22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Effects of non-thermal microwave argon plasma on bacterial pathogens and skin
cells
E. Sysolyatina1,2, I. Trusova1, M. Vasiliev3, E. Petersen1, A. Melerzanov1, O. Petrov3, E. Fortov3, A. Gintsburg2
and S. Ermolaeva2
1
2
Moscow Institute of Physics and Technology, Moscow, Russia
Gamaleya Research Centre of Epidemiology and Microbiology, Moscow, Russia
3
Joint Institute for High Temperatures RAS, Moscow, Russia
Abstract: Microwave argon plasma is bactericidal for Staphylococcus aureus and
Pseudomonas aeruginosa, while it shows the absence of cytotoxicity for keratinocytes and
human fibroblasts up to 140 h post treatment and stimulates the proliferation of
keratinocytes starting at 60 h post treatment.
Keywords: non-thermal argon plasma, infected wounds, fibroblasts, keratinocytes
1. Introduction
Treatment of infected wounds and ulcers is still a
challenge for medicine.
Usage of antibiotics and
antibacterial toppings, remaining the main method of
treatment of infected wounds and ulcers, is often
inefficient due to both the spread of resistance among
pathogens and the decline of the immune status of
patients. In this regard the search of new methods having
both antibacterial and wound-healing effect remains
actual. One of the most new and promising methods in
this direction is the use of non-thermal atmospheric
pressure plasma (NTP).
In the last five years, it was shown that NTP possesses
antibacterial and wound healing properties [1-3]. An
absence of toxic effects upon NTP treatment of chronic
wounds and ulcers was shown in clinical trials on human
volunteers [1]. An increase in wound closure rate was
demonstrated in experiments with animals [2, 3].
The aim of this work was to evaluate the influence of
microwave argon plasma onto bacterial and eukaryotic
cells, coexisting in infected wounds.
2. Materials and Methods
The microwave generator of argon plasma “Plasma
200” (JIHT RAS, Russia) was used in all experiments
(Fig. 1). It is based on the source of microwave
electromagnetic waves with a frequency of 2.45 GHz.
The plasma torch is an axial electrode, which is placed in
a grounded metal cylinder with a nipple, supplying of
working gas (argon). The flow of microwave plasma is
formed, when argon is pumped through the discharge gap
at a rate of 1 - 10 /min, and feeding power from the
magnetron is from 10 to 200 W. The diameter of the
plasma jet is 16 mm. The rate of argon flow determines
the length of the plasma flow, it could vary from 15 to
45 mm.
Bacterial pathogens, that often colonize and infect
wound surfaces, Staphylococcus aureus ATCC 25923 and
Pseudomonas aeruginosa PA103 with multiple antibiotic
P-III-10-29
Fig. 1. Plasma torch of plasma generator “Plasma 200”.
resistance were used as model organisms. Bacteria were
plated onto Petri dishes in a concentration of 105 cells/ml
and then were treated by argon plasma for 60 and 120 sec
in a distance of 7 and 20 mm from plasma torch and the
speed of gas flow of 1, 3, 6, 9 l/min. The amount of
survived bacteria was counted in 24 h incubation (37 °C).
For the estimation of cytotoxic effect and the influence
of NTP on cells proliferation human lung fetal fibroblasts
and human keratinocytes HaCat were used. They form
wound surface and play the key role in wound healing.
Cells were cultivated in DMEM/F12, containing
10% vol/vol
fetal
bovine
serum
(FBS)
and
2 mM l-glutamine. Culture cells (20000 cells/well) were
exposed to argon plasma for 2, 3 and 5 min in a distance
of 7 mm (direct treatment). Only 1 mm of medium was
remained in wells before treatment, and immediately after
that the medium with FBS was added. Additionally, the
medium, containing FBS and without FBS, was treated
and then added to cells (indirect treatment).
The cytotoxicity and the proliferation index (cell index)
was evaluated by means of xCELLigence (Roche).
1
3. Results
The bacterial load of gram-negative P. aeruginosa
decreased up to 4 logs after 120 sec and 7 mm distance to
plasma torch (Fig. 2).
2 min
5 min
control
Fig. 4. The cell index of fibroblasts after direct plasma
treatment.
The arrow shows the time of plasma
application.
Fig. 2. Bactericidal effect of NTP to P. aeruginosa. The
top line – at a 20 mm distance, the low line – at 7 mm.
The speed of gas flow is 1, 3, 6, 9 l/min.
The bactericidal effect of NTP to gram-positive
S. aureus was less pronounced: only 100 - 1000 reduction
of initial bacterial load was observed at the distance of
7 mm and 120 sec time treatment.
The direct and indirect treatment of human keracytes
HaCat during 120 and 300 sec did not induce immediate
cell death (Fig. 3), though in 60 h after plasma application
the delay of cells prolifiration and plato stage was
observed.
Fig. 3. The cell index of HaCat cells after plasma
treatment.
The arrow shows the time of plasma
application.
Human fibroblasts showed the increasing of cell
proliferation in 60 h after 2 min and 5 min the direct and
indirect plasma treatment (Fig. 4 and 5). In control wells
the slow cells death was observed.
4. Conclusions
Taking into account that fact, that healing of infected
wounds requires pathogens elimination and the creation
of favourable conditions for skin cells, pathogenic
microorganisms should be killed without harm for
fibroblasts and keratinocytes. We used argon plasma
source “Plasma 200” to investigate its bactericidal effect
2
medium 2 min
medium 5 min
contro
Fig. 5. The cell index of fibroblasts after indirect plasma
treatment.
The arrow shows the time of plasma
application.
and potential cytotoxicity to human cells.
It was established, that:
- Argon microwave plasma was bactericidal for
P.aeruginosa and S.aureus, though the last grampositive bacteria was more resistant to plasma
treatment, that is in agreement with our previous data
[2].
- The treatment of human fibroblasts and keratinocytes
(HaCat) cells for 2 and 5 min did not cause cell death
either immediately or after 60 h after plasma
application.
- Direct (treatment of cells) and indirect (treatment of
medium) plasma application induced the increase of
fibroblast’s proliferation.
5. Acknowledgments
This work was supported by RFBI grant №15-04-06932а
6. References
[1] G. Isbary, G. Morfill, H.U. Schmidt, et al. “A first
prospective randomized controlled trial to decrease
bacterial load using cold atmospheric argon plasma
on chronic wounds in patients”. Br. J. Dermatol.,
163, 78-82 (2010)
[2] S.A.
Ermolaeva,
A.F.
Varfolomeev,
M.Y. Chernukha, et al. “Bactericidal effects of nonthermal argon plasma in vitro, in biofilms and in the
P-III-10-29
[3]
animal model of infected wounds”.
J. Med.
Microbiol., 60, 75-83 (2011)
C. Bender, A. Kramer and N. Hübner. “Synergistic
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