Negative-pressure wound therapy is an established treatment used to enhance wound healing in human medicine and is gaining popularity in veterinary medicine,1–5 although its feasibility for clinical use in veterinary medicine is just beginning to be researched. In equine medicine, published literature6–10 regarding the use of NPWT is sparse, and most of it consists of case reports involving very few horses. Negative-pressure wound therapy could be valuable in equine practice because it successfully addresses common problems associated with wound healing in horses such as massive tissue loss, poor tissue perfusion, and a weak but protracted inflammatory response.
Negative-pressure wound therapy increases blood flow, promotes angiogenesis, induces cell proliferation, reduces wound area in certain types of wounds, and modulates inhibitory substances in wound fluid such as matrix metalloproteinases.3 Nevertheless, the effect of NPWT on wound contamination or infection with various bacterial species is controversial in both human and veterinary medicine.3,11–16 In 1 study,11 the bacterial load decreased quicker for wounds treated with NPWT than for wounds treated with gauze soaked with saline (0.9% NaCl) solution. Conversely, results of other studies indicate that the bacterial load in a wound increased13 or remained fairly stable, albeit with a decrease in the number of nonfermentative gram-negative rods and an increase in the number of Staphylococcus aureus organisms12 during NPWT. Results of yet another study14 suggest that NPWT did not enhance bacterial clearance from wounds. Thus, it is impossible to formulate an unequivocal conclusion regarding the effect of NPWT on the bacterial load of wounds.
Data regarding the effect of dressing type used with NPWT on the bacterial load of wounds are also sparse. To our knowledge, only 1 study15 has been published in which the bacterial load was compared between 2 different dressings used for NPWT. In that study,15 the bacterial load after NPWT in black polyurethane foam was significantly lower than that in white polyvinyl alcohol foam. However, the in vivo bacterial load in the wounds was not measured in that study,15 and the investigators could only hypothesize that the bacterial load in the dressing foams was representative of the bacterial load in the wounds.
The study reported here was conducted to compare the bacterial load among wounds treated by means of NPWT and 1 of 3 types of dressings, or foams (silver-impregnated polyurethane foam, standard polyurethane foam, and polyvinyl alcohol foam), with wounds treated by use of a standard dressing without NPWT. Our hypotheses were that the bacterial load of wounds treated with NPWT would be lower than that for wounds treated with a standard dressing without NPWT, regardless of the type of foam used for NPWT, and that the greatest decrease in bacterial load would be achieved for wounds treated with NPWT with silver-impregnated polyurethane foam, which was developed specifically for the treatment of heavily contaminated wounds.
Materials and Methods
Ex vivo equine perfused wound model
We developed an ex vivo perfused wound model that was based on a previously described in vitro model.17 The model allowed us to perform sequential biopsies of the ex vivo wound specimens to monitor bacterial load. All study procedures were approved by the Ghent University Animal Care and Use Committee, and all owners consented to the use of their horses' cadavers for scientific purposes. One musculocutaneous flap was obtained from the abdomen of each of 6 horses after they were euthanized for clinical reasons other than colic or septicemia. Immediately after a euthanasia solution containing embutramide was injected, each horse received heparin (150 U/kg, IV) to prevent the blood from coagulating in the capillaries of the intended flap. The cadaver was positioned in lateral recumbency as soon as it was determined that the horse was dead. The hair over the rectus abdominis muscle was clipped, and the underlying skin was then shaved with a razor blade. The shaved area was thoroughly scrubbed with chlorhexidine digluconate for 10 minutes until the gauzes used for scrubbing were free from visible contamination and then disinfected with 70% ethanol. A 25 × 35-cm musculocutaneous flap that included the rectus abdominis muscle and its overlying skin was aseptically harvested at the level of the superficial epigastric vein and artery and placed on a sterile plate. Care was taken to ensure that the linea alba was not included in the flap. The flap was transported to the laboratory within 5 minutes after harvesting to minimize contamination.
The flap was kept at room temperature (approx 22°C) in the laboratory. An 18-gauge catheter was inserted into the superficial epigastric artery and secured with a Chinese finger-trap suture. The catheter was connected to an infusion bag that contained sterile saline (0.9% NaCl) solution at room temperature. Saline solution was flushed through the catheter into the flap vasculature to identify patent blood vessels at the periphery and innermost muscular surface of the flap, which were then ligated with simple interrupted sutures. The infusion rate was adjusted to 0.5 mL/min to prevent tissue desiccation.
Pure strains of MRSA and Pseudomonas aeruginosa (2 common pathogens of equine wounds) that were originally isolated from the wounds of horses were used to prepare 2 separate bacterial stock solutions. For each pathogen, the designated culture was diluted with sterile PBS solution to achieve a stock solution with a bacterial concentration of approximately 106 CFUs/mL.
Four circular wounds, each with a diameter of 5 cm, were created in each flap by removing the skin, cutaneous trunci muscle, and outer layer of the underlying rectus abdominis sheath. An air-displacement micropipette was used to inoculate each wound with 1 mL of the MRSA stock solution and 1 mL of the P aeruginosa stock solution. The flap was incubated for 1 hour at 37°C and 5% CO2. Saline perfusion of the flap was temporarily discontinued during bacterial inoculation and the subsequent 1-hour incubation period.
Wound treatments
Saline perfusion of the flap was resumed following the 1-hour incubation period. The wounds of each flap were randomly assigned by means of a random number generator to 1 of 4 treatment groups, which included NPWT with silver-impregnated polyurethane foama (NPWT-AgPU), NPWT with a standard polyurethane foama (NPWT-PU), NPWT with polyvinyl alcohol foama (NPWT-PVA), or application of a nonadherent dressing that contained polyhexamethylene biguanideb without NPWT (control). Thus, each treatment was replicated 6 times.
Before treatment application, excess bacterial solution was removed from the wounds with dry sterile gauze, and the skin around each wound was disinfected with alcohol to prevent contamination. Each foam or dressing was cut into a circle 5 cm in diameter and placed in the wound designated for that treatment. The skin surrounding each wound was degreased with ether, and an adhesive spray was applied. The 3 wounds designated for NPWT were covered with an occlusive polyurethane foil.a Then, a 2-cm opening was cut in each occlusive foil, and a suction pada was applied over the opening. The suction pads were connected to the canister of the NPWT system by means of Y-connectors. The NPWT system was set at 125 mm Hg in continuous mode. The wound assigned to the control treatment was covered with an adhesive fabricb to keep the dressing in place (Figure 1).
Representative photograph of the ex vivo equine perfused wound model developed for a study to compare the bacterial loads of Pseudomonas aeruginosa and MRSA among wounds treated with 4 treatments. A 25 × 35-cm musculocutaneous flap that included the rectus abdominis muscle and its overlying skin was aseptically harvested at the level of the superficial epigastric vein and artery from each of 6 horses after they were euthanized for clinical reasons other than colic or septicemia. Immediately after injection of the euthanasia solution, each horse received heparin (150 U/kg, IV) to prevent blood from coagulating in the capillaries of the intended flap. An 18-gauge catheter (black arrow) was inserted into the superficial epigastric artery so that the flaps could be continuously infused with saline (0.9% NaCl) solution during the observation period. Four 5-cm circular wounds were created in each flap and contaminated with 106 CFUs of both P aeruginosa and MRSA. After a 1-hour incubation period, 1 of 4 treatments (from left to right: NPWT with polyurethane foam [NPWT-PU], polyvinyl alcohol foam [NPWT-PVA], or silver-impregnated polyurethane foam [NPWT-AgPU], or a nonadherent dressing containing polyhexamethylene biguanide without NPWT [control]) was randomly applied to each wound. For the NPWT-treated wounds, the designated foam dressing was cut into a circle 5 cm in diameter and placed in the assigned wound. The skin surrounding the wound was degreased with ether, and an adhesive spray was applied. The foam was covered with an occlusive polyurethane foil, then a 2-cm opening was cut in the occlusive foil and a suction pad was applied over the opening. The suction pads were connected to the canister of the NPWT system by means of Y connectors. The NPWT system was set at 125 mm Hg in continuous mode. For the control wound, the nonadherent dressing was covered with an adhesive fabric to keep it in place. An 8-mm punch biopsy specimen was obtained from each wound immediately before (0 hours) and 6, 12, 18, and 24 hours after treatment application to determine the bacterial load for both P aeruginosa and MRSA.
Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1325
Microbiological analysis
An 8-mm punch biopsy specimen was obtained from each wound immediately before (0 hours; baseline) and 6, 12, 18, and 24 hours after treatment application for determination of bacterial load. Each biopsy procedure required temporary removal and repositioning of the wound dressings. All biopsy specimens were stored at 4°C and processed for standard quantitative bacteriologic analysis in the laboratory within 12 hours after collection. Briefly, each biopsy specimen was aseptically weighed and homogenized in 1 mL of PBS solution by use of a disposable tissue grinder.c Serial dilutions of the homogenized sample were spot plated on Columbia agar supplemented with colistin and nalidixic acid and MacConkey agar for isolation of MRSA and P aeruginosa, respectively. The plates were incubated for 24 hours at 37°C and 5% CO2, after which the number of CFUs per gram of tissue was determined for both plates.
Statistical analysis
For each treatment group, the mean bacterial load at each sampling time after treatment application was compared with that at baseline (immediately before treatment application). Bacterial load data were subjected to a base-10 logarithmic (log10) transformation so that the distributions approximated normality. The transformed data were assessed for normality by use of Q-Q plots and for sphericity by use of the Mauchly test of sphericity. When sphericity could not be assumed, P values were corrected by use of the Greenhouse-Geisser method. A repeated-measures ANOVA was performed with log10 bacterial load as the dependent variable and bacteria (MRSA or P aeruginosa), treatment group (NPWT-AgPU, NPWT-PU, NPWT-PVA, or control), sample acquisition time (0, 6, 12, 18, or 24 hours), and all possible 2-way interactions as fixed effects. If the bacterial load for a biopsy specimen was less than the limit of detection for the quantitative bacteriologic method used, the missing value was replaced with the product of 0.5 × the limit of detection for the bacteria in question as recommended.18 When an interaction was significant, the dataset was stratified on the basis of the categories for one of the variables included in the significant interaction, and repeated-measures ANOVA was performed for the data within each stratum by use of the other variable that contributed to the significant interaction term as the explanatory variable. The Tukey method was used for all multiple comparisons. Values of P ≤ 0.05 were considered significant. All statistical analyses were performed with statistical software.d
Results
The mean bacterial load of MRSA differed significantly (P < 0.001) from the mean bacterial load of P aeruginosa at baseline (immediately before treatment application; 0 hours); therefore, all analyses were performed separately for the 2 pathogens. For each pathogen, the mean bacterial load at baseline did not differ significantly (P = 0.58) among the 4 treatment groups; thus, the mean bacterial load for each subsequent sampling time could be compared with that at baseline without having to convert the data into percentages.
For P aeruginosa, the mean bacterial load for the NPWT-PVA group was significantly lower than that for the other 3 treatment groups at 6, 12, 18, and 24 hours after treatment application (Figure 2). Although the mean bacterial load for the NPWT-AgPU group was numerically lower than that for the NPWT-PU and control groups at all 4 sampling times after treatment application, it was significantly (NPWT-PU group, P = 0.033; control group, P = 0.029) lower only at 12 hours after treatment application. The mean bacterial load for the NPWT-AgPU group did not differ significantly from baseline at any time after treatment application. The mean bacterial load for the NPWT-PU group was significantly higher than that at baseline at 12 (P = 0.002) and 18 (P = 0.008) hours after treatment application. The mean bacterial load for the NPWT-PVA group was numerically lower than that at baseline for the duration of the observation period but was significantly (P = 0.026) lower than baseline only at 18 hours after treatment application. The mean bacterial load for the control group increased steadily for the duration of the observation period and was significantly greater than that at baseline at 18 (P = 0.007) and 24 (P = 0.001) hours after treatment application.
Mean ± SD bacterial load of P aeruginosa (A) and MRSA (B) over time for wounds treated with the NPWT-PU (squares), NPWT-PVA (circles), NPWT-AgPU (triangles), and control (diamonds) treatments described in Figure 1. There were 6 replicates/treatment. *Within a sampling time, value for the NPWT-PVA treatment differs significantly (P ≤ 0.05) from the corresponding values for the other 3 treatments. †Within a sampling time, value for the NPWT-AgPU treatment differs significantly (P ≤ 0.05) from the corresponding values for the NPWT-PU and control treatments.
Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1325
For MRSA, the mean bacterial load for the NPWT-PVA group was significantly (P < 0.001 for all comparisons) lower than that for the other 3 treatment groups at 6, 12, 18, and 24 hours after treatment application (Figure 2). The mean bacterial loads for the NPWT-AgPU, NPWT- PU, and control groups increased steadily for the duration of the observation period but did not differ significantly at any time. The mean bacterial load for the NPWT-AgPU group was significantly greater than that at baseline at 18 (P = 0.002) and 24 (P = 0.003) hours after treatment application. The mean bacterial load for the NPWT-PU group was significantly (P = 0.001 for all comparisons) greater than that at baseline at 12, 18, and 24 hours after treatment application, whereas the mean bacterial load for the control group was significantly greater than baseline at only 12 (P = 0.039) and 24 (P = 0.019) hours after treatment application. The mean bacterial load for the NPWT-PVA group did not differ significantly from baseline at any time during the observation period.
Discussion
Results of the present study indicated that, of the 4 treatments evaluated, NPWT-PVA had the greatest antibacterial effect against both P aeruginosa and MRSA. For wounds treated with NPWT-PVA, the bacterial load of P aeruginosa decreased significantly during the 24-hour observation period, and although the bacterial load of MRSA likewise decreased from baseline over time, the magnitude of that decrease was not significant and remained fairly stable. The antibacterial effect of NPWT-AgPU against P aeruginosa was fairly consistent for the duration of the observation period and was significantly better than that of the NPWT-PU and control treatments at 12 hours after treatment application. However, the antibacterial effect of NPWT-AgPU against MRSA did not differ from that of the NPWT-PU and control treatments, and in fact, the bacterial load of MRSA increased steadily during the observation period for all 3 of those treatments. The NPWT-PU and control treatments had only a limited antibacterial effect against both P aeruginosa and MRSA, and the bacterial loads of both pathogens increased during the observation period for both of those treatments. This was an unexpected finding, but it is unknown what the bacterial loads would have been in untreated wounds.
It was interesting that the antibacterial effect of the NPWT-PVA treatment was greater than that of the NPWT-AgPU treatment because the silver-impregnated polyurethane foam used in the NPWT-AgPU treatment was developed specifically for the treatment of heavily contaminated wounds.19 It is possible that the continuous negative pressure exerted on the wounds assigned to the NPWT-AgPU treatment cleared out the silver ions before they had sufficient time to destroy a substantial number of bacteria. Additionally, the MRSA strain used in the present study might have been resistant to silver, a property that is becoming increasingly prevalent in MRSA strains isolated from both human and veterinary patients.20,21
The manufacturera of the polyvinyl alcohol foam used in the NPWT-PVA treatment informed us that the foam contains low concentrations of formaldehyde, which might have contributed to its antibacterial effects. Formaldehyde is not an intentional component of the foam but is formed during the γ radiation–sterilization process. Formaldehyde interacts with primary amines and amides to form Schiff bases and hydroxymethyl compounds (protein denaturation),22 respectively; it also has a low pH (2.8 to 4.0), which enhances its detrimental effect against bacteria. Formaldehyde is toxic to both bacteria and cells in general. Therefore, the presence of formaldehyde in the polyvinyl alcohol foam could negatively affect the formation of granulation tissue and epithelization of wounds. To our knowledge, no studies have been performed to compare the rate of granulation tissue formation between wounds treated with polyurethane and polyvinyl alcohol foams or to evaluate the effect of polyvinyl alcohol foams on wound healing in a controlled manner. The manufacturer of the polyvinyl alcohol foam also mentioned that when the foam was placed in a solution containing Pseudomonas organisms, the bacterial concentration of the solution decreased while the bacterial concentration of the foam increased. That observation was consistent with the results of another study15 in which the bacterial load of polyvinyl alcohol foams was significantly greater than that of polyurethane foams after application to chronic wounds in human patients.
The fact that the NPWT-PU and control treatments appeared to have only limited ability to slow bacterial growth strengthens the supposition that the antibacterial effect of NPWT is not the result of suctioning bacteria out of the wound but rather is a reflection of the antibacterial effect of the primary dressing used or the possible immune system modulation and increase in wound blood flow induced by negative pressure. Almost all studies11,12,14,17,23 conducted to investigate the effect of NPWT on bacterial clearance of wounds used normal polyurethane foams as the primary dressing with varying results. Results of the present study suggested that polyvinyl alcohol foam was a better choice than normal polyurethane foam as the primary dressing for heavily contaminated wounds, although in vivo studies are necessary to further investigate the effect of polyvinyl alcohol foam on wound healing in a clinical setting.
We chose to use an ex vivo wound model instead of experimentally induced or clinical wounds for the present study because of ethical considerations. Although some might consider that to be a study limitation, the ex vivo model allowed us to evaluate the antibacterial effect of NPWT in conjunction with various foams without interference from the immune system. The perfused wound model we used in this study was based on a model developed for another study17 that involved the use of raw porcine muscle tissue without skin. The musculocutaneous flaps used for the present study were obtained immediately after horses were euthanized, so tissue decay was minimized and the integrity of the subcutaneous connection and patency of the capillaries were preserved. The skin was preserved and the tissues of the musculocutaneous flaps were perfused to mimic a potential flushing effect of wound fluid in an effort to make the model as representative of clinical wounds as possible. The fluid retrieved from the 3 NPWT-treated wounds on each musculocutaneous flap was collected into 1 container, which prohibited us from calculating the exact amount of fluid retrieved from each wound. However, the estimated mean amount of fluid retrieved from each NPWT-treated wound was 15 mL/d.
In the original study11 on NPWT, the authors attributed the enhanced bacterial clearance of wounds treated with NPWT to an increase in wound blood flow, which ameliorates the resistance of compromised tissue to infection and improves oxygenation. They postulated that an increase in local tissue oxygenation diminishes the growth of anaerobes and enhances the oxidative bursts of neutrophils, thus destroying bacteria.11 Investigators of another study17 reported that the bacterial load of wounds was not associated with the mere suction effect of NPWT. In that study,17 the bacterial load did not differ significantly between polyurethane dressing applied to wounds with and without NPWT, and the authors concluded that the apparent effect of NPWT on bacterial clearance of wounds was the result of immune system modulation. Although the ex vivo model used in the present study negated, or eliminated, the role of the immune system on wound bacterial clearance, it did not allow us to differentiate between the antibacterial effects associated with dressing type and those associated with the suction effect of the NPWT system. To do that, it would have been necessary to evaluate the bacterial loads for wounds that were dressed with each of the 3 foams without NPWT as described in a previous study17; however, that would not be clinically relevant because the dressings evaluated in this study are always used in conjunction with NPWT. We chose to use a nonadherent dressing that contained polyhexamethylene biguanide without NPWT as the control treatment for this study because that is the wound treatment most frequently used at our institution, and we considered it more clinically relevant than application of an NPWT dressing to a wound without the actual implementation of NPWT.
Because of practical reasons, the antibacterial effects of each treatment against only 2 bacterial pathogens were evaluated in the present study. Although it might be beneficial to investigate the antibacterial effects of each treatment against other pathogens in the future, the 2 bacterial species (P aeruginosa and MRSA) evaluated in this study are common pathogens of wounds in horses. Those 2 species were purposely selected to be representative of typical gram-positive (MRSA) and gram-negative (P aeruginosa) wound flora. It was interesting that, even though only 2 pathogens were evaluated, clear differences in the antibacterial effect were observed among the 3 NPWT treatments. The reported antibacterial effect of NPWT varies among in vivo studies11–14; however, the discrepancies in the results of those studies can be attributed to differences in study design, methods of sample collection, and types of wounds. For example, the first study11 that described the use of NPWT involved an in vivo animal model in which wounds were experimentally induced and inoculated with 2 gram-positive bacterial species, and wound biopsy specimens were evaluated to determine quantitative bacterial load. In that study,11 the bacterial load for wounds treated with NPWT-PU decreased at a significantly faster rate than did the bacterial load for control wounds that were treated with gauze moistened with saline solution. Conversely, results of a retrospective study13 in which the bacterial load for both chronic and acute wounds treated with NPWT was determined by use of quantitative culture methods on swab specimens obtained before and during or after treatment indicate that NPWT (type of dressing not reported) was associated with an increase in bacterial load. In a prospective randomized trial12 involving human patients with acute or chronic wounds that required open management before surgical closure, quantitative bacterial culture methods were used to determine the bacterial load of wound biopsy specimens, and results were compared between wounds that were treated with NPWT-PU and control wounds treated with saline solution–moistened gauze. Compared with control wounds, the wounds treated with NPWT-PU had a fairly stable overall bacterial load, but there was a decrease in the number of nonfermentative gram-negative rods and an increase in the number of S aureus.12 Results of another similar prospective randomized trial14 in which wound bacterial load was determined by a semiquantitative procedure (bacteria were classified as either present or absent) indicate that bacterial clearance did not differ between control wounds and wounds treated with NPWT-PU. Results of the present study indicated that the overall bacterial load for wounds treated with NPWT-PU (the most common treatment used in the cited in vivo studies11–14) increased over time and were most consistent with the findings of the retrospective study.13 However, comparing the results of an ex vivo wound model with those for in vivo studies is difficult. Although the results of the present study provided initial information regarding the antibacterial effects for 3 different dressings commonly used in conjunction with NPWT, these findings need to be substantiated in an in vivo study in which the effect of each dressing on wound healing can also be evaluated. This is particularly important for the silver-impregnated polyurethane foam and polyvinyl alcohol foam dressings because they have yet to be used in randomized controlled trials.
Findings of the present ex vivo perfused wound model suggested that the capacity of NPWT to enhance bacterial clearance was dependent on the primary dressing used. Wounds treated with NPWT-PVA had the greatest decrease in bacterial load for both P aeruginosa and MRSA. Bacterial loads of P aeruginosa and MRSA over time remained fairly stable and increased, respectively, for wounds treated with NPWT-AgPU. The bacterial loads of P aeruginosa and MRSA increased over time for wounds treated with NPWT-PU and control wounds treated with a nonadherent dressing that contained polyhexamethylene biguanide without NPWT. Although the results of the present study suggested that polyvinyl alcohol foam was better than silver-impregnated polyurethane foam for use with NPWT, those foams need to be evaluated in controlled in vivo trials to objectively determine whether the type of foam is associated with wound healing.
Acknowledgments
This manuscript represents a portion of a dissertation submitted by Dr. Van Hecke to the Department of Surgery and Anaesthesiology of Domestic Animals, Ghent University, as partial fulfillment of the requirements for a Doctor of Philosophy in Veterinary Sciences degree.
Supported by the Special Research Fund, Ghent University (grant No. 01D25512).
The authors declare that there were no conflicts of interest. KCI Medical had no role in the study design; data collection, analysis, and interpretation; or the decision to submit the manuscript for publication.
Presented in abstract form at the 24th Annual Scientific Meeting, European College of Veterinary Surgeons, Berlin, July 2015.
ABBREVIATIONS
| MRSA | Methicillin-resistant Staphylococcus aureus |
| NPWT | Negative-pressure wound therapy |
Footnotes
KCI Medical, Houten, The Netherlands.
Instrulife, Oostkamp, Belgium.
Pestle and microtube combo, VWR international BVBA, Heverlee, Belgium.
SPSS statistics, version 20, IBM Corp, Armonk, NY.
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