Analysis of biological responses to plasma-treated sodium chloride solutions
Mareike A. Ch. Hänsch, Klaus-Dieter Weltmann, Thomas von Woedtke
INP - Leibniz Institute of Plasma Science and Technology, D-17489 Greifswald, Germany
Abstract: Increasing resistances of germs to established antiseptics demand a continuous
development of new antimicrobial agents. Therefore, atmospheric pressure plasma was
used to produce antimicrobial NaCl-solutions. Its short- and long-term germicidal effects
have been investigated by inactivation of Escherichia coli in batches. Liquid changes by
plasma exposure that correlate with bacteria inactivation have been monitored for nitrite,
nitrate, hydrogen peroxide and pH value. Acidified nitrite has been identified as key
molecule in biochemical mechanisms of short-term bactericidal effects.
Keywords: Atmospheric pressure plasma, surface DBD, antimicrobial, inactivation,
Escherichia coli, short- and long-term effects, sodium chloride solution, nitrite, nitrate,
hydrogen peroxide, acidification.
1. Introduction
Today, increasing resistance of microorganisms to
common available antibiotics and antiseptics is a major
problem in medicine to treat and prevent nosocomial
infections. Such infections lead to longer duration of
treatment, increasing mortality, and higher treatment costs
[1]. There are only a few therapy options still available.
Consequently, the finding of novel antiseptic agents is
strongly wanted.
In the past few years, atmospheric pressure plasmas
appeared as promising technology in the field of
disinfection [2]. Recent investigations have also
demonstrated that simple liquids like water and saline
solutions get germicidal properties by plasma treatment
[3, 4]. Such bactericidal effects are assumed to be caused
by a pH change to the acidic range and the formation of
low-molecular compounds such as nitrate, nitrite,
hydrogen peroxide and peroxynitrite [3-7].
This study was conducted to assess short- and longterm germicidal effects of plasma treated liquids. Plasma
generated low-molecular compounds were qualitatively
and quantitatively determined to detect changes in liquid
composition that correlate with antimicrobial effects.
Furthermore, to estimate the development of bacteria
resistance against plasma-treated liquid, re-exposition
experiments had been carried out.
2. Experimental Setup
In this study, experiments were performed by using a
ceramic based surface dielectric barrier discharged
(sDBD), which is schematically shown in Fig. 1. The
electrode system is mounted in a lit of a gas-tight chamber
that fits to a lower shell of a petri dish. Both together form
a gas-tight chamber.
A non-structured Silver / Platinum Conductor Paste
layer (d=35 µm) served as counter electrode and is placed
on the upper side of a dielectric ceramic disc (Ø 55 mm,
d=1.5 mm). The high-voltage (HV) electrode is set on the
bottom and consists of four concentric placed rings of
Silver / Platinum Conductor Paste. A protective dielectric
glass ceramic film covers the HV-electrode.
The sDBD is driven in atmospheric air by a sinusoidal
voltage with 6 kHz frequency. A maximum signal
amplitude of ~17 kV(peak-to-peak) is achieved by
amplification and transformation of a function generator
signal (Agilent 33120A).
Figure 1:
3D model of sDBD with petri dish containing a liquid
sample.
3. Methology
Basic bactericidal activity of plasma treated liquids
was assessed using EN 1040:2005 “Chemical
disinfectants and antiseptics-Quantitative suspension test
for the evaluation of basic bactericidal activity of
disinfectants and antiseptics-test method and requirements
(phase 1)” representing a European standard method for
establishing whether a chemical disinfectant or antiseptic
does or does not have a basic bactericidal activity.
Therefore, overnight cultures of Escherichia coli (K12)
NCTC 10538 were used as reference strain. Initially ~108
colony forming units per milliliter (cfu/ml) vegetative
bacteria were exposed to plasma-treated NaCl-solutions.
Plasma treatment time, application time after plasma
4. Results
108
initial concentration
107
E. coli in 5 mL
NaCl-Solution
Max
Min
cfu*ml-1
106
105
10
4
10
3
3 min plasma treated
NaCl-solution
min
max
106
105
104
103
102
detection limit
101
100
0
1
2
3
4
5
exposure time*min-1
Figure 3: Inactivation kinetics of E.coli by plasma treated NaClsolution immediately applied after plasma treatment (PT). All presented
data are means with max and min values resulting from at least 3
independent experiments.
By comparing the results of both approaches, it
becomes obviously that antimicrobial effects are depend
remarkably on changed liquid phase.
In order to determine the bactericidal stability of
plasma activated antimicrobial NaCl-solutions the time of
application to reference bacteria was 30 minutes delayed.
No bacteria inactivation was observed with 3 and 4
minutes plasma treated NaCl-solutions even if the
exposure time on bacteria was increased up to 60 minutes.
However, a combination of longer duration of plasma
treatment (up to 6 minutes) and exposure times (up to
60 min) resulted in 6-7 log10 bacteria growth reduction
and therefore in stable antimicrobial NaCl-solutions (see
Fig. 4).
3 min PT
108
4 min PT
5 min PT
6 min PT
107
106
cfu*ml-1
This work was conducted to determine bactericidal
effects of plasma treated NaCl-solutions and their
antimicrobial stability compared to direct sDBD treatment
on planktonic bacteria suspended in NaCl.
Direct sDBD treatment of Escherichia coli (K12) in
batch culture resulted in a 6-7 log10 reduction within 5
minutes plasma treatment time (Fig. 2). By comparison, 3
minutes plasma treatment were sufficient to generate
antimicrobial active NaCl-solutions which needed only 5
minutes exposure time to result also in 6-7 log10 reduction
in bacteria growth (Fig. 3).
initial concentration
107
cfu*ml-1
treatment, and exposure time to bacteria was
systematically varied to estimate the bactericidal effects.
For re-exposition experiments, NaCl-solution was treated
with ceramic sDBD to get an activated antimicrobial
probe liquid sample and the exposure time to bacteria was
adjusted to get a 50% growth reduction. Aliquots of
plasma treated bacteria suspension were spread on agar
plates and incubated at 37° C for ~18h. Surviving
colonies of bacteria were collected and recultivated in
culture media overnight. Then, they were prepared and reexposed to plasma-treated NaCl-solution. Such cycles
were repeated for at least ten times.
In order to detect changes in liquid composition that
correlate with antimicrobial effects, the generation of lowmolecular compounds by plasma treatment, such as
nitrite, nitrate, and hydrogen peroxide, were qualitatively
and quantitatively monitored. For that purpose plasma
treatment time and time of detection was systematically
varied. Nitrite and nitrate were determined by using
Spectroquant® test kids according to DIN EN 26 777 D10
and DIN EN 38405 D9. Hydrogen peroxide was detected
based on the reaction with titanyl sulfate in sulfuric acid
solution. For quantification of those molecules absorption
spectra for all reaction products were recorded with a
UV/Vis SPECORD® S 600 Spectrophotometer (Analytic
Jena GmbH, Jena, Germany) in corresponding absorption
ranges [3,5].
105
104
103
102
detection limit
101
100
0
10
20
30
40
50
60
exposure time*min-1
102
detection Limit
101
100
0
1
2
3
4
5
6
7
8
9
10
Figure 4: Inactivation kinetics of E. coli by 3-6 min plasma treated
NaCl-solutions applied 30 min after plasma treatment (PT). All
presented data are means with max and min values resulting from at
least 3 independent experiments.
Treatment Time [min]
Figure 2: Inactivation kinetics of E. coli suspended in NaCl-solution
by direct plasma treatment. All presented data are means with max and
min values resulting from at least 3 independent experiments.
To detect changes in liquid composition that correlate
with antimicrobial effects, plasma generated lowmolecular
compounds
were
qualitatively
and
quantitatively determined. The
experiments are shown in Fig. 5.
results
of
10
Nitrate
Nitrite
Hydrogen Peroxide
pH
100
these
8
6
pH
conc. mg*l-1
10
observed time frame. The amount of nitrite decreases
drastically within the first 30 minutes after plasma
treatment. Afterwards, nitrite concentration decline slow
but continuously.
Besides short- and long-term antimicrobial effects as
well as changes in liquid composition, the possible
development of acquired resistance of treated bacteria has
been estimated. Fig. 7 depicts the reduction factors (Rf)
of each treated bacteria generation by plasma activated
antimicrobial NaCl-solutions (1-4 minutes PT).
1
4
1 min PT
2 min PT
3 min PT
4 min PT
9
2
0,1
8
0
1
2
3
4
5
6
treatment time / min
Figure 5: Chemical changes in liquid composition by plasma
treatment. All shown data are means with standard deviation.
reduction factor
7
0
6
5
4
3
2
Surface DBD treatment of physiological NaCl-solutions
resulted in a time depending formation of nitrite, nitrate
and hydrogen peroxide molecules. Hydrogen peroxide
and nitrite have been formed only in very low
concentrations <10 mg/l, whereas, nitrate represents the
dominant species within the liquid phase. In line with the
changed liquid composition, surface DBD treatment also
resulted in a fast acidification from pH 7 to 3.
As 5 minutes plasma treated NaCl-solution excels in
stabile antimicrobial activity, liquid composition of 5
minutes plasma treated NaCl-solutions have been
monitored at frequent intervals after plasma exposure.
Fig. 6 represents the results of this systematic liquid
analysis.
Nitrate
Nitrite
Hydrogen Peroxide
100
1
0
1
2
7
4
3
5
9
6
8
number of treated bacteria generation
10
Figure 7: Reduction factors of each treated bacteria generation by
plasma activated antimicrobial NaCl-solutions (1-4 min PT, 5 min
exposure time) and the average reduction factors of all treated bacteria
generation. Data are shown as means with standard deviation.
PT for 2 and 3 minutes produced germicidal NaClsolutions with acceptable Rf of 5-6. Those Rf remain
stable for each treated bacteria generation. An increase of
PT time up to 4 minutes resulted in antimicrobial NaClsolutions with higher Rf up to ~ 9 which stayed stable for
the first 4 bacteria generations. Subsequent conducted reexposure experiments have shown unexpected
fluctuations in bacteria inactivation but they are
discontinuously and mainly in the range of the standard
deviations.
4. Summary
conc. mg*l-1
10
The present work describes results of a systematic
investigation of biological responses to plasma-treated
NaCl-solutions. In this preliminary analysis,
1
0.1
0
30
45
35
60
time after plasma treatment / min
90
Figure 6: Liquid composition at certain times after plasma treatment.
All shown data are means with standard deviation.
Plasma treatment of 5 mL NaCl-solution yielded in stable
nitrate and hydrogen peroxide concentrations over the
effectivity of plasma-treated NaCl-solutions
compared to direct sDBD treatment,
bactericidal stability of plasma activated
antimicrobial NaCl-solutions, and
changes in liquid composition
were considered to assess plasma-treated liquids as
antiseptic agents.
It is shown that non-thermal plasma can be used to
generate NaCl solutions with short- and long-term
germicidal effects. When using plasma above a liquid
reactive species interact with the liquid surface and lead
to a lower pH that is attributed to an enrichment of
nitrogen containing compounds such as nitrite and nitrate.
Surface DBD treatment also yielded in low hydrogen
peroxide concentrations. Therefore, antimicrobial plasma
effects are contributed to a changed liquid composition
and are mainly mediated by the liquid phase. As nitrite is
the sole detected variable parameter of monitored species
within the plasma treated liquid it seems to play a key role
at least in fast antimicrobial effects. Unfortunately, this
reaction mixture implies very complex reaction cascades
that further liquid analytics are needed to identify all
antibacterial reactive species.
Longer plasma treatment times are needed to generate
antimicrobial active NaCl-solutions with adequate longterm bactericidal activity, which dependents also on
longer exposure times to bacteria. With the used
analytical methods in this study, it was not possible to
identify clearly the chemical compound that is responsible
for lasting antimicrobial effects. As nitrate and hydrogen
peroxide are stable compounds within the plasma treated
liquids they need to be also considered as cause for
prolonged germicidal properties. Therefore, to identify
further reactive species or/and clarify ambiguous reaction
mechanisms more sophisticated methods are required.
In addition to above described analysis of plasma
activated antimicrobial NaCl-solutions, the possible
occurrence of secondary/acquired bacteria resistance has
been systematically investigated.
Re-exposure of plasma activated germicidal NaClsolutions to E. coli resulted in persistent antimicrobial
effects. Some temporary, discontinues fluctuations in
bacteria inactivation have been observed but no bacteria
resistance in particularly has been detected. Such
fluctuation can be caused by application-related
differences such as initial bacteria concentrations as well
as due to sensitivity of test procedure.
For future work, further investigations are required to
estimate the medical applicability of plasma treated
liquids that must include gram-positive and gram negative
as well as multi resistant germs.
5. Acknowledgement
Part of this work was realized within the joint research
project “Campus PlasmaMed” (grants no. 13N9779 and
13N11188). The financial support of the German
Federal Ministry of Education and Research is gratefully
acknowledged.
6. References
[1] ECDC-European Center for Disease Prevention and
Control, Annual Report of the European
Antimicrobial Resistance Surveillance Network
(ERAS-Net), Stockholm (2011)
[2] J. Ehlbeck et al., Journal of Physics D: Applied
Physics, 44, 013002 (2011)
[3] K. Oehmigen et al., Plasma Processes and Polymers,
8, 904 (2011)
[4] M. Traylor et al., Journal of Physics D: Applied
Physics, 44, 472001 (2011)
[5] K. Oehmigen et al., Plasma Processes and Polymers,
7, 250 (2010)
[6] J. L. Brisset et al., Plasma Chemistry and Plasma
Processing, 32, 655, (2012)
[7] Z. Machala et al., Plasma Processes and Polymers,
DOI: 10.1002/ppap.201200113 (2013)