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3/11/2017 What’s in that wound bed? Slough, Eschar, or Biofilm? Linda J. Cowan, PhD, ARNP, FNP-BC, CWS ©2017 National Pressure Ulcer Advisory Panel | www.npuap.org Disclosures • Employed as a Research Health Scientist, North Florida/South Georgia Veterans Health System, Gainesville, FL. • Research funding received from: – VA – Biomonde – Healthpoint – Smith & Nephew – Hollister – Medline • This material is the result of work supported with resources and the use of facilities at the VA. 1 3/11/2017 Disclaimers • Speaker does not endorse any one particular company’s products, is not employed by industry, has no financial interest in the listed commercial companies. • Contents of this presentation do not represent the views of the U.S. Department of Veterans Affairs or the United States Government Participants will describe: • Key characteristics of chronic nonhealing wounds • Impediments to wound healing • Characteristics of slough, eschar, and biofilm in open wounds • Evidence-based approaches to address or remove slough, eschar, and biofilm from open wounds • Potential antibiofilm treatment strategies 2 3/11/2017 Chronic Wounds vs. Acute Wounds • All chronic wounds begin as acute wounds • Common chronic wounds – – – – Venous ulcers of the lower extremities Diabetic foot ulcers Pressure ulcers Complex trauma and surgical wounds Key characteristics of chronic wounds • Imbalanced at microcellular level • Stuck in inflammatory phase1-4 – – – – High MMPs / Low TIMPs (inverse correlation) High inflammatory cytokines Low growth factors Fibroblast inhibition5 • Does not follow expected pathway to healing (less than 50% improvement in 4 weeks)6 3 3/11/2017 Percent wound healing in 4 weeks 58% of pts who had >50% reduction in wound size at 4 weeks, healed at 12 weeks6 Only 9% of pts with <50% reduction in wound size at 4 weeks, healed at 12 weeks6 6Sheehan, P., Jones, P., Caselli, A., Giurini, J.M., Veves, A. (2003). Percent Change in Wound Area of Diabetic Foot Ulcers Over a 4-Week Period Is a Robust Predictor of Complete Healing in a 12-Week Prospective Trial. Diabetes Care, 26(6), 1879-1882. Figure courtesy of G. Schultz, taken from: http://care.diabetesjournals.org/content/diacare/26/6/1879/F1.large.jpg?width=800&height=600&carousel=1 Intrinsic impediments to wound healing • Physiological, potentially modifiable: – Nutrition – Blood sugar control – Immune compromise (HIV, Sickle Cell) • Improved management of certain conditions – Pain, psychological stress – Edema – e.g. lymphedema – Tissue viability, perfusion & oxygenation • Physiological, not modifiable: – Advanced age – Certain comorbid conditions (CVA, SCI, neurodegenerative, cancer, etc.) 4 3/11/2017 Extrinsic impediments to wound healing - potentially modifiable14 – Medications • chemotherapy, steroids, anticoagulants – Persistent or repetitive trauma • immobility - failure to off-load, inappropriate shoes or mobility devices, wet-to-dry dressings – Exposure • Smoking/nicotine, alcoholism • Environmental, toxic chemicals, hygiene (personal & environmental), parasites, pets, etc. – Physical barriers • Rolled wound edges, non-viable tissue (slough, fibrin, eschar) – Invasion – virulent pathogens (biofilm) T-I-M-E-(s) Principle for WBP1,8,9 T - remove non-viable Tissue in wound I - address Infection (prevent, treat, remove problematic organisms/biofilm) M – manage Moisture34 E – address wound Edges S – address Surrounding Skin 5 3/11/2017 Documenting wound assessments • • • • • • • • Location Suspected etiology, contributing factors Size (W X L X D in cm) Undermining, tunneling (clock method) Exudate (color, amount, odor) Wound bed tissue (color, amount viable) Wound edges and surrounding tissue Last treatments used, compliance, wound response, patient/CG education Describing wound tissue • • • • • Color of wound bed (in percentages) Viable (living tissue with good perfusion) Non-viable (dead/dying host tissue) Boggy (wet spongy consistency) Fluctuant (moving in waves, movable & compressible, variable/unstable) • Friable (bleeds easily with light touch) • Hypergranulating (overgrowing baseline) • Pale (anemic looking) 6 3/11/2017 Characteristics of slough in wounds • What it is13 – Non-viable host tissue (or “avascular fat”) – Typically it is moist, white, yellow, grey, or tan dead tissue; loose or adherent; includes white blood cells, fibrin, and other proteins – May have “chicken fat” appearance • What it is not – Alive – slough by itself is not living tissue • Slough will not “grow” on dressings – Biofilm - may have bacteria/biofilm on it – ?Blood clot, dried exudate, softened scab? Fibrin35 • Fibringen is a glycoprotein in vertebrates that helps in formation of blood clots. • Fibrin is an insoluble, non-globular protein formed from fibrinogen during the clotting of blood. It is formed by the action of the protease thrombin (clotting enzyme) on fibrinogen which causes it to polymerize. • The polymerized fibrin, together with platelets form a hemostatic plug or clot over a wound site. 35Laurens, N., Koolwijk, P., DeMaat, M/P. (2006). Fibrin Structure and wound healing. Journal of Thrombosis and Haemostasis, 4:932-939. 7 3/11/2017 Slough • Best ways to remove Characteristics of eschar in wounds • What it is – Dry, dead host tissue • What it is not – Scab / crust (dried exudate) – Dry Gangrene (condition where tissue dies caused by ischemia due to underlying illness, injury, and/or infection). Fingers, toes, & limbs most often affected. • When not to remove – If providing reliable protective barrier 8 3/11/2017 Eschar • When to remove – Integrity is compromised (no longer acting as body “bandaid”) – Impediment to healing • Best ways to remove Biofilm – What is it? • “Any group of microorganisms in which cells stick to each other and often these cells adhere to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS).” Wikipedia • “Van Leeuwenhoek, using his simple microscopes, first observed microorganisms on tooth surfaces and can be credited with the discovery of microbial biofilms.” Rodney Donlan (2002). Biofilms: Microbial Life on Surfaces. Emerging Infectious Diseases, 8(9), 881-890. 9 3/11/2017 Biofilm – what it is not • Not the same as surface or “free-floating” (planktonic) bacteria • Not typically identified using traditional culture swab techniques (identifies mostly planktonic bacteria) • Not easy to eradicate! – Exhibits increased tolerance to antimicrobial, immunological & chemical attack compared to planktonic bacteria Biofilms in >80% of Biopsies of Chronic Wounds7 Versus 6% of Acute Wounds A B Very likely more prevalent in chronic wounds than we think! 7 Panels A & B: G. James, E. Swogger, R. Wolcott, E. Pulcini, P. Secor, J. Sestrich, J. Costerton, P. Stewart. Wound Rep Regen, 16:37-44, 2008 7M. Malone, T. Barjnsholt, A. McBain, G. James, P. Stoodley, D. Leaper, M. Tachi, G. Schultz, T. Swanson, R. Wolcott. Prevalence of biofilms in chronic wounds: a systematic review and meta-analysis of published data, J wound Care, JWC 2017; 26:20-25. 10 3/11/2017 Characteristics of biofilm in open wounds • Mostly* unable to see it with naked eye • Polymicrobial – Aerobic + non-aerobic bacteria – gram pos + gram neg – Fungus + virus • • • • Hydrophilic polymeric protective coating Quorum sensing Attached 2mm below wound bed surface Grows back in 48 hours *Hurlow J. Blanz E. Gaddy JA. Clinical investigation of biofilm in non-healing wounds by high resolution microscopy techniques. J Wound Care. (2016). 25(9). S11-S22. Distribution of aerotolerance of bacterial populations in chronic wounds Aerobes Facultative Anaerobes Strict Anaerobes Scott Dowd, et al. (2008). BioMed Central Microbiology, 8(43). Courtesy of G. Schultz. 11 3/11/2017 Why are bacteria in biofilms so difficult to kill? 1. Extracellular polymeric substance (EPS) of biofilm • • • Dense matrix impairs diffusion of large antibodies EPS materials chemically react (neutralize) microbicides Negative charges of polysaccharides and DNA bind cationic molecules like Ag+, antibiotics, PHMB+ 2. Persister bacteria have low metabolic activity • Antibiotics only kill metabolically active bacteria 3. Oxygen diffusion to center of biofilm is limited • Promotes growth of anaerobic bacteria 4. Synergism between different bacteria • • MRSA secrete resistance proteins Pseudomonas secrete catalase that destroys H2O2 Slide material: Courtesy of G. Schultz, PhD Biofilm • When to remove – When biofilm presence has negative consequences – When interferes with wound healing – When especially virulent (β hemolytic streptococci) – To prevent re-growth • Best ways to remove – DEBRIDEMENT – Total kill 12 3/11/2017 Evidence for debridement methods • Sharp – Scalpel, scissors, curette • Enzymatic (collagenase) – Pros (gentle) Cons (slow) • Autolytic (exudate/MMPs) – Pros (gentle) Cons (slow) • Ultrasonic (low and high frequency) – With and without forced water • Mechanical (debriding gauze, wet-to-dry) – Surfactants (w/wo mechanical wiping) • Larval/biological – medicinal maggots Ultrasonic debridement: PA biofilm 3 day old PA biofilm 3 day old PA biofilm after low frequency non-contact, nonthermal ultrasound 13 3/11/2017 Gauze debridement of biofilm bacteria on pig skin explants Slide material: Courtesy of G. Schultz, PhD Effect of wiping only: total and biofilm PA bacteria Wiping with gauze only Yang Q, Larose C, Porta AD, Della Porta AC, Schultz GS, Gibson DJ. A surfactant-based wound dressing can reduce bacterial biofilms in a porcine skin explant model. Int Wound J 2016; Slide material courtesy of G. Schultz, PhD 14 3/11/2017 Daily wiping + surfactant gel vs. total & biofilm PA bacteria Wiping + Surfactant Yang Q, Larose C, Porta AD, Della Porta AC, Schultz GS, Gibson DJ. A surfactant-based wound dressing can reduce bacterial biofilms in a porcine skin explant model. Int Wound J 2016; Slide material courtesy of G. Schultz, PhD Effect of Daily Wiping + Surfactant gel on PA Bacteria Yang Q, Larose C, Della Porta AC, Schultz GS, Gibson DJ. A surfactant-based wound dressing can reduce bacterial biofilms in a porcine skin explant model. Int Wound J 2016; Slide material courtesy of G. Schultz, PhD 15 3/11/2017 Wiping only time course: A. baumannii Total Bacteria Biofilm Bacteria 1.0E+08 Viable Bacteria (CFU) 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 After Wiping Day 1 Day 2 Day 3 Before Wiping ©2017 National Pressure Ulcer Advisory Panel | www.npuap.org Slide material courtesy of G. Schultz, PhD Wiping + surfactant gel: A. baumannii Total Bacteria Biofilm Bacteria 1.0E+08 Viable Bacteria (CFU) 1.0E+07 1.0E+06 1.0E+05 1.0E+04 Not impressive 1.0E+03 1.0E+02 1.0E+01 1.0E+00 After Wiping Day 1 Day 2 Day 3 Before Wiping Slide material courtesy of G. Schultz, PhD 16 3/11/2017 Total Acinetobacter baumannii treated with Surfactant gel + antibiotics made daily Gauze Surfactant Gel Surfactant Gel with 2% Mupirocin Surfactant Gel with 8.5% Mafenide Acetate 1.E+08 Colony Forming Units 1.E+07 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 1.E-01 0 24 48 72 96 Hours LDT Evidence? • Gray, M. (January 01, 2008) Systematic Review 14 – Is maggot debridement effective for removal of necrotic tissue from chronic wounds? – January 1960 to February 2008: 4 studies (pooled n=193); • 3 studies <60 subjects – Compared MDT (LDT) to autolytic/other debridement • pressure ulcers, leg ulcers, burn wounds – Concluded: “evidence base for the efficacy of maggot debridement therapy (MDT) in the management of necrotic wounds is sparse.” – “Even though clinical evidence supporting the use of MDT for debridement of wounds is lacking, clinical experience strongly suggests that this technique is an effective and safe method of debridement for selected patients.” • Increased evidence from 2008 to 2017 – more than 300 studies! 17 3/11/2017 Evidence for larval debridement • Multiple actions of Lucilia sericata larvae in hard-to-heal wounds: Larval secretions contain molecules that accelerate wound healing, reduce chronic inflammation and inhibit bacterial infection. – Cazander, G., Pritchard, D. I., Nigam, Y., Jung, W., & Nibbering, P. H. (2013). Bioessays, 35(12), 1083-1092. • A randomized controlled trial of larval therapy for the debridement of leg ulcers: Results of a multicenter, randomized, controlled, open, observer blind, parallel group study. – Mudge, E., Price, P., Neal, W., & Harding, K. G. (2014). Wound Repair and Regeneration, 22, 1, 43-51. • Selective Antibiofilm Effects of Lucilia sericata Larvae Secretions / Excretions against Wound Pathogens. Bohova, Jana, Majtan, Juraj, – Majtan, Viktor, & Takac, Peter. (2014). Hindawi Publishing Corporation. • Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria. – Pöppel, A. K., Vogel, H., Wiesner, J., & Vilcinskas, A. (2015). Antimicrobial Agents and Chemotherapy, 59(5), 2508-14. LDT – mechanisms of action • Larval enzymes: protease, collagenase, ammonia, allantoin and urea, lysozymes20,22,25,26,28,33 • Increase in alkalinity - breaks down necrotic tissues26-28,31,33 • Antimicrobial action of LDT secretions: peptides (diptericins, lucifensin); chymotrypsin disrupts protein adhesionmediated biofilm formation; crude methanol extract21-25, 29-31 • Improved antibiotic effectiveness (re-susceptibility to antimicrobials observed after LDT)29 • Stimulate fibroblast proliferation and promote fibroblast motility; may improve angiogenesis (amino acid derivatives), vascular perfusion, and tissue oxygenation; may reduce scarring; reduces inflammation20, 26-28, 32, 33 18 3/11/2017 Larval Debridement PA01 biofilm culture before LDT PA01 biofilm culture 24 hours after LDT Potential antibiofilm strategies • • • • • Prevention – reduce risk factors Debridement Selecting suitable topical products Selecting suitable systemic products Combined approaches – – – – “one-two” punches LDT + advanced therapies / skin grafts Ultrasonic treatments + antimicrobials Surfactant gels + mechanical disruption 19 3/11/2017 Summary What is in that chronic wound bed? Slough, Eschar, Biofilm? • • • • • Examine – not only with naked eye Determine - what is it? Address – targeted treatment Evaluate treatment effectiveness, wound progress Prevent regrowth Special Thanks To our US Veterans and their families Micah Flores, PhD Gregory Schultz, PhD Dan Gibson, PhD Qingping Yang, MS Josh Yarrow, PhD Gary Wang, MD, PhD Randall Wolcott, MD Cynthia Garvan, PhD Alessandra Della Porta (UF Student) Meg Kincaid, BS Casey Bopp, RN 20 3/11/2017 Questions? • Contact information: – [email protected] – [email protected] Specific References 1. Schultz, G. S., Sibbald, R. G., Falanga, V., Ayello, E. A., Dowsett, C., Harding, K., Romanelli, M., ... Vanscheidt, W. (2003). Wound bed preparation: a systematic approach to wound management. Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society, 11, 1-28. 2. Trengove, N.J., Stacey, M.C., Macaulley, S., Bennett, N., Gibson, J., Burslem, F., Murphy, G., Schultz, G. (1999). Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair and Regeneration, 7, 442-452. 3. Demidova-Rice, T., Hamblin, M.R., Herman, I.M. (2012). Acute and Impaired Wound Healing: Pathophysiology and Current Methods for Drug Delivery, Part 1: Normal and Chronic Wounds: Biology, Causes, and Approaches to Care. Adv Skin Wound Care, 25(7), 304–314. 4. Schultz, G. (2014). Molecular and cellular regulation of wound healing: What goes wrong when wounds fail to heal or heal too much?. London: Henry Stewart Talks. http://hstalks.com/lib.php?t=HST186.3834&c=252. 5. Harding, K. G., Moore, K., & Phillips, T. J. (2005). Wound chronicity and fibroblast senescence - implications for treatment. International Wound Journal, 2(4), 364-368. 21 3/11/2017 Specific References 6. Sheehan, P., Jones, P., Caselli, A., Giurini, J.M., Veves, A. (2003). Percent Change in Wound Area of Diabetic Foot Ulcers Over a 4-Week Period Is a Robust Predictor of Complete Healing in a 12-Week Prospective Trial. Diabetes Care, 26(6), 1879-1882. 7. Malone, M., Barjnsholt, T., McBain, A.J., James, G.A., Stoodley, P., Leaper, D., Tachi, M., Shultz, G., Swanson, T., Wolcott, R.D. (2017). The prevalence of biofilms in chronic wounds: a systematic review and metaanalysis of published data. Journal of Wound Care, 25(12), 1-5. 8. Schultz, G. S., Barillo, D. J., Mozingo, D. W., & Chin, G. A. (April 01, 2004). Wound bed preparation and a brief history of TIME. International Wound Journal, 1(1), 19-32. 9. Leaper, D. J., Schultz, G., Carville, K., Fletcher, J., Swanson, T., & Drake, R. (January 01, 2012). Extending the TIME concept: what have we learned in the past 10 years? International Wound Journal, 9, 1-19. 10. Granick, M., Boykin, J., Gamelli, R., Schultz, G., & Tenenhaus, M. (January 01, 2006). Toward a common language: surgical wound bed preparation and debridement. Wound Repair and Regeneration, 14. Specific References 11. Cowan, L., Phillips, P., Stechmiller, J., Yang, Q., Wolcott, R. & Schultz, G. (2013). Antibiofilm Strategies and Antiseptics (Chapter 4) in Antiseptics in surgery: Scientific basis, indications for use, evidence based recommendations, vacuum instillation therapy; Willy, C., & Alt, V. (editors), 33 tables. Berlin: Lindqvist Book Publ. 12. Cowan, L., Phillips, P., Liesenfeld, B., Mikhaylova, A., Moore, D., Stechmiller, J., & Schultz, G. (June 01, 2011). Caution: When Combining Topical Wound Treatments, More Is Not Always Better. Wound Practice & Research: Journal of the Australian Wound Management Association, 19(2), 60-64. 13. Swanson, T., Hurlow, J., Schultz, G., & Fletcher, J. (2014). Slough: What is it? How do we manage it? International Wound Infection Institute. http://www.woundinfection-institute.com/wpcontent/uploads/2014/11/Slough_AWMA_2014.pdf 14. Gray, M. (2008). Systematic Review: Is maggot debridement effective for removal of necrotic tissue from chronic wounds? Journal of Wound, Ostomy, and Continence Nursing, 35(4). 22 3/11/2017 Additional Resources & References Wound Bed Preparation: 15. Doughty, D. & McNichol, L. (2016). Wound, Ostomy and Continence Nurses Society® Core Curriculum: Wound Management 1st Edition. Chapter 2: Wound healing (Janice Beitz). Lippincott Williams & Wilkins, Philadelphia, PA. 16. Enoch, S., Harding, K. (2003). Wound Bed Preparation: The Science Behind the Removal of Barriers to Healing. Wounds, 15(7). http://www.medscape.com/viewarticle/459733_5 17. Moffat, C., Falanga, V, Vowden, P. European Wound Management Association (EWMA). Position Document: Wound Bed Preparation in Practice. London: MEP Ltd, 2004. Available at: http://www.woundsinternational.com/media/issues/87/files/content_49.pdf Larval Debridement Therapy: 18. Cowan, L. J., Stechmiller, J. K., Phillips, P., Yang, Q., & Schultz, G. (2013). Chronic Wounds, Biofilms and Use of Medicinal Larvae. Ulcers, 2013, 1, 1-7. 19. Sherman et al. (2013). Chapter 2 (Maggot Therapy) in Biotherapy-History, Principles, and Practice: A Practical Guide to the Diagnostics and Treatment of Disease Using Living Organisms, Springer Publishers: Netherlands. 20. Cazander, G., Schreurs, M. W. J., Renwarin, L., Dorresteijn, C., Hamann, D., & Jukema, G. N. (2012). Maggot excretions affect the human complement system. Wound Repair and Regeneration, 20, 6, 879-886. 21. Kawabata, T., Mitsui, H., Yokota, K., Ishino, K., Oguma, K., & Sano, S. (2010). Induction of antibacterial activity in larvae of the blowfly Lucilia sericata by an infected environment. Medical and Veterinary Entomology, 24, 4, 375-381. 22. Harris, L. G., Nigam, Y., Sawyer, J., Mack, D., & Pritchard, D. I. (2013). Lucilia sericata chymotrypsin disrupts protein adhesion-mediated staphylococcal biofilm formation. Applied and Environmental Microbiology, 79, 4, 1393-5. 23. Teh, C. H., Nazni, W. A., Lee, H. L., Fairuz, A., Tan, S. B., & Sofian-Azirun, M. (2013). Antibacterial activity and physicochemical properties of a crude methanol extract of the larvae of the blow fly (Lucilia cuprina). Medical and Veterinary Entomology, 27, 4, 414-420. 24. Bohova, Jana, Majtan, Juraj, Majtan, Viktor, & Takac, Peter. (2014). Selective Antibiofilm Effects of Lucilia sericata Larvae Secretions/Excretions against Wound Pathogens. Hindawi Publishing Corporation. 25. Valachova, I., Takac, P., & Majtan, J. (2014). Midgut lysozymes of Lucilia sericata new antimicrobials involved in maggot debridement therapy. Insect Molecular Biology, 23, 6, 779-787 26. Cazander, G., Pritchard, D. I., Nigam, Y., Jung, W., & Nibbering, P. H. (2013). Multiple actions of Lucilia sericata larvae in hard-to-heal wounds: Larval secretions contain molecules that accelerate wound healing, reduce chronic inflammation and inhibit bacterial infection. Bioessays, 35, 12, 1083-1092. 27. Li, P.-N., Li, H., Zhong, L.-X., Sun, Y., Yu, L.-J., Wu, M.-L., Zhang, L.-L., ... Lv, D.-C. (2015). Molecular events underlying maggot extract promoted rat in vivo and human in vitro skin wound healing. Wound Repair and Regeneration, 23, 1, 65-73. 23 3/11/2017 28. Bexfield, A., Bond, A. E., Morgan, C., Wagstaff, J., Newton, R. P., Ratcliffe, N. A., Dudley, E., ... Nigam, Y. (2010). Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis. British Journal of Dermatology, 162, 3, 554-562. 29. Arora, Shuchi, Baptista, Carl, & Lim, Chu. (2011). Maggot metabolites and their combinatory effects with antibiotic on Staphylococcus aureus. BioMed Central Ltd. 30. Beasley, W. D., & Hirst, G. (2004). Making a meal of MRSA—the role of biosurgery in hospital-acquired infection. Journal of Hospital Infection, 56, 1, 6-9. 31. Bohova, Jana, Majtan, Juraj, Majtan, Viktor, & Takac, Peter. (2014). Selective Antibiofilm Effects of Lucilia sericata Larvae Secretions/Excretions against Wound Pathogens. Hindawi Publishing Corporation. 32. Grassberger, M. (2013). Biotherapy-- History, principles and practice: A practical guide to the diagnosis and treatment of disease using living organisms. Dordrecht: Springer. 33. Horobin, A. J., & University of Nottingham. (2004). Maggots and wound healing: The effects of Lucilia sericata larval secretions upon interactions between human dermal fibroblasts and extracellular matrix proteins. Nottingham: University of Nottingham. 34. Sibbald, R.G., Elliott, J.A., Ayello, E.A., Somayaji, R. (2015). Optimizing the moisture management tightrope with wound bed preparation 2015. Advances in Skin & Wound, 28(10), 466-476. 35. Laurens, N., Koolwijk, P., DeMaat, M/P. (2006). Fibrin Structure and wound healing. Journal of Thrombosis and Haemostasis, 4:932-939. 24