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.

In Vitro Activity of a Hypochlorous Acid-Generating Electrochemical Bandage against Yeast Biofilms.

The antibiofilm activity of a hypochlorous acid (HOCl)-producing electrochemical bandage (e-bandage) was assessed against 14 yeast isolates in vitro. The evaluated e-bandage was polarized at +1.5 VAg/AgCl to allow continuous production of HOCl. Time-dependent decreases in the biofilm CFU counts were observed for all isolates with e-bandage treatment. The results suggest that the described HOCl-producing e-bandage could serve as a potential alternative to traditional antifungal wound biofilm treatments.

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.

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

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.

In Vitro Antibiofilm Activity of Hydrogen Peroxide-Generating Electrochemical Bandage against Yeast Biofilms.

Wound infections are caused by bacteria and/or fungi. The presence of fungal biofilms in wound beds presents a unique challenge, as fungal biofilms may be difficult to eradicate. The goal of this work was to assess the in vitro antibiofilm activity of an H2O2-producing electrochemical bandage (e-bandage) against 15 yeast isolates representing commonly encountered species. Time-dependent decreases in viable biofilm CFU counts of all isolates tested were observed, resulting in no visible colonies with 48 h of exposure by plate culture. Fluorescence microscopic analysis showed extensive cell membrane damage of biofilm cells after e-bandage treatment. Reductions in intracellular ATP levels of yeast biofilm cells were recorded post e-bandage treatment. These results suggest that exposure to H2O2-producing e-bandages reduces in vitro viable cell counts of yeast biofilms, making this a potential new topical treatment approach for fungal wound 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.

in vitro Activity of Hydrogen Peroxide and Hypochlorous Acid Generated by Electrochemical Scaffolds Against Planktonic and Biofilm Bacteria.

Hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are biocides used for cleaning and debriding chronic wound infections, which often harbor drug resistant bacteria. Here, we evaluated the in vitro activity of H2O2 and HOCl against 27 isolates of eight bacterial species involved in wound infections. Minimum inhibitory concentrations (MICs) and minimum biofilm bactericidal concentrations (MBBCs) were measured. When compared to their respective MICs, MBBCs of isolates exposed to H2O2 were 16- to 1,024-fold higher and those exposed to HOCl were 2- to 4-fold higher. We evaluated selection of resistance after exposure of Staphylococcus aureus and Pseudomonas aeruginosa biofilms to 10 iterations of electrochemically generated HOCl or H2O2 delivered using electrochemical scaffolds (e-scaffolds), observing no decrease in anti-biofilm effects with serial exposure to e-scaffold-generated H2O2 or HOCl. 24-hour exposure to H2O2-generating e-scaffolds consistently decreased colony forming units (CFUs) of S. aureus and P. aeruginosa biofilms by ∼5.0-log10 and ∼4.78-log10 through 10 iterations of exposure, respectively. 4-hour exposure to HOCl-generating e-scaffolds consistently decreased CFUs of S. aureus biofilms by ∼4.9-log10, and 1-hour exposure to HOCl-generating e-scaffolds consistently decreased CFUs of P. aeruginosa biofilms by ∼1.57-log10 These results suggest that HOCl has similar activity against planktonic and biofilm bacteria, whereas the activity of H2O2 is less against biofilm than planktonic bacteria, and that repeat exposure to either biocide, generated electrochemically under the experimental conditions studied, does not lessen antibiofilm effects.

An Integrated HOCl-Producing E-Scaffold Is Active against Monomicrobial and Polymicrobial Biofilms.

Oxidizing agents like hypochlorous acid (HOCl) have antimicrobial activity. We developed an integrated electrochemical scaffold, or e-scaffold, that delivers a continuous low dose of HOCl aimed at targeting microbial biofilms without exceeding concentrations toxic to humans as a prototype of a device being developed to treat wound infections in humans. In this work, we tested the device against 33 isolates of bacteria (including isolates with acquired antibiotic resistance) grown as in vitro biofilms alongside 12 combinations of dual-species in vitro biofilms. Biofilms were grown on the bottoms of 12-well plates for 24 h. An integrated e-scaffold was placed atop each biofilm and polarized at 1.5 V for 1, 2, or 4 h. HOCl was produced electrochemically by oxidizing chloride ions (Cl-) in solution to chlorine (Cl2); dissolved Cl2 spontaneously dissociates in water to produce HOCl. The cumulative concentration of HOCl produced at the working electrode in each well was estimated to be 7.89, 13.46, and 29.50 mM after 1, 2, and 4 h of polarization, respectively. Four hours of polarization caused an average reduction of 6.13 log10 CFU/cm2 (±1.99 log10 CFU/cm2) of viable cell counts of monospecies biofilms and 5.53 log10 CFU/cm2 (±2.31 log10 CFU/cm2) for the 12 dual-species biofilms studied. The described integrated e-scaffold reduces viable bacterial cell counts in biofilms formed by an array of antibiotic-susceptible and -resistant bacteria alone and in combination.

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.

Electrochemically Active Biofilms as an Indicator of Soil Health.

Soil health is a complex phenomenon that reflects the ability of soil to support both plant growth and other ecosystem functions. To our knowledge, research on extracellular electron transfer processes in soil environments is limited and could provide novel knowledge and new ways of monitoring soil health. Electrochemical activities in the soil can be studied by inserting inert electrodes. Once the electrode is polarized to a favorable potential, nearby microorganisms attach to the electrodes and grow as biofilms. Biofilms are a major part of the soil and play critical roles in microbial activity and community dynamics. Our work aims to investigate the electrochemical behavior of healthy and unhealthy soils using chronoamperometry and cyclic voltammetry. We developed a bioelectrochemical soil reactor for electrochemical measurements using healthy and unhealthy soils taken from the Cook Agronomy Farm Long-Term Agroecological Research site; the soils showed similar physical and chemical characteristics, but there was higher plant growth where the healthy soil was taken. Using carbon cloth electrodes installed in these soil reactors, we explored the electrochemical signals in these two soils. First, we measured redox variations by depth and found that reducing conditions were prevalent in healthy soils. Current measurements showed distinct differences between healthy and unhealthy soils. Scanning electron microscopy images showed the presence of microbes attached to the electrode for healthy soil but not for unhealthy soil. Glucose addition stimulated current in both soil types and caused differences in cyclic voltammograms between the two soil types to converge. Our work demonstrates that we can use current as a proxy for microbial metabolic activity to distinguish healthy and unhealthy soil.

Hydrogen Peroxide-Generating Electrochemical Scaffold Activity against Trispecies Biofilms.

The antibiofilm activity of a hydrogen peroxide-generating electrochemical scaffold (e-scaffold) was determined against mono- and trispecies biofilms of methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, and Candida albicans Significant time-dependent decreases were found in the overall CFU of biofilms of all three monospecies and the trispecies forms. Confocal laser scanning microscopy showed dramatic reductions in fluorescence intensities of biofilm matrix protein and polysaccharide components of e-scaffold-treated biofilms. The described e-scaffold has potential as a novel antibiotic-free strategy for treating wound biofilms.

Three-dimensional biofilm image reconstruction for assessing structural parameters.

Parameters representing three-dimensional (3D) biofilm structure are quantified from confocal laser-scanning microscope (CLSM) images. These 3D parameters describe the distribution of biomass pixels within the space occupied by a biofilm; however, they lack a direct connection to biofilm activity. As a result, researchers choose a handful of parameters without there being a consensus on a standard set of parameters. We hypothesized that a select 3D parameter set could be used to reconstruct a biofilm image and that the reconstructed and original biofilm images would have similar activities. To test this hypothesis, an algorithm was developed to reconstruct a biofilm image with parameters identical to those of the original CLSM image. We introduced an objective method to assess the reconstruction algorithm by comparing the activities of the original and reconstructed biofilm images. We found that biofilm images with identical structural parameters showed nearly identical activities and substrate concentration profiles. This implies that the set containing all common structural parameters can successfully describe biofilm structure. This finding is significant, as it opens the door to the next step, of finding a smaller standard set of biofilm structural parameters that can be used to compare biofilm structure.

Responses of Acinetobacter baumannii Bound and Loose Extracellular Polymeric Substances to Hyperosmotic Agents Combined with or without Tobramycin: An Atomic Force Microscopy Study.

In this work, contributions of extracellular polymeric substances (EPS) to the nanoscale mechanisms through which the multidrug-resistant Acinetobacter baumannii responds to antimicrobial and hyperosmotic treatments were investigated by atomic force microscopy. Specifically, the adhesion strengths to a control surface of silicon nitride (Si3N4) and the lengths of bacterial surface biopolymers of bound and loose EPS extracted from A. baumannii biofilms were quantified after individual or synergistic treatments with hyperosmotic agents (NaCl and maltodextrin) and an antibiotic (tobramycin). In the absence of any treatment, the loose EPS were significantly longer in length and higher in adhesion to Si3N4 than the bound EPS. When used individually, the hyperosmotic agents and tobramycin collapsed the A. baumannii bound and loose EPS. The combined treatment of maltodextrin with tobramycin collapsed only the loose EPS and did not alter the adhesion of both bound and loose EPS to Si3N4. In addition, the combined treatment was not as effective in collapsing the EPS molecules as when tobramycin was applied alone. Finally, the effects of treatments were dose-dependent. Altogether, our findings suggest that a sequential treatment could be effective in treating A. baumannii biofilms, in which a hyperosmotic agent is used first to collapse the EPS and limit the diffusion of nutrients into the biofilm, followed by the use of an antibiotic to kill the bacterial cells that escape from the biofilm because of starvation.

Design and Finite Element Model of a Microfluidic Platform with Removable Electrodes for Electrochemical Analysis.

A microfluidic platform for hydrodynamic electrochemical analysis was developed, consisting of a poly(methyl methacrylate) chip and three removable electrodes, each housed in 1/16" OD polyether ether ketone tube which can be removed independently for polishing or replacement. The working electrode was a 100-µm diameter Pt microdisk, located flush with the upper face of a 150 µm × 20 µm × 3 cm microchannel, smaller than previously reported for these types of removable electrodes. A commercial leak-less reference electrode was utilized, and a coiled platinum wire was the counter electrode. The platform was evaluated electrochemically by oxidizing a potassium ferrocyanide solution at the working electrode, and a typical limiting current behavior was observed after running linear sweep voltammetry and chronoamperometry, with flow rates 1-6 µL/min. While microdisk channel electrodes have been simulated before using a finite difference method in an ideal 3D geometry, here we predict the limiting current using finite elements in COMSOL Multiphysics 5.3a, which allowed us to easily explore variations in the microchannel geometry that have not previously been considered in the literature. Experimental and simulated currents showed the same trend but differed by 41% in simulations of the ideal geometry, which improved when channel and electrode imperfections were included.

Structural and metabolic responses of Staphylococcus aureus biofilms to hyperosmotic and antibiotic stress.

Biofilms alter their metabolism in response to environmental stress. This study explores the effect of a hyperosmotic agent-antibiotic treatment on the metabolism of Staphylococcus aureus biofilms through the use of nuclear magnetic resonance (NMR) techniques. To determine the metabolic activity of S. aureus, we quantified the concentrations of metabolites in spent medium using high-resolution NMR spectroscopy. Biofilm porosity, thickness, biovolume, and relative diffusion coefficient depth profiles were obtained using NMR microimaging. Dissolved oxygen concentration was measured to determine the availability of oxygen within the biofilm. Under vancomycin-only treatment, the biofilm communities switched to fermentation under anaerobic condition, as evidenced by high concentrations of formate (7.4 ± 2.7 mM), acetate (13.1 ± 0.9 mM), and lactate (3.0 ± 0.8 mM), and there was no detectable dissolved oxygen in the biofilm. In addition, we observed the highest consumption of pyruvate (0.19 mM remaining from an initial 40 mM concentration), the sole carbon source, under the vancomycin-only treatment. On the other hand, relative effective diffusion coefficients increased from 0.73 ± 0.08 to 0.88 ± 0.08 under vancomycin-only treatment but decreased from 0.71 ± 0.04 to 0.60 ± 0.07 under maltodextrin-only and from 0.73 ± 0.06 to 0.56 ± 0.08 under combined treatments. There was an increase in biovolume, from 2.5 ± 1 mm3 to 7 ± 1 mm3 , under the vancomycin-only treatment, while the maltodextrin-only and combined treatments showed no significant change in biovolume over time. This indicated that physical biofilm growth was halted during maltodextrin-only and combined treatments.

Osmotic Compounds Enhance Antibiotic Efficacy against Acinetobacter baumannii Biofilm Communities.

Biofilm-associated infections are a clinical challenge, in part because a hydrated matrix protects the bacterial community from antibiotics. Herein, we evaluated how different osmotic compounds (maltodextrin, sucrose, and polyethylene glycol [PEG]) enhance antibiotic efficacy against Acinetobacter baumannii biofilm communities. Established (24-h) test tube biofilms (strain ATCC 17978) were treated with osmotic compounds in the presence or absence of 10× the MIC of different antibiotics (50 µg/ml tobramycin, 20 µg/ml ciprofloxacin, 300 µg/ml chloramphenicol, 30 µg/ml nalidixic acid, or 100 µg/ml erythromycin). Combining antibiotics with hypertonic concentrations of the osmotic compounds for 24 h reduced the number of biofilm bacteria by 5 to 7 log (P < 0.05). Increasing concentrations of osmotic compounds improved the effect, but there was a trade-off with increasing solution viscosity, whereby low-molecular-mass compounds (sucrose, 400-Da PEG) worked better than higher-mass compounds (maltodextrin, 3,350-Da PEG). Ten other A. baumannii strains were similarly treated with 400-Da PEG and tobramycin, resulting in a mean 2.7-log reduction in recoverable bacteria compared with tobramycin treatment alone. Multivariate regression models with data from different osmotic compounds and nine antibiotics demonstrated that the benefit from combining hypertonic treatments with antibiotics is a function of antibiotic mass and lipophilicity (r2 > 0.82; P < 0.002), and the relationship was generalizable for biofilms formed by A. baumannii and Escherichia coli K-12. Augmenting topical antibiotic therapies with a low-mass hypertonic treatment may enhance the efficacy of antibiotics against wound biofilms, particularly when using low-mass hydrophilic antibiotics.IMPORTANCE Biofilms form a barrier that protects bacteria from environmental insults, including exposure to antibiotics. We demonstrated that multiple osmotic compounds can enhance antibiotic efficacy against Acinetobacter baumannii biofilm communities, but viscosity is a limiting factor, and the most effective compounds have lower molecular mass. The synergism between osmotic compounds and antibiotics is also dependent on the hydrophobicity and mass of the antibiotics. The statistical models presented herein provide a basis for predicting the optimal combination of osmotic compounds and antibiotics against surface biofilms communities.

Hyperosmotic Agents and Antibiotics Affect Dissolved Oxygen and pH Concentration Gradients in Staphylococcus aureus Biofilms.

Biofilms on wound surfaces are treated topically with hyperosmotic agents, such as medical-grade honey and cadexomer iodine; in some cases, these treatments are combined with antibiotics. Tissue repair requires oxygen, and a low pH is conducive to oxygen release from red blood cells and epithelialization. We investigated the variation of dissolved oxygen concentration and pH with biofilm depth and the variation in oxygen consumption rates when biofilms are challenged with medical-grade honey or cadexomer iodine combined with vancomycin or ciprofloxacin. Dissolved oxygen and pH depth profiles in Staphylococcus aureus biofilms were measured using microelectrodes. The presence of cadexomer iodine with vancomycin or ciprofloxacin on the surface of the biofilm permitted a measurable concentration of oxygen at greater biofilm depths (101.6 ± 27.3 µm, P = 0.02; and 155.5 ± 27.9 µm, P = 0.016, respectively) than in untreated controls (30.1 µm). Decreases in pH of ∼0.6 and ∼0.4 units were observed in biofilms challenged with medical-grade honey alone and combined with ciprofloxacin, respectively (P < 0.001 and 0.01, respectively); the number of bacteria recovered from biofilms was significantly reduced (1.26 log) by treatment with cadexomer iodine and ciprofloxacin (P = 0.002) compared to the untreated control. Combining cadexomer iodine and ciprofloxacin improved dissolved oxygen concentration and penetration depth into the biofilm, while medical-grade honey was associated with a lower pH; not all treatments established a bactericidal effect in the time frame used in the experiments.IMPORTANCE Reports about using hyperosmotic agents and antibiotics against wound biofilms focus mostly on killing bacteria, but the results of these treatments should additionally be considered in the context of how they affect physiologically important parameters, such as oxygen concentration and pH. We confirmed that the combination of a hyperosmotic agent and an antibiotic results in greater dissolved oxygen and reduced pH within an S. aureus biofilm.

Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.

Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, oxygen microsensors to measure oxygen transects through in vitro cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens was used. The results showed that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17 to 72 mmHg on live mice and from 6.4 to 1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds was performed. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results supported the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes.

Staphylococcus aureus induces hypoxia and cellular damage in porcine dermal explants.

We developed a porcine dermal explant model to determine the extent to which Staphylococcus aureus biofilm communities deplete oxygen, change pH, and produce damage in underlying tissue. Microelectrode measurements demonstrated that dissolved oxygen (DO) in biofilm-free dermal tissue was 4.45 ± 1.17 mg/liter, while DO levels for biofilm-infected tissue declined sharply from the surface, with no measurable oxygen detectable in the underlying dermal tissue. Magnetic resonance imaging demonstrated that biofilm-free dermal tissue had a significantly lower relative effective diffusion coefficient (0.26 ± 0.09 to 0.30 ± 0.12) than biofilm-infected dermal tissue (0.40 ± 0.12 to 0.48 ± 0.12; P < 0.0001). Thus, the difference in DO level was attributable to biofilm-induced oxygen demand rather than changes in oxygen diffusivity. Microelectrode measures showed that pH within biofilm-infected explants was more alkaline than in biofilm-free explants (8.0 ± 0.17 versus 7.5 ± 0.15, respectively; P < 0.002). Cellular and nuclear details were lost in the infected explants, consistent with cell death. Quantitative label-free shotgun proteomics demonstrated that both proapoptotic programmed cell death protein 5 and antiapoptotic macrophage migration inhibitory factor accumulated in the infected-explant spent medium, compared with uninfected-explant spent media (1,351-fold and 58-fold, respectively), consistent with the cooccurrence of apoptosis and necrosis in the explants. Biofilm-origin proteins reflected an extracellular matrix-adapted lifestyle of S. aureus. S. aureus biofilms deplete oxygen, increase pH, and induce cell death, all factors that contribute to impede wound healing.