Evaluation of treatment of methicillin-resistant Staphylococcus aureus biofilms with intermittent electrochemically generated H2O2 or HOCl.

Chronic wound infections can be difficult to treat and may lead to impaired healing and worsened patient outcomes. Novel treatment strategies are needed. This study evaluated the effects of intermittently produced hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), generated via an electrochemical bandage (e-bandage), against methicillin-resistant Staphylococcus aureus biofilms in an agar membrane biofilm model. By changing the working electrode potential, the e-bandage generated either HOCl (1.5 VAg/AgCl) or H2O2 (-0.6 VAg/AgCl). The degree of biocidal activity of intermittent treatment with HOCl and H2O2 correlated with HOCl treatment time; HOCl treatment durations of 0, 1.5, 3, 4.5, and 6 hours (with the rest of the 6-hour total treatment time devoted to H2O2 generation) resulted in mean biofilm reductions of 1.36 ± 0.2, 2.22 ± 0.16, 3.46 ± 0.38, 4.63 ± 0.74, and 7.66 ± 0.5 log CFU/cm2, respectively, vs. non-polarized controls, respectively. However, application of H2O2 immediately after HOCl treatment was detrimental to biofilm removal. For example, 3 hours HOCl treatment followed by 3 hours H2O2 resulted in a 1.90 ± 0.84 log CFU/cm2 lower mean biofilm reduction than 3 hours HOCl treatment followed by 3 hours non-polarization. HOCl generated over 3 hours exhibited biocidal activity for at least 7.5 hours after e-bandage operation ceased; 3 hours of HOCl generation followed by 7.5 hours of non-polarization resulted in a biofilm cell reduction of 7.92 ± 0.12 log CFU/cm2 vs. non-polarized controls. Finally, intermittent treatment with HOCl (i.e., interspersed with periods of e-bandage non-polarization) for various intervals showed similar effects (approximately 6 log CFU/cm2 reduction vs. non-polarized control) to continuous treatment with HOCl for 3 hours, followed by 3 hours of non-polarization. These findings suggest that timing and sequencing of HOCl and H2O2 treatments are crucial for maximizing biofilm control when using an e-bandage strategy.

HOCl-producing electrochemical bandage for treating Pseudomonas aeruginosa-infected murine wounds.

The growing threat of antibiotic-resistant bacterial pathogens necessitates the development of alternative antimicrobial approaches. This is particularly true for chronic wound infections, which commonly harbor biofilm-dwelling bacteria. A novel electrochemical bandage (e-bandage) delivering low-levels of hypochlorous acid (HOCl) was evaluated against Pseudomonas aeruginosa murine wound biofilms. 5 mm skin wounds were created on the dorsum of mice and infected with 106 colony-forming units (CFU) of P. aeruginosa. Biofilms were formed over 2 days, after which e-bandages were placed on the wound beds and covered with Tegaderm. Mice were administered Tegaderm-only (control), non-polarized e-bandage (no HOCl production), or polarized e-bandage (using an HOCl-producing potentiostat), with or without systemic amikacin. Purulence and wound areas were measured before and after treatment. After 48 hours, wounds were harvested for bacterial quantification. Forty-eight hours of polarized e-bandage treatment resulted in mean biofilm reductions of 1.4 log10 CFUs/g (P = 0.0107) vs non-polarized controls and 2.2 log10 CFU/g (P = 0.004) vs Tegaderm-only controls. Amikacin improved CFU reduction in Tegaderm-only (P = 0.0045) and non-polarized control groups (P = 0.0312) but not in the polarized group (P = 0.3876). Compared to the Tegaderm-only group, there was less purulence in the polarized group (P = 0.009). Wound closure was neither impeded nor improved by either polarized or non-polarized e-bandage treatment. Concurrent amikacin did not impact wound closure or purulence. In conclusion, an HOCl-producing e-bandage reduced P. aeruginosa in wound biofilms with no impairment in wound healing, representing a promising antibiotic-free approach for addressing wound infection.

Hydrogen peroxide-producing electrochemical bandage controlled by a wearable potentiostat for treatment of wound infections.

Chronic wound infections caused by biofilm-forming microorganisms represent a major burden to healthcare systems. Treatment of chronic wound infections using conventional antibiotics is often ineffective due to the presence of bacteria with acquired antibiotic resistance and biofilm-associated antibiotic tolerance. We previously developed an electrochemical scaffold that generates hydrogen peroxide (H2 O2 ) at low concentrations in the vicinity of biofilms. The goal of this study was to transition our electrochemical scaffold into an H2 O2 -generating electrochemical bandage (e-bandage) that can be used in vivo. The developed e-bandage uses a xanthan gum-based hydrogel to maintain electrolytic conductivity between e-bandage electrodes and biofilms. The e-bandage is controlled using a lightweight, battery-powered wearable potentiostat suitable for use in animal experiments. We show that e-bandage treatment reduced colony-forming units of Acinetobacter buamannii biofilms (treatment vs. control) in 12 h (7.32 ± 1.70 vs. 9.73 ± 0.09 log10 [CFU/cm2 ]) and 24 h (4.10 ± 12.64 vs. 9.78 ± 0.08 log10 [CFU/cm2 ]) treatments, with 48 h treatment reducing viable cells below the limit of detection of quantitative and broth cultures. The developed H2 O2 -generating e-bandage was effective against in vitro A. baumannii biofilms and should be further evaluated and developed as a potential alternative to topical antibiotic treatment of wound infections.

HOCl-producing Electrochemical Bandage is Active in Murine Polymicrobial Wound Infection.

Wound infections, exacerbated by the prevalence of antibiotic-resistant bacterial pathogens, necessitate innovative antimicrobial approaches. Polymicrobial infections, often involving Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), present formidable challenges due to biofilm formation and antibiotic resistance. Hypochlorous acid (HOCl), a potent antimicrobial agent produced naturally by the immune system, holds promise as an alternative therapy. An electrochemical bandage (e-bandage) that generates HOCl in situ was evaluated for treatment of murine wound biofilm infections containing both MRSA and P. aeruginosa with "difficult-to-treat" resistance. Previously, the HOCl-producing e-bandage was shown to reduce wound biofilms containing P. aeruginosa alone. Compared to non-polarized e-bandage (no HOCl production) and Tegaderm only controls, the polarized e-bandages reduced bacterial loads in wounds infected with MRSA plus P. aeruginosa (MRSA: vs Tegaderm only - 1.4 log10 CFU/g, p = 0.0015, vs. non-polarized - 1.1 log10 CFU/g, p = 0.026. P. aeruginosa: vs Tegaderm only - 1.6 log10 CFU/g, p = 0.0015, vs non-polarized - 1.6 log10 CFU/g, p = 0.0032), and MRSA alone (vs Tegaderm only - 1.3 log10 CFU/g, p = 0.0048, vs. non-polarized - 1.1 log10 CFU/g, p = 0.0048), without compromising wound healing or causing tissue toxicity. Addition of systemic antibiotics did not enhance the antimicrobial efficacy of e-bandages, highlighting their potential as standalone therapies. This study provides additional evidence for the HOCl-producing e-bandage as a novel antimicrobial strategy for managing wound infections, including in the context of antibiotic resistance and polymicrobial infections.

Hypochlorous acid produced at the counter electrode inhibits catalase and increases bactericidal activity of a hydrogen peroxide generating electrochemical bandage.

Previously, an electrochemical bandage (e-bandage) that uses a three-electrode system to produce hydrogen peroxide (H2O2) electrochemically on its working electrode was developed as a potential strategy for treating biofilms; it showed activity in reducing biofilms in an agar biofilm model. Xanthan gum-based hydrogel, including NaCl, was used as the electrolyte. While H2O2 generated at the working electrode in the vicinity of a biofilm is a main mechanism of activity, the role of the counter electrode was not explored. The goal of this research was to characterize electrochemical reactions occurring on the counter electrode of the e-bandage. Counter electrode potential varied between 1.2 and 1.5 VAg/AgCl; ∼125 µM hypochlorous acid (HOCl) was generated within 24 h in the e-bandage system. When HOCl was not produced on the counter electrode (achieved by removing NaCl from the hydrogel), reduction of Acinetobacter baumannii BAA-1605 biofilm was 1.08 ± 0.38 log10 CFU/cm2 after 24 h treatment, whereas when HOCl was produced, reduction was 3.87 ± 1.44 log10 CFU/cm2. HOCl inhibited catalase activity, abrogating H2O2 decomposition. In addition to H2O2 generation, the previously described H2O2-generating e-bandage generates HOCl on the counter electrode, enhancing its biocidal activity.