Clinical Experience with Wound Biofilm and Management: A Case Series
Abstract: Biofilm is a relatively new concept in the fields of infectious disease, wound infection, and healing. Although scientific research and “noise” regarding wound biofilm is increasing, little is known about the presentation, diagnosis, potential implications, and management strategies regarding wound biofilms. A series of four clinical cases is utilized to demonstrate the existence of wound biofilm. All patients presented with or developed a film on the wound bed that appeared to be distinct from slough; wounds also were failing to progress. Although the slough in some of the wounds was easily removed with traditional debridement methods, removal of the film required physical disruption with a curette or dry gauze. All wounds eventually progressed to healing. Considering the biofilm concept and available preclinical research, it is evident from this small case series that the appearance of biofilm in wounds is quite different from slough and requires different management strategies for its control. The evolving biofilm paradigm could profoundly change approaches to wound management. Additional research is needed in this evolving aspect of wound management. The need to consider wound biofilm is likely to be new to a majority of wound care practitioners. However, they all may have encountered wound biofilm without being aware of its potential implications. Biofilm is found everywhere — in the slime growing on the inside of a flower vase, as plaque growing on the surface of unclean teeth, and as the gunk that clogs household drainpipes. Biofilm can form on virtually any natural or man-made surface; in medicine, infections such as periodontal disease, endocarditis, and otitis media all involve biofilm, as do infections associated with foreign bodies such as contact lenses, sutures, and catheters.1 All of these infections involve bacteria sticking to a tissue surface and the subsequent formation of a complex polymicrobial community within a micro-environment that provides protection from the outside world. This is biofilm, an entity most likely to form where substrate, moisture, nutrition, and stasis combine. A skin ulcer potentially provides an ideal environment for biofilm development. The concept of biofilm was first described in detail in 1978.2 Although bacteria are perhaps most widely thought of as free-living (planktonic) or floating single cells that exist within the air or in aqueous environments, the most natural environment for a bacterium involves attaching to a surface and existing within a community of bacterial cells. It is now recognized that the physical and behavioral (phenotypic) characteristics of bacteria within a surface-attached biofilm community are very different from those exhibited by free-living bacterial cells. Where free-living bacteria are metabolically active and often highly susceptible to antimicrobial agents (including antibiotics and biocides) and immune cells, biofilm bacteria often adopt a sessile behavior with a significantly reduced growth rate that has been found in in vitro studies3 to result in a slower uptake of antimicrobial agents.3Additionally, scientific studies have shown that once attached to a surface, biofilm bacteria produce an outer protective matrix (exopolymeric substance [EPS]) that acts as a physical barrier to permeation and action of antimicrobial agents. The biofilm environment not only provides physical protection to bacteria from a potentially hostile external environment, but it also provides a habitat where bacteria can communicate with each other (quorum sensing), which may lead to an increase in virulence and propensity to cause infection.4 Results of scientific and clinical studies have shown that an elevated and persistent inflammatory response may lead to the over-production of potentially destructive enzymes (eg, matrix metalloproteinases [MMPs] and pro-inflammatory cytokines) as well as oxygen metabolites, promoting tissue destruction.5 Although the existence of biofilm now is well accepted in the field of medical microbiology and infectious disease, knowledge relative to the characteristics and physical presentation of biofilm in wounds presently is limited. However, based on current scientific understanding, several leads from clinical experience are evident that may help to visually characterize and anticipate chronic wound biofilm. First, studies have shown that bacteria that are attached to a tissue surface often are encased within an extensive polysaccharide matrix,6 which contributes to their stickiness and protection. Polysaccharide is a complex carbohydrate consisting of smaller sugar units and forms a significant part of biofilm EPS.7 Laboratory studies have shown unbound polysaccharide released by biofilm cells is responsible for an often slimy, viscous matrix.8 The polysaccharide polymers also can be bound together by metal ions such as calcium and magnesium; these enhance the viscous, gel-like properties of EPS.8 These characteristics may be associated with the stubborn, slimy film often evident on the surface of chronic wounds and frequently overlying granulation tissue. Pseudomonas aeruginosa, a common wound pathogen, is a potent producer of alginate (polysaccharide) slime; laboratory studies have shown that over-expression of this substance is associated with a structurally highly differentiated and robust biofilm.7,8 Starkey et al8 have postulated that low oxygen tension and anaerobic metabolism may be a key signaling mechanism for alginate production, which may well explain the often viscous, slimy exudate that accompanies P. aeruginosa in poorly perfused chronic wounds. It is also believed that biofilm bacteria that produce significant amounts of exopolysaccharide matrix are more pathogenic than their non-exopolysaccharide-producing counterparts as demonstrated by an increased resistance to antimicrobial agents.8 In industry, biofilms can be removed successfully by strong acids or surfactants or by vigorous mechanical interventions (eg, ultrasonification). However, removing biofilm from body tissue can be much more challenging because of the obvious risk of toxicity, pain, and tissue damage. The purpose of this case series is to stimulate thought, insight, and further clinical research into wound biofilm as it may appear in vivo and to relate this to the authors’ early scientific understanding in the hope that it will encourage the development and communication of strategies to control this potentially formidable barrier to wound healing.
Background. The authors’ consideration of chronic wound biofilm was first prompted by observance of viscous green slime developing in several ulcers managed with a silver-containing alginate dressing (see Figures 1a,b). These ulcers were generally free of devitalized tissue and exhibited at least moderate wound drainage. Several of the patients were receiving systemic antibiotics. Most wound care practitioners understand that green slime and a sweet odor in a wound are clinical indicators of the presence of Pseudomonas aeruginosa (the green color is caused by production of the pigments pyocyanin and fluorescein). Two swab samples confirmed the presence of P. aeruginosa in one wound and a Gram-negative bacterium in the other. The anomaly here was that this bacterium was growing in the presence of both systemic antibiotics and a silver-containing dressing that is reported to release silver ions that kill a variety of pathogenic bacteria, including P. aeruginosa. Case 1. Mr. Q was 58 years old with a history of diabetes, hypertention, coronary artery disease, a coronary artery bypass graft in 1999, and left lower extremity femoropopliteal bypass graft in 2006. Most recently, he had surgical intervention to release compartment syndrome of his lower left leg. During the initial postoperative period, sharp debridement and papain-urea ointment were used to remove dead sloughy tissue from the ulcer that had developed as a consequence of ischemia resulting from the compartment syndrome. After about 2 weeks, when the incision sites were 80% to 90% free of slough, the treatment strategy was changed to a silver-containing alginate dressing in an attempt to control exudate and minimize the opportunity for infection to develop. Careful inspection of the wound revealed a cloudy, shiny, thin slime material on the ulcer bed that persisted despite daily cleaning with pulsed lavage. A protein-degrading papain-urea ointment was effective at digesting the noticeable yellow slough but it did not prevent nor seem to facilitate removal of the shiny slime. A noticeable edge to the slime (see Figure 2a) at the outer aspect of the wound could be lifted with forceps. The ability to remove the slime as a continuous sheet appeared to be dependent on its thickness and tensile strength, both of which were variable over the wound. A disposable curette was found to be the most effective in gently disrupting and removing the film while minimizing trauma to the underlying wound bed, which was a deeper red color with more defined wound base contours (see Figure 2b). Yellow slough present on the wound bed was adhered and could not be removed in this fashion. Slough was loosened by pulsed lavage during the initial postoperative treatment period but the physical force of this mechanical debridement strategy was seen to be completely ineffective in disrupting the film. The film redeveloped daily but areas of yellow slough decreased in size over time. Despite the reduction in slough, the wound failed to show signs of healing as indicated both by the amount of exposed tendon and unchanged wound dimensions (see Figure 2b) A transcutaneous oxygen measurement (TcOM) was taken to help determine the probability of healing. Tissue oxygen tensions (PO2) of 5 mm Hg to 20 mm Hg have been recorded in nonhealing wounds; whereas, 25 mm Hg to 35 mm Hg have been measured in healing wounds9 and a TcOM value above 40 mm Hg has been shown to be indicative of wound healing.10 Mr. Q’s TcOM values were 14 mm Hg at the dorsal foot and 24 mm Hg at the level of the ulcer. He was referred for hyperbaric oxygen therapy but did not tolerate treatment due to impaired cardiac function; he was re-hospitalized at a different facility for cardiac intervention. Subsequent improvement in cardiac output led to a rapid decrease in wound size over approximately 2 weeks with minimal evidence of film redevelopment in the wound bed. Final closure was achieved using split-thickness graft that fully took. Case 2. Ms. R was 83 years old, thin-framed and ambulatory with a history of venous stasis ulcerations for which she had been treated for several months as an outpatient. She was admitted to acute care for infection of a stasis ulcer on her left calf that measured approximately 5 cm in diameter. At the time of her admission, she was wearing a compression wrap. The wound was managed topically with a silver-containing alginate dressing. Within the first week of her hospitalization, her lower extremity edema resolved due to more prolonged supine positioning. During this transition, she became less tolerant of compression and developed an area of pressure necrosis. Compression was stopped and necrotic areas were sharp debrided and treated with collagenase ointment for enzymatic debridement and polysporin powder for antimicrobial protection (see Figure 3a). Over approximately the next 3 weeks, Ms. R completed her systemic antibiotics. Topical wound management cleared devitalized tissue from the wound bed and pink granular buds began to develop. However, as her ambulation increased, moisture management became an issue, leading to maceration and circumferential ulceration. Because she was unable to tolerate compression, absorptive cotton pads and elevation were added to her protocol of care. As the exudate increased, a thick film layer over the wound bed was noticed. This film persisted after daily pulsed lavage and reformed daily despite application of collagenase. The film could be partially removed by gently rubbing the wound with a sterile gauze pad, revealing an area of red granulation tissue at the distal edge of the ulcer (see Figure 3b). On the following day, a significant amount of sanguinous drainage was noted on the cover dressing and the film had reformed over the wound bed. A silver-containing alginate dressing was applied for two consecutive days and changed daily before performing pulsed lavage. Because the pale green film reformed each day, this strategy was discontinued. A silver hydrofiber dressing was used for the next 3 days. This dressing seemed to provide better absorption, leaving a healthier pink/red wound bed with clearly defined contours but further treatment decisions were compromised by this product’s limited availability (this particular product is not available on facility formulary). Hydrofiber and alginate are formally considered by regulators to be interchangeable but in vivo activity appeared distinctly different in this instance. No further study of vessel perfusion pressure was provided. During this period of time, exudate management was a problem. The wound bed film reoccurred daily and was physically removed as much as Ms. R was able to tolerate. Wound maceration was leading to increased ulcer size, now nearly circumferential. The most absorptive formulary product, a silver-containing alginate, had demonstrated inadequate absorption for Ms. R’s wounds. Therefore, negative pressure wound therapy was initiated in an attempt to more effectively manage exudate. Ms. R tolerated -50 mm Hg continuous suction with dressing changes on Monday, Wednesday, and Friday. This strategy was effective in promoting wound progression and all ulcers became bright red and granular. Split-thickness graft was performed for final wound closure. Case 3. Seventy-eight-year-old Mr. D’s medical history included congestive heart failure, enlarged prostate, hypertension, and chronic lower extremity edema that had been diagnosed as venous stasis disease based on presentation. No formal venous studies had been performed. The referring physician had attempted a procedure to remove or ligate veins but this procedure did not solve Mr. D’s problem. At his first office visit, he had 3 to 4+ bilateral lower extremity pitting edema with hemosiderin staining and three full-thickness stasis ulcers, one on his right shin and one on his proximal right calf measuring approximately 0.1 cm depth and one on his distal right calf with depth of approximately 0.3 cm. Specific chronicity was uncertain but Mr. D reported reoccurring problems with weeping ulcerations. Initial treatment included silver alginate dressing and compression. Close contact was maintained with his cardiologist during the time that his lower extremity edema was initially mobilized. At first, all ulcers demonstrated significant improvement using 20 mm Hg to 30 mm Hg compression provided by elastic wrap. Although the more superficial wounds closed during the first several weeks, the deeper shin ulcer stalled, with a clinically significant film build-up noted on the wound bed. The film appeared as a cloudy, translucent film on the wound surface through which larger granular buds protruded. Mr. D tolerated careful sharp removal of the film during each visit. However, the film redeveloped between monthly visits and the ulcer showed minimal decrease in diameter or depth. Initially, the visible slime was removed with scalpel and sharp forceps. Mr. D was instructed to cleanse the wound daily with a 0.057% sodium hypochlorite antiseptic before applying a new dressing. At Mr. D’s appointment the following month, the wound was without significant film and the ulcer depth had begun to decrease. At Mr. D’s next monthly visit, the ulcer depth had reduced to skin level and the wound had begun to re-epithelialize. Case 4. Mr. V was 54 years old with a history of hypertension, chronic venous stasis disease, and deep vein thrombosis. He was admitted to acute care for management of lower extremity cellulitis. Initial assessment revealed bilateral lower extremity 4+ pitting edema with actively exudating ulcerations on both calves and his left dorsal foot. The ulcers were covered with a thick, opaque, wet scab-like layer. The left dorsal foot ulcer is seen in Figure 4a. Mr. V was receiving systemic antibiotics, diuretics, and warfarin sodium (a potent blood thinner). An arterial ultrasound ruled out significant impairment to distal arterial flow. Initial wound therapy included application of mafenide acetate covered with oil emulsion gauze, absorbent cotton pad, gauze wrap, and compression wrap. Neither this management strategy nor rubbing a sterile gauze pad on the wound facilitated film removal or prevented its redevelopment. However, the film could be removed with forceps and scalpel. Removal was somewhat akin to trying to pull a suction-pad bath mat from a wet tub — the film appeared to be stuck to the wound bed without being a part of it (as slough is). Interestingly, the wound areas cleared of film with this method of sharp debridement but did not bleed despite the patient being anti-coagulated; this is very different from how this same wound would appear after sharp debridement of adherent slough (see Figure 4c). Skillful film removal can inflict minimal harm to the wound bed, and in some wounds, thick film can be removed completely (see Figure 4d).
Through this small case series of patients, it has been shown that links may exist between biofilm, ischemia, persistent inflammation, moisture, and wound recalcitrance and that signs of biofilm in chronic wounds may be visually evident. Biofilm is bacteria-derived living material (as opposed to slough being host-derived dead tissue) that often has a cloudy, translucent and viscous, gel-like appearance. It often forms above granulation tissue, and as such it may interfere with epithelialization. Despite attaching firmly to wound tissue, biofilm can be carefully peeled away without causing damage to underlying tissue. Although biofilm will reform, a protocol of care that includes its repeated removal is likely to support wound progression over time. Biofilm and alginate. In the cases described, the use of a silver-containing alginate dressing coincided with the rapid development of viscous, green-colored exudate; P. aeruginosa commonly was implicated. Although silver is used for antimicrobial protection, in this study it was observed that use of the silver alginate dressing was associated with the formation of thick, green exudate. Clinicians may want to consider 1) why silver (and in some cases, systemic antibiotics) appears to be ineffective as an antimicrobial agent in certain situations; 2) whether exudate may be too viscous to trigger release of the silver ions from the alginate dressing; and 3) whether exudate absorption is compromised by the quantity of the fluid (in each case the dressing looked like a wet blanket over the wound, but even a wet blanket can bleed its dye) or the presence of biofilm (ie, viscous alginate exopolysaccharide produced by P. aeruginosa). Possibly, dressing components (eg, alginate and calcium) could have contributed to formation of the viscous exudate. In the authors’ scientific experience, alginate has been found to be an important component of biofilm EPS (notably in the presence of P. aeruginosa), rendering it hydrophobic, robust, and difficult to disperse. Calcium likely strengthens the bonds between alginate polysaccharide chains in the EPS8 and enhances pyocyanin production in P. aeruginosa.11 A base wound pH likely enhances fluorescence of the P. aeruginosa-produced fluorescein dye, inducing a bright green-blue-yellow coloration in the wound. Interestingly, with regard to silver, one in vitro study12 demonstrated the ability of silver ions to disrupt the biofilm (EPS) structure formed by Staphylococcus epidermidis at low concentration (50 parts per billion). However, regarding the current case studies, clinical wound conditions such as viscosity of the EPS produced by P. aeruginosa may have compromised the ability of the silver dressing to interact with and combat biofilm bacteria. Biofilm and MMPs. In the cases described, the slimy, cloudy film often seemed to be associated with wounds that did not exhibit clinical signs of infection but appeared to be fighting something that manifested as persistent inflammation. This observation may relate to the state of “silent infection” or “critical colonization,” a concept described as a state in which bacteria are established within a biofilm community and compromise wound healing without inducing clear signs of clinical infection.13 Inflammation, a natural and essential part of any healing process, manages potential infection and clears the wound of devitalized tissue. However, clinical studies have shown that chronic wounds often exist in a prolonged inflammatory state involving elevated pro-inflammatory cytokine, proteinase, and oxidative activity — all of which may play a role in delayed wound healing.5 It is widely acknowledged that pathogenic bacteria stimulate inflammation through activation and proliferation of macrophages and pro-inflammatory cytokines.14,15 Recently, biofilm has been considered to be a primary cause of the prolonged inflammation that exists in chronic wounds16 and the cause of silent chronic inflammation in hemodialysis patients due to repeated stimulation of macrophages.17 The persistence of biofilm in chronic wounds may be associated with elevated production of proteinases (MMPs) and subsequent delayed healing. Biofilm-induced chronic inflammation also is likely to lead to increased fluid production which, in turn, is conducive to biofilm development. If this is the case, adequate biofilm control may help abate elevated and destructive proteinases and reactive oxygen species associated with chronic inflammation as well as fluid production in wounds. Biofilm and moisture. In Case 2, stasis disease and infection led to significant wound drainage. The authors believe that moisture management may be an important factor in biofilm control. Too much absorption is likely to dry a wound bed which supports neither bacterial proliferation nor wound healing. The authors have observed that inadequate wound absorption may provide a moist, somewhat static wound environment that may contribute to the development of wound biofilm. Biofilm and slough. Slough is commonly associated with chronically inflamed, bacteria-colonized, malodorous wounds. Clear differences between slough and biofilm became evident in the current clinical cases primarily due to the observed variable effectiveness of treatment modalities such as enzymatic debridement and pulsed lavage in the management of slough and biofilm. Slough is moist, dead, fibrinous material, comprised essentially of protein that tends to be recurrent particularly in poorly perfused chronic wounds. Enzymatic debridement and pulsed lavage were somewhat effective at removing slough but not the film. In Case 2, the yellow slough was denatured and diminished quickly with papain-urea ointment; whereas, the cloudy biofilm slime redeveloped daily. Because papain-urea is designed to facilitate debridement through its non-specific proteolytic enzyme activity and moisture-holding capabilities, better clinical outcomes may be expected against slough than against biofilm. Based on the authors’ scientific knowledge, slough’s composition and moisture content may provide an ideal medium for the proliferation of bacteria and may enable bacteria to exist as susceptible “planktonic” cells within the proteinaceous matrix. In contrast, bacteria attempting to stick to the surface of a wound are presented with a greater challenge, encouraging development of a biofilm state to provide protection from a relatively hostile inflammatory environment. The appearance of biofilm was quite different from slough, often appearing as a cloudy, translucent, slimy layer across the wound surface. Although biofilm has numerous chemical constituents (eg, proteins, nucleic acids, and lipids),18 polysaccharides are a major component, particularly within EPS. Hence, biofilm’s physical form and characteristics in wounds are different from slough. In this case series, both pulsed lavage and enzymatic (proteolytic) debriding agents demonstrated some efficacy in removing slough, yet they were ineffective against biofilm. Other treatment modalities such as physical debridement (sharp or use of a sterile gauze pad) were more effective in removing biofilm and the daily application of a nontoxic antiseptic solution was shown to prevent biofilm redevelopment. In Case 3, biofilm removal and wound depth reduction coincided with the daily application of a hypochlorite-based antiseptic solution. Oxidizing biocides such as sodium hypochlorite have been shown to reduce wound bioburden19 and remove biofilm from a nonviable surface material.20 Clearly, the challenge is to prevent or remove biofilm using agents that do not induce toxicity, pain, or significant tissue damage. Biofilm and peripheral arterial disease (PAD). In Case 1, biofilm elimination was clearly associated with increased perfusion and oxygenation of wound tissue following cardiac intervention. The lack of oxygen (hypoxia) in wounds may stimulate the production of biofilm slime (exopolysaccharide)8 and increase probability of wound infection.21 Effective autolytic debridement relies on optimal functioning of host immune cells (neutrophils); in a low-oxygen, poorly perfused environment, this process is likely to be impaired, potentially contributing to the accumulation of biofilm. This case series demonstrated that some management strategies used therein were more successful than others. Often a combination of strategies was required to overcome biofilm and encourage healing. Physical devices (curette, scalpel, forceps) effectively removed biofilm; increasing tissue oxygen tension and applying an oxidizing cleansing solution also were beneficial in specific cases. The use of antimicrobial agents alone may be ineffective against biofilm bacteria as evidenced in Case 4 where biofilm re-appeared daily despite treatment with systemic antibiotics and mafenide acetate followed by silver alginate dressing.
From this small case series of patients and based on limited clinical experience, the authors have determined that with a trained eye, biofilm can be visualized in chronic wounds and that its appearance is quite different from that of slough. Due to the differing biochemical compositions of biofilm and slough, different management strategies are required for these substances’ removal and control. Biofilm is often difficult to remove; perseverance is necessary to encourage wound progression. Although some clinical options to facilitate biofilm control currently exist, the authors recognize much remains to be learned. There are many challenges ahead in this evolving aspect of chronic wound management that well may lead to profound changes in clinical practice. Further clinical research relative to biofilm observation and management strategies in chronic wounds is needed. Ms. Hurlow is a nurse practitioner, Plastic Surgery Group, Memphis, TN. Mr. Bowler is Director, ConvaTec Global Research and Development, Wound Therapeutics, Deeside, UK. Please address correspondence to: Philip G. Bowler, ConvaTec, Wound Therapeutics Global Development Centre, First Avenue, Deeside Industrial Park, Deeside, Flintshire CH5 2NU, UK; email:email@example.com.
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