Biofilms and Chronic Wounds: Winning the War in Wounds. Prof. Gregory Schultz, Ph.D.

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1 1 Professor of Obstetrics/Gynecology Institute for Wound Research University of Florida 2 Overview of topics Review the four sequential phases of normal wound healing and recognize the beneficialeffects of controlledinflammation and protease activities Understand the link between chronic inflammation caused by planktonic and biofilm bacteria and elevated protease activitiesthat destroyproteins that are essential to healing (extracellular matrix, growth factors, receptors) Recognize the high toleranceof biofilmbacteria to most antibiotics, antiseptics and disinfectants Integrate biofilm based wound care into wound bed preparation (TIME)by debriding biofilms andpreventing planktonic bacterial from reforming biofilm Sequence of molecular and cellular events in skin wound healing Four Phases of Healing 1. Hemostasis 2. Inflammation 3. Repair 4. Remodeling 1 Clotting 2 Vascular Response 3 Inflammation 4 Scar Formation 5 Epithelial Healing 6 Contraction 7 Scar Remodeling 3 1

2 Controlled wound inflammation is beneficial Macrophages Neutrophils Elastase MMPs O 2 - H 2 O 2 HOCl 4 Inflammatory cells kill planktonic bacteria by phagocytosis and reactive oxygen species They also release proteases (MMPs, elastase) that remove denatured ECM components and permit wound healing to proceed. Inflammatory cells are not effective against bacteria in biofilms Is there a common molecular pathology of chronic wounds? Diabetic foot ulcer Arterial ulcer 5 Pressure ulcer Venous ulcer Hypothesis of chronic wound pathophysiology Repeated Tissue Injury, Ischemia and Bacteria - Biofilms TNF-α IL-1β, IL-6 Prolonged, elevated inflammation neutrophils macrophages mast cells Imbalanced Proteases & Inhibitors, Reactive Oxygen Species Proteases (MMPs, elastase, plasmin), inhibitors (TIMPs, α1pi), ROS Destruction of Essential Proteins (off-target) growth factors / receptors, ECM degradation cell migration, cell proliferation 6 Chronic Non-Healing Wound B.A. Mast and G.S. Schultz. Wound Rep Reg4: ,

3 7 Basic background of bacterial biofilms Planktonic bacteria single, non-attached bacteria Biofilm bacteria a structured community of bacteria cells enclosed in a self-produced exopolymeric matrix this is tightly adherent (sessile) to living or inert surface Quorum sensing process by which bacteria molecules shift growth from planktonic to biofilm phenotypes Exopolymeric matrix of biofilms consists of predominately of polysaccharides along with bacterial DNA and proteins that are extremely inflammatory to innate and acquired immune systems Biofilms provide a protected mode of growth evolutionary defense against natural predators: bacterial viruses, amoeba, and microbicides. Also protects against phagocytosis (inflammatory cells), antibodies, natural reactive oxygen species (ROS), antibiotics, antiseptics, and disinfectants Persister bacteria are quiescent (not metabolically active) and are not killed by antibiotics that only act on metabolically active bacteria 20 µm A B May or may not be Biofilm accompanied by the classic signs of infection and inflammation Confocal laser scanning microscopy (top view) of (A) Planktonic Pseudomonas aeruginosa (B) Biofilm community (C) Schematic representation of polymicrobial bacterial biofilm formation (side view) 8 P. Phillips, R. Wolcott, J. Fletcher, G. Schultz. Biofilms Made Easy. Wounds International, 1(3): 1-6, 2010 Free download from Wounds International 9 P. Phillips, R. Wolcott, J. Fletcher, G. Schultz. Biofilms Made Easy. Wounds International, 1(3): 1-6,

4 How does the immunological response to biofilms cause tissue damage and impair healing? A B C D In Panel A, planktonic bacteria can be cleared by antibodies, phagocytosis, and are susceptible to antibiotics Adherent bacterial cells (Panel B) form biofilms preferentially on inert surfaces or devitalized tissue, and these sessile communities are resistant to antibodies, phagocytosis and antibiotics Neutrophils (Panel C) are attracted to the biofilms, but cannot engulf biofilm. Neutrophils still release proteases and reactive oxygen species Phagocytic enzymes (Panel D) damage tissue around the biofilm, and planktonic bacteria are released from the biofilm, causing dissemination and acute infection in neighboring tissue 10 Costerton, Stewart, Greenberg, Science 284, 1999 High levels of MMP activity in chronic wounds decrease as wounds heal Chronic VLUs 11 Trengove, Stacey, Macauley, Bennett, Gibson, Burslem, Murphy, Schultz. Wound Rep Reg 7: , 1999 Low protease activity in chronic wound fluids of pressure ulcers predicts the rate and extent of healing Ratio of MMP-9 (pro+active):timp-1 (ng/ml) Good healing >95% area healed; n=12 Intermediate healing <95% & >65% area healed; n=36 Poor healing < 65% area healed; n= Day-0 Day-10 Day-36 Ladwig, Robson, Liu, Kuhn, Muir, Schultz. Ratios of activated matrix metalloproteinase-9 to tissue inhibitor of matrix metalloproteinase-1 in wound fluids are inversely correlated with healing of pressure ulcers. Wound Repair Reg 10:26-37,

5 MMP-9 activity correlates with wound healing time course 13 D. Gibson, G. Schultz, unpublished data Fibronectin is degraded by chronic wound fluids Fibronectin is absent in the base of chronic venous ulcers Fibronectin reappears in ulcer base during healing Plasma Venous ulcer 1 Venous ulcer 2 Diabetic ulcer 1 Diabetic ulcer 2 Mastectomy Fluid 1 Mastectomy Fluid 2 Plasma + CWF 1 Plasma + CWF 2 Fibronectin is degraded in non-healing ulcer 14 Fibronectinprofile in plasma shows a single intact band at 250 kda. In contrast, fibronectin is degraded to lower molecular weight fragments in venous stasis ulcers and in diabetic ulcers. Wysockiand Grinnell. Lab Invest 63:825, 1990 Fibronectin reappears (stable) as ulcer heals Fibronectin plays a key role in epidermal cell migration. It is degraded by proteases in chronic ulcers, but is stable when inflammation and protease levels decrease, allowing epithelial cell migration. Herrick, Sloan, McGurk, Freak, McCollum and Ferguson. Am J Pathol 141, 1992 Free download from Wounds International 15 D. Gibson, B. Cullen, R. Legerstee, K.G. Harding, G. Schultz. MMPs Made Easy. Wounds International, 1(1): 1-6,

6 Biofilms identified in 60%of biopsies of chronic wounds but in only 6% of acute wounds 6 microns 16 G.A. James, E. Swogger, R. Wolcott, E.D. Pulcini, P. Secor, J. Sestrich, J.W. Costerton, P.S. Stewart. Biofilms in chronic wounds. Wound Repair Regen, 16: 37-44, 2008 Q.P. Yang, P. Phillips, E. Sampson, A. Progulske-Fox, P. Antonelli, S. Jin, G. Schultz. Development of anovel in vitro porcine skin explant model for the assessment of mature bacterial biofilms. Wound Repair Regen, 21: , 2013 Question: Does formation of biofilm colonies in a wound retard healing? Answer: YESor NO 17 Biofilm formation by staphylococcal species delays healing of mouse cutaneous wounds 18 Schierleet al., Wound Rep Reg17: 354, 2009 Biofilm Formation in Cutaneous Mouse Wounds. (A) Gross appearance of uninfected wounds. (B) Gross appearance of wound colonized by biofilm. (C) Gram stain of uninfected control wound. (D) Gram stain of biofilm wound bed. (E) Uninfected control wound stained with fluorescein-labeled antibody to biofilm matrix. (F) Infected control wound stained with fluorescein-labeled antibody to biofilm matrix 6

7 19 Question: Why are bacteria in biofilms hard to kill? Answer: Exopolymeric material (EPM) of the biofilm Dense matrix impairs diffusion of large antibodies EPM materials chemically react (neutralize) microbicides Negative charges of polysaccharides and DNA bind cationic molecules like Ag +, antibiotics, PHMB + Persister bacteria have low metabolic activity Antibiotics only kill metabolically active Oxygen diffusion to center of biofilm is limited Promotes growth of anaerobic bacteria Synergism between different bacteria MRSA secrete resistance proteins Pseudomonas secrete catalase that destroys H 2 O 2 Hypochlorous acid very slowly penetrates biofilm matrix reaction-diffusion problem 90 minutes Dead Live 25 minutes 20 After 60 minutes of exposure to dilute bleach (Dakin s solution), many bacteria in this biofilm were dying (green cells), but many cells in the interior of the biofilm were still alive (orange cells) Costerton, SciAm, sec Reaction-Diffusion Problem Hypochlorous acid rapidly reacts with molecules that form the biofilm exopolymeric matrix, which limits its diffusion into the center of the biofilm colony Phil Stewart, MSU Center for Biofilm Engineering Biofilms are highly tolerant to antibiotics Tobramycin vs. P. aeruginosa biofilm biofilm planktonic 21 Tobramycin rapidly kills planktonic Pseudomonas aeruginosa( )very effectively, but is not effective against biofilm ( ). Walters et al., AntimicrobAgents Chemother 47: 317,

8 Topical antibiotics effectively kill planktonic bacteria in pig skin wounds but only reduce bacteria in biofilms 2-logs after 48 hours 22 Davis et al., Wound Rep Regen16: 23-29, 2008 Thicker biofilm requires higher concentrations of antibiotic to inhibit growth S. pneumoniae isolate 23 Serotype Planktonic IC*(µg/ml) BiofilmIC (µg/ml) Fold Increase Number of replicates BS 68 9V < 2 4 > 2 6 BS < >64 8 BS 71 3 <2 4 >2 6 BS F < > BS 73 6A <2 4 >2 6 BS F < 2 62 >32 8 *Highest concentration of azithromycin producing no turbidity after overnight incubation with antibiotic with planktonic cells Highest concentration producing no turbidity after overnight incubation with antibiotic with biofilm cells. Turbidity was scored by eye as well as by determining the highest concentration that was within the standard deviation of the optical density measurements of 6 8 replicate untreated controls (no antibiotic) + Indicates high Biofilm Index strain produces extensive exopolymeric matrix Hall-Stoodley et al., BMC Microbiology 2008, 8: 173 Metabolic activity of Pseudomonas aeruginosa in mature biofilms is limited to the surface layers 24 Only fluorescent bacteria are metabolically active Only located in outer layers of the biofilm matrix Antibiotics only kill metabolically active bacteria Phil Stewart, Montana State University, Center for Biofilm Engineering 8

9 Dissolved oxygen gradients measured in biofilm Depth (µm) Horizontal distance (µm) 25 Z. Lewandowski, D. De Beer, P. Stoodley Distribution of aerotolerance of bacterial populations in chronic wounds 100% Aerobes Percentage of population 80% 60% 40% 20% 0% Venous Diabetic Pressure Facultative Anaerobes Strict Anaerobes 26 Scott Dowd, et al., BioMed Central Microbiology, 8: 43, 2008 Biofilm based wound care 27 9

10 28 Principles of biofilm based wound care 1.Frequent debridement of wounds to physically remove biofilm communities 2.Use an effective microbicidal dressing after debridement to prevent reformation of biofilms 3.Alter topical & systemic antimicrobial treatments to prevent emergence of dominant bacteria from polymicrobial populations; utilize bacterial DNA identification techniques 4.Biofilm Based Wound Care is part of Wound Bed Preparation (TIME) Wolcott,R.D.; Rhoads,D.D. A study of biofilm-based wound management in subjects with critical limb ischaemia. J. Wound Care 17: , 2008 Free download from International Wound Journal 29 Leaper DJ, Schultz G, Carville K, Fletcher J, Swanson T, Drake R. Extending the TIME concept: what have we learned in the past 10 years? Int Wound J, 2012;9(Suppl. 2):1 19 What is this filmy wound slough? Mainly fibrin - surrogate biomarker for inflammation 30 Dr Randy Wolcott 10

11 Question: How quickly can planktonic bacteria form protective biofilms in wounds after debridement? Which answer is true? 1.7 days 2.5 days 3.33 days 4.1 day 31 Biofilm maturity studies indicate sharp debridement opens a time-dependent therapeutic window Biopsies from three patients with large (>10 cm 2 ) venous ulcer were split into two tubes containing saline (control) or saline with 200 µg/ml gentamicin (treatment), and after 24 hours of incubation, samples were disperse biofilm into microcolonies and CFU/5 gm were measured. Total levels of bacteria at 0, 1, 2, and 3 days after initial debridement remained consistently high. However, in two of the three wounds, all bacterial were planktonic at 1 and 2 days after debridement (full kill by exposure to gentamicin), but by 3 days post-debridement, all three wounds had re-established substantial levels of biofilm bacteria ( CFU/5 gm) 32 R.D. Wolcott, K.P. Rumbaugh, G. James, G. Schultz, P. Phillips, Q. Yang, C Watters, P.S. Stewart, S.E. Dowd. J Wound Care 19: , 2010 Debridement - biofilm will reform No matter how good the debridement, fragments will always remain embedded in the wound Those fragments will reattach quickly, and become metabolically active They will start propagating and coalescing, quorum sensing will then take place Within 24 hours, early biofilm will have formed Within 2-3 days formal, mature biofilm will be present 33 11

12 Question: Do all antimicrobial wound dressings effectively kill biofilm colonies grown on pig skin explants? Answer: YES or NO 34 Can dressings disrupt & kill mature biofilms? 35 PL Phillips, Q Yang, E Sampson, GS Schultz. Effects of antimicrobial agents on an in vitro biofilm model of skin wounds. AdvWound Care 2010; 1: hr continuous exposure of mature PAO1 biofilm on porcine explants 36 P.L. Phillips, Q. Yang, E. Sampson, G. Schultz. Effects of Antimicrobial Agents on an In Vitro Biofilm Model of Skin Wounds, Advances Wound Care, 1: ,

13 Free download from International Wound Journal 37 Priscilla L Phillips, Qingping Yang, Stephen Davis, Edith M Sampson, John I Azeke, Afifa Hamad, Gregory S Schultz. Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants Int Wound J, 2013, doi: /iwj Larval debridement therapy Before treatment After 24hr treatment 38 Staphylococcus aureus L. Cowan, J. Stechmiller, P. Phillips, Q.P. Yang and G. Schultz. Chronic Wounds, Biofilms and Use of Medicinal Larvae, Ulcers, Article ID , 7 pages; Question: What effect does NPWT alone or combined with instillation of antimicrobial solutions have on killing biofilm colonies grown on pig skin explants? Answer: It depends on the instillation solution used 39 13

14 Assessment of NPWT + instillation on biofilms grown on pig skin explants Instill NP port Fix center 8mm biopsies cut from under both NP and instill ports for SEM analysis; Trump fixative NP port Check for leaks, NP pressure, Instillation, Saturation of wound and foam before NPWT and final collection canister volume to verify completion of 6 cycles. 40 P.L. Phillips, Q. Yang, G.S. Schultz. Effect of Negative Pressure Wound Therapy with Periodic Instillation Using Antimicrobial Solutions on Pseudomonas aeruginosabiofilm on Porcine Skin Explants. International Wound J, in press Effects of 6-cycles of NPWT-instill. treatments over 24 hours on P. aeruginosa biofilm grown on pig skin explants Log CFU/ml * VAC Port * * Instillation Port *** * 41 * P-Value <0.005 compared to saline control P.L. Phillips, Q. Yang, G.S. Schultz. The Effect of Negative Pressure Wound Therapy with Periodic Instillatio Using Antimicrobial Solutions on Pseudomonas aeruginosa Biofilm on Porcine Skin Explants. International Wound J, 10 (suppl. 1) 48-55, 2013 Summary of NPWT + instillation on mature biofilms on pig skin explants NPWT alone has minimal effects on reducing mature P. aeruginosabiofilms when tested using an in vitropig skin explant model 2. Combining NPWT with instillation of some wound cleansing solutions significantly reduces CFUs of Pseudomonas aeruginosa biofilms 1% Povidone iodine kills 99% 0.1% Polyhexamethylene biguanide kills 99.99% 0.05% Chlorhexidine gluconate kills 99.9% 14

15 43 Summary 1. Biofilmsare communities of bacteria encased in a self-produced matrix of polysaccharides, protein and DNA that provides high levels of tolerance to antibodies, antibiotics and antiseptics 2. Biofilms are present in a high percentage of chronic wounds and they impair healing by stimulating chronic inflammation, leading to elevated levels of proteases and ROS that degrade proteins that are essential for healing 3. Topical dressings can reduce biofilm CFUs ~1 to 2 logs except sustained release cadexomer iodine dressings - kills biofilm 4. NPWT alone has minimal effectson reducing mature biofilms when tested using an in vitropig skin explant model 5. NPWT + Instillation of some wound cleansing solutions significantly reduced CFUs of P. aeruginosa biofilms compared to NPWT control 6. Larval debridement reduce biofilms in vitro 7. Biofilm Based Wound Care is part of Wound Bed Preparation (TIME) 44 15