Background: Multidrug-resistant bacteria and biofilms (i.e. microbial communities encased in a protective matrix) pose a threat to human health worldwide. Best medical care with antibiotics increasingly fails to cure infections, stressing the need for more effective treatments.
Aim: Validation of plasma-activated water (PAW) as an antibacterial treatment. Cold plasma-activation is a physical process to enrich water with energetic particles, such as reactive oxygen and nitrogen species, which has the potential to treat wound infections without using antibiotics.
Hypothesis: PAW is a potent antibiofilm agent that significantly reduces biofilm load of ESKAPE pathogens.
Methods: In ESKAPE pathogens the minimum inhibitory concentration of PAW was determined and the antibiofilm activity of PAW was assessed in the AlamarBlue viability assay in vitro. Focussing on methicillin-resistant Staphylococcus aureus (MRSA), the antibiofilm activity of PAW was determined in a 3D-biofilm model with real-time imaging (bioflux flow system), in a wound infection model using an artificial dermis and in an in vivo infection model using Galleria mellonella larvae. Cytotoxicity of PAW was evaluated in human keratinocytes (HaCaT) by the lactate dehydrogenase assay. Statistical analysis: one-way ANOVA.
Results: PAW inhibited ESKAPE pathogens at 25% concentration (in media) and killed 99% of biofilm for all bacteria tested (p<0.0001). Results were confirmed by real-time imaging, showing substantial killing and partial removal of 3D-MRSA biofilms under flow conditions. In a wound infection model using an artificial dermis, PAW treatment resulted in a 4 log10 CFU reduction of MRSA biofilms compared to untreated controls (equal to 99.99% biofilm eradication). Galleria mellonella larvae infected with MRSA and treated with PAW survived the deadly infection compared to infected, untreated controls. PAW showed no toxicity in human keratinocytes.
Conclusions: PAW exhibited high efficacy against multidrug-resistant bacteria and biofilms. Cold plasma technology has the potential to produce effective antimicrobial treatments that could negate using conventional antimicrobial drugs, which are susceptible to resistance. PAW could serve as an antibiotic-free irrigation for wounds or surgical procedures.