Treatment of Antifungal Resistant Candida Biofilms And Clinical
Dermatosis Using A Direct-Current, Atmospheric-Pressure Cold Plasma
Micro-Jet
Peng Sun, Shuang Yu, Haiyan Wu, Jue Zhang, Jing Fang
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Yi Sun, Wei Liu
Department of Dermatology and Venereolgy, Peking University First Hospital, Beijing, China
WeiDong Zhu
Saint Peter’s College, Jersey City, New Jersey, USA
Abstract: In this paper, a direct-current, atmospheric-pressure, He/O2 (2%) cold plasma microjet (PMJ) was
applied to Trichophyton rubrum (the most frequent dermatophyte), Candida spp (which causes thrush and vaginal
candidiasis) and three other types of fungi. Effective inactivation was achieved both in air and in water within 5
min of plasma treatment [1]. Ten dermatosis biofilms (C. glabrata ,C. albicans, C. krusei) were also performed,
effective inactivation was also observed. The inactivation was verified by a XTT test. Severely deformation of the
biofilms was observed after PMJ treatment through SEM. The existence of ·OH, ·O2-, 1O2 was verified by
Electron Spin Resonance (ESR) spectroscopy. Optical emission spectroscopy also showed strong atomic oxygen
emissions in air. The sessile minimal inhibitory concentrations (SMICs) of three antifungal medicines against the
Candida spp. biofilms were decreased to 2-6 fold dilutions in PMJ treated group in comparison with untreated
controls. This novel approach holds potential in clinical treatment of dermatosis.
Keywords: Antifungal; Candida biofilm; Electron Spin Resonance; Non-thermal plasma jet; Minimal
inhibitory concentrations
1. Introduction
Candidiasis, caused by Candida species, is the most
common fungal infection in humans [2, 3]. Beside
invasive disease including candidemia and
candidiasis in deep-seated organ, mucocutaneous
disorders such as oral candidiasis, vaginal and
vulvovaginal candidiasis, have become the major
clinical problem [4, 5]. Although Candida species
are the microorganism exhibiting planktonic
unicellular form, filamentous growth or complex
multicellular structure is observed mainly in the
infected tissues [6]. The structured microbial
communities attached to surfaces and encased within
a matrix of exopolymeric materials is now defined as
biofilms [6, 7], which can form on various implanted
medical devices such as vascular and urinary
catheters, joint prostheses, cardiac valves, artificial
vascular bypass devices, and those being topically
used including contact lens and dentures[7-10]. The
biofilm with the complex structure is resistant to
both host defense and commonly used antifungal
drugs [7, 11, 12]. The cells of Candida species,
dropped constantly from the structured microbial
communities, can spread and further cause
antifungal treatment failure, devices failure or
persistent infections [9]. And the contribution of
biofilm to the increasing of candidiasis has been now
confirmed [13]. Thereby, to develop novel
approaches inactivating candidal biofilm has great
significance in treating candidiasis, especially those
related to Candida biofilm.
Compared with traditional therapeutic methods, nonthermal plasma as a physical method could provide a
more economic and effective method. In addition,
the simple generation system of plasma and its
gaseous form provide the possibility to penetrate in
inhomogeneous surfaces, cavities and fissures down
to the micrometer scale, and allow combining this
technique with minimally invasive surgery or with
antimicrobial chemotherapy. In addition to several
papers reporting the plasma fungicidal ability against
Candida albicans [14, 15], our group [1] have
shown that non-thermal plasmas can effectively
inactive the strains of Candida species including
fluconazole-resistant C. albicans, Candida glabrata
and Candida krusei. We also found that the
antifungal susceptibility of Candida species strains
to common antifungal drug is enhanced after they
were treated with plasma. These results indicated
that non-thermal plasma could be a potential
treatment method or supplementary treatment
method for candidiasis. In the present study, we
further investigated the fungicidal effect of nonthermal plasma on Candida biofilms (which are
considered more difficult to inactive than their
planktonic counterparts), and its effect on antifungal
susceptibility of Candida biofilms to common
antifungal drug.
the biofilms lost their original morphologic
characteristics and were degraded to clusters of cell
fragments, which included rupture, distortion and
shrinking of the outer layer. This degradation led to
the leakage of cell inclusion. Above morphological
changes of cell wall are considered detrimental for
the survival of the fungi, which were not seen in the
negative control samples where only He/O2 flow
was introduced. In summary, highly structured
community of Candida biofilm was destroyed by
PMJ gradually.
2. Materials and Methods
Ten strains of Candida species used in this study
were isolated and stored in our laboratory as shown
in Tab 1. RPMI-1640 medium was used to resuspend the pellet and adjust the final density to
1.0×106 cells/ml for all strains. One hundred
microliter of prepared suspension was pipetted into
selected wells of 96-well microtiter plates and every
replication was separated by an empty well. After
incubating statically for 24 hours at 37℃, the
biofilm was formed.
3. Results
Fungal elements embedded in Candida biofilm
were severely damaged after treatment with
plasma
From SEM, healthy yeast cell and pseudohyphae
with smooth surfaces were observed in biofilm
treated with He/O2 gas flow (without PMJ), as
shown in Fig1-a. Samples treated with PMJ for 10s
show crude shape of sessile cells and some fractured
cells (Fig1-b). When the treatment time was
extended to 20s, deformed and ruptured cells were
observed (Fig1-c). Noticeably, the majority of
components were fragile, fissile yeast cells and
pseudohyphea after 30s PMJ treatment (Fig1-d).
When the treatment time reached to 1min (Fig1-e),
Figure.1. A picture of plasma microjet (PMJ)
Enhancement of fungicidal effect of common
antifungal drugs on plasma-treated Candida
biofilm
The minimal inhibitory concentrations (MICs) of
amphotericin B, fluconazole, and caspofungin
against the planktonic cells of above-mentioned 10
strains were performed.
The SMICs value of fluconazole, amphotericin B,
and caspofungin for the groups treated with PMJ
(treatment with plasma for 10s, 20s and 30s) showed
a significant reduction, compared with those for the
untreated candidal biofilm. After being treated with
plasma for 20s or 30s, small part of the Candida
species cells in the Candida biofilm were survived
while most of them were inactivated, as shown by
incubation of the plasma-treated biofilm in
RPMI1640 medium for 24h at 37℃. Since being
completely inactivated within 1 min, the antifungal
activity of the common antifungal drugs against
those Candida biofilm treated with plasma for 60s
was not tested. Detailed information about the
antifungal results will be discussed on the
conference.
[2] Concia E, Azzini AM, Conti M. Epidemiology,
incidence and risk factors for invasive candidiasis in
high-risk patients. Drugs 2009;69:5-14.
Free radicals were generated by He/O2 (2%)
[4] Rowen JL. Mucocutaneous candidiasis. Semin
Perinatol 2003;27:406-13.
non-thermal plasma
Since we showed in the previous study [1] that
He/O2 (2%) cold plasma inactivated the plantonic
cells of Candida species in both air and water via
partly producing reactive oxygen species (ROS), we
also observed that [16] the oxidative stress pathway
of Eukaryotic cells Saccharomyces cerevisiae could
lead to their hypersensitivity to plasma treatment. It
is supposed that ROS generated by plasma in the
present study contribute partly to the damage of
candidal biofilms. Further ESR assaying showed
·OH, ·O2-, and 1O2 were detected in He/O2 (2%) cold
plasma treatment, confirming above inference. The
ROS including ·OH, ·O2-, and 1O2 were proven to
cause lipids and proteins of cytomembrane suffering
from oxidative damage which lead to impediment of
ion transition. Furthermore, the oxidative molecular
could combine with DNA causing additional cell
damage [17].
Funding
This research is supported by National Natural
Science Foundation of China (30970131) and
Beijing Natural Science Foundation (7102149) to Dr.
Wei Liu, and is also sponsored by Bioelectrics Inc.
(U.S.A.), MST Program of International Science and
Technology
Cooperation
(under
Grant
#
2009DFB30370: ‘‘Cold Plasma induced biological
effect and its clinical application studies’’) and
National
Basic
Research
Program
(No.
2007CB935602)
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Figure. 2. SEM results of SC5314 biofilm before and after plasma treatment, from left to right, the magnificent scale increases from×2000
to ×22000: (a), a negative control treated with He/O2 flow; (b), that treated with PMJ for 10 seconds; (c), that treated with PMJ for 20
seconds; (d), that treated with PMJ for 30 seconds; (e), that treated with PMJ for 60 second
Tab 1 Ten strains of Candida species used in this study
Isolate
BMU02971
BMU03213
BMU04801
SC5314
ATCC6258
BMU05102
BMU05137
BMU00271
BMU01689
BMU04388
Source
Pharynx
Oral
mucosa
Oral
mucosa
Blood
Sputum
Oral
mucosa
Oral
mucosa
Blood
Knee
Intraperitoneal
fluid
Species
C.albicans
C.albicans
C.albicans
C.albicans
C..krusei
C. krusei
C. krusei
C.glabrata
C.glabrata
C.glabrata