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. 2023 Sep 4;134(9):lxad194. doi: 10.1093/jambio/lxad194

In vitro activity of hypochlorous acid generating electrochemical bandage against monospecies and dual-species bacterial biofilms

Joseph Kletzer 1,2,#, Yash S Raval 3,#, Abdelrhman Mohamed 4, Jayawant N Mandrekar 5, Kerryl E Greenwood-Quaintance 6, Haluk Beyenal 7, Robin Patel 8,9,✉
PMCID: PMC10508963  PMID: 37667489

Abstract

Aims

As antimicrobial resistance is on the rise, treating chronic wound infections is becoming more complex. The presence of biofilms in wound beds contributes to this challenge. Here, the activity of a novel hypochlorous acid (HOCl) producing electrochemical bandage (e-bandage) against monospecies and dual-species bacterial biofilms formed by bacteria commonly found in wound infections was assessed.

Methods and results

The system was controlled by a wearable potentiostat powered by a 3V lithium-ion battery and maintaining a constant voltage of + 1.5V Ag/AgCl, allowing continuous generation of HOCl. A total of 19 monospecies and 10 dual-species bacterial biofilms grown on polycarbonate membranes placed on tryptic soy agar (TSA) plates were used as wound biofilm models, with HOCl producing e-bandages placed over the biofilms. Viable cell counts were quantified after e-bandages were continuously polarized for 2, 4, 6, and 12 hours. Time-dependent reductions in colony forming units (CFUs) were observed for all studied isolates. After 12 hours, average CFU reductions of 7.75 ± 1.37 and 7.74 ± 0.60 log10 CFU/cm2 were observed for monospecies and dual-species biofilms, respectively.

Conclusions

HOCl producing e-bandages reduce viable cell counts of in vitro monospecies and dual-species bacterial biofilms in a time-dependent manner in vitro. After 12 hours, >99.999% reduction in cell viability was observed for both monospecies and dual-species biofilms.

Keywords: electrochemical bandage, hypochlorous acid, biofilm, wearable potentiostat, wound infection

Impact Statement.

The described e-bandages reduced cell viability in in vitro biofilms (both monospecies and dual-species); results suggests that HOCl producing e-bandages should be further developed as a potential strategy to treat wound infections.

Introduction

Treatment of chronic wound infections represents a complex healthcare issue. The estimated prevalence of chronic wounds is 2.21 per 1000 population, ∼1%–2% of the population is anticipated to experience a chronic wound infection in their lifetime (Crovetti et al. 2004, Martinengo et al. 2019). Bacteria with acquired antibiotic resistance pose a challenge, as does the presence of bacteria in biofilm states, making it especially difficult to treat wound infections using traditional antibiotics (Bertesteanu et al. 2014, Orazi and O'toole 2019).

Wounds go through stages during normal wound healing, including hemostasis, inflammation, proliferation, and remodeling. Chronic wounds arise if one or more of these stages is disrupted (Singer and Clark 1999, Gosain and Dipietro 2004, Broughton et al. 2006). The presence of biofilms in wounds may disrupt wound healing by causing excessive inflammation and mechanically impeding reepithelialization; an estimated almost 60% of infected chronic wounds harbor biofilms (Zhao et al. 2013, Gompelman et al. 2016, Versey et al. 2021). There are several mechanisms through which biofilm recalcitrance to antibiotics occurs (Zhang and Mah 2008, Orazi and O'toole 2019). Due to the low oxygen availability, nutrient deficiency, and the presence of extracellular polymeric substance (EPS) secreted by biofilm-producing microorganisms, metabolism and growth are slowed in bacterial cells in biofilms (Evans et al. 1991, Brown et al. 1988, Nguyen et al. 2011). This increases resistance and tolerance to many antibiotics. Moreover, EPS reduces efficiency and slows transport of some antibiotics (Orazi and O'toole 2019). Persister cells, which are dormant, nonmetabolic but genetically nondistinct cells, also increase biofilm tolerance to antibiotics (Yan and Bassler 2019). Traditional treatment of chronic wound infections consists of debridement, and application of antiseptic dressings, antimicrobial solutions, and antibiotics (Bertesteanu et al. 2014, Han and Ceilley 2017, Liu et al. 2022). The naturally occurring biocide hypochlorous acid (HOCl) has been used in wound care. It is produced by neutrophils during the respiratory burst (Andres et al. 2022). Beyond antimicrobial activity, HOCl may promote wound healing at low concentrations (Loo et al. 2012, Sakarya et al. 2014, Gold et al. 2017, Murphy and Friedman 2019). HOCl may also reduce scar formation and pruritus (Gold et al. 2017). Dose-dependent favorable effects on fibroblast and keratinocyte migration have been noted compared to the commonly used antiseptic wound care agent povidone-iodine (Sakarya et al. 2014). Unfortunately, currently used HOCl solutions are volatile, and therefore stable, low, and biocidal concentrations are difficult to maintain over prolonged periods (Ishihara et al. 2017). In earlier work, construction and application of a novel electrochemical scaffold (e-scaffold) system that continuously delivers low concentrations of HOCl—a predecessor to the e-bandage system, was described (Sultana et al. 2015, Kiamco et al. 2019). The HOCl-generating e-scaffold system was active against monospecies and dual-species bacterial biofilms and yeast biofilms in vitro (Zmuda et al. 2020, Flurin et al. 2021, Raval et al. 2021a).

While e-scaffolds and e-bandages operate on the same principles, they differ in that e-scaffolds are immersed in liquid, use an external reference electrode, and require a large bench-top potentiostat, such that they are not useful in in vivo settings (Sultana et al. 2015, Kiamco et al. 2019). In contrast, e-bandages are designed to be placed onto surfaces of infected wounds and are controlled by wearable potentiostats. Operational principles for the e-bandage and electrochemistry underlying HOCl production have been described (Mohamed et al. 2022), and e-bandages evaluated against yeast biofilms (Kletzer et al. 2023). Here, activity of HOCl producing e-bandages against monospecies and dual-species bacterial biofilms was assessed. Monospecies biofilms formed by 19 bacterial isolates alone and 10 dual-species bacterial biofilm combinations selected based on their frequency in polymicrobial wound infections (Flurin et al. 2021), were evaluated to assess in vitro anti-biofilm activity of HOCl producing e-bandages.

Materials and methods

e-Bandage

Construction of the HOCl producing e-bandage and wearable potentiostat have been recently described (Mohamed et al. 2022). The e-bandage was made of a working, counter, and quasi reference electrode (Fig. 1) (Mohamed et al. 2022). The working and counter electrodes consisted of 1.77 cm2 circular carbon fabric patches (Panex 30 PW-09, Zoltek Companies Inc., St. Louis, MO, USA). For the quasi reference electrode, a silver/silver chloride (Ag/AgCl) wire was used. The working electrode potential was controlled at + 1.5VAg/AgCl using the potentiostat. When polarized, oxidation of chloride ions (Cl-) to chlorine (Cl2) leads to HOCl generation near the working electrode surface (Kiamco et al. 2019). To separate the counter and working electrodes, cotton fabric separators were placed between the electrodes. Another cotton fabric layer was placed on top of the counter electrode for additional moisture retention. Additional details about e-bandage construction are found in (Mohamed et al. 2022). e-Bandages were autoclaved at 121oC for 20 minutes prior to use. Sterile e-bandages were then soaked in sterile 1 × phosphate buffer saline (PBS) for 15 minutes. To preserve moisture during treatment, 100 μl of 1.8% w/v sterile xanthan gum (Namaste Foods LLC, Coeur d'Alene, ID, USA) based hydrogel in PBS was injected atop membrane biofilms, between the cotton fabric separator layers, and atop the e-bandage itself. Finally, a 5 × 4 cm piece of sterile Tegaderm TM transparent film ( 16002, 3 M) was placed over the e-bandage.

Figure 1.

Figure 1.

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Experimental setup and e-bandage composition; created with BioRender.com.

Growth of monospecies and dual-species biofilms on polycarbonate membranes

Table 1 lists the bacterial strains investigated. Monospecies biofilms were grown by placing 2.5 µl of freshly grown bacteria in tryptic soy broth (TSB) onto the center of sterile 13 mm polycarbonate membranes (Whatman® Cat. No. 110 406, GE Healthcare) placed on a 5% sheep blood agar plate. Before being transferred, bacteria for monospecies biofilms were grown to 0.5 McFarland, except for Pseudomonas aeruginosa and Staphylococcus epidermidis (P. aeruginosa: 0.5 McFarland, then reduced to ∼104 CFU/ml through dilution; S. epidermidis: 3.0 McFarland). Additional protocol details are found in (Raval et al. 2021b, Mohamed et al. 2022). To grow dual species biofilms, adjustments were made to achieve equal amounts of each species in final biofilms. Additional details on dual-species biofilm pairs, inocula, and other details are found in Table 2.

Table 1.

Bacterial isolates, identification number, origin, antibiotic resistance, and starting inoculum of each studied strain.

Species Isolate designation Source Resistance phenotype Starting inoculum for dual-species biofilms
Staphylococcus aureus IDRL-6169 Periprosthetic hip infection Methicillin and mupirocin 1.25 μl of 0.5 McFarland
S. aureus IDRL-8660 Clinical Methicillin Only used for monospecies biofilms
S. aureus IDRL-4284 Clinical Methicillin Only used for monospecies biofilms
P. aeruginosa IDRL-11442 Groin infection Piperacillin/tazobactam, cefepime, ceftazidime, meropenem, aztreonam, ciprofloxacin, and levofloxacin 1.25 μl of 0.5 McFarland reduced to ∼104 CFU/ml
P. aeruginosa IDRL-7262 Periprosthetic knee infection Only used for monospecies biofilms
P. aeruginosa PAO1, ATCC 47085 Wound Only used for monospecies biofilms
S. epidermidis ATCC 35984 Catheter sepsis Methicillin 1.25 μl of 3 McFarland
S. epidermidis Xen 43 Catheter Only used for monospecies biofilms
S. epidermidis IDRL-6461 Knee infection Only used for monospecies biofilms
Acinetobacter baumannii ARLG-1268 Wound Amikacin, ampicillin, cefepime, ceftazidime, ciprofloxacin, and tobramycin 1.25 μl of 0.5 McFarland
A. baumannii ATCC-17978 Meningitis
A. baumannii ATCC BAA-1605 Sputum Ceftazidime, gentamicin, ticarcillin, piperacillin, aztreonam, cefepime, ciprofloxacin, imipenem, and meropenem Only used for monospecies biofilms
Escherichia coli IDRL-10366 Clinical microbiology laboratory bla KPC-positive; ceftolozane/tazobactam, imipenem, meropenem, ertapenem, ceftriaxone, and cefepime 1.25 μl of 0.5 McFarland
E. coli IDRL-6199 Periprosthetic knee infection Only used for monospecies biofilms
Enterococcus faecium IDRL-11790 Abscess Vancomycin and penicillin 1.25 μl of 0.5 McFarland
Klebsiella pneumoniae IDRL-10377 Clinical microbiology laboratory bla KPC-positive; ceftolozane/tazobactam, imipenem, meropenem, ertapenem, ceftriaxone, and cefepime 1.25 μl of 0.5 McFarland
Enterococcus faecalis IDRL-12374 Periprosthetic hip infection Vancomycin and levofloxacin 1.25 μl of 1 McFarland
E. faecalis IDRL-7327 Urine Only used for monospecies biofilms
E. faecalis IDRL-7107 Periprosthetic knee infection Only used for monospecies biofilms
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Table 2.

Dual-species bacterial biofilm combinations studied, and selective media used for colony forming unit (CFU) quantification.

Dual-species combination Selective media for quantification
A. baumannii ARLG-1268 and S. epidermidis ATCC 35984 Eosin methylene blue (EMB) and colistin nalidixic acid (CNA)
P. aeruginosa IDRL-11442 and S. epidermidis ATCC 35984 EMB and CNA
P. aeruginosa IDRL-11442 and S. aureus IDRL-6169 EMB and CNA
E. coli IDRL-10366 and E. faecium IDRL-11790 EMB and CNA
A. baumannii ARLG-1268 and P. aeruginosa IDRL-11442 ChromAgar A. baumannii and ChromAgar P. aeruginosa
E. coli IDRL-10366 and K. pneumoniae IDRL-10377 ChromAgar Orientation
S. aureus IDRL-6169 and E. faecium IDRL-11790 ChromAgar MRSA and ChromAgar VRE
S. aureus IDRL-6169 and S. epidermidis ATCC 35984 Mannitol salt
P. aeruginosa IDRL-11442 and E. faecium IDRL-11790 EMB andCNA
S. epidermidis ATCC 35984 and E. faecalis IDRL-12374 Mannitol salt agar and ChromAgar VRE
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e-Bandage treatment

Figure 1 visually summarizes the treatment procedure. For treatment, biofilms on membranes were transferred to TSA plates to prevent biofilm growth from spreading beyond e-bandages. After placing e-bandages on top of bacterial biofilms grown on membranes, working, counter, and reference electrodes were connected to a wearable potentiostat, and a 3V lithium-ion battery inserted into its socket. After ten minutes, a voltmeter was used to measure working electrode potential to verify operation. Both monospecies and dual-species biofilms were treated (polarized treatment group) for 2, 4, 6, and 12 hours: monospecies biofilms of Gram-positive bacteria were treated for 2, 6, and 12 hours whereas monospecies biofilms of Gram-negative bacteria were treated for 2, 4, and 6 hours. At the end of treatment, potential of the working electrode potential relative to the quasi-reference electrode was measured again as a quality check. Control groups comprised biofilms exposed to nonpolarized e-bandages (i.e., e-bandages not connected to wearable potentiostats). Experiments for each timepoint tested were performed on separate days with newly grown biofilms.

Post-treatment processing

After treatment, e-bandages were removed from the surface of membrane biofilms and biofilm cells attached to e-bandages scraped into 5 ml of 1 × PBS in a sterile Petri dish using a sterile pipette tip. The PBS rinse was transferred to a 15-ml test tube. Membrane biofilms were added to test tubes containing their corresponding PBS rinse. Tubes were vortexed for 30 seconds, sonicated for 5 minutes, and then vortexed again for 30 seconds, following which they were centrifuged for 5 minutes at 5000 rpm at 20oC and the supernatant removed. A total of 1 ml of fresh 1 × PBS was added to each tube and tubes gently vortexed until the cell pellet was dispersed in solution. A total of 100 μl of this suspension was serially diluted in 1 × PBS (10-fold dilutions). CFUs for monospecies biofilm were determined by spread plating 100 µl of each dilution onto sheep blood agar plates. Dual-species biofilm CFUs were determined by spread-plating 100 µl of each dilution on appropriate selective agar plates (Table 2). Plates were incubated at 37oC for 24 hours. In addition, 100 μl of the original undiluted suspension was added to 5 ml of TSB and incubated under shaking conditions overnight to check for bacterial growth. The streak-plate method was calculated to have a limit of detection of 0.87 log10 CFU/cm2, whereas for broth culture it was 0.71 log10 CFU/cm2. Figure 1 illustrates the experimental procedure.

Statistical analysis

For each time period, three replicate treatments were performed on different days. Means and SDs were calculated. To compare experimental groups, a one-sided Kruskall Wallis test was used. Further comparisons were performed using a one-sided Wilcoxon rank-sum test. Nonparametric tests were used as normal distribution of the data cannot be assumed. P-values ≤ 0.05 were deemed statistically significant. Analysis was performed using SAS software version 9.4 (SAS Inc., Cary, NC, USA). Graphs were created in GraphPad Prism (software version 9.0, GraphPad software).

Results

Monospecies biofilms

Treatment (polarized group) of monospecies bacterial biofilms with HOCl producing e-bandages led to significant (P < 0.05) time-dependent reductions in CFU counts when compared with nonpolarized control groups (Fig. 2). For Gram-positive bacterial biofilms, treatment results were as follows: 2 hours of exposure led to a mean reduction of 0.95 ± 0.38 log10 CFU/cm2 (P < 0.05); after 6 hours of exposure, biofilms were reduced by 3.38 ± 0.67 log10 CFU/cm2 (P < 0.05); and 12-hour treatments reduced cell counts by 7.75 ± 1.37 log10 CFU/cm2 (P < 0.05). For Gram-negative bacterial biofilms, 2 hours of treatment reduced cell counts by 3.58 ± 0.74 log10 CFU/cm2 (P < 0.05); 4-hour exposure to e-bandages reduced cell counts by 5.76 ± 1.10 log10 CFU/cm2 (P < 0.05); and 6-hour treatment resulted in reductions of 8.54 ± 0.11 log10 CFU/cm2 (P < 0.05). For Gram-positive biofilms, no growth was observed on any quantification plate, or in broth cultures tubes after 12-hour e-bandage treatment (except for S. epidermidis Xen 43), with the same observed for Gram-negative biofilms exposed to e-bandages for 6 hours. Based on these results, Gram-positive and -negative monospecies biofilms were affected by HOCl producing e-bandage treatment, with Gram-negative biofilms being more sensitive compared to Gram-positive biofilms based on the faster CFU reduction.

Figure 2.

Figure 2.

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e-Bandage treatment of monospecies biofilms after 2, 4, 6, and 12 hours (hr). Data points represent means and error bars represent SDs (n = 3). Data showing statistical significance (P-value < 0.05) are denoted by (*). Red solid symbols represent the nonpolarized (control) and green open symbols the polarized (active treatment) groups.

Dual-species bacterial biofilms

Ten dual-species bacterial biofilms were assessed (Table 2). As with monospecies biofilms, dual-species biofilms were reduced in a time-dependent manner when exposed to HOCl producing e-bandages (Fig. 3, P < 0.05). After 4 hours of exposure, an average reduction of 4.59 ± 1.5  log 10 CFU/cm 2 (P < 0.05) was achieved. A 6-hour treatment led to a mean reduction of 7.09 ± 0.84 log 10 CFU/cm 2 (P < 0.05). A total of 12-hour treatment reduced viable cell counts by 7.74 ± 0.60  log 10 CFU/cm 2 (P < 0.05). A total 12 hours of exposure also led to no colonies on the quantification plates or growth from broth cultures. After 12-hours of treatment, Gram-positive bacteria were reduced by 7.52 ± 0.63 log 10 CFU/cm 2 (P < 0.05), whereas Gram-negative bacteria were reduced by 8.10 ± 0.36  log 10 CFU/cm 2 (P < 0.05), on average.

Figure 3.

Figure 3.

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e-Bandage treatment of dual-species biofilms after 2, 4, 6, and 12 hours (hr). Data points represent means and error bars represent SDs (n = 3). Data showing statistical significance (P-value < 0.05) are denoted by (*). Solid symbols represent the non-polarized (control) and open symbols the polarized (active treatment) groups.

Discussion

In this work, the anti-biofilm activity of HOCl producing e-bandages was demonstrated against monospecies and dual-species bacterial biofilms grown in vitro. Biofilms were grown on polycarbonate membranes on agar plates. With their ability to continuously deliver low concentrations of HOCl over extended periods, the described e-bandages can overcome rapid dissipation of HOCl when bulk solutions are used as wound disinfectants. Low concentrations of HOCl are suggested to improve wound healing but cannot be maintained over time when using conventional HOCl solutions (Sakarya et al. 2014, Sultana et al. 2015, Gold et al. 2017, Ishihara et al. 2017). Previously, HOCl-based e-scaffolds were tested against in vitro monospecies and dual-species bacterial and yeast biofilms, showing that electrochemically generated HOCl could eliminate biofilms (Kiamco et al. 2019, Flurin et al. 2021). To enable direct application to wounds, the HOCl-generating e-scaffold was transformed into an HOCl-generating e-bandage, and a wearable potentiostat that controls working electrode potential and associated electrochemical reactions incorporated (Mohamed et al. 2022). This study demonstrates in vitro anti-biofilm activity of the HOCl producing e-bandage against 19 monospecies and 10 dual-species bacterial biofilms, adding to recent work demonstrating in vitro activity against yeast biofilms (Kletzer et al. 2023).

In earlier studies, HOCl producing e-scaffolds reduced bacterial or yeast in a time-dependent manner. Specifically, activity against S. aureus, P. aeruginosa, and A. baumannii, among other monospecies and dual-species biofilms, as well as candidal biofilms, was shown (Kiamco et al. 2019, Zmuda et al. 2020, Flurin et al. 2021). In this study, in vitro activity against biofilms formed by the bacteria and bacterial combinations listed in Tables 1 and 2 was demonstrated. As with e-scaffolds, and e-bandages with yeast biofilms, time-dependent reductions of viable bacterial cells were observed. Moreover, activity was observed regardless of bacterial species or combination. Biofilms formed by Gram-negative bacterial species were more responsive to HOCl e-bandage treatment compared to those formed by Gram-positive species, possibly as a result of differences in cell wall composition. HOCl exerts antimicrobial activity after it enters cells, leading to rapid ATP hydrolysis. It likely enters cells through passive diffusion, as it is electrically neutral (Barrette et al. 1987, Fukuzaki 2006, da Cruz Nizer et al. 2020). As Gram-positive bacteria have thicker cell walls (20–80 nm) than Gram-negative bacteria (<10 nm), HOCl penetration may take longer for the former, hypothetically explaining observed differences in effects between Gram-positive and -negative biofilms (Mai-Prochnow et al. 2016). Although prior studies suggest that complex molecular interactions in dual-species biofilms may negatively affect survival of certain individual bacterial species (Flurin et al. 2021, Raval et al. 2021a), CFU reductions were numerically higher for dual-species biofilm combinations, although this difference was not statistically significant.

Previously, an e-bandage operated at −0.6VAg/AgCl for H2O2 production was shown to reduce the numbers of viable bacterial and fungal cells in biofilms below the limit of detection when used for 48 hours (Mohamed et al. 2021, Raval et al. 2021b, Raval et al. 2022). Instead of H2O2 production via partial reduction of dissolved oxygen, the higher working electrode potential used here leads to oxidation of chloride ions, resulting in HOCl generation (Sultana et al. 2015, Kiamco et al. 2019). A reduction of CFU counts below the detection limit occurred after only 12 hours, regardless of bacterial species or bacterial combination investigated (except for S. epidermidis Xen 43). Compared to previous work examining the H2O2 producing e-bandage, this could mean a potential four-fold reduction in treatment time (Raval et al. 2021a).

Wound care can be challenging, especially in the presence of biofilms, which hinder healing (Zhao et al. 2013, Gompelman et al. 2016, Versey et al. 2021). The 10 dual species biofilm combinations investigated here were selected based on species commonly found in polymicrobial wound infections and to include antibiotic-resistant bacteria (Serra et al. 2015, Mende et al. 2019). In addition to the modes of antimicrobial recalcitrance associated with biofilms in general, the presence of multiple species may enhance biofilm resilience against antimicrobial strategies (Roy et al. 2014, Bessa et al. 2015, Kean et al. 2017). This work shows that, regardless of whether there is one or more bacterial species in a biofilm, the HOCl producing e-bandage can reduce the numbers of viable bacteria in biofilms.

As mentioned previously, only S. epidermidis Xen 43 was not reduced below the limit of detection after 12 hours of treatment. The reason for this strain being an outlier is unclear. Various defense systems of bacteria against HOCl have been described (Gundlach and Winter 2014, Sultana et al. 2020). However, there is no established single enzyme that degrades HOCl, such as catalase can with H2O2 (Sen and Imlay 2021). Most defences against HOCl focus on minimizing damage. For example, heat shock proteins, which convert into the active chaperone holdase can limit protein denaturation caused by HOCl. Additionally, general resistance against oxidative stress caused by expression of certain regulons can decrease the effectiveness of HOCl (Gundlach and Winter 2014). Therefore, decreased vulnerability to oxidative stress caused by HOCl might confer tolerance to the described e-bandage. Previously, no evidence for emergence of resistance to HOCl in bacterial biofilms continuously exposed to an HOCl-generating e-scaffold was found (Raval et al. 2021a).

As this work was done in vitro, it does not fully represent conditions present in actual wound infections. Discoloration of the agar in the vicinity of e-bandages was occasionally observed, especially at the 12 hour timepoint; the cause of this and whether it will occur in vivo remains to be determined. Wounds are in a state of continual change as they progress through different phases of healing. The formation of a scab could potentially reduce the effectiveness of e-bandage treatment by creating a physical barrier for HOCl (Li et al. 2007, Martin and Nunan 2015, Rodrigues et al. 2019). Moreover, actual wounds differ in size and shape. The e-bandage is designed and can be reconfigured to be even more flexible, to conform to wound surfaces. To further understanding of how physiological processes of wound healing might affect e-bandage activity, in vivo studies are necessary. Biocompatibility of the HOCl producing e-bandage needs assessment. Previous work done with the e-bandage and literature on stability of HOCl suggest that the amount of HOCl produced during e-bandage treatment should not reach the toxicity threshold of 286 μM due to it dissipating before reaching high concentrations (Ishihara et al. 2017, Flurin et al. 2021). Studies done on porcine explants support this notion (Kiamco et al. 2019, Zmuda et al. 2020).

In summary, this work shows that HOCl producing e-bandages reduce numbers of viable bacteria in monospecies and dual-species biofilms below the limit of detection of the methods used. This reduction occurs in a shorter timeframe than noted with a previously-described H2O2 producing e-bandage (Raval et al. 2021b). Future work is needed to assess the antimicrobial efficacy of HOCl producing e-bandage in vivo.

Acknowledgements

The content presented in this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Henry Chambers III (University of California, San Francisco) for providing S. aureus IDRL-8660; Caliper Life Sciences for providing S. epidermidis Xen 43; Daniel Hassett (University of Cincinnati) for providing P. aeruginosa PAO1; and the Antibacterial Resistance Leadership Group (supported by a grant from the National Institutes of Health through Duke University) for providing A. baumannii ARLG-1268.

Contributor Information

Joseph Kletzer, Paracelsus Medical University, Salzburg 5020, Austria; Division of Clinical Microbiology, Mayo Clinic Rochester, Rochester, MN 55905, United States.

Yash S Raval, Division of Clinical Microbiology, Mayo Clinic Rochester, Rochester, MN 55905, United States.

Abdelrhman Mohamed, The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, United States.

Jayawant N Mandrekar, Department of Quantitative Health Sciences, Mayo Clinic, Rochester, MN 55905, United States.

Kerryl E Greenwood-Quaintance, Division of Clinical Microbiology, Mayo Clinic Rochester, Rochester, MN 55905, United States.

Haluk Beyenal, The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, United States.

Robin Patel, Division of Clinical Microbiology, Mayo Clinic Rochester, Rochester, MN 55905, United States; Division of Public Health, Infectious Diseases, and Occupational Medicine, Mayo Clinic, Rochester, MN 55905, United States.

Conflict of interest

H.B. holds a patent (US20180207301A1) “Electrochemical reduction or prevention of infections”, which refers to the electrochemical scaffold described herein. R.P. reports grants from ContraFect, TenNor Therapeutics Limited, and BioFire. R.P. is a consultant to PhAST, Torus Biosystems, Day Zero Diagnostics, Mammoth Biosciences, and HealthTrackRx; monies are paid to Mayo Clinic. Mayo Clinic and R.P. have a relationship with Pathogenomix. R.P. has research supported by Adaptive Phage Therapeutics. Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics. R.P. is also a consultant to Netflix, Abbott Laboratories, Oxford Nanopore Technologies, and CARB-X. In addition, R.P. has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. R.P. receives honoraria from the NBME, Up-to-Date and the Infectious Diseases Board Review Course.

Funding

This research was supported by the National Institute of Allergy and Infectious Diseases of the National Institute of Health under the grant number R01 AI091594.

Author contributions

Joseph Kletzer (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft), Yash S. Raval (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft), Abdelrhman Mohamed (Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing), Jayawant N. Mandrekar (Data curation, Writing – review & editing), Kerryl E. Greenwood-Quaintance (Supervision, Writing – review & editing), Haluk Beyenal (Resources, Supervision, Writing – review & editing), and Robin Patel (Conceptualization, Project administration, Supervision, Writing – review & editing)

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

References

  1. Andres CMC, de la Lastra JMP, Juan CAet al. The role of reactive species on innate immunity. Vaccines (Basel). 2022;10:1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrette WC, Albrich JM, Hurst JK. Hypochlorous acid-promoted loss of metabolic energy in Escherichia coli. Infection and Immununity. 1987;55:2518–25.. 10.1128/iai.55.10.2518-2525.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bertesteanu S, Triaridis S, Stankovic Met al. Polymicrobial wound infections: pathophysiology and current therapeutic approaches. Int J Pharm. 2014;463:119–26.. 10.1016/j.ijpharm.2013.12.012. [DOI] [PubMed] [Google Scholar]
  4. Bessa LJ, Fazii P, Di Giulio Met al. Bacterial isolates from infected wounds and their antibiotic susceptibility pattern: some remarks about wound infection. Int Wound J. 2015;12:47–52.. 10.1111/iwj.12049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broughton G, Janis JE, Attinger CE. the basic science of wound healing. Plast Reconstr Surg. 2006;117:12S–34S.. 10.1097/01.prs.0000225430.42531.c2. [DOI] [PubMed] [Google Scholar]
  6. Brown MRW, Allison DG, Gilbert P. Resistance of bacterial biofilms to antibiotics: a growth-rate related effect?. J Antimicrob Chemother. 1988;22:777–80.. 10.1093/jac/22.6.777. [DOI] [PubMed] [Google Scholar]
  7. Crovetti G, Martinelli G, Issi Met al. Platelet gel for healing cutaneous chronic wounds. Transfus Apher Sci. 2004;30:145–51.. 10.1016/j.transci.2004.01.004. [DOI] [PubMed] [Google Scholar]
  8. Da Cruz Nizer WS, Inkovskiy V, Overhage J. Surviving reactive chlorine stress: responses of gram-negative bacteria to hypochlorous acid. Microorganisms. 2020;8:1220. 10.3390/microorganisms8081220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Evans DJ, Allison DG, Brown MRWet al. Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. J Antimicrob Chemother. 1991;27:177–84.. 10.1093/jac/27.2.177. [DOI] [PubMed] [Google Scholar]
  10. Flurin L, Raval YS, Mohamed Aet al. An integrated HOCl-producing e-scaffold is active against monomicrobial and polymicrobial biofilms. Antimicrob Agents Chemother. 2021;65:e02007–20.. 10.1128/AAC.02007-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fukuzaki S. Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes. Biocontrol Sci. 2006;11:147–57.. 10.4265/bio.11.147. [DOI] [PubMed] [Google Scholar]
  12. Gold MH, Andriessen A, Dayan SHet al. Hypochlorous acid gel technology-its impact on postprocedure treatment and scar prevention. J Cosmet Dermatol. 2017;16:162–67.. 10.1111/jocd.12330. [DOI] [PubMed] [Google Scholar]
  13. Gompelman M, Van Asten SAV, Peters EJG. Update on the role of infection and biofilms in wound healing: pathophysiology and treatment. Plast Reconstr Surg. 2016;138:61S–70S.. 10.1097/PRS.0000000000002679. [DOI] [PubMed] [Google Scholar]
  14. Gosain A, Dipietro LA. Aging and wound healing. World J Surg. 2004;28:321–6.. 10.1007/s00268-003-7397-6. [DOI] [PubMed] [Google Scholar]
  15. Gundlach J, Winter J. Evolution of Escherichia coli for maximum HOCl resistance through constitutive expression of the OxyR regulon. Microbiology (Reading). 2014;160:1690–704.. 10.1099/mic.0.074815-0. [DOI] [PubMed] [Google Scholar]
  16. Han G, Ceilley R. Chronic wound healing: A review of current management and treatments. Adv Ther. 2017;34:599–610.. 10.1007/s12325-017-0478-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ishihara M, Murakami K, Fukuda Ket al. Stability of weakly acidic hypochlorous acid solution with microbicidal activity. Biocontrol Sci. 2017;22:223–27.. 10.4265/bio.22.223. [DOI] [PubMed] [Google Scholar]
  18. Kean R, Rajendran R, Haggarty Jet al. Candida albicans mycofilms support Staphylococcus aureus colonization and enhances miconazole resistance in dual-species interactions. Front Microbiol. 2017;8:1–11.. 10.3389/fmicb.2017.00258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kiamco MM, Zmuda HM, Mohamed Aet al. Hypochlorous-acid-generating electrochemical scaffold for treatment of wound biofilms. Sci Rep. 2019;9:2683. 10.1038/s41598-019-38968-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kletzer J, Raval YS, Mohamed Aet al. In vitro activity of a hypochlorous acid-generating electrochemical bandage against yeast bofilms. Antimicrob Agents Chemother. 2023;67:e0116622. 10.1128/aac.01166-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol. 2007;25:9–18.. 10.1016/j.clindermatol.2006.09.007. [DOI] [PubMed] [Google Scholar]
  22. Liu Y-F, Ni P-W, Huang Yet al. Therapeutic strategies for chronic wound infection. Chinese J Traumatol. 2022;25:11–16.. 10.1016/j.cjtee.2021.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Loo AEK, Wong YT, Ho Ret al. Effects of hydrogen peroxide on wound healing in mice in relation to oxidative damage. PLoS One. 2012;7:e49215. 10.1371/journal.pone.0049215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mai-Prochnow A, Clauson M, Hong Jet al. Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma. Sci Rep. 2016;6:38610. 10.1038/srep38610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martin P, Nunan R.. Cellular and molecular mechanisms of repair in acute and chronic wound healing. Br J Dermatol. 2015;173:370–8.. 10.1111/bjd.13954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Martinengo L, Olsson M, Bajpai Ret al. Prevalence of chronic wounds in the general population: systematic review and meta-analysis of observational studies. Annals in Epidemiology. 2019;29:8–15.. 10.1016/j.annepidem.2018.10.005. [DOI] [PubMed] [Google Scholar]
  27. Mende K, Stewart L, Shaikh Fet al. Microbiology of combat-related extremity wounds: trauma Infectious Disease Outcomes Study. Diagn Microbiol Infect Dis. 2019;94:173–79.. 10.1016/j.diagmicrobio.2018.12.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mohamed A, Anoy MMI, Tibbits Get al. Hydrogen peroxide-producing electrochemical bandage controlled by a wearable potentiostat for treatment of wound infections. Biotechnol Bioeng. 2021;118:2815–21.. 10.1002/bit.27794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mohamed A, Raval YS, Gelston Set al. Antibiofilm activity of a tunable hypochlorous acid-generating electrochemical bandage controlled by a wearable potentiostat. Adv Eng Mater. 2022;25:2200792. 10.1002/adem.202200792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Murphy EC, Friedman AJ. Hydrogen peroxide and cutaneous biology: translational applications, benefits, and risks. J Am Acad Dermatol. 2019;81:1379–86.. 10.1016/j.jaad.2019.05.030. [DOI] [PubMed] [Google Scholar]
  31. Nguyen D, Joshi-Datar A, Lepine Fet al. Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science. 2011;334:982–6.. 10.1126/science.1211037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Orazi G, O'toole GA. “It takes a village”: mechanisms underlying antimicrobial recalcitrance of polymicrobial biofilms. J Bacteriol. 2019;202:e00530–19.. 10.1128/JB.00530-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Raval YS, Flurin L, Mohamed Aet al. In vitro&nbsp; antibacterial activity of hydrogen peroxide and hypochlorous acid, including that generated by electrochemical scaffolds. Antimicrob Agents Chemother. 2021a;65:e01966–20.. 10.1128/AAC.01966-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Raval YS, Mohamed A, Flurin Let al. Hydrogen-peroxide generating electrochemical bandage is active in vitro against mono- and dual-species biofilms. Biofilm. 2021b;3:100055. 10.1016/j.bioflm.2021.100055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Raval YS, Mohamed A, Mandrekar JNet al. in vitro antibiofilm activity of hydrogen peroxide-generating electrochemical bandage against yeast biofilms. Antimicrob Agents Chemother. 2022;66:e0179221. 10.1128/aac.01792-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rodrigues M, Kosaric N, Bonham CAet al. Wound healing: A cellular perspective. Physiol Rev. 2019;99:665–706.. 10.1152/physrev.00067.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roy S, Elgharably H, Sinha Met al. Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function. J Pathol. 2014;233:331–43.. 10.1002/path.4360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sakarya S, Gunay N, Karakulak Met al. Hypochlorous acid: An ideal wound care agent with powerful microbicidal, antibiofilm, and wound healing potency. Wounds. 2014;26:342–50. [PubMed] [Google Scholar]
  39. Sen A, Imlay JA. How microbes defend themselves from incoming hydrogen peroxide. Front Immunol. 2021;12:1–19.. 10.3389/fimmu.2021.667343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Serra R, Grande R, Butrico Let al. Chronic wound infections: The role of Pseudomonas aeruginosa and Staphylococcus aureus. Expert Rev Anti Infect Ther. 2015;13:605–13.. 10.1586/14787210.2015.1023291. [DOI] [PubMed] [Google Scholar]
  41. Singer AJ, Clark RAF. Cutaneous wound healing. N Engl J Med. 1999;341:738–46.. 10.1056/NEJM199909023411006. [DOI] [PubMed] [Google Scholar]
  42. Sultana S, Foti A, Dahl J-U. Bacterial defense systems against the neutrophilic oxidant hypochlorous acid. Infect Immun. 2020;88:e00964–19.. 10.1128/IAI.00964-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sultana ST, Atci E, Babauta JTet al. Electrochemical scaffold generates localized, low concentration of hydrogen peroxide that inhibits bacterial pathogens and biofilms. Sci Rep. 2015;5:14908. 10.1038/srep14908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Versey Z, Da Cruz Nizer WS, Russell Eet al. Biofilm-innate immune interface: contribution to chronic wound formation. Front Immunol. 2021;12:648554. 10.3389/fimmu.2021.648554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yan J, Bassler BL. Surviving as a community: Antibiotic tolerance and persistence in bacterial biofilms. Cell Host Microbe. 2019;26:15–21.. 10.1016/j.chom.2019.06.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang L, Mah T-F. Involvement of a novel efflux system in biofilm-specific resistance to antibiotics. J Bacteriol. 2008;190:4447–52.. 10.1128/JB.01655-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhao G, Usui ML, Lippman SIet al. Biofilms and inflammation in chronic wounds. Adv Wound Care (New Rochelle). 2013;2:389–99.. 10.1089/wound.2012.0381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zmuda HM, Mohamed A, Raval YSet al. Hypochlorous acid-generating electrochemical scaffold eliminates Candida albicans biofilms. J Appl Microbiol. 2020;129:776–86.. 10.1111/jam.14656. [DOI] [PMC free article] [PubMed] [Google Scholar]

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