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Efficacy of Wound Cleansers on Wound-Specific Organisms Using In Vitro and Ex Vivo Biofilm Models

Empirical Studies

Efficacy of Wound Cleansers on Wound-Specific Organisms Using In Vitro and Ex Vivo Biofilm Models

Index: Wound Management & Prevention 2020;66(11):31–42 doi: 10.25270/wmp.2020.11.3142

Abstract

Biofilms are believed to be a source of chronic inflammation in non-healing wounds. PURPOSE: In this study, the pre-clinical anti-biofilm efficacy of several wound cleansers was examined using the Calgary minimum biofilm eradication concentration (MBEC) and ex vivo porcine dermal explant (PDE) models on Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans biofilms. METHODS: A surfactant-based cleanser and antimicrobial-based cleansers containing ionic silver, hypochlorous acid (HOCl), sodium hypochlorite (NaOCl), and polyhexamethylene biguanide (PHMB) were tested on the MBEC model biofilms with a 10-minute application time. Select cleansers were then tested on the mature PDE biofilms with 10-minute applications followed by the application of cleanser-soaked gauze. The PDE model was further expanded to include single and daily applications of the cleansers to mimic daily and 72-hour dressing changes. RESULTS: In the MBEC model, PHMB- and HOCl-based cleansers reduced immature MRSA, C albicans, and P aeruginosa  biofilm regrowth by > 3× when compared with silver, surfactant, and saline cleansers. The major differences could be elucidated in the PDE model in which, after daily application, 1 PHMB-based cleanser showed a statistically significant reduction (3–8 CFU/mL log reduction) in all mature biofilms tested, while a NaOCl-based cleanser showed significant reduction in 2 microorganisms (3–5 CFU/mL log reduction, P aeruginosa and MRSA).The other PHMB-based cleanser showed a statistically significant 3 log CFU/mL reduction in P aeruginosa. The remaining cleansers showed no statistically significant difference from the saline control. CONCLUSION: Results confirm that there are model-dependent differences in the outcomes of these studies, suggesting the importance of model selection for product screening. The results indicate that 1 PHMB-based cleanser was effective in reducing mature P aeruginosa, MRSA, and C albicans biofilms and that sustained antimicrobial presence was necessary to reduce or eliminate these mature biofilms. 

Introduction

A meta-analysis of clinical studies on chronic wounds from 2008 through 2015 showed that biofilms are present in 78.2% of chronic wounds,1 yet many clinicians believe that biofilms are ubiquitous in chronic wounds.2,3 These biofilms may be implicated in the protracted inflammatory response that contributes to delayed wound healing based on clinical observations of chronic wounds.4,5 They are polymicrobial, and the species interact to affect distribution, biomass, functionality, and, ultimately, survivability.6–9 The chemical composition of the extracellular polymeric substances (EPS) is dependent on the species present, environment, and stage of the biofilm,8 whereas the spatial organization of organisms within a biofilm is influenced by numerous parameters including wound stage and type. For example, facultative anaerobes and aerobes are localized based on the availability of oxygen; the aerobes are closer to the wound surface, while the anaerobes are deeper within the wound bed.10–12

The first step in wound biofilm formation is the attachment of planktonic organisms to the wound surface. A review of microbiological analysis conducted by Bowler, Duerden, and Armstrong13 discusses how microbes migrate to attach to the wound surface from the periwound area, environment (exogenous microorganisms), and endogenous sources (gastrointestinal, oropharyngeal, and genitourinary mucosa). The microbes then start forming microcolonies, signaling for additional microorganisms to join the colony, and synthesizing EPS, which provides protection and secure attachment to the wound surface and to other microbes.14,15 In this phase, the biofilms are still immature, less organized, more metabolically active, and susceptible to antibiotics and antimicrobials. Mature biofilms, which form in 24 to 72 hours, are resistant to eradication by the host immune system and antibiotics due to reduced metabolic rates and other protective mechanisms, such as the increased EPS accumulation and formation of water channels.10,11,16

Although there is increasing consensus recognizing wound biofilms and the associated delays in wound healing,2,3 an accepted protocol for their treatment is lacking. Wound cleansing, defined as the removal of contaminants, microbes, loose debris, slough, and remnants of previous dressing from the wound surface and periwound area,17–19 is a critical component of wound bed preparation.5 The ideal wound cleanser would inflict minimal damage to healthy tissues, cause no sensitivity reactions, remain effective in the presence of organic material, reduce or eliminate the biofilm and number of microorganisms, and remain shelf-stable and cost-effective.19–22 When addressing hard-to-heal wounds, it is crucial that the wound cleansers can penetrate and remove the biofilm even if the microorganisms within the biofilms are dead. If the EPS, which comprises more than 90% of the biofilm volume as postulated by Flemming and Windgender in their biofilm matrix review,23,24 is still present, increased inflammation, heightened risk of infection, and delayed wound healing can still occur.25 Historically, water, saline, or, more recently, surfactant solutions26 were selected to address the concern regarding the potential cytotoxic effect of the routine use of antimicrobial solutions to cleanse wounds.27,28 However, the recommendation for use of topical antimicrobial cleansers is growing.2,29,30 Specifically, these recommendations are for wounds that have an increased microbial burden, obvious infection, biofilm, excess exudate, necrotic tissue, or debris in the wound bed based on the published principles of best practice by the International Wound Infection Institute as well as cited reviews within.2,29,30 Most decisions regarding the use of wound cleansers are based on experience, personal preference, or institutional policy.31 This can be attributed to the shortage of, or conflicting evidence linking, specific types of cleansers to a reduction in bioburden or increased rates of healing.31 

The purpose of this in vitro study was to examine pre-clinical antibiofilm efficacy of several commercial wound cleansers against Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Candida albicans biofilms using the in vitro Calgary minimum biofilm eradication concentration (MBEC) and ex vivo porcine dermal explant (PDE) models. The microorganisms selected represent important gram-negative and gram-positive bacteria as well as fungi (P aeruginosa, MRSA, and C albicans, respectively). These microbes are commonly found in chronic non-healing wounds.10,32,33 A 2019 meta-analysis on antimicrobial effectiveness of several antibiotics and antimicrobials against in vitro biofilms showed that the experimental method used was the most important factor determining the outcome of these tests, as different test methods can produce varied results for the same antimicrobial agent.34 With this knowledge, 2 biofilm models were used to evaluate several commercially available wound cleansers for their effectiveness in the reduction of bacterial and fungal biofilms, namely the MBEC and ex vivo PDE. The MBEC model was selected as a high-throughput biofilm method to screen many cleansers for effectiveness.34 The cleanser classes that showed efficacy in the MBEC model were then tested in the ex vivo PDE model, which provides a more clinically relevant wound-like substrate for biofilm attachment and nutrition31 and produces mature biofilms that are more challenging to eliminate. 

Materials and Methods

Microorganisms. The microorganisms and biofilm culture conditions used in the MBEC study are listed in Table 1. The microorganisms and biofilm culture environments used in the ex vivo porcine skin explant study are listed in Table 2

Modified MBEC model. The Calgary biofilm MBEC model (MBEC Assay; Innovotech, Edmonton, Alberta, Canada), was used as described previously,35 with modifications to include biofilm regrowth. This method allows for biofilm formation on the surface of the pegs molded to the lids of microtiter plates. The 96 individual biofilms are then simultaneously exposed to rinse solutions, cleansers, neutralizers, or growth media by placing the lid on new microtiter plates (Figure 1). Commercial wound cleansers, designated PHMB-1, PHMB-2, HOCl-1, surfactant, silver, and saline (negative control) (Table 3), were tested to determine their antibiofilm efficacy against P aeruginosa, MRSA, and C albicans biofilms. For screening purposes, cleansers were selected and classified based on each product’s published mode of action (surfactant or antimicrobial). The investigator was blinded to the treatment groups.

Establishment of biofilm. In brief, MRSA, P aeruginosa, and C albicans were grown in tryptic soy broth media with or without glucose supplement (growth media are summarized in Table 1). The growing organisms were mixed with an equal volume of their appropriate growth medium in tubes and then used for MBEC-High Throughput (Innovotech) peg biofilm seeding (100 µL/well) in a 96-well format (biofilm growth conditions are summarized in Table 1). 

Wound cleanser treatments and regrowth measurements. After 24- to 72-hour incubations at the appropriate temperature for biofilm growth on the pegs (Table 1), the pegs were transferred into a 96-well saline rinse plate for 2 minutes (200 µL/well), followed by a 10-minute exposure to each wound cleanser (200 µL/well). The plates were rinsed again with saline (200 µL/well) for 2 minutes with rocking, neutralized with Dey/Engley (DE) Broth (Hardy Diagnostics; Santa Maria, CA; 200 µL/well), and sonicated for 30 seconds to remove planktonic microorganisms. The pegs were then placed back into the appropriate growth media (200 µL/well) at 37°C to allow for regrowth of living microorganisms for 72 hours before quantification with optical density at a wavelength of 600 nm (OD 600) readings. Each wound cleanser and control were evaluated using 12 replicates.

Ex vivo porcine dermal explant model. A schematic diagram of this method is shown in https://www.o-wm.com/sites/default/files/2020-10/McMahon%20Figure%202.jpeg. This method is generally considered to produce mature biofilms that more closely resemble the characteristics of biofilms found in wounds, compared with biofilms grown on abiotic surfaces such as in the MBEC model.36 Commercial wound cleansers, PHMB-1, PHMB-2, HOCl-2, NaOCl-1, NaOCl-2, and saline (Table 3), were blind tested using the ex vivo PDE model to determine their efficacy against preestablished biofilms of P aeruginosa, MRSA, and C albicans. Based on their success in the MBEC model, the chlorine-containing cleanser class was expanded to examine multiple types (HOCl and NaOCl) and concentrations of active ingredient for testing in the mature biofilm study. Given that HOCl-1 is a combination of both HOCl and NaOCl components, it was not selected for inclusion in the PDE model to isolate which chlorine-based component was more effective.

Preparation of the explants and establishment of biofilm. Porcine dermal tissue was obtained from Midwest Research Swine (Gibbon, MN), a facility licensed by the United States Department of Agriculture that uses precision leveling technology to prepare porcine dermis with a thickness of approximately 2.5 mm. Using a punch biopsy, the tissue was cut into circular explants approximately 12 mm in diameter and artificially wounded using a Dremel tool (Robert Bosch Tool Corp., Mount Prospect, IL) to create a wound roughly 2 mm in diameter with a 1.5-mm deep cavity. The explants were then extensively washed with phosphate-buffered saline and sterilized using chlorine gas. Before inoculation, explants were placed on 0.5% trypticase soy agar in an incubator at 37°C for approximately 2 hours to equilibrate. The explants were then inoculated with 15 to 20 µL of log phase cultures, at approximately 105 CFU per explant, and allowed to incubate for 3 days on the explants, allowing mature biofilm to form. The trypticase soy agar was supplemented with the appropriate antibiotic or antifungal agent (Table 2). The explants were transferred daily to fresh agar plates to prevent overgrowth of microorganisms on agar and washed twice in 2 mL of phosphate-buffered saline for 2 minutes before treatment with wound cleansers. There were 4 replicates for each cleanser and control for each experiment to allow for statistical analysis.

Wound cleanser treatments. The 3-day-old explants (prepared as described above) were divided into 2 groups: 1 that received a single application and 1 that received daily applications (Figure 2). The explants were subjected to the following series of treatments:

1. A 10-minute soak in wound cleanser solution: 2 mL in a 24-well plate.

2. Transferring explants into clean wells of a 24-well plate and wiping the “wound” using a sterile cotton swab.

3. Application of gauze presoaked with a wound cleanser to the wound. The gauze was 4-ply, approximately 1 cm2, and presaturated with the appropriate cleanser (approximately 0.5 mL). 

Before applying the gauze, the explants were placed in individual wells of 24-well plates containing soft agar supplemented with an antimicrobial agent (Table 2). The explants that were treated only once (at t = 0; ie, single treatment) were transferred every 24 hours to new 24-well plates containing fresh agar with the appropriate antibiotic or antifungal agent without reapplying the cleansers. The explants that were designated for daily treatment received 3 applications of wound cleanser in total (at t = 0, 24 hours, and 48 hours) using the same procedure as outlined above and transferred to fresh agar daily. At 72 hours, surviving bioburden was recovered from all explants, from both groups, and enumerated via serial dilution and plating.

Recovery and enumeration of surviving bacteria. After the 72-hour incubation, the surviving bacteria and yeasts were recovered from the tissue and enumerated. Each washed explant was placed in a 15-mL centrifuge tube containing 2 mL of DE broth, a broad-spectrum neutralizer that neutralizes PHMB, iodine, and chlorine-based compounds. The explants were then vortexed for 10 seconds and subjected to a series of 5 sonication debridement steps: 90-second sonication/60-second rest intervals. The samples were vortexed again to ensure homogeneity, serially diluted, and spot plated (10 µL sample/spot, triplicate) up to the 10-6 dilution and spread plated where necessary (200 µL of undiluted recovery solution). 

Wound cleansers. The wound cleansers tested are listed in Table 3

Statistics. For the MBEC studies, the reported data indicate multiplicative new growth. This transformation was performed before evaluation to remove the baseline (time zero [0]) growth values; ODs were normalized by dividing 72-hour data by the time zero data. For ex vivo studies, all bacterial counts (CFU/mL) were log-transformed prior to analysis. Data are presented as mean ± standard error of the mean (SEM). For both studies, a one-way analysis of variance was conducted on the transformed groups to identify the existence of statistically significant differences between test groups (P < .05). Post hoc comparisons were performed using Tukey’s honestly significant difference test to identify differences between groups. Statistical analysis was performed using the SPSS software package.

Results

Modified MBEC model. After a 10-minute treatment with PHMB-1, PHMB-2, HOCl-1, or surfactant cleansers followed by 72 hours to allow for regrowth of the surviving P aeruginosa microorganisms, the normalized growth assessment showed approximately 2.56 ± 0.85, 3.40 ± 0.77, 2.67 ± 0.10, and 3.83 ± 1.10 times the time zero growth readings,  (Figure 3). Regrowth of silver and saline cleanser-treated groups were 10.52 ± 0.19 and 10.25 ± 0.13 times the time zero readings, respectively. The differences in biofilm regrowth between the saline control and PHMB-1 (mean difference = 8.03; 95% CI, 4.20-11.86; P < .001), PHMB-2 (mean difference = 6.93; 95% CI, 3.10-10.76; P < .001), HOCl-1 (mean difference = 7.11; 95% CI, 3.29-10.95; P < .001), or surfactant cleansers (mean difference = 6.42; 95% CI, 2.59-10.25; P < .001) were statistically significant; however, differences between the PHMB-1, PHMB-2, HOCl-1, and surfactant groups were not significant.

The 72-hour regrowth of MRSA was 1.92 ± 0.62, 1.31 ± 0.34, or 1.59 ± 0.34 times the initial growth values after biofilms were treated with PHMB-1, PHMB-2, or HOCl-1, respectively, whereas the regrowth values for immature MRSA biofilm treated with surfactant, silver, or the saline control were 7.54 ± 1.09, 7.72 ± 0.21, and 6.36 ± 0.70 times higher than the time zero optical density measurements, respectively, as seen in Figure 3. Differences between the PHMB-1, PHMB-2, and HOCl-1 groups were not significant, but differences between the saline control and the PHMB-1 (mean difference = 4.44; 95% CI, 2.02-6.86; P < .001), PHMB-2 (mean difference = 5.04; 95% CI, 2.56-7.52; P < .001), or HOCl-1 (mean difference = 4.82; 95% CI, 2.45-7.20; P < .001) cleanser groups were statistically significant. 

For C albicans, the 72-hour regrowth measurements were 2.44 ± 0.91, 4.01 ± 1.31, and 2.71 ± 1.12 times the time zero growth values after treatment with PHMB-1, PHMB-2, or HOCl-1, respectively; treatment with surfactant, silver, or saline resulted in a 13.22 ± 0.46, 13.11 ± 0.06, and 13.36 ± 0.09 times the initial growth values, respectively (Figure 3). The differences in biofilm regrowth between the saline control and the PHMB-1 (mean difference = 10.91; 95% CI, 7.47–14.36; P < .001), PHMB-2 (mean difference = 9.34; 95% CI, 5.89-12.79; P < .001), or HOCl-1 (mean difference = 10.66; 95% CI, 7.21-14.10; P < .001) groups were once again significant, whereas differences between the PHMB-1, PHMB-2, and HOCl-1 groups were not statistically significant.

Ex vivo porcine dermal explant studies 

Effect of wound cleansers on P aeruginosa. After a single 10-minute application of the wound cleansers on P aeruginosa biofilms followed by a 72-hour incubation period (ie, to simulate a Friday to Monday dressing change), the PHMB-1 cleanser significantly reduced the mature P aeruginosa biofilm by 0.58 ± 0.45 log CFU/mL (95% CI, 0.30-0.85 CFU/mL; P < .001), whereas differences in the remaining cleansers were not statistically significant (Figure 4). The PHMB-1 treatment resulted in a mean log reduction of 0.63 ± 0.46 (95% CI, 0.35-0.90 CFU/mL; P < .001), 0.69 ± 0.46 (95% CI, 0.41-0.96 CFU/mL; P < .001), 0.59 ± 0.45 (95% CI, 0.31-0.87 CFU/mL; P < .001), and 0.48 ± 0.46 CFU/mL (95% CI, 0.20-0.75 CFU/mL; P < .001) compared with NaOCl-1, NaOCL-2, HOCl-2, and PHMB-2, respectively. These between-group results were statistically significant based on post hoc analysis.

Following 3 daily applications over 72 hours to simulate daily dressing changes, PHMB-2, PHMB-1, and NaOCl-2 showed significant reductions in P aeruginosa biofilms of approximately 2.27 ± 0.10 (95% CI, 1.49-3.05 CFU/mL; P < .001), 3.11 ± 0.82 (95% CI, 2.33-3.89 CFU/mL; P < .001) , and 4.42 ± 0.43 (95% CI, 3.64-5.19 CFU/mL; P < .001) log CFU/mL, respectively, compared with the saline control, whereas differences between HOCl-2 and NaOCl-1 and saline were not statistically significant (Figure 4). Post hoc analysis showed that the 0.84 ± 0.65 log CFU/mL reduction between PHMB-2 and PHMB-1 (95% CI, 0.06-1.62 CFU/mL; .01 < P < .05) groups, and 1.3 ± 0.93 CFU/mL mean log reduction between the PHMB-1 and NaOCl-2 (95% CI, 0.53-2.08 CFU/mL; P < .001), were both significant. 

Effect of wound cleansers on MRSA. After a single application of wound cleanser followed by a 72-hour incubation period to mimic a Friday through Monday dressing change, the differences in average log CFU/mL of all test cleansers and the control, saline, were not significant, though PHMB-1 sustained an 1.40 ± 0.81 log CFU/mL reduction in comparison with saline (Figure 5). 

Daily applications to simulate daily dressing changes resulted in PHMB-1 effectively eliminating MRSA biofilms with a 8.1 log CFU/mL reduction, whereas NaOCl-2 solution resulted in an approximately 3.22 ± 0.65 mean log reduction (95% CI, 2.04-4.39 CFU/mL; P < .001) compared with saline (Figure 5). PHMB-1 resulted in a 4.88 ± 0.68 mean log reduction (CFU/mL) in MRSA mature biofilms in comparison to NaOCl-2 (95% CI, 3.71-6.05 CFU/mL; P < 0.001), and a 7.49 ± 0.31 (95% CI, 6.32-8.66 CFU/mL; P < .001), 8.13 ± 0.311 (95% CI, 6.96-9.30 CFU/mL; P < .001), or 8.33 ± 0.29 CFU/mL (95% CI, 7.16-9.50 CFU/mL; P < .001) mean log reduction in comparison to the PHMB-2, HOCl-2, or NaOCl-1, respectively. 

Effect of wound cleansers on C albicans. PHMB-1 showed a statistically significant 1.36 ± 0.14 CFU/mL mean log reduction (95% CI, 0.51-2.20 CFU/mL; .001 < P < .01) in C albicans mature biofilm compared with the saline control after a single application followed by 72 hours of incubation to simulate a 3-day dressing change (Figure 6). Differences between saline and the HOCl-2, NaOCl-1, NaOCl-2, and PHMB-2 treatment groups were not statistically significant. PHMB-1 also showed statistically significant (.001 ≤ P < .01) differences compared with other groups according to post hoc analysis.

Daily application for 3 days with PHMB-1 resulted in a 7.12 ± 0.21 CFU/mL (95% CI, 5.41-8.84 CFU/mL; P < .001) mean log reduction of C albicans mature biofilm compared with the saline control (Figure 6). Differences between the PHMB-1 cleansers and other groups were 7.27 ± 0.26, 7.18 ± 0.21, 5.66 ± 0.41, and 6.11 ± 0.23 in comparison to NaOCl-1 (95% CI, 5.55-8.98 CFU/mL; P < .001), HOCl-2 (95% CI, 5.46-8.89 CFU/mL; P < .001), NaOCl-2 (95% CI, 3.94-7.37 CFU/mL; P < 0.001), and PHMB-2 (95% CI, 4.40-7.82 CFU/mL; P < .001) groups, respectively. PHMB-2 and NaOCl-2 showed 1.01 ± 0.10 and 1.46 ± 0.35 CFU/mL log reduction of mature C albicans, respectively, whereas the other groups were comparable to the saline control; no statistical differences were observed between these groups. 

Discussion

Wound cleansing is an integral part of the management of acute and chronic wounds.21,30,37–41 The selection of an appropriate wound cleanser is confounded by conflicting evidence about effectiveness in infection reduction and wound healing. These reviews span clinical, in vitro, and ex vivo studies that, depending on study type, organism type, biofilm growth substrate, age of biofilm, or application times, can produce large fluctuations in reported efficacy.21,31,42,43,52 

The current study compared different types of commercially available wound cleansers in 2 biofilm models for 3 prevalent, difficult-to-treat pathogens found in chronic wounds, namely P aeruginosa, MRSA, and the fungi C albicans, with the intention of providing clarity in cleanser selection for chronic wound management. Several types of wound cleansers were studied including saline, a surfactant-based cleanser, and antimicrobial-based cleansers with ionic silver, hypochlorous acid, sodium hypochlorite, or PHMB. In the MBEC model, the 2 PHMB-based cleansers (PHMB-1 and PHMB-2) and chlorine-containing wound cleanser (HOCl-1) showed effectiveness in limiting regrowth of P aeruginosa, MRSA, and the fungi C albicans. However, the largest differences in regrowth of P aeruginosa, MRSA, and the fungi C albicans biofilms was observed with the ex vivo PDE model, with the cleanser applied once daily over 72 hours to mimic daily dressing changes. The PHMB-based cleanser (PHMB-1) had the largest biofilm reductions when compared to all other cleansers after a single application and significantly lower reductions of MRSA and C albicans biofilms following daily treatments over 72 hours compared to all other test articles. However, NaOCl-2 had the lowest biofilm reductions following daily applications for P aeruginosa

In vitro and ex vivo explant studies have shown that wound cleansers containing surfactants can disrupt the attachment of microorganisms to wound surfaces, interfere with aggregation of microbes to create biofilms, or solubilize the EPS.44–48 In an in vivo study of different types of wound irrigation solutions, a surfactant solution was superior to saline or antibiotic solution in removing adherent bacteria from metallic surfaces, bone, and bovine muscle; in addition, sequential applications of surfactant irrigation solutions effectively cleansed wounds with established polymicrobial (MRSA and P aeruginosa) infection.44 In the MBEC model, the surfactant-based cleanser (Surfactant) was effective against P aeruginosa but ineffective against MRSA or C albicans biofilms, so it was not explored in the ex vivo porcine dermal model.

Silver, used topically, systemically, or in clinical tools, may be the antimicrobial with the longest history of use; the ancient Greeks used silver preparations to treat ulcers and to stimulate wound healing.49 Silver ions kill microbes by interacting with the cytoplasmic membrane and compromising electron transfer and proton motive force,50 mainly through interaction with thiol groups.51 In the current study, a silver-based cleanser containing silver microparticles that release ions (Silver) was ineffective in inhibiting regrowth of immature biofilms. These results contrast with a recent systemic review in which, in part, the focus was on 39 in vitro studies, involving 78 microbial species, the primary microbe studied being MRSA, that showed an average 2.18 ± 1.81 log reduction after 24 hours across all studies of in vitro biofilm studies microbes.52 These differences could be due to the short application times (10 minutes) used in the current study. In 2015, Phillips et al53 compared antimicrobial dressing efficacy on mature P aeruginosa biofilms using the ex vivo porcine explant model,  which reinforces the necessary time duration for efficacy of silver. In that study, a silver gel reduced mature P aeruginosa by 2.66 log CFU/mL after 24 hours of exposure, and 5.633 log CFU/mL by 72 hours of continuous exposure, which suggests that the 10-minute wound cleanser application is not sufficient when using silver-based cleanser products. The silver-based cleanser was not used in the ex vivo porcine model due to the low effectiveness in the MBEC testing.

Hypochlorous acid and sodium hypochlorite-based antimicrobial cleansers kill microbes using free radical and ionic reaction with pendant nitrogen- and sulfur-containing groups found on the surface of human and microbial tissues, including cells.54 Historically, results with HOCl and sodium hypochlorite-based cleansers at various concentrations have been inconsistent. An HOCl-based cleanser was tested by Davis et al55 using an in vivo partial-thickness wound model inoculated with MRSA, which resulted in a 0.67-log reduction after twice-daily irrigation for 6 days. Conversely, Day et al56 published results in which a 1% HOCl solution successfully killed S aureus and P aeruginosa biofilms in vitro on collagen, whereas Robson57 demonstrated a > log 6 CFU/cm3 reduction in MRSA after a 10-minute treatment with HOCl in an in vitro study on medical tubing.

Several hypochlorous acid (HOCl) and sodium hypochlorite (NaOCl)-based cleansers were tested in the current study, delineated as HOCl-1, HOCl-2, NaOCl-1, and NaOCl-2. The study showed efficacy of HOCl-1 in the immature model (MBEC), demonstrating a statistically significant decrease in the normalized growth assessment in all of the microorganisms tested. This product class was then expanded into additional products that covered both HOCl (HOCl-1) and NaOCl (NaOCl-1, NaOCl-2) for the mature biofilm model (ex vivo). NaOCl-2 was the standout with greater than 3-log reduction of mature MRSA and P aeruginosa with daily applications. Based on generation doubling times, a 3-log reduction in the ex vivo porcine explant biofilm model may be appropriate for planktonic colonization or low load (immature) biofilms, whereas greater than 5-log reduction in mature biofilm is likely required for an effective antimicrobial dressing,53 suggesting NaOCl-2 may be effective against planktonic microbes and immature clinical biofilms. However, clinically, NaOCl-2 would be diluted to quarter-strength (quarter-strength Dakin’s solution; 0.125% sodium hypochlorite solution) due to concerns about cytotoxicity,58 and this diluted solution would likely be less effective against mature biofilms. In this study, however, the aim was to test the unadulterated cleanser given no other test subjects were altered for the study.

PHMB interacts with negatively charged phospholipids in bacterial membranes, resulting in loss of integrity and ultimately death. A systematic clinical and literature review compiled by Hübner and Kramer59 found it also disrupts microbe metabolism when it is transferred to the cytoplasm. In a systematic review of in vitro studies, PHMB has been shown to be effective in biofilm efficacy studies with an average 3.33 ± 2.28 log reduction of biofilms with 24-hour treatment.52 

Two (2) PHMB-based cleansers were tested in the current study. PHMB-2 showed statistically significant difference to the control in the immature biofilm model and demonstrated similar normalized regrowth assessment to PHMB-1; both of which were > 3 times lower than the control. In the mature biofilm model, PHMB-2 showed limited efficacy with the only statistically significant difference from the control showing in one organism, P aeruginosa. PHMB-1 showed efficacy in the MBEC model, through a statistically significant decrease in the normalized growth assessment versus the control, and higher efficacy than PHMB-2 in the ex vivo porcine biofilm study which showed a statistically significant decrease compared to the control in mature biofilm in all microorganisms tested. PHMB-1 was more effective than HOCL-2, NaOCL-1, and PHMB-2, showing a greater than 3-log reduction of P aeruginosa mature biofilm. PHMB-1 was more effective than HOCL-2, NaOCl-1, NaOCl-2, and PHMB -2 against MRSA and C albicans mature biofilm with daily applications showing greater than 7-log reduction; this suggests that PHMB-1 may be effective against mature biofilms based on population doubling times.53

In general, the HOCl- and PHMB-based cleansers were effective in the MBEC model, whereas only PHMB-1 showed effectiveness and NaOCl-2 showed limited effectiveness in the PDE model. There are several factors that could explain these differences. The MBEC model is grown on an abiotic surface, and these peg-based biofilms have high surface area/volume microorganisms that are relatively easy to kill.30 The biofilms formed in the PDE model use the skin as the main source of nutrition, similar to a wound environment, and these biofilm microorganisms are able to penetrate the skin.36 Thus, in this model, there is competition for activity of the antimicrobial because it can react with components of the biofilm and the porcine skin tissue. Additionally, it can be expected that the differences in results between the MBEC and ex vivo pig skin models are due in part to biofilm maturity. Wolcott et al61 demonstrated that biofilms are more susceptible when immature. Much of the antimicrobial resistance in a biofilm is due to protection from the EPS matrix.62,63 Therefore, it is reasonable to assume that the differences in pre-clinical antimicrobial biofilm efficacy in different biofilm models34 is due, at least in part, to the maturity of the biofilm EPS. The biofilms used in the PDE model were more mature31,64 and, thus, more clinically relevant than those formed using the MBEC method. A chlorine-based (HOCl or NaOCl) cleanser will react with the surface of the EPS as well as the surrounding human tissue, which will limit penetration and effectiveness in a mature biofilm.65 The 2 PHMB-based cleansers had different results in the ex vivo PDE study, which indicates that the PHMB-1 cleanser containing not only PHMB, but also EDTA salts and vicinal diols, may be disrupting the biofilm EPS structure and allowing for penetration of the antimicrobials and ultimate kill of the biofilm microbes.60

Overall, these promising in vitro and ex vivo results warrant controlled clinical testing in chronic wounds to confirm that they are clinically relevant. As the PHMB-1, PHMB-2, and NaOCl-2 cleansers showed increased pre-clinical efficacy with daily applications in the ex vivo porcine model of mature biofilm, it seems likely that sustained antimicrobial presence may be required to reduce or eliminate biofilms in human wounds, which is shown in other studies, particularly with silver.52 This should be considered when designing clinical studies by focusing on continuous irrigation or advanced antimicrobial dressings to provide sustained biofilm disruption and/or antimicrobial activity. 

The control and mitigation of microbial infection is pivotal in optimizing the wound healing process as established in two clinical meta-analysis performed in 2008 and 2016.1–5 Wound cleansing is an integral part of wound management and leads to decreased risk of infection and improved wound healing as stated by Lewis and Pay40 in their clinical instructions on wound care. Given the structural and biochemical changes that occur as a biofilm forms and matures, it is crucial that clinicians are familiar with the capabilities and shortfalls of the various classes of cleansers available. This study used immature and mature biofilm models of common wound pathogens with the intent that the results will assist clinicians in making more informed treatment choices.

Limitations

The current study tested many classes of commercially available wound cleansers for pre-clinical antibiofilm efficacy, but not every possible cleanser. The Calgary biofilm MBEC model provides a high-throughput method for screening pre-clinical antibiofilm efficacy, but the results are less relevant for clinical translation as biofilm phenotype and maturity are dependent on their substrate and nutritional environment.36,66–69 The ex vivo PDE model was selected as a more clinically relevant biofilm model, and although these biofilms are more mature, there are still differences between them and a wound, perhaps the most relevant being the lack of the host immune response. Additionally, in nature, biofilms are heterogenous and polymicrobial. Monospecies biofilms were selected for wound cleanser tests to improve reproducibility, but this is a limitation for translation of the results to clinical biofilms. Polymicrobial biofilms and studies with simulated wound fluid should be considered for future investigation. 

Conclusion

This study was designed to test pre-clinical antibiofilm efficacy of commercial wound cleansers against immature and mature biofilms composed of gram-positive, gram-negative, and fungal microbes. Several wound cleansers were effective against immature biofilms (in vitro MBEC model); these included PHMB-1, PHMB-2, and HOCl-1 with greater than 4× reduction in immature biofilm regrowth. Few, however, demonstrated high efficacy against mature biofilms (ex vivo porcine model); PHMB-1 showed efficacy against all 3 organisms (3–8 CFU/mL mean log reduction) while NaOCl-2 showed efficacy against 2 (3–5 CFU/mL mean log reduction of MRSA and P aeruginosa) with daily application. It should be noted that NaOCl-2 was used in higher concentrations than usual for clinical usage. PHMB-2 showed limited efficacy (3 CFU/mL mean log reduction) against mature P aeruginosa biofilms. This outcome may be due, in part, to the inability of certain wound cleansers to penetrate the biofilms and disrupt the EPS or reach the microorganisms that thrive within the biofilms. This finding is of particular interest when comparing data from other biofilm studies because it indicates there are model-specific differences in results that should be considered. It is clear from this study that the ex vivo porcine biofilms of MRSA, P aeruginosa, and C albicans biofilms are more challenging to eliminate than those from the MBEC model given maturity and substrate used. PHMB-1 was the only wound cleanser that was highly effective against both immature and mature MRSA, P aeruginosa, and C albicans biofilms. Daily applications were required to reduce or eliminate the mature biofilms, which suggests that sustained antimicrobial presence may be required to eliminate clinical biofilms. Although the MBEC and PDE models used indicated initial efficacy against immature and mature biofilms, respectively, further research, including clinical studies will be needed to best ascertain optimal usage in a clinical settings. 

Acknowledgments

Bradley Rodier, PhD, and Carlyn Abbott assisted with editing and preparing the final draft of the manuscript. Cindy Taylor provided editing support, and Jane Andrews, PhD, assisted with composing an early draft.

Affiliations

Dr. McMahon is president, Rochal Industries, San Antonio, TX; Ms. Salamone is chair of the board, Rochal Industries, San Antonio, TX; Ms. Poleon was a scientist I, Rochal Industries, San Antonio, TX; Dr. Bionda is a research scientist/study director II, iFyber, Ithaca, NY; Dr. Salamone (deceased) was chief science officer, Rochal Industries, San Antonio, TX. Address all correspondence to: Rebecca McMahon, MBA, PhD, Rochal Industries, 12000 Network Boulevard, Suite B200, San Antonio, TX 78249; tel: (518) 428-9496; email: remcmahon@rochalindustries.com.

Potential Conflicts of Interest

This work was sponsored by Rochal Industries, LLC, San Antonio, TX, the inventors of the BIAKŌS Antimicrobial Skin and Wound Cleanser, which is licensed by Sanara MedTech (Fort Worth, TX). Rochal Industries paid to design and conduct the studies at the following independent laboratories using blinded samples: INCELL, San Antonio, TX (MBEC), and iFyber LLC, Ithaca, NY (ex vivo porcine dermal explant).