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.

Activity of a hypochlorous acid-producing electrochemical bandage as assessed with a porcine explant biofilm model.

The activity of a hypochlorous acid-producing electrochemical bandage (e-bandage) in preventing methicillin-resistant Staphylococcus aureus infection (MRSA) infection and removing biofilms formed by MRSA was assessed using a porcine explant biofilm model. e-Bandages inhibited S. aureus infection (p = 0.029) after 12 h (h) of exposure and reduced 3-day biofilm viable cell counts after 6, 12, and 24 h exposures (p = 0.029). Needle-type microelectrodes were used to assess HOCl concentrations in explant tissue as a result of e-bandage treatment; toxicity associated with e-bandage treatment was evaluated. HOCl concentrations in infected and uninfected explant tissue varied between 30 and 80 µM, decreasing with increasing distance from the e-bandage. Eukaryotic cell viability was reduced by an average of 71% and 65% in fresh and day 3-old explants, respectively, when compared to explants exposed to nonpolarized e-bandages. HOCl e-bandages are a promising technology that can be further developed as an antibiotic-free treatment for wound biofilm infections.

Efficacy and toxicity of hydrogen peroxide producing electrochemical bandages in a porcine explant biofilm model.

AIMS: Effects of H2 O2 producing electrochemical-bandages (e-bandages) on methicillin-resistant Staphylococcus aureus colonization and biofilm removal were assessed using a porcine explant biofilm model. Transport of H2 O2 produced from the e-bandage into explant tissue and associated potential toxicity were evaluated. METHODS AND RESULTS: Viable prokaryotic cells from infected explants were quantified after 48 h treatment with e-bandages in three ex vivo S. aureus infection models: (1) reducing colonization, (2) removing young biofilms and (3) removing mature biofilms. H2 O2 concentration-depth profiles in explants/biofilms were measured using microelectrodes. Reductions in eukaryotic cell viability of polarized and nonpolarized noninfected explants were compared. e-Bandages effectively reduced S. aureus colonization (p = 0.029) and reduced the viable prokaryotic cell concentrations of young biofilms (p = 0.029) with limited effects on mature biofilms (p > 0.1). H2 O2 penetrated biofilms and explants and reduced eukaryotic cell viability by 32-44% compared to nonpolarized explants. CONCLUSIONS: H2 O2 producing e-bandages were most active when used to reduce colonization and remove young biofilms rather than to remove mature biofilms. SIGNIFICANCE AND IMPACT OF STUDY: The described e-bandages reduced S. aureus colonization and young S. aureus biofilms in a porcine explant wound model, supporting their further development as an antibiotic-free alternative for managing biofilm infections.

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 effects of intermittently produced H2O2 and 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.

DUAL ACTION ELECTROCHEMICAL BANDAGE OPERATED by a PROGRAMMABLE MULTIMODAL WEARABLE POTENTIOSTAT.

Electrochemical bandages (e-bandages) can be applied to biofilm-infected wounds to generate reactive oxygen species, such as hypochlorous acid (HOCl) or hydrogen peroxide (H 2 O 2 ). The e-bandage-generated HOCl or H 2 O 2 kills biofilms in vitro and in infected wounds on mice. The HOCl-generating e-bandage is more active against biofilms in vitro , although this distinction is less apparent in vivo . The H 2 O 2 -generating e-bandage, more than the HOCl-generating e-bandage, is associated with improved healing of infected wounds. A strategy in which H 2 O 2 and HOCl are generated alternately-for dual action-was explored. The goal was to develop a programmable multimodal wearable potentiostat (PMWP) that could be programmed to generate HOCl or H 2 O 2 , as needed. An ultralow-power microcontroller unit managed operation of the PMWP. The system was operated with a 260-mAh capacity coin battery and weighed 4.6 grams, making it suitable for small animal experiments or human use. The overall cost of a single wearable potentiostat was $6.50 (USD). The device was verified using established electrochemical systems and functioned comparably to a commercial potentiostat. To determine antimicrobial effectiveness, PMWP-controlled e-bandages were tested against clinical isolates of four prevalent chronic wound bacterial pathogens, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter baumannii , and Enterococcus faecium , and one fungal pathogen of emerging concern, Candida auris . PMWP-controlled e-bandages exhibited broad-spectrum activity against biofilms of all study isolates tested when programmed to deliver HOCl followed by H 2 O 2 . These results show that the PMWP operates effectively and is suitable for animal testing.

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 challenges due to biofilm formation and antibiotic resistance. Hypochlorous acid (HOCl), a potent antimicrobial agent, holds promise as an alternative therapy. An electrochemical bandage (e-bandage) that generates HOCl in situ via precise polarization controlled by a miniaturized potentiostat was evaluated for the treatment of murine wound biofilm infections containing both P. aeruginosa with "difficult-to-treat" resistance and MRSA. Previously, HOCl-producing e-bandage was shown to reduce murine wound biofilms containing P. aeruginosa alone. Here, in 5-mm excisional skin wounds containing 48-h biofilms comprising MRSA and P. aeruginosa combined, polarized e-bandage treatment reduced MRSA by 1.1 log10 CFU/g (P = 0.026) vs non-polarized e-bandage treatment (no HOCl production), and 1.4 log10 CFU/g (0.0015) vs Tegaderm only controls; P. aeruginosa was similarly reduced by 1.6 log10 CFU/g (P = 0.0032) and 1.6 log10 CFU/g (P = 0.0015), respectively. For wounds infected with MRSA alone, polarized e-bandage treatment reduced bacterial load by 1.1 log10 CFU/g (P = 0.0048) and 1.3 log10 CFU/g (P = 0.0048) compared with non-polarized e-bandage and Tegaderm only, respectively. The e-bandage treatment did not negatively impact wound healing or cause tissue toxicity. The addition of systemic antibiotics did not enhance the antimicrobial efficacy of e-bandages. 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. IMPORTANCE: New approaches are needed to combat the rise of antimicrobial-resistant infections. The HOCl-producing electrochemical bandage (e-bandage) leverages in situ generation of HOCl, a natural biocide, for broad-spectrum killing of wound pathogens. Unlike traditional therapies that may exhibit limited activity against biofilms and antimicrobial-resistant organisms, the e-bandage offers a potent, standalone solution that does not contribute to further resistance or require adjunctive antibiotic therapy. Here, we show the ability of the e-bandage to address polymicrobial infection by antimicrobial resistant clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa, two commonly isolated, co-infecting wound pathogens. Effectiveness of the HOCl-producing e-bandage in reducing pathogen load while minimizing tissue toxicity and avoiding the need for systemic antibiotics underscores its potential as a tool in managing complex 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.

Anti-Biofilm Activity of a Tunable Hypochlorous Acid-Generating Electrochemical Bandage Controlled By a Wearable Potentiostat.

Chronic wound biofilm infections represent a major clinical challenge which results in a substantial burden to patients and healthcare systems. Treatment with topical antibiotics is oftentimes ineffective as a result of antibiotic-resistant microorganisms and biofilm-specific antibiotic tolerance. Use of biocides such as hypochlorous acid (HOCl) has gained increasing attention due to the lack of known resistance mechanisms. We designed an HOCl-generating electrochemical bandage (e-bandage) that delivers HOCl continuously at low concentrations targeting infected wound beds in a similar manner to adhesive antimicrobial wound dressings. We developed a battery-operated wearable potentiostat that controls the e-bandage electrodes at potentials suitable for HOCl generation. We demonstrated that e-bandage treatment was tunable by changing the applied potential. HOCl generation on electrode surfaces was verified using microelectrodes. The developed e-bandage showed time-dependent responses against in vitro Acinetobacter baumannii and Staphylococcus aureus biofilms, reducing viable cells to non-detectable levels within 6 and 12 hours of treatment, respectively. The developed e-bandage should be further evaluated as an alternative to topical antibiotics to treat wound biofilm 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.