Evidence-Based Review of Antibiofilm Agents for Wound Care

Abstract

Significance: Biofilms in vivo are small densely packed aggregations of microbes that are highly resistant to host immune responses and treatment. They attach to each other and to nearby surfaces. Biofilms are difficult to study and identify in a clinical setting as their quantification necessitates the use of advanced microscopy techniques such as confocal laser scanning microscopy. Nonetheless, it is likely that biofilms contribute to the pathophysiology of chronic skin wounds. Reducing, removing, or preventing biofilms is thus a logical approach to help clinicians heal chronic wounds.

Recent Advances: Wound care products have demonstrated varying degrees of efficacy in destroying biofilms in in vitro and preclinical models, as well as in some clinical studies.

Critical Issues: Controlled studies exploring the beneficial role of biofilm eradication and its relationship to healing in patients with chronic wounds are limited. This review aims to discuss the mode of action and clinical significance of currently available antibiofilm products, including surfactants, dressings, and others, with a focus on levels of evidence for efficacy in disrupting biofilms and ability to improve wound healing outcomes.

Future Directions: Few available products have good evidence to support antibiofilm activity and wound healing benefits. Novel therapeutic strategies are on the horizon. More high-quality clinical studies are needed. The development of noninvasive techniques to quantify biofilms will facilitate increased ease of research about biofilms in wounds and how to combat them.

Maximillian A. Weigelt, MD

Scope and Significance

The role of biofilms in the pathophysiology of chronic wounds has become increasingly recognized. Although evidence directly demonstrating the benefits of biofilm eradication on wound healing is scarce, targeting biofilms remains a logical approach to help the clinician heal patients with chronic wounds. There are many available products with purported antibiofilm activity. In this study, we discuss the mode of action and clinical significance of available antibiofilm products, with a focus on levels of evidence for efficacy in destroying biofilms and facilitating positive wound healing outcomes.

Translational Relevance

Current biofilm research is limited by a lack of standardized models capable of accurately recapitulating the complex human wound microenvironment. This restricts the generalizability of results, limiting the translation of antibiofilm treatments into the clinical setting. Highlighting these gaps in research will provide valuable future directions for researchers to improve biofilm models and engage in focused study design to ultimately improve clinical outcomes and quality of life for patients with wounds.

Clinical Relevance

Chronic wounds are a burden on public health and the economy, with estimated annual Medicare costs of over U.S. $30 billion.¹ They are frequently recalcitrant to standard of care (SOC) treatments² and thus present a challenge to clinicians. With the increasing appreciation of the role of biofilms in maintaining a deleterious chronic wound microenvironment, biofilm eradication is a new tool in the clinician's armory in the battle against chronic wounds. Examining levels of evidence for antibiofilm agents and their ability to promote wound healing and eliminate biofilm will help guide treatment protocols to the benefit of patients with chronic wounds.

Background

Introduction to biofilms

With the appreciation that bacteria form biofilms, it has come to light that various diseases, including wounds, exhibit biofilms that may contribute to their pathogenesis. In all environments, microorganisms naturally exist in one of two main states: planktonic state (free living) and an attached or sessile state.³ Biofilms in vivo are composed of many small densely packed aggregations of microbes, segregated by intervening host tissue, that tightly attach to each other and to nearby surfaces.³ Encased in an extracellular polymeric substance (EPS), which may be self-synthesized or adapted from host extracellular matrix materials, biofilms are composed of 75–95% EPS and only 5–25% bacteria.⁴ In contrast to biofilms, planktonic bacteria, the traditional focus of microbiology research, are free-floating microorganisms in an aqueous environment. The transition of planktonic microorganisms to a biofilm lifestyle may be damaging to a host as biofilms notoriously resist elimination by host defenses and antibiotics.⁵

Today, it is known that bacteria exist predominantly in biofilms in both natural and clinical settings.⁶ Biofilms are seen more commonly in chronic (60%) than acute (6%) wounds⁷; the fact that chronic wounds harbor biofilms more commonly has been part of the rationale linking their presence to delayed healing. Biofilms have been shown to prevent wound contracture and epithelialization, disrupt the host immune response, induce chronic inflammation, and prevent normal epidermal differentiation after healing.⁸ Conversely, the chronic wound microenvironment favors biofilm formation.⁹ Whether biofilms cause chronic wounds or vice-versa remains unclear, although both are likely true (Fig. 1). Even in the absence of delayed healing in some acute wound models, biofilms may delay or prevent restoration of skin barrier and might be a risk factor for ulcer recurrence.¹⁰

Figure 1.

The chicken or the egg—do biofilms cause chronic wounds or vice versa? The exact answer remains nebulous, but likely includes a combination of both. There is also evidence to suggest that biofilm formation impairs healing in acute wounds, thereby converting them into a chronic phenotype.

Identification and treatment of biofilms presents a challenging conundrum for clinicians. Biofilm develops quickly, reforms as soon as 10 h after sharp debridement, and displays mature characteristics between 24 and 72 h, making it resilient to current SOC measures.¹¹ Biofilm architecture cannot be assessed with routine culture techniques (swabbing or tissue culture).¹² Chronic wounds often lack overt clinical signs of infection, making low clinical suspicion a confounding problem in wound biofilm identification (Fig. 2).¹² Many studies have shown that clinical signs of infection lack adequate sensitivity and specificity to reliably identify infection in patients with chronic wounds. Experimental techniques such as 16S rRNA and fluorescence in situ hybridization (FISH) with confocal laser scanning microscopy (CLSM) to detect biofilms are not routine.

Figure 2.

Venous leg ulcer in a patient with chronic venous insufficiency. Is there biofilm present in this wound? Clinical inspection alone is not sufficient to detect biofilms, which contribute to impaired healing in up to or more than 60% of chronic wounds. The routine use of antibiofilm agents in wound care protocols has potential to improve wound healing.

Current approaches to biofilm management

Recent consensus guidelines for the treatment of biofilms in chronic wounds are available.¹² Sharp debridement is the most important strategy as it removes both the biofilm, as well as niduses, for attachment and bacterial growth (e.g., dead tissue), but should not be used alone as it does not remove all biofilms.¹² System antibiotics and topical antiseptics are of little utility in combating biofilms due to their intrinsic resistance against such treatments; however, the timely application of topical antiseptics may prevent spread or reforming of a biofilm after debridement, and thus, they remain recommended as a first-line therapy for stalled wounds.¹²

Other conventional wound therapies have purported antibiofilm efficacy, but strong evidence to support them is generally lacking. Silver, for example, has demonstrated significant antimicrobial efficacy in vitro and in vivo; however, evidence for its antibiofilm activity either alone or when incorporated into dressings (e.g., alginates) is poor.¹³ Hyperbaric oxygen therapy may be used as an adjunct therapy for chronic wounds, but evidence for its ability to destroy biofilms is sparse,¹⁴ and in addition, it is known to achieve inconsistent results in improving wound healing despite being Food and Drug Administration approved for the treatment of diabetic foot ulcers (DFUs). Thus, the need for high-quality research demonstrating the efficacy of available agents in combating biofilms and promoting wound healing, as well as the continued development of novel agents for this purpose, cannot be overstated.

Challenges in wound biofilm research

High quality studies regarding the beneficial role of biofilm eradication and the subsequent relationship to wound healing in patients with chronic wounds are lacking and, thus, represent an unfulfilled need. Many experiments studied biofilm using in vitro or animal models, which have inherent limitations in their generalizability and clinical applicability, which should be considered by the reader.

Although in vitro methods have been used extensively to study biofilms, in vitro biofilms are different than their in vivo counterparts.¹⁵ In vitro biofilms form large, single three-dimensional structures and exhibit classic “mushroom-like” multicellular structures.¹⁵ In contrast, in vivo biofilms lack the mushroom-like structures, are significantly smaller in diameter, and form many small aggregates segregated by host tissue rather than single large structures.¹⁵ In addition, it is known that biofilm aggregates can form without attachment to a surface, and conventional in vitro biofilm models miss these populations due to their requisite abiotic surfaces.¹⁵ Most importantly, in vitro models fail to accurately recapitulate the complex microenvironment of the human skin wound, that is, the host immune response and metabolic factors, which shape the phenotypic and genotypic profiles of the invading bacteria.¹⁵

Animal models provide a more accurate medium for the study of biofilms, but are limited by obvious differences between animal and human physiology, for example, differences in murine and human sensitivity to lipopolysaccharide and murine wound healing occurring primarily through contraction.¹⁶ The fact remains that there is no gold standard in vivo model, and each may provide answers to different specific questions.¹⁶ Standardized biofilm models which can more accurately replicate the human wound microenvironment are needed so that researchers and clinicians can more easily compare their results and guide therapeutic strategies for patients with wounds.¹⁶ In addition, the methods used to measure biofilms are not created equal; although there is no gold standard, CLSM combined with FISH may be the most powerful tool available.¹⁷ Scanning electron microscopy (SEM) is also often used, but is limited by superficial depth penetration and other factors.¹⁷ Many studies, including some discussed in this article, do not directly measure biofilm architecture with imaging. Often biofilm formation is not measured pretreatment but is nonetheless assumed, while plating of recoverable bacteria (PRB) from the surface or wound model is used as a surrogate indicator of antibiofilm activity. Ideally, biofilm architecture would be directly visualized by high-powered imaging techniques before and after treatment. The many biofilm models, measurement techniques, and critiques thereof can be found in two excellent recent reviews.^17,18

Despite these significant limitations, reducing, removing, or preventing biofilms remains a logical approach to help clinicians heal chronic wounds (Fig. 3). Recent consensus guidelines for research on biofilms state that evidence from in vitro and animal models is important to consider when choosing treatments and is helpful in screening for their antibiofilm efficacy.¹² Furthermore, in the absence of randomized controlled trial (RCT) level evidence, the choice of such interventions should be supported by their evidence in promoting beneficial wound healing outcomes.¹²

Figure 3.

Role of antibiofilm agents in a treatment paradigm for wounds. Antibiofilm agents may be used to prevent biofilm formation or to prevent reformation after debridement, thus improving wound healing. It is important to note that resolution of biofilm is not necessarily sufficient to resolve poor wound healing as other factors may need to be corrected (e.g., chronic venous insufficiency, malnutrition).

As such, this review aims to discuss the mode of action and clinical significance of currently available antibiofilm products, with a focus on levels of evidence for efficacy in disrupting biofilms and ability to improve wound healing outcomes (Table 1).

Table 1.

Summary of antibiofilm agents—mechanisms and levels of evidence

Product	Mechanism of Antibiofilm Action	Antibiofilm Evidence (Method, Model)	Healing Outcomes Evidence (Outcome Measure, Wound Type)
Polyhexanide (polyhexamethylene biguanide or PHMB)	Disruption and increased permeability of bacterial cell membranes	Level VI21 (PRB, porcine in vivo)	Level IV22 (% size reduction, varied)
Poloxamer-based surfactants (PluroGel®, Medline Industries, Inc., Northfield, IL)	Inhibition of bacterial surface adherence Reduces cohesion of constituent biofilm molecules	Level VI25 (PRB, Porcine ex vivo)	Level IV26 (complete healing, varied) Level IV24(complete healing, varied)
AWG (BlastXTM, Next Science, St. Paul, MN)	Dissolves the extracellular polysaccharide matrix, exposing encapsulated bacteria for removal Osmotic lysis of cell wall	Level VI8 (MTP and murine in vivo, CLSM)	Level I28 (50% size reduction at 4 weeks, varied) Level I27 (% size reduction or complete closure, varied)
Cadexomer Iodine (IODOSORB™; Smith and Nephew, London, United Kingdom)	Directly destroys biofilms Collapses bacterial glycocalyx Traps bacteria within beads.	Level VI29 (MTP, CLSM) Level VI30 (porcine in vivo and ex vivo, HPT) Level IV31 (clinical DFUs, SEM/FISH)	Level I32 (Complete healing at 4–12 weeks, VLUs)
Honey	High osmolarity Low pH Peroxide produced by breakdown of glucose	Level VI35 (MTP, CVS, and CLSM)	Level I34 (Various, partial thickness burns)
Hypochlorous acid	Chemical inactivation of various cellular processes, including amino acid modification and protein synthesis	Level VI36 (MTP, PRB, and CVS)	Low-to-strong based on wound type37 (Expert consensus panel)
Lasers and phototherapy	Induction of oxidative stress Impaired polysaccharide production	Level VI38 (CLSM, flow chamber) (HPT, murine)	None
Low frequency ultrasound	Microstreaming and cavitational effects	Level VI41 (CLSM, plated biofilms)	Varies40
Electroceuticals	Disruption of electrostatic adhesion forces Superoxide production Bacterial membrane enzyme disruption	Level VI43 (CVS + HPT, flow chamber) Level VI42 (SEM, polycarbonate filter) Level VI46 (SEM, porcine in vivo)	Level VI46 (acute wound closure time, porcine in vivo) Level III45 (time to complete closure and rate of size reduction, varied)

Mechanisms of action and levels of evidence for available antibiofilm agents for wound care.

AWG, antimicrobial wound gel; CLSM, confocal laser scanning microscopy; CVS, crystal violet staining; DFUs, diabetic foot ulcers; FISH, fluorescence in situ hybridization; HPT, histopathology; MTP, microtiter plate; PRB, plating of recoverable bacteria; SEM, scanning electron microscopy; VLUs, venous leg ulcers.

Methodology

PubMed and Embase searches were performed. Search terms used included biofilm, antimicrobial, surfactant, and the specific names of products described in this review. A level of evidence was assigned to each intervention according to the Oxford Center for Evidence-based Medicine Levels of Evidence table, with one level of evidence (level VI) added for in vitro and animal studies¹⁹ (Table 2).

Table 2.

Modified Oxford Levels of Evidence¹⁷

Level of Evidence	Study Types
I	Randomized controlled trials
II	Nonrandomized prospective
III	Retrospective cohort; case–control
IV	Case series, uncontrolled cohort
V	Case reports
VI	In vitro, animal models

Levels of evidence used in this review were adapted from the Oxford Levels of Evidence, with an extra level (VI) added to account for in vitro and animal studies.

Results and Discussion

Polyhexamethylene biguanide

Polyhexamethylene biguanide (PHMB) is a broad-spectrum biocide with potent bactericidal activity.²⁰ It is a cationic polymer, which disrupts bacterial cell membranes, increasing their permeability.²⁰ PHMB has been studied in vivo; 39 partial thickness wounds in a porcine wound model were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and covered with a polyurethane dressing for 24 h to allow for presumed biofilm formation (Level VI evidence).²¹ The six wounds treated with PHMB demonstrated the smallest amount of recoverable bacteria, with 99.64% reduction of MRSA at 6 days.²¹

An uncontrolled cohort study evaluated the efficacy of a polyhexanide-containing biocellulose dressing (Suprasorb X + PHMB, Lohmann & Rauscher, Regensdorf, Germany) in 28 subjects with nonhealing wounds of varied etiologies, who were followed prospectively for 24 weeks or until healing (Level IV evidence). They observed a mean reduction in wound area of 61% and a significant increase in mean granulation tissue in the wound beds.²² Limitations included small sample size and heterogenous wound etiologies. Biofilm was not measured appropriately in this study.

A randomized controlled double-blinded clinical trial evaluated the antibiofilm activity of PHMB in 44 patients with venous leg ulcers (VLUs).²³ Punch biopsies were collected pre- and postcleansing to evaluate microbial load and biofilm burden using SEM. PHMB was equally as effective as saline in reducing microbial load, and neither treatment demonstrated the ability to reduce biofilms in wounds.²³ Limitations of this study include small sample size and significant dropout of patients in the PHMB group (14 of 22 enrolled).

PMHB's potential as an antibiofilm agent remains unclear given the conflicting evidence and thus may benefit from further exploration in higher powered clinical studies with a focus on direct measurement of biofilm burden using FISH/CLSM.

Poloxamer-based surfactants

Surfactants are commonly used cleaning agents often used to remove dirt from skin, clothes, and other materials. They are amphiphilic compounds which reduce the surface tension between two substances, thus increasing the solubility of materials that would otherwise be nonmiscible. They interfere with the ability of microorganisms to adhere to surfaces and to one another, and their resulting ability to disrupt biofilms and improve wound healing outcomes has thus far been observed in several studies.

Poloxamers are nonionic surfactants composed of amphiphilic polymer chains, with a central hydrophobic core and two hydrophilic ends. They tend to form sphere-like micelles with surfactant/detergent properties, creating a moist healing environment that promotes autolytic debridement. The hydrophilic surface of the micelles softens and loosens wound debris, which is then trapped by the hydrophobic inner core.²⁴

The efficacy of one such concentrated surfactant gel (CSG, PluroGel^®, Medline Industries, Inc., Northfield, IL) in killing sessile bacteria within biofilms has been demonstrated in porcine skin explants using PRB (level VI evidence).²⁵

There is some evidence to support positive effects of poloxamer-based surfactants on wound healing outcomes. Zölß and Cech used CSG with 1% silver sulfadiazine (SSD) on 226 patients with chronic wounds of various etiologies at an outpatient tertiary care wound center. Eighty-eight patients had previously failed SOC treatments, and 138 patients began receiving CSG at the onset of wound treatment. The rates of complete healing or wound improvement were 74% and 85% for each group, respectively, with median times to heal of 17 and 12 weeks for each group. Improved compliance, reduced pain, and reduced costs were noted across the cohort (Level IV evidence).²⁶ Biofilms were not measured in these patients.

A European multicenter cohort study used CSG with 1% SSD on 1,036 patients with chronic wounds of varied etiologies, greater than 3 months in duration and not responding to SOC. Seventy percent of patients achieved wound closure. Since comparison to SOC was not directly possible given the nature of the study design, the authors established a baseline of expected wound outcomes for comparison by analysis of five other RCTs: they found mean cross-trial closure rate of 43.8% (n = 645) for SOC and 58.9% (n = 727) for various other experimental therapies (Level IV evidence). Biofilm was not measured.²⁴ Limitations of this study include the lack of direct comparison to standard of care, as well as the fact that each center used their own individual SOC protocols, which may vary significantly between institutions. The results of this and the previously mentioned study should be interpreted with caution as CSG was not evaluated on its own.

Antimicrobial wound gel

Antimicrobial Wound Gel (BlastX™, AWG, Next Science, St. Paul, MN) is a new intervention composed of benzalkonium chloride 0.13%, polyethylene glycol, sodium citrate, citric acid, and water. Also referred to as a “high osmolarity surfactant,” AWG has been shown to significantly reduce bacterial loads through in vitro microtiter plate assay (MTP), particularly of common wound colonizers Pseudomonas aeruginosa and MRSA.⁸ AWG has been approved by the U.S. Food and Drug Administration as a medical device.²⁷

The benzalkonium chloride 0.13% within AWG serves as a cationic surfactant, which is responsible for directly lysing the bacterial cell wall by osmotically drawing proteins into the solution. The polyethylene glycol-based hydrogel base serves to maintain a moist wound environment optimal for granulation, epithelialization, and prevention of tissue necrosis.²⁸ AWG should not be used in conjunction with calcium alginate dressings as AWG destabilizes the biofilm matrix, in part, from chelation of calcium, and consequently, both may neutralize each other's effects.^27,28

AWG was first shown by CLSM to destroy mature biofilms of S. aureus and P. aeruginosa in vitro using MTP. It was also observed to prevent novel biofilm formation by these same organisms in a murine model of wound biofilm infection, also by CLSM (Level VI evidence).⁸

Wolcott conducted a randomized controlled clinical trial of 45 patients in which AWG was found to act synergistically with SOC.²⁸ For this trial, a 50% reduction in wound volume over 4 weeks was considered successful treatment, and wounds of >30-day duration were included. SOC consisted of weekly debridement and application of a topical antibiotic thrice weekly. When AWG was used as an adjunct to SOC, 93% of wounds were successfully treated, while those with SOC alone showed 53% of wounds successfully treated and AWG alone showed 80% of wounds successfully treated (Level I evidence).²⁸ This study was limited by relatively small sample size and lack of direct biofilm measurement as the authors relied on clinical signs of biofilm which are now known not to be valid.

Finally, Kim et al. conducted a randomized open-label clinical trial, in which they compared AWG (experimental group) to a triple antibiotic ointment (control group) as adjuncts to sharp debridement for chronic refractory wounds.²⁷ Over a 12-week period, the investigators observed a median 72% wound area reduction in the experimental group, which was a statistically significant difference compared to the 24% wound area reduction in the control group. Similarly, 52% of AWG-treated patients showed complete wound closure compared to 17% in the control group. Although the authors refer to biofilm analysis, biofilm was not measured using high-powered techniques (FISH/CLSM).

None of the studies has reported adverse effects associated with AWG use other than occasional mild stinging. Although AWG certainly has evidence to support its use as a powerful antimicrobial surfactant gel that can destroy both biofilm and planktonic bacteria, further studies should be performed that specifically assess its antibiofilm efficacy (using FISH + CLSM) and the subsequent relationship to wound healing in a clinical setting. Level I evidence has shown that AWG works to reduce wound size most effectively in conjunction with sharp debridement. Therefore, AWG appears to work as a safe and effective adjunct to SOC.

AWG is currently being evaluated in two randomized clinical trials involving below-knee surgical amputations (NCT04053946) and chronic wounds of various etiologies (NCT03686904).

Cadexomer iodine

Cadexomer iodine (CDI, IODOSORB™, Smith And Nephew, London, United Kingdom) is a spherical starch bead lattice containing 0.9% iodine by weight and boasting a high absorptive capacity.²⁹ Iodine is slowly released over time as the beads come into contact with exudate.²⁹ The efficacy of CDI in disrupting mature biofilms of S. aureus and P. aeruginosa has been demonstrated in vitro²⁹ (CLSM of MTP), as well as in ex vivo porcine skin explants³⁰ through histopathology (HPT) and in vivo murine (using CLSM) and porcine (HPT) wound models (Level VI evidence).^29,30 Malone et al. used CDI on a cohort of 17 patients with chronic diabetic foot ulcers complicated by biofilms (confirmed through SEM or FISH). Eleven patients exhibited 1–2 log reductions in bacterial load after treatment with CDI, and a significant decrease in biofilm size and complexity was noted between pre- and post-treatment wound samples (Level IV evidence).³¹ A Cochrane review evaluated four RCTs involving CDI for VLUs: pooled estimate for complete healing at 4–12 weeks was relative risk 2.17 (CI 1.30–3.60) compared with SOC (Level I Evidence).³² No differences in complete healing were observed when comparing CDI with hydrocolloid dressing, paraffin gauze dressing, dextranomer, or silver dressings.

Honey

The antibacterial properties of honey have been known for centuries.³³ Comprised mainly of sugar and water, honey also contains various amino acids, minerals, vitamins, and enzymes.³³ Honey stimulates cellular processes important for physiologic wound healing, including macrophage activity and fibroblast proliferation.³³ The antimicrobial properties of honey are chiefly due to its high osmolarity and low pH (3.2–4.5).³³ Peroxide produced during the breakdown of glucose further exerts antimicrobial effects and promotes angiogenesis.³³ Although many honey-based wound care products are commercially available, there is a lack of definitive evidence to support honey's efficacy in improving wound healing.³³ A recent review found RCT-based evidence of limited quality to suggest honey's benefit for the healing of partial thickness burns (level I evidence), but inconclusive evidence regarding the use of honey for other wound-related indications.³⁴ Nonetheless, honey has been observed to inhibit biofilm formation and eradicate mature biofilms of P. aeruginosa in vitro through MTP as assessed by crystal violet staining (CVS) and CLSM (level VI evidence).³⁵ As such, honey would benefit from prospective clinical studies assessing antibiofilm activity and the subsequent relationship to wound healing outcomes.

Hypochlorous acid

Hypochlorous acid (HOCL) is a commonly used antiseptic agent with significant killing activity against a wide variety of pathogens, including bacteria, fungi, and viruses.³⁶ It exerts these effects through the disruption of various chemical linkages, such as by the oxidation of sulfhydryl enzymes or ring chlorination of amino acids.³⁶ In doing so it inhibits cellular processes, including protein synthesis, oxygen metabolism, and DNA synthesis.³⁶ HOCL has been found to disrupt biofilm structures from in vitro MTP assays at high concentrations (3–5%), using CVS and PRB (Level VI evidence).³⁶ No studies have yet looked at the biofilm-dispersing ability of HOCL in vivo or in a clinical setting. An expert consensus panel found the following evidence for improvement in wound healing by adjunct HOCL use: strong evidence for DFUs, moderate evidence for septic surgical wounds, and low evidence for VLUs, wounds of mixed etiologies, and chronic wounds.³⁷

Lasers and phototherapy

Recent data suggest that laser and phototherapy may serve as an innovative approach to combat bacterial overgrowth and biofilm formation. Blue laser light has proven efficacious in destroying P. aeruginosa biofilms in vitro (CLSM of a drip flow chamber model) and in vivo (HPT of a murine wound model), with minimal cytotoxicity to surrounding tissues and cells (Level VI evidence); the death of HaCaT cells (an immortalized keratinocyte cell line) in vitro was induced only by exposure to light fluences in significant excess of those necessary to attain optimal antibiofilm activity.³⁸ Blue light antimicrobial and antibiofilm effects are due to induction of oxidative stress.³⁸ The use of phototherapy as an antibiofilm therapeutic agent is not limited to bacterial biofilms. Exposure to red and blue light twice daily has been found to be efficacious in preventing in vitro Candida albicans biofilm formation through PRB from MTP assay (Level VI evidence).³⁹ Impairment of polysaccharide production is thought to be the mechanism of action behind fungal biofilm impairment.³⁹ These results merit further exploration in prospective clinical studies.

Low-frequency ultrasound

Low-frequency ultrasound (LFUS) (20–50 kHz) has been used as a debridement modality for chronic wounds. Through microstreaming and cavitational effects, it emulsifies dead and dying tissues with gas microbubbles, stimulates the cell membranes of nearby cells to promote healing, and increases bacterial antibiotic susceptibility.⁴⁰ LFUS was found to disrupt biofilm architecture of in vitro plated biofilm models through CLSM without significant bacterial killing and significantly enhanced the bactericidal activity of PHMB in this model (level VI evidence).⁴¹ Evidence to support the use of LFUS as an adjunct treatment or alternative to sharp debridement for chronic wounds exists and is summarized in an recent review.⁴⁰ It remains difficult to draw conclusions about the clinical utility of LFUS due to the high number of confounding variables between studies, small sample sizes, and inconsistent ultrasound strengths and treatment protocols used.⁴⁰ Nonetheless, LFUS is a promising adjunct treatment for chronic wounds whose antibiofilm activity and potential to improve wound healing outcomes deserve further exploration in well-designed RCTs with large sample sizes.

Electroceuticals

Energy-based technologies (“electroceuticals”) use electrical currents or fields to treat medical problems.⁴² They represent a novel domain of therapeutic options, which have been explored for the treatment of wounds and eradication of bacteria within them.⁴³ A physiological electric current is necessary for normal wound healing and migration of cells within the wound site—augmentation of this current may thus be beneficial for wound healing.⁴³ Indeed, a recent review found 14 RCTs demonstrating that the use of electrical stimulation was associated with greater rates of complete wound closure and greater wound area reduction in DFUs, VLUs, pressure ulcers, and mixed ulcers.⁴⁴

A wireless bioelectric dressing (WED) capable of generating electric potential without an external power source is available (Procellera, Vomaris Wound Care, Inc., Tempe, AZ) and has demonstrated potential to eliminate biofilms and improve wound healing.⁴³ WED is a matrix of alternating silver and zinc dots on a polyester substrate, capable of generating a direct current of 0.5–0.9 volts in the presence of an electrolyte such as wound exudate.^43,45 Its antibiofilm effects may be due to disruption of the weak electrostatic forces necessary for bacterial adhesion, as well as production of superoxide and disruption of bacterial membrane enzymes.^42,46

The wireless electroceutical dressing has been observed to destroy mature biofilms in vitro, assessed through CVS, and microscopy of a drip flow reactor model⁴³ and SEM of a polycarbonate filter model (level VI evidence).⁴² Barki et al. found that WED prevented and disrupted biofilms as measured with SEM in an in vivo porcine model of chronic biofilm infection (level VI evidence).⁴⁶ They also observed that WED accelerated functional wound closure in the same model (level VI evidence).⁴⁶ A two-center retrospective chart review of 38 patients with chronic wounds of various etiologies found significantly faster time to closure and higher wound volume reduction per day in the group treated with WED compared to SOC (level III evidence).⁴⁵ Thus WED demonstrates potential to destroy biofilms and improve wound healing outcomes which merits further exploration in prospective randomized trials.

Conclusion

It has become increasingly appreciated that bacteria in biofilms play a significant role in the pathophysiology of chronic skin wounds. Very few available products have stronger evidence than in vitro and animal studies for their ability to destroy biofilms; notably, AWG™ (level I evidence) has the strongest evidence in this domain; however, limitations in the study methods confound results. In general, evidence for promoting good wound healing outcomes is more robust but these studies do not demonstrate healing linked to biofilm reduction. Many of the newer products understandably do not yet have evidence to support their ability to ameliorate wound healing. Overall, there is a need for more high-quality clinical studies demonstrating the antibiofilm activity and wound healing benefits of existing antibiofilm agents.

Increasing our understanding of the complex interactions between multispecies biofilms and the microcellular environment of chronic wounds will allow for more effective development of targeted therapies. Indeed, the development of various promising novel techniques to destroy biofilm is ongoing, including: nanoparticle delivery systems,⁴⁷ enzymatic wound spray,⁴⁸ gallium dressings (SBIR Grant #2R44AR073710-02), photosensitizers,⁴⁹ bacteriophages,¹⁴ and quorum sensing inhibitors.¹⁴ These technologies are not yet available to clinicians but have demonstrated varying degrees of ability to eradicate biofilm in preclinical studies. A chief limitation of current research is the lack of accurate wound biofilm models that accurately recapitulate the complex wound microenvironment. Furthermore, the most powerful test for biofilm detection (FISH + CLSM of deep tissue biopsy) is invasive and time consuming. There are not currently any biofilm-specific biomarkers or noninvasive detection techniques. This may change in the future as recent research has focused on developing noninvasive techniques for biofilm appraisal, for example, biosensors and others; these are summarized in another recent and excellent review.⁵⁰ These tools are still in the early stages of development and, after clinical validation with comparison to FISH + CLSM, would facilitate increased ease of research about biofilms in wounds and how to combat them.

Summary

Biofilms in vivo are small densely packed aggregations of microbes, encased in an EPS, which tightly adhere to surfaces and each other. Biofilms confer resistance to host defenses and conventional antimicrobial therapy. Chronic wounds are a major burden to public health and the economy with estimated annual costs to Medicare exceeding U.S. $30 billion. The role of biofilms in the pathogenesis of chronic wounds is becoming increasingly apparent. Although evidence demonstrating the benefits of biofilm removal as it pertains to positive wound healing outcomes is limited, destruction of biofilms nonetheless remains a logical approach to help clinicians heal chronic wounds.

In this article we have reviewed the antibiofilm wound care products currently available on the market, with a focus on levels of evidence for their efficacy in destroying biofilms and improving the healing of chronic wounds. The products reviewed herein include surfactants, dressings, lasers, and more. Overall, few products have strong evidence to support their antibiofilm activity in human patients. The body of evidence to support positive wound healing outcomes is more robust, but the need to demonstrate a definitive link between healing and biofilm reduction remains. There is a need for more high-quality clinical studies to characterize the antibiofilm activity of these agents and link the destruction of biofilm to improved healing. The development of simpler, faster, and less-invasive techniques for measuring biofilm, as well as more accurate and standardized biofilm research models, is ongoing and will both simplify and accelerate research about biofilms in wounds and how to combat them.

Take-Home Messages

• Biofilms are three-dimensional structures consisting of tightly packed aggregations of microbes encased in an EPS.

• The role of biofilms in driving the pathogenesis of chronic skin wounds has become increasingly apparent.

• Whether biofilms cause chronic wounds, are sequelae of the same, or both, is yet to be fully understood.

• Although evidence linking biofilm removal to improved wound healing is scant, destroying biofilms remains a logical approach to help clinicians heal chronic wounds.

• Currently available wound care products are reviewed, with a focus on levels of evidence for ability to destroy biofilm and capacity to improve wound healing outcomes.

• Products discussed include surfactants, dressings, and more.

• Overall, evidence to support antibiofilm activity and improved wound healing outcomes for most agents is limited.

• More high-quality clinical studies are needed to demonstrate these qualities and to show a definitive link between biofilm removal and improved wound healing.

Abbreviations and Acronyms

AWG

antimicrobial wound gel

CDI

cadexomer iodine

CLSM

confocal laser scanning microscopy

CSG

concentrated surfactant gel

CVS

crystal violet staining

DFUs

diabetic foot ulcers

ECM

extracellular matrix

EPS

extracellular polymeric substance

FISH

fluorescence in situ hybridization

HOCL

hypochlorous acid

HPT

Histopathology

LFUS

low-frequency ultrasound

MRSA

methicillin-resistant Staphylococcus aureus

MTP

microtiter plate

PHMB

polyhexamethylene biguanide

PRB

plating of recoverable bacteria

PUs

pressure ulcers

RCT

randomized controlled trial

SEM

scanning electron microscopy

SOC

standard of care

SSD

silver sulfadiazine

VLUs

venous leg ulcers

WED

wireless bioelectric dressing

Acknowledgments and Funding Sources

No acknowledgments. This work was not supported by any funding.

Author Disclosure and Ghostwriting

No competing financial interests exist. The content of this article was expressly written by the authors listed. No ghostwriters were used to write this article.

About the Authors

Maximillian A. Weigelt, MD, is a clinical research fellow in Wound Healing in the Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery at the University of Miami.

Stephanie A. McNamara, MD, is a clinical research fellow in Wound Healing in the Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery at the University of Miami.

Daniela P. Sanchez is a medical student at Boston University School of Medicine.

Penelope A. Hirt, MD, is a first-year medical resident at Aventura Hospital in Hollywood, FL.

Robert S. Kirsner, MD, PhD, is the Chairman and Harvey Blank Professor of the Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Professor of Public Health Sciences at the University of Miami Miller School of Medicine and Director of the University of Miami Hospital and Clinics Wound Center.