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Biofilms made

Volume 1 | Issue 3 | May 2010 www.woundsinternational.comeasy

tend to attach to surfaces and eventually form Introduction biofilms1,9 (Figure 1). The initial attachment is reversible. This article describes what biofilms are and the Stage two: permanent surface attachment important roles they appear to play in disrupting As the multiply, they become more firmly attached . In addition, it discusses potential (sessile) and differentiate, changing gene expression patterns interventions aimed at removing/reducing biofilms in ways that promote survival6,9. This is usually the result of a 10 and preventing their reformation in wounds. type of bacterial communication known as (see glossary page 5).

Stage three: slimy protective matrix/biofilm Authors: Phillips PL, Wolcott RD, Fletcher J, Schultz GS Once firmly attached, the bacteria begin to secrete a surrounding Full author details can be found on page 5. matrix known as extracellular polymeric substance (EPS)11. This is a protective matrix or ‘slime’. Small bacterial colonies then form What are biofilms? an initial biofilm2,6. Biofilms are complex microbial communities containing bacteria and fungi. The microorganisms synthesise and secrete The exact composition of EPS varies according to the micro- a protective matrix that attaches the biofilm firmly to a living or organisms present, but generally consists of , non-living surface1. , glycolipids and bacterial DNA2.9.11. Bacterial DNA released by living or dead bacteria is thought to provide an Biofilms are dynamic heterogeneous communities that are important structural component for biofilm EPS matrix12. Various continuously changing2. They may consist of a single bacterial secreted proteins and enzymes help the biofilm to become firmly or fungal species, or more commonly, may be polymicrobial, attached to the wound bed9. ie contain multiple diverse species3,4. At the most basic level a biofilm can be described as bacteria embedded in a thick, Fully mature biofilms continuously shed planktonic bacteria, slimy barrier of sugars and proteins. The biofilm barrier protects microcolonies and fragments of biofilm, which can disperse the microorganisms from external threats. and attach to other parts of the wound bed or to other wounds, forming new biofilm colonies5,6. How are biofilms relevant to wounds? Biofilms have long been known to form on surfaces of medical devices, such as urinary , endotracheal and Figure 1 Schematic representation of polymicrobial biofilm tympanostomy tubes, orthopaedic and breast implants, contact formation (adapted from13) lenses, intrauterine devices (IUDs) and sutures5,6. They are a major contributor to diseases that are characterised by an underlying bacterial infection and chronic inflammation, eg periodontal Dispersion of planktonic bacteria and biofi lm fragments from mature biofi lm disease, , chronic and osteomyelitis2,5,7.

Biofilms are also found in wounds and are suspected to delay healing in some. Electron microscopy of biopsies from chronic wounds found that 60% of the specimens contained biofilm structures in Quorum sensing comparison with only 6% of biopsies from acute wounds8. Since biofilms are reported to be a major factor contributing to multiple Bacterial differentiation chronic inflammatory diseases, it is likely that almost all chronic Reversible  permanent attachment wounds have biofilm communities on at least part of the wound bed.

Biofi lm development Contamination Colonisation Spreading  systemic infection How do biofilms form? Infl ammatory host response Stage one: reversible surface attachment Microorganisms are commonly perceived to be free-floating and solitary (ie planktonic). However, under natural conditions most

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Biofilm Kathy ill1.indd 1 28/04/2010 14:37 made Biofilms easy

Living in the mixed microbial standard clinical laboratory biofilm in a wound. However, such a link communities typical of biofilms allows assays18. However, standard clinical between slough and biofilms in chronic microorganisms to share their individual microbiology tests are optimised to wounds is yet to be fully defined. ‘skills and abilities’ for the survival of culture planktonic bacteria, and do not the group14,15. This gives them many adequately measure biofilm bacteria, Currently, the most reliable method to protective advantages. which require special cultivation confirm the presence of microbial biofilm techniques19,20. is specialised microscopy, eg confocal laser scanning microscopy. How quickly do biofilms The term critical colonisation was form? developed in an attempt to acknowledge Experimental laboratory studies16,17 the concept that bacteria play a How do mature biofilms have shown that planktonic bacteria, eg critical role in the failure of wounds ‘protect’ bacteria? Staphylococci, Streptococci, that do not have obvious infection to Biofilms greatly enhance the tolerance of and , typically: heal21. In reality, the concept of critical microorganisms embedded in the matrix n attach within minutes colonisation/localised infection probably to the , n form strongly attached describes the presence of a biofilm in a and environmental stresses (eg microcolonies within 2–4 hours . nutritional or oxygen limitation). This n develop initial EPS and become tolerance may approach complete increasingly tolerant to biocides, resistance to factors that would easily kill eg , antiseptics and Can we see biofilms? these same microbes when growing in disinfectants, within 6–12 hours Biofilms are microscopic structures. an unprotected, planktonic state9,25. n evolve into fully mature biofilm However, in some situations, when colonies that are extremely allowed to grow undisturbed for an Blocking resistant to biocides and shed extended period of time, biofilms One simple way that EPS protects planktonic bacteria within 2–4 can become thick enough to be seen microbes is by preventing large days, depending on the species and with the naked eye. For example, molecules (eg antibodies) and growth conditions plaque can accumulate and inflammatory cells from penetrating n rapidly recover from mechanical become clearly visible within a day. deeply into the biofilm matrix. Mature disruption and reform mature Some bacteria in biofilm phenotype biofilm may also act as a diffusion biofilm within 24 hours. produce pigments, which may barrier even to small molecules like aid visual detection of biofilm. For agents26. This suggests that serial wound example, debridement/disruption could provide produces the quorum sensing molecule Mutual protection only a brief window of opportunity, ie pyocyanin, which is green, when in Another unique property of less than 24 hours, in which antimicrobial biofilm phenotype22. Even so, green polymicrobial biofilms is the cooperative treatments are more effective in discolouration of a wound is not always protective effects that different species reducing both planktonic and biofilm indicative of a Pseudomonas biofilm. of bacteria can provide to each other. microorganisms in wounds. For example, resistant bacteria Can biofilms be distinguished may secrete protective enzymes or from slough? antibiotic binding proteins that can Why have we just found Wound slough has been described as a protect neighbouring non-antibiotic out about biofilms in viscous, yellow, and relatively opaque resistant bacteria in a biofilm2, as well wounds? layer on wound beds, while biofilm found as transfer genes to other bacteria It is only relatively recently that biofilms in wounds has been suggested to appear that confer antibiotic resistance, even have been generally accepted as a factor more gel-like and shiny23. Nevertheless, between different species27. Studies that can contribute to delay in healing in there may be a link between biofilms and have also shown that the specific skin wounds7,8. slough. Biofilms stimulate inflammation, characteristics of the EPS of biofilms which increases vascular permeability established by one species can play a Chronic skin wounds often lack overt and production of wound exudate and significant role in the ability of other clinical signs of infection and often have the build up of fibrin slough24. Therefore, species to attach and incorporate into an low bacterial burdens as measured by slough may indicate the presence of existing biofilm28.

2 Hibernation (quiescent bacteria) the response is in the interest of the biofilm. By inducing an Another survival strategy that many bacteria in biofilms have ineffective inflammatory response, the biofilm protects the developed is for a subpopulation to become metabolically quiescent, microorganisms it contains and increases exudate production, ie to hibernate2,29,30. Because bacteria need to be metabolically active which provides a source of nutrition and helps to perpetuate the for antibiotics to act, hibernating bacteria in biofilms are unaffected biofilm36. by antibiotics that would normally kill active bacteria2,31.

Research has shown that the lowest concentration required to Are there conditions that predispose a kill or eliminate bacterial biofilm for many antibiotics actually wound to develop a biofilm? exceeds the maximum prescription levels for the antibiotics31-34. It is not known whether there are conditions that predispose Thus, standard oral doses of those antibiotics, which effectively wounds to developing a biofilm. However, general conditions kill the normally susceptible bacteria when grown planktonically that impair the immune system or reduce the effectiveness in a clinical laboratory, may have little or no antimicrobial effect of antibiotic drugs may favour the development of biofilms on the same type of bacteria in biofilm form in the patient. in wounds. These include tissue ischaemia or necrosis, poor nutrition and comorbidities that impair immune function. How do biofilms delay wound healing? Biofilms stimulate a chronic inflammatory response in an What are the principles of managing attempt to rid the wound of the biofilm (Figure 2). This response biofilms? results in abundant neutrophils and macrophages surrounding Even when a wound is strongly suspected of containing a biofilms. These inflammatory cells secrete high levels of reactive biofilm, there is no one-step for treatment. A proactive oxygen species (ROS) and proteases (matrix metalloproteinases approach using a combination strategy based on elements of (MMPs) and elastase). The proteases can help to break down the wound bed preparation37 may be helpful (Figure 3) and aims to: attachments between biofilms and the tissue, dislodging the n reduce the biofilm burden biofilms from the wound35. However, the ROS and proteases also n prevent reconstitution of the biofilm. damage normal and healing tissues, proteins and immune cells and have ‘off target’ effects that impair healing. This approach is sometimes called ‘biofilm-based wound care’.

The chronic inflammatory response is not always successful in removing the biofilm and it has been hypothesised that How can biofilm burden be reduced? Evidence to date suggests that physical removal, ie debridement or vigorous physical cleansing, are the best methods for reducing biofilm burden37. Figure 2 Delayed wound healing: host factors, microbial factors Debridement involves the removal of necrotic and Antibodies and topical and systemic antibiotics are unable to penetrate the biofilm, contaminated tissue and matter from a wound so that healing which also acts as a microbial reservoir for infection of neighbouring tissue can occur. There are numerous methods of debridement, ranging from sharp debridement to techniques usually thought of as wound cleansing, eg wound irrigation38,39. Macrophages unable to clear biofilm Choice of method of debridement or cleansing by a clinician will be heavily influenced by knowledge, training and competency, and must take into account safety and ethical considerations40.

Chronic inflammation and release of proteases and Bacterial cytotoxins kill Research into the management of wound biofilms has so far reactive oxygen species result in tissue damage host immune cells used sharp debridement and ultrasonic debridement with Dispersed biofilm Host secreted the aim of opening all tunnels and removing undermining, fragments and Macrophages Antibiotics proteases and reactive 41 planktonic bacteria oxygen species all devitalised tissue, slough and discoloured or soft bone . Biofilm bacteria However, because of the difficulties of visualising biofilms, the Bacterial embedded in Neutrophils Antibodies cytotoxins impact of debridement and the best method of debridement for protective EPS biofilm management is not yet clear.

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Biofilm2 Kathy.indd 1 29/04/2010 12:00 Frequency of debridement/ cleansing No form of debridement or cleansing is Figure 3 Principles of wound biofilm management likely to remove all of a biofilm, and so any remaining bacteria/biofilm has the Chronic wound potential to regrow and form mature Static healing, moderate improvement with repeated rounds of oral antibiotics biofilm within a matter of days. As a result it is suggested that debridement in Suspected biofilm a wound suspected of containing biofilm needs to be performed regularly. As Reduce biofilm burden yet, the ideal frequency of debridement  debridement/vigorous cleansing is not clear; in a study of patients with critical limb ischaemia, debridement was Prevent recontamination with microorganisms  barrier dressing weekly41. AND Suppress biofilm reformation sequential topical antimicrobials Some products are suggested to have additional roles in wound cleansing Reassess healing by aiding removal of bacteria and debris, and disturbing biofilm. A Healed promising technology, for example, lies in the surfactant properties of some polyhexamethylene biguanide (polyhexanide or PHMB) wound cleansing formulations (eg Prontosan®). contamination (ie the use of dressings), How will I know when The surfactant component (betaine) and the use of antimicrobial agents to the biofilm has gone? of the cleansing agent reduces surface kill planktonic microorganisms. The lack of definitive signs and readily tension and aids removal of debris and available laboratory tests for biofilms bacteria by irrigation42,43. The polymicrobial nature of many means that it is not possible to state biofilms indicates that a topical broad categorically when a wound has become If a wound is still not progressing spectrum antimicrobial that kills rather biofilm free. The clearest clinical indicator following regular debridement with one than inhibits microorganisms is the is likely to be healing progression, along method, it may be necessary to consider most appropriate. Details of the effects with a reduction in the production of a more ‘aggressive’ form of debridement of antimicrobials on biofilm reformation exudate and slough. with specialist referral as appropriate. are not yet known. However, the broad spectrum microbicidal antimicrobials Until clear guidance becomes most widely used in wound care are available, clinical judgement will be How can biofilm silver, iodine, honey and PHMB. These are required to decide when and how to reconstitution be available in a range of formulations. modify the management of wounds prevented? with suspected biofilm. For example, Biofilm may reform in a wound by: An emerging principle for the use of when healing is progressing well, it may n growth of fragments left behind topical antimicrobials is changing to a be appropriate to change debridement following debridement/cleansing different antimicrobial if there is lack of method or reduce debridement n spread of planktonic bacteria progress. As yet, there is no evidence to frequency, and/or reconsider whether released from the remaining suggest which antimicrobial is preferable the use of a topical antimicrobial is biofilm first line; choice will be dependent on necessary. n growth of biofilm by newly how the antimicrobial will be used. introduced microorganisms. For example, is it to be left in place for Additional important concepts include several days? If so, a sustained release frequent wound reassessment and Principles involved in preventing formulation will be required to cover a holistic approach to patient health reconstitution of the biofilm include the period of use. Patient sensitivities/ to boost the immune system and to prevention of further wound allergies must also be taken into account. promote wound healing.

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Future developments dressings against mature Pseudomonas a combination of debridement and/ There is a need to develop methods or aeruginosa biofilm cultured on porcine or cleansing to remove the biofilms, devices to quickly detect the presence skin has revealed significant biofilm application of dressings to block new of biofilm before and after selected eliminating properties of cadexomer bacteria from reaching the wound, and treatments. Initially, this would help iodine44. However, the complex the use of antimicrobials to kill bacteria guide researchers and healthcare ever changing polymicrobial nature left in the wound bed. Patients should providers to develop effective wound of biofilm, complicated by biofilm be told that treatment needs to be management strategies. Later, it would bacterial phenotypic heterogeneity repeated and regular because biofilms aid monitoring of treatment progress. means that antibiofilm efficacy of can reform within a day and prevent agents must be verified on a patient by wound healing. Both currently available and novel patient basis. antimicrobial agents and treatment methods are being scrutinised for their Supported by an educational grant from efficacy against biofilm, both as biofilm How do we explain B. Braun. The views expressed in this ‘Made eliminators and biofilm inhibitors. biofilms to patients? Easy’ section do not necessarily reflect For example, recent studies of the Patients can be reassured that those of B. Braun. Prontosan® is a registered antimicrobial efficacy of various wound biofilms can be effectively treated by trademark of B.Braun.

Glossary Author details Biofilm phenotype Biofilm phenotype microorganisms express genes optimal for developing an PL Phillips1, RD Wolcott2, J Fletcher3, attached community embedded in a protective self-secreted 'slime' matrix and surviving environmental stresses. GS Schultz4. Commensal A that lives on/in a tissue, usually without causing disease. 1. Postdoctoral Fellow, Institute for Wound Research, Department of Obstetrics Extracellular The 'slime' produced by microorganisms in a biofilm comprising and Gynecology, University of Florida, polymeric polysaccharides, proteins, glycolipids and bacterial DNA; it protects the Gainesville, Florida, USA substance (EPS) microorganisms living in the biofilm from the host's immune system and 2. Medical Director, Southwest Regional antimicrobial agents. Wound Care Center, Lubbock, Texas, USA Phenotype The characteristics of a microorganism that result from an interaction between 3. Principal Lecturer, School of Nursing, the environment and the microorganism's genes. Midwifery and Social Work, University of Hertfordshire, Hatfield, UK and Senior Planktonic Refers to free-floating microorganisms that are expressing genes optimal for Professional Tutor, Department of single, unattached growth. Dermatology and Wound Healing, School of Medicine, Cardiff University, Cardiff, UK Quorum sensing A mechanism used by microorganisms to communicate within and between bacterial species. It is used to detect and respond to changes in 4. Professor, Institute for Wound the environment (including the presence of other microbes or nutritional Research, Department of Obstetrics limitations). Quorum sensing induces changes in bacterial gene expression that and Gynecology, University of Florida, Gainesville, Florida, USA aim to promote survival of the microorganisms. With thanks to the Bedfordshire and Sessile Microorganisms that are firmly attached to a surface by means of receptors Hertfordshire Tissue Viability Nurses’ Forum. and/or proteins called adhesins.

Summary Bacterial biofilms are known to contribute to numerous chronic inflammatory diseases and recent evidence suggests that biofilms also play important roles in impairing healing in chronic wounds. Biofilms have high levels of tolerance to antibodies, antibiotics, disinfectants and phagocytic inflammatory cells. Current understanding of biofilms suggests that management of suspected wound biofilm should involve frequent debridement along with interventions such as dressings and antimicrobials to prevent recontamination of the wound and suppress biofilm reformation. To cite this publication Phillips PL, Wolcott RD, Fletcher J, Schultz GS. Biofilms Made Easy.Wounds International 2010; 1(3): Available from http://www.woundsinternational.com

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Further reading Bryers JD. Medical Biofilms. Biotechnology and Bioengineering 2008: 100: 1-18. Davies D. Understanding biofilm resistance to antibacterial agents. Nature 2003; 2: 114-22 Costerton JW, Stewart PS. Battling biofilms. Scientific American 2001; 285: 74-81. Gibson D, Cullen B, Legerstee R, et al. MMPs Made Easy. Wounds International 2009; 1(1). Available from http://www.woundsinternational.com/article.php?issueid=1&contentid=123&articleid=21. 6