Effect of Mechanical Debridement and Irrigation With Hypochlorous Acid Wound Management Solution on Methicillin-resistant Staphylococcus aureus Contamination and Healing Deep Dermal Wounds in a Porcine Model

Patients with diabetes mellitus, among other medical conditions, have a high likelihood to develop chronic wounds, such as foot ulcers.¹ These chronic wounds are difficult to heal; the patients’ comorbidities inhibit the wound’s ability to move through each phase of the wound healing process, potentially creating complications, such as amputations.² These complications increase mortality rates to 39% to 80% after 5 years of wound-related amputations. Hence, proven methods to manage chronic and acute wounds and prevent and manage infections are needed.

According to a literature review specific to burn infection by Norbury et al,³  many infections in burns wounds are caused by antibiotic-resistant organisms such as methicillin-resistant Staphylococcus aureus (MRSA). Numerous animal and human studies have shown the presence of biofilms and have demonstrated delayed healing and aberrant inflammatory responses.⁴ Bacteria within biofilm have been shown to have a 10- to 1000-fold increase in resistance toward conventional antibiotics and antiseptics.^5-7 In addition, the close proximity of cells within the biofilm facilitates lateral gene transfer, which aids the passage of resistance genes from cell to cell in bacteria and results in a more uniformly resistant bacterial population.^8,9 We have demonstrated in our porcine model that once biofilms are formed they are much more difficult to eradicate as compared to planktonic bacteria.¹⁰ Furthermore, an in vivo porcine study by Nusbaum et al¹¹ showed that surgical debridement (curette) had limited ability to mechanically remove bacteria from deep partial-thickness wounds in pigs, suggesting that adjunctive thereapies are needed after debridement. Snyder et al¹² advocate that appropriate topical antimicrobial application be used after debridement to suppress biofilm reformation.

Wilkins et al¹³ found that appropriate wound cleaning of clinical wounds removes necrotic tissue and can enhance wound healing. Various irrigation methods have been shown to provide both mechanical and chemical activity. Wound irrigation using hypochlorous acid (HOCl) has been shown, in a clinical study (n = 12) for skin split graft in acute wounds, to have antimicrobial activity without irritating the skin.¹⁴ A preliminary clinical study¹⁵ testing the ocular, skin, and wound sensitivity against HOCl found that low-level concentrations of the agent are not cytotoxic. In an in vitro study by Sakarya et al,¹⁶ all pathogenic microorganisms were killed within 12 seconds after HOCl exposure.

The polyatomic compound derived from HOCl (hypochlorite [ClO^-]), has been reported and shown to have positive results in the presence of biofilm in dentistry^17,18 and plastic surgery.^19,20  The protonated form found in HOCl has been found in 30 clinical case studies treating chronic nonhealing wounds to be 80 to 100 times more effective as an antimicrobial than its ClO^- (hypochlorite anion) counterpart.²¹ In addition, the force created by the irrigation and the antimicrobial capabilities of HOCl have been shown to decrease the bacterial count on both biofilm and planktonic forms against pathogens on surfaces.²²

The purpose of this in vivo study was to determine the effect of an HOCl wound irrigation solution on MRSA and healing when used in conjunction with debridement utilizing a well-established biofilm porcine model.²³

Experimental animals. Three (3) female specific pathogen-free pigs were used as the experimental research animal because swine skin is morphologically similar to human skin.²⁴ The protocol was approved by the University of Miami Animal Use Committee. Animals were monitored daily for any observable signs of pain or discomfort. The animals were provided analgesics during the entire study to help minimize possible discomfort.

Protocol.

Animal preparation and wounding technique. The hair on the flanks and backs of the pigs was trimmed with standard animal clippers, and the areas washed with a nonantibiotic soap (Neutrogena) and deionized sterile water. A specialized electrokeratome was used to create 19 deep reticular dermal wounds (22 mm × 22 mm × 3 mm deep) on the paravertebral and thoracic area on each animal. The wounds were separated by at least 5 cm to 7 cm of unwounded skin. Immediately after wounding, wounds were randomly (block randomization) divided into 2 groups of 8 wounds on each animal; 3 additional wounds were created to obtain baseline counts before treatment.

Wound inoculation. A fresh culture of pathogenic isolate of MRSA was used in these studies. The frozen bacterium was recovered from glycerol stock (15% glycerol in tryptic soy broth [TSB], -80° C). All inoculum suspensions were made by scraping the overnight growth from a culture plate into 5 mL of normal saline. This resulted in a suspension concentration of approximately 10⁸ colony forming units/mL (CFU/mL). The 10⁸ CFU/mL suspension was serially diluted to make an inoculum suspension with a concentration of 10⁶ CFU/mL as determined by optical density at 570 nm. A small amount of the inoculum suspension was plated onto culture media to quantify the exact concentration of viable organisms. A 25 µL aliquot of this suspension was deposited into the center of each wound. Each aliquot then was lightly scrubbed into the test site for 10 seconds using a sterile Teflon spatula and left for 3 minutes before the wounds were covered with a polyurethane film dressing (each wound was dressed individually). Wounds remained covered for 72 hours to allow the biofilms to become established before treatment.¹⁰

Treatment regimen. Wounds were debrided once every 72 hours after wound creation and MRSA inoculation using a sterile curette. Using a block randomization method, sets of wounds were randomly assigned to saline (0.9% normal saline, Baxter Healthcare Model) or HOCl (Puracyn Plus, Innovacyn, Inc.) treatment groups. Wounds were rinsed twice using 10-mL syringes with 1.5-inch long, 21-gauge needles held at a 45° angle over the wounds, receiving a total of 20 mL fluid. During wound treatment, the adjacent wounds were covered with sterile 1.5-inch metal caps to prevent the rinse from flowing into the other wounds. After rinsing the wounds, each wound bed was encircled with a sterile cylinder measuring 1¾ inches and the cylinder was simultaneously filled with 4 mL of treatment substance (sterile saline or HOCl) for 5 minutes to fill the entire wound. Any excess treatment was pipetted out. Sterile gauze soaked with 2 mL of treatment of either saline or HOCl then was placed over the wound. All treatments were covered with polyurethane film to retain moisture. Wounds received twice-daily treatments for the first 5 days after debridement and once-daily treatment for the remainder of the study.

Microbiology assessment. Three (3) wounds were cultured 72 hours after wounding and inoculation for baseline enumeration of bacteria using a sterile 6-mm punch biopsy. On days 4, 8, and 11 (after debridement and initial treatment), 2 or 3 additional wounds per group were biopsied from the center of the wound area (6-mm biopsy). Treatment application was not performed on wound to be biopsied. After biopsy recoveries, the remaining wounds not recovered would be then treated.

Biopsy specimens were weighed and immediately placed in 1 mL of all-purpose neutralizing solution. The sample was combined with an additional 4 mL of neutralizing solution and homogenized in a sterile homogenization tube. Serial dilutions were made from all culture samples and the extent of microbiological contamination assessed using the Spiral Plater System (Spiral Biotech). This system deposits a 50 µL aliquot of the scrub bacterial suspension over the surface of a rotating agar plate. Oxacillin resistance screening agar (ORSAB) was used to isolate MRSA USA300. All plates were incubated aerobically overnight (24 hours) at 37° C, after which the number of viable colonies were counted. This method has been used for more than 30 years to evaluate the antimicrobial efficacy of various topical agents and/or dressings.^10,25,26

Histological assessment. After treatment application on days 4, 8, and 11, incisional biopsies were obtained through the center of the wounds and included normal adjacent skin on both sides. In a similar fashion to microbiology analysis, treatment application was not performed on wound to be biopsied. After biopsy recoveries, the remaining wounds not recovered then would be treated. These specimens were placed in formalin and then stained with hematoxylin and eosin. One section per block containing the tissue specimens fixated in wax was analyzed. The specimens were evaluated via light microscopy and examined by researchers blinded²⁷ as to treatment groups for the following wound healing elements to determine a potential treatment response:

• Percent of wound epithelialized: measurement of the length of the wound surface covered with epithelium compared with the whole wounded area. The percent of reepithelialization represented the percent of the wound area covered by newly formed epidermis with one or more layers of keratinocytes,²⁷ which has been shown to be a good index for the speed of keratinocyte migration and the first step of the reepithelialization. Percent of epithelialization was calculated dividing length of epithelialized surface by the total length of the wound bed surface multiplied by 100;
• Epithelial thickness (cell layers µm): epithelial thickness may vary from area to area within the biopsy. The thickness of the epithelium in µm was measured on 5 equidistant points from each other in the biopsy and averaged;
• White cell infiltrate: this was a measurement of the presence and amount of subepithelial mixed leukocytic infiltrates. Mean score: 1 = absent, 2 = mild, 3 = moderate, 4 = marked, 5 = exuberant;
• Granulation tissue formation: the approximate amount of new granulation tissue formation (dermis) was graded as follows: 1 = ≤ 5%; 2 = 6% to 25%; 3 = 26% to 50%; 4 = 51% to 75%; and 5 = 76% to 100%.

Statistical analysis. Microbiology and histology results (n = 8) were analyzed for significant differences between treatments and assessment days using one-way analysis of variation (ANOVA) using SPSS, version 22 (IBM), followed by student t-tests. A P value ≤ .05 was considered significant. All treatment results were compared to the baselines and among groups using ANOVA. Student t-test was used to determine the statistical significance among assessment days.

Microbiology analysis. Biopsies that were recovered after debridement alone had significantly lower MRSA counts (6.69 ± 0.38 Log CFU/g) than the biopsies recovered before debridement (8.10 ± 0.60 Log CFU/g) (P ≤ .05). On day 4, wounds irrigated with HOCl had an MRSA count of 5.36 ± 0.17 Log CFU/g, which was significantly lower than wounds irrigated with saline at 6.26 ± 0.31 Log CFU/g (P ≤ .05 (Figure 1).

On day 8, wounds irrigated with HOCl had an MRSA count of 3.66 ± 0.56 Log CFU/g, which was significantly lower than the saline control (5.52 Log CFU/g; P ≤ .05). A bacterial reduction of 98.61% was noted in all assessment times when comparing WMS to saline control. By the end of the study on day 11, wounds irrigated with HOCl exhibited MRSA counts of 3.40 ± 0.51 Log CFU/g, which were significantly (P ≤ .05) lower than saline control (5.21 Log CFU/mL). During all assessment days (4, 8, and 11) wounds treated with the HOCl had significantly lower bacterial counts (P ≤ .05) compared to baseline whereas wounds treated with saline control showed counts lower than baseline wounds only on days 8 and 11 (Figure 2).

Histology results.

Epithelialization. On day 4, all wounds treated with HOCl and saline reached less than 40% of reepithelialization, showing no statistical (P ≤ .05) difference among treatment groups. On day 8, wounds irrigated with HOCl exhibited 78% reepithelialization versus 67% for saline (P ≤ .1). By day 11, the difference in reepitalization was not statistically significant (Figure 3).

Epithelial thickness. Epithelial thickness measurements ranged from 129 µm to 142 µm during the various assessment days, with no significant differences noted between treatment groups (Figure 4).

White cell infiltrate and granulation. When HOCl and saline data were compared, no statistical differences were noted with regard to white cell infiltrate or granulation tissue formation scores during the entire study (Figure 5 and and Figure 6).

The results of this study showed that HOCl solution irrigation was superior to saline irrigation of dermal wounds, reducing bacterial counts and increasing the amount of epithelialization when used in conjunction with sharp debridement.

Debridement methods in chronic wounds have proven to be reliable in removing necrotic tissue, and bacterial biofilms from a wound bed.²⁸ A survey²⁹ involving 706 health care professionals specializing in wound care found the vast majority (96%) were completely satisfied with the healing results after performing mechanical debridement on their patients. Hydromechanical therapy (ie, mechanical intervention via debridement with a curette and then spraying a solution into each wound bed) has demonstrated to reduce bacterial presence in previous clinical studies of infected chronic wounds.³⁰  A clinical case³¹ involving a patient whose nonhealing wound had been treated unsuccessfully for more than 3 months using negative pressure wound therapy showed wound irrigation and mechanical debridement improved wound healing. In the current study, positive outcomes were achieved using either HOCl or saline when compared to baseline bacterial counts (pre and post debridement); HOCl irrigated wounds had lower bacterial levels and higher reepithelialization rates. Other clinical studies have demonstrated similar results; the challenge is to utilize the correct amount of hypochlorite anion concentration within the solution to provide an optimal wound care.^32-34

In a clinical study (n = 17) comparing HOCl and saline irrigation in chronic wounds,³⁵ patients whose wounds were treated with HOCl had lower bacterial growth than wounds treated with saline. An in vitro study by Granick et al³⁶  using surfaces of metallic discs composed of titanium and stainless steel, comparable with the alloys used in surgical implants and infected with Staphylococcus epidermidis, showed that ultrasound and HOCl combined treatment completely eradicated S epidermidis when compared to saline.

The current study also demonstrated that wounds irrigated with HOCl achieved more than 97% epithelialization by day 11, and at day 8 the rate of reepithelialization was significantly higher than wounds irrigated with saline (P ≤ .01). In short, wounds irrigated with HOCl had significantly lower bacterial counts and higher healing rates than saline-irrigated wounds. Although the current study was limited to treatment of MRSA-inoculated wounds, an ex vivo study³⁷ using porcine dermal explants found HOCl demonstrated potential antifungal capabilities against C albicans.

Various studies have demonstrated the germicidal properties of HOCl.^38-43 A common disinfectant used all over the world, HOCl has theoretical efficacy against bacteria, yeasts, and even viruses such as SARS-CoV.⁴⁴ The mechanisms by which HOCl affects bacteria has been widely researched.^45-47 The interaction of HOCl with sulfur- and heme-containing membrane enzymes and structural proteins⁴⁸ causes a respiratory loss in the bacterial cell membrane, which leads to a cell death and non-viability.^44,45,47,49 An in vitro study by Harriott et al⁵⁰ testing the reaction of clinical isolates of gram-negative and gram-positive bacteria and Candida (12 species total) to HOCl-based solutions found HOCl to be effective in eliminating bacterial and fungal biofilms. Biofilm disaggregation has been cited as one mechanism responsible for its efficiency against bacteria.⁵¹ Biofilm tube experiments with S aureus⁵² showed that bacterial counts were reduced by >5 logs after a 1-minute exposure to HOCl and 6 logs after 10 minutes; about 70% of the biofilm polysaccharide and > 90% of the biofilm protein also were removed after a 10-minute exposure. Additionally, an in vitro study using human red blood cells⁵³ showed that a low HOCl dosage (300 nmol of HOCl/mL of cell suspension) was enough to be effective against microorganisms without the risk of cytotoxicity. Recent literature confirms the efficacy for HOCl to provide desirable results against gram-positive and gram-negative bacteria, and yeasts during in vitro and in vivo studies.^54,55 A retrospective clinical study⁵⁶ involving 897 patients (44% diagnosed with diabetes mellitus) with 1249 venous ulcers found HOCl can reduce the effects of some comorbidities while accelerating healing times. Most importantly, this clinical study demonstrated the crucial benefits provided when the in tandem protocol involving debridement and HOCl irrigation caused all ulcers to close completely.

One of the limitations of this study and all animal wound models is that they tend to be short term, do not address underlying comorbidities, and do not necessarily replicate a clinical low-grade wound infection.^57,58 However, it is widely accepted that porcine skin wound healing most closely resembles the human healing process. A review of 25 wound therapies revealed that porcine studies were in agreement with human-related results 78% of the time compared with 53% and 57% with rodents and in vitro, respectively.²⁶ Consequently, evidence from animal studies is important for clinicians to consider when choosing treatments for chronic wounds, although clinical evidence is still required to validate these findings.

The results of this in vitro study showed that the combination of debridement and HOCl wound irrigation can significantly reduce MRSA contamination and facilitate the healing process. This preclinical study suggests that HOCl irrigation in conjunction with surgical debridement techniques may have important clinical benefits in controlling heavily colonized wounds. Clinical studies are needed to elucidate the long-term effect of this treatment strategy on chronic wound healing.

Mr. Davis is a professor; Mr. Gil is a lab research manager; Dr. Li is an associate professor; and Mr. Simms, Mr. Valdes, Mr. Solis, and Mr. Higa are research associates, University of Miami Miller School of Medicine, Department of Dermatology and Cutaneous Surgery, Miami, FL. Address all correspondence to: Stephen C. Davis, BS, University of Miami Miller School of Medicine, Department of Dermatology and Cutaneous Surgery, PO Box 016250 (R 250), Miami, FL 33101; email: sdavis@med.miami.edu.