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.

ABSTRACT

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.

INTRODUCTION

Chronic wound infections pose a burden to patients and wound clinics. More than 3 million people are treated for chronic wound infections every year in the United States, with estimated costs of treatment exceeding $10 billion (1, 2). Most of these wound infections are associated with microorganisms in biofilms, a growth mode in which microorganisms exhibit decreased susceptibility to many available antimicrobial agents (3, 4). Microorganisms in biofilms are encased in an extracellular polymeric matrix made of glycoproteins, lipopolysaccharides, and extracellular DNA (eDNA), which contributes to the reduced treatment efficacy of many available antimicrobial agents (5). Moreover, the presence of biofilms in wounds may negatively affect the overall wound repair process (6, 7). Chronic wound infections are generally polymicrobial in nature, consisting of multiple species of microorganisms (8, 9). Some of the commonly found microorganisms that coinhabit wound beds are Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Candida albicans, among others (10). Although medical biofilms are typically studied as single species, mixed-species biofilms more closely recapitulate the clinical situation.

Topical antimicrobials, and antiseptics are current first-line agents for treatment of chronic wound infections. Agents used for cleaning and debriding wounds include benzalkonium chloride, chlorhexidine, sodium hypochlorite, and hydrogen peroxide (H₂O₂) (11, 12). The presence of biofilms can, however, confer decreased susceptibility to these agents. H₂O₂ is among the most interesting biocides for targeting wound infections and augmenting wound healing. Several studies have shown that small amounts of H₂O₂ can improve the overall wound-healing process by increasing fibroblast and keratinocyte differentiation and their migration to wound sites (13–15). However, a limitation in using H₂O₂ is that it is rapidly oxidized, thereby losing potency. To address this issue, in our previous work, we developed and assessed the antimicrobial activity of electrochemical scaffolds (e-scaffolds) that continuously generate H₂O₂ (∼45 mM) against monospecies biofilms (13, 16). However, for clinical success, it is necessary to show that such e-scaffolds have activity against biofilms formed of multiple species likely to be present in wound environments. In this work, we therefore evaluated the antibiofilm activity of our e-scaffolds against trispecies biofilms of methicillin-resistant S. aureus, P. aeruginosa, and C. albicans. In addition, we visualized the trispecies biofilms using confocal laser scanning microscopy to assess effects of e-scaffold treatment on components of the biofilm matrix.

For the monospecies and trispecies biofilm experiments, S. aureus and P. aeruginosa biofilms were grown as described in our prior study (17). C. albicans biofilms were grown similarly but were incubated at 30°C. Additional biofilm growth details are provided in the supplemental material. A custom-made e-scaffold comprised of carbon fabric was fabricated and assembled as previously described (13). The e-scaffold was polarized at −0.6 V_Ag/AgCl in phosphate-buffered saline to continuously generate low concentrations of H₂O₂ by reducing oxygen. Biofilms were exposed to e-scaffolds for 6, 12, 24, or 48 h. Control experiments were performed by exposing biofilms to nonpolarized e-scaffolds (i.e., with no H₂O₂ generation). After e-scaffold treatment, CFUs were quantified (for details, see the supplemental material) using spread plating onto sheep blood agar for monospecies experiments and selective media for bacterial and candidal enumeration, with results reported as log₁₀ CFU/cm². Each of the triplicate experiments was performed on 3 different days. As evident in Fig. 1, there was an overall time-dependent decrease in CFU counts of biofilms after exposure to polarized e-scaffolds. Significant differences in CFU counts were observed compared with control groups for all microorganisms tested, alone and in combination. Mono- and trispecies biofilm CFU counts were below the detection limit of quantitative culture after 48 h of continuous polarized e-scaffold exposure (P < 0.001, analysis of variance [ANOVA]). Interestingly, for the trispecies biofilm experiment, 6 h exposure to e-scaffolds did not yield a significant reduction in CFU counts compared with the control group (Fig. 1D). In contrast, CFU counts of monospecies biofilms of all three organisms were reduced by ∼1 to ∼2 logs after 6 h of e-scaffold treatment. As noted, by 48 h, CFU counts of all components of the trispecies biofilm were below the limit of detection by quantitative culture (10 CFU/ml).

FIG 1.

CFU/cm² of Staphylococcus aureus USA100 biofilms (A), Pseudomonas aeruginosa IDRL-11442 biofilms (B), Candida albicans IDRL-7033 biofilms (C), and trispecies biofilms of isolates from panels A to C (D) after e-scaffold treatment for 6, 12, 24, and 48 h compared with no treatment. Data are means ± SD (n = 3). Statistical analysis was performed using one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

To determine the effect of H₂O₂ generated by e-scaffolds on matrix proteins, eDNA, and polysaccharides in the biofilm matrix, confocal laser scanning microscopy (CLSM) was performed after exposing trispecies biofilm to polarized e-scaffolds for 48 h. Trispecies biofilms were stained with FilmTracer SYPRO ruby biofilm matrix stain to assess protein, wheat germ agglutinin Oregon green 488 conjugate to assess polysaccharide, and DAPI (4′,6-diamidino-2-phenylindole) to assess DNA (18, 19). Additional details regarding CLSM experimentation and image analysis are found in the supplemental material. Figure 2 shows images of trispecies biofilms after 48 h of polarized e-scaffold treatment. Biofilm matrix is typically enclosed by extracellular polymeric substances (EPS) comprised of polysaccharides, proteins, and nucleic acids. As evident from Fig. 2B and E, e-scaffold treatment reduced the relative amounts of matrix proteins and polysaccharides compared with control levels. We also quantified the relative fluorescent units of the staining dyes and observed statistically significant decreases (P < 0.05, unpaired Student's t test) in the intensities of the SYPRO ruby and Oregon green stains, which correspond to reduced relative amounts of matrix proteins and polysaccharides in biofilms, respectively (Fig. 2C and F). However, we did not observe differences for the DAPI channel intensities, between the experimental and control groups (Fig. 2G, H, and I). These results suggest that e-scaffold treatment disrupted the biofilm matrix of trispecies biofilms.

FIG 2.

Staining of biofilm components of trispecies biofilm of Staphylococcus aureus USA100, Pseudomonas aeruginosa IDRL-11442, and Candida albicans IDRL-7033. (A, B) Biofilm protein matrix stained with SYPRO ruby. (D, E) Membrane glycoprotein stained with Oregon green. (G, H) DNA stained using DAPI. Total magnification, 400×. (C, F, I) Fluorescent staining intensity of SYPRO ruby, Oregon green, and DAPI. Data are means ± SD (n = 4).

Treating mixed-species biofilm within wounds is a challenge in clinical settings. Complex antimicrobial regimens are often used to treat such infections. H₂O₂ is one of the commonly used biocides for wound debridement and is an effective antimicrobial agent. High concentrations of H₂O₂ are toxic to underlying tissues. Long-term H₂O₂ depot delivery is not possible because of its autocatalytic decomposition. The described e-scaffold solves these problems by continuously generating low concentrations of H₂O₂. This low concentration has antibiofilm activity and, as previously shown, results in minimal tissue toxicity (13). Another advantage of H₂O₂ over some other antimicrobial treatments is that H₂O₂ is active against bacteria and fungi, abrogating the need for coadministration of antibacterial and antifungal agents (20).

Several studies have documented that the frequency of S. aureus, P. aeruginosa, and C. albicans in mixed-species biofilm-associated infections in wounds is high (8, 21). The presence of biofilms in wounds affects the overall efficacy of antimicrobial strategies. Low pH, low water and oxygen availability, low growth rates, the presence of persister cells, biofilm thickness, and EPS render antimicrobials poorly active against biofilms (22, 23); these effects may be amplified in mixed-species biofilms (12, 24). Biofilm killing tended to be time-dependent with e-scaffold exposure (Fig. 1). Interestingly, killing by e-scaffolds was more pronounced with mono- than with trispecies biofilms at 6, 12, and 24 h. At 6 h of exposure, for example, no significant reduction in CFU counts was observed for trispecies biofilms.

For the same effectiveness of e-scaffold treatment, trispecies biofilms needed to be treated longer than monospecies biofilms. This is probably because mixed-species biofilms are less susceptible to antimicrobials and biocides than single-species biofilms (12, 24). Several in vitro studies have described a protective role of C. albicans in colonization of S. aureus and P. aeruginosa biofilms and a reduction in drug susceptibility due to the complex molecular interactions occurring in mixed-species biofilms formed by these microorganisms (25–27). The three-dimensional biofilm images showed a considerable decrease in protein and polysaccharide components after e-scaffold treatment. A few studies involving antimicrobial peptides, ginger extracts, and polymer dressings have reported decreases in protein and carbohydrate contents of S. aureus and P. aeruginosa biofilms (18, 28, 29). Interestingly, DAPI staining remained unchanged. DAPI may stain eDNA, DNA in cells, and DNA released from cells into the matrix during e-scaffold treatment. Our previous work demonstrated extensive cell membrane damage of S. aureus biofilms after e-scaffold treatment (17). This may potentially release DNA into the matrix as well as provide intracellular DNA for staining. E-scaffold treatment may promote release of DNA in the matrix. In a study by Das and Manefield (30), P. aeruginosa mutants that produced excess pyocyanin had increased eDNA in their matrices. In another study, physicochemical stress due to H₂O₂ treatment of Staphylococcus epidermidis biofilm induced DNA release into the matrix (31). Another potential explanation for the DAPI findings may be that the low concentration of H₂O₂ (∼45 mM) produced by our e-scaffold was not enough to affect eDNA found in the matrix.

In conclusion, our results demonstrate that an H₂O₂-producing e-scaffold is active against mono- and trispecies biofilms, a strategy that may potentially be leveraged as a novel antimicrobial-free approach to treatment of chronic wound infections.