Wound Biofilm Research Articles

A Novel Organo-Selenium Bandage that Inhibits Biofilm Development in a Wound by Gram-Positive and Gram-Negative Wound Pathogens.

Biofilm formation in wounds is a serious problem which inhibits proper wound healing. One possible contributor to biofilm formation in a wound is the bacteria growing within the overlying bandage. To test this mechanism, we used bandages that contained a coating of organo-selenium that was covalently attached to the bandage. We tested the ability of this coating to kill bacteria on the bandage and in the underlying tissue. The bandage material was tested with both lab strains and clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa and Staphylococcus epidermidis. It was found that the organo-selenium coated bandage showed inhibition, of biofilm formation on the bandage in vitro (7–8 logs), with all the different bacteria tested, at selenium concentrations in the coating of less than 1.0%. These coatings were found to remain stable for over one month in aqueous solution, 15 min in boiling water, and over 6 years at room temperature. The bandages were also tested on a mouse wound model where the bacteria were injected between the bandage and the wound. Not only did the selenium bandage inhibit biofilm formation in the bandage, but it also inhibited biofilm formation in the wound tissue. Since selenium does not leave the bandage, this would appear to support the idea that a major player in wound biofilm formation is bacteria which grows in the overlying bandage.

Multispecies biofilm in an artificial wound bed—A novel model for in vitro assessment of solid antimicrobial dressings

Wound infections represent a major problem, particularly in patients with chronic wounds. Bacteria in the wound exist mainly in the form of biofilms and are thus resistant to most antibiotics and antimicrobials. A simple and cost-effective in vitro model of chronic wound biofilms applied for testing treatments and solid devices, especially wound dressings, is presented in this work. The method is based on the well-established Lubbock chronic wound biofilm transferred onto an artificial agar wound bed. The biofilm formed by four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa) was stable for up to 48h post-transplant. The applicability of the model was evaluated by testing two common iodine wound treatments. These observations indicate that this method enables assessing the effects of treatments on established resilient wound biofilms and is clinically highly relevant.

Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function.

In chronic wounds, biofilm infects host tissue for extended periods of time. This work establishes the first chronic preclinical model of wound biofilm infection aimed at addressing the long-term host response. Although biofilm-infected wounds did not show marked differences in wound closure, the repaired skin demonstrated compromised barrier function. This observation is clinically significant, because it leads to the notion that even if a biofilm infected wound is closed, as observed visually, it may be complicated by the presence of failed skin, which is likely to be infected and/or further complicated postclosure. Study of the underlying mechanisms recognized for the first time biofilm-inducible miR-146a and miR-106b in the host skin wound-edge tissue. These miRs silenced ZO-1 and ZO-2 to compromise tight junction function, resulting in leaky skin as measured by transepidermal water loss (TEWL). Intervention strategies aimed at inhibiting biofilm-inducible miRNAs may be productive in restoring the barrier function of host skin.

Bacterial biofilm in chronic venous ulcer

presence of bacterial biofilm in chronic wounds may explain Venous leg ulcer (VLU) is a type of chronic wound that can take a long time to heal, being the most advanced presentation of chronic venous insufficiency (CVI).1 VLUs are usually recurrent, affecting work productivity, quality of life and high public health care treatment costs. It is estimated that VLUs affect up to 1% of the adult population.1 These chronic wounds are usually infected by a diverse microbial flora, which may probably contribute to the nonhealing phenotype.2 A study developed at the university hospital of the Federal University of Minas Gerais, Belo Horizonte, Brazil, included patients with VLU to investigate the presence of bacterial biofilm. A VLU biopsy was performed in a total of 45 patients who had not used antibiotics and presented signs of CVI, such as edema, varicose veins, hyperpigmentation, and lipodermatosclerosis (Fig. 1A). The samples were submitted to bacteriological tests to characterize the bacterial flora and examined by transmission electron microscopy

Biofilm-infected wounds in a dog

A 4-year-old spayed female Mastiff was evaluated for treatment of chronic nonhealing pressure wounds over both elbow regions resulting from attempts at hypertrophic callus excision. The wound bed granulation tissue was mottled red and yellow with hyperemic, rolled epithelial edges. The right wound communicated with a large fluid pocket along the thoracic wall. The dog had an inflammatory leukogram with a left shift. The wounds were debrided, and tissue specimens were collected for histologic evaluation, microbial culture, and bacterial identification by means of molecular diagnostic techniques. The left wound was closed immediately. Calcium alginate rope with silver was packed into the right wound. Vacuum-assisted closure was applied for 6 days. Debridement was repeated, and a thoracodorsal axial pattern flap was used to cover the wound. Systemic treatment with antimicrobials was initiated, and pressure over the elbow regions was relieved. Bacterial biofilms were identified histologically in tissue specimens from both wounds. Staphylococcus intermedius, Staphylococcus epidermidis, and Streptococcus canis were cultured and identified by 16S rRNA fragment sequencing. Pyrosequencing identified multiple bacterial species and no fungal organisms. Both wounds healed successfully. Biofilms are implicated in infected orthopedic implants in veterinary patients; however, this is the first report of a bacterial biofilm in chronic wounds in a dog. In human wound care, extensive debridement is performed to disrupt the biofilm; a multimodal treatment approach is recommended to delay reformation and help clear the infection. In this case, biofilm reformation was prevented by systemic treatment with antimicrobials, by reducing local pressure on the wounds, and by wound closure.

A clinical algorithm for wound biofilm identification

Recognition of the existence of biofilm in chronic wounds is increasing among wound care practitioners, and a growing body of evidence indicates that biofilm contributes significantly to wound recalcitrance. While clinical guidelines regarding the involvement of biofilm in human bacterial infections have been proposed, there remains uncertainty and lack of guidance towards biofilm presence in wounds. The intention of this report is to collate knowledge and evidence of the visual and indirect clinical indicators of wound biofilm, and propose an algorithm designed to facilitate clinical recognition of biofilm and subsequent wound management practices.

Springing into action

M arch is a very busy month here at the Journal of Wound Care. In addition to preparing this bumper March issue for you to enjoy, we are also in the middle of organising our 7th National Wound Care Conference, our annual Journal of Wound Care Awards, and our first-ever Journal of Wound Care Technology Forum. The theme of this year’s National Wound Care Conference is ‘the way forward for tissue viability’. Topics discussed include the practicalities of working with CCGs, the implications of the Department of Health policy for tissue viability practice, and the development of tools to measure patient experience and patient outcomes. The conference will be a valuable platform to facilitate the share and exchange of ideas between leading tissue viability practitioners. Our second-ever Journal of Wound Care awards will be held at the Midland Hotel in Manchester. These annual awards aim to recognise the wound care practitioners and researchers who, through demonstrating initiative and original thinking, have made a significant impact in his or her area of wound care, benefitting patient outcomes. We received an incredibly high calibre of entries for this year’s awards from more than 10 countries worldwide, and I’m sure our judging panel, made up of members of our editorial board, had a hard time choosing three of the top entries in each category. Congratulations to the shortlisted candidates, and thank you to everyone who submitted an entry. A final shortlist of applicants for the 2014 awards can be found on page 124 of this issue. Finally, we will also be hosting the 1st Journal of Wound Care Technology Forum, with the aim of unveiling the current trends and innovations in tissue viability scientific research. This forum will provide an opportunity for scientists, clinicians and industry representatives to discuss the latest developments in wound care research, and will cover topics including the wound microenvironment, wound biofilms, in vitro and in vivo wound models, and biomaterial concepts. An article by Forbes last month drew attention to the increasing appeal of wound care-related medical advances to healthcare analysis and investors.1 This is fantastic news for wound care researchers; increased research funding will hasten the development of innovative new treatments. Our technology forum will ensure that clinicans and researchers are kept up-to-date with the latest in wound care treatment breakthroughs. References 1 Marcial, G. Overlooked advanced wound care market catches attention of healthcare analysts, investors. Available from http:// www.forbes.com/sites/ genemarcial/2014/02/03/ overlooked-advancedwound-care-marketcatches-attention-ofhealthcare-analystsinvestors/ [Accessed March 2014].

Inhibition of biofilms of Pseudomonas aeruginosa by Medihoney in vitro

Pseudomonas aeruginosa has been linked to chronic wound infections, where its ability to form biofilms and to tolerate antimicrobial agents helps to facilitate its persistence. This study aimed to investigate the susceptibility of biofilms of Pseudomonas aeruginosa to Medihoney in vitro. Biofilms were cultivated in microtitre plates with and without a range of concentrations of Medihoney, and effects on biofilm were monitored by optical density (at 650nm), biomass (by staining with crystal violet), metabolic activity (using an esterase assay) and viability (by determining total cell counts). Structural effects on established biofilms were examined by scanning electron microscopy and epifluorescence following staining by LIVE/DEAD® BacLight, which also showed effects on vitality. The lowest concentration of Medihoney found to prevent biofilm formation was 17%(w/v), whereas on average 35.5%(w/v) of Medihoney was required to inhibit established biofilms. Susceptibility did not vary with length of biofilm establishment between 24 and 72 hours. Extensive structural changes in established biofilms were seen in the sample with less than or equal to 30%(w/v) Medihoney using scanning electron microscopy and loss of viability was found in test samples with less than or equal to 20%(w/v) Medihoney concentration using fluorescent staining, together with loss of biofilm structure. Using a range of methods to evaluate biofilm integrity, this study demonstrates that Medihoney inhibits Pseudomonas aeruginosa biofilms in vitro at concentrations that are attainable in clinical use. Whether Medihoney has the potential to disrupt Pseudomonas aeruginosa biofilms in cutaneous wounds must now be tested in patients. This study was sponsored by Derma Sciences Inc, NJ. An unrestricted grant was provided and the sponsors were not involved in the design of the experiments or the preparation of this manuscript.

The effects of pH on wound healing, biofilms, and antimicrobial efficacy

It is known that pH has a role to play in wound healing. In particular, pH has been shown to affect matrix metalloproteinase activity, tissue inhibitors of matrix metalloproteinases activity, fibroblast activity, keratinocyte proliferation, microbial proliferation, and also immunological responses in a wound; the patient's defense mechanisms change the local pH of a wound to effect microorganism invasion and proliferation; this pH change has been found to affect the performance of antimicrobials, and therefore the efficacy in biological environments directly relevant to wound healing. Based on the available body of scientific evidence to date, it is clear that pH has a role to play in both the healing of and treatment of chronic and acute wounds. It is the purpose of this review to evaluate the published knowledge base that concerns the effect of pH changes, the role it plays in wound healing and biofilm formation, and how it can affect treatment efficacy and wound management strategies.

The effect of negative pressure wound therapy with periodic instillation using antimicrobial solutions on Pseudomonas aeruginosa biofilm on porcine skin explants

Negative pressure wound therapy with instillation (NPWTi) is increasingly used as an adjunct therapy for a wide variety of infected wounds. However, the effect of NPWTi on mature biofilm in wounds has not been determined. This study assessed the effects of NPWTi using saline or various antimicrobial solutions on mature Pseudomonas aeruginosa biofilm using an ex vivo porcine skin explant biofilm model. Treatment consisted of six cycles with 10-minute exposure to instillation solution followed by 4 hours of negative pressure at -125 mm Hg over a 24-hour period. NPWTi using saline reduced bacterial levels by 1-log (logarithmic) of 7-log total colony-forming units (CFUs). In contrast, instillation of 1% povidone iodine (2-log), L-solution (3-log), 0·05% chlorhexidine gluconate (3-log), 0·1% polyhexamethylene biguanide (4-log), 0·2% polydiallyldimethylammonium chloride (4-log) and 10% povidone iodine (5-log), all significantly reduced (P < 0·001) total CFUs. Scanning electron micrographs showed disrupted exopolymeric matrix of biofilms and damaged bacterial cells that correlated with CFU levels. Compared with previous studies assessing microbicidal effects of topical antimicrobial dressings on biofilms cultured on porcine skin explants, these ex vivo model data suggest that NPWTi with delivery of active antimicrobial agents enhances the reduction of CFUs by increasing destruction and removal of biofilm bacteria. These results must be confirmed in human studies.

Antimicrobial and antiseptic strategies in wound management

Wounds, especially chronic wounds, represent a global problem costing millions of dollars per year in developed countries and are characterised by microbial complications including local or overt infection, delayed healing and spread of multiresistant germs. Therefore, antimicrobial wound management is a major challenge that continues to require new solutions against microbes and their biofilms. As systemic antibiotics can barely penetrate into wound biofilms and topically applied ones can easily lead to sensitisation, antisepsis is the method of choice to treat germs in wounds. This brief review discusses the role of antiseptics in reducing bioburden in chronic wounds. Balancing antimicrobial potency and tolerability of antiseptic procedures is critical in wound therapy. However, antiseptics alone may not be able to achieve wound healing without addressing other factors regarding the patient's general health or the wound's physical environment. Although the precise role of bioburden in chronic wounds remains to be evaluated, planktonic as well as biofilm-bound microbes are indications for antiseptic intervention. Octenidine dihydrochloride and polyhexanide are the most effective, as well as best tolerated, antiseptics in wound management today, and new strategies to reduce bacterial wound burden and support the body's immune response are being developed.

An Update on Chronic Wounds and The Role of Biofilms

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. 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. PubMed searches were performed to identify publications on chronic wounds and biofilms. Biofilms contribute to chronic wound nonhealing. There is an abundance of research into novel antibiofilm strategies for chronic wounds. Current research is being targeted at antibiofilm strategies needed to restore an optimal wound-healing environment. A combined treatment approach involving aggressive débridement and the addition of antibiofilm agents is needed.

Antimicrobial Blue Light: An Emerging Alternative to Antibiotics

Antimicrobial Blue Light: An Emerging Alternative to Antibiotics

Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic, biofilm‐containing chronic wounds

Diabetic patients exhibit dysregulated inflammatory and immune responses that predispose them to chronic wound infections and the threat of limb loss. The molecular underpinnings responsible for this have not been well elucidated, particularly in the setting of wound biofilms. This study evaluates host responses in biofilm-impaired wounds using the TallyHo mouse, a clinically relevant polygenic model of type 2 diabetes. No differences in cytokine or Toll-like receptor (TLR) expression were noted in unwounded skin or noninoculated wounds of diabetic and wild-type mice. However, diabetic biofilm-containing wounds had significantly less TLR 2, TLR 4, interleukin-1β, and tumor necrosis factor-α expression than wild-type wounds with biofilm (all p < 0.001). Both groups had similar bacterial burden and neutrophil infiltration after development of biofilms at 3 days postwounding, but diabetic wounds had significantly less neutrophil oxidative burst activity. This translated into a log-fold greater bacterial burden and significant delay of wound epithelization for biofilm-impaired diabetic wounds at 10 days postwounding. These results suggest that impaired recognition of bacterial infection via the TLR pathway leading to inadequate cytokine stimulation of antimicrobial host responses may represent a potential mechanism underlying diabetic susceptibility to wound infection and ulceration.

Biofilms and Inflammation in Chronic Wounds

The incidence, cost, morbidity, and mortality associated with non-healing of chronic skin wounds are dramatic. With the increasing numbers of people with obesity, chronic medical conditions, and an increasing life expectancy, the healthcare cost of non-healing ulcers has recently been estimated at $25 billion annually in the United States. The role played by bacterial biofilm in chronic wounds has been emphasized in recent years, particularly in the context of the prolongation of the inflammatory phase of repair. Rapid high-throughput genomic approaches have revolutionized the ability to identify and quantify microbial organisms from wounds. Defining bacterial genomes and using genetic approaches to knock out specific bacterial functions, then studying bacterial survival on cutaneous wounds is a promising strategy for understanding which genes are essential for pathogenicity. When an animal sustains a cutaneous wound, understanding mechanisms involved in adaptations by bacteria and adaptations by the host in the struggle for survival is central to development of interventions that favor the host. Characterization of microbiomes of clinically well characterized chronic human wounds is now under way. The use of in vivo models of biofilm-infected cutaneous wounds will permit the study of the mechanisms needed for biofilm formation, persistence, and potential synergistic interactions among bacteria. A more complete understanding of bacterial survival mechanisms and how microbes influence host repair mechanisms are likely to provide targets for chronic wound therapy.

Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilms

Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms.

First Evidence of Sternal Wound Biofilm following Cardiac Surgery

Management of deep sternal wound infection (SWI), a serious complication after cardiac surgery with high morbidity and mortality incidence, requires invasive procedures such as, debridement with primary closure or myocutaneous flap reconstruction along with use of broad spectrum antibiotics. The purpose of this clinical series is to investigate the presence of biofilm in patients with deep SWI. A biofilm is a complex microbial community in which bacteria attach to a biological or non-biological surface and are embedded in a self-produced extracellular polymeric substance. Biofilm related infections represent a major clinical challenge due to their resistance to both host immune defenses and standard antimicrobial therapies. Candidates for this clinical series were patients scheduled for a debridement procedure of an infected sternal wound after a cardiac surgery. Six patients with SWI were recruited in the study. All cases had marked dehiscence of all layers of the wound down to the sternum with no signs of healing after receiving broad spectrum antibiotics post-surgery. After consenting patients, tissue and/or extracted stainless steel wires were collected during the debridement procedure. Debrided tissues examined by Gram stain showed large aggregations of Gram positive cocci. Immuno-fluorescent staining of the debrided tissues using a specific antibody against staphylococci demonstrated the presence of thick clumps of staphylococci colonizing the wound bed. Evaluation of tissue samples with scanning electron microscope (SEM) imaging showed three-dimensional aggregates of these cocci attached to the wound surface. More interestingly, SEM imaging of the extracted wires showed attachment of cocci aggregations to the wire metal surface. These observations along with the clinical presentation of the patients provide the first evidence that supports the presence of biofilm in such cases. Clinical introduction of the biofilm infection concept in deep SWI may advance the current management strategies from standard antimicrobial therapy to anti-biofilm strategy.

Biofilm inhibitory and eradicating activity of wound care products againstStaphylococcus aureusandStaphylococcus epidermidisbiofilms in anin vitrochronic wound model

Although several factors contribute to wound healing, bacterial infections and the presence of biofilm can significantly affect healing. Despite that this clearly indicates that therapies should address biofilm in wounds, only few wound care products have been evaluated for their antibiofilm effect. For this reason, we developed a rapid quantification approach to investigate the efficacy of wound care products on wounds infected with Staphylococcus spp. An in vitro chronic wound infection model was used in which a fluorescent Staph.aureus strain was used to allow the rapid quantification of the bacterial burden after treatment. A good correlation was observed between the fluorescence signal and the bacterial counts. When evaluated in this model, several commonly used wound dressings and wound care products inhibited biofilm formation resulting in a decrease between one and seven log CFU per biofilm compared with biofilm formed in the absence of products. In contrast, most dressings only moderately affected mature biofilms. Our model allowed the rapid quantification of the bacterial burden after treatment. However, the efficacy of treatment varied between the different types of dressings and/or wound care products. Our model can be used to compare the efficacy of wound care products to inhibit biofilm formation and/or eradicate mature biofilms. In addition, the results indicate that treatment of infected wounds should be started as soon as possible and that novel products with more potent antibiofilm activity are needed.

Wound biofilms: Lessons learned from oral biofilms

Biofilms play an important role in the development and pathogenesis of many chronic infections. Oral biofilms, more commonly known as dental plaque, are a primary cause of oral diseases including caries, gingivitis, and periodontitis. Oral biofilms are commonly studied as model biofilm systems as they are easily accessible; thus, biofilm research in oral diseases is advanced with details of biofilm formation and bacterial interactions being well elucidated. In contrast, wound research has relatively recently directed attention to the role biofilms have in chronic wounds. This review discusses the biofilms in periodontal disease and chronic wounds with comparisons focusing on biofilm detection, biofilm formation, the immune response to biofilms, bacterial interaction, and quorum sensing. Current treatment modalities used by both fields and future therapies are also discussed.

Biofilms in Chronic Wounds and the Potential Role of Negative Pressure Wound Therapy

Biofilms are communities of microbes that exist in a variety of environments. The extracellular substances secreted by biofilms make them difficult to eradicate, giving the bacterial community in the biofilm a resistance advantage over freefloating (planktonic) microbes. Biofilms are particularly problematic in chronic wounds because of their resistance to conventional therapies and tendency to delay healing. Multimodal strategies to combat wound biofilms are necessary, including wound debridement, antimicrobial treatment, and continued disruption of biofilms. Negative pressure wound therapy with irrigation or instillation may lower the bacterial burden in chronic wounds and prevent the biofilm formation. This article provides an overview of biofilms and evolving strategies to counteract them.