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Biofilm And DFUs: What The Literature Reveals
Biofilm is a complex surface-attached community of bacteria, viruses, protozoa and fungi, both planktonic and integrated, encased in an extracellular polymeric substance (EPS) composed of hydrated polymers and debris.1-6 Biofilm composition is approximately 70 to 90 percent EPS and five to 30 percent microorganisms.1,3 Biofilm growth occurs as microorganisms detach from the biofilm and become planktonic.1 Reports reveal the presence of biofilms in at least 60 percent of chronic wounds.2 Fifty-nine to 85 percent of diabetic foot ulcerations (DFUs) are polymicrobial due to the ability of various pathogens to form their own microcolonies within the biofilm.6-8
Gram-positive organisms alone account for 13 to 29 percent of biofilms, gram-negative organisms alone account for 39 to 79 percent of biofilms and 31 to 48 percent of biofilms are mixed gram-positive and gram-negative organisms.8,9 Longer wound duration also positively correlates with increased bacterial diversity.10-13 Wound depth also results in variation of pathogens present.2,10-24 Bacteria located more superficially within the biofilms are typically aerobic and skilled at producing the protective biofilm cover and propagating a continued inflammatory response. These include Staphylococci and Corynebacterium, both normal cutaneous flora of the foot.2,10-23 Pathogens located deep within biofilms are more likely to be Pseudomonas and anaerobes due to local hypoxemia in the wound bed.11,13,23,24
How Do Biofilms Impact Wound Healing?
Biofilms may propagate delayed healing through their: structure; resistance to antibiotics; and interaction between the microorganisms residing within the biofilm and of the biofilm itself with the host.1,13,21-23 The EPS structure of biofilms prevents neutrophil and antibiotic penetration, leading to persistence of the protective covering and propagation of microcolonies of pathogens within.1,13
Pathogens within biofilms can display an increase in antibiotic resistance by a factor of up to 1,000 compared to planktonic pathogens.13 Biofilms convey antibiotic resistance through multiple methods including: preferential selection of antibiotic-resistant bacteria; senescent or slow-growing bacteria which make antibiotic therapy targeted at actively dividing bacteria ineffective; and the transfer of genes conveying antibiotic resistance between bacteria within the biofilm.1,13,21-23 Interaction of the biofilm with the host depends on factors that can alter a host’s ability to mount an immune response, such as the presence of diabetes.5,13,20,23,25-27
Symbiosis between the host’s cutaneous immune system and resident cutaneous bacterial flora help prevent infection by less virulent opportunistic or pathogenic bacteria.5,13,20,23,25,26Staphylococci species account for up to 70 percent of the normal cutaneous flora of the foot.15- 21Staphylococcus epidermidis and diphtheroids, which include the genuses Corynebacterium and Enterococcus, being the most common normal cutaneous bacterial residents.15-21 The presence of Staphylococcus aureus on the foot keeps infection related to the less virulent and slower growing Staphylococcus epidermidis and Corynebacterium from becoming pathogenic.21,26 However, higher sweat glucose concentrations, reduced tissue perfusion and oxygenation, altered cutaneous thermoregulatory function due to peripheral neuropathy and reduction of Langerhans cells (the resident immune cells in the epidermis), result in the cutaneous structures of patients with diabetes being in a constant subclinical inflammatory state, making them more prone to infection from less virulent and opportunistic pathogens.5,13,20,23,25-27
As the plantar aspect of the foot harbors the majority of normal pedal cutaneous bacterial flora, it is not surprising that the most prevalent bacteria reported in DFUs are Staphylococci, Corynebacterium and Pseudomonas aeruginosa.5,7,10,13,15,19,24,26 While there are higher concentrations of more virulent Staphylococcus aureus, Staphylococcus epidermidis remains the most commonly isolated bacteria, occurring 5.4 times more often in patients with diabetes than those without diabetes.19,26 As these bacteria are normally resident flora of this foot, some may deem them skin contaminants on culture, downplaying their pathogenic role.
A recent study using advanced molecular diagnostic techniques for bacteria identification of bone samples with suspected or confirmed osteomyelitis identified Corynebacterium as the most prevalent pathogen.28 Studies also note Streptococcus, Enterococcus, Acinetobacter, Proteus, Klebsiella, Citrobacter, Stenotrophomonas, Enterobacter, Escherichia and enterococci in DFUs.5,7,10,13,15,19,24,26 One report cited Clostridium as the most common anaerobe encountered in DFUs across all Wagner grades, most prevalent in wounds of long duration.10
Fungal pathogens belonging to the phylum Ascomycota, Cladosporidium herbarum and Candida albicans (the most prevalent in DFUs) can also be present, particularly in patients who have already received antibiotic therapy or are experiencing a complication.29 A statistically significant delay in wound healing existed in DFUs when these fungi were present versus when they were not.29 The structure and extracellular matrix of Candida albicans mycofilms support bacterial growth and confer reciprocal protection from antibiotic therapy.30 There is evidence that Candida albicans protects Staphylococcus epidermidis from vancomycin and that Staphylococcus epidermidis can inhibit biofilm penetration by fluconazole.30 Dysbiosis of the commensal flora on the diabetic foot may also contribute to the preferential selection of bacteria resistant to β-lactams, aminoglycosides, macrolides and minocyclines.31 Methicillin-sensitive and methicillin-resistant coagulase-negative Staphylococci and Pseudomonas are notably present more often on the feet of patients with diabetes as opposed to those without diabetes.31 Methicillin-sensitive and methicillin-resistant Staphylococcus epidermidis persisted in patients with diabetes underdoing elective procedures (intact skin) and urgent/emergent procedures (presence of a DFU) even after use of a specific surgical preparation.32 The ability of biofilms to delay healing, combined with the systemic effects diabetes has on commensal bacteria of the foot, may explain the importance of identification and treatment of biofilms to accelerate wound resolution.
Key Considerations In Organism Identification And Biofilm Disruption
The ideal pathway of biofilm treatment is difficult to ascertain given the paucity of literature in which there is employment of standardized and optimal methods of identifying offending pathogens prior to and following treatment. However, recent consensus recommendations consist of three steps:33
1) identification of offending microorganisms;
2) biofilm disruption; and
3) topical and/or systemic treatment.33
The diversity of bacteria within a biofilm makes it difficult to accurately determine through a swab culture of the wound, which reportedly only identifies one percent of bacteria present.6,10,24 This is due to capture of more superficial, planktonic, aerobic and actively dividing bacteria. Thus, more resistant, slower-growing and anaerobic pathogens residing deeper within the biofilm may be missed. Tissue samples obtained from the depths of the wound may be more predictive of the true offending organism.4 Use of advanced molecular diagnostics can also help identify offending pathogens.5,9,10 Molecular diagnostic methods identified 338 different species in 168 wounds, compared to identification of only 17 different species detected by swab culture.10 Of the 338 different species detected with molecular diagnostic methods, nine of the top 20 identified were anaerobic. However, access to these methods, the time required to perform them and their cost can limit their use.5,9
Use of novel, point-of-care fluorescence imaging may assist in identification of bacteria present within a wound, including targeting treatment of pathogenic bacteria in DFUs.34-36 This device utilizes safe, violet light to cause bacterial fluorescence. Staphylococcus, Proteus and Klebsiella will fluoresce red, while Pseudomonas will fluoresce cyan. A study of 350 wounds compared clinical examination findings with fluorescence imaging and microbiological culture analysis of wound punch biopsies.36 Thirty-seven percent of wounds have bacterial loads greater than 107 CFU/g, an amount that exceeds the point where a wound is considered infected. Clinical assessment correlated with less than 15 percent of wounds meeting this level of bacterial load. The added use of point-of-care fluorescence imaging was able to alert clinicians of areas of increased bacterial load not evident on visual examination, guide debridement and provide further input on initiation and monitoring of antibiotic therapy effectiveness.
Debridement is the most clinically and cost-effective method of disrupting biofilms.23 Significant reduction in bacterial load, particularly anaerobic bacteria, takes place following debridement.6,11 However, frequent debridement is necessary as biofilms can re-form within 24 hours.6 Use of cleansers containing surfactants and enzymes can also disrupt biofilms, resulting in the potential for increased susceptibility to antibiotic therapy.4,24,33 While several solutions are used in practice for wound disinfection, published literature on their efficacy for biofilm management are lacking. The effects of 15 minutes of exposure of four cleansers – povidone-iodine (PVP-I), chlorhexidine (CHX), a surfactant-based solution with polyhexamethylene biguanide (PHMB) and a superoxided solution of sodium hypochlorite, hypochlorous acid, sodium chloride and oxidized water (Microcyn®, Sonoma Pharmaceuticals, Woodstock, GA) – on Staphylococcus aureus and Pseudomonas aeruginosa biofilms found complete killing of Staphylococcus aureus biofilms with PVP-I and reduction with PHMB, Microcyn and CHX. PVP-I, PMHB and Microcyn also resulted in complete killing of Pseudomonas aeruginosa biofilms while CHX only resulted in a reduction of Pseudomonas aeruginosa biofilms.37 Another modality of biofilm disruption is the use of low-frequency (20 to 60 kHz) ultrasound. The micro-sized gas bubbles formed with these devices and their cavitation effects are theorized to remove non-viable tissue and cause bacteria to be more susceptible to antibiotic therapy.38 A recent systematic review of the use of low-frequency ultrasound debridement in the treatment of chronic wounds determined that use of this modality at least three times per week may be a beneficial adjunctive treatment for chronic wounds, although the body of evidence was poor.38
A Closer Look At Topical Treatment Options For Biofilms
Topical treatment of biofilms includes the use of cadexomer iodine, silver and antimicrobial peptides. While some may use other topical agents clinically for treatment of DFU biofilms, specific support in the published literature does not exist. The three topical agents mentioned above have some clinical evidence, beyond the bench top, to support their potential use. However, results of these studies are limited by small sample sizes and inconsistent protocols for disinfection, debridement and methods of bacterial identification.
Cadexomer iodine is an iodophor of iodine, allowing for sustained release. It has broad spectrum antimicrobial activity, anti-inflammatory effects and microorganisms show no resistance to it.39 A pilot study of cadexomer iodine used for two or six weeks, applied every other day, found reduction in total microbial loads, composition and richness of diversity of biofilms, although no statistically significant difference in total microbial load was evident between the two treatment durations.40 Larger studies with standardized debridement frequency, tissue samples from the depths of the wound as opposed to the wound margin and use of advanced molecular diagnostic techniques for pathogen identification may also help elucidate the benefit of cadexomer iodine use.
Silver also has a broad spectrum of antimicrobial activity, including activity against resistant strains. There is evidence that it is more effective against Pseudomonas aeruginosa than Staphylococcus aureus.41 A pilot study of 16 DFUs treated with disinfection, debridement and application of silver sulfadiazine every 72 hours showed reduction of inflammation and bacteria present and deposition of new collagen at two weeks.41 At thirty days, all culture results, obtained from punch biopsies of the wound bed, were negative. Larger studies are required to determine if the effect of silver sulfadiazine on DFU biofilms is due to the topical agent alone or the combined effect of wound disinfection and debridement performed every 72 hours.
Use of topical antimicrobial peptides is an up-and-coming treatment modality. Antimicrobial peptides are part of the innate defense mechanisms of the skin and include βbeta-defensins, cathelicidins, ribonuclease and chenerin.42 Antimicrobial peptides exhibit a broad spectrum of activity against bacteria, fungi, viruses and protozoa.24,42 These peptides function to disrupt bacterial and biofilm growth, modulating the host immune response, resulting in more effective pathogen killing.24 They also promote wound healing through modulation of angiogenesis and reepithelization.24 More research is necessary to determine their efficacy in biofilm reduction and to establish an appropriate carrier to enable sustained, long-term release and prevention of degradation by pathogens and/or substances within the wound environment.24
Systemic And Adjunctive Approaches To Biofilm Treatment
Systemic treatment of biofilms involves the administration of antibiotic therapy. Specific to DFU biofilms, systematic antibiotic therapy noted to be most effective against Staphylococcus aureus and Staphylococcus epidermidis, including MRSE and MRSA, are ciprofloxacin, clindamycin, gentamycin, co-trimoxazole, doxycycline, tetracycline, vancomycin and linezolid.7,8,14,20 Enterococci show sensitivity to high levels of aminoglycosides.7 Reports reveal susceptibility of gram-negative organisms, including Pseudomonas aeruginosa and Klebsiella pneumoniae, to piperacillin/tazobactam, amikacin, imipenem, polymyxin-E and tigecycline.7,8,14,20 The major disadvantage of systemic antibiotic therapy is its limited ability to penetrate biofilm in levels sufficient to provide a positive antimicrobial effect while avoiding systemic adverse and serious adverse events. The ability to have sustained release of high local concentrations of antibiotics may be better suited at treating biofilm pathogens while mitigating the potential for adverse events.14,43 One of these modalities is the use of antibiotic impregnated beads. Absorbable or non-absorbable beads impregnated with heat-stable antibiotics, such as vancomycin and/or aminoglycosides, can reduce bacterial load when combined with aggressive surgical debridement (see photos above).43,44 One study reported a two to eight-log reduction of antimicrobial load with adjunctive antibiotic impregnated bead use.44
Concluding Thoughts
While the clinical evidence basis within the literature for the optimal treatment pathway is lacking, the recommendation for identification of offending microorganisms, biofilm disruption, and topical and/or systemic treatment remains.33 Use of these three steps has been shown to not only expedite wound resolution but to reduce health care expenditure, as well. A retrospective review comparing the economic impact of treating patients with DFUs with the standard-of-care for DFUs (n = 189) compared to those treated with a biofilm-based system, in which offending organisms were identified using advanced molecular diagnostics (n = 215) noted a 73 percent reduction in total cost to wound resolution.45 Reduced health care expenditure occurred primarily due to a dramatic decrease in the use of systemic antibiotics and the number of debridements required for healing. This latter part seems counterintuitive given that debridement encompasses the second of the three basic steps in biofilm treatment pathway. However, the ability to target treatment to the offending pathogen may contribute to the reduced presence of non-viable tissue which previously accumulated due to persistent biofilm presence.
Biofilms are present in at least 60 percent of chronic wounds.1 A recent systematic review and meta-analysis of biofilm presence in chronic wounds reported a prevalence of 78.2 percent.2 Biofilms present in DFUs are most often polymicrobial with increased diversity in wounds of greater duration and severity.6-8 Microorganisms present can vary throughout the biofilm based on location and depth.2,10-24 The most common offending pathogens are commensal flora of the foot, now able to exhibit virulence due to the dysbiotic effect of diabetes.5,7,10,13,15,19,20,23-27 Clinical pathways should focus on pathogen identification, disruption and treatment. Deep tissue samples obtained after disinfection and debridement, along with advanced molecular diagnostic techniques and point-of-care fluorescence imaging can help identify pathogens and guide treatment. One can then optimally select and implement topical and systemic treatments for increased effectiveness. Future large studies using standardized protocols for disinfection, debridement and pathogen identification will continue to optimize the evidence basis for management of biofilms associated with DFUs.
Dr. Marmolejo is the Western Region Clinical Wound Specialist with LifeNet Health and a freelance medical writer with Scriptum Medica.
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