Taking A Closer Look At The Impact Of Wound Bioburden
Bioburden can have an adverse effect on the environment of the chronic wound and may lead to infection and delayed healing. Accordingly, this author examines the literature on wound bioburden and offers insights on the use of antimicroibial wound dressings and larvae therapy to target biofilms.
Chronic skin ulcerations of the lower extremities affect millions of patients in the United States and impose a tremendous medical, psychosocial and financial impact.1 Chronic ulcerations may be secondary to a myriad of etiologies, including pressure, metabolic, trauma, venous, arterial and diabetic neuropathy.2
The Wound Healing Society defines chronic skin ulcerations as wounds that have “failed to proceed through an orderly and timely process to produce anatomic and functional integrity, or proceeded through the repair process without establishing a sustained anatomic and functional result.”3 This prolonged, sometimes interrupted healing process affects the patient’s quality of life because of impaired mobility and substantial loss of productivity, and is a significant management challenge to healthcare professionals.4
The relapsing course may also be reflected in the astounding economic burden chronic ulcerations have placed on healthcare. The U.S. spends over $25 billion a year on the treatment of chronic non-healing ulcers.5 Medicare expenditures for patients with lower extremity ulcers were, on average, three times higher than those for Medicare patients in general.6 Furthermore, a lack of immediate attention to these wounds can often serve as a prelude to significant health problems because of associated infections that may lead to amputations or induce life-threatening situations.2,7
Over the past decade, there have been numerous biotechnological advances in wound healing modalities. These advances include the development of acellular-matrix based materials, cytokines and bioengineered tissue replacements. Many of these modalities show some degree of promise in healing both simple and complex diabetic foot wounds.
However, results from a myriad of published and unpublished industry-sponsored, randomized trials that evaluated the efficacy of these advanced wound healing agents only yielded healing rates of 45 to 55 percent.7-12 Since the objective healing rates from these studies are considerably less than ideal, it stands to reason that some diabetic foot ulcers may be more recalcitrant to treatment than others.12
How Bioburden Impacts The Chronic Wound Environment
Wound repair is an orchestra of highly integrated cellular and biochemical responses to injury.13 Certain pathophysiologic and metabolic conditions can alter this normal course of events so that healing is impaired or delayed. This results in chronic, non-healing wounds, including the contamination of bacterial pathogens.13,14 In recent years, there has been an increase in recognition of the concept of critical colonization or local infection that may result in wound healing delays despite the absence of the gross clinical features of infection.14
The progression from wound colonization to infection therefore depends on several factors. Needless to say, the progression is dependent on the bacterial count and the species present. However, other important factors to take into consideration include the host immune response, the number of different species present, the virulence of the organisms and synergistic interactions between the different species.14
There is also increasing evidence that bacteria within chronic wounds live within biofilm communities, in which the bacteria are protected from host defenses. Biofilms are ubiquitous, complex structures consisting of microbial-associated cells embedded in a self-produced extracellular matrix of hydrated extrapolymeric substances, which are irreversibly attached to a biological or non-biological surface.15-17
James and colleagues collected and analyzed chronic wound specimens, and acute wound specimens from various subjects, and noted a statistical difference in biofilm formation between the two types of wounds.18 Of the 50 chronic wound specimens evaluated, 30 (60 percent) contained biofilm whereas only one of the 16 (6 percent) acute wound specimens contained biofilm. The authors’ molecular analyses of chronic wound specimens revealed diverse polymicrobial communities and the presence of bacteria, including strictly anaerobic bacteria that cultures did not fully reveal.
Research has shown that most chronic wound biofilms have significant populations of anaerobes.19 Sun and colleagues developed a rapid model to simulate polymicrobial chronic wound biofilms. Their model incorporated three common bacteria; methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and Pseudomonas aeruginosa. The researchers noted that anaerobic bacteria proliferate through integration into the biofilm under aerobic conditions. They also used electron microscopy to demonstrate a close association between aerobes and anaerobes within the biofilm. This suggests a synergistic relationship between the aerobes and anaerobes.
Researchers have speculated that the persistent biofilm communities may prevent ulcerations from healing and predispose the wound to infections and amputations.16
Bacteriae that reside as biofilms are often resistant to traditional antibiotic therapy and studies have demonstrated in murine wound models that bacterial biofilms significantly delayed wound re-epithelialization.1,16,17
Studies have postulated that biofilm producing bacteria contribute to intractable inflammatory process, secrete matrix metalloproteinases (MMPs) and secrete tissue inhibitors. Researchers believe these processes have a potentially negative impact for wound healing and may help explain why chronic wounds are so refractory to healing.1,15,20
Further, in vitro studies have demonstrated that Staphylococcus aureus biofilms significantly reduce viability and increase apoptosis in human keratinocytes.21
Ngo and co-workers assessed 12 chronic wound samples from routine debridements and noted bacterial biofilms in 60 percent of the wounds.22 They noted that the necrotic, superficial layer of wounds may serve as niduses for continual bacterial seeding and were more conducive to biofilm formation than deeper viable tissues.
In a study of clinically uninfected diabetic foot ulcers, Edmonds and Foster noted that antibiotic therapy helped reduce hospitalization and amputation in wounds that lacked overt signs and symptoms of infection.23
Moreover, in their assessment of chronic, non-healing wounds, Xu and colleagues found positive correlation between quantitative measurements of bacterial load and the rate of diabetic wound healing.24
What The Research Reveals About Possible Treatments To Target Biofilms
Biofilms are often recalcitrant to routine surgical debridements. Several researchers have assessed the effect of antimicrobial wound dressings and larvae therapy on destroying existing biofilms and preventing the formation of new biofilms.25,26
Cadexomer iodine. Cadexomer iodine is a preparation that releases iodine (0.9% weight/weight) slowly from beads of dextrin and epichlorohydrin. It is an effective debridement and antiseptic agent for chronic exudative wounds.
Aklyama and colleagues examined the influence of cadexomer iodine against glycocalyx production of Staphylococcus aureus in vitro and in a mice model.27 Based on their findings, the authors suggested that cadexomer iodine soaked up Staphylococcus aureus cells encircled by glycocalyx, directly destroyed biofilm structures, collapsed glycocalyx during dehydration and subsequently killed the Staphylococcus aureus cells within biofilm.
Silver. For centuries, silver has been recognized for its antimicrobial properties and is a popular component of a plethora of wound dressings. An in vitro study assessed the antimicrobial effect of silver-containing dressings on both monomicrobial and polymicrobial biofilms.25 The biofilms were composed of either Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus or a mixture of the bacteria. The researchers noted that the application of silver dressings was bactericidal to 90 percent of the bacteria within the biofilm in 24 hours and killed 100 percent of the bacteria in vitro after 48 hours.
A similar study has not been conducted in vivo. Bjarnsholt and colleagues investigated the action of silver on mature biofilms of Pseudomonas aeruginosa in vitro, a primary pathogen of chronic infected wounds.28 The authors found silver to be very effective against mature biofilms of Pseudomonas aeruginosa but noted that the silver concentration is important. For example, a concentration of 5-10 µg/mL of silver sulfadiazine eradicated the biofilm whereas a lower concentration (1 µg/mL) had no effect.
Further, the bactericidal concentration of silver required to eradicate the bacterial biofilm was 10 to 100 times higher than that used to eradicate planktonic bacteria that were not attached to a surface.28 The study suggested that the concentration of silver in some currently available wound dressings may be much too low for treatment and prevention of biofilms in chronic wounds.
Honey. Therapeutic honey has been advocated by many clinicians to be an alternative treatment for chronic wounds. Merckoll and co-workers assessed the effects of different concentrations of honey on bacteria by incubating methicillin-resistant Staphylococcus epidermidis (MRSE), MRSA, extended-spectrum beta-lactamases (ESBL), Klebsiella pneumoniae and Pseudomonas aeruginosa with dilution series of the honeys in microtiter plates for 20 hours.1 The authors found honey to be bactericidal against all strains of bacteria and contained bactericidal substances that penetrated the biofilms.
Maggots. Another form of wound debridement that has been gaining attention to help treat and prevent biofilm is maggot therapy. Studies have shown that both the excretions and secretions of Lucilia sericata larvae (maggots) contain bioactive compounds that may contribute to the efficacy of maggot therapy.29
Harris and co-workers evaluated the effect of Lucilia sericata excretions and secretions on the formation and disruption of Staphylococcus epidermidis biofilms.26 The authors noted that the maggot secretion and excretions not only inhibited the formation of nascent Staphylococcus epidermidis biofilm but disrupted pre-formed biofilms as well.
The maggot secretions and excretions may interfere with Staphylococcus epidermidis biofilm formation specifically via degrading factors employed in biofilm accumulation to help increase bacterial susceptibility to antibiotics and the host's immune system. Interestingly, the authors noted that maggot secretion and excretion activity depended on temperature and time. They proposed that biofilm disruption depended on the mechanism of intercellular adhesion.26
Van der Plas and colleagues assessed the effect of maggot excretions and secretions on Staphylococcus aureus and Pseudomonas aeruginosa biofilm.29 The authors noted that as little as 0.2 µg of maggot excretions and secretions prevented S. aureus biofilm formation in vitro while 2 µg of maggot excretions and secretions were required for rapidly degraded biofilms.
In contrast, the researchers found that maggot secretions and excretions initially promoted Pseudomonas aeruginosa biofilm formation, but noted the collapse of the biofilm after 10 hours of exposure.29 This led the authors to conclude that degradation of Pseudomonas aeruginosa biofilms started after 10 hours and required 10-fold more maggot secretions and excretions than Staphylococcus aureus biofilms. Further, the researchers found that boiling of maggot secretions and excretions abrogated their effects on Staphylococcus aureus, but not on Pseudomonas aeruginosa biofilms. This indicates that different molecules within maggot secretion and excretions are responsible for the observed effects.
In Conclusion
The future of chronic wound healing may lie in a better understanding of bacterial contaminants, the formation of biofilms and the development of appropriate therapies. Most biofilm infection related research findings have not yet reached clinical practice. However, a better understanding and appreciation of the factors affecting the progression from colonization to infection can help clinicians with the interpretation of clinical findings and microbiological investigations in patients with chronic wounds.
Researchers have developed animal and in vitro models to better study biofilms. This will allow a venue for therapeutic intervention and further knowledge of the physiology and interactions within multi-species biofilms, which may help develop more effective modalities and methods to treat infected and poorly healing wounds.
Preventing bacterial attachment and biofilm formation, or disrupting biofilm formation may allow penetration of topical antimicrobial agents. Other options are interference with quorum sensing and enhancement of bacteria dispersion from biofilms to a more easily destroyed state. These options can help improve clinical outcomes in soft tissue and bone infections, and in the treatment of wounds.
Dr. Wu is an Associate Professor of Surgery at the Dr. William M. Scholl College of Podiatric Medicine and Associate Professor of Stem Cell and Regenerative Medicine at the School of Graduate Medical Sciences at Rosalind Franklin University of Medicine and Science in Chicago. She is also the Director for Educational Affairs and Outreach at the Center for Lower Extremity Ambulatory Research (CLEAR) in Chicago.
Editor’s note: For related articles, see “How Biofilm Affects Healing In Diabetic Foot Wounds” on page 20, “Biofilms And Infection: What You Should Know” in the November 2006 issue and “Current Concepts In Wound Debridement” in the July 2009 issue.
References:
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