Bioburden-Biofilms in Inflammation

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1 Bioburden-Biofilms in Inflammation Gregory Schultz, h.d. UF Research Foundation rofessor of Obstetrics/Gynecology Director, Institute for Wound Research University of Florida Learning Objectives Review the four sequential phases of normal wound healing and recognize the BENEFICIAL effects of CONTROLLED INFLAMMATION and ROTEASE ACTIVITIES Understand the link between CHRONIC INFLAMMATION caused by LANKTONIC and BIOFILM BACTERIA and ELEVATED ROTEASE ACTIVITIES that DESTROY proteins that are essential to healing (extracellular matrix, growth factors, receptors) Recognize the high TOLERANCE of BIOFILM bacteria to most antibiotics, antiseptics and disinfectants Describe key principles of BIOFILM-BASED WOUND CARE that emphasize DEBRIDING BIOFILMS and REVENTING REFORMATION OF BIOFILMS as part of the STE-DOWN approach for effective therapies Sequence of Molecular and Cellular Events in Skin Wound Healing Controlled Wound Inflammation Is Beneficial Macrophages Neutrophils ELASTASE Four hases of Healing 1. Hemostasis 2. Inflammation 3. Repair 4. Remodeling MMs O 2 - H 2 O 2 HOCl Vascular Scar Epithelial Scar Clotting Response Inflammation Formation Healing Contraction Remodeling Inflammatory cells kill planktonic bacteria by phagocytosis and reactive oxygen species. They also release proteases (MMs, elastase) that remove denatured ECM components and permit wound healing to proceed. Inflammatory cells are not effective against bacteria in biofilms. Respiratory Burst In Neutrophils & Macrophages roduces Reactive Oxygen Species (ROS) That Kill Bacterial & Fungi Question: What happens when the respiratory burst is impaired? In the membranes of neutrophils, NADH oxidase generates superoxide (O 2- ), which spontaneously dismutates to H 2 O 2, and is converted to hypochlorous acid (HOCl) by myeloperoxidase (MO). These reactive oxygen species (ROS), especially HOCl, participate in the killing of bacteria. The right panels show a bacteria being phagocytized and production of ROS (red color) surrounding the yeast cell. Answer: Severe impairment of host resistance to occurs. Clinical condition known as Chronic Granulomatous Disease is due to mutated NADH oxidase. 1

2 Chronic Granulomatous Disease Characterized by predisposition to bacterial and fungal s Associated with decreased oxygen consumption and defective microbial killing Due to defective mutation in components of NADH oxidase complex Reduced levels of superoxide anion (O 2- ) which is converted to bactericidal reactive oxidants results in decreased levels of: hydroxyl radical (OH - ) hydrogen peroxide (H 2 O 2 ) peroxynitrite anion (ONOO - ) oxyhalides (HOCl, hypochlorus acid) Controlled MMs Are Necessary for Wound Healing Debridement, Angiogenesis, Contraction, Epithelial Migration, Remodeling MMs ARE NECESSARY FOR SEVERAL KEY ROCESS IN WOUND HEALING 1. removing denatured matrix 2. degrading capillary basement membrane for angiogenesis 3. contraction of ECM by myofibroblasts 4. migration of epidermal cells 5. remodeling of scar MMs in Normal Wound Healing MMs are essential for normal wound healing, BUT must be: At the right places At the right times At the right amounts Major Cytokines Involved in Wound Healing CYTOKINE CELL SOURCE BIOLOGICAL ACTIVITY Tumor Necrosis Factor alpha TNF-a Interleukin-1 IL-1 Interleukin-6 IL-6 Interleukin-8 IL-8 Interferon-g INF-g Interleukin-4 IL-4 Interleukin-10 IL-10 RO-INFLAMMATORY CYTOKINES macrophages macrophages, keratinocytes macrophages, keratinocytes, MNs macrophages, fibroblasts macrophages, T-lymphocytes ANTI-INFLAMMATORY CYTOKINES T-lymphocytes, basophils, mast cells T-lymphocytes, macrophages, keratinocytes MN margination and cytotoxicity MM synthesis fibroblast and keratinocyte chemotaxis, MM synthesis fibroblast proliferation macrophage and MN chemotaxis collagen synthesis macrophage and MN activation collagen synthesis MM synthesis TNF-a, IL-1, IL-6 synthesis fibroblast proliferation, collagen synthesis TNF-a, IL-1, IL-6 synthesis macrophage and MN activation Is There a Common Molecular athology Of Chronic Wounds?? Hypothesis Of Chronic Wound athophysiology Repeated Tissue Injury, Ischemia and Bioburden lanktonic & Biofilms TNF-a IL-1, IL-6 Diabetic foot ulcer Arterial ulcer rolonged, elevated inflammation neutrophils macrophages mast cells Imbalanced roteases & Inhibitors roteases (MMs, elastase, plasmin), inhibitors (TIMs, a1i), ROS Destruction of Essential roteins (off-target) growth factors / receptors, ECM degradation cell proliferation, cell migration, ressure ulcer Venous ulcer Acute Wound Chronic Non-Healing Wound B.A. Mast and G.S. Schultz. Wound Rep Reg 4: ,

3 Chronic Infection by Medical Biofilms chronic sinusitis CNS shunt contact lens associated keratitis chronic otitis media cochlear implant burn Wound biofilms are linked to delayed healing breast implant ventilator associated pneumonia pulmonary in CF patient intravascular stent catheter prosthetic valve endocarditis pacemaker biliary stent peritoneal dialysis catheter urinary stent prosthetic joint peritoneal dialysis catheter Biofilm-Associated Bacterial Infections. Clin harm 18 Clinical athologies del ozo and atel. The Challenge of Treating Ther 82:204-20, 2007 Mouse model showed presence of S. aureus and S. epidermidis biofilms significantly delayed re-epithelialisation. 1 Negative impact of biofilm on healing verified by other studies 2,3 1. Schierle, C. F., et al.. Wound Repair Regen. 17, (2009). 2. Zhao, G. et al. Wound Repair Regen. 20, (2012). 3. Roche, E. D. et al.. Wound Repair Regen. 20, (2012). Effect of Staphylococcus aureus and Staphylococcus epidermidis biofilms on wound re-epithelialization.1 Biofilms Identified in >80% of Biopsies of Chronic Wounds but in Only 6% of Acute Wounds A D B C E Heterogeneous Distribution Of Bacteria In Chronic wounds 6 9 qcr seudomonas aeruginosa C 12 3 osition Wound 1 Wound 2 C 510±18% 920±9% 3 No sample 300±13% 6 760±7% 8200±8% 9 47±9% 800±10% ±3% 15±5% anels A, B & C: G. James, E. Swogger, R. Wolcott, E. ulcini,. Secor, J. Sestrich, J. Costerton,. Stewart. Wound Rep Regen, 16:37-44, 2008 anel D: HC Flemming, J Wingender The Biofilm Matrix, Nature Rev Microbiol, 8: , 2010 anel E: SR Schooling, A Hubley, TJ Beveridge. J Bacteriol 191: , 2009 M. Malone, T. Barjnsholt, A. McBain, G. James,. Stoodley, D. Leaper, M. Tachi, G. Schultz, T. Swanson, R. Wolcott. revalence of biofilms in chronic wounds: a systematic review and meta-analysis of published data, J wound Care, in press icture from homepage of Montana State University with permission Thomsen TR, Aasholm MS, Bjarnsholt T, Givskov M, Kirketerp-Møller K, and Nielsen H. The bacteriology of chronic venous leg ulcer examined by culture-independent molecular methods. Wound Repair Regen, 18(1):38-49, 2010 Distribution of Species S. aureus biofilm. aeruginosa biofilm S. aureus biofilm Mono-species Biofilms Verses Multi-species Infections A 25 mm B. aeruginosa biofilm Fazli and Bjarnsholt et al: J Clin Microbiol Dec;47(12): Images from rof Bjarnsholt with permission Confocal Laser Scanning Microscopic (CLSM) images of 48-h in situ dental biofilms stained simultaneously with allbacterium-specific EUB338 probe (red), a Streptococcus-specific STR405 probe (yellow-green), and Actinomycesspecific ACT476 probe (blue) and red represent streptococci. (A) Maximum projection image of relative thin 48-h biofilm showing complete surface coverage with the dominance of streptococci. Well-defined microcolonies of large coccoid non-streptococci are observed as well as microcolonies of A. naeslundii. Scale bar = 25 mm. (B) Sagittal (x-z, y-z) section of a multilayered dental biofilm. Note that A. naeslundii (blue) is predominantly located in the inner part of the biofilms next to the surface (bottom of the images). Some microcolonies of A. naeslundii extended almost throughout the entire thickness of the biofilm. Burmølle, M. et al. Biofilms in chronic s a matter of opportunity monospecies biofilms in multispecies s. FEMS Immunol. Med. Microbiol. 59, (2010). 3

4 Ratio of MM-9 (pro+active):tim-1 (ng/ml) rhmm-9 Equivalent Activity (µg/ml) % of Area at First resentation (%) Question: How do biofilms impair healing of skin wounds? Chronic Venous Ulcers Have High Levels Of IL-1β and TNFa That Decrease With Healing TNFa IL1 Answer: Biofilms stimulate chronic inflammation by increasing release of proinflammatory cytokines that leads to highly increased levels of proteases and reactive oxygen species that degrade proteins that are essential for healing. TNF-alpha (pg/ml) Non-healing Healing IL-1 u/ml Non-healing Healing Trengove, Stacey, Macauley, Bennett, Gibson, Burslem, Murphey, Schultz. Wound Rep Reg 7: , 1999 How Does The Immunological Response to Biofilms Cause Tissue Damage and Impair Healing? High Levels of MM Activity in Chronic Wounds Decrease as Wounds Heal A B C D In anel A, planktonic bacteria can be cleared by antibodies, phagocytosis, and are susceptible to antibiotics. Adherent bacterial cells (anel B) form biofilms preferentially on inert surfaces or devitalized tissue, and these sessile communities are tolerant to antibodies, phagocytosis and antibiotics. Neutrophils (anel C) are attracted to the biofilms, but cannot engulf biofilm. Neutrophils still release proteases and reactive oxygen species. hagocytic enzymes (anel D) damage tissue around the biofilm, and planktonic bacteria are released from the biofilm, causing dissemination and acute in neighboring tissue. Costerton, Stewart, Greenberg, Science 284, 1999 Trengove, Stacey, Macauley, Bennett, Gibson, Burslem, Murphy, Schultz. Wound Rep Reg 7: , 1999 Healing of ressure Ulcers is redicted by rotease Activity in Wound Fluids Good Healing Good Responders >95% area healed; n=12 Intermediate Healing Intermediate <95% Responders but >65% area healed; n=36 oor Healing <65% area healed; n=8 oor Responders MM-9 Activity Correlates With Wound Healing Time Course MM Time Line Wound Area Time Line Day-0 Day-10 Day-36 Time Course Ladwig, Robson, Liu, Kuhn, Muir, Schultz. Ratios of Activated MM-9/TIM-1 in Wound Fluids Are Inversely Correlated With Healing of ressure Ulcers. Wound Rep Reg 26-37, Day Within Trial (day #) G. Bohn, B. Liden, G. Schultz, Q. Yang, D.J. Gibson. Ovine-Based Collagen Matrix Dressing: Next- Generation Collagen Dressing for Wound Care. Advances Wound Care 6(1):1-6,

5 Conclusion: Inflammation in chronic wounds must be reduced to levels that lead to low protease activities that allow wounds to heal. Action: Bacterial levels (both planktonic and biofilm) must be reduced for healing Free download from Wounds International D. Gibson, B. Cullen, R. Legerstee, K.G. Harding, G. Schultz. MMs Made Easy. Wounds International, 1(1): 1-6, Question: Why are bacteria in biofilms hard to kill? Answer: Exopolymeric material (EM) of the biofilm Dense matrix impairs diffusion of large antibodies EM materials chemically react (neutralize) microbicides Negative charges of polysaccharides and DNA bind cationic molecules like Ag +, antibiotics, HMB + ersister bacteria have low metabolic activity Antibiotics only kill metabolically active bacteria Oxygen diffusion to center of biofilm is limited romotes growth of anaerobic bacteria Synergism between different bacteria MRSA secrete resistance proteins seudomonas secrete catalase that destroys H 2 O 2 Hypochlorous Acid Very Slowly enetrates Biofilm Matrix Reaction-Diffusion roblem dead live 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, Sci Am, minutes 25 minutes 47 sec Reaction-Diffusion roblem Hypochlorous acid rapidly reacts with molecules that form the biofilm exopolymeric matrix, which limits its diffusion into the center of the biofilm colony. Stewart,.S. et al. Biofilm penetration and dis efficacy of alkaline hypochlorite and chlorosulfamates. J Applied Microbiol 91: , Biofilms are Highly Tolerant to Antibiotics Metabolic Activity of seudomonas aeruginosa in Mature Biofilms is Limited to the Surface Layers dead control biofilm planktonic Tobramycin rapidly kills planktonic seudomonas aeruginosa ( ) very effectively, but is not effective against biofilm ( ). Walters et al, Contributions of antibiotic penetration oxygen limitation metabolic activity to antibiotic tolerance of aeruginosa. Antimicrob Agents Chemother 47: , Only fluorescent bacteria are metabolically active -- Only located in outer layers of the biofilm matrix -- Antibiotics only kill metabolically active bacteria. Stewart, Controlling Biofilms, Chapter 7, in The Biofilms Hypertextook, published by Montana State University, A.B. Cuningham, J.E. Lennox & R.J. Ross, eds,

6 rinciples of Biofilm Based Wound Care 1. Frequent sharp debridement of wounds to physically remove biofilm communities 2. Use an effective, fast acting microbicidal dressing after debridement to manage residual biofilm bacteria and to prevent reformation of biofilms e.g. Cadexomer Iodine 3. Alter topical & systemic antimicrobial treatments to prevent emergence of dominant bacteria from polymicrobial populations; utilize DNA bacterial identification techniques 4. Step-down treatment should be used to rapidly decrease biofilms and proteases that impair healing Wolcott,R.D.; Rhoads,D.D. A study of biofilm-based wound management in subjects with critical limb ischaemia. J.Wound.Care 17: , 2008 Question: Can you see biofilms on the surface of wound beds? Answer: YES or NO Most biofilms are NOT visible on the surface of a wound bed, and much of the biofilm is beneath the surface of the wound bed where it is very inflammatory! What is This Filmy Wound Slough? Mainly Fibrin - Surrogate Biomarker for Inflammation Can you see a biofilm in this wound? Dr Randy Wolcott hoto provided by Dr Matthew Malone hoto provided by Dr Matthew Malone hoto provided by Dr Matthew Malone 6

7 seudomonas aeruginosa Staphylococcus aureus Gauze Debridement of Biofilm Bacteria on ig Skin Explants Effect of Wiping Only on Total and Biofilm Bacteria Biofilm grown on pig skin explant Biopsy biofilm on pig skin explant ig skin explant after gauze debridement Gauze used to debride pig skin explant Effect of Wiping Only on Total and Biofilm Bacteria Effect of Daily Wiping + lurogel on Total & Biofilm Bacteria Wiping only Yang Q, Larose C, orta AD, Della orta AC, Schultz GS, Gibson DJ. A surfactant-based wound dressing can reduce bacterial biofilms in a porcine skin explant model. Int Wound J 2016; Wiping + Surfactant Yang Q, Larose C, orta AD, Della orta AC, Schultz GS, Gibson DJ. A surfactant-based wound dressing can reduce bacterial biofilms in a porcine skin explant model. Int Wound J 2016; Effects of Non-Contact Ultrasonic Wound Cleansing on Biofilms silver solution 1.E+08 1.E+07 1.E+06 Larval Debridement Therapy CFU / 5mm Biopsy Before treatment Before treatment After 24hr treatment 1.E+05 1.E+04 iodine solution bleach solution 1.E+03 1.E+02 1.E+01 A01 SA E+00 L. Cowan, J. Stechmiller,. hillips, Q.. Yang and G. Schultz. Chronic Wounds, Biofilms and Use of Medicinal Larvae, Ulcers, Article ID , 7 pages;

8 Question: How quickly can planktonic bacteria form protective biofilms in wounds after sharp debridement? Biofilm Maturity Studies Indicate Sharp Debridement Opens a Time-Dependent Therapeutic Window Which answer is true? 1. 7 days 2. 5 days 3. 3 days 4. 1 day Biopsies from three patients with large (>10 cm 2 ) venous ulcer were split into two tubes containing saline (control) or saline with 200 ug/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). R.D. Wolcott, K.. Rumbaugh, G. James, G. Schultz,. hillips, Q. Yang, C Watters,.S. Stewart, S.E. Dowd, J Wound Care 19: , Can Dressings Disrupt & Kill Mature Biofilms? Question: Do all antimicrobical wound dressings effectively kill biofilm colonies grown on pig skin explants? Answer: YES or NO L hillips, Q Yang, E Sampson, GS Schultz. Effects of antimicrobial agents on an in vitro biofilm model of skin wounds. Adv Wound Care 2010; 1: Effects of Antimicrobial Agents on Mature Biofilms on ig Skin Explants 24 hours of continuous exposure Step-Down Treatment Strategy for Chronic Wounds.L. hillips, Q. Yang, E. Sampson, G. Schultz. Effects of Antimicrobial Agents on an In Vitro Biofilm Model of Skin Wounds, Advances Wound Care, 1: ,

9 Summary Free download from Wounds International. hillips, R. Wolcott, J. Fletcher, G. Schultz. Biofilms Made Easy. Wounds International, 1(3): 1-6, Biofilms are communities of bacteria encased in a 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. Biofilm based wound care emphasizes effective debridement combined with effective topical and systemic treatments that effectively prevent reformation of biofilms 4. Step-down therapy is based on starting with the therapies that most effectively reduce biofilms, inflammation, and proteases (debridement, biofilm killing agents, protease inhibitors) then shifting to advanced therapies that further reduce proteases and provide biological support for repairing the wound bed (granulation tissue, fibroblasts and epithelial cell proliferation and migration, collagen synthesis) such as growth factors, collagen dressings, biological membranes, and NWT. 9