Plasma activated water pre-treatment substantially enhances phage activity against Proteus mirabilis biofilms

Abstract

The ongoing antimicrobial resistance crisis has incentivised research into alternative antibacterial and antibiofilm agents. One of them is plasma-activated water (PAW), which is produced by exposing water to a cold plasma discharge. This process generates a diverse array of reactive oxygen and nitrogen species (ROS/RNS) with antimicrobial properties. Another intensively studied class of alternative antimicrobials are bacteriophages, attracting attention due to their specificity and strong antibacterial activity. As combinations of different types of antimicrobials are known to often exhibit synergistic interactions, in this study we investigated the combined use of cold atmospheric-pressure plasma-activated water and the bacteriophage vB_PmiS_PM-CJR against Proteus mirabilis biofilms as a potential option for treatment of catheter-associated urinary tract infections (CAUTIs). We compared the effect of two cold plasma discharge setups for PAW production on its antimicrobial efficacy against P. mirabilis planktonic and biofilm cultures. Next, we assessed the stability of the phage vB_PmiS_PM-CJR in PAW. Finally, we tested the antimicrobial activity of the phages and PAW against biofilms, both individually and in combinations. Our findings demonstrate that the combination of PAW with phage is more effective against biofilms compared to individual treatments, being able to reduce the number of biofilm-embedded cells by approximately 4 log. We were also able to show that the order of treatment plays an important role in the anti-biofilm activity of the phage-PAW combination, as the exposure of the biofilm to PAW prior to phage administration results in a stronger effect than the reverse order. This research underlines PAW's ability to potentiate phage activity, showcasing a considerable reduction in biofilm viability and biomass. Additionally, it contributes to the growing body of evidence supporting the use of phage-based combinatorial treatments. Overall, this sequential treatment strategy demonstrates the potential of leveraging multiple approaches to address the mounting challenge of antibiotic resistance and offers a promising avenue for enhancing the efficacy of CAUTI management.

Keywords: Bacteriophage; Biofilms; Cold plasma; Phage; Plasma activated water; Proteus mirabilis; Urinary tract infections.

Fig. 1

Experimental setup for cold plasma generation. (A) Spark discharge setup with a 4 kV power supply connected to a high voltage electrode, producing a plasma plume directed at the target surface, and (B) Glow discharge configuration utilizing a 2 kV power supply with the high voltage electrode and associated plasma directed towards a liquid medium, both systems grounded through a secondary electrode. This schematic illustrates the differing electrical arrangements and plasma generation methods for experimental applications. Adapted from Lu et al., 2017 [22].

Fig. 2

Experimental design for the investigation of the effect of combinatorial treatments with PAW and phage on P. mirabilis biofilms. Phage PM-CJR was added in the same growth medium that was used to grow biofilms (either LBB or artificial urine).

Fig. 3

Identification and quantification of ROS/RNS generated in deionised water following treatment exposure to Spark and Glow discharges, hydrogen peroxide (A), nitrite (B), nitrate (C), and pH (D). The error bars represent the mean ± standard deviation from three biological replicates. Asterisks indicate significant differences between the relevant exposure times compared to baseline (time = 0), ∗ (p < 0.05), ∗∗(p < 0.01), ∗∗∗, (p < 0.001), and ∗∗∗∗ (p < 0.0001) using one-way ANOVA and Dunnett's post-test analysis (n = 3).

Fig. 4

Bactericidal effects of Spark and Glow discharge generated PAW against P. mirabilis in planktonic (A, B) and biofilm (C, D) states. The error bars represented the mean ± standard deviation from three biological replicates. Asterisks indicate significant differences between the relevant exposure times, ∗ (p < 0.05), ∗∗(p < 0.01), ∗∗∗, (p < 0.001), and ∗∗∗∗ (p < 0.0001) using one-way ANOVA and Dunnett's post-test analysis (n = 3).

Fig. 5

Impact of chemical scavengers on the antimicrobial activity of Spark 30 (A) and Glow 30 (B) PAW treatments against P. mirabilis. The bars represent the mean bacterial count following treatment with scavengers targeting hydrogen peroxide (sodium pyruvate), singlet oxygen and other longer-living ROS (L-histidine), ozone (uric acid), superoxide anions (O₂⁻) (Tiron), and nitric oxide (haemoglobin). The error bars represent the mean ± standard deviation from three biological replicates, asterisks indicate significant differences between the control (bacteria only) and treated groups, ∗ (p < 0.05), ∗∗(p < 0.01), ∗∗∗, (p < 0.001), and ∗∗∗∗ (p < 0.0001) using one-way ANOVA and Dunnett's post-test analysis (n = 3).

Fig. 6

Stability of phage PM-CJR in Spark 30 (●) and Glow 30 (■) discharge treated PAW. The error bars represent the mean ± standard deviation from three biological replicates, asterisks indicate significant differences between the relevant exposure times compared to baseline (time = 0), ∗ (p < 0.05), ∗∗(p < 0.01), ∗∗∗, (p < 0.001), and ∗∗∗∗ (p < 0.0001) using one-way ANOVA and Dunnett's post-test analysis (n = 3).

Fig. 7

The combination treatment results of Spark 30 PAW and phage against the biofilm formed in LBB (top panels) and artificial urine (bottom panels). (A, C) Bacterial titre reduction. (B, D) Biofilm biomass reduction measured by crystal violet assay. PAW followed by phage treatment exhibited superior efficacy of reduction of bacterial cells within the biofilm than both the individual and phage followed by PAW treatment. Similarly, biofilm biomass decrease was most notable in the PAW followed by phage treatment group. The error bars represent the mean ± standard deviation from three biological replicates, asterisks indicate significant differences between the untreated (bacteria only) and treated groups ∗ (p < 0.05), ∗∗(p < 0.01), ∗∗∗, (p < 0.001), and ∗∗∗∗ (p < 0.0001) using one-way ANOVA and Tukey's post-test analysis (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)