22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Resistance of Pseudomonas aeruginosa biofilms to cold atmospheric pressure
plasma treatment is linked to the redox-active molecule phenazine
A. Mai-Prochnow1, M. Bradbury2, K. Ostrikov1,3 and A.B. Murphy1
1
CSIRO Manufacturing Flagship, Sydney, NSW, Australia
CSIRO Food and Nutrition Flagship, Sydney, NSW, Australia
3
Institute for Health and Biomedical Innovation, School of Chemistry, Physics and Earth Sciences, Queensland
University of Technology, Brisbane, QLD, Australia
2
Abstract: In this study we report on significant advances in understanding the response of
biofilms of P. aeruginosa to cold atmospheric plasma (CAP). Our study shows that CAP
has the potential to eradicate antimicrobial-resistant biofilm bacteria. We also discover that
low doses may lead to the surviving bacteria being more resistant to subsequent plasma
exposure. We provide strong evidence that the resistance is associated with production of
the redox-active pigment phenazine by the bacteria.
Keywords: Cold plasma, Biofilm, Pseudomonas, Resistance
1. Introduction
A number of studies show very promising results for
cold atmospheric plasma (CAP)-mediated killing and
removal of biofilms, including pathogenic bacteria such
as Pseudomonas aeruginosa [1, 2]. Biofilms are the
predominant mode of growth for bacteria. They are cell
clusters encapsulated by an extracellular matrix attached
to a surface. It is now widely recognized that more than
60% of all infections are caused by biofilm-forming
bacteria. These biofilm infections can become resistant to
treatment with traditional antibiotics and often develop
into a chronic state.
2. CAP effect on biofilms
Biofilms were grown in a CDC biofilm reactor [3] and
treated using the kINPen med (Neoplas tools GmbH,
Greifswald, Germany) [4]. The kINPen hand-held nozzle
was connected to a base unit with a gas feeding bottle.
Argon was used as a feeding gas and plasma pulses are
generated at a frequency of 1.82 MHz at a gas flow rate of
4.2 slm. After a range of treatment times (1, 3, 5 and
10 min), biofilms were subjected to viable cell counts
(Table 1) and confocal microscopy (Fig. 1) to investigate
the effect of CAP on biofilms. The untreated control
biofilms reached cell numbers of 4.5 x 105 CFU per ml
(Table 1). The gas control showed a slight decrease in
CFU numbers (3.9 x 104 per ml), possibly due to a drying
effect from the gas flow. Plasma treatments after 1 and
3 minutes produced similar results as the gas control with
a reduction of viable cell numbers by only 1 log compared
to the control. However, no viable cells were detected
after 5 min of plasma treatment (>5 log reduction),
indicative of complete biofilm removal (Table 1).
In addition to CFU numbers, a second set of biofilms
grown on glass coupons was observed with confocal laser
scanning microscopy. The untreated control showed
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Table 1. CFU counts of P. aeruginosa biofilms cells after
argon plasma treatment.
Treatment
CFU
(ml-1)
counts
Untreated
4.5 (± 0.6) x 105
control
Gas control
3.9 (± 0.6) x 104
1 min CAP
1.8 (± 0.3) x 104
3 min CAP
2.1 (± 0.4) x 104
5 min CAP
*
10 min CAP
*
* indicates counts were below detection limit (1 x 102)
attached,
viable
cells
forming
microcolonies
approximately 10 µm thick (Fig. 1A). The gas control
showed similar size microcolonies. A few single dead
cells were observed in the gas control, presumably due to
a drying effect from the gas flow. After only 1 min of
plasma treatment, microcolonies were significantly
smaller and some dead cells appeared.
Large
microcolonies are removed after only 3 min of plasma
treatment with most of the biofilm removed after 5 min
and no cells left after 10 min (Fig. 1).
3. Bacterial resistance to CAP
To investigate a possible bacterial resistance
mechanism to plasma treatment, a lawn of P. aeruginosa
cells was spread onto agar plates and exposed to argon
plasma treatment. Surviving bacterial colonies from
plasma and gas control plates were selected for whole
genome sequencing. Comparison between surviving cells
and control cells revealed 10 distinct polymorphic
regions, including four belonging to the redox-active,
1
P aeruginosa biofilms of about 15 µm thick can be
completely eradicated using a 10 min plasma treatment
with a kINPen med. However, a low dose (3 min) plasma
treatment allowed for some cells to survive, which
subsequently exhibited a higher resistance to further
plasma treatment. DNA sequencing revealed that this
higher resistance is related to mutations in phenazine
biosynthesis genes. Phenazines were previously shown to
increase resistance to antibiotics [9] and promote
anaerobic survival via extracellular electron transfer [10],
and are involved in protection to environmental oxidative
stress [11]. To our knowledge this is the first report
describing a role for phenazines in the response to CAP.
Fig. 1. Confocal images of P. aeruginosa biofilms cells
after argon plasma treatment stained with BacLight
Live/Dead. Viable cells are stained green and dead cells
are stained red. A) Untreated control, B) 10 min argon
gas control, C) 1 min plasma, D) 3 min plasma, E) 5 min
plasma and F) 10 min plasma treatment.
antibiotic pigment phenazine.
Subsequently, the
interaction between phenazine production and CAP
resistance was demonstrated in biofilms of transposon
mutants disrupted in different phenazine pathway genes,
which exhibited significantly altered sensitivity to CAP
(Fig. 2). Mutants whose ability to produce phenazine is
compromised (ΔphzD, ΔphzE and ΔphzF) show
significantly reduced survival under CAP exposure.
Fig. 2. Survival of P. aeruginosa PAO1 phenazine
mutants upon plasma exposure. Percentage CFU counts
of Δphz mutant biofilms exposed to 3 min argon plasma.
* denotes statistically significant difference (p ≤ 0.05)
4. Discussion
Pathogenic bacteria exhibiting resistance to antibiotic
agents have become an area of great concern. Whilst
antibiotic resistance in bacteria occurs naturally to some
extent [5, 6], inappropriate use and overuse of these drugs
has increased this process at an alarming rate [7]. In
particular biofilms, which account for over 60% of
infections, show a high degree of resistance [8].
Results of our study clearly demonstrate that
2
5. Acknowledgements
We would like to thank Debra Birch from the
Microscopy Unit, Faculty of Science, Macquarie
University (Sydney, Australia) for help with biofilm
imaging and the Ramaciotti Centre for Genomics
(UNSW, Sydney, Australia) for performing the
ILLUMINA MiSeq sequencing.
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