore peaks in the UVC region
125
of the spectrum resulting from the NO γ system can be detected. The UV power
126
density measured with a power meter (HAMAMATSU UV-Power Meter C8026,
127
Japan) equalled 25 nW/cm2. This value is far below ICNIRP (International 6
128
Commission Non-Ionizing Radiation Protection) safety levels. The measurement of
129
ozone was performed by using an UV absorption spectroscopy, NO2 concentration
130
was measured using a gas detector (Dräger AG, Multiwarn II, Germany) (table 1).
131
The power consumption for the plasma discharge was approximately 0.02 W/cm2
132
measured with the Lissajous method using a 1 μF capacitance.
133 134
Microorganism
135
In order to obtain well isolated discrete colonies, Candida albicans were streaked on
136
a Sabouraud dextrose agar plate and cultured at 37°C. A single colony of Candida
137
albicans (ATCC-MYA-273) was picked up using a sterile inoculation loop and
138
suspended in 5 ml of Sabouraud dextrose broth (SDB) (Sigma Chemical Co., St.
139
Louis, Mo, USA). The suspension was cultured overnight at 37°C on a shaker
140
platform (200 rpm). When the cultures reached the stationary phase of growth, the
141
cells were harvested by centrifugation (1800 rcf, 5 min) and washed once with PBS
142
(Dulbecco's phosphate-buffered saline without Ca2+ and Mg2+, PAA Laboratories
143
GmbH, Austria).
144 145
Biofilm formation
146
The Candida cell density was determined using a microscope counting chamber. For
147
biofilm formation, C. albicans were diluted to 106 cells ml-1 in 25% of fetal bovine
148
serum (FBS) and 2.5 ml were added to sterile flat-bottomed 6-well polystyrene plates
149
for cell culture and incubated at 37°C for 24 h without shaking.
150 151
Cold atmospheric plasma treatment
152
In a first step a proof of concept study was adopted from Shimizu et al. (29).
153
Suspensions of free-floating Candida cells with a density of 2 x 106/ml were prepared 7
154
in PBS. 3 x 20 µl of serially diluted samples were dropped out on Sabouraud
155
dextrose agar plates and kept at RT under laminar flow for 30 min to dry the surface.
156
After treatment with plasma, the agar plates were incubated at 37°C for 24 h until
157
surviving Candida colonies were counted.
158
Second, 24 h after biofilm generation, the samples were washed twice with PBS to
159
remove free-floating Candida cells. The 6-well plates containing the biofilm were
160
placed inside the device and treated for different time intervals as follows: 0 min, 2, 5,
161
6, 7, 8, 9, 10 and 15 min. As controls, non plasma-treated samples were placed as
162
well in the SMD device for the respective time points.
163
Furthermore the disinfectant efficacy of 70% Ethanol was tested on the Candida
164
biofilm as recently published by Théraud et al. (30).
165 166
Quantification of inactivation efficacy
167
Post plasma treatment the biofilm was scraped out of the 6-well plate using a sterile
168
cell scraper (Sarstedt, Newtonk, NC, USA). Each sample was sonicated for 3 min in
169
an ultrasonic bath sonicator (Merck Eurolab, USR 30H, Germany) at a frequency of
170
35kHz to disrupt the biofilm, because the colony forming unit assay was used to
171
determine the survivors by the Miles and Misra technique for viable counts (24).
172
Serially diluted aliquots (20 µl) of treated and untreated samples were plated on
173
Sabouraud dextrose agar, and the number of CFU per millilitre was counted after 24
174
h of incubation at 37°C.
175 176
Statistical methods
177
All results are shown as medians, including the 25% and 75% quartiles, which were
178
calculated from the values of at least three independent experiments. Each
179
experiment was conducted in triplicates (three wells of a 6-well-plate corresponding 8
180
to a biofilm area of 28.9 cm2), with Prism 4 for Windows (GraphPad Software Inc.,
181
San Diego, CA, U.S.A). The calculation (reduction of CFU/ml) was then compared
182
with the untreated controls (non plasma treated). In figures 3-4, medians on or below
183
the dotted horizontal line in red or green represent ≥ 99.9% efficacy or ≥ 99.999% of
184
Candida cell killing corresponding to at least more than three magnitudes or five
185
magnitudes of log10 reduction compared with the matching untreated controls (non
186
plasma treated). A reduction of at least three magnitudes of log10 of viable median
187
numbers of Candida cells was stated as biologically relevant with regard to the
188
guidelines of hand hygiene (3).
189 190
9
191
Results
192
SMD Plasma device
193
A SMD plasma device was used for all experiments. During the experiments, the
194
door (as shown in figure 1) of the plasma device was closed, i.e. almost no gas
195
exchange was able to take place. Thermal effects on the Candida biofilm can be
196
ruled out in this study because the increase of the gas temperature during 10 min
197
application did not exceed 4°C (data not shown). As mentioned in Materials and
198
Methods the UV light emission was mainly observed from the N2 positive system
199
between 280 and 420 nm (Fig. 2). Furthermore, emission in the UVC region resulting
200
from the NO γ system was detected. Nevertheless the main content of the measured
201
UV radiation refers to the UV-A wavelength range between 320 to 400 nm. The UV
202
power density of the plasma equalled 25 nW/cm2. The main components produced
203
by the SMD plasma device are shown in table 1 which are relevant for biomedical
204
applications. The ozone concentration in the device (inside the box, door closed)
205
after 60 seconds of plasma production was approximately 500 ppm. The NO2
206
concentration was approximately 3 ppm.
207 208
Plasma rapidly kills planktonic Candida cells: A proof of concept
209
First the susceptibility of the SMD plasma device was determined by applying plasma
210
to planktonic Candida cells plated on Sabouraud Dextrose agar plates. The fungicidal
211
effect of plasma is shown in figure 3. A killing efficacy of 99.9% of viable Candida
212
cells was achieved upon a plasma treatment time of 40 sec. The reduction of viable
213
Candida cells was increased further up to 5 log10 steps when the plasma treatment
214
time was 5 min. No fungicidal effect was observed for the untreated controls.
215 216
Killing efficacy of Plasma against Candida biofilms 10
217
The Candida biofilms formed on inanimate surfaces were exposed to different
218
plasma treatment times (Fig. 4). A biofilm surface area of 28.9 cm2 was treated with
219
plasma corresponding to three single wells of a 6-well plate. The plasma application
220
of Candida biofilms resulted in a successful inactivation of Candida cells within the
221
biofilm, which increased with increasing treatment time (Fig. 4). After 7 min, more
222
than 99.9% of Candida cells were inactivated. 8 min results in a reduction of 6 log10
223
steps which corresponds to a killing efficacy of 99.9999%. Again no fungicidal effect
224
was observed for the untreated controls. Here we also want to mention, that the
225
inactivation efficacy also depends on the distance between the electrode and the
226
biofilm (data not shown). The tested distances ranged from 6 to 10 mm. All the
227
results in this study refer to the most effective distance of 6 mm. In a second set of
228
experiments, 70% ethanol was tested on biofilm inactivation. A reduction efficacy of
229
only 1.5 log10 , 2.8 log10 and >3 log10 was achieved within a treatment time of 5 min,
230
7min and 10 min, respectively (data not shown).
231 232 233
11
234
Discussion
235
Candida growing as a biofilm produces a broad range of infections, ranging from non
236
life-threatening mucocutaneous illnesses to severe invasive diseases (4, 27). This
237
study demonstrates the efficacy of SMD plasma for killing biofilm growing C.
238
albicans. The results of this study clearly show, that cold atmospheric plasma
239
treatment is an attractive potential approach for Candida biofilm inactivation growing
240
on inanimate surfaces. A reduction of up to 99.9999% was achieved in a time-
241
dependent manner (8 min) without any direct contact to the biofilm. A biofilm surface
242
area of 28.9 cm2 was efficiently inactivated. This area is 72-times greater than the
243
biofilm surface area of a 96-well plate, which was used in other studies (19). The
244
possibility of removing large areas of biofilm in one step, without screening the
245
surface, clearly exhibits advantages for industrial applications. Koban et al. measured
246
a reduction factor of 5.2 log10 steps of a Candida biofilm grown in a 96-microtitre
247
plate upon 10 min plasma treatment using the dielectric barrier discharge (DBD)
248
plasma device (19). As already stated in the introduction, the great advantage of
249
SMD plasma compared to DBD is that there is no direct contact to the surface
250
necessary and no current passes through the plate and the biofilm.
251
The main species which contribute to the inactivation of the biofilm using SMD
252
plasma in ambient air are reactive species and charged particles. As indicated
253
earlier, the density of the charged particles which interact with the biofilm is low due
254
to excitation, dissociation, attachment and recombination processes on their way
255
from the electrode to the sample (25). Therefore the main impact on the biofilm
256
results from the produced reactive species: Measurements of the produced ozone in
257
the device after a treatment time of 60s showed a value of approximately 500 ppm.
258
The NO2 concentration was approximately 3 ppm. Concerning the ozone
259
concentration, Kowalski et al. reported that 15 s with 1500 ppm of ozone were 12
260
necessary for a 3 log10 reduction of E. coli plated on agar (21). In our study, the
261
reduction of even Candida albicans (and E.coli (data not shown)) is faster with a
262
smaller amount of ozone. This leads to the conclusion that the produced plasma
263
agents have synergetic effects and therefore result in a faster inactivation of
264
pathogens. Various inactivation experiments with bacteria showed that the UV
265
photons (gained by treating a planktonic bacteria sample with plasma filtered by a
266
quartz glass) do not have any bactericidal property up to 120 s (data not shown).
267
Therefore we conclude that the UV radiation alone does not contribute to the
268
fungicidal property as well. The electric field produced by the plasma can cause
269
stress on the cell wall of fungi. However, we could not observe any inactivation of
270
fungi due to the electric field produced by the plasma (data not shown). As stated
271
earlier the electrical current through the sample is negligibly small.
272
In recent years opportunistic pathogens from the genera Candida are the main cause
273
of fungal infections, especially as biofilm-mediated infections (4, 23). Therefore
274
contamination of inanimate surfaces by Candida may lead to the formation of
275
Candida biofilm and if left untreated or not disinfected it may become a risk in health
276
care services. Candida biofilms are more resistant to standard antifungal agents than
277
free-floating Candida cells (7). This study supports this statement as we clearly
278
showed that planktonic Candida cells can be easier killed using shorter plasma
279
treatment times than biofilm growing cells. Therefore we conclude that not only the
280
amount of cells per ml limits the inactivation but also the growing conditions:
281
planktonic or biofilm. Single Candida cells can survive for a longer time embedded in
282
a biofilm and grow with slower rates, and their metabolic and antifungal responses
283
are often different from their planktonic counterpart. Furthermore we were able to
284
show that the disinfectant ethanol (70%) was less effective in killing the Candida
285
biofilm which is in agreement with already published data (30). Théraud et al. 13
286
demonstrated that the overall efficacy of antiseptics and disinfectants on yeast
287
isolates is different when the cells are grown in planktonic or biofilm conditions (30).
288
Eight out of nine agents investigated (10% iodine polyvinylpyrrolidone, 2% sodium
289
lauryl sufate, 0.5% and 0.05% chlorhexidine digluconate, 3% hydrogen peroxide,
290
70% ethanol, 0.5% alkylamine, 0.5% alkylamine + sodium hypochlorite and UV
291
radiation at 365 nm were ineffective against biofilm growing Candida (30).
292
Chlorhexidine digluconate at a concentration of 0.5% was the only antifungal agent
293
which was active both on planktonic Candida cells suspension and biofilm growing
294
Candida albicans. Today chlorhexidine digluconate is used as a “gold standard”
295
antiseptic in the oral cavity, but some risks must be considered (12). Chlorhexidine
296
has a mutagenic potential, possesses a neurotoxic side effect and can induce
297
hypersensitiveness (22). The Japanes Ministry of Health recommended in 1984 to
298
avoid the use of chlorhexidine. Therefore the long-term use of chlorhexidine has to
299
be questioned (1).
300
As stated in the introduction, the high antifungal resistance of biofilms is a multifactor
301
process where an antifungal drug with just one single mechanism of action is unlikely
302
to be effective (2). In contrast, the mechanism of action of cold atmospheric plasma is
303
different from that of an antifungal agent. Typically an antifungal drug acts using the
304
lock-and-key principle. This means that a specific antifungal agent has to fit in a
305
certain way inside or outside of a Candida cell to induce an antifungal effect. In
306
contrast cold atmospheric plasma produced by this device results in the generation of
307
reactive oxygen and nitrogen species as well as atomic O and N and hydrogen
308
peroxide interacting with water vapour. All these species are capable to induce
309
oxidative and radical damage of the biofilm immediately during the plasma treatment.
310
Therefore a rapid fungicidal disinfection is possible. In this case no specific
311
interaction is necessary to induce the antifungal effect. 14
312
Overall the use of cold atmospheric plasma for disinfection of inanimate surfaces
313
could lead to a major development preventing biofilm-associated Candida infections
314
in the future. Therefore the advantage of a concept of a contact-free application of
315
plasma to sterilize Candida biofilm contaminated surfaces (and items) in health care
316
settings might have a positive impact on preventing community-acquired and
317
nosocomial infections in the medical field, but also in foodstuff industry where time
318
saving is a critical point to achieve efficient disinfection in the future.
15
319
Acknowledgement
320
The excellent technical assistant of Judith Heider is gratefully acknowledged. Dr. Tim
321
Maisch was supported by a grant (M.TT.A.EXT00002) of the Max Planck Institute for
322
Extraterrestrial Physics, Garching, Germany.
323 324
16
325
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
329
Figure 1. A sketch of the plasma device.
330
a) The plasma device contains one SMD electrode and the sample to treat is placed
331
below the electrode. In this study the distance between the electrode and the sample
332
was fixed to 6 mm.
333 334
Figure 2. Spectra of the plasma produced by the SMD device
335
The spectrum of the produced SMD plasma was measured in front of the electrode at
336
a distance of 6 mm. The main UV components are in the wavelength range between
337
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
341
plasma
342
20 µl of serial diluted free-floating C. albicans suspensions according to the Miles and
343
Misra technique were applied to agar-plates and dried for 45 min. Cold atmospheric
344
plasma treatment was performed using different time intervals. Surviving colonies
345
were counted 24 h later. Black dotted line: baseline of viable C. albicans per ml; red
346
dotted line: reduction of three log10 steps of viable C. albicans (99.9%); green dotted
347
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
351
C. albicans biofilm was grown on the surface of 6-well-plates for 24 h. After washing
352
the biofilm to remove free floating cells, the biofilm was dried for 30 min. Cold
353
atmospheric plasma treatment was done using different time intervals [20sec to 18
354
10min].
A CFU assay was performed immediately after the plasma treatment.
355
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
357
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
362
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