H A RD -TO-HEAL WO UNDS | MANAGEM ENT O F B IOFIL M

WORLD UNION OF WOUND HEALING SOCIETIESPOSITION DOCUMENT

MANAGEMENT OFBIOFILMThe role of biofilm in delayed
wound healing
Biofilm management in practice
How is research advancing theunderstanding of biofilm

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PUBLISHER
Clare Bates
MANAGING DIRECTOR
Rob Yates

florence, ITAlY

WUWHS
2016
www.wuwhs.net
How to cite this document
World Union of Wound Healing
Societies (WUWHS), Florence
Congress, Position Document.
Management of Biofilm.
Wounds International 2016

Supported by an educational grant from B Braun
The views expressed in this publication are those of the authors and do not necessarilyreflect those of B Braun
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H A RD -TO-HEAL WO UNDS | MANAGEM ENT O F B IOFIL M

Antimicrobial and multi-drug resistance loom large on the global
healthcare landscape, in particular in the treatment of chronic, hard-toheal wounds where current figures put the presence of biofilm in
60%–100% of non-healing wounds. While the role that biofilm playin the chronicity of wounds is still in infancy, it is becoming widely
accepted that hard-to-heal wounds contain biofilm — and that somehow theirpresence delays or prevents healing.

Management of biofilm in chronic wounds is rapidly becoming a primary objective ofwound care. However management of biofilm is an undeniably complex task. Beyond
the basic steps of initial prevention (use of anti-biofilm agents), removal (debridement,
desloughing) and prevention of reformation (use of antimicrobial agents), there are
myriad patient, environmental and clinical parameters that must be considered whenidentifying a tailored solution.
Detection and localisation of biofilms in chronic wounds provide useful clinicalinformation that helps assess and direct the effectiveness of debridement. Yet gaps in
the knowledge base remain in detecting and localising biofilm. While existing guidelines(e.g. ESCMID 2015) do offer direction in diagnosis and treatment of biofilm infection,questions remain unanswered, including whether there are visual signs that might beuseful in deciding whether or not to take a biopsy.
As the debate around whether or not biofilm can be seen with the naked eye gathers paceand new techniques (e.g. Nagakami and colleagues’ ‘biofilm wound map’) come to light,
there still reminas a critical need for a ‘point-of-care’ biofilm detector that can detect thepresence of biofilm in minutes, not hours or days.
While significant progress has been made in prevention, detection and managementof biofilm, more research is needed to reduce the impact on patients and on healthcare
systems alike.
In this Position Document, leading clinicians look at the role biofilm plays in delayed
wound healing; the management of biofilm in practice, and how research — existing andyet to come — will further understanding of these bacterial communities.

Authors
Thomas Bjarnsholt, Costerton Biofilm Center, Department of Immunology & Microbiology,
Faculty of Health and Medical Sciences, The University of Copenhagen, Denmark; Departmentof Clinical Microbiology, Rigshospitalet, Denmark
Rose Cooper, Cardiff School of Health Sciences, Cardiff Metropolitan University, Cardiff, UK
Jacqui Fletcher, Independent Nurse Consultant, UK
Isabelle Fromantin, Wounds and Healing Expert, Institut Curie, France
Klaus Kirketerp-Møller, Copenhagen Wound Healing Center, Bispebjerg University Hospital,
Copenhagen, Denmark
Matthew Malone, MSc, PhD candidate, FFPM RCPS (Glasg), Head of Department PodiatricMedicine, High Risk Foot Service, Liverpool Hospital Research Fellow, LIVE DIAB CRU,
Inghams Institute of Applied Medical Research, Sydney, Australia
Greg Schultz, Institute for Wound Research, Department of Obstetrics & Gynecology,
University of Florida,USA
Randall D Wolcott, President, Professional Association and Research and Testing Lab of the South
Plains, Texas, USA
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The role of biofilms indelayed wound healing

B
Thomas Bjarnsholt,
Costerton Biofilm Center,
Department of Immunology& Microbiology, Facultyof Health and Medical
Sciences, The Universityof Copenhagen and
Department of ClinicalMicrobiology, Rigshospitalet,
Denmark; Greg Schultz,
Institute for WoundResearch, Department of
Obstetrics & Gynecology,
University of Florida, USA;
Klaus Kirketerp-Møller,
Copenhagen Wound
Healing Center, Bispebjerg
University Hospital,
Copenhagen, Denmark;Jacqui Fletcher, Independent
Nurse Consultant, UK and
Matthew Malone, MSc,
PhD candidate, FFPM RCPS
(Glasg), Head of Department
Podiatric Medicine, High
Risk Foot Service, Liverpool
Hospital Research Fellow,
LIVE DIAB CRU, Inghams
Institute of Applied Medical
Research, Sydney, Australia

acteria are often viewed as being single cells that multiply rapidly whenin exponential growth, and are susceptible to antibiotics if not inherently
resistant. Antimicrobial resistance and multi-drug resistance are an
increasing problem across the globe, and are a current hot topic subject
to much debate. Most clinicians involved in the treatment of wounds will
utilise susceptibility patterns they receive from the clinical microbiology laboratory as
a guide to determine which antibiotic(s) a patient requires. These decisions are often
aided by international consensus guidelines, which are sufficient when managing
acute infections[1,2,3,4]. However, in cases of chronic infection, such as those seen for
implantable medical devices, pulmonary infections of cystic fibrosis (CF) patients andchronic non-healing wounds, these guidelines may be inadequate. Why is this? How
can we explain the quick resolution of infective symptoms using antimicrobial agentsin patients with acute wounds, in comparison to the lethargic or non-response often
noted in non-healing chronic wounds?

The answer is both complicated and also rather simple (Box 1, page 7). Bacteria can exist
in at least two different phenotypic growth forms: the first being single, fast-growing cells
i.e. the planktonic form; the second as aggregated communities of slow-growing cells
in a biofilm form. All classic microbiology and development of antimicrobials have been
based solely on planktonic paradigms, through methods developed in the early 1800s.
It is considerably easier to grow bacteria using these methods, through shaken cultures
or by spreading on an agar plate — and it is how bacteria presumably exist during acuteinfections. These methods are still widely accepted as ‘gold standard’ for depicting thepathogens of acute infections.
The picture for chronic infections is the complete opposite, however. In this case, asubstantial amount of the bacteria reside in biofilms, where they are surrounded by adense matrix of polysaccharides, free DNA (eDNA) of either bacterial or host origin,and proteins that attach tightly to the biofilm community and structures, protecting
them from being engulfed and killed by neutrophils and macrophages. In addition, manyof the bacteria are not dividing or metabolising rapidly, which causes them to become
tolerant — almost all antibiotics kill only metabolically active bacteria by inhibiting criticalbacterial enzymes. It is important to realise that most chronic infected wounds harbour
several different bacterial species requiring different treatments, such as antibiotics[5,6,7].
However, the different species are not necessarily within the same biofilm but rather
scattered around in small, sovereign, single-species islands[8,9,10].
In this review we will explore the implications of biofilms in human chronic, non-healing
wounds, presenting evidence or hypothesis of how biofilms delay wound healing. We will
also address the clinical conundrum of how to diagnose biofilm within wounds and thebest methods in their treatment.
DEFINITION OF BIOFILM
Biofilms are frequently defined based on in vitro observations. Classic definitions often
describe biofilms as bacteria attached to surfaces, encapsulated in a self-produced
extracellular matrix and tolerant to antimicrobial agents (this includes antibiotics andantimicrobials). In addition, biofilm development is often described as a three-to-five-stage
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H A RD -TO-HEAL WO UNDS | MANAGEM ENT O F B IOFIL M“Antimicrobial resistance andmulti-drug resistance are an
increasing problem across theglobe, and are a current hot
topic subject to much debate”

scenario, beginning with single cells attaching to a surface, maturation of the biofilm and,lastly, dispersal of bacteria from the biofilm[11,12,13]. In vitro observations, based on flow cell
models utilising glass surfaces and fresh oxygenated culture media continuously flowing
over the bacterium, differ greatly when compared to conditions within chronic wound
infections[14]. Here, the bacteria are not exposed to a continuous flow of fresh media and are
not attached to a glass surface (or indeed any surface)[6,10]. In vivo chronic wound biofilm areoften encapsulated in a matrix, which includes host material, making dispersal problematic.
Therefore, using in vitro observations to define, diagnose and treat biofilms in chronic
infections may provide a misguided impression[15]. There are, however, commonalities
between in vitro and in vivo evidence that can help in providing a definition of a biofilm.
These include:n Aggregation of bacterian Some sort of matrix that is not restricted to self-produce as it canalso be of host origin
n Extreme tolerance and protection against most antimicrobial agentsand the host defence.
We suggest following this simplified definition in order to define biofilms in chronic
infections: an aggregate of bacteria tolerant to treatment and the host defence.
HOW DO BIOFILM COMMUNITIES DIFFER FROM PLANKTONIC BACTERIA?
All planktonic bacteria are single cells that are usually fast growing and are rarely
observed directly in infections, except during severe conditions such as sepsis[14].
However, we assume that during acute infections bacteria are of the planktonic
phenotype, since they are susceptible to antimicrobial agents with targeted treatments
causing an abrupt resolution of symptoms.
In vivo evidence has suggested biofilm phenotypes differ markedly in both their physiology
and activity when compared with planktonic cells. The bacteria are aggregated anddifficult to treat, if not impossible, somehow evading host defences[3,14]. Often the bacteria
are embedded in a matrix which can be produced by the bacteria or is of host origin. The
exact composition of extracellular polymeric substance (EPS) varies according to themicroorganisms present, but generally comprise polysaccharides, proteins, glycolipids
and extracellular DNA (eDNA)[16, 17, 18].
Microelectrode studies have further identified anoxic regions within a biofilm, resulting
in lower bacterial cell metabolic activity[19,20,21]. This contributes in part to the inherent
resilience of biofilms to antimicrobial treatments.
PREVALENCE OF BIOFILMS IN CHRONIC WOUNDS
Less than 10 studies have visualised biofilms in non-healing chronic wounds using theaccepted approaches of microscopy with or without molecular analysis[6, 9,10,21-24]. Thesestudies identified the presence of biofilms in 60% to 100% of samples. In light of theheterogeneity and spatial distribution of biofilms within chronic wounds, the failure ofsampling techniques to capture tissue ‘housing’ biofilm could potentially see the ‘true’
prevalence being closer to 100%[7,10].
DETECTING BIOFILMS IN CHRONIC WOUNDS
We have addressed these issues in reverse, for which our rationale will become
apparent. Current accepted methods to visualise biofilm from tissue samples have been
confined primarily to the use, by researchers, of high-powered microscopes (scanningelectron microscopy — SEM; confocal laser scanning microscopy — CLSM) alone or in
combination with molecular DNA sequencing techniques that use fluorescent probes
to determine the presence or absence and location of bacteria. Even these approaches
have limitations, in particular the heterogeneous distribution of bacteria within a wound.

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This makes the choice of wound sampling challenging; a tissue biopsy is ‘gold standard’ butwill only collect bacteria from a small area, significantly increasing the chances that somerelevant bacteria will be missed completely[7]. In comparison, the use of superficial swabs
using the Levine technique can sample a broad area but will only collect the bacteria on thewound surface, and this may not necessarily reflect the microbiota[25,26].

Picture 1: Tissue biopsy froma chronic, non-healing DFU
complicated by biofilm viewed underscanning electron microscopy. Notethe aggregates of microbial cells withproduction of extracellular polymeric
substance (EPS) which resembles alattice or spider web appearance

Picture 2: Scanning electron
microscopy viewed from a DFU with
biofilm. Coccoid microorganisms are
surrounded by EPS which resembles alattice network

There has been much debate over whether biofilms, which are microscopic in nature, canbe seen with the naked eye. In differing human health and disease conditions biofilms, whenleft to thrive, may show evidence at a macroscopic level, one example being oral plaque[27].
However, the picture is less clear for chronic wounds. Some clinicians have used rhetoric
to promote what they believe are ‘clinical cues’ of biofilm presence through naked-eye
observations that are not based on scientific rigour[2,28,29]. Such signs have included; a shiny,translucent, slimy layer on the non-healing wound surface[28,29]; the presence of slough or
fibrin and gelatinous material reforming quickly following removal, in contrast to slough andother devitalised tissue or fibrin that often takes longer to reform[29,30,31].
Currently, there is no ‘gold standard’ diagnostic test to define the presence of wound biofilm
and no quantifiable biomarkers. This may pose a significant clinical challenge given that
distinguishing between planktonic or biofilm phenotype pathogenicity in chronic wound
infection is a major barrier to effective treatment.
Based on our previous statement that ‘all non-healing chronic wounds potentially harbour
biofilms’, relying on anecdotal visual cues is unnecessary. We propose that clinicians should‘assume all non-healing, chronic wounds that have failed to respond to standard care havebiofilm’ and, therefore, treatments should be targeted towards this. We suggest that clinicalsuspicion of the presence of biofilm be raised in those patients where chronic wound
infections have failed to respond adequately to antimicrobial agents and standard woundcare treatment, or where chronic wound infections experience periods of quiescence thatalternate with acute episodes[32]. These signs and symptoms are based on current evidence
identifying that biofilms cannot be eradicated by antimicrobial agents, so it is fair to assume
that a non-healing, chronic wound contains bacteria in the biofilm phenotype.
HOW DO BIOFILMS INHIBIT WOUND HEALING?
The exact mechanisms by which biofilm impairs the healing processes of wounds remain
ambiguous. Current data suggest the wound is kept in a vicious inflammatory statepreventing normal wound healing cycles from occurring. The pathways behind this are notclear, but several systemic and local factors contribute to the occurrence and maintenanceof a chronic wound. At the systemic level, physiological factors include diabetes mellitus,
venous insufficiency, malnutrition, malignancy, oedema, repetitive trauma to the tissue andimpaired host response. The majority of chronic wounds will heal if the predisposing factors
are treated properly; for example, oedema reduction in venous leg ulcers, off-loading indiabetic foot ulcers and pressure ulcers, along with moist wound healing principles.
At local level bacteria colonise all chronic wounds; the most commonly reported are
Staphyloccocus aureus and Pseudomonas aeruginosa — two renowned biofilm formers. In a
paper by Gjødsbølk et al[33], 93.5% of chronic leg ulcers contained S. aureus and 52.2 %harboured P. aeruginosa, but only the ulcers with P. aeruginosa were characterised by larger
wound sizes and slower healing rates. This could be explained by the ability of P. aeruginosato eliminate polymorphonuclear leucocytes (PMN) by secreting rhamnolipid[34]. This
glycolipid is controlled though the quorum sensing system and is probably one of the main
mechanisms behind the lack of eradication of P. aeruginosa in chronic wounds.
In expanding further on the role of PMN, Ennis et al (2000) [26] stated that chronic wounds
were ‘stunned in the inflammatory phase of healing’. In normal wound healing trajectories
this phase would be proceeded by a proliferative phase, where the function of PMN are
gradually overtaken by macrophages, and fibroblasts begin to rebuild the tissue[26].

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H A RD -TO-HEAL WO UNDS | MANAGEM ENT O F B IOFIL M“We propose that clinicians
should ‘assume all nonhealing, chronic wounds thathave failed to respond to
standard care have biofilm’and, therefore, treatments
should be targeted towards
this”

Box 1: Biofilms — challenging current wound management practices
Biofilms present several challenges for traditional wound management and wound healing.
Firstly, locating biofilms in wound beds can be difficult, and clinicians are usually limited todebriding areas that have secondary signs of biofilms — ‘wound slough’ and other surface signsof local inflammation.
Secondly, optimal sampling of both the surface and subsurface regions of wound beds is difficult
and the bacteria are very heterogenously distributed. Subsequent identification of biofilm
bacteria is therefore a challenge because a standard clinical microbiology lab is not aware of themore complicated nature of biofilms and does not process wound samples to disperse biofilms
adequately in order that bacteria can be cultured by standard plate growth assays.
The biofilms interfere with normal wound healing, apparently by ‘locking’ the wound bed into achronic inflammatory state that leads to elevated levels of proteases (matrix metalloproteaseand neutrophil elastase) and reactive oxygen (ROS) that damage proteins and molecules thatare essential for healing. A large percentage of bacteria in biofilm communities are metabolicallydormant, which generates tolerance to antibiotics. Highly chemically reactive disinfectant
molecules frequently react with the components of the biofilm exopolymeric matrix, depleting
their concentration and impeding their penetration deep into the biofilm matrix.
The consequences, therefore, of sustained, in situ necrosis by bacterial cells could
explain both the constant influx of PMN into chronic wounds containing P. aeruginosaand the resulting localised release of proteolytic enzymes that are pro-inflammatory[35].
Unfortunately, we cannot postulate the mechanism responsible for this phenomenon in
non-Pseudomonas infested wounds[36].
In 2015, Marano et al[37] identified that migration and proliferation of human epidermal
keratinocytes were decreased by derivatives from biofilms of P. aeruginosa and S. aureus.
Employing proteomic analysis allowed Marano et al to map S. aureus activity to a protein,while P. aeruginosa activity was more likely due to a small molecule[37]. The several
proteins revealed through proteomic analysis had putative links to delayed wound healing.
These included α-haemolysin, alcohol dehydrogenase, fructose-bisphosphate aldolase,
lactate dehydrogenase and epidermal cell differentiation inhibitor.
A second research area of interest has suggested that infecting bacterial biofilms
contribute to a localised low oxygen tension within the wound. Early in vitro studies using
microelectrodes identified discrete areas of significant oxygen depletion within biofilms[38].
Further studies employing microelectrodes with CLSM, identified micro-domains withdifferent areas of the biofilm harbouring alternate biochemical environments, including
alterations in pH and oxygen[39]. The creation of anoxic areas within biofilm may explain
the presence of anaerobes in mixed-species biofilms. The anoxic conditions have also been
seen in chronic pulmonary infections in patients with CF[40]. Within a chronically infected CF
lung, PMN have been shown primarily to consume oxygen resulting in oxygen depletion that
suffocates the bacteria causing lower metabolic activity[19,20].
In 2016 data by James and colleagues provided further evidence to support a conceptof localised low oxygen tensions contributing to wound chronicity[21]. Using oxygen
microsensors and transcriptomics (examining microbial metabolic activities) to study insitu biofilms, James and colleagues identified steep oxygen gradients and induced oxygenlimitation stress responses from bacteria. Taken collectively, these data support theconcept that biofilm helps to maintain localised low oxygen tensions in the wound, thus
contributing to chronicity[21].
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The presence of the highly persistent biofilms results in a chronic inflammatory state within the wound
bed that leads to elevated levels of proteases (matrix metalloprotease and neutrophil elastase) andreactive oxygen species (ROS) that damage the proteins and molecules that are vital for healing[41]. By‘locking’ the wound bed into a chronic inflammatory state, biofilms disrupt normal wound healing.
Our current understanding of how biofilms inhibit wound healing remains scarce, but the two
examples above postulate how wound healing can be delayed. It is also apparent that systemic
factors contribute to a paradoxical state of play. It is possible that in some cases the bacterial
biofilm is the primary inhibitor of wound healing. Yet in other circumstances some of thesewounds will heal if the original cause of the wound is addressed (e.g. compression therapy fora venous leg ulcer or off-loading a diabetic foot ulcer). Certainly some chronic wounds will not
heal, despite proper treatment of local impairment. These wounds may prove to have especiallyvirulent bacterial content.
The wound treadmill (Figure 1) illustrates this paradox. The force driving clockwise momentum isthe sum of the virulence of the bacteria while the figure in the centre that is driving counterclockwise
movement represents the sum of the healing capacity of the patient. The healthier the patient (local
and systemically), the more virulent the bacteria need to be to prevent or halt healing. This implies
that ‘weak’ patients will suffer from even the most opportunistic infections. The current treatment ofchronic wounds aims at reducing local impairment by modalities such as compression, off-loading
and moist wound dressings. In addition, the systemic impairments are managed by correcting themalnourished patient or by adjusting glycosylated haemoglobin (HbA1c) levels.
CONCLUSION
It is apparent from this review that diagnosing, treating and understanding the role biofilms play inthe chronicity of wounds is still in its infancy. Scientific endeavour into this niche area is gatheringpace with mounting evidence suggesting we are on the right track. It is becoming widely acceptedthat non-healing, chronic wounds contain biofilms, and that these somehow delay or prevent
wound healing. More focused research ensuring standardisation between study methodologies,
such as optimal sampling techniques, will ensure comparability between studies. New treatment
paradigms are required, but in order to achieve this the development of in vitro models that mimic
the actual wound environment are required.
Lastly, more interdisciplinary collaborations between front-line clinicians and basic scientists
are needed to bridge the gap between what is clinically relevant to patients suffering with
biofilm-related complications.

Figure 1 | Wound Treadmill
The Wound Treadmill (Figure 1)
illustrates the paradox in chronic
wounds: why do some patientsdevelop chronic wounds whileothers do not? The person in thecentre is forcing the wheel toturn counterclockwise and thedriving force on the outer rim is
the combined virulence of thebacteria. Hence the ‘stronger’
the person the more virulenceis required from the bacteria
to prevent healing. See text for
further explanation.

Polymicrobialcolonisation

Skin defectBacterialcolonisation

Tissue damage
Necrosis

PMN elimination

Biofilm
Quorum sensing

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Biofilm management in practiceT

he prevention and management of biofilm in chronic wounds is rapidly
becoming a primary objective of wound care, with the presence ofbiofilm acknowledged as a leading cause of delayed wound healing[1-4].

Figure 1 depicts the basic principles of wound management for cases where
wounds have stalled during healing in spite of repeated antibiotic treatment, and sopresence of biofilm may be suspected. This article looks at when to treat a suspectedbiofilm, various strategies for its prevention and treatment, how these strategies may be
combined for optimum success, and principles for monitoring this success.

Figure 1 |Principles ofwound biofilm
management[5]CHRONIC WOUND
Static healing, moderate improvement with repeated rounds of oral antibiotics

Suspected biofilm

Reduce biofilm burden
Debridement/vigorous cleansing

Prevent recontamination with microorganisms
barrier dressing
AND
Suppress biofilm reformation
sequential topical antimicrobials

Reassess healing

HealedJacqui Fletcher, Independent
Nurse Consultant, UK;
Randall D Wolcott,President, Professional
Association and Research
and Testing Lab of the South
Plains, Texas, USA andIsabelle Fromantin, Wounds
and Healing Expert, Institut
Curie, France

While acute infections tend to produce the classic signs and symptoms of wound
infection, such as inflammation, pain, heat, redness and swelling[6], microbes growing as
biofilm produce a distinctly different pattern, often recognised as chronic infection[7].
Systemic treatment strategies are required for infected chronic wounds, whereas in
non-infected wounds where the presence of biofilm is impeding healing, strategies canbe adopted to break up the biofilm. Alternately, attempts can be made to prevent initial
biofilm formation in patients or wounds judged to be at high risk [8].
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Targeted therapies could be used to improve healing in cases where microbial biofilm isa causal component of chronic wounds as opposed to non-pathogenic colonisation; forexample:n Early use of systemic antibiotics directed at planktonic bacterian Unique strategies to make microbes more susceptible to antimicrobials
for clearance by the host immune systemn Therapies directed at preventing a prolonged inflammatory componentof wound healing[9].
With this in mind, it is important that novel strategies to prevent and treat biofilm aredeveloped[3], which confer:
n Preventative action, interfering with either microbial attachment or processes involved
in biofilm maturation or removal, and/or disruption of mature biofilmn Action against existing biofilm, removing or disruption of the biofilm andprevention of reformation.
WHEN TO TREAT A BIOFILM
Expertise in chronic wound treatment, particularly strategies for treating infected wounds
and recognition of biofilm, is vital in order to ensure patients receive optimum treatment.
The Wounds at Risk (WAR) score was devised to aid decision-making in antimicrobial use(specifically polyhexanide) where there was previously no method to accurately predict
infection risk in chronic wounds. The scoring system considers the quantity and virulenceof a wound’s pathogenic bioburden and the patient’s immune competence, but provides
no support for recognition of biofilm or suggestions for debridement. The existence ofdiagnostics to support detection of biofilm may render the WAR score more helpful[10].
The actual identification of biofilm requires sophisticated laboratory techniques suchas confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM)or molecular techniques for definition[11]. Standard culture microbiology procedures
only detect planktonic bacteria, so a different process must be used to detect bacteria
in biofilms; typically, samples are treated initially to kill all planktonic bacteria, then thebiofilm is physically dispersed with ultrasonic energy and cultured on nutrient agar plates
to determine the extent of biofilm presence[5].
Identification of biofilm in clinical practice is also difficult, with few guidelines available
to facilitate its recognition. Keast et al (2014)[5] propose four main features that may
increase suspicion of the biofilm presence, as follows:
1. Antibiotic failure
2. Infection of >30 days’ duration
3. Friable granulation tissue
4. A gelatinous material easily removed from wound surface that quickly rebuilds.
A recent study that collated current data regarding appearance, behaviour and clinicalindicators associated with biofilm suggested that, on occasion, there may be visualcues suggestive of the presence of biofilm in the wound bed. A number of ‘non-visual’clinical cues were also identified: signs of local infection, failure of antimicrobials, culturenegative swabs or recalcitrance of the wound despite all other factors being addressed.
The authors suggested an algorithm incorporating both visual and non-visual cues could
facilitate more effective biofilm-based wound management[12].
However, there is no evidence to date that biofilm appears as a ‘layer of slime’ on thewound surface, so Percival et al (2015)[13] argue that in the absence of any such scientific
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evidence, manifestation of a slimy, translucent layer can be a crude and often misleadingvisual marker. They propose an approach to biofilm identification similar to Keast et al[5],based on the hierarchical questions below. Where the answer is ‘no’, standard care should
be continued; where the answer is ‘yes’, there is progression to the next question. If theanswer to 5 is ‘no’, then biofilm-based wound management should be initiated (Figure 2)[13].
1. Is the wound failing to heal as expected?
2. Have all appropriate clinical diagnostic and therapeutic procedures been
properly undertaken?
3. Is there evidence of slough or necrotic tissue in the wound?
4. Does the wound show signs of a local infection or inflammation?
5. Is the wound responding to topical or systemic antimicrobial interventions?

Figure 2 |Algorithm todetect suspectedbiofilm[13]Yes

Is the wound failing to heal
as expected?

No

Yes

Have all appropriate clinicaldiagnostic and therapeutic
procedures been properly
undertaken?

No

Yes

Is there evidence ofslough or necrotic tissue inthe wound?

No

Yes

Does the wound show
signs of local infection or
inflammation?

No

No

Is the wound responding
to topical or systemic
antimicrobial interventions?

Yes

Continue withstandard protocols
of careInitiate biofilm based wound
managementl Periodic debridementl Appropriate antimicrobial
treatmentNo

Is the wound showing
evidence of granulation?

Yes

HOW TO TREAT A BIOFILM
Strategies for prevention and treatment of biofilm
Once the likelihood of biofilm presence is established, an appropriate treatment
strategy should be determined, taking into account that there are several stages of biofilm
formation. A proactive approach to treatment recognises that there is noone-step solution for treatment of biofilm, but aims to reduce burden and prevent
its reconstitution[14].
Wolcott (2015)[15] states that: ‘Biofilm-based wound care is predicated on using multiple
different treatment strategies simultaneously including antibiotics, anti-biofilm agents,
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Table 1: Potential anti-biofilm agents
Mode of action

ExamplesFurther details

Interference with biofilm
surface attachment

Lactoferrin
Ethylenediaminetetraacetic acid (EDTA)
Xylitol
Honey

As part of the innate human response mechanism, lactoferrinbinds to cell walls causing destabilisation, leakiness and,ultimately, cell death[17]. EDTA has been used as a permeating
and sensitising agent for biofilm conditions in dentistry and otherfields[18]. Xylitol (an artificial sweetener) and honey have also beenshown to block attachment[17]Interference with quorum
sensing, a mechanismof chemical signalling orcommunication between thecells within the biofilm

Farnesol
IberinAioeneManuka honey

Several agents block or interfere with quorum sensing, including:• Farnesol• Iberin (from horseradish)• Ajoene (from garlic)
Manuka honey has also been shown to down-regulate 3 of the 4
genes responsible for the quorum sensing process[17]Disruption of the extracellular EDTA
polymeric substance (EPS), a
protective matrix secreted byand surrounding the biofilm

EDTA supports and enhances topical antimicrobials by disrupting
the EPS in which microorganisms are encased[18]. Proprietaryproducts also exist that claim, among their actions, to disrupt the
EPS[19]

False metabolites

Gallium, Xylitol

Low doses of Gallium and Xylitol have been shown to interferewith biofilm formation[20]Disruption of existing
biofilm

Betaine(combination of
PHMB and betaine)

Current solutions favoured in the disruption of biofilm containsurfactants, such as betaine, which lower the surface tension of themedium in which they are dissolved, allowing dirt and debris to belifted and suspended in the solution[21,22]

selective antimicrobials and frequent debridement.’ Moreover, Hurlow et al (2015)[16] cautionthat while focused activity against the biofilm is paramount, maximising the host responsemust also be addressed with attention paid to all local and underlying causes of delayed
wound healing.
Potential anti-biofilm agents
In practice, physical biofilm disruption in the form of debridement and/or cleansing,followed by use of antimicrobial agents (such as PHMB or silver) to prevent its reformation,is the primary anti-biofilm option available to clinicians at present; this is discussed in moredetail below[4]. However, various potential anti-biofilm agents that interfere with elements of
their formation or support and enhance the effect of antimicrobials have been investigated;these are summarised in Table 1, categorised by their modes of action. Where such anagent is chosen, this choice should be based on factors including the biocidal capability andlength of activity of the active agent, and the capability of the carrier dressing to manage
presenting symptoms, such as increased levels of exudate.
The importance of wound bed preparation
Preparation of the wound bed, including cleansing and debridement, are importantprinciples of wound management, since wounds must be clean to heal[23]. The concept
of TIME (Tissue, Infection/Inflammation, Moisture, Edge of wound) is a widely accepted
standard of wound management. In the intervening 10 years there have been importantdevelopments including, understanding of biofilm presence (and the need for a simple
diagnostic), the importance of clinical recognition of infection, and the value in repetitiveand maintenance debridement and cleansing of wounds, which is paramount[11].
Where either slough or necrosis is present in a wound, this non-viable tissue should be
removed as it may support the attachment and development of biofilm[24]. The speedof tissue removal should be conducted according to the patient’s ability to undergo theprocedure, the skill and competence of the practitioner, and the safety of the environment
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in which the technique is to be performed[25]. A distinction has recently been drawn
between removal of slough (‘desloughing’)[24] and that of necrotic tissue (debridement).
In order to ensure effectiveness, it is proposed that neither therapy be conducted as aone-off, with both maintenance debridement and desloughing recommended.
Various debridement techniques are available, from surgical (performed in theatre, backto healthy bleeding tissue), and autolytic (use of dressings to facilitate removal of necrotic
tissue[23-24]) through to debridement pads and cloths[26,27]. The current cleansing solutions
favoured to assist in the disruption of biofilm contain surfactants, which lower the surface
tension of the medium in which they are dissolved, making it easier to lift off dirt/debris
and suspend this in solution, to avoid re-contamination of the wound[21,22]. Solutions may
be added directly to the wound, used as soaks on gauze or used as part of an instillation
alongside negative pressure wound therapy[28].
Based on current literature, the combination of polyhexanide and betaine, a surfactant,
has been identified as effective for autolytic wound debridement. In a randomised
controlled trial conducted across six Italian centres (June 2010 — December 2013),
the solution was found to promote wound bed preparation, reduce inflammatory signs,
and accelerate healing of vascular leg ulcers, as well as having a lasting barrier effect.
Indeed, compared with normal saline, the solution was statistically significantly superior(p<0.001) in terms of both wound improvement and reduction in inflammatory signs[23].
Using antimicrobials following debridement to prevent reformation
Once the wound has been appropriately cleansed and as much non-viable tissue removed
as is comfortable for the patient, it is suggested that an antimicrobial product be used
to prevent reformation of the biofilm (Figure 1)[5]; for example, anti-biofilm dressings
containing antimicrobials agents like PHMB, silver and a surfactant[29]. A number of active
antimicrobial agents have been linked to biofilm treatment:n Acetic acid30]
n Honey[3,31]
n Iodine[18,32-34]
n PHMB[18,35]
n Silver[18,33-36].
Importantly, these must be used following physical disruption of the biofilm by cleansing(i.e. with a solution containing a surfactant combined with an antimicrobial, such as
PHMB with betaine) and debridement, in order to ensure antimicrobial efficacy (Figure 1).
In addition, other products without an active antimicrobial agent have demonstrated antibiofilm activity, such as products that work by irreversibly binding the bacteria to dialkyl
carbamoyl chloride (DACC)-coated dressings[37]; the microorganisms are removed alongwith the dressing and there is no cell debris remaining in the wound[38].
HOW TO MONITOR SUCCESS
It is not possible to say definitely when a biofilm has been removed, since there is alack of categorical signs and tests for its identification. As such, clinicians must use
healing progression as a marker for success, also taking into account reduction inother parameters such as levels of slough and production of exudate[14]. Indeed, whenmeasuring the outcome of biofilm-based wound management, the primary factors that
led to initial suspicion of biofilm should be reviewed:
n Is the wound failing to heal despite appropriate treatment?
n Is the wound demonstrating signs and symptoms of infection, which do not resolvewith appropriate antimicrobial agents?
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n Is there a gelatinous material on the wound surface, which does not resolve?
If these have been resolved then it may be assumed that the treatment plan has beensuccessful.
Any product selected should be used for an appropriate length of time and continued for aminimum of 7–10 days before a decision is made to continue or discontinue use. A recent
consensus recommended utilising a ‘2-week challenge’ to determine the efficacy of an
antimicrobial (specifically silver dressings). After 2 weeks it should be determined whether
the wound has improved and whether there are any continuing signs of infection[5].
It is suggested that a wound with suspected biofilm should be debrided and cleansed
regularly, since it is difficult to remove all of the biofilm, which has the potential to regrow
and form mature biofilm within just days. If a wound is not progressing following regulartreatment, a more aggressive approach to biofilm removal may be required, with specialist
referral as appropriate[39].
CONCLUSION
Appropriate management of biofilm is arguably a complex task, with various solutions,gels and dressings for its management supported by the literature and clinical experience.
The basic steps of initial prevention (with anti-biofilm agents), removal (clean, deslough,debride) and prevention of reformation (use of an antimicrobial agent) provide aframework for the treatment of biofilm; beyond this, myriad patient, environment andclinical parameters must be considered to reach a tailored solution for each patient[40].
The combination of anti-biofilm agents and antimicrobial agents for biofilm management
may occur within the same dressing or their actions may be synergised at dressingchange (e.g. using Prontosan solution/gel and Calgitrol Ag). Understanding and keeping
up to date with evidence may be challenging, but is a crucial part of any clinician’s role if
he is to deliver optimal wound care, while being mindful of biofilm management. Caring
for the patient holistically and addressing any underlying systemic, psychological or
psychosocial issues is also important in underpinning a ‘gold standard’ care.

REFERENCES
1. James GA, Swogger E, Wolcott R et al. Biofilms in chronic wounds. Wound Repair Regen 2008; 16(1): 37–44.
2. Percival S, McCarty S and Lipsky B. Biofilms and wounds: An overview of the evidence Advances in Wound Care 2015; 4(7):373–381.
3. Cooper R, Bjarnsholt T and Alhede M. Biofilms in wounds: A review of present knowledge. Journal of Wound Care 201423(11): 570–582.
4. Thomson CH. Biofilms: do they affect wound healing? Int W J 2011; Feb 8(1):63–7. doi: 10.1111/j.1742-481X.2010.00749.x.
Epub 2010 Dec.
5. Keast D, Swanson T, Carville K, Fletcher J, Schultz G and Black J. Top Ten Tips: Understanding and managing woundbiofilm. Wound International 2014; 5(20): 1–4.
6. Swanson T, Grothier L, Schultz G. Wound Infection Made Easy. Wounds International 2014. Available from:www.woundsinternational.com.
7. Kim M, Ashida H, Ogawa M, Yoshikawa Y, Mimuro H, Sasakawa C. Bacterial interactions with the host epithelium. Cell
Host Microbe 2010; 8(1):20–35.
8. Zhoa GE, Usui ML, Lippmann SI, et al. Biofilms and Inflammation in Chronic Wounds. Adv Wound Care (New Rochelle)
2013; Sep 2(7): 389–399.
9. Wolcott. The role of Biofilm: Are we hitting the Right target? Plast Reconstr Surg 2011; January Suppl 127: 28S-35S).
10. Leaper D. Practice development – Innovations. Expert commentary. Wounds International 2010; 3(1): 19

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11. Leaper DJ, Schultz G, Carville K, Fletcher J, Swanson T and Drake R. Extending the TIME concept: what have we learnedin the past 10 years? Int W J 2010; 9(Suppl 2): 1–19.
12. Metcalf D, Bowler P, Hurlow J. Development of an algorithm for wound biofilm identification. Poster presented at EWMA
2013. Available at: http://old.ewma.org/fileadmin/user_upload/EWMA/P314.pdf
13. Percival SL, Vuotto C, Donelli G, Lipsky BA. Biofilms and Wounds: An identification algorithm and potential treatment
options. Advances in Wound Care 2015; 4(7): 389–397.
14. Phillips PL, Wolcott RD, Fletcher J, et al. Biofilms Made Easy. Wounds International 2010; 1(3): S1–S6.
15. Wolcott R. Economic aspects of biofilm based wound care in diabetic foot ulcers. Journal of Wound Care 2015; 24(5):189–194.
16. Hurlow J, Couch K, Laforet K, Bolton L, Metcalf D and Bowler P. Clinical Bioﬁlms: A Challenging Frontier in Wound Care.
Advances in Wound Care 2015; 4(5:) 295–301.
17. Cooper R, Jenkins L and Hooper S. Inhibition of biofilms of Pseudomonas aeruginosa by medihoney in vitro. J Wound
Care 2014; 23(3): 93–104.
18. Finnegan S and Percival SL. EDTA: An antimicrobial and antibiofilm agent for use in wound care. Advances in Wound
Care 2015; 4(7): 415–421.
19. Wolcott R. Disrupting the biofilm matrix improves wound outcomes. J Wound Care 2015; 24(8): 366–371.
20. Rhoads DD, Wolcott RD, Percival SL. Biofilms in wounds: management strategies. J Wound Care 2008; Nov 17(11):50–8.
21. Bradbury S and Fletcher J. Prontosan Made Easy. Wounds International 2011; 2(2).
22. JWC Wound Care Handbook 2016–2017. London 2016. MA Healthcare Limited.
23. Bellingeri A et al. Effect of a wound cleansing solution on wound bedpreparation and inflammation in chronic Wounds:
a single-blind RCT. J Wound Care 2016; 25: 3, 160–168.
24. Percival and Suliman. Slough and biofilm: removal of barriers to wound healing by desloughing. Journal of Wound Care
2015; 24(11): 498–510.
25. Vowden K and Vowden P. Debridement Made Easy. Wounds UK 2011; 7(4).
26. Debrisoft: Making the case. Wounds UK 2015. London.
27. Downe A. How wound cleansing and debriding aids management and healing. Journal of Community Nursing 2014;
28(4): 33–37.
28. Rycerz A, Vowden K, Warner V, Jørgensen BF. VAC Ulta NPWT System Made Easy. Wounds International 2012; 3(3).
29. Metcalf DG, Parsons D, Bowler PG. Clinical safety and effectiveness evaluation of a new antimicrobial wound dressing
designed to manage exudate, infection and biofilm. IWJ 2016; Mar 1.
30. Bjarnsholt T, Alhede M, Østrup Jensen P, Nielsen AK, Krogh Johansen H, Homøe P, Høiby N, Givskov M and KirketerpMøller K. Antibiofilm properties of acetic acid. Advances in Wound Care 2015; 4(7): 363–372.
31. Halstead FD, Webber MA, Rauf M, Burt R, Dryden M, and Oppenheim BA. In vitro activity of an engineered honey,
medical grade honeys and antimicrobial wound dressings against biofilm producing clinical bacterial isolates. Journal of
Wound Care 2016; 25(2): 93–102.
32. Hoekstra MJ, Westgate SJ, Mueller S. Povidone-iodine ointment demonstrates in vitro efficacy against biofilm
formation. International Wound Journal 2016; doi: 10.1111/iwj.12578. [Epub ahead of print]
33. Percival SL, Finnegan S, Donelli G, Vuotto C, Rimmer S, Lipsky BA (2016) Antiseptics for treating infected wounds:
Efficacy on biofilms and effect of pH. Crit Rev Microbiol 2016; 42(2): 293–309.
34. Thorn RM, Austin AJ, Greenman J, Wilkins JP, Davis PJ (2009). In vitro comparison of antimicrobial activity of iodine
and silver dressings against biofilms. Journal of Wound Care 2009; 18(8): 343–6.
35. Lenselink E, Andriessen A. A cohort study on the efficacy of a polyhexanide-containing biocellulose dressing in thetreatment of biofilms in wounds. Journal of Wound Care 2011; 20(11): 534–539.
36. Percival S and McCarty S. Silver and alginates: Role in wound healing and biofilm control. Advances in Wound Care 2015;
4(7): 407–414.
37. Cooper R and Jenkins L. Binding of two bacterial biofilms to dialkyl carbamoyl chloride (DACC) – coated dressings invitro. Journal of Wound Care 2016; 25(2): 6–82.
38. Butcher M. Catch or kill. How DACC technology redefines antimicrobial management. Br J Comm Nurs 2011 (Suppl).
39. Wolcott RD, Rhoads DD. A study of biofilm based wound management in subjects with critical limb ischaemia. J
Wound Care 2008; 17(4): 145–55.
40. International consensus. Appropriate use of silver dressings in wounds. An expert working group consensus. London:
Wounds International, 2012. Available to download from: www.woundsinternational.com.

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Biofilm research: filling in the gaps
in knowledge in chronic woundsT

he initial hypothesis that bacteria in biofilm structures was an importantfactor that contributed to chronic refractory infections originatedfrom studies in the early 1980s of diseases including endocarditis,
osteomyelitis, periodontitis and cystic fibrosis[1]. Following these initialstudies, extensive laboratory and clinical research publications haveconfirmed that bacterial biofilms are a critical factor in multiple diseases that arecharacterised by persistent bacterial infections that are tolerant to a patient’s own
immune system (antibodies and phagocytic inflammatory cells) and to standard
duration regimens of oral (or topical, IV) antibiotics[2-4].

Greg Schultz,
Institute for WoundResearch, Department of
Obstetrics & Gynecology,
University of Florida, USA;
Thomas Bjarnsholt,
Costerton Biofilm Center,
Department of Immunology& Microbiology, Facultyof Health and Medical
Sciences, The Universityof Copenhagen and
Department of ClinicalMicrobiology, Rigshospitalet,
Denmark; Klaus KirketerpMøller, Copenhagen Wound
Healing Center, Bispebjerg
University Hospital,
Copenhagen, Denmark andRose Cooper, Cardiff Schoolof Health Sciences, Cardiff
Metropolitan University,
Cardiff, UK

This important concept was expanded in a landmark paper published in Science[5] in 1999.
The paper integrated the concept of biofilm-stimulated chronic inflammation leading
to elevated proteases and Reactive Oxygen Species (ROS) that damaged surrounding
tissue, which could lead to tissue destruction as in periodontal disease or impairment oforgan function by scar formation (fibrosis) as in cystic fibrosis. Recognising that chronicskin wounds have many of the same clinical manifestations as most other diseases withchronic inflammation associated with bacterial biofilms, James and colleagues (2008[6]published the initial report of biofilm structures in chronic wounds. Using light andscanning electron microscopy to examine specimens from 66 subjects, they found biofilm
structures present in a high percentage (~60%) of 50 chronic wound biopsies in contrast
to only 1 of 16 (6%) acute wound specimens. This paper helped to draw attention to thepossible critical roles that bacterial biofilms could play in development and maintenanceof chronic skin wounds.
RELATIONSHIP BETWEEN BIOFILMS AND CHRONIC WOUND PATHOPHYSIOLOGY
Independent of the research on bacterial biofilms in chronic wounds, multiple laboratories
were actively investigating the molecular difference between healing and chronic
wounds. Among the first major molecular differences that were identified was thesubstantial elevation in two major families of proteases in chronic wounds, the matrix
metalloproteases (MMPs) and the neutrophil elastase (NE), a member of the serine
protease superfamily[7-13]. Several detrimental effects on healing were attributed to theelevated protease activities in chronic wounds. These included:n Destruction of important extracellular matrix (ECM) proteins including the multidomain adhesion protein, fibronectin[7,14], that is important in epithelial cell migrationn Destruction of important growth factors including platelet derived growth factor(PDGF)[15]n Degradation of key membrane receptor proteins for growth factors[16].
Similarly, elevations in proinflammatory cytokines, including tumor necrosis factor
alpha (TNFα) and interleukin-1 alpha (IL1α), were also reported in chronic wound
fluid samples or biopsies compared to healing wounds.(17) All these data pointed to acommon pathological pathway in which the development of bacterial biofilms in acutewounds stimulates chronic inflammation, indicated by persistently elevated levelsof proinflammatory cytokines (TNFα and IL1α). These proinflammatory cytokines
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chemotactically draw inflammatory cells (neutrophils, macrophages and mast cells)
into the wound bed where they secrete proteases (MMPs and NE) and release ROS. The
chronically elevated levels of proteases and ROS eventually have ‘off target’ effects thatdamage or degrade proteins that are essential for healing, converting a healing wound intoa stalled, chronic wound (Figure 1)[18].
In addition, the presence of Pseudomonas aeruginosa in biofilms have been hypothesisedby Bjarnsholt and colleagues[19] to produce a ‘shielding’ mechanism that offers protection
from the phagocytic activity of PMNs by synthesising and secreting virulence factors,
including a rhamnoilipid that efficiently eliminates PMNs (by lysis) and the enzyme
catalase that degrades hydrogen peroxide, a major ROS produced by PMNs, to non-toxic
products oxygen and water.

Hypothesis of chronic wound pathophysiology
Repeated tissue injury, ischaemia and bacteria – biofilms

TNFα

IL-1β, IL-6
Prolonged, elevated inflammation
neutrophils
macrophages mast cells

Imbalanced proteases and inhibitors
proteases (MMPs, elastase, plasmin) inhibitors (TIMPs, α1PI)

ROS

Destruction of essential proteins (off target)
growth factors/receptors
ECM degradation
cell migration
cell profileration

Chronic, non-healing wound

Figure 1 | | Hypothesis of chronic wound pathophysiology and biofilms[18]Development of biofilms in acute wounds leads to chronic inflammation characterised by elevated levelsof proinflammatory cytokines that leads to increased numbers of neutrophils, macrophages and mast
cells that secrete proteases and ROS that become chronically elevated and accidently (off-target) destroy
proteins that are essential for healing, leading to a chronic, non-healing wound
A GAP IN THE KNOWLEDGE BASE
Detection and measurement of biofilm bacteria in wounds
Based on apparent correlation between chronic wound pathophysiology and the presenceof biofilms in a high percentage of chronic wounds, the detection and localisation of
biofilms in chronic wound beds provides useful clinical information, especially forassessing and directing the effectiveness of debridement. In addition, assessing thebiofilm status of a chronic wound could potentially indicate when a chronic wound bed
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is adequately prepared to be able to respond to advanced therapies such as growth
factors, advanced matrix dressings, cell-based therapies or skin grafts[20,21]. However, mostclinical microbiology and pathology laboratories use conventional techniques (scanning,sequencing and sampling) that are not able to distinguish between bacteria that wereexisting either planktonically or within a biofilm[22]. Thus, clinicians should assume thatthe reported bacteria are biofilms and should treat them accordingly.
Furthermore, multiple studies have reported that conventional culture methods used byclinical microbiology laboratories to assess the bioburden in wound samples are biased todetecting easily cultured planktonic organisms and fail to detect many bacterial species,
especially anaerobic bacteria, as well as fungal and yeast species[23-26]. For example, Dowd
and colleagues (2008)[23] reported that standard culturing techniques identified only 1% of
all microorganisms present in samples of 30 chronic wounds, especially strict anaerobes.
Thomsen and colleagues (2010)[26] reported similar results when using DNA-based
identification techniques and fluorescence in situ hybridisation to identify bacterial species
in 14 ulcers undergoing skin graft operations. They found substantial differences between
results obtained by standard culture-based methods and molecular-biology-based methods.
Expanding their initial study, Wolcott and colleagues (2016)[27] used 16S rDNA
pyrosequencing to analyse the microbiota of 2,963 samples from chronic venous leg ulcers(n=916), diabetic foot ulcers (910), decubitus ulcers (767) and non-healing surgical wounds(370). They found similar profiles for the 20 bacterial species most frequently identifiedin each of the four types of chronic wounds, with Staphylococcus and Pseudomonas species
comprising the most prevalent genera. In addition, strict anaerobes comprised 4 of the top
10 genera detected in the chronic wound samples. Commensal microorganisms, including
coagulase-negative Staphylococcus, Corynebacterium and Propionibacterium, were present innearly half of the chronic wound samples tested, but further research is needed to assess
whether the presence of these organisms affect the healing of chronic wounds.
It is important to understand that using both culture and DNA-based methods to detectbacterial species present in wound samples does not differentiate between bacteria
growing planktonically or growing in biofilm communities. This can only be accomplishedby microscopy or by selective culturing for biofilms as described below.
Does biofilm-based wound care improve healing of chronic wounds?
An important question to ask is: ‘Does more accurately knowing the actual bacterial,
fungal and yeast species present in chronic wounds, including bacteria in biofilms,
actually provide important information that a clinician can use to improve healing
outcomes?’ In a large, level A, retrospective cohort study, implementation of personalised
topical therapeutics guided by molecular diagnosis of bacterial species resulted instatistically and clinically significant improvements in healing[28].
In the standard of care (SOC) group, 48.5% of patients (244/503) healed completely
during the 7-month study period; this increased to 62.4% (298/479) in the treatmentgroup that received SOC plus systemic antibiotics based on the results of molecular
identification of wound bacteria. Completed healing further increased to 90.4%(358/396) in the treatment group that received SOC plus topical therapeutics (including
antibiotics) based on the results of molecular diagnostics (p<0.001 compared to SOC or
SOC + systemic antibiotics, Cox proportional hazards analysis). More recently, Wolcott(2015)[29] reported significantly increased healing of wounds treated with SOC combinedwith a hydrogel that contained topical antibiotics and agents that disrupted biofilms.
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How and where to sample a chronic wound bed for biofilm
Currently, detecting and localising biofilms in chronic skin wound beds is one of the mostimportant ‘gaps in the knowledge base’ for biofilm-based wound care, especially sincemature, tolerant biofilms can reform within three days following effective debridement ofchronic skin wounds[30,31].
In May 2015, the European Society for Clinical Microbiology and Infectious Diseases(ESCMID) published guidance for diagnosis and treatment of biofilm infections[32]. It includes
information and guidelines on detecting and treating biofilm infections in multiple conditions.
These included tissues/mucus infections, such as in patients with chronic lung infections(cystic fibrosis), and in chronic infections where biofilms form on devices inside the body(orthopaedic implants, breast implants) or on devices that connect between the inner (sterile)and outer surface of the body such as intravenous catheters, indwelling urinary catheters
or endotracheal tubes. The target readers for this guideline are clinical microbiologists andinfectious disease specialists involved in diagnosis and management of biofilm infections.
The ESCMID guideline states: ‘Biopsy tissues are considered the most reliable samples toreveal biofilm in wounds. The use of swabs to collect biofilm samples from the wound surface
is considered an inadequate method, due to contamination from the skin flora, the strong
adherence of biofilm to the host epithelium and the growth of anaerobes in the deep tissues.
If a moderate to severe soft tissue infection is suspected and a wound is present, a soft tissue
sample from the base of the debrided wound should be examined. If this cannot be obtained, asuperficial swab may provide useful information on the choice of antibiotic therapy’[33,34].
However, assuming a biopsy or curettage wound sample can be obtained from the chronicskin wound, this guideline leaves several important questions unanswered, including:n Where in the wound bed should a single sample be taken?
n Is one biopsy sufficient to confidently assess if a chronic wound has (or does not have)a mature biofilm? It is highly unlikely that biofilms are uniformly present over the entire
wound bed and edge of the wound, so what should guide the clinician?
n Are there any visual signs that might be useful in deciding where to take the single biopsy?
Several papers have reported that the distribution of biofilm aggregates throughout
chronic wound beds is not uniform[35,36]. For example, as shown in Figure 2, aggregates of P.
aeruginosa biofilms are not homogenously distributed on the chronic wound bed[36].

Figure 2 | Biofilms of P. aeruginosa in a chronic wound visualised using a specific peptide nucleic
acid-fluorescence in situ hybridisation probe (red) with confocal laser scanning microscopy. The rightimage shows an enlargement of the middle image. The distribution of biofilm colonies on the wound bed
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4.0
3.5
3.0
2.5
2.0
1.5
1.0

60

-60
50

-50
40

-40
30

-30
20

-20
10

0-1

0

0.0

-70

0.5

Figure 3 | Distribution of the distances from the wound surface to the centre of mass of S. aureus
aggregates (light grey shading) or P. aeruginosa aggregates (dark grey shading). The distances areaverage values obtained from the analysis of 15 images for each of 9 chronic wound samples.
In addition, aggregates of biofilms are not necessarily present only on the surface of woundbeds[35]. As shown in Figure 3, biofilm structures were identified beneath the surface of
9 chronic wound beds, with S. aureus aggregates nearer the surface of the wound bed(~20-30 micron depth) compared to P. aeruginosa aggregates (50–60 micron depth). It is
most likely that different species or phenotypes of bacteria prefer environmental niches.
Also, distribution of bacteria and biofilms could also be dependent on competition orcollaboration with other microorganisms[26,36].
As explained in the accompanying article by Bjarnsholt et al (pages 4–8)[37], there has been
considerable debate over whether biofilms in chronic wound beds can be visually observedby clinicians. While large formations of biofilms on the enamel surface of teeth can bevisualised by ‘disclosing dyes’, it is less clear if all biofilm formations can be visualisedin chronic wounds. Some clinicians have proposed that ‘clinical signs’ such as a shiny,translucent, slimy layer on non-healing wound beds that quickly reform when debrided,
may be easier to remove by fabric pads, and may be less responsive to enzymatic or
autolytic debridement are likely to be biofilms[38,39]. However, these observations need to besupported by rigorous analysis of these types of materials on wound beds for biofilms.
A new ‘biofilm wound map’ technique described by Nakagami and colleagues[40] may
provide useful information on localising biofilms in the surface of a wound bed. A clinician
presses a highly positively charged nylon membrane onto the wound bed for a few minutes,
which produces a ‘molecular imprint’ of the molecules on the wound bed surface that arevery tightly bound to the membrane. The ‘blot’ is then submerged for a few seconds in asolution containing a positively charged dye molecule (such as ruthenium red) that ionicallybinds to highly negatively charged molecules bound on the membrane, and then briefly
rinsed. Most bacterial biofilms contain substantial amounts (~20%) of free bacterial DNA,
which is highly negatively charged[41].
Laboratory experiments demonstrated that areas of the membrane that retain the dyecorrespond to areas on a wound bed surface that have an exopolymeric matrix of biofilm
communities. Furthermore, the amount of surface area of a wound bed that generated
staining on the membrane predicted the extent of slough that developed on the chronic
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Figure 4 | Biofilm Wound Map. Orange-red staining present on the membrane (panel B)
suggests the presence of biofilm exopolymeric matrix of chronic wound (panel A) blotted
onto a positively charged membrane

A

B

1cm

1cm

wound bed during the following week. A weakness of this technique is that would
preferentially detect biofilm exopolymeric matrix located on the surface of the wound bed,
and not detect biofilm exopolymeric matrix buried deep in the wound bed matrix. Clearly,
there is a need to develop a rapid, inexpensive, and easy-to-use biofilm detector that canbe used at the point-of-care in a few minutes.
WHAT IS THE OPTIMAL BIOFILM ASSAY(S) FOR CHRONIC WOUNDS?
Several different assays are used to determine if a wound sample contains a mature
tolerant biofilm. The most common approach is to visualise biofilm-like structures using
either light microscopy, often with antibodies that detect a unique component of the
exopolymeric matrix of some biofilms such as alginate synthesised by P. aeruginosa, or
fluorescence in situ hybridisation (FISH). However, it can take several days to process
tissue samples through paraffin embedding. Cryosectioning may offer faster processing
and evaluation. Both techniques require expensive microscopes and trained technicians,
and cannot be done during a clinic visit.
Most standard clinical microbiology laboratories can adopt a relatively simple andstandard approach to measuring bacteria in protective biofilms[42].Briefly, wound samples
are placed in phosphate buffered saline (PBS) containing 5 ppm Tween 20 (5 ml/ml).
They are vortexed to suspend tissue after which dilute bleach to a final concentration of
0.03% is added.
The samples are then incubated for 10 minutes to kill all planktonic bacteria and thebleach is neutralised with sodium metabisulfite (0.3% final concentration). The biofilm
aggregate is then dispersed in to single bacteria by five, 1.5-minute cycles of sonication
with a 1 minute cooling pause between sonication cycles. Samples are plated by 10-folddilutions onto selective growth agar plates and colonies counted after 24 hours and 48
hours of incubation at 37°C.
Alternatively, wound samples can be placed into solutions containing antibiotics(gentamicin, moxifloxacin, penicillin) for 24 hours at 37°C to kill susceptible planktonic
bacteria then washed twice in Dey-Engley neutralising broth, vortexed (30 seconds),
sonicated (two minutes), and vortexed (30 seconds) three times, to disperse biofilms
into single cell suspensions that are then serially diluted with PBS, plated on TSB and theplates were incubated at 37°C for 24–48 hours[43].
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CAN WOUNDS HEAL WITH A SMALL AMOUNT OF BIOFILM?
Many acute wounds can heal despite bacterial colonisation. This is a paradox that may
be explained by hypothesising that the immune system of most patients (sometimes
supplemented by systemic antibiotics or topical antiseptic dressings) can kill planktonic
bacteria before they develop into biofilms that are very difficult to kill. Most chronic
wounds have become chronic due to maltreatment, and they undoubtedly have substantial
amounts of bacterial biofilm, but when many chronic wounds receive correct treatment
such as compression and/or off-loading, they start to heal, even without adding antibiotics
or antiseptics. It is possible that this may be explained by the fact that some bacteria are
more virulent like Pseudomonas and some Staphylococcus strains[19], but many of the bacteria
in the wounds are opportunistic infectious agents. The immune response might create
opportunities for less virulent bacteria, fighting for the same space, to influence the bacteria
in the biofilm. Clearly, this is an important question that further research needs to address.
CONCLUSION
Bacterial biofilm can play a pivotal role in the development and maintenance of chronic
wounds. The detection and localisation of biofilms in chronic wounds provide useful clinicalinformation, in particular in assessing and directing the effectiveness of debridement.
However, gaps in the knowledge base remain when it comes to detecting and localising
biofilms in chronic wounds. The ESCMID guideline[32] published in 2015 offers direction
on diagnosis and treatment of biofilm infection but leaves some questions unanswered,including whether visual signs might be useful in deciding where to take a biopsy.
The debate surrounding whether or not biofilm can be seen with the naked eye continues.
New techniques, such as the ‘biofilm wound map’ from Nagakami and colleagues[40]may provide useful information on localising biofilms in the surface of the wound bed.
However, as with other existing techniques, this has its weaknesses and there remains aneed to develop a point-of-care biofilm detector that can provide results in a few minutes
not a few days. More focused research is needed to accurately and effectively detect andlocalise biofilms in chronic wounds.

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