Biofilms: possible strategies for suppression in chronic wounds

Percival SL, Cutting KF and Williams, D

Summary

The aim of this paper is to provide a brief overview of strategies that could be applied to chronic wounds harbouring biofilms.  The importance of biofilms in chronic wounds is highlighted in respect of them preventing the normal wound healing processes and with regards to their resistance to removal by conventional antimicrobial agents.  An array of anti-biofilm agents is provided, and the mode of action of these agents together with their efficacy in suppressing biofilms is discussed.  It is hoped this article will provide foresight into wound management and promote consideration of future anti-biofilm strategies.


[keywords] Biofilms; Chronic wound infections; Wound healing


Chronic wounds can be defined as those wounds that do not heal within an expected time frame.  Delayed wound healing is a worldwide problem and not only serves to increase patient morbidity, but also significantly increases costs to the healthcare provider.  To highlight this management of chronic wounds by the NHS has been conservatively estimated  at ?2.3-?3bn per annum, at 2005-2006 costs (Posnett & Franks 2008).    

The causes of chronic wounds are varied with many contributing factors described including systemic factors such as old age, poor nutrition and diabetes as well as local factors like the presence of a foreign body and microbial colonisation (Percival, 2009, Percival and Dowd 2010).  The latter has recently received much attention and it?s important at this stage is to accept that no wound environment is free of microorganisms although the invariable presence of microorganisms in a wound does not necessarily lead to a wound that clinically appears infected. 

The numbers of microorganisms in a chronic wound is often thought to be an important factor in determining whether wound healing can progress normally (White and Cutting 2006, Ryan 2007, Tan et al 2007, Percival and Dowd 2010). Historically, standard microbiological testing procedures used routinely in wound care studies have reported only on those bacteria that are freely removed from the wound bed to help guide management strategies (Rhoads et al 2007, Percival and Dow 2010).  Such free-living microorganisms are often termed as being in a planktonic form.  Today, research has highlighting that microorganisms attached to a surface express different outward (phenotypic) characteristics to those in a planktonic state (Clutterbuck et al 2007, Rhoads et al 2008, Percival et al 2010a).  This is an important distinction and the presence of such attached bacteria within the wound effectively means two differently behaving groups of microorganisms are present (Percival and Bowler 2004a, Rhoads et al 2007, James et al 2008).  The attached microorganisms are often described as being in a biofilm state.

A biofilm can be defined as a community of microorganisms, attached to each other, or a surface and encased within an extracellular polymeric substance (EPS) or ?slime matrix?.   A key property of biofilms is that compared with their planktonic equivalents they demonstrate increased resistance to both host defences and chemical attack (Woods et al 2010, Percival et al 2010b).  As such once established, biofilms are highly resistant to removal.  It is these resilient biofilms that are likely to cause a significant delay in healing and clinicians need to take into account their increased resistance to antimicrobials if healing is to be achieved (Percival et al 2010b,c).

A misunderstanding over the role microorganisms play in modulating wound healing may result in sub-optimal management of chronic wounds resulting in delayed healing in some acute wounds (Costerton et al 1999, Costerton and Stewart 2001, Wolcott et al 2008).  

The article highlights possible approaches to suppress biofilm growth in chronic wounds, thus allowing the host defence mechanisms to prevail (Woods et al 2010).

[A HEAD] The biofilm concept 
Over 99% of microorganisms naturally found in their native habitats persist within a biofilm state (Percival et al 2010a). Consequently, it is unsurprising that this mode of microbial growth is now known to occur in the wound environment (Percival and Rogers 2005, James et al 2008, Ngo et al 2007, Percival et al 2010a).  Both planktonic bacteria and fragments of biofilms can initially attach to a surface producing clusters or aggregates of bacteria which then grow into a mature biofilm (Percival et al 2000, Thomas 2008, Westgate et al 2010,). 

[B HEAD BOLD] Medical biofilms [BOLD ENDS]
Up to 80% of human infections are thought to be related to pathogenic biofilms (Anon 1997). However, it is only relatively recently that clinicians have begun to appreciate that many persistent infections are caused by biofilms.  Examples of chronic infections influenced or induced by a biofilm, include prostatitis, endocarditis and osteomyelitis, all conditions which are often found to persist indefinitely (Costerton and Stewart 2001). It seems plausible therefore to hypothesise that biofilms have a fundamental role in chronic wound infections. 
[B HEAD BOLD] Implications of biofilms [BOLD ENDS]
As microorganisms within a biofilm multiply they continually produce chemical signals or ?pheromones?.  These chemical messages are also called quorum sensing molecules (Box 1) and deemed important in controlling the formation of a biofilm and also the behaviour of its microbial community.  Quorum sensing mediated changes within a chronic wound biofilm, can enhance the tolerance of a biofilm to antimicrobial agents (Sauer et al 2002).  

With an armory of physical and biochemical defenses, biofilms have reduced susceptibility to many antimicrobials including antibiotics, antiseptics as well as host defense mechanisms (Costerton et al 2003, Costerton and Stewart 2001, Marion et al 2006, Burm?lle et al 2006, Percival et al 2010c).  The physical properties of the biofilm are largely dictated by the EPS that is present which can effectively trap and impede passage of an antimicrobial through the biofilm.  Since a biofilm is often comprised of several different microbial species, cooperation between these organisms in terms of sharing biochemical features (including those related to antimicrobial resistance) can occur (Xu et al 2000, Fux et al 2005, Burm?lle et al 2006 Shen et al 2006, Chang et al 2007). 

The microbial activity within a biofilm can be complex with highest activity and reproduction occurring towards the biofilm surface.  It is these active microorganisms that are constantly dividing that will disperse from the biofilm at a high rate. Metabolically active microorganisms have also been shown to be the most vulnerable subpopulation of a biofilm to antimicrobials. In contrast, those microorganisms that are located deeper in the biofilm matrix are less active and appear to be less susceptible to external perturbations, including the presence of antimicrobials (Xu et al 2000, Lewis 2007, Percival et al 2010c). It is these microorganisms that can reconstitute the community of the biofilm following periods of extreme stress (Lewis 2007). 

Based on the above findings, management of a biofilm community is significantly more challenging than combating a planktonic community.  As a consequence, alternative management strategies that are directed towards biofilm for the treatment of chronic wounds may be beneficial. 

[A HEAD] Theory and practice of management of wound biofilms
When skin is broken the primary bacterial defense barrier is compromised and a relatively ?immature? wound is formed (Niyonsaba et al 2006, Woods et al 2010). The primary objective of host defence mechanisms in this situation is to prevent the microorgansms that have contaminated the wound from increasing in numbers and inducing infection.  Typically, the host easily fends off potential pathogens through inflammation (proinflammatory cytokines, matrix metalloproteases, phagocytosis, and degranulation of neutrophils).  In addition, a number of factors can promote the establishment of a chronic wound and infection, including poor perfusion, malnutrition, foreign body presence, pressure, repetitive trauma, hyperglycemia, and white blood cell dysfunction.  If a biofilm becomes established in a wound, it will be difficult to eradicate, particularly in an immunocompromised individual (Costerton et al 2003).  Consequently, the microorganisms and their exuded products within the biofilm will prolong the state of acute inflammation indefinitely, delaying the normal healing process.  Clinicians often focus on the number of culturable bacteria present in the wound as this often correlates with the degree of immune stimulation (i.e. classic signs of acute infection) seen in the patient (Dow et al 2001).  However, many biofilm bacteria may be unculturable using traditional microbiological methods and these bacteria are therefore not detected (Costerton et al 2003, Davies et al 2004, Cochrane et al 2008). 
Sharp debridement of devitalized tissue promotes wound healing by removing tissue that not only supports microbial (i.e. the biofilm) proliferation but also reduces the efficacy of topical therapies.  Consequently, debridement should be performed at weekly intervals in chronic wounds (Wolcott et al 2009). Although admittedly this is a skill that few nurses have acquired. Debridement is thought to not only remove microorganisms but also exposes deeper host defenses therefore enhancing their efficacy (Wolcott et al 2009).  However, debridement alone in the authors? opinion is not sufficient to manage the majority of chronic wounds because of biofilm removal and re-establishment. Other concomitant strategies such as administration of antimicrobials should be considered as an adjunct therapy (Schultz et al 2004). 
Some studies have shown that antimicrobial agents may sometimes suppress the metabolically active cells within a biofilm. However, it is important to acknowledge that presently no single strategy has proven to be consistently effective at suppressing the entire biofilm.  For example, following the administration of a course of antibiotics infection often reoccurs.  This is because antibiotics have been shown only to be effective in transiently suppressing rapidly growing cells, which are generally located towards the outermost surface of the biofilm.  Recalcitrant microbial cells found deeper in the biofilm will persist.
  
Shortcomings exist in the use of single or sequential treatment strategies.  In the authors? experience, concurrent management strategies will increase the likelihood of prolonging biofilm suppression, and as a consequence help foster healing (Costerton et al 2003). 

[A HEAD] Management strategies 
Many commercially available wound dressings are not inherently antimicrobial.  However, some wound dressings have been shown to passively reduce the bacterial load at a wound surface through sequestration (binding) of bacteria.  (Mertz and Eaglstein 1984, White et al 2006).  Consequently, wound dressings should be selected carefully as they may help reduce the risk of biofilm growth and therefore further microbial proliferation on, or within the wound.  
Whilst the modes of action of antimicrobial agents differ, their fundamental effects are similar (Percival et al 2010b).  Antimicrobials can impair microbial metabolism or structural integrity leading to the prevention of growth (microbiostatic) or direct killing (microbiocidal).  The most recently designed anti-biofilm agents often function not by these mechanisms, but by disrupting the biofilm structure, removing essential nutrients, metal ions (Kite et al 2004) or by interfering with microbial community interactions (Singh et al 2002, Costerton et al 2003, Kaneko et al 2007). 
Systemic antibiotic treatment for chronic wounds is warranted in situations where there is significant deep tissue wound infection or a risk of septicaemia. Whilst, systemic antibiotics may suppress the cells at the outermost region of the biofilm (Xu et al 2000), such antibiotics generally have poor efficacy when biofilms are present (Moss et al 1990, Marr et al 1997).  Furthermore, the effectiveness of antibiotics appears to be reduced in ischaemic wounds since therapeutic and effective concentrations are not reached at the site of infection. 

Topical antiseptics can also help to reduce the wound bioburden and inhibit biofilms, particularly where there is a concurrent infection or an increased risk of infection. 

In the authors? opinion, the efficacy of antimicrobial agents can be substantially enhanced when combined with other management strategies, specifically debridement and antimicrobial wound cleansers, in conjunction with an appropriate wound dressing. 

[B HEAD BOLD] Antimicrobial agents [BOLD ENDS]
It is plausible to suggest that antimicrobial agents could be employed prophylactically to suppress biofilm development (Percival et al 2010c).  However, some non-selective antimicrobials may be detrimental to wound healing by harming the cells of the host, as well as the existing commensal or ?normal? microbial population.  Such non-selective antimicrobials include alcohols, hydrogen peroxide, carbolic acid, sodium hypochlorite and acetic acid as examples.  Selective antimicrobials such as molecular iodine and ionic silver may therefore be more suitable for chronic wounds by targeting particular microorganisms. 
[C HEAD ITALIC] Iodine [ITALIC ENDS]
Iodine has been used for many years as a wound antiseptic (Cooper 2007).  However, high doses of iodine can be detrimental to host healing (Kramer 1999, Wilson et al 2005).  Cadexomer iodine can be used to suppress biofilms without causing significant host cell damage (Akiyama et al 2004).  
[C HEAD ITALIC] Ionic silver [ITALIC ENDS]
Ionic silver is a beneficial antimicrobial for use in wound care, particularly for biofilm-based management strategies. Ionic silver has a broad range of efficacy against many microorganisms (Russell and Hugo 1994, Lansdown et al 1997, Bradford et al 2009).  In addition, a number of silver dressings have been shown to prevent biofilm formation in vitro (Percival et al 2007) and also have in vivo benefits (Fong and Wood 2006, Beele et al 2010). 
[C HEAD ITALIC] Honey [ITALIC ENDS]
In vitro evidence shows that honey is effective against a range of multi-resistant microorganisms including Meticillin-resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE) and multi-resistant Gram negative microorganisms including Pseudomonas aeruginosa (George and Cutting 2007).  The effectiveness of honey has even been shown to surpass that of traditional antibiotics/antiseptics in previously unresponsive wounds (Dunford et al 2000).  Irish et al (2006) also demonstrated the effectiveness of honey in preventing biofilm formation whilst Okhiria et al (2006) found that the application of honey caused disruption of pseudomonal biofilms (in vitro).
[B HEAD BOLD] Antibiofilm agents [BOLD ENDS]
The use of antibiofilm agents is considered by many to be beneficial to wound management as they have been shown to be less cytotoxic than many of the traditionally used antimicrobials.  These antibiofilm agents include lactoferrin (Singh et al 2002), xylitol (Katsuyama et al 2005), gallium (Kaneko et al 2007), chitosan and Dispersin B (Lu et al 2007) (Table 1).  Evidence regarding the benefits of such agents to wound healing is slowly increasing.  Other agents that have been used for anti-biofilm purposes in wound care can also be found in Table 1. Currently, such agents are in an evaluative stage and not available to general clinical practice.   


Table 1 Potential antibiofilm agents
AgentMode of actionUsageLactoferrinBlocks the attachment of planktonic bacteria to a surface which is the first step in biofilm formationBovine lactoferrin is a protein used in the meat packing industry to protect exposed meat from bacterial biofilm formationXylitolInterferes with biofilm formation  5-carbon alcohol sugar used in chewing gum. It has been shown to reduce the incidence of dental carries (Burt 2006).Gallium Interferes with bacterial iron metabolism pathwaysGallium nitrate is currently an Food and Drug Administration (FDA)-approved drug that can be used intravenouslyDispersin BTargets the extracellular polymeric substance of some types of biofilm and works to degrade the community structure of the biofilm (Itoh et al 2005)Dispersin B is a bacterial enzyme, but is not presently used commercially  HoneyPossess antibacterial activity (Molan 1999, 2006) and modulates monocytic cell activity (Tonks et al 2001, 2003)Honey has been found to have activity against in vitro biofilms. 
[A HEAD] Conclusion
The aim for the wound care provider is to establish the best management strategies possible for each individual patient.  To achieve this, clinicians need to be familiar with both antimicrobial and antibiofilm agents.  The basis of managing biofilms is the frequent removal of the biofilm from the surface.  This can be achieved with either sharp debridement (curette), or formal surgical debridement.  As biofilms reconstitute quickly following invasive debridement, suppressing their re-growth and reconstitution using multiple antimicrobial management strategies is warranted.  Management strategies that could be used include the application of wound cleansers, topical antimicrobials and advanced wound dressings.  Such strategies may work synergistically to help suppress and reduce the re-growth of biofilms.  A ?rotating? regimen of selective antimicrobials may be advantageous in biofilm-based wound management.  For effective management of the wound biofilm, systemic antibiotics may also help to further suppress the biofilm.

For positive clinical outcome, it is important that all concurrent barriers to healing should be addressed.  This will help to augment the host?s defenses which, when working optimally, provide the best strategies to help manage a wound.  Consequently, biofilm-based management of chronic wounds is becoming fundamental to treating these non-healing wounds.

[BOX 1 BEGINS]
Glossary 

Biofilm ? a community of microorganisms, attached to each other, or a surface and encased within an extracellular polymeric substance (EPS) and demonstrating increased resistance to cellular and chemical attack 

Contamination ? presence of bacteria within a wound without inducing a host reaction

Colonisation ? the presence of bacteria in increasing numbers within the wound without inducing a host reaction.  It is not possible to differentiate clinically between contamination and colonisation.

Planktonic - microorganisms which are free-floating and not attached to a surface

Quorum sensing ? measuring the bacterial population concentration through the expression of bacterial signaling molecules.  When a ?quorum? population threshold is reached, specific biological activities, for example, expression of virulence factors are activated

Sessile ? microorganisms attached to a surface

Proinflammatory cytokines ? immune system derived protein molecules that amplify the inflammatory response

Matrix metalloproteases (MMPs) ? proteases that may be endogenous or exogenous in origin and break down protein e.g. collagen.


[BOX 1 ENDS]



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