WOUND HEALING Update on the Role of Infection and Biofilms in Wound Healing: Pathophysiology and Treatment Michelle Gompelman, MD, MSc Suzanne A. V. van Asten, MD, MSc Edgar J. G. Peters, MD, PhD Amsterdam, The Netherlands; and Dallas, Texas

Background: Chronic wounds, and among these infected diabetic foot ulcers, are a worldwide problem. The poor treatment outcomes result in high healthcare costs, amputations, a decreased quality of life, and an increased mortality. These outcomes are influenced by several factors, including biofilm formation. A biofilm consists of pathogenic bacteria that are encased in an exopolysaccharide layer and communicate through secretion of signaling molecules. Bacteria that live in a biofilm are refractory to host responses and treatment. Methods: We performed a nonsystematic review of the currently published to-date medical biofilm literature. The review summarizes the evidence of biofilm in chronic wounds, the role of biofilm in wound healing, detection of biofilm, and available antibiofilm treatments. Articles containing basic science and clinical research, as well as systematic reviews, are described and evaluated. The articles have variable levels of evidence. All articles have been peer reviewed and meet the standards of evidence-based medicine. Results: Both animal and human studies have identified biofilm in chronic wounds and have suggested that healing might be influenced by its presence. A promising development in biofilm detection is rapid molecular diagnostics combined with direct microscopy. This technique, rather than classic culture, might support individualized treatment in the near future. A wide range of treatments for chronic wounds also influence biofilm formation. Several agents that specifically target biofilm are currently being researched. Conclusions: Biofilm formation has a substantial role in chronic wounds. Several diagnostic and therapeutic methods against biofilm are currently being developed.  (Plast. Reconstr. Surg. 138: 61S, 2016.)

T

he biofilm phenotype of bacteria has not been satisfactorily studied in chronic wounds.1 However, it has been hypothesized that the characteristics of biofilm-related diseases are expressed in chronic wound infections as well.2 Chronic wounds typically develop slowly, are rarely resolved by the immune response of the host, and do not always respond to antimicrobial therapy.3 Most chronic wounds can be classified into 3 major types: pressure ulcers, venous ulcers, and diabetic (foot) ulcers. Infected diabetic foot ulcers [diabetic foot infections (DFIs)] are common and are often complicated lesions to heal.4 The role of tissue hypoxia, repetitive ischemia, aging, and From the Department of Internal Medicine, VU University Medical Center; and Department of Plastic Surgery, University of Texas Southwestern Medical Center . Received for publication February 15, 2016; accepted May 20, 2016. Copyright © 2016 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000002679

bacterial colonization in chronic wounds5 has been well documented, but the importance of biofilms herein has only recently been given attention. The poor treatment outcomes of DFIs might be influenced by the virulence factors expressed by the variety of microorganisms in the ulcers, including this biofilm formation. Because diabetic ulcers are colonized by a wide variety of bacterial species,6 synergies that occur in these polymicrobial communities7 need to be considered when selecting an antimicrobial therapy. Only a few studies have reported on biofilm formation and evaluated its presence in chronic wounds. In this review, we aim to summarize the evidence of biofilm in chronic wounds (emphasizing on diabetic foot ulcers), the role of biofilm in wound healing, detection of biofilm, and currently available antibiofilm treatment options. Disclosure: The authors have no financial interest in any of the products, devices, or drugs mentioned in this article.

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61S

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Plastic and Reconstructive Surgery • September Supplement 2016 PATHOPHYSIOLOGY A microbial biofilm is a structured consortium of microbial cells surrounded by a self-produced polymer matrix.8 Biofilms typically consist of 3 components: the biofilm-growing microorganisms, the planktonically (nonaggregated) growing organisms, and the integrated host component, such as fibrin, platelets, or immunoglobulins.9 Biofilm formation may prolong the healing time of chronic wounds and affect clinical outcomes in a negative way. First, biofilm has the capability to seize and concentrate a number of environmental nutrients in the extracellular matrix, such as carbon, nitrogen, and phosphate.10 Second, biofilm facilitates the resistance to antimicrobial factors by mediation of low metabolic levels of entrenched organisms, down-regulation of cell division, and the capability to act as a diffusion barrier.11 Finally, there is a potential for dispersion through detachment, allowing an enduring bacterial source population.12 Figure  1 illustrates the biofilm life cycle in 3 sequential steps. Biofilm-growing organisms may also be difficult to extract from clinical samples with conventional culture techniques and are physiologically more resistant to the effects of disinfectants.13 When harbored within a biofilm, pathogenic bacteria are protected against host defenses and antimicrobials. The stability of this environment allows the bacteria to grow and contribute to recalcitrant infections in tissues (skin, sinuses, gallstones, urinary tract, or respiratory tract)14,15 or on temporary or permanent biomedical devices (endotracheal tubes, intravascular and

urinary catheters, orthopedic implants, and arterial stents or prosthetic grafts).16,17 Within days of adherence to a substrate surface, multiple bacterial layers have formed, microcolony aggregation has occurred, and an extracellular matrix of exopolysaccharide (EPS) has formed an antimicrobial glycocalyx or “slime” layer.18 Bacteria use cell to cell communication through molecular signals to detect that the colony is large and dense enough to produce such a glycocalyx. This process is called quorum sensing (QS).19 Many proteins and virulence factors are produced by bacteria in response to QS, including the genes responsible for biofilm formation. A large part of the interconnected microbial population exists in a down-regulated state because of the low nutrient and oxygen gradient within the biofilm. Host responses with granulocytes and immunoglobulins and antimicrobial therapy seem to have no effect because they fail to penetrate the biofilm.20 The release of waste products, oxygen radicals, antigens, enzymes, and toxins into the surrounding tissues creates a chronic, low-grade immune response.18,21 Biofilm in Wound Healing When a bacterial biofilm is established within a wound, the healing process is inhibited by the physical barrier that biofilm creates for reepithelization22 and by the opsonization of bacteria.23 Also, the constant release of waste products induces a chronic inflammatory response in the surrounding tissue that interferes with wound healing.24 The ability of the host, to control the growth of

Fig. 1. The biofilm life cycle in 3 steps: attachment, growth of colonies, and detachment. Reprinted with permission from © 2003 Center for Biofilm Engineering at MSU-Bozeman, Bozeman, Mont.

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Volume 138, Number 3S • Infection and Biofilms in Wounds the biofilm aggregates in the wound, decreases as the biofilm community matures. One of the first studies presenting clear evidence of the presence of biofilm in wounds was published in 1996.25 In this murine model, serial tissue biopsies from Staphylococcus aureus–infected cut wounds were taken at different time points. Membrane-like structures surrounding clusters of bacterial colonies were found after several hours of inoculation. Furthermore, inflammatory cells were seen around, but outside, the membranelike structures. The tissues surrounding the structures were degenerated and nearly necrotic. The authors concluded that S. aureus had formed a biofilm in the dermal and subcutaneous tissues in the wounds of the mice. After this report, a lot of experimental mice, rat, and rabbit models studying the effects of biofilm in wound healing appeared.22,26–29 In 2010, Zhao et al30 reproduced a chronic wound model by applying Pseudomonas aeruginosa biofilm on punch biopsy wounds in diabetic mice 2 days after wounding. None of the biofilmchallenged wounds closed, whereas most of the control wounds were epithelialized 28 days after wounding. Extensive tissue necrosis and epidermal hyperplasia were seen adjacent to the biofilm-challenged wounds with histological analysis, indicating a delayed wound healing. The impaired healing was confirmed in a biofilm with P. aeruginosa in a diabetic mouse model a year later.31 The author also reported an increased density of biofilm when the mice were treated with insulin. These results suggested an additional effect of insulin treatment on the development of P. aeruginosa biofilm in diabetic wounds. More recently, mixed species biofilms have been investigated,28,32 demonstrating the synergistic effects of the presence of several bacterial species. This results in delayed wound healing and increased antimicrobial resistance. In 2008, the first clinical studies of biofilm in human wounds were published.1,2,33 Ulcers infected with P. aeruginosa have significantly more accumulation of active neutrophils, suggesting a persistent inflammatory response and indirect impaired wound healing.34 In a recent study, biofilm formation on 95 diabetic foot ulcer isolates was studied at 3 different time points (24, 48, and 72  hours), using a microtiter plate assay and a multiplex fluorescent in situ hybridization.35 All isolates were biofilm positive at 24 hours, and the biofilm production increased with incubation time and in the presence of Pseudomonas. Even more striking was that the biofilm formation

increased when certain synergies between bacteria occurred. The combination of Pseudomonas and Enterococcus and Acinetobacter and Staphylococcus produced denser biofilms compared with combinations of other species.

DIAGNOSIS Detection of Biofilm For decades, microscopic examination was the only method for biofilm detection. A wide range of these visualization techniques, including confocal scanning laser microscopy, transmission electron microscopy, scanning electron microscopy, and fluorescence microscopy, have been developed. The process of S. aureus biofilm formation on the surface of a platinum electrode is illustrated by the scanning electron micrographs in Figures 2 and 3. More recently, molecular diagnostics based on nucleic acid sequence analysis have been introduced. These DNA-based pathogen diagnostics revealed new information on how bacteria are organized in biofilms.36,37 However, these techniques are time consuming and expensive. Direct microscopy is the observation of potentially infecting organisms in an unstained (wet mount) preparation by light or electron microscopy. With direct microscopy, the clinician can visualize if and where a biofilm is present, shortly after sample collection and well before the results of conventional cultures become available. This could offer a faster, more appropriate and effective selection of therapeutic options.8,38,39 Because of this, interest in developing more rapid and effective DNA-based molecular techniques in combination with direct microscopy is growing.40,41 Although promising for future applications, more in vitro and in vivo studies are needed to reliably prove the representation of biofilm by molecular techniques. Biofilm Susceptibility Testing and Use of New Molecular Techniques Because all wounds are colonized with bacteria, differentiating colonizers from the real pathogens is a challenge. Identifying bacteria with traditional culture methods is still the reference standard. However, these culture results do not report the full bacterial diversity in the biofilm as the previously mentioned, newer genotypic (molecular) techniques.42 These techniques showed that only 1% of bacteria are cultured using standard methods, especially the identification of anaerobes is

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Plastic and Reconstructive Surgery • September Supplement 2016

Fig. 2. High-resolution scanning electron micrograph of a Staphylococcus aureus biofilm forming on the surface of a platinum electrode. In this area, the extracellular matrix has just begun to form, and the individual bacteria are easily discerned. Courtesy of K. Pawlowski, Assistant Professor, University of Texas Southwestern Medical Center, Dallas, Tex.

Fig. 3. Scanning electron microscopic image of a Staphylococcus aureus biofilm forming on the surface of a platinum electrode. The bare electrode surface can be seen in the foreground (lower left). The bacteria-filled biofilm can be seen in the distance. As the matrix of the biofilm thickens, the individual bacteria become harder to discern. Courtesy of K. Pawlowski, Assistant Professor, University of Texas Southwestern Medical Center, Dallas, Tex.

difficult.43 Molecular tools such as nucleic acid amplification, rapid DNA sequencing, and development of 16S ribosomal clone libraries have enhanced our ability to understand the microbiology in biofilm formation.36,44 Because biofilms are

composed of diverse polymicrobial communities, many bacterial species can be identified only with polymerase chain reaction.2 Therefore, such techniques can help us to select more effective therapeutic strategies.

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Volume 138, Number 3S • Infection and Biofilms in Wounds TREATMENT Less than 10% of acute wounds contain biofilm, compared with 60% of chronic wounds.2 As a result, treatments targeting biofilm are now being widely researched. Finding a highly effective treatment is challenging, because microorganisms that grow in a biofilm are mostly polymicrobial and are more resistant to host defenses and antimicrobial therapy.21 Approaches for reducing the biofilm in nonhealing wounds include the use of debridement, negative pressure wound therapy, topical and antiseptic dressings, systemic antibiotics, antibiofilm agents, and newer methods using molecular analysis techniques.45 Although most of the treatment modalities seem promising, evidence of improved outcomes with specific biofilm

treatment is scarce. An earlier published45 potential algorithm for the detection and treatment of biofilm might help in distinguishing the best approach for chronic wounds without the direct need for a biopsy (Fig.  4). Recently, systematic reviews have been published by the International Working Group on the Diabetic Foot on both ulcer healing and cure of DFI of these treatment modalities.46 Studies that directly link biofilm reduction to improved ulcer or infection outcomes could not be identified in these systematic reviews. A summary of treatment options is listed below. Off-loading Biomechanical Pressure Although not directly targeting the biofilm, long-term off-loading is crucial for healing of

Fig. 4. A potential algorithm for the detection and treatment of biofilms without the necessity to undertake biopsy samples.45

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Plastic and Reconstructive Surgery • September Supplement 2016 diabetic foot ulcers and decubitus ulcers.47 Most available topical or systemic wound treatments do not eliminate the underlying biomechanical etiology of such ulcers. Abnormal pressure and shear stress remain present. In case of diabetic foot ulcers, wearing protective footwear can reduce repetitive injury, providing the existing wounds the opportunity to heal. Furthermore, it protects high-risk areas from recurrent ulcers.48 To our knowledge, there has not been a study on a direct change in microbiota or biofilm and off-loading diabetic foot ulcers with total contact casts. Debridement Debridement methods include surgical (or sharp), autolytic (eg, hydrogel), enzymatic, mechanical, and biosurgery (eg, larval therapy). Surgical debridement remains the mainstay of adequate wound care and is performed to stimulate the healing process.49–51 It works by removing nonviable tissue and debris at the wound margins. It is a way to decrease the microbial bioburden while enabling the stimulation of contraction and epithelization.52 The difficulty of this procedure is to distinguish the right extent of debridement, including eradication of the biofilm.53,54 Repopulation of biofilm within 24 hours is common.24 However, frequent debridement disrupts biofilm and may explain its success in wound healing.55 There is evidence suggesting that hydrosurgical debridement decreases biofilm by 1 to 3 log, although the remaining biofilm remains tolerant to antibiotics and will regrow.56 Biosurgical debridement with larvae results in a significant reduction of the wound area57 as larvae seem capable of dissolving biofilm and inhibit the growth of new biofilm.58,59 Future development of molecular markers to identify necrotic tissue will likely make debridement more accurate and efficacious. The generation of gene expression profiles of specific wound regions quickly helps identifying the exact location of a wound biopsy and can determine how accurate the debridement was performed.60 Negative-Pressure Wound Therapy Negative-pressure wound therapy (NPWT) is a therapeutic technique that uses a vacuum dressing to promote healing in acute or chronic wounds. NPWT is often used in the treatment of complex wounds. The contribution of negative pressure therapy in reducing biofilm is controversial. An animal study involving swine wounds demonstrated that wounds treated with NPWT resulted in faster reduction in bacterial colonization.61 In contrast, Weed et al62 retrospectively reviewed

25 patients with chronic wounds and found a significant increase of bacterial colonization. The possible synergistic effect on biofilm by combining NPWT with topical dressings as reported in some small preclinical studies is promising.63–65 In one of these studies,65 the authors used an in vitro P. aeruginosa biofilm model and demonstrated a significant reduction of biofilm bacteria when these were exposed to topical negative pressure. This reduction was even stronger when topical negative pressure was combined with silver-impregnated foam. In addition, it was microscopically visible that NPWT compressed the biofilm architecture with a reduction in thickness and diffusion distance. For diabetic wounds, there are clinical reports suggesting that NPWT supports bacterial clearance,66,67 although a recent systematic review on bacterial load and NPWT showed variable results.68 NPWT is still an evolving technology, and new additions such as instillation therapy, nanocrystalline adjuncts, and portable systems are currently being developed. The use of NPWT may further improve outcomes in infected wounds if used for the right indication.69 Further research is needed to define the position of NPWT in routine clinical practice. Antiseptic and Topical Antimicrobial Dressings Although antiseptic dressings (eg, chlorhexidine, iodine, or silver) decrease the amount of bacteria in a wound, there is little evidence that application of antimicrobials or antiseptics lead to better outcomes of chronic, infected wounds.46,70,71 Topical antimicrobials (eg, fusidic acid and clindamycin cream) have the disadvantage that they contribute to the formation of resistant strains and lead to sensitization. Modern topical therapies, such as medicinal honey and peptide-based treatments such as pexiganan, are expected to be beneficial as an additional treatment because these therapies do not directly rely on the inherently decreased metabolic activity of bacteria in biofilm. This would potentially make them more effective against biofilm,72,73 although robust data to support their use in preference to any other therapies are missing.74 Over the years, several antiseptic dressings, including compounds with chlorhexidine, silver, povidone or cadexomer iodine, polyhexanide, or hypochlorite solutions, have been developed. None of these agents have demonstrated superior outcomes compared with traditional use of simple gauze and saline solution alone.75 In general, DFIs with heavy exudate need a dressing that absorbs moisture, whereas dry wounds need topical treatments that add moisture.76

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Volume 138, Number 3S • Infection and Biofilms in Wounds Systemic Antibiotics Systemic antibiotic treatment is useful in clinically infected wounds. Available clinical and epidemiologic data should form the basis of empiric antibiotic regimens. Such regimens should preferably contain antibiotics that can achieve high tissue concentrations. In most regions of the world, the empiric antibiotic regimens cover staphylococci and streptococci. Broad-spectrum antimicrobial therapy with gram-negative coverage may be considered in case of severe infections, after previous treatment failure, or in warmer climates. If there is a substantial risk of infection with methicillin-resistant S. aureus, an agent with methicillin-resistant S. aureus coverage is recommended. Several studies showed that the results of superficial wound cultures do not represent the diversity of bacteria present in chronic wounds encasing biofilm.77–79 Therefore, it is important that definitive therapy is based on well-performed cultures of the infected tissue. Other key elements for narrowing the antibiotic regimen are adjustments based on the antibiotic susceptibility profile of the bacteria and the clinical response of the patient to the empiric regimen.80 Knowledge of systemic antimicrobial therapy for biofilm-related infections has mostly been gained from experience in treatment of lung infections in cystic fibrosis patients, periprosthetic joint– associated infections, and central venous catheter infections. Oral antimicrobial agents with high bioavailability, such as clindamycin, rifamp(ic)in, fluoroquinolones, and trimethoprim/sulfamethoxazole, are frequently used. The effectiveness of oral therapy with such medications is comparable with parenteral therapy.81 In particular, fluoroquinolones can achieve high tissue concentrations.82 Earlier studies demonstrated that rifamp(ic)in exhibits high activity against staphylococcal biofilms, specifically in periprosthetic joint–associated infections.83,84 This effect can best be achieved by combining another antibiotic such as daptomycin or flucloxacillin with rifamp(ic)in, which would enhance biofilm penetration by synergism.85 It is important to note that clinical evidence for the antibiofilm effect of such combinations for chronic wounds is not yet available.86 Other, less commonly used agents that appear to penetrate well in antimicrobial biofilms include linezolid, fosfomycin, daptomycin, and possibly ceftaroline.87–89 Adjunctive Treatments A Cochrane review has been performed to use granulocyte colony-stimulating factors for treating DFI.90 Five studies were identified and included in

a meta-analysis. Adding granulocyte colony-stimulating factors did not significantly change wound healing, infection resolution, or the duration of systemic antibiotic therapy. However, it was associated with a significantly reduced likelihood of lower extremity surgical interventions (including amputation) and a reduced duration of hospital stay. No high-quality studies have been published that provide evidence for the efficacy of other growth factors or any physical therapy. Antibiofilm Agents Agents Targeting Exopolysaccharide A characteristic phenomenon of biofilm is the thick layer of EPS that protects bacteria from the host’s immune system and antimicrobials. Several studies have researched therapeutic modalities targeting and degrading this EPS layer in vitro.91–93 Future prospective studies are needed to demonstrate whether these influence healing time of chronic wounds. Quorum Sensing Inhibitors Where other antibiofilm techniques mechanisms of action include killing or inhibition of the growth of bacterial cells, quorum sensing inhibitors (QSIs) act by influencing biofilm formation and/ or matrix production. In vivo and in vitro models were used to compare QSIs with conventional antimicrobial agents.94 The researchers concluded that QSIs may increase the success of antibiotic treatment by increasing the susceptibility of bacteria in the biofilms. Although a lot about the impact of QS in biofilm formation remains to be studied, it is expected that QSI can lead to efficacious treatments with lower doses of antimicrobials needed. Targeted Therapy Based on Molecular Microbiology Diagnostics A promising approach is antibiofilm therapy based on results of molecular microbiology (genotyping). Authors of 1 study95 compared healing outcomes of patients with chronic wounds before and after the implementation of molecular pathogen diagnostics. Before implementation, 48.5% of the patients healed completely, whereas this was 62.4% after implementation. In addition, the healing time was reduced. Finally, expenses of firstline antibiotics declined because of increased use of cheaper targeted antibiotics. In another study, authors compared 3 treatments: culture-driven standard-of-care treatment, molecular diagnostic– based treatment, and personalized topical treatment specific to the microbial burden of each patient.96 The authors demonstrated that the

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Plastic and Reconstructive Surgery • September Supplement 2016 percentage of subjects who achieved wound closure increased from 48.5% to 62.4% and 90.4%. Further techniques that might have potential in targeting biofilm formation include laser therapy, photodynamic therapy, and cold plasma therapy. These techniques are still experimental.

FUTURE Most importantly, it still needs to be determined whether specific therapy of biofilm improves wound healing and cures or prevents infection. Molecular diagnostics appear to demonstrate the composition and organization of the biofilm reliably. It seems a promising tool to understand the local environment of chronic wounds better. Future research is needed to differentiate the real pathogens from the colonizers, especially in DFIs. This will give the clinicians the opportunity to provide a more rapid, effective, and accurate treatment. Emphasize must be put on individualized treatment targeting the polymicrobial nature, the interindividual differences and epidemiological differences of a biofilm containing wound. There are several interesting treatment options available, which—provided specific biofilm therapy is needed in the first place— might add specific advantages in the treatment of biofilm-associated infections. Edgar J. G. Peters, MD, PhD Department of Internal Medicine VU University Medical Center Room ZH-4A35 PO Box 7057 NL-1007 MB Amsterdam, The Netherlands [email protected]

acknowledgment

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Plastic and Reconstructive Surgery • September Supplement 2016 68. Patmo AS, Krijnen P, Tuinebreijer WE, et al. The effect of vacuum-assisted closure on the bacterial load and type of bacteria: a systematic review. Adv Wound Care (New Rochelle) 2014;3:383–389. 69. Hasan MY, Teo R, Nather A. Negative-pressure wound therapy for management of diabetic foot wounds: a review of the mechanism of action, clinical applications, and recent developments. Diabet Foot Ankle 2015;6:27618. 70. Nelson EA, O’Meara S, Golder S, et al; DASIDU Steering Group. Systematic review of antimicrobial treatments for diabetic foot ulcers. Diabet Med. 2006;23:348–359. 71. Gottrup F, Apelqvist J, Bjarnsholt T, et al. EWMA document: antimicrobials and non-healing wounds. Evidence, controversies and suggestions. J Wound Care. 2013;22:1–89. 72. Moghazy AM, Shams ME, Adly OA, et al. The clinical and cost effectiveness of bee honey dressing in the treatment of diabetic foot ulcers. Diabetes Res Clin Pract. 2010;89:276–281. 73. Lipsky BA, Holroyd KJ, Zasloff M. Topical versus systemic antimicrobial therapy for treating mildly infected diabetic foot ulcers: a randomized, controlled, double-blinded, multicenter trial of pexiganan cream. Clin Infect Dis. 2008;47:1537–1545. 74. Jull AB, Walker N, Deshpande S. Honey as a topical treatment for wounds. Database Syst Rev. 2013;2:CD005083. 75. Uçkay I, Gariani K, Pataky Z, et al. Diabetic foot infections: state-of-the-art. Diabetes Obes Metab. 2014;16:305–316. 76. Lipsky BA, Aragón-Sánchez J, Diggle M, et al. IWGDF guidance on the diagnosis and management of foot infections in persons with diabetes. Diabetes Metab Res Rev. 2016;32(Suppl 1):45–74. 77. Slater RA, Lazarovitch T, Boldur I, et al. Swab cultures accurately identify bacterial pathogens in diabetic foot wounds not involving bone. Diabet Med. 2004;21:705–709. 78. Melendez JH, Frankel YM, An AT, et al. Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds. Clin Microbiol Infect. 2010;16:1762–1769. 79. Sharp CS, Bessmen AN, Wagner FW Jr, et al. Microbiology of superficial and deep tissues in infected diabetic gangrene. Surg Gynecol Obstet. 1979;149:217–219. 80. Lipsky BA, Berendt AR, Cornia PB, et al; Infectious Diseases Society of America. 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections. Clin Infect Dis. 2012;54:e132–e173. 81. Grayson LM, Crowe, SM, McCarthy, et al. The Use of Antibiotics Sixth Edition: A Clinical Review of Antibacterial, Antifungal and Antiviral Drugs. Boca Raton, FL: CRC Press; 2010. 82. Kuck EM, Bouter KP, Hoekstra JB, et al. Tissue concentrations after a single-dose, orally administered ofloxacin in patients with diabetic foot infections. Foot Ankle Int. 1998;19:38–40.

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Update on the Role of Infection and Biofilms in Wound Healing: Pathophysiology and Treatment.

Chronic wounds, and among these infected diabetic foot ulcers, are a worldwide problem. The poor treatment outcomes result in high healthcare costs, a...
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