wraps and devices to better assist in edema control, venous return, and wound healing. Support surface technology continues

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1 Understanding the Presence of Biofilms in Wound Healing: Opportunities for Intervention During the past several decades, major advances have been made in the practice of wound care. Research has generated insights that have lead to the realization that chronic wounds have molecular, microbiological, and clinical characteristics that distinguish them from acute, healing wounds. As a result, practitioners have actively looked at coordinated cellular and biochemical events that take place in wound healing, while manufacturers of wound care products have partnered with practitioners to identify materials that assist in the management of simple and complex wounds. At the same time, standards for describing and documenting skin and wounds are being disseminated in many forms to assist the practitioner in documenting what is assessed. When reflecting on the advances that have been made over the years, one can see the progress made to accelerate wound healing. Consider: Today, dressings are available to manage wounds with various exudate amounts, to support autolytic debridement, to promote granulation tissue, and, ultimately, to promote wound closure. Clinicians know that select dressing types, such as occlusive dressings, assist in pain management one additional assessment parameter in wound care. Growth factors and bioengineered skin products are now available to jump-start and stimulate the wound-healing process in hard-toheal or stalled wounds. Some of the bioengineered constructs are available as dermal replacements as well as epidermal/dermal replacements. Devices are now available to manage the wound through the removal of wound fluid. Compression therapy has advanced. Although still of value, the Unna Boot has now been joined by multilayered, sustained, graduated compression wraps and devices to better assist in edema control, venous return, and wound healing. Support surface technology continues to promote optimal environments for pressure redistribution. Documentation systems for wound care continue to be refined and available to the practitioner in specialty electronic medical records. Novel dressings aimed at biochemical alterations have been developed, including those containing slow and steady state release of iodine and silver in an effort to decrease the bioburden in the wound while minimizing the cytotoxicity seen in commonly used antiseptics. Development of multidisciplinary wound centers that serve as a resource for patients with nonhealing wounds. However, on a cellular and molecular level, wounds possess features that oppose healing. Infection, prolonged inflammation and hypoxia, unavailability or decreased amount of growth factors and progenitor cells, excess fibrin, Cathy Thomas Hess, BSN, RN, CWOCN; and Robert S. Kirsner, MD, PhD and senescent fibroblasts all may lead to the chronicity of a wound despite the interventions employed in the woundhealing process. More recently, knowledge from other fields such as dentistry has led to the concept that biofilms also may inhibit healing. We have learned that bacteria maintain a complex role in the microenvironment of a chronic wound. In this article, we focus on biofilms and their role in the chronic woundhealing process as well as opportunities for interventions. WoundHealingandHemostasis Although alteration in skin integrity causes a healing process to commence, we most often consider the development of a wound as when healing begins. This processisgenerallywell-orchestrated,leading to repair of the injury. However, chronic wounds do not follow this well-orchestrated plan. Because of an impediment to the healing process, the wound is often thought to be stuck in the inflammatory or proliferative phase. Over time, key cells become senescent. Understanding and correcting the barriers to healing will spark the formation of granulation tissue, leading to the next stage of healing. 1 Hemostasis occurs soon after initial injury. The key cell responsible for this function is the platelet, which causes the body to form a clot to prevent further bleeding. In addition, platelets also release key cytokines, such as platelet-derived growth fac- 12 April 2012 Today s Wound Clinic

2 tor, that attract cells to participate in later phases of healing. Following hemostasis, the inflammatory phase begins. InflammaTORy PHASE The inflammatory (defensive or reaction) phase is characterized by a variety of host cells infiltrating the wound sites. Many of these are inflammatory cells, such as leukocytes and macrophages. The inflammatory phase begins immediately following injury and typically lasts 4 to 6 days. After bleeding is controlled by hemostasis, the inflammatory phase ensues and any bacteria present are destroyed by leukocytes, particularly the polymorphonuclear neutrophils (PMNs). About 4 days after the injury, macrophages (tissue cells derived from circulating monocytes that migrate to the area) also work to destroy bacteria, cleansing the wound of cellular debris. Macrophages replace the leukocytes (which phagocytize bacteria in the wound, stimulate the inflammatory response, and trigger other biochemical actions) and produce a host of cytokines and growth factors that act as chemoattractants to other cells needed for tissue repair. Macrophages also convert macromolecules into the amino acids and sugars necessary for wound healing. Proliferative Phase The proliferative (fibroblastic, regeneration, or connective-tissue) phase typically lasts several weeks. In an open wound, granulation tissue is generated, which can be seen clinically as the production of red, beefy buds (or granules) of tissue. Granulation tissue consists of macrophages, fibroblasts, immature collagen, blood vessels, and ground substance. As this type of tissueproliferates,fibroblastsstimulatethe production of collagen, which gives the tissue its tensile strength and, ultimately, its structure. As the wound site fills with granulation tissue, its margins contract, or pull together, decreasing the wound s surface. During epithelialization, the final step of this phase, keratinocytes migrate from the wound margins. Subsequently, they divide and, ultimately, become contiguous. Metalloproteinases (MMPs) such as collagenase-1 are critical in epidermal migration, while other MMPs, such as MMP 8 and 9, are important in the normal healing process. The proteins are regulated by a set of inhibitors. Epithelialization can occur only in the presence of viable vascular tissue. The inflammatory process in the dermis leads to scar formation. Maturation Phase During the maturation (remodeling) phase, which can last from 21 days to several months or even a year, collagen fibers reorganize, remodel, and mature, gaining tensile strength. Fibroblasts, MMPs, and their inhibitors play a crucial role in this process, as do certain growth factors such as transforming growth factor beta. This process continues until the scar tissue has regained about 80% of the skin s original strength. This tissue will always be at risk for breakdown because its tensile strength is less than that of uninjured skin. Wound repair occurs by primary intention, secondary intention, or tertiary intention. Many acute wounds, such as surgical wounds, are closed by primary intention, meaning that the skin edges are approximated (brought close together). Such wounds have a lower risk of infection, involve minimal tissue loss, and heal with light scarring after 4 to 14 days. Chronic wounds, such as pressure ulcers, heal by secondary intention; the skin edges are not approximated. Due to the delay in healing, chronic wounds are at a greater risk of becoming infected. 2 The burden of chronic wounds is high. Associated morbidity, decreased quality of life, loss of limbs, and, in some cases, mortality are among the many reasons significant interest has been focused on the prevention and treatment of the most common types of these wounds. 3 Despite best clinical coordination of efforts, wounds stall in the healing process and become chronic wounds. It has been hypothesized that chronic wounds occur due to a variety of reasons including, but not limited to, decreased low cell mitosis, high inflammatory cytokines, high proteases (MMPs), decreased fibroblast production, and an abnormal production of growth factors, and bacteria. 4-6 BaCTerialBalanceandBiofilms Wounds contain a variety of organisms. The notion of bacterial balance stresses the need for the clinician to recognize when the bacterial load has increased through a change in granulation tissue appearance and exudate amount. In a healing chronic wound, the level of bacteriapresentisreferredtoascontamination or colonization. This is a steady state of replicatingorganismsthatmaintainapresence in the wound but don t cause injury or delay the wound-healing process. The next level of bacterial load is critical colonization. This level is characterized as replicating (infectious) organisms that cause a change in wound status. The clinician may observe understated clinical features in the wound s appearance, including: foul or excessive odor; absent or abnormal granulation tissue; change in color of the wound bed from previous evaluations; delayed healing; friable granulation tissue; severe or increased pain at the wound site; excessive or increased serous exudate; serous exudate with concurrent redness of surrounding periwound wound edges; and/or tunneling or pocketing of the wound. A wound infection can be characterized as an invasion of organisms into the wound and surrounding soft tissue that results in a host response and leads to nonhealing or worsening of the wound. Classic clinical signs and symptoms include: periwound and soft-tissue edema; periwound and soft-tissue erythema; fever; foul odor; severe or increasing pain at the wound site; tenderness at the wound and periwoundsiteandsurroundingsofttissue excessive or purulent drainage; warmth of the surrounding soft tissue and periwound skin; and/or evaluated white blood cell count Today s Wound Clinic April

3 Figure 1. Scanning electron micrograph of Staphylococcus aureus biofilm in a partialthickness wound after 72 hours. Used with permission from Stephen C. Davis. with an increase in newly developed cells (bands). Knowing that all wounds are colonized with microorganisms, it has been questioned whether the bacterial colonization is a causative factor in the failure of the chronic wounds to heal and whether the biofilm state contributes to this pathology. 7 Bacteria live in several forms, including planktonic (free-floating) and biofilms. Focusing on biofilms, Bjarnsholt et al, 6 defined the development of a biofilm in a chronic infected wound as: Small numbers of surviving planktonic bacteria develop over time into microcolonies, with the formation of larger aggregations referred to as biofilms. The biofilm bacteria are encased in a self-produced polymeric matrix. The ability to form biofilms is believed to be one of the main survival strategies of bacteria in a hostile environment. In this state, the bacteria tolerate antimicrobial compounds such as antibiotics and the action of the host cells. Biofilm formation also facilitates the buildup of bacterial cell-to-cell signaling molecules used in a process termed quorum sensing (QS). When a certain cell density is reached, the QS system dictates the production of virulence factors, some of which offer a shield against the attended PMNs. According to the National Institutes of Health (NIH), biofilm is defined as an accumulation of bacterial, fungi, or protozoa on solid surfaces (such as tissue cells, teeth, and artificial implants). Biofilms are medically important because we are learning that fewer diseases are caused by planktonic microbes, ie, nonadherent and free-floating, than previously thought. 8 NIH further documents that more than 80% of microbial infections in the body are caused by bacteria growing as a biofilm. 8 Examples of biofilms include the layer of Pseudomonas aeruginosa that forms in the trachea of cystic fibrosis patients, the Escherichiacolibacterialbiofilmthatforms in urinary tract and intestinal infections, Staphylococcusspinfectionsfrombiofilms on indwelling catheters, and eye infections from biofilms that form on contact lenses. In the oral cavity, microbial biofilms (commonly referred to as dental plaque) are involved in the pathogenesis of caries, periodontitis, dental implant failures, denture stomatitis, and oral yeast infections (such as candidiasis) 8 Biofilms have also been noted in chronic wounds 8 (see Figure 1). IDENTIFYING BIOFILMS It is well documented that bacteria can exist in multiple forms such as biofilms, different phenotypes including freefloating/planktonic organisms, dispersed bacteria, or microcolonies. 3,4 Armed with the knowledge that wounds may stall because of biofilms, how can the healthcare professional truly know if a biofilm exists? This is a true enigma, as routinely used techniques are not available for clinicians to detect biofilms, and no clinical characteristics of a wound have been established to date to distinguish wounds with biofilms from wounds without biofilms. Experimentally, scientists take advantage of the two major features of a biofilm to help establish the presence of a biofilm. The first is the presence of an extrapolysachharide matrix (EPS) that biofilms form. Using this information, special staining techniques and electron microscopy has been utilized to detect biofilms. The second is that biofilms are tightly adherent to underlying tissue. Therefore, scientists can flush away free-floating or planktonic bacteria while they need to scrape away the tightly adherent biofilm bacteria. 9 Understanding that biofilms disrupt the wound healing process, Davis et al 4 provide an excellent review of ways to accomplish biofilm disruption (see Table 1). BiofilmInterventionPaTHWay As clinicians, we like direction and process. We like algorithmic thinking that leads to interventions, such as wound-healing pathways. We are comfortable with knowing that if the characteristics of the wound change, we can review the wound-healing pathway for alternate care options. Typically, a wound-healing pathway is designed for a specific diagnosis, documentation, intervention processes, and expected outcome. Remember: Assessment, both initial and ongoing, describes the overall condition of the patient, including wound status. Documentation, whether handwritten, electronic, and/or photographic, becomes the foundation for management decisions, evaluation of the wound-healing process, and reimbursement decisions. It also serves as a defense in litigation. 14 April 2012 Today s Wound Clinic

4 TABLE 1. 5 Potential Methods to Prevent, Reduce, or Treat Biofilms 1. Preventing the Bacterial Attachment The study of preventing bacterial attachment has been around for a long time, especially in the dentistry field. The attachment and colonization of subgingival sites by bacteria is a critical step in the initiation of periodontal diseases and various compounds have been shown to prevent attachment of bacteria. 10 In the orthopedic surgery area, treating implanted devices with certain compounds to reduce bacterial adhesion has been successfully examined. 11 It is plausible to imagine topical agents being applied to the diabetic ulcer after wound bed preparation to reduce bacterial colonization. However, the effect of these agents on the wound healing process would also need to be examined. 2. Preventing Biofilm Formation We have recently studied the effect of lactoferrin, a glycoprotein that belongs to the iron transporter family. This glycoprotein is found in milk and exocrine secretions, released by neutrophils during degranulation, and has been shown to have antimicrobial and anti-inflammatory properties. 12 It has recently been investigated as a possible treatment for biofilm formation. Interestingly, although early studies have demonstrated its use in preventing Pseudomonas aeruginosa, S. aureus, and Epidermidis biofilm formation, its effect on already formed biofilms is less impressive. 13 The proposed mechanism by which lactoferrin inhibits biofilm formation is by chelating iron, which in turn causes a low iron state that triggers bacteria to twitch, which inhibits bacterial adhesion. 14 We examined the effect of lactoferrin, apolactoferrin (which is the iron-depleted form of lactoferrin), and lysozyme (an enzyme that can destroy bacterial cell walls and can be found in the mucosal membranes) on S. aureus biofilm mass. The lactoferrin we used is a recombinant human supplied by Ventria Biosciences (Sacramento, CA). 15 Using optical density as a measure of biomass, we found both recombinant human lactoferrin and apolactoferrin reduced biomass material in vitro by 45.5% and 49.1%, respectively. Lysozyme-treated bacteria showed lower biofilm formation than the control; however, this difference was not significant. Both lactoferrin and apolactoferrin showed a small but significant decrease in cell counts as well. This indicates that these substances, in addition to bactericidal effects, appear to have an inhibitory effect on biofilm production of S. aureus. However, it is unclear if the reduction in biofilm mass was due to the decrease in cell counts or a direct effect on the biofilm. One conclusion might be that lactoferrin would be effective for the treatment of chronic wounds, specifically diabetic foot ulcers; controlled clinical trials are currently ongoing. 3. Disrupting the Biofilm to Allow Penetration of Topical Antimicrobial Agents The idea of disrupting a biofilm that is already formed is attractive. This could be accomplished in a number of ways, including physical methods and/or application of topical substances. Potential physical methods include debridement, electrical stimulation, and ultrasound. Debridement may not only remove the bacteria and biofilm, but it also may aid in the removal of necrotic tissue on which the bacteria thrive. Electrical stimulation has been used over the years to assist penetration of various topical agents (eg, electroporation and electrophoresis have been shown to enhance the penetration of a photosensitizer). 16 It is conceivable that electrical currents could aid in the delivery of topical antimicrobial agents through the physical barrier that is a biofilm; however, well-controlled studies are needed. The use of enzymatic agents is another way in which biofilms could be penetrated and disrupted. In a recent nonpublished study, we evaluated the effect of an enzyme cocktail on S. aureus biofilms in acute partial-thickness porcine wounds (Welsh et al, unpublished data). The enzyme cocktail was tested by itself and in combination with a topical antibiotic (mupirocin 2% cream). The enzyme, when used in combination with the mupirocin 2% cream, yielded increased reduction of the bacteria compared with the mupirocin 2% cream alone. This suggests that enzymatic therapies could help facilitate the antimicrobial action on the bacteria within the biofilm. 4. Interfering With Quorum Sensing Interfering with bacterial communication to one another (quorum sensing) may interfere biofilm development. For example, part of the way P. aeruginosa imparts disease is through many virulence factors using various quorum sensing systems, including the Pseudomonas quinolone signal (PQS). Studying PQS s ability to alter gene regulation and virulence in P. aeruginosa, could lead to promising target agents for therapeutics aimed at decreasing the pathogenesis of this pathogen. 17 Finally, in addition to interfering with quorum sensing, factors that control bacteria dispersion are now being closely studied. Cells that become dispersed are, therefore, not living in a biofilm state are easier to eradicate. A recent study has shown that dispersal of P. aeruginosa (PAO1) from biofilms is induced by a sudden increase in carbon substrate availability. 5. Enhancing Dispersion of Bacteria From Biofilms Additional knowledge of signal transduction in biofilm dispersion by screening for differential gene expression and phosphorylated proteins associated with dispersion may offer new targets for the treatment of biofilm infections. This could ultimately guide us to novel approaches for biofilm control that could be used clinically. 18 Interventions, guided by the multidisciplinary wound care team, include topical treatments, use of support surfaces, adjunctive therapies and products, and nutritional supplements. Expectedoutcomedescribestheoverall condition of the patient that should result from all the processes performed on or for that patient. Although the wound care community has written, researched, and reviewed the etiology of a chronic wound, we have a lot more work to do to understand how biofilms affect chronic wounds and what interventions are appropriate to eradicate them from the wound. To that end, the authors share an idealized Biofilm Intervention Pathway for your consideration. This pathway has been created as a clinical tool to assist the clinician in understanding the ideal steps necessary to remove and prevent biofilms from occurring (see Figure 2). This idealized algorithm shares the following fundamental thoughts: Clinical characteristics of wounds 16 April 2012 Today s Wound Clinic

5 infection 2. Hess CT, Kirsner RS. Orchestrating wound healing: assessing and preparing the wound bed. Advances in skin and wound care. Volume 15, Number 5. September/October 2003: Wolcott R, Dowd S. The role of biofilms: are we hitting the right target? Plast Reconstr Surg Jan;127 Suppl 1:28S-35S. 4. Davis SC, Martinez L, Kirsner R. The diabetic foot: theimportanceofbiofilmsandwoundbedpreparation. Curr Diab Rep Dec;6(6): James G, Swogger E, Wolcott R, Secor P, Sestrich J, Costerton J, Stewart P. Biofilms on chronic wounds. Wound Repair Regen 2008; 1: Bjarnsholt T, Kirketerp-Moller K, Jensen P, Kit M, Krogfelt K, Phipps R, Holby N, Glyskov M. Why chronic wounds won t heal: a novel hypothesis. Wound Repair Regen 2008; 1: Singh, V, Barbul, A. Bacterial biofilms in wounds. Wound Rep Regen 2008: 1: Minutes of the National Advisory Dental and Craniofacial Research Council - 153rd Meeting Report. National Institutes of Health. 9. Davis S, Ricotti C, Cazzaniga A, Welsh E, Eaglstein W, Mertz P. Microscopic and physiologic evidence for biofilm-associated wound colonization in-vivo. Wound Repair Regen 2008; 1: Labrecque J, Bodet C, Chandad F, Grenier D. Effects of a high-molecular-weight cranberry fraction on growth, biofilm formation and adherence ofporphyromonasgingivalis.jantimicrobchemother 2006, 58: Kustos I, Kustos T, Kilar F, et al. Effect of antibiotic treatment on bacterial attachment to a DePuy enduronorthopedicimplant.chemotherapy2005, 51: Valenti P, Berlutti F, Conte M, et al. Lactoferrin functions: current status and perspectives. J Clin Gastroenterol 2004, 38(suppl 2):s127 s Weinberg E: Suppression of biofilm formation by ironlimitation.medhypotheses2004,63: Singh P, Parsek M, Greenberg P, Welsh M.A component of innate immunity prevents bacterial biofilm formation. Nature 2002, 417: Nandi S, Yalda D, Lu S, et al. Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain. Transgenic Res 2005, 14: Johnson PG, Oseroff AR. Electrically enhanced percutaneousdeliveryofdelta-aminolevulinicacid using electric pulses and dc potential. Photochem Photobiol 2002, 75: Wade DS, Calfee MW, Rocha ER, et al. Regulation of pseudomonas quinolone signal synthesis in pseudomonas aeruginosa. J Bacteriol 2005, 187: Sauer K, Cullen MC, Rickard AH, et al. Characterization of nutrient-induced dispersion in pseudomonasaeruginosapao1biofilm.jbacteriol 2004, 186: D U PL IC A TE Figure 2. Biofilm Intervention Pathway: An Indealized Algorithm Wound Photo Analysis generated from TPSTM EMR AutoCalculate TM ly does not provide a clear method to prevent biofilm reformation. The burden of chronic wounds is high. Associated morbidity, decreased quality of life, and, in some cases, mortality are among the many reasons substantial interest has been focused on the prevention and treatment of the most common types of these wounds. We know that impediments to the wound healing process exist. Biofilms are one of the impediments that lead to a stalled wound. What we are still pondering is how to detect and eradicate them.n D O N O T that have biofilm have not been validated, and currently routine testing to confirm the presence of a biofilm does not currently exist. It is envisioned that diagnostic testing via tissue or fluid analysis will be available in the future. Removal of biofilm by surgery is theoretically possible, but the depth needed for debridement is not currently known. Other techniques including nonsurgical debridement, and use of energy sources. Physical or enzymatic means to eradicate biofilms may also prove to be useful in the future. Without adequate prevention reformation of biofilm is likely to occur after removal. Opportunities for biofilm preventionexist,butevidencecurrent- 18 References 1. Hess CT. Clinical guide to skin and wound care (Seventh edition in press). Lippincott Williams & Wilkins, Philadelphia, PA April 2012 Today s Wound Clinic Additional Resources Mertz, P. Cutaneous biofilms: friend or foe? Wounds. 2003; 15(5): Cooper R. Biofilms and wounds: much ado about nothing? Wounds UK. 2010, 6.4. Percival S. Assessing the effect of an antimicrobial hydrofiber wound dressing on biofilms. Wound Repair Regen. 2008, 1: Cathy Thomas Hess, BSN, RN, CWOCN, is President and Director, Clinical Operations of Well Care Strategies.Pleaseaddresscorrespondencetocathy@ wcscare.com.