Delivery and quantification of hydrogen peroxide generated via cold atmospheric pressure plasma through biological material

The ability of plasma-generated hydrogen peroxide (H 2O 2) to traverse bacterial biofilms and the subsequent fate of the generated H 2O 2 has been investigated. An in vitro model, comprising a nanoporous membrane impregnated with artificial wound fluid and biofilms of varying maturity was treated with a helium-driven, cold atmospheric pressure plasma (CAP) jet. The concentration of H 2O 2 generated below the biofilms was quantified. The results showed that the plasma-generated H 2O 2 interacted significantly with the biofilm, thus exhibiting a reduction in concentration across the underlying nanoporous membrane. Biofilm maturity exhibited a significant effect on the penetration depth of H 2O 2, suggesting that well established, multilayer biofilms are likely to offer a shielding effect with respect to cells located in the lower layers of the biofilm, thus rendering them less susceptible to plasma disinfection. This may prove clinically significant in the plasma treatment of chronic, deep tissue infections such as diabetic and venous leg ulcers. Our results are discussed in the context of plasma-biofilm interactions, with respect to the fate of the longer lived reactive species generated by CAP, such as H 2O

On-demand cold plasma activation of acetyl donors for bacteria and virus decontamination

Antibiotics are commonly used as the first line of defense in the treatment of infectious diseases. However, the rise of antimicrobial resistance (AMR) is rendering many antibiotics less effective. Consequently, effective non-antibiotic antimicrobial strategies are urgently needed to combat AMR. This paper presents a strategy utilizing cold plasma for the "on-demand"activation of acetyl donor molecules. The process generates an aqueous-based antimicrobial formulation comprising a rich mixture of highly oxidizing molecules: peracetic acid, hydrogen peroxide, and other reactive oxygen and nitrogen species. The synergistic potent oxidative action between these molecules is shown to be highly effective at eradicating common wound pathogenic bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) and at inactivating a virus (SARS-CoV-2)

Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model

The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2 h post-infection (treatment) or 2 h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24 h for quantification of bacteria and phages. Survival rates at 24 h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI = 10; 47% vs. 20%, MOI = 1; and 33% vs. 7%, MOI = 0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI = 10; 53% vs. 13%, MOI = 1; 67% vs. 7%, MOI = 0.1). By 24 h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses

Cold Plasma Generation of Peracetic Acid for Antimicrobial Applications

This study compares how a helium plasma jet activates peracetic acid (PAA) from tetraacetylethylenediamine (TAED) and acetic acid (AA). Hydrogen peroxide (H 2 O 2 ) generated from the plasma jets reacts with TAED resulting in the formation of PAA which further dissoci-ates into AA. The by-product AA can also react with H 2 O 2 to form PAA, which might also be use-ful for antimicrobial applications when coupled with plasma. Equivalent concentrations of TAED and AA solutions are used to compare the formation of PAA after activation with a helium plasma jet. Our results showed that the concentrations of both H 2 O 2 and PAA in plasma-activated TAED (PAT) are higher than plasma-activated AA (PAAA), and that PAT is more efficient in reducing the growth of Pseudomonas aeruginosa and Staphylococcus aureus; the pathogens commonly found in wounds. The results are attributed to the presence of more acetyl donor groups in TAED, resulting in the formation of higher concentrations of PAA and H 2 O 2

The influence of a second ground electrode on hydrogen peroxide production from an atmospheric pressure argon plasma jet and correlation to antibacterial efficacy and mammalian cell cytotoxicity

This study investigates how addition of a 2nd ground electrode in an argon plasma jet influences the production of hydrogen peroxide (H2O2) in deionised water (DIW). Briefly, plasma is ignited by purging argon gas through a quartz tube at 1 l min-1 and applying a sinusoidal voltage of 7 kV (peak-peak) at 23.5 kHz to a high voltage stainless steel needle electrode sealed inside the quartz tube surrounded by one or two copper ring(s) that served as the ground electrode(s) situated downstream of the high voltage electrode. The mechanisms of H2O2 production are investigated through the electrical and optical plasma properties and chemical analysis of the treated DIW. We discover that the addition of a 2nd ground electrode results in higher accumulation of charges on the inner wall surface of the quartz tube of the plasma jet assembly resulting in an increase in the discharge current and dissipated power. This further leads to an increase in the electron temperature that more than doubles the H2O2 production through dissociative recombination of water vapour molecules, whilst still maintaining a biological tissue tolerable gas temperature. The double ground electrode plasma jet is shown to be highly effective at reducing the growth of common wound pathogens (Pseudomonas aeruginosa and Staphylococcus aureus) in both planktonic and biofilm states whilst inducing a low level of cytotoxicity in HaCaT keratinocyte skin-like cells under certain conditions. The information provided in this study is useful in understanding the complex physicochemical processes that influence H2O2 production in plasma jets, which is needed to optimise the development of plasma sources for clinical applications

A small-molecular inhibitor against Proteus mirabilis urease to treat catheter-associated urinary tract infections

Infection and blockage of indwelling urinary catheters is significant owing to its high incidence rate and severe medical consequences. Bacterial enzymes are employed as targets for small molecular intervention in human bacterial infections. Urease is a metalloenzyme known to play a crucial role in the pathogenesis and virulence of catheter-associated Proteus mirabilis infection. Targeting urease as a therapeutic candidate facilitates the disarming of bacterial virulence without affecting bacterial fitness, thereby limiting the selective pressure placed on the invading population and lowering the rate at which it will acquire resistance. We describe the design, synthesis, and in vitro evaluation of the small molecular enzyme inhibitor 2-mercaptoacetamide (2-MA), which can prevent encrustation and blockage of urinary catheters in a physiologically representative in vitro model of the catheterized urinary tract. 2-MA is a structural analogue of urea, showing promising competitive activity against urease. In silico docking experiments demonstrated 2-MA’s competitive inhibition, whilst further quantum level modelling suggests two possible binding mechanisms

Assessment of mutations induced by cold atmospheric plasma jet treatment relative to known mutagens in Escherichia coli

Abstract The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV-radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test, respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, which did not resemble that for UV implying that CAP-produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences