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

ABSTRACT

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.

INTRODUCTION

The growing threat of antibiotic-resistant bacterial pathogens has created a need for alternative antimicrobial strategies. This is particularly true in the case of chronic wound infections; it has been estimated that nearly 90% of wound isolates may harbor resistance to at least one antibiotic, with nearly 30% being resistant to at least six antibiotics (1). Among these resistant isolates, Pseudomonas aeruginosa, a Gram-negative bacterium that is intrinsically resistant to a variety of antibiotics and exhibits high rates of acquired resistance (2), is a commonly identified chronic wound pathogen (3, 4).

Further augmenting antibiotic resistance is the ability of P. aeruginosa and other pathogens to form biofilms, communities of microorganisms protected by a heterogeneous matrix of polysaccharides, proteins, DNA, and other molecules, termed extracellular polymeric substance. Biofilm-associated infections can be recalcitrant to current therapeutics and impede wound closure, leading to a perpetual state of inflammation and delaying healing (5, 6). In the United States, nearly 7 million patients experience chronic wounds annually (7), with an estimated 60% of those wounds associated with microbial biofilms (8). Given the recalcitrance of chronic wound infections, and the common involvement of antibiotic-resistant P. aeruginosa, novel antibiofilm strategies that do not contribute to further resistance are necessary.

Hypochlorous acid (HOCl) is a biocide that is naturally produced by phagocytes (9, 10) and has activity against both bacteria and fungi (11–13). A challenge in its clinical use as an anti-infective has been the inability to deliver it continuously. In previous work, we developed an electrochemical platform for generating HOCl in situ. This platform was active against both bacterial and fungal biofilms in vitro (11–14). Here, we show, for the first time, that an HOCl-producing electrochemical bandage (e-bandage), controlled by a customized ‘wearable’ potentiostat, effectively treats P. aeruginosa biofilm infections in an in vivo environment. We evaluated HOCl-producing e-bandage activity against P. aeruginosa-infected murine wounds by quantifying viable bacterial cell reduction in the wound bed, evaluating wound healing factors (wound area reduction, purulence score, histopathology profile, and inflammatory cytokines), and measuring HOCl concentrations in the wound bed. Lastly, we compared HOCl-producing e-bandage treatment with that of systemically administered amikacin.

MATERIALS AND METHODS

Electrochemical bandage

The e-bandage and wearable potentiostat are described in previous studies (15, 16). Briefly, the e-bandage comprises three integrated electrodes: functional circular carbon fabric working and counter electrodes, each with surface areas of 1.77 cm² (Panex 30 PW-06, Zoltek Companies Inc., St. Louis, MO), along with a silver/silver chloride (Ag/AgCl) wire that functions as a quasi-reference electrode (QRE). The operational potential of the working electrode is maintained at +1.5 V_Ag/AgCl using a wearable potentiostat. Working and counter electrodes are separated by two layers of cotton fabric, with an additional layer placed above the counter electrode to facilitate moisture retention. To secure the fabric, silicone adhesive is employed, which partially covers the external borders of the electrodes and cotton layers. The QRE is affixed between the two cotton fabric layers that isolate the carbon electrodes. Titanium wires (TEMCo, Amazon.com, catalog #RW0524) are connected to opposing ends of the e-bandage using nylon sew-on caps (Dritz, Spartanburg, SC, item#85). When the working electrode of the e-bandage is polarized under physiological conditions, HOCl generation occurs via the following reactions:

$${\displaystyle 2{\mathrm{C}\mathrm{l}}^{-}⟺\mathrm{C}{\mathrm{l}}_2+2{\mathrm{e}}^{-}}$$

$${\displaystyle {\mathrm{E}}_0=+1.119{\mathrm{V}}_{\mathrm{A}\mathrm{g}/\mathrm{A}\mathrm{g}\mathrm{C}\mathrm{l}}}$$

$${\displaystyle \mathrm{C}{\mathrm{l}}_2+{\mathrm{H}}_2\mathrm{O}⟺\mathrm{C}{\mathrm{l}}^{-}+\mathrm{H}\mathrm{O}\mathrm{C}\mathrm{l}+{\mathrm{H}}^{+}}$$

At pH 7.4 and 25°C, the conditions under which e-bandage was employed, HOCl dissociates to ~57% HOCl and ~43% ClO⁻.

Mice skin wound infection model

All animal experiments were approved by the Mayo Clinic Institutional Animal Care and Use Committee under protocol approval number, A00003272-20. To generate full-thickness skin wounds on Swiss Webster mice (Charles River, Wilmington, MA), animals were anesthetized by intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Buprenorphine ER-Lab (1 mg/kg) was administered subcutaneously for analgesia. Mature wound biofilms were generated as in our previous studies (17, 18). The dorsal surface was shaved and disinfected, and a 5 mm biopsy punch (Acuderm Inc., Fort Lauderdale, FL) was utilized to create a circular, full thickness skin wound. Wounds were infected with 10⁶ colony-forming units (CFUs) of a clinical P. aeruginosa isolate (IDRL-11442) suspended in 0.9% sterile saline. P. aeruginosa IDRL-11442 is a wound isolate that exhibits “difficult-to-treat resistance,” in that it is resistant to first-line antibiotics piperacillin/tazobactam, cefepime, ceftazidime, meropenem, aztreonam, ciprofloxacin, and levofloxacin (19). Uninfected control mice were administered 10 µL of saline as a vehicle control. Bacterial suspensions were allowed to settle into wound beds for 5 minutes, after which wounds were covered with semi-occlusive transparent Tegaderm (3M, St. Paul, MN) using liquid adhesive Mastisol (Eloquest Health care, Ferndale, MI). Wound photographs were taken, and wound diameters were recorded every other day with a Silhouette wound imaging system (Aranz Medical Ltd, Christchurch, NZ). Purulence was scored before treatment and before sacrifice to gauge immune response to biofilm infection and treatment. The scoring system was based on our previous work (17) using the following scaling model: 0 = no exudate in the wound bed; 1 = slight turbid exudate at the wound site; 2 = mild amount of white exudate at the wound site; 3 = moderate amount of white exudate at the wound site; 4 = moderate amount of yellowish exudate at the wound site; 5 = large amount of turbid yellow exudate extending beyond the wound bed.

e-Bandage treatment

After wound bed infections were established for 48 hours, mice were anesthetized, Tegaderm removed, and wearable potentiostats affixed to the skin at the scruff of the neck. Sterile e-bandages pre-hydrated in sterile 1× phosphate buffer saline (1× PBS) and 200 µL of sterile hydrogel [1.8% (wt/vol) xanthan gum in 1X PBS] were injected between the fabric layers. Another 200 µL of hydrogel was added on top of the wound beds, and e-bandages were placed on top, sutured in place (two sutures, one on each lateral side of the bandage to ensure continuous contact with the wound bed with mouse activity) and attached to the female socket of the wearable potentiostats. An additional 200 µL of hydrogel was added on top of the e-bandages, and the entire bandage and wound were covered with Tegaderm. 3V coin cell batteries (Ecr1220 Energizer, St. Louis, MO) were inserted into each potentiostat to start polarized e-bandage treatment. Treatment proceeded for 48 hours with hydrogel refreshment, and battery changes every 24 hours. Potentials of the working electrodes relative to the QREs were measured at the start of treatment, before and after each battery change, and before euthanasia, to ensure device operation. Control groups included mice with hydrogel and Tegaderm only, and mice with non-polarized e-bandages (i.e., no potentiostat). Additionally, the experimental and control groups were tested with concurrent amikacin dosing. Amikacin was chosen due to the resistance status of the P. aeruginosa isolate studied, as the amikacin mimimum inhibitory concentration was 16 mg/ml. Previously performed pharmacokinetic studies of amikacin in Swiss Webster mice were used to select a treatment dose of 15 mg/kg subcutaneously every 6 hours (18). Subcutaneous amikacin injections were delivered in the flank of the animals to avoid the potentiostat/e-bandage set-up and to limit local effects on the dorsal wounds. At least 8 mice were included in each experimental and control group.

Wound biofilm quantification

Following treatment, Tegaderm and e-bandages were removed from the wound bed, and wound tissue was excised with a 10 mm biopsy punch tool (Acuderm Inc.). Skin tissue was weighed, homogenized (Omni International, Kennesaw, GA) in sterile PBS, sonicated in for 5 minutes in a water bath, and vortexed for 30 seconds. 100 µL of the resulting homogenate was serially diluted (10-fold dilutions) in 0.9% saline, and CFUs were determined by spread-plating 100 µL of each dilution onto tryptic soy agar with 5% sheep blood. After 24 hours of incubation at 37°C, colonies exhibiting typical P. aeruginosa morphology were counted, and results were reported as log₁₀ CFU/g. Occasional contaminant colonies were present, usually at lower quantities than P. aeruginosa.

Total wound HOCl measurement

Following euthanasia, the unused portion (900 µL) of the wound homogenate not used for quantification of bacterial load was utilized to measure total wound HOCl content with fee chlorine spectrophotometer test kits (TNT866; Hach Company, Ames, IA), as per manufacturer instructions. Briefly, homogenized wound contents were mixed with 4.1 mL of 1× PBS and centrifuged at 5,000 x g for 15 minutes. The resulting supernatants were passed through syringe filters (0.22 micron pore size), and 4 mL was added to free chlorine test tubes and allowed to react for 1 minute before being read at 515 nm in a Hach DR 1,900 portable spectrophotometer (Hach Company). Free chlorine content was then converted to HOCl content via the following equation and adjusted for volume:

$${\displaystyle \mathrm{C}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{H}\mathrm{O}\mathrm{C}\mathrm{l}\left[\mathrm{M}\right]=\frac{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{e}\left[\frac{\mathrm{m}\mathrm{g}}{\mathrm{L}}\right]\times \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\left[\frac{\mathrm{g}}{\mathrm{m}\mathrm{g}}\right]}{\mathrm{M}\mathrm{o}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{r}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{e}\left[\frac{\mathrm{g}}{\mathrm{m}\mathrm{o}\mathrm{l}}\right]}}$$

The molecular weight of chlorine, 70.906 g/mol, and a conversion factor of 0.001 g/mg were used, and full conversion of HOCl from free chlorine was assumed.

Histopathology

Wounds from a subset of animals (n = 3) from each group underwent an assessment of histopathology. Wound beds were removed via a 10 mm biopsy punch and fixed in 10% formalin. Fixed samples were stained with hematoxylin and eosin (H&E), and slides were blindly analyzed by a board-certified pathologist for overall inflammation (0 = none, 1 = mild, 2 = moderate, and 3 = severe), abscess formation (yes or no), ulceration (Y/N), tissue necrosis (Y/N), and neutrophilic infiltration (Y/N).

Toxicity screen analysis and inflammatory panel screening

Following euthanasia, blood was obtained through cardiac puncture and subjected to centrifugation. The resulting plasma samples were assessed using a Piccolo Xpress Chemistry Analyzer at the Mayo Clinic Central Clinical Laboratory. Concentrations of glucose, amylase, blood urea nitrogen, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, lactate dehydrogenase, C-reactive protein, total bilirubin, creatinine, uric acid, albumin, total protein, calcium, chloride, magnesium, potassium, sodium, and total carbon dioxide levels were determined. Additionally, plasma samples were subjected to analysis using a MesoScale Discovery SQ 120 to assess the levels of pro-inflammatory biomarkers, including IFN-γ, IL-10, IL-12p70, IL-2, IL-4, IL-5, IL-6, TNF-α, and KC/GRO (CXCL1).

Statistical analysis

Initial comparisons among experimental groups were conducted through the Kruskal-Wallis test. Subsequent pairwise comparisons between groups were carried out using the Wilcoxon rank sum test. Non-parametric tests were selected due to limited sample sizes and the absence of support for the assumption of a normally distributed data set. All tests were two-tailed, with statistical significance considered for P-values below 0.05. Corrections for False Discovery Rate (Benjamin-Hochberg) were performed for all comparisons with group sizes greater than three. For histological comparisons of wound samples, Fisher’s exact test was used to compare observed Y/N conditions. The analytic process utilized SAS software (version 9.4, SAS Institute), with GraphPad Prism (software version 8.0, GraphPad Software) employed for generating graphs.

RESULTS

Polarized e-bandages produced HOCl in situ

Previously, microelectrodes were used to confirm that the e-bandages produce HOCl at the working electrode, and that HOCl can penetrate into biofilms and explant tissue (15, 20). Here, free chlorine spectrophotometer test kits were used to measure total HOCl content in wounds. Wounds harvested from mice treated with polarized e-bandages had higher levels of HOCl than those treated with non-polarized e-bandages (P = 0.0170) or Tegaderm alone (P = 0.0045; Table 1).

TABLE 1.

Endpoint HOCl content (µM) of wounds

Group	N	Mean	SD
Tegaderm-only	8	66.5	16.8
Polarized e-bandage	9	161.7	59.8
Non-polarized e-bandage	10	80.7	43.2

Polarized e-bandage treatment reduced bacterial load

To test if HOCl-producing e-bandage treatment reduces wound bacterial burden in vivo, 5 mm mouse dermal wounds were infected with P. aeruginosa, and biofilms were allowed to be established for 48 hours before treatment. Wounds were treated with either polarized or non-polarized e-bandages and compared to Tegaderm alone. After 48 hours of treatment, endpoint wound CFUs were quantified. Treatment with polarized e-bandages resulted in lower bacterial loads than non-polarized e-bandages (P = 0.0048) or Tegaderm alone (P = 0.004; Fig. 1A). Treatment with non-polarized e-bandages resulted in lower bacterial loads than the Tegaderm-only group, possibly due to physical forces associated with e-bandage application and removal, drying of the wound bed, which was more frequently witnessed as formation of a crust on top of the wounds exposed to either polarized or non-polarized e-bandages than Tegaderm alone, and/or antimicrobial activity of the QRE of the e-bandage.

Fig 1.

Colony forming units (CFU) counts in wounds treated with e-bandages and/or amikacin (AMK). 48-hour P. aeruginosa wound bed biofilms were treated for 48 hours with either polarized (HOCl-producing) or non-polarized e-bandages, with or without systemic AMK and compared to Tegaderm-only controls, without (A) or with (B) systemic AMK. Statistical analysis was performed using the Wilcoxon rank sum test with correction for false discovery rate. Individual data points with the means (bars) are shown. Bolded significance bars highlight the differences between polarized and non-polarized e-bandages. N ≥ 8. *P ≤ 0.05 and **P ≤ 0.01.

To test if the e-bandages synergize with a systemic antibiotic against established wound biofilms, additional mice from all three groups were administered concurrent systemic amikacin for the duration of the e-bandage treatment window (Fig. 1; Table 2). Although amikacin alone was effective in reducing CFU counts in Tegaderm only (P = 0.031) and non-polarized groups (P = 0.006), no additional reduction in CFUs was found when comparing polarized treatment with and without amikacin (P = 0.388).

TABLE 2.

The effects of concurrently administered systemic amikacin on e-bandage treatment efficacy^a

	Log10 CFU/g			ΔPurulence score			% Wound closure
	-−AMK	+ AMK		− AMK	+ AMK		−AMK	+ AMK
Tegaderm only	9.78	7.83	**c	+0.5	−1.14	NS	0.86%	23.13%	NS
Non-polarized e-bandage	8.97	8.11	*b	−1.0	−2.5	NS	26.83%	14.80%	NS
Polarized e-bandage	7.71	7.29	NS	−2.55	−3.14	NS	39.79%	12.15%	NS

^a Values represent means (N ≥ 8). Statistical analysis was performed using the Wilcoxon rank sum test. AMK = amikacin. NS = not significant.

^b P ≤ 0.05.

^c P ≤ 0.01.

Treatment of infected wounds with e-bandages did not negatively impact wound healing

To determine the effect of e-bandage and/or amikacin treatment on wound healing, wound areas were measured before and after treatment. No significant differences in overall wound closure percentage were observed between any group (Fig. 2; Table 2).

Fig 2.

Closure of wounds treated with e-bandages and/or amikacin (AMK). 48-hour P. aeruginosa wound bed biofilms were treated with either polarized (HOCl-producing) or non-polarized e-bandages, without (A) or with (B) systemic AMK and compared to Tegaderm-only controls, with or without systemic AMK. Wound area was measured before and after 48 hours of treatment. Individual data points with means (bars) are shown. Statistical analysis was performed using the Wilcoxon rank sum test with correction for false discovery rate. N ≥ 8.

Treatment of infected wounds with polarized e-bandages reduced purulence

To determine the effect of e-bandage and/or amikacin treatment on purulence in the wound beds, wounds were scored for purulence before and after treatment. Treatment with polarized e-bandages led to improvement in purulence compared to either Tegaderm-only or non-polarized controls (P = 0.009 and 0.048, respectively; Fig. 3). Purulence reduction within the non-polarized group was not different than in the Tegaderm-only group (P = 0.064). Concurrent amikacin did not improve purulence reduction in any group (Table 2).

Fig 3.

Reduction in purulence in wounds treated with e-bandages and/or amikacin (AMK). 48-hour P. aeruginosa wound bed biofilms were treated with either polarized (HOCl-producing) or non-polarized e-bandages, with or without systemic AMK and compared to Tegaderm-only controls, without (A) or with (B) systemic AMK. Wound purulence was scored before and after 48 hours of treatment. Individual data points with the means (bars) are shown. Statistical analysis was performed using the Wilcoxon rank sum test with correction for false discovery rate. N ≥ 8. *P ≤ 0.05 and **P ≤ 0.01.

No tissue toxicity was observed with polarized e-bandage treatment

To determine if e-bandage treatment resulted in additional tissue toxicity beyond that occurring from infection alone, three wounds from each non-antibiotic treated group were harvested and fixed in 10% formalin before being processed for H&E staining (Table 3). Moderate to acute inflammation of the epidermal and dermal layers and ulceration were observed in all tissue samples. Abscess formation was noted in all samples except for one from the non-polarized group. The same sample was also the only one to not exhibit significant neutrophilic inflammation.

TABLE 3.

Histopathological profiles

Group	Average inflammation score (0–3)	% Necrosis	% Abscess	% Ulceration	% Neutrophilic inflammation
Polarized e-bandage	3	100	100	100	100
Non-polarized e-bandage	2	100	66	100	66
Tegaderm-only	3	100	100	100	100

Assessment of blood biomarkers

A subset (n = 3) of animals treated with polarized e-bandages, non-polarized e-bandages, and Tegaderm-only controls was utilized to examine the immune response and general health of infected animals based on an assessment of blood parameters. Although minor differences were observed between animals treated with either polarized or non-polarized e-bandages and Tegaderm-only controls (Table Sl ), no differences were observed between polarized and non-polarized e-bandage groups, and mean analyte levels for all groups were within normal healthy ranges for mice.

DISCUSSION

P. aeruginosa is a commonly isolated chronic wound pathogen that is notorious for its intrinsic resistance and propensity for acquisition of resistance to multiple classes of antibiotics (21). Additionally, P. aeruginosa is a prominent biofilm producer, further promoting its recalcitrance to traditional antibiotic treatment and clearance by the host immune system, delaying healing when present in wounds (5, 22). Here, a novel e-bandage was tested for the in situ delivery of the biocide, HOCl. Utilizing HOCl as a biocidal agent circumvents the issue of resistance development resulting from the use of traditional antimicrobial agents. As stated in a recent publication, “There has not been a single verified claim of clinical resistance (to HOCl) over more than 100 years of careful evaluation” (23); this statement is corroborated by our inability to select HOCl resistance in bacterial pathogens (24). Additionally, HOCl produced by the e-bandage is delivered at concentrations below the cytotoxic limit (10).

HOCl-producing e-bandages were tested for 48 hours against 2-day P. aeruginosa biofilms in a murine dermal wound model. In confirmation of previous in vitro and ex vivo tests of this HOCl-producing electrochemical approach (12, 13, 15, 24), the polarization of e-bandages resulted in more HOCl within wounds in comparison to non-polarized and Tegaderm-only controls (161 vs 81 and 67 µM, respectively) after 48 hours of treatment.

HOCl-producing e-bandages reduced P. aeruginosa populations within wound beds. 48 hours of polarized treatment resulted in mean bacterial loads 2.2 log₁₀ CFU/g lower than Tegaderm-only controls (P = 0.004), and 1.4 log₁₀ CFU/g lower than non-polarized bandage controls (P = 0.0048). Combinatorial treatment with e-bandages and amikacin was also tested, with no added microbicidal activity when compared to amikacin alone or polarization alone. Combinatorial treatment with amikacin and polarized e-bandages did, however, result in reduced bacterial counts compared to non-polarized e-bandages with amikacin (P = 0.0195).

HOCl-producing e-bandages did not obviously hinder wound healing. Unlike our previous work investigating hydrogen-peroxide- (H₂O₂-) producing e-bandages (18), however, HOCl-producing e-bandages did not improve wound healing under the conditions studied. HOCl e-bandage treatment did, however, result in a reduction in wound purulence compared to both Tegaderm-only and non-polarized controls (P = 0.009 and 0.048, respectively), similar to findings with H₂O₂-producing e-bandages (18). This suggests that the overall severity of infection after e-bandage treatment was reduced, resulting in a tempered inflammatory response and conditions favorable for ultimate wound healing.

As expected, concentrations of HOCl produced by the e-bandages showed no apparent toxicity. Blind review of wound sections from polarized, non-polarized, and Tegaderm-only groups revealed no differences in overall inflammation, abscess formation, ulceration, tissue necrosis, or neutrophilic infiltration between infected tissues in the different groups. Furthermore, assay of inflammatory cytokines/chemokines and blood biochemical analytes commonly measured in clinical infection to investigate immune response and findings of sepsis revealed that, although there were minor differences between e-bandage (polarize and non-polarized)-treated animals and Tegaderm-only controls, no significant differences were found between the polarized and non-polarized groups across all analytes. Notably, all analyte levels fell within the normal healthy ranges for mice, indicating no apparent toxicity. It should also be noted that throughout the course of the experiments, minor to moderate discoloration of the skin surrounding the wound was observed with polarized treatment. Discolored matter was physically removable by cleansing with an alcohol swab, suggesting that it related to bandage debris.

While the findings outlined here show promise, there are several limitations. Only a single treatment timepoint and a single bacterial strain were evaluated. Extension of duration of treatment should be explored in the future; this could hypothetically improve antibacterial efficacy, as well as wound healing. Furthermore, future endeavors could be aimed at enhancing the overall performance of the e-bandage system by integrating hydrogels that enhance moisture retention, essential for optimal e-bandage operation, as well as by assessing alternative electrolytes that may augment HOCl production at comparable potentials. Concurrent treatment with other systemic and/or topical antimicrobial agents could be explored. Finally, debridement of the wound prior to treatment might improve e-bandage efficacy, as mature, densely populated biofilms are generally more difficult to eradicate than thinner, less populated biofilms, and treatment of thinner biofilms may allow for greater delivery of HOCl to the entirety of the biofilm matrix.

In conclusion, HOCl-producing e-bandages reduced P. aeruginosa biofilms in a murine dermal wound infection model, with no evidence of toxicity and with reduced wound purulence. Based on these results, HOCl-producing e-bandages are a promising non-antibiotic strategy to treat P. aeruginosa wound infections, including those caused by P. aeruginosa exhibiting difficult-to-treat resistance.

AFTER EPUB

[This article was published on 12 January 2024 with Kerryl E. Greenwood-Quaintance's name misspelled in the byline. The spelling was corrected in the current version, posted on 19 January 2024.]

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.01216-23.

Supplemental material. aac.01216-23-s0001.pdf.

A supplemental table and associated references.

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