REVIEW
An Update on Chronic Wounds and the Role of Biofilms Christina Scali and Brian Kunimoto Background: Chronic wounds cause significant morbidity and mortality and cost our health care system millions of dollars each year. A major impediment to wound healing is the formation of bacterial biofilms. Biofilms are communities of bacteria associated with chronic infections. Objective: This article reviews the literature on chronic wounds and biofilms. The role of biofilms in chronic wounds is not widely known. The purpose is to increase awareness of their role and to discuss research into novel therapeutic options. Methods: PubMed searches were performed to identify publications on chronic wounds and biofilms. Results: Biofilms contribute to chronic wound nonhealing. There is an abundance of research into novel antibiofilm strategies for chronic wounds. Conclusion: Current research is being targeted at antibiofilm strategies needed to restore an optimal wound-healing environment. A combined treatment approach involving aggressive de´bridement and the addition of antibiofilm agents is needed. Contexte: Les plaies chroniques sont une cause importante de morbidite´ et de mortalite´, et elles couˆtent annuellement des millions de dollars au syste`me de soins de sante´. Un obstacle important a` la cicatrisation des plaies est la formation de biofilms bacte´riens, qui sont des populations de bacte´ries associe´es aux infections chroniques. Objectifs: Nous passons en revue, dans le pre´sent article, la documentation sur les plaies chroniques et les biofilms. Leur roˆle dans les plaies chroniques n’est pas bien connu. L’e´tude avait pour buts d’approfondir le roˆle des biofilms et de faire e´tat de la recherche sur les nouveaux moyens the´rapeutiques. Me´thode: Des recherches ont e´te´ mene´es dans PubMed afin de relever les publications sur les plaies chroniques et les biofilms. Re´sultats: Les biofilms participent a` la non-cicatrisation des plaies chroniques, et un grand nombre de recherches portent sur de nouvelles interventions de lutte contre les biofilms dans le contexte des plaies chroniques. Conclusions: La recherche actuelle cible surtout les interventions de lutte contre les biofilms afin de re´tablir un milieu propice a` la cicatrisation des plaies, mais le traitement appelle une approche mixte, associant de´bridement e´nergique et agents antibiofilm.
HE DURATION of a chronic wound is not universally agreed upon. Some experts define chronic wounds as wounds that have not healed in 4 to 6 weeks1 or in 3 months.2 Chronic wounds include diabetic, venous stasis, arterial and pressure ulcers, and those related to hematologic disorders and malignancy. Although there are few data on their prevalence in Canada, they are not uncommon. Chronic wounds have an enormous economic impact on health care systems. On a global scale, they cost $13 to $15 billion annually.3 The cost is also increased by complications such as wound infection. Chronic wounds
T
From the Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC. Address reprint requests to: Christina Scali, BSc, MSc, PhD, MD, 835 West 10th Avenue, Vancouver BC V5Z 4E8; e-mail: [email protected].
DOI 10.2310/7750.2013.12129 # 2013 Canadian Dermatology Association
cause significant morbidity and mortality. Considering that they mainly affect older individuals, their incidence will only continue to increase due to the aging population.
Risk Factors for Nonhealing of Chronic Ulcers Risk factors for nonhealing of chronic venous leg ulcers include advanced age, increased body mass index, a history of deep vein thrombosis, noncompliance with compression therapy, a large ulcer area, and more severe venous reflux.4,5 Nonhealing has also been associated with longer ulcer duration, increased ulcer complexity, the presence of lipodermatosclerosis, a history of deep vein thrombosis, and thrombophlebitis.6 General risk factors for wound healing also include the presence of arterial/venous disease, diabetes mellitus, nutritional deficiencies, and smoking.5 Biofilms, which are communities of bacteria often found in chronic infections, have been shown to be risk factors for nonhealing in animal models.7,8 Biofilms lead to a
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chronic inflammatory phase and prevent healing,9 as discussed herein.
Colonization of Chronic Wounds Acute wounds follow an orderly repair process that includes hemostasis, inflammation, proliferation, and remodeling.10 In contrast, chronic wounds are slower to heal and have defective reepithelialization and remodeling. They usually remain in an inflammatory state and often have high bacterial loads. Bacteria in wounds exist in a continuum from contamination to colonization to critical colonization to infection.11 Contamination involves low numbers of nonreplicating bacteria and occurs from the surrounding skin and the environment.11,12 Colonization involves proliferating bacteria in the absence of a host response. Colonization may impair wound healing, especially if the bacterial load is greater than 105 organisms per gram of tissue13,14 and particularly in the presence of Pseudomonas, Streptococcus, or Staphylococcus.13 Critical colonization involves a nonhealing inflammatory phase in which breakdown and discoloration of granulation tissue, increased friability, and discharge can occur.11,14,15 Significant colonization in the form of biofilm is very problematic for chronic wounds as it delays wound healing. Infection is the final stage where bacteria overcome the host immune response and invade deeper compartments, resulting in damage to tissues.16,17 The transition to infection is dependent on factors such as the amount of bacteria present and their virulence, as well as the host immune response.18
Microbiology of Chronic Wounds The colonizing flora of chronic wounds is complex and dynamic. The more chronic an ulcer, the more likely it is to have an increased proportion of anaerobes and multiple aerobic species.19,20 The growth of anaerobes is enhanced in chronic wounds because there is a low tissue oxygen level.21 The most commonly isolated organism from chronic venous leg ulcers using culturing techniques is Staphylococcus aureus (64.3–93.5%).20,22,23 The next most commonly isolated organisms using culturing techniques include Enterococcus faecalis (71.7–74%), Pseudomonas aeruginosa (32.6–52.2%), coagulase-negative staphylococci (45.7%), and Proteus species (16.1–41.3%). Finally, anaerobes, Enterobacter cloacae, Peptostreptococcus magnus, Corynebacterium striatum, fungi, Helcococcus kunzii, and Finegoldia magna are also found (Table 1). 372
The culture methods used to determine the microbiology of wounds can have an impact on the species identified. Wound biopsy is considered the gold standard to obtain tissue for culturing; however, the majority of studies use wound swabs. These have the potential to identify only surface bacteria. The microbiologic results obtained by culture may also represent bacterial species that are the most readily propagated in vitro under aerobic conditions.24 Using molecular techniques, Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia species were identified in chronic wounds of all types.24 The most commonly identified organisms from chronic venous leg ulcers include Pseudomonas, Enterobacter, Serratia, Proteus, and Stenotrophomonas species (see Table 1). This study also found that only diabetic ulcers had the same species identified via culturing as identified by molecular methods.
Role of Biofilms in Chronic Wounds Biofilms are communities of bacteria attached to a surface, embedded in a self-produced extracellular polysaccharide matrix.25 The matrix provides stability and protection to the biofilm. There are water channels that allow nutrient delivery and metabolic waste removal. Biofilms are formed in two stages, beginning with adhesion of planktonic bacteria to a surface mediated by adhesins (adhesion stage).26 This is followed by proliferation and maturation of the attached cells (maturation stage). The maturation stage is dependent on quorum sensing, a microbial cell-tocell communication system.27,28 Quorum sensing involves the release of small signaling molecules that increase in concentration in response to an increase in cell density. These signaling molecules then regulate gene expression of the entire community, allowing behavior to be coordinated. This allows the population to change its phenotype to form a biofilm when a certain density of bacteria is reached. Biofilms can occur in the skin, gut, and genitourinary and respiratory tracts and are the mode of growth of many chronic infections.25,29–31 According to the Centers for Disease Control and Prevention, biofilms are thought to be involved in 65% of all infections. Examples where biofilms can be found include P. aeruginosa chronic bronchitis in cystic fibrosis patients, infective endocarditis, chronic wounds, chronic otitis media, chronic sinusitis, chronic osteomyelitis, and prosthetic joint and catheter infections. Biofilms offer several survival advantages to bacteria. Bacteria within biofilms exhibit phenotypic and genotypic plurality.32 Phenotypic plurality allows bacteria to adapt to
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Table 1. Most Commonly Isolated Organisms from Chronic Venous Leg Ulcers Using Culturing and Sequencing Techniques Study
Wounds
Technique
22
Gjodsbol et al, 2006
Chronic venous leg ulcers
Culture
Hansson et al, 199523
Chronic venous leg ulcers
Culture
Moore et al, 201020
Chronic venous leg ulcers
Culture
Dowd et al, 200824
Chronic venous leg ulcers
Shotgun Sanger sequencing
Pyrosequencing
differences in nutrient availability, pH, and oxidizing potential in microenvironments within the biofilm. Genotypic plurality results in part from horizontal gene transfer, which allows for virulence and drug resistance genes to be passed between members of the biofilm community.33 Other survival advantages include a greater resistance to the host innate and adaptive immune responses. During wound healing, the extracellular polysaccharide allows evasion of neutrophil phagocytosis by preventing the detection of opsonins on the bacterial cell wall.34–36 There is also attenuation of neutrophil degranulation and formation of reactive oxygen species.37 The treatment of bacterial biofilms is further complicated by the fact that growth within biofilms is slow, rendering bactericidal antibiotics relatively insensitive.26,36 In addition, minimum inhibitory concentrations (MICs) are much higher (100- to 1,000-fold) than for planktonic bacteria, making biofilms much more difficult to eradicate.29,36,38 The higher MIC is in part related to the fact that the matrix decreases the effective drug concentration.
Species
%
Staphylococcus aureus Enterococcus faecalis Pseudomonas aeruginosa Coagulase negative staphylococci Proteus species Anaerobes Staphylococcus aureus Enterococcus faecalis Enterobacter cloacae Peptostreptococcus magnus Fungi Staphylococcus aureus Corynebacterium striatum Pseudomonas aeruginosa Helcococcus kunzii Finegoldia magna Proteus mirabilis Pseudomonas species Enterobacter species Stenotrophomonas maltophilia Proteus species Staphylococcus aureus Clostridium species Enterobacter species Serratia species Stenotrophomonas species Proteus species Staphylococcus species
93.5 71.7 52.2 45.7 41.3 39.1 88.0 74.0 29.0 29.0 11.0 64.3 60.6 32.6 22.0 21.4 16.1 49.0 14.9 7.2 6.2 3.6 3.1 44.8 19.2 11.1 11.1 0.5
Bacterial dormancy within the biofilm also plays a role. Genotypic plurality and transfer of resistance genes encoding multidrug resistance pumps, as well as target receptor blockers and drug degradation contribute to higher MICs. Colonization is very common, and significant colonization in the form of biofilm is very problematic for chronic wounds as it delays wound healing. Biofilms are more common in chronic than acute wounds (60% versus 6%).39 Chronic wounds remain open longer, thus contributing to their increased colonization. Chronic wounds also provide a perfect environment for biofilm formation because proteins such as fibronectin and collagen, which facilitate attachment, are present.26 Furthermore, the relatively poor blood flow and hypoxia make host defenses more challenging.40 Chronic wounds contain senescent host cells, proinflammatory cytokines, and increased matrix metalloproteinases and neutrophils. Biofilms lead to a chronic nonhealing inflammatory phase.9 Biofilms were shown to induce extensive inflammatory cell
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infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged wounds and delay reepithelialization in murine models.7,8 S. aureus, Streptococcus agalactiae, P. aeruginosa, coliforms, and anaerobes have been associated with delayed healing and venous leg ulcers of larger area.8 Although biofilms are microscopic structures, clinically, they can be detected if allowed to grow large enough. They have been described as gel-like and shiny and differ from slough, which is yellow and opaque. Biofilms also require more physical disruption to remove when compared to slough.41
Treatment Biofilm-based wound care involves multiple strategies to target biofilms.42 These include de´bridement and antibiofilm and antimicrobial agents such as disinfectants, antiseptics, and antibiotics.26 The main component involves aggressive de´bridement of the wound to restore an optimal wound-healing environment.43,44 De´bridement is a quick, easy, safe, and relatively inexpensive way to remove biofilms from chronic wounds. There are many problems associated with current wound care practices. Antiseptics such as silver compounds, which are commonly used topically for chronic wounds, are toxic to keratinocytes in in vitro studies.45 Topical antibiotics for the treatment of colonized or infected wounds can induce contact allergy and delayed hypersensitivity reactions.46,47 The use of topical and systemic antibiotics contributes to the growing problem of antimicrobial resistance. Furthermore, systemic antibiotics are recommended only when wound infection cannot be controlled with local treatments such as in cellulitis, lymphangitis, and sepsis.48 Finally, costs begin to increase when antimicrobials, cadexomer iodine, and the many silver-containing products are used. Given the drawbacks of current therapies, alternative therapies are needed. Targeting biofilms will lead to an improvement in healing outcomes, which theoretically will lead to a reduction in the significant morbidity and mortality as well as the economic burden chronic wounds place on health care systems.
Current Research into Antibiofilm Agents Much has been learned over the past few years about the importance of biofilms in chronic infections. Many antibiofilm products are being developed and are currently under investigation. However, the production of antibiofilm agents is a long process. We are still awaiting the 374
appearance of the first antibiofilm agent on the market. The development of in vivo biofilm wound infection models will facilitate the development and testing of novel antibiofilm agents.49,50 Some examples of recent publications into antibiofilm agents include a microbicidal polyhexanide-containing biocellulose dressing, which was shown to be effective for the eradication of biofilms in nonhealing wounds.51 An attempt to use a near-infrared laser on monomicrobial biofilms of S. aureus and P. aeruginosa and polymicrobial biofilms in vitro did not demonstrate a significant effect.52 The use of topical negative pressure demonstrated a small but statistically significant decrease in wound biofilms in an in vitro model.53 The effects were much greater when combined with a silver-impregnated foam. Bismuth thiol preparations were shown to have bactericidal activity against Pseudomonas and methicillin-resistant Staphylococcus aureus (MRSA) biofilms in an in vitro wound model.54 Natural and synthetic cathelicidin peptides have shown antimicrobial and antibiofilm activity against S. aureus.55 The antibiofilm efficacy of a lactoferrin/xylitol hydrogel used in combination with silver-based wound dressings has been demonstrated.56 A Staphylococcus epidermidis serine protease Esp has demonstrated activity in destroying and preventing the formation of S. aureus biofilms in vitro as well as the elimination of S. aureus nasal colonization in vivo.57 Novel antibiofilm control agents that are currently under development include antiadhesion molecules, quorum sensing inhibitors, and selectively targeted antimicrobial peptides (STAMPs).26 Given that adhesion is the first stage in biofilm formation, antiadhesion molecules should keep bacteria in the planktonic state, making them more susceptible to the host immune system and antibiotics. The sortase of gram-positive bacteria is an antiadhesion candidate because this protein is important in covalent anchoring of surface proteins to peptidoglycan and is shared by most of the surface proteins.26 Quorum sensing is very important in the maturation stage of biofilm formation. Quorum sensing inhibitors theoretically can keep cells planktonic by causing a breakdown in cell-to-cell communication and therefore make them more susceptible to the immune system and antibiotics.58 Two classes of leading candidates for quorum sensing inhibitors include the furanones and ribonucleic acid (RNA) III inhibiting peptide (RIP).26 Species-specific control of biofilms can be achieved with STAMPs.26 Antimicrobial peptides are an essential part of the innate immune system. STAMPs are modified and recombinant versions of antimicrobial peptides that contain a species-specific binding domain.
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Other novel strategies include matrix-degrading enzymes, which degrade the protective polysaccharide layer and make cells more susceptible to antibiotics.59 Dispersin B is an enzyme able to degrade the polysaccharide matrix of staphylococci.60 It is currently being commercially developed as a wound care gel. Other matrix-degrading enzymes include deoxyribonuclease, lysostaphin, proteinase K, and trypsin.59 Finally, there is great interest in developing passive immunotherapy or vaccines for bacterial infections such as staphylococci.59
5.
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8.
Conclusion Chronic wounds lead to significant patient morbidity and mortality and cost health care systems millions of dollars each year. Given that chronic wounds mainly affect individuals over the age of 60 years, their prevalence will only continue to increase with our aging population. One of the major problems associated with chronic wounds is bacterial biofilm formation. Bacterial biofilms delay reepithelialization and prevent wound healing by inducing a chronic inflammatory state. Mechanical de´bridement is a safe, quick, and relatively inexpensive technique to remove biofilms from chronic wounds. A combined approach involving aggressive de´bridement and the addition of antibiofilm agents is needed. Ideally, a treatment would be applicable to many different types of bacterial biofilms, have low potential for the development of resistance, be easy to administer, have minimal side effects, and have sufficient activity against the slowgrowing organisms in a biofilm. There is a need to reevaluate traditional treatment methods of chronic wounds and apply our current knowledge of biofilms to design new strategies to combat them.
9. 10.
11. 12.
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14. 15.
16. 17. 18.
Acknowledgment Financial disclosure of authors and reviewers: None reported.
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Canadian Dermatology Association | Journal of Cutaneous Medicine and Surgery, Vol 17, No 6 (November/December), 2013: pp 371–376
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An Update on Chronic Wounds and the Role of Biofilms Christina Scali and Brian Kunimoto Background: Chronic wounds cause significant morbidity and mortality and cost our health care system millions of dollars each year. A major impediment to wound healing is the formation of bacterial biofilms. Biofilms are communities of bacteria associated with chronic infections. Objective: This article reviews the literature on chronic wounds and biofilms. The role of biofilms in chronic wounds is not widely known. The purpose is to increase awareness of their role and to discuss research into novel therapeutic options. Methods: PubMed searches were performed to identify publications on chronic wounds and biofilms. Results: Biofilms contribute to chronic wound nonhealing. There is an abundance of research into novel antibiofilm strategies for chronic wounds. Conclusion: Current research is being targeted at antibiofilm strategies needed to restore an optimal wound-healing environment. A combined treatment approach involving aggressive de´bridement and the addition of antibiofilm agents is needed. Contexte: Les plaies chroniques sont une cause importante de morbidite´ et de mortalite´, et elles couˆtent annuellement des millions de dollars au syste`me de soins de sante´. Un obstacle important a` la cicatrisation des plaies est la formation de biofilms bacte´riens, qui sont des populations de bacte´ries associe´es aux infections chroniques. Objectifs: Nous passons en revue, dans le pre´sent article, la documentation sur les plaies chroniques et les biofilms. Leur roˆle dans les plaies chroniques n’est pas bien connu. L’e´tude avait pour buts d’approfondir le roˆle des biofilms et de faire e´tat de la recherche sur les nouveaux moyens the´rapeutiques. Me´thode: Des recherches ont e´te´ mene´es dans PubMed afin de relever les publications sur les plaies chroniques et les biofilms. Re´sultats: Les biofilms participent a` la non-cicatrisation des plaies chroniques, et un grand nombre de recherches portent sur de nouvelles interventions de lutte contre les biofilms dans le contexte des plaies chroniques. Conclusions: La recherche actuelle cible surtout les interventions de lutte contre les biofilms afin de re´tablir un milieu propice a` la cicatrisation des plaies, mais le traitement appelle une approche mixte, associant de´bridement e´nergique et agents antibiofilm.
HE DURATION of a chronic wound is not universally agreed upon. Some experts define chronic wounds as wounds that have not healed in 4 to 6 weeks1 or in 3 months.2 Chronic wounds include diabetic, venous stasis, arterial and pressure ulcers, and those related to hematologic disorders and malignancy. Although there are few data on their prevalence in Canada, they are not uncommon. Chronic wounds have an enormous economic impact on health care systems. On a global scale, they cost $13 to $15 billion annually.3 The cost is also increased by complications such as wound infection. Chronic wounds
T
From the Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC. Address reprint requests to: Christina Scali, BSc, MSc, PhD, MD, 835 West 10th Avenue, Vancouver BC V5Z 4E8; e-mail: [email protected].
DOI 10.2310/7750.2013.12129 # 2013 Canadian Dermatology Association
cause significant morbidity and mortality. Considering that they mainly affect older individuals, their incidence will only continue to increase due to the aging population.
Risk Factors for Nonhealing of Chronic Ulcers Risk factors for nonhealing of chronic venous leg ulcers include advanced age, increased body mass index, a history of deep vein thrombosis, noncompliance with compression therapy, a large ulcer area, and more severe venous reflux.4,5 Nonhealing has also been associated with longer ulcer duration, increased ulcer complexity, the presence of lipodermatosclerosis, a history of deep vein thrombosis, and thrombophlebitis.6 General risk factors for wound healing also include the presence of arterial/venous disease, diabetes mellitus, nutritional deficiencies, and smoking.5 Biofilms, which are communities of bacteria often found in chronic infections, have been shown to be risk factors for nonhealing in animal models.7,8 Biofilms lead to a
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chronic inflammatory phase and prevent healing,9 as discussed herein.
Colonization of Chronic Wounds Acute wounds follow an orderly repair process that includes hemostasis, inflammation, proliferation, and remodeling.10 In contrast, chronic wounds are slower to heal and have defective reepithelialization and remodeling. They usually remain in an inflammatory state and often have high bacterial loads. Bacteria in wounds exist in a continuum from contamination to colonization to critical colonization to infection.11 Contamination involves low numbers of nonreplicating bacteria and occurs from the surrounding skin and the environment.11,12 Colonization involves proliferating bacteria in the absence of a host response. Colonization may impair wound healing, especially if the bacterial load is greater than 105 organisms per gram of tissue13,14 and particularly in the presence of Pseudomonas, Streptococcus, or Staphylococcus.13 Critical colonization involves a nonhealing inflammatory phase in which breakdown and discoloration of granulation tissue, increased friability, and discharge can occur.11,14,15 Significant colonization in the form of biofilm is very problematic for chronic wounds as it delays wound healing. Infection is the final stage where bacteria overcome the host immune response and invade deeper compartments, resulting in damage to tissues.16,17 The transition to infection is dependent on factors such as the amount of bacteria present and their virulence, as well as the host immune response.18
Microbiology of Chronic Wounds The colonizing flora of chronic wounds is complex and dynamic. The more chronic an ulcer, the more likely it is to have an increased proportion of anaerobes and multiple aerobic species.19,20 The growth of anaerobes is enhanced in chronic wounds because there is a low tissue oxygen level.21 The most commonly isolated organism from chronic venous leg ulcers using culturing techniques is Staphylococcus aureus (64.3–93.5%).20,22,23 The next most commonly isolated organisms using culturing techniques include Enterococcus faecalis (71.7–74%), Pseudomonas aeruginosa (32.6–52.2%), coagulase-negative staphylococci (45.7%), and Proteus species (16.1–41.3%). Finally, anaerobes, Enterobacter cloacae, Peptostreptococcus magnus, Corynebacterium striatum, fungi, Helcococcus kunzii, and Finegoldia magna are also found (Table 1). 372
The culture methods used to determine the microbiology of wounds can have an impact on the species identified. Wound biopsy is considered the gold standard to obtain tissue for culturing; however, the majority of studies use wound swabs. These have the potential to identify only surface bacteria. The microbiologic results obtained by culture may also represent bacterial species that are the most readily propagated in vitro under aerobic conditions.24 Using molecular techniques, Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia species were identified in chronic wounds of all types.24 The most commonly identified organisms from chronic venous leg ulcers include Pseudomonas, Enterobacter, Serratia, Proteus, and Stenotrophomonas species (see Table 1). This study also found that only diabetic ulcers had the same species identified via culturing as identified by molecular methods.
Role of Biofilms in Chronic Wounds Biofilms are communities of bacteria attached to a surface, embedded in a self-produced extracellular polysaccharide matrix.25 The matrix provides stability and protection to the biofilm. There are water channels that allow nutrient delivery and metabolic waste removal. Biofilms are formed in two stages, beginning with adhesion of planktonic bacteria to a surface mediated by adhesins (adhesion stage).26 This is followed by proliferation and maturation of the attached cells (maturation stage). The maturation stage is dependent on quorum sensing, a microbial cell-tocell communication system.27,28 Quorum sensing involves the release of small signaling molecules that increase in concentration in response to an increase in cell density. These signaling molecules then regulate gene expression of the entire community, allowing behavior to be coordinated. This allows the population to change its phenotype to form a biofilm when a certain density of bacteria is reached. Biofilms can occur in the skin, gut, and genitourinary and respiratory tracts and are the mode of growth of many chronic infections.25,29–31 According to the Centers for Disease Control and Prevention, biofilms are thought to be involved in 65% of all infections. Examples where biofilms can be found include P. aeruginosa chronic bronchitis in cystic fibrosis patients, infective endocarditis, chronic wounds, chronic otitis media, chronic sinusitis, chronic osteomyelitis, and prosthetic joint and catheter infections. Biofilms offer several survival advantages to bacteria. Bacteria within biofilms exhibit phenotypic and genotypic plurality.32 Phenotypic plurality allows bacteria to adapt to
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Table 1. Most Commonly Isolated Organisms from Chronic Venous Leg Ulcers Using Culturing and Sequencing Techniques Study
Wounds
Technique
22
Gjodsbol et al, 2006
Chronic venous leg ulcers
Culture
Hansson et al, 199523
Chronic venous leg ulcers
Culture
Moore et al, 201020
Chronic venous leg ulcers
Culture
Dowd et al, 200824
Chronic venous leg ulcers
Shotgun Sanger sequencing
Pyrosequencing
differences in nutrient availability, pH, and oxidizing potential in microenvironments within the biofilm. Genotypic plurality results in part from horizontal gene transfer, which allows for virulence and drug resistance genes to be passed between members of the biofilm community.33 Other survival advantages include a greater resistance to the host innate and adaptive immune responses. During wound healing, the extracellular polysaccharide allows evasion of neutrophil phagocytosis by preventing the detection of opsonins on the bacterial cell wall.34–36 There is also attenuation of neutrophil degranulation and formation of reactive oxygen species.37 The treatment of bacterial biofilms is further complicated by the fact that growth within biofilms is slow, rendering bactericidal antibiotics relatively insensitive.26,36 In addition, minimum inhibitory concentrations (MICs) are much higher (100- to 1,000-fold) than for planktonic bacteria, making biofilms much more difficult to eradicate.29,36,38 The higher MIC is in part related to the fact that the matrix decreases the effective drug concentration.
Species
%
Staphylococcus aureus Enterococcus faecalis Pseudomonas aeruginosa Coagulase negative staphylococci Proteus species Anaerobes Staphylococcus aureus Enterococcus faecalis Enterobacter cloacae Peptostreptococcus magnus Fungi Staphylococcus aureus Corynebacterium striatum Pseudomonas aeruginosa Helcococcus kunzii Finegoldia magna Proteus mirabilis Pseudomonas species Enterobacter species Stenotrophomonas maltophilia Proteus species Staphylococcus aureus Clostridium species Enterobacter species Serratia species Stenotrophomonas species Proteus species Staphylococcus species
93.5 71.7 52.2 45.7 41.3 39.1 88.0 74.0 29.0 29.0 11.0 64.3 60.6 32.6 22.0 21.4 16.1 49.0 14.9 7.2 6.2 3.6 3.1 44.8 19.2 11.1 11.1 0.5
Bacterial dormancy within the biofilm also plays a role. Genotypic plurality and transfer of resistance genes encoding multidrug resistance pumps, as well as target receptor blockers and drug degradation contribute to higher MICs. Colonization is very common, and significant colonization in the form of biofilm is very problematic for chronic wounds as it delays wound healing. Biofilms are more common in chronic than acute wounds (60% versus 6%).39 Chronic wounds remain open longer, thus contributing to their increased colonization. Chronic wounds also provide a perfect environment for biofilm formation because proteins such as fibronectin and collagen, which facilitate attachment, are present.26 Furthermore, the relatively poor blood flow and hypoxia make host defenses more challenging.40 Chronic wounds contain senescent host cells, proinflammatory cytokines, and increased matrix metalloproteinases and neutrophils. Biofilms lead to a chronic nonhealing inflammatory phase.9 Biofilms were shown to induce extensive inflammatory cell
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infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged wounds and delay reepithelialization in murine models.7,8 S. aureus, Streptococcus agalactiae, P. aeruginosa, coliforms, and anaerobes have been associated with delayed healing and venous leg ulcers of larger area.8 Although biofilms are microscopic structures, clinically, they can be detected if allowed to grow large enough. They have been described as gel-like and shiny and differ from slough, which is yellow and opaque. Biofilms also require more physical disruption to remove when compared to slough.41
Treatment Biofilm-based wound care involves multiple strategies to target biofilms.42 These include de´bridement and antibiofilm and antimicrobial agents such as disinfectants, antiseptics, and antibiotics.26 The main component involves aggressive de´bridement of the wound to restore an optimal wound-healing environment.43,44 De´bridement is a quick, easy, safe, and relatively inexpensive way to remove biofilms from chronic wounds. There are many problems associated with current wound care practices. Antiseptics such as silver compounds, which are commonly used topically for chronic wounds, are toxic to keratinocytes in in vitro studies.45 Topical antibiotics for the treatment of colonized or infected wounds can induce contact allergy and delayed hypersensitivity reactions.46,47 The use of topical and systemic antibiotics contributes to the growing problem of antimicrobial resistance. Furthermore, systemic antibiotics are recommended only when wound infection cannot be controlled with local treatments such as in cellulitis, lymphangitis, and sepsis.48 Finally, costs begin to increase when antimicrobials, cadexomer iodine, and the many silver-containing products are used. Given the drawbacks of current therapies, alternative therapies are needed. Targeting biofilms will lead to an improvement in healing outcomes, which theoretically will lead to a reduction in the significant morbidity and mortality as well as the economic burden chronic wounds place on health care systems.
Current Research into Antibiofilm Agents Much has been learned over the past few years about the importance of biofilms in chronic infections. Many antibiofilm products are being developed and are currently under investigation. However, the production of antibiofilm agents is a long process. We are still awaiting the 374
appearance of the first antibiofilm agent on the market. The development of in vivo biofilm wound infection models will facilitate the development and testing of novel antibiofilm agents.49,50 Some examples of recent publications into antibiofilm agents include a microbicidal polyhexanide-containing biocellulose dressing, which was shown to be effective for the eradication of biofilms in nonhealing wounds.51 An attempt to use a near-infrared laser on monomicrobial biofilms of S. aureus and P. aeruginosa and polymicrobial biofilms in vitro did not demonstrate a significant effect.52 The use of topical negative pressure demonstrated a small but statistically significant decrease in wound biofilms in an in vitro model.53 The effects were much greater when combined with a silver-impregnated foam. Bismuth thiol preparations were shown to have bactericidal activity against Pseudomonas and methicillin-resistant Staphylococcus aureus (MRSA) biofilms in an in vitro wound model.54 Natural and synthetic cathelicidin peptides have shown antimicrobial and antibiofilm activity against S. aureus.55 The antibiofilm efficacy of a lactoferrin/xylitol hydrogel used in combination with silver-based wound dressings has been demonstrated.56 A Staphylococcus epidermidis serine protease Esp has demonstrated activity in destroying and preventing the formation of S. aureus biofilms in vitro as well as the elimination of S. aureus nasal colonization in vivo.57 Novel antibiofilm control agents that are currently under development include antiadhesion molecules, quorum sensing inhibitors, and selectively targeted antimicrobial peptides (STAMPs).26 Given that adhesion is the first stage in biofilm formation, antiadhesion molecules should keep bacteria in the planktonic state, making them more susceptible to the host immune system and antibiotics. The sortase of gram-positive bacteria is an antiadhesion candidate because this protein is important in covalent anchoring of surface proteins to peptidoglycan and is shared by most of the surface proteins.26 Quorum sensing is very important in the maturation stage of biofilm formation. Quorum sensing inhibitors theoretically can keep cells planktonic by causing a breakdown in cell-to-cell communication and therefore make them more susceptible to the immune system and antibiotics.58 Two classes of leading candidates for quorum sensing inhibitors include the furanones and ribonucleic acid (RNA) III inhibiting peptide (RIP).26 Species-specific control of biofilms can be achieved with STAMPs.26 Antimicrobial peptides are an essential part of the innate immune system. STAMPs are modified and recombinant versions of antimicrobial peptides that contain a species-specific binding domain.
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Other novel strategies include matrix-degrading enzymes, which degrade the protective polysaccharide layer and make cells more susceptible to antibiotics.59 Dispersin B is an enzyme able to degrade the polysaccharide matrix of staphylococci.60 It is currently being commercially developed as a wound care gel. Other matrix-degrading enzymes include deoxyribonuclease, lysostaphin, proteinase K, and trypsin.59 Finally, there is great interest in developing passive immunotherapy or vaccines for bacterial infections such as staphylococci.59
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Conclusion Chronic wounds lead to significant patient morbidity and mortality and cost health care systems millions of dollars each year. Given that chronic wounds mainly affect individuals over the age of 60 years, their prevalence will only continue to increase with our aging population. One of the major problems associated with chronic wounds is bacterial biofilm formation. Bacterial biofilms delay reepithelialization and prevent wound healing by inducing a chronic inflammatory state. Mechanical de´bridement is a safe, quick, and relatively inexpensive technique to remove biofilms from chronic wounds. A combined approach involving aggressive de´bridement and the addition of antibiofilm agents is needed. Ideally, a treatment would be applicable to many different types of bacterial biofilms, have low potential for the development of resistance, be easy to administer, have minimal side effects, and have sufficient activity against the slowgrowing organisms in a biofilm. There is a need to reevaluate traditional treatment methods of chronic wounds and apply our current knowledge of biofilms to design new strategies to combat them.
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Acknowledgment Financial disclosure of authors and reviewers: None reported.
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Canadian Dermatology Association | Journal of Cutaneous Medicine and Surgery, Vol 17, No 6 (November/December), 2013: pp 371–376
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