The presence of biofilm can complicate the treatment of chronic wounds. Accordingly, these authors survey the literature for physiological insights on biofilm development, discuss diagnostic challenges and emphasize judicious use of antibiotics to help ensure optimal outcomes.
Treatment of chronic skin ulcerations can pose significant problems for the clinician. In the United States, there are millions of patients who are affected with the attendant medical, financial and psychological impact of ulcers.1
Up to 20 percent of people with diabetes will develop a diabetic foot ulcer in their lifetime. As the incidence of diagnosed diabetes rises, the number of diabetic foot ulcers continues to increase. It is estimated that the number of patients with diabetes will double in the United States by the year 2050 to approximately 48 million people.2 This would indicate that 9.6 million diabetic foot ulcers would require treatment.
Many of these wounds would become chronic in spite of appropriate treatment. There are many contributing factors including metabolic factors, peripheral arterial disease, venous disease, neuropathy and/or deformity and trauma. A chronic wound is any wound that fails to proceed through an orderly and timely process to produce anatomical and functional integrity.3 Difficulty in the timely treatment of these chronic wounds can lead to infections, which open the pathway for amputation and life-threatening sepsis.1,4 The prolonged healing can be a financial burden on the healthcare system with an expenditure of over $25 billion a year in the United States.5
The long-term healing associated with a chronic diabetic ulcer certainly affects a person’s quality of life. This can result in depression, social changes and financial loss due to decreased mobility and changes in lifestyle.6
There have been many recent advances in wound care and wound healing modalities. Numerous studies have examined the treatment of diabetic foot ulcers with advanced modalities such as cultured human dermis, skin equivalents, recombinant human platelet-derived growth factors and advanced wound dressings.4,7-11
Any physiologic or metabolic factor that adversely affects the normal healing pathway can lead to a delayed, non-healing chronic wound. These wounds often develop bacterial contamination, which frequently goes unrecognized due to the lack of the cardinal signs of gross infection.12 This bacterial contamination in chronic wounds can lead to biofilm structures that develop a matrix that offers protection against the host defenses.13-15 Biofilms can adhere to either living tissue or inert substances such as implants, screws or plates.
A biofilm is a polymicrobial collection of microorganisms that develop on the surface of chronic wounds. These microorganisms produce an extra-polymeric substance that contributes to the structure of the biofilm. This matrix forms a barrier around itself, making it difficult for antimicrobial agents to penetrate.
The study of bacterial biofilms has gained increased attention in recent years due to the implication of biofilms in delayed chronic wound healing. Numerous studies have linked biofilms with infection with one study attributing biofilms to up to 80 percent of human infections.13,16,17 The study of biofilms is a relatively new field. There is no unanimous method of diagnosis nor do we know the most effective treatment.
Certainly, we are learning that the chronic wound commands special notice and treatment. Acute wounds usually harbor bacteria in the free floating or planktonic form without a complex matrix or biofilm covering. Unfortunately, clinicians commonly utilize a swab culture for diagnosis. It is much more difficult to diagnose biofilms in chronic wounds. A routine swab culture may be adequate for sampling of planktonic forms but might not be adequate in assessing the growth of biofilm, which may go undetected. Lack of identification of a biofilm infection in a chronic wound can lead to amputation and life-threatening sepsis.
Unfortunately, the patients who develop chronic wounds are often immunocompromised in some way (e.g. poor circulation or hyperglycemia), which facilitates the establishment of bacterial biofilm communities and makes elimination of the biofilm all the more difficult. Wounds in limbs with poor perfusion are among the most difficult to heal.18 Patients with a chronic wound in a diabetic limb with a TcPO2 less than 20 mmHg often must undergo a major limb amputation.
The healing of wounds, especially in patients with diabetes, can be a life or death situation. Apelqvist and colleagues showed that after a major limb amputation, patients with diabetes underwent contralateral limb amputation in 48 percent of cases within five years.19 Pohjolainen and co-workers demonstrated that the five-year mortality rate for patients with diabetes after a major limb amputation is 80 percent.20
A wound care specialist, who understands that biofilms exist in chronic wounds, needs to seek reliable detection and treatment methods. Unfortunately, satisfactory solutions do not yet exist.
The current cultivation methods that are in routine use in most diagnostic labs to isolate and identify microbial species do not support the development of biofilms. Unlike their planktonic natural form, biofilms coaggregate, which contributes to a resistance as well as a metabolic advantage, allowing the survival of anaerobic species in the presence of an aerobic environment.20,21 The adherence of biofilm to host tissue, combined with the possibility that biofilm may form in deep tissue, makes the use of swabs on wound surfaces an unreliable method for recovery.
One should never interpret the presence of slimy material within a wound as the presence of biofilm since the arrangement of cells within that slime is only visible with high power magnification rather than the naked eye. Slough in a wound is not an indicator of biofilm. Although one can easily remove slough by conventional debridement, the clinician cannot remove biofilm in this manner.22
At present, the detection of biofilm in wounds depends on the examination of biopsy tissue using sophisticated research techniques. Scanning electron microscopy and laser scanning microscopy are appropriate means to observe biofilm, but are usually costly, time-consuming and unavailable in most microbiology labs. Microbial DNA-based studies or molecular diagnostics are very promising tools for biofilm evaluation. Molecular diagnosis is currently in use to personalize topical therapeutic treatment of biofilm infections.23
There is much controversy concerning the treatment of biofilm infections although there have been great strides recently as we gain a better understanding of biofilms. According to the Center for Biofilm Engineering at Montana State University, bacteria initially adhere to a surface utilizing weak bonds called van der Waals forces. If these bonds are not separated, the bacteria utilize cell adhesion molecules to bind other cells.24 They begin to build the matrix that holds the biofilm together. If there are species that are unable to attach to a surface on their own, they often attach to the matrix or the earlier cells.
During this time, pathogens inside the biofilm can communicate thanks to a phenomenon called quorum sensing. Bacteria must reach a certain concentration before quorum sensing allows bacteria to sense its concentration and release enzymes and/or toxins into the wound environment.25 This can occur within a single bacterial species as well as between diverse species.
The development of a biofilm allows for its cells to become more resistant to antibiotics administered in a standard fashion. Biofilm bacteria can be up to 1,000 times more resistant to antibiotics than free floating or planktonic bacteria of the same species found in an acute wound.21
Biofilms grow slowly and are often slow to produce overt symptoms. Biofilm bacteria can move in numerous ways that allow them to infect new tissues easily.26 Research on the molecular and genetic basis of biofilm development has made it clear that when cells switch from planktonic to community mode, they also undergo a shift in behavior that involves alterations in the activity of numerous genes.27 A study by Cho and colleagues states: “When bacteria are under stress, they team up and form this collective called a biofilm. If you look at naturally occurring biofilms, they have a very complicated architecture. They are like cities with channels for nutrients to go in and waste to go out.”28
The clinician must have an understanding of biofilm physiology in order to render adequate treatment. The application of topical antimicrobials may aid in the treatment of biofilm buildup when one uses the topicals in the appropriate manner. Iodine, especially iodine-impregnated dressings, is effective against biofilms. According to Hill and colleagues, iodine is more effective against biofilms than silver products.21 In fact, most silver dressings contain a low concentration of silver, which is ineffective in treating bacterial biofilm. For the best results, one should combine sharp excisional debridement with topical antimicrobial therapy for treating biofilms. Debridement allows prevention of biofilm formation, interference of the quorum sensing and facilitates better penetration of topical antimicrobial agents.13
Traditional administration of antibiotics is often ineffective when biofilms are present in the wound. Patients with biofilm infections frequently hear from mainstream doctors that they have an untreatable infection and that biofilms in the lower extremity can often lead to an amputation. Unfortunately, when one administers an antibiotic in high doses, the antibiotic may temporarily weaken the biofilm but is incapable of destroying it as certain cells inevitably persist and allow the biofilm to regenerate.
To be effective, physicians must administer antibiotics in a specific manner. The Marshall Protocol states that biofilm pathogens succumb to specific bacteriostatic antibiotics given in very low, pulsed doses.29,30 With traditional high-dose antibiotic regimens, the first onslaught of antibiotics does not eliminate some cells called “persisters,” which form the biofilm again. Persister cells form with particular ease in immunocompromised patients.31
There is some controversy about the Marshall Protocol although there are numerous treatment successes utilizing low pulsed doses of bacteriostatic antibiotics such as minocycline, azithromycin (Zithromax), clindamycin, trimethoprim-sulfamethoxazole (Bactrim DS, AR Scientific) and demeclocycline (Declomycin).32
Specifically, Starner and colleagues found that subinhibitory concentrations of azithromycin significantly decreased biomass and maximal thickness in both forming and established biofilms.33 These concentrations of azithromycin inhibited biofilms in all but the most highly resistant isolates. In contrast, subinhibitory concentrations of gentamicin, which is not a bacteriostatic antibiotic, did not affect biofilm formation. The authors found that biofilms actually became resistant to gentamicin at concentrations far above the minimum inhibitory concentration.
In cases of limb salvage, we suggest patient education and co-management with a knowledgeable infectious disease specialist.
The bacterial bioburden in chronic wounds is very difficult to manage due to their highly resistant state in comparison to their planktonic cousins. There is no set protocol in treating a patient with a chronic wound that has bacterial biofilm production.
It is our experience to treat chronic diabetic wounds that demonstrate delayed healing as if they harbor biofilm infections. Aggressive debridement of the wound followed by application of topical antimicrobials is necessary to prevent biofilm and aid in wound closure. With delayed healing of diabetic foot wounds, it is important to fully re-evaluate the wound and change the treatment protocol. If one has ruled out all causative factors, the clinician may need to address bacterial bioburden to facilitate wound closure.
Dr. Pupp is a member of the Residency Training Committee at Providence Hospital in Southfield, Mich. He is a Fellow of the American College of Foot and Ankle Surgeons, and is board certified in foot and ankle surgery by the American Board of Podiatric Surgery.
Dr. Koivunen is an attending physician at Providence Hospital in Southfield, Mich. He is an Associate of the American College of Foot and Ankle Surgeons.
1. Lee KH. Tissue-engineered human living skin substitutes: development and clinical application. Yonsei Med J. Dec 2000;41(6):774-779.
2. Available at http://www.cdc.gov/diabetes/pubs/factsheet07  . Published 2007. Accessed May 31, 2012.
3. Lazarus GS, Cooper DM, Knighton DR, Percoraro RE, Rodeheaver G, Robson MC. Definitions and guidelines for assessment of wounds and evaluation of healing. Wound Repair Regen. 1994;2(3):165-170.
4. Veves A, Falanga V, Armstrong DG, Sabolinski ML. Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial. Apligraf Diabetic Foot Ulcer Study. Diabetes Care. 2001; 24(2):290-295.
5. Harrington C, Zagari MJ, Corea J, Klitenic J. A cost analysis of diabetic lower-extremity ulcers. Diabetes Care. 2000;23(9):1333-1338.
6. Paquette D, Falanga V. Leg ulcers. Clin Geriatr Med. 2002; 18(1):77-88, vi.
7. Steed DL. Clinical evaluation of recombinant human platelet-derived growth factor for the treatment of lower extremity diabetic ulcers. Diabetic Ulcer Study Group. J Vasc Surg. 1995;21(1):71-78.
8. Veves A, Sheehan P, Pham HT. A randomized, controlled trial of Promogran (a collagen/oxidized regenerated cellulose dressing) vs standard treatment in the management of diabetic foot ulcers. Arch Surg. 2002; 137(7):822-827.
9. Gentzkow GD, Iwasaki SD, Hershon KS. Use of Dermagraft, a cultured human dermis, to treat diabetic foot ulcers. Diabetes Care. 1996; 19(4):350-354.
10. Cavanagh PR, Lipsky BA, Bradbury AW, Botek G. Treatment for diabetic foot ulcers. Lancet. 2005; 366(9498):1725-1735.
11. Armstrong DG, Boulton AJ. Pressure offloading and “advanced” wound healing: isn’t it finally time for an arranged marriage? Int J Low Extrem Wounds. Dec 2004;3(4):184-187.
12. Edwards R, Harding KG. Bacteria and wound healing. Curr Opin Infect Dis. 2004; 17(2):91-96.
13. Davis SC, Martinez L, Kirsner R. The diabetic foot: the importance of biofilms and wound bed preparation. Curr Diab Rep. 2006;6(6):439-445.
14. Harrison-Balestra C, Cazzaniga AL, Davis SC, Mertz PM. A wound-isolated Pseudomonas aeruginosa grows a biofilm in vitro within 10 hours and is visualized by light microscopy. Dermatol Surg. 2003; 29(6):631-635.
15. Schierle CF, De la Garza M, Mustoe TA, Galiano RD. Staphylococcal biofilms impair wound healing by delaying reepithelialization in a murine cutaneous wound model. Wound Repair Regen. 2009; 17(3):354-359.
16. Percival SL, Cutting KF. Biofilms: possible strategies for suppression in chronic wounds. Nursing Standard 2009; 23(32):64-72.
17. Brady RA, Leid JG, Calhoun JH, Costerton W, Shirtliff ME. Osteomyelitis and the role of biofilms in chronic infection. FEMS Immunol Med Microbiol. 2008 Jan; 52(1):13-22.
18. Marston WA, Davies, SW, Armstrong B, et al. natural history of limbs with arterial insufficiency and chronic ulceration treated without revascularization. J Vasc Surg 2006; 44(1):108-114.
19. Apelqvist J, Larsson J, Agardh CD. Long-term prognosis for diabetic patients with foot ulcers. J Intern Med 1993; 233(6):485-491.
20. Pohjolainen T, Alaranta H. Ten-year survival of Finnish lower limb amputees. Prosthet Orthot Int 1998; 22(1):10-16.
21. Hill KE, Malic S, McKee R, et al. An in vitro model of chronic wound biofilms to test wound dressings and assess antimicrobial susceptibilities. J Antimicrobial Chemotherapy. 2010; 65(6):1195–1206.
22. Dowd SE, Sun Y, Secor PR, et al. Survey of bacterial diversity in chronic wounds using pyrosequencing DDGE and full ribosome shotgun sequencing. BMC Microbiology. 2008; 43(3):1471–2180.
23. Hurlow J, Bowler P. Clinical experience with wound biofilm and management: a case series. Ostomy Wound Management. 2009; 55(4):38–49.
24. Data on file, PathoGenius Laboratories, Lubbock, Texas.
25. Hoiby N, Ciofu O, Johansen HK, et al. The clinical impact of bacterial biofilms. Int J Oral Sci. 2011; 3(2):55-65.
26. Stoodley P, Purevdorj-Gage B, Costerton JW. Clinical significance of seeding dispersal in biofilms: a response. Microbiology. 2005; 151(11):34-53.
27. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999; 284(5418):1318-22.
28. Cho H, Jönsson H, Campbell K, Melke P, Williams JW, Jedynak B, et al. Self-organization in high-density bacterial colonies: efficient crowd control. PLoS Biology. 2007; 5(11):e302.
29. Marshall TG. A new approach to treating intraphagocytic CWD bacterial pathogens in sarcoidosis, CFS, Lyme and other inflammatory diseases. Available at http://mpkb.org/home/publications/marshall_american_academy_of_environme...  . Published 2006. Accessed May 31, 2012.
30. Marshall TG, Marshall FE. Sarcoidosis succumbs to antibiotics–implications for autoimmune disease. Autoimmunity Reviews. 2004; 3(4):295-300.
31. Marshall TG. Bacterial capnine blocks transcription of human antimicrobial peptides. Nature Precedings. Available at http://precedings.nature.com/documents/164/version/1  . Published Oct. 29, 2006. Accessed May 31, 2012.
32. The Marshall Protocol Knowledge Base, Autoimmunity Research Foundation. Available at http://mpkb.org/home/mp  . Posted June 22, 2007. Accessed May 31, 2012.
33. Starner TD, Shrout JD, Parsek MR, Appelbaum PC, Kim G Subinhibitory concentrations of azithromycin decrease nontypeable Haemophilus influenzae biofilm formation and diminish established biofilms. Antimicrob Agents Chemother. 2008; 52(1):137-45.
For further reading, see “What You Should Know About Biofilms And Chronic Wounds” in the July 2008 issue of Podiatry Today, “How Biofilm Affects Healing In Diabetic Foot Wounds” in the April 2010 issue of Podiatry Today or “Biofilms And Infection: What You Should Know” in the November 2006 issue.
To access the archives, visit www.podiatrytoday.com .