Is Antimicrobial Efficacy Sufficient? A Question Concerning the Benefits of New Dressings

Portions of this manuscript were presented at the 2001 Wound Healing Society Educational Symposium in Albuquerque, New Mexico. Disclosures: All authors currently have or had an employment/consultancy agreement with the manufacturer of one of the products. Introduction Chronic ulceration of the skin, regardless of etiology, causes a prolonged breach of one of the primary host defenses against bacterial infection. As a result, wounds are prone to bacterial contamination and subsequent infection.[1,2] A significant number of reports have documented the impact of increased bacterial bioburden on the progression of wound healing. Perhaps the most cited papers are those by Robson, et al., that suggest that only when the bacterial bioburden rises above a certain level (105 CFU/g of tissue) or when specific species are present is there a significant impediment to wound healing.[2–4] Robson’s observations have largely been translated into a parameter by which to determine whether or not a wound requires antimicrobial therapy. A more complete understanding of the negative aspects of wound contamination is now emerging. This understanding focuses on the relationship between the number and virulence of wound bacteria relative to host resistance. This is a shift from the former reliance on a determination of absolute numbers[5]—itself difficult to determine accurately. One of the consequences of this shift has been the design and marketing of many new products, primarily dressings, with claims of being bacterial barriers or otherwise being able to control wound bioburden. The intent behind these dressings is to minimize the colonization of wounds by bacteria and, thereby, enhance wound “healability,” assuming there is at least a partial amelioration of the underlying condition(s) that precipitated the development of the wound. Experimental models and clinical observations support the utilization of various antimicrobial products that have positive impacts on wound healing. These include observations that suggest bacterial contamination may result in more problems than an increased chance of overt infection. For example, a prolonged host inflammatory response against the wound microflora may contribute to the excessively proteolytic environment of chronic wounds, compared to healing wounds, as observed by several authors.[6–9] The elevated proteolytic environment of chronic wounds, connected with the prolonged inflammatory response, has led to an association of these events to the bioburden of the wound. The relationship between elevated bioburden and a more intense inflammatory response has been made both intuitively and in combination with various wound culture observations. The use of antimicrobial products, therefore, seems to be targeted towards a general reduction in the wound bioburden. The assumption that has been made is that a reduction in bioburden will result in a wound environment more conducive to healing. As a result of the increased attention to controlling bacterial populations in wounds, several new antimicrobial dressings have been introduced. The dressings, although utilizing different active ingredients or different presentations of a particular active ingredient, all attempt to deliver a similar outcome, which is protection of the wound from bacterial colonization during healing. As some topical antimicrobials are falling into disfavor due to their negative impact on wound healing,[10–12] it was of interest to determine the impact of the different antimicrobial dressings on wound healing. Two new dressing types, a nanocrystalline silver-coated dressing (SCD) and an absorbent gauze dressing impregnated with polyhexamethylene biguanide (PHMB), were tested in this study. Both of these dressings are intended for use as primary wound dressings in exuding wounds where protection from bacterial contamination is desired. The dressings were tested for their in-vitro efficacy and examined in a porcine model of wound healing for their impact on wound healing. Materials and Methods Dressing materials. The SCD was composed of five alternating layers of absorbent rayon/polyester and nano-crystalline silver-coated high-density polyethylene (HDPE) mesh layers spot welded together (Acticoat® 7, Smith & Nephew, Inc., Largo, Florida). The silver-coated layers of the dressing were present as the two outermost and the middle layers of the dressing. The dressings were sterile and were used as supplied by the manufacturer. The PHMB dressing was composed of absorbent cotton impregnated with 0.2-percent polyhexamethylene biguanide (Kerlix® AMD, Tyco-Kendall Healthcare, Mansfield, Massachusetts). The manufacturer supplied the dressings sterile as a double-layered product. The dressings were used as a double-layered product in the in-vitro tests and as a four-layered (folded in half) product in the in-vivo tests. Control SCDs were made of the same materials in an identical configuration as the SCD except that the HDPE was not silver coated; the control PHMB dressing was nonimpregnated gauze dressing (Kerlix®, Tyco-Kendall Healthcare). These dressings were also sterilized prior to use. Bacteria. The bacterial strains used in the in-vitro tests were clinical isolates obtained from either Calgary (Alberta) Laboratory Services or from University of Toronto (Ontario) Hospital. The bacteria are listed in Table 1, along with the antibiotics to which they have tested resistant. In the majority of cases, the bacteria are resistant to multiple antibiotics. Stock cultures of all strains were maintained at -80 degrees C. Prior to use, the bacterial strains were recovered from the frozen stock by overnight growth in tryptic soy broth (TSB) (Difco, Detroit, Michigan). Following overnight growth in TSB, Gram-negative bacteria were harvested by centrifugation (2000 x g, 20 min.), washed once with physiological saline (Baxter Corporation, Toronto, Ontario), and resuspended in physiological saline to an optical density (625nm) of 0.15 to 0.30. For the in-vivo work, Pseudomonas aeruginosa, coagulase-negative staphylococci, and Fusobacterium sp. were employed. These isolates were obtained from contaminated porcine wounds and maintained at -80 degrees C until grown in preparation for the test. The isolates used were not used beyond five subcultures from the original isolate. In preparation for the in-vivo tests, the bacteria were grown in appropriate liquid media under either aerobic (P. aeruginosa, staphylococci) or anaerobic (Fusobacterium sp.) conditions. Reagents. All reagents, unless otherwise specified, were obtained from Fisher Scientific Company (Nepean, Ontario, Canada). In-vitro inhibition of growth test (corrected zone of inhibition or CZOI). To determine the antibacterial properties of the two dressing materials, a CZOI test was employed. This test was, in essence, the same as a standard zone of inhibition test except the size of the zones were corrected to take into account the differences in shape and size of the hand-cut pieces of test material. In these cases, the dressings were cut into 2.5cm x 2.5cm squares and placed upon lawns of bacterial culture (P. aeruginosa or S. aureus) seeded on Mueller Hinton Agar (MHA, Difco, Chicago, Illinois). The bacterial lawns were incubated overnight and the CZOI was determined. The CZOI was determined by measuring the zone of clearing across one direction of the test article and subtracting the exact width of the test article. This was done in two perpendicular directions across the dressing and the results averaged to yield the final value. Thus, the CZOI reflects only the width of the zone of clearing surrounding the test article and is corrected for variances in the exact size of the individual test articles. Following measurement of the CZOI, the dressing was transferred to a freshly seeded lawn of bacteria and incubated for an additional 24 hours. The CZOI was measured and the process repeated for three days. This test is a modification of the standard Kirby-Bauer test for susceptibility of the test organism to the tested antimicrobial agent. Due to the nature of the test, it also provides a measure of the ability of the antimicrobial agent to diffuse into the surrounding agar medium, producing larger or smaller zones of inhibition dependent upon the release and migration of the antimicrobial agent in the medium. By transferring the dressing to freshly inoculated plates every 24 hours, this method also provided an indication of the duration of the antimicrobial effect, after repeated challenge. In-vitro bactericidal efficacy determination. To determine the ability of the test articles to kill microbes, the test articles and their respective control dressings were prepared in triplicate and placed, individually, on pieces of plastic sheet slightly larger than the test article. The dressings were inoculated with an aliquot of bacterial suspension to yield approximately 3 x 107 colony-forming units per test article. Each dressing piece was covered with an additional piece of plastic sheet and pressed down with a 35g weight to ensure contact of the inoculum with the active components of the dressing. The inoculated dressings were incubated for 30 minutes at 37 degrees C. Following incubation, the dressings were removed from the incubator and carefully immersed in a salt, polysorbate, sodium thioglycollate (SPS) bacterial recovery solution (SPS: 0.85% [w/v] NaCl, 1% [v/v} polysorbate 20, and 0.1% [w/v] sodium thioglycoate). In the case of the staphylococci and enterococci, the NaCl concentration of the SPS was increased to six percent to facilitate recovery of the bacteria from the dressings. The dressings and SPS were vigorously vortexed and serially diluted in phosphate-buffered saline (PBS; pH 7.0) containing 8.5g/L NaCl, 0.61g/L KH2PO4, and 0.96g/L K2HPO4. The number of viable bacteria remaining following exposure to the different dressings was determined by plating the serial dilutions on MHA and enumerating the number of colony forming units after 24 to 48 hours of incubation at 37 degrees C. This method has previously been demonstrated to yield very good recovery of the bacteria from the test articles and their vehicle controls over the period of time examined in the assays.[13] Porcine model of wound healing. The porcine wound healing model that was employed in these studies is described in detail in a recent paper by Wright, et al.6 Briefly, for each dressing type, an experiment was conducted using three female, Yorkshire-cross, farm-raised swine (15–20kg). A total of twenty 20mm-diameter, full-thickness, excisional wounds were created on the dorsum of each pig. The wounds were contaminated with a mixed culture of P. aeruginosa, coagulase-negative staphylococci, and Fusobacterium sp. The wounds on one side of the dorsal mid-line of each animal were covered with moistened PHMB dressings; the other side of each animal was dressed with moistened SCD dressings. The dressings were held in place with tape placed along the dressing edges to help hold the dressing in place. An occlusive drape was placed over the dressings on each animal and secured in place with elastic tape. This dressing scheme prevented dressing movement on the animals and also helped maintain a moist wound environment during the period between dressing changes (at least two times per week). On a regular basis, the wounds were photographed, carefully measured, and samples obtained by biopsy for quantitative microbiology. Wound assessment. On a regular schedule, usually twice per week, the dressing materials were removed and the wounds were measured using digital calipers. The wounds were measured across the greatest and smallest (perpendicular to each other) distances of nonreepithelialized defect, as determined by clinical assessment, and the wound area was calculated based on the formula for calculating the area of an ellipse. The wound area was expressed as a percentage of the original wound area, measured immediately after wound creation. Following measurement and any other requisite wound manipulation (e.g., sampling), the wounds were redressed with the same type of dressing material as was originally used on the wound site. Also, on a weekly basis, the wounds were sampled for quantitative microbiology. This was accomplished by using a 4mm trephine to recover a section of tissue that was placed into a preweighed vial containing PBS. The tissue section was weighed, homogenized, diluted, plated on TSA, and incubated for 48 hours. Following incubation, the number of resultant colonies were determined and expressed as the number of colony-forming units per gram of tissue. The wounds that were sampled at any given time point were predetermined prior to commencement of the experiment. Wounds were regularly sampled for histopathology, as well. A mid-wound biopsy was collected with a sterile 4mm biopsy punch. The tissue was fixed in 10-percent neutral buffered formalin, embedded in methacrylate, and sectioned (2–5mm thick). The sections were stained with Lee’s methylene blue and basic fuschin to show the cellular organization and bacteria.[6] A pathologist, blinded to the treatments, scored the sections based on the presence of fibroblasts, polymorphonuclear leukocytes, and bacteria. Sampled wounds were excluded from area calculations or assessment of epithelialization for one week following wounding. This length of time has been found appropriate, in control (moist wound healing in the absence of any antimicrobial or other agent) experiments, to allow the small defects caused by biopsying to visually appear identical to the surrounding tissue in most wounds. Results In-vitro antimicrobial efficacy. Two different types of antimicrobial tests were conducted. The first test involved the determination of the ability of each antimicrobial dressing to inhibit the growth of bacteria in the area immediately surrounding the dressing. The results, shown in Table 2, indicated that the SCD was able to produce a zone of inhibition around each dressing piece and that the activity was maintained for at least three days. These results are consistent with those obtained with a closely related product.[14] The results also demonstrated that the PHMB dressing was not able to produce a zone of inhibition. This suggests that the antimicrobial activity associated with this dressing product is tightly bound to the dressing and not released into the surrounding medium under the conditions of this study. In addition to the zone of inhibition experiments, experiments were conducted to determine if bacterial suspensions applied directly to the dressing surfaces would be killed (Table 3). The SCD demonstrated an ability to reduce the viable population of all of the tested bacteria to levels below detection. The PHMB dressing demonstrated potent bactericidal efficacy against several strains of bacteria with a trend towards greater efficacy against Gram-negative organisms than against Gram-positive ones. Porcine model experiments. The remainder of the tests involved the performance of the dressings in a porcine model of contaminated wound healing. The healing of the full-thickness wounds was monitored over time, as assessed by measurement of the wound area not completely re-epithelialized. Although neither dressing is occlusive, moist wound healing conditions were achieved by covering all wounds with occlusive pads. This aspect of the dressing protocol ensured that all wounds remained moist between dressing changes. The results of this study (Figure 1) indicate that over the 21-day period of the study, more than 95 percent of the wounds achieved complete reepithelization when they were covered with the SCD product. However, the wounds dressed with the PHMB dressings showed a markedly different response with only approximately 20 percent of the wounds achieving complete reepithelialization over the same period of time. These differences are apparent in the photographs of the wounds over the period of the study (Figures 2–4). At least over the first seven days, the PHMB-dressed wounds demonstrated a propensity to bleed easily. This may be explained, in part, by the adherence of the PHMB dressing to the wound bed. The dressings were always difficult to remove even when soaked with saline in an effort to fully loosen the dressings from the wound bed. As granulation tissue formed under the PHMB dressing, it remained friable over the treatment period. This is contrasted with the wounds dressed with the SCD that appeared to rapidly develop firm, healthy granulation tissue. By Day 21 (Figure 4), a large percentage of the PHMB wounds had considerable areas of open wound whereas the wounds covered by the SCD were largely closed, except in a few instances where very small areas of the wound were not completely reepithelialized. Figure 5 shows representative histopathology of the wounds at Day 4. The PHMB wounds demonstrate evidence of a pronounced inflammatory response with many polymorphonuclear cells evident in each field. The fibroblasts that are present in these sections appeared to be largely necrotic. By contrast, the SCD-treated wounds showed an abundance of healthy fibroblasts with very few polymorphonuclear cells present in each field. By Day 21 (Figure 6), the PHMB wounds continued to demonstrate the presence of inflammatory cells in a highly cellular granulation tissue. The SCD wounds did not demonstrate any evidence of continued inflammatory cell infiltration and the healing wounds also demonstrated a marked reduction in cellularity by this time. This study also attempted to quantitate the ability of the dressings to control the wound bioburden throughout the experimental period. Following inoculation of the wounds, a bacterial load of approximately 104 CFU/g tissue was achieved. After the first 24-hour period of contact with the antimicrobial dressings, the average wound bioburden dropped significantly with both treatments (Figure 7). The bioburden remained below detectable levels with the SCD over the first week and then gradually increased up to Day 21. The wound bioburden in the SCD-dressed wounds remained well below the initial inoculation level and the average values demonstrated a high standard deviation due to occasional wounds demonstrating a bioburden that was high compared to the majority of similarly treated wounds. For the wounds dressed with the PHMB dressings, a similar scenario was observed except that the wound bioburden increased much more rapidly and peaked at close to 106 CFU/g, almost two orders of magnitude higher than the initial inoculum. The smaller standard deviation for these wounds reflects the greater similarity in the extent of colonization in each PHMB-dressed wound. These results suggested that the bacterial cells that did not contact the dressing were not adversely affected by the dressing; the antimicrobial properties of the dressing were tightly associated with the dressing material. Discussion In recent years, there has been considerable interest in the development of new antimicrobial therapies for chronic wounds. This is evidenced by the number of novel antimicrobial agents being marketed for use in wounds. These materials range from various topical ointments to dressings having claims of antimicrobial activity. One of the prime concerns involved in the development of many of these products is the increased spectrum of antibiotic resistance demonstrated by many species of bacteria, including those commonly associated with wounds.[13,15–17] This has resulted in the quest for antimicrobial products that are less likely to promote the development of resistance as well as having the ability to kill resistant organisms. However, with the development of many new antimicrobial products and the abundance of literature about the antimicrobial natures of these products, comparatively little literature documents the antimicrobial activity of the various products and their impact on well-defined wounds under controlled conditions. The absence of these data, combined with a dearth of controlled clinical studies, complicates the selection of an appropriate product. This study sought to determine the relative antimicrobial activity of two antimicrobial dressings and to correlate these findings with the rate of wound healing. The results demonstrated that both PHMB and SCD dressings convey powerful antimicrobial activities when tested against various isolates, including clinical wound isolates, in vitro. This was best demonstrated in the bactericidal assay wherein the test dressings effected significant reductions in the number of viable bacteria recoverable from the test articles after only a 30-minute exposure. The PHMB dressing demonstrated a trend towards being more effective against Gram-negative organisms (although not universally true), but the results against the Gram-positive organisms were also generally favorable. However, the activity of the PHMB dressing appeared dependent on the microbes being in close proximity to the dressings. This is suggested by the results of the zone of inhibition experiments that indicate that there is little apparent diffusion of the antimicrobial activity away from the dressing. The in-vitro antimicrobial effectiveness translated well to the in-vivo situation. When applied to contaminated wounds, both dressings demonstrated a rapid and pronounced bactericidal effect on the wound-associated organisms, reducing recoverable bacteria by more than three logs over the first 24-hour period. It was notable, however, that the SCD dressing was able to provide much greater long-term control over the bacterial population. The SCD dressing kept the bacterial population minimal over the course of the experiment. However, the PHMB dressing, after showing the early and rapid decline in culturable wound organisms, allowed the bacterial population to increase. Interestingly, this was in spite of twice-weekly dressing changes. This combination of observations leads to the suggestion that bacterial cells that were not in close proximity to the PHMB dressing over the first 24 hours and that did not subsequently contact the replacement dressing over the duration of the experiment were able to multiply within the wounds. The data suggested that the PHMB dressing is an effective microbial barrier but that microbes already present within a wound could continue to proliferate unless they came into contact with the dressing. The proliferation of bacteria in wounds covered with the SCD product was markedly less than that observed under the PHMB dressing. The most profound difference between the two antimicrobial dressings is in their impact on wound healing. The PHMB dressings appeared to be associated with prolonged bleeding of the wounds. Even though hemostasis was achieved prior to contaminating or dressing the wounds, the PHMB-dressed wounds continued to exhibit considerable bleeding even seven days post wounding. Bleeding was likely due, at least in part, to the trauma caused by removing the dressing from the wound. The PHMB dressing was very resistant to removal in spite of attempts to soak the dressing off of the wound bed after contact times as short as 24 hours, in a continuously moist environment. The three-week in-vivo experiments demonstrated that wounds dressed with the SCD dressing healed considerably faster than those dressed with the PHMB dressing. This is particularly notable when one considers that over 90 percent of the wounds dressed with SCD are completely reepithelialized by Day 18 and yet only 25 percent of the PHMB-dressed wounds are completely re-epithelialized by Day 21. Previous work with this model has shown that over 60 percent of wounds dressed with moistened gauze dressings are healed by Day 21.[18] Part of the reason for the relative delay in healing of wounds dressed with PHMB as compared to those dressed with SCD may be due to the prolonged inflammatory response apparent in wounds dressed with PHMB-impregnated dressings. The histopathology samples recovered from both wound types showed that inflammatory cells were more prevalent in the PHMB-dressed wounds over the duration of the experiment than in the SCD-dressed wounds. A prolonged inflammatory reaction is recognized as a common contributor to delayed wound healing in other models6 and in clinical medicine.[19] Further, a longer period of time is required to get a healthy population of fibroblasts present in the wound bed after exposure to PHMB than after SCD. Again, this observation may be related, in part, to the localized trauma caused by the removal of the adherent PHMB dressing. This study serves to demonstrate that although some dressings may be distinguished based on their antimicrobial properties, the impact of the dressing itself on wound healing must also be assessed. This study concludes that both the PHMB and SCD dressings are excellent topical antimicrobials. The PHMB dressings appear to require much more intimate association with the bacteria in order to manifest their microbicidal activity; SCD dressings have the ability to mediate antimicrobial effects over some distance from the dressing. However, the most important difference between the two dressings lies in the ability of the dressings to promote wound healing. The PHMB material appears to enhance or prolong the inflammatory response of the wound. This may lead to the greater propensity for bleeding during the bandage changes as well as the overall decrease in the healing of the wounds. These observations may both be related to the adherence of the dressing fabric to the wound, indicating that direct wound contact with this gauze dressing is not its optimal use. This situation is analogous to that previously described for topical antiseptics. Many topical antiseptics have demonstrated efficacy against a broad spectrum of bacteria but have also been demonstrated to retard healing by virtue of their cytotoxicity.[5,20] Conversely, agents do exist that have been demonstrated to be effective against wound microbes without demonstrating toxicity towards mammalian cells, in vivo. A body of literature is now being developed for silver, in particular suggesting that it might play a role in promoting wound healing in addition to being antimicrobial.[6,21,22] In summary, the results of this study indicate that as much care needs to be taken in evaluating antimicrobial dressing products for their impact on wound healing as has been focused on topical antiseptics. Some dressings that may have good antimicrobial activity may not be well suited for direct wound contact. Although protecting open wounds from contamination by environmental bacteria is certainly desirable, it normally should not to be sought at the expense of wound healing.