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. 2023 Sep 19:6:100154.
doi: 10.1016/j.bioflm.2023.100154. eCollection 2023 Dec 15.

Plasma activated water as a pre-treatment strategy in the context of biofilm-infected chronic wounds

Heema K N Vyas  1   2 , Binbin Xia  1 , David Alam  1 , Nicholas P Gracie  3 , Joanna G Rothwell  3 , Scott A Rice  4   5 , Dee Carter  2   3 , Patrick J Cullen  1 , Anne Mai-Prochnow  1
Affiliations

Plasma activated water as a pre-treatment strategy in the context of biofilm-infected chronic wounds

Heema K N Vyas et al. Biofilm. .

Abstract

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of plasma activated water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular reactive oxygen and nitrogen species accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

Keywords: Antiseptics; Biofilm; Chronic wounds; Escherichia coli; In vitro; Plasma activated water.

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Conflict of interest statement

Patrick J. Cullen is the CEO of PlasmaLeap Technologies, the supplier of the plasma power source and BSD reactor utilised in this study.

Figures

Fig. 1
Fig. 1
PAW generation and treatment of biofilms. (A) Schematic representation of the BSD reactor used to generate the PAW with photograph of PAW generation (left) and control generated without plasma discharge (right). (B) PAW was added directly onto the 24 h E. coli biofilms formed on either the plastic well surface (left) or a fixed keratinocyte monolayer (in vitro biofilm-skin epithelial cell model; right). PAW was applied for 15 min as a pre-treatment, biofilms were then challenged with clinically relevant topical antiseptics routinely used for the treatment of chronic wounds.
Fig. 2
Fig. 2
PAW pre-treatment greatly increases the in vitro antimicrobial susceptibility of E. coli biofilms. Effect on biofilm viability of PAW + PHMB/PI/MediHoney ( × ), control + PHMB/PI/MediHoney (■), PAW (purple dotted line), and control (blue dotted line) on (A–C) plastic and (D-F) keratinocyte monolayer is demonstrated. Data represents mean ± SEM; n = 3 biological replicates, with 2 technical replicates each.
Fig. 3
Fig. 3
RONS primarily contribute to the anti-biofilm activity of PAW. (A) PAW with the addition of tiron, uric acid, and ascorbic acid to scavenge superoxide, ozone, and general ROS, respectively. Removal of these species from the PAW resulted in significant increases in biofilm viability when compared to biofilms treated with unmodified PAW. (B) Intracellular ROS was measured using DCFDA staining (left) and intracellular RNS was measured using DAF-FM staining (right). Biofilms treated with PAW have significantly higher accumulation of RONS compared to biofilms treated with control. Data represents mean ± SEM, * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001), and **** (P ≤ 0.0001); n = 3 biological replicates, with 2 technical replicates each.
Fig. 4
Fig. 4
PAW disrupts the integrity of the E. coli cell membrane. (A) Membrane depolarisation was determined for E. coli incubated with 2 μmol/L DiSC3(5). E. coli cells were challenged with PAW (▾), control (i.e., a negative control of Milli-Q water subjected to air flow without plasma discharge, ●), and gramicidin (positive control) (■). (B) Inner membrane permeability was determined by the addition of 1.5 mM ONPG to the E. coli cells and cytoplasmic b-galactosidase leakage was determined with o-nitrophenol detection (405 nm) upon challenge with PAW (▾), control (●), and gramicidin (positive control) (■). (C) Outer membrane permeability was evaluated by incubating E. coli with 10 μM NPN and subsequently challenged with PAW (▾), control (●), and colistin (positive control) (×). NPN uptake was expressed as a percentage (%). (D) SEM was utilised to visualise morphological changes induced by PAW, particularly on E. coli biofilm cell membranes. Biofilms were treated for 1 or 15 min with PAW and control. Gramicidin and colistin positive controls were also included. Morphological changes included cell flattening (black arrows), cell membrane blebbing (white arrows), and collapsing/concaving inward of individual cell ends (grey arrows).
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