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Biofilm Induction of Cellular Senescence
- Dr. Matthew Regulski DPM
Cellular Senescence Dr. Matthew Regulski DPM Director, The Wound - - PowerPoint PPT Presentation
Cellular Senescence Dr. Matthew Regulski DPM Director, The Wound - - PowerPoint PPT Presentation
Biofilm Induction of Cellular Senescence Dr. Matthew Regulski DPM Director, The Wound Institute of Ocean County NJ Partner, Ocean county Foot and Ankle Surgical Associates Toms River NJ APMA National Meeting 2018, Washington D.C. Don t
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INFECTIONS COST THE HEALTHCARE SYSTEM
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- 9. The Committee to Reduce Infection Deaths. The cost of infection. Preventing Infections Makes Hospitals More Profitable. http://www.hospitalinfection.org/cost_of_infection.shtml.
Last accessed January 25, 2017.
- 10. Kaiser Health News. Medicare Fines 2,610 Hospitals In Third Round Of Readmission Penalties. Jordan Rau. http://khn.org/news/medicare-cuts-payments-to-721-hospitals-with-
highest-rates-of-infections-injuries/. Last updated October 2, 2014. Last accessed January 25, 2017.
Estimated cost of hospital-acquired infections in the United States9. 2,000,000 estimated infections per year X $15,275 (Average additional costs for contracted infections) = $30.5 billion In 2014, 721 hospitals had their Medicare reimbursement lowered 1%—roughly $373 million in penalties—for having high hospital-acquired infection rates.10 In 2014, 18 percent of Medicare patients who had been hospitalized were readmitted within one month. Roughly two million patients are readmitted every year, costing Medicare $26 billion. Officials estimate $17 billion of that comes from potentially avoidable readmissions.10 Healing wounds quickly to full closure will not only save the healthcare system millions of dollars every year but improve the quality of life for millions of people living with chronic wounds.
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Medical Biofilms
Medical Biofilm US Incidence Annual Cost Diabetic foot ulcers (P) 3 M 50,000 deaths, 30% of hospital cost for diabetics Venous leg ulcers (P) 2.5 M Decubitus ulcers (P) 3-5 M (P) 27%NH > 50,000 Surgical site infections 500,000 $5-10 B, 5,000 deaths Burn wounds 1.1 M 15,000 deaths Chronic meningitis 1,400-2,800 140-390 deaths Bacterial prostatitis (P) 162,800 All odontogenic infections Chronic tonsillitis 11,000 $121.5 M Gallstones 430,000 $5 B Crohn’s disease 36,000-60,000 Ulcerative colitis 24,000-40,000 COPD (P) 30 M $37.2 B, 120,000 deaths Bronchiolectasia 110,000
General Infections
Pneumonia (non-VAP) 1.2 M $14-$25 B, 54,000 deaths Medical Biofilm US Incidence Annual Cost Vascular graft infection 16,000 $640 M Cardiac pacemakers 4,000-20,000 Peritoneal dialysis peritonitis ~20-25,000 on CPD Ventilator acquired pneumonia 135,000 $1.5 B, 61,000 deaths Endotracheal tubes 100s of thousands* $5 B Urinary catheter cystitis Millions 4,500 deaths
Nosocomial
Central venous catheters 250,000 $296 M-$2.3 B, 30-62.5 K deaths
Total 20 Million $100 B, >500k deaths
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Disease Incidence Annual Cost Cardiovascular Disease 2.28 M per year $431.8 B, 650,000 deaths Cancer 1.5 M per year $206.3 B, 550,000 deaths Diabetes 1.5 M per year, ages 20+ $132 B, 73,000 deaths Medical Biofilm > 10M per year > $200 B, > 500,000 deaths
Medical Biofilms
Context
For Comparison
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Biofilm EPS Structure (P. aeruginosa) – Ca+ Bridging
– These polymers are water-soluble – they should go into solution in saline! – This material has calcium-ion bridging in it to produce gelling
- In effect, this bridging works as cross-links would work in a traditional
thermoset polymer.
- As such, even if a good solvent for this material were found, it would not
be able to bring the EPS into solution – it would swell the polymer, but the bridging would prevent the individual polymeric strands from going into solution.
7 Ca ion
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Biofilm Development
Masako,K Journal of Dermatologic Science June 2005
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Biofilm Detachment
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Biofilm Infection
- (a) Bacteria adhered to surface
Surface selects (but is not necessary) for biofilm formation
- (a) Direct visualization of biofilm morphology The current “gold standard” for diagnosing biofilm
- (a) Confined to a particular location Biofilm seems to limit its size (quorum sensing)
- (a) Resistant to appropriate antibiotics A hallmark of biofilm is high resistance to antibiotics
- (b)Resistant to biocides
A hallmark of biofilm is high resistance to biocides
- (b)Large number with high diversity in a host lesion
- (b)Infections that wax and wane with exacerbations
- (b)Secondary signs of infection
(a)Parsek Annu. Rev. Microbiol. Vol57, 2003 (b) Wolcott JWC Vol19(2), 2010
Costerton and Stewart Sci Am Vol 285, 2001
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Neutrophils
Hartl, D Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease Nature Medicine Vol 13, 2007
Biofilms and Chronic Wound Inflammation JWC Vol 17, 2008 Diegelmann RF Wound Repair Regen Vol 11 2003
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Host Defenses
Leid, JG Infect Immun Vol 70, 2002
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Current Antimicrobial Wound Solutions are Ineffective Against Microbial Biofilms - in-vitro testing against biofilms1
2 4 6 8 Log 10 Viable Bacteria (cfu/mL)
CDC Reactor Biofilm Model, 72 hour biofilm, 15 minute treatment
- S. aureus
- P. aeruginosa
1: Johani, K., et al. "Evaluation of short exposure times of antimicrobial wound solutions against microbial biofilms: from in vitro to in vivo." Journal of Antimicrobial Chemotherapy (2017). *: Chlorhexidine: 0.015% chlorhexidine + 0.15% cetrimide **: 10% povidone-iodine
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Current Antimicrobial Wound Solutions are Ineffective Against Microbial Biofilms – ex-vivo testing against biofilms on porcine skin explants1
1: Johani, K., et al. "Evaluation of short exposure times of antimicrobial wound solutions against microbial biofilms: from in vitro to in vivo." Journal of Antimicrobial Chemotherapy (2017). 2 4 6 8 Total Bacteria Biofilm NPWT Saline Installatoin Microcyn Installation
10 Viable Bacteria (cfu/mL)
Treatment of Porcine Explants, 108 cfu of P. aeruginosa inoculation, 3 days growth before or after 12 cycles of 10 min installation
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Current Antimicrobial Wound Solutions are Ineffective Against Microbial Biofilms – in-vivo testing of chronic wounds and Key Findings1
- Key Findings
– The performance of these solutions is poor when challenged against mature biofilms using short exposure times that mimic real clinical use (i.e. 15 min application) – Clinicians using topical antimicrobials to cleanse chronic wounds as a single therapy under the assumption of removing biofilm may therefore experience poor clinical outcomes – Clinicians should consider multifaceted strategies that include sharp debridement as the gold standard
1: Johani, K., et al. "Evaluation of short exposure times of antimicrobial wound solutions against microbial biofilms: from in vitro to in vivo." Journal of Antimicrobial Chemotherapy (2017).
Effects of Melaleuca Oil pre- and post- treatment of 10 chronic non-healing diabetic foot ulcers. Box-and-whisker plots show the median log<sub>10</sub> 16S copies/mg of tissue values for all 10 patients
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Slow Penetration
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Biochemical Impairment of Chronic Wounds
Elevated proinflammatory cytokines Elevated proteinase activity – MMPs Diminished activity of growth factors Degraded receptor sites (degradation blocked by the addition of MMP inhibitors)
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Table 4. Functional Group: Immune responses
Conclusions
S. aureus interferes with the wound healing process by reducing the expression of several cytokines and chemokine genes
Table 3. Functional Group: Recruitment, activation of immune
cells
Innate immune responses Adaptive immune responses
Alteration in cytokine and chemokine expression during Staphylococcus aureus wound infections
Kayla Bounds1,Cassandra Kruczek2, Matt Myntti3, Jane A. Colmer-Hamood4,5, Randall Jeter1, and Abdul N. Hamood2,5
1Biology Dept., Texas Tech University Lubbock, TX; 2Dept. Of Surgery, Texas Tech University Health Sciences Center; 3Next Science, Jacksonville, FL; 4Dept. of MedicalEducation, TTUHSC, Lubbock, TX; 5Dept of Immunology and Molecular Microbiology, TTUHSC Lubbock, TX
Abstract
Chronic wounds, which include pressure ulcers, diabetic foot ulcers, and venous ulcers, affect approximately 6.5 million persons with an annual cost for treatment that may reach as high as $25 billion dollars. Wound healing occurs through specific overlapping steps that involve interactions of different cell types, extracellular matrix proteins, and their receptors. These interactions are mediated by cytokines and growth factors. Infection prevents or slows wound healing, yet the influence of specific microorganisms on these interactions is not well defined. Staphylococcus aureus is one of the microorganisms commonly isolated from infected chronic wounds. Using the murine model of wound infection, we examined the level of cytokine expression in S. aureus-infected full-thickness excision wounds compared with uninfected wound tissues. Tissues excised from the wounds at 24 hours were homogenized and total bacterial RNA was isolated. Cytokines expression was determined using RT² Profiler™ PCR Array Mouse Cytokines and Chemokines kit (QIAGEN), which measures the expression of 94 mouse cytokines and chemokines. In uninfected wounds, the expression of numerous cytokines belonging to the following functional four groups was greatly enhanced: 1) response to injury and tissue homeostasis; 2) production of immune cells and hematopoiesis; 3) recruitment and activation of immune cells; and 4) immune responses. However, the level of these cytokines was either reduced or only slightly increased in wounded/infected tissue. For example, the level of expression of Ccl20, a cytokine associated with wound healing, was increased in wounded tissues by 63-fold but decreased by 1.27-fold in wounded/infected tissues. Additionally, while the expression of Cxcl5, a cytokine involved in the activation of immune cells, was increased in wounded tissues by 2000-fold, it was increased by only two-fold in wounded/infected- tissue. These results suggest that wound infection by S. aureus interferes with the expression of
Hypothesis
- S. aureus infection of wounded tissues alters the
expression of different cytokines and chemokines
Chronic wounds are defined as those that fail to proceed through an orderly and timely reparative process to produce anatomic and functional integrity of the injured site. Chronic wounds constitute a serious threat to the public health
- worldwide. In United States, it is estimated that chronic wounds affect 6.5 million
patients. In addition, due to the increase in; health care costs, the incidence of diabetes and obesity, the cost of treating chronic wounds is growing very rapidly. In the US, treatment of chronic wounds may reach as high as $25 billion annually. Major chronic wounds include diabetic foot ulcers, venous leg ulcers, pressure ulcers, and ulcers resulting from peripheral vascular disease. Chronic wounds contain diverse bacterial species of pathogenic bacteria that changes over time. Using molecular amplifications and pyrosequencing, investigators examined bacterial species present in chronic wounds of diabetic foot ulcers, venous leg ulcers, and pressure ulcers. In all these wound types bacterial populations were frequently polymicrobial and included Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. It has been shown that hospitalized patients, patients with surgical procedures, as well as those on prolonged or broad-spectrum antibiotic therapy are predisposed to colonization or infection, or both, with resistant organisms, including methicillin resistant S aureus (MRSA). In general, the wound healing process is divided into four overlapping stages; hemostasis, inflammation, proliferation, and remodeling. The hemostasis stage begins as the tissues are injured and when blood moves into the site of injury. The inflammation occurs after hemostasis. In this stage, the phagocytotic process is initiated by the appearance of the neutrophils and macrophages. This leads to an increase in the secretion of secretion of growth factors and inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. In addition, neutrophils activate fibroblasts and epithelial cells. The proliferation stage involves migration of fibroblasts to the wounded tissues. The fibroblasts perform several functions including; the deposition of a new extracellular matrix, stimulation of protease inhibitors, promotion of angiogenesis, and the release of cytokines such as interleukins, fibroblast growth factor and TNF-α. During the remodeling stage, the wound becomes re-epithelized. In addition, the extracellular matrix becomes cross-linked and the healed wound becomes less vascular. Each one of the above described wound healing stage would likely involve significant variations in the expression of different cytokines, chemokines, and other wound healing related genes. Bacterial infection prevents wound healing by interfering with one
- r more of these four healing stages.
Such interference may occur through alteration of the expression of genes that code for essential cytokines and chemokines. In this study, we utilized the murine model of wound infection to examine the effect
- f S. aureus on the expression of different cytokines and chemokines within the
wound/infected tissues.
Introduction
- Fig. 1. Diagram illustrating the murine model of wound infection.
Table 2. Functional Group: Hematopoiesis and production of
immune cells
Materials and Methods
1 group – no further procedures: Uninjured Remove hair from mice 1 group – 1.5 x 1.5 cm wound, covered with OPSITE, and injected with 200-250 CFU of S. aureus under dressing: Injured infected Euthanize mice and collect tissue 1 group – 1.5 x 1.5 cm full- thickness surgical wound covered with OPSITE dressing: Injured or Injured uninfected- Fig. 2. Flow chart illustrating different steps involved in tissue
homogenization, RNA extraction, and microarray analysis.
5 mm-tissue punch from site adjacent to wound RNA later Homogeni ze tissue in RNA Bee Extract RNA Used CT values for data analysis Rever se trans cribe to cDNA Perform real-time PCR using primers for various cytokines to check RNA quality Perform real-time PCR using RT² Profiler™ PCR Array Mouse Cytokines and Chemokines kit (QIAGEN)Results
Legend for all tables: Colors indicate changes in gene expression relative to
- control. Reds: decreased; yellow: little or no change; greens: increased
Table 1. Functional Group: Response to Injury
Protein Function Fold Change Injured / Uninjured Fold Change Injured infected / Injured uninfected Adipoq Metabolism (fat cells) 1.58- 3.23
- 4.00
- 3.23
- 4.00
- 4.00
- 4.00
- 4.00
- 1.27
- 2.50
- 1.59
- 1.59
- 3.23
- 2.00
- 1.28
- 2.56
- 2.04
- 1.27
- 1.59
- 4.00
- 1.05
- 3.23
- 2.00
- 1.28
- 1.59
- 1.27
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Compared to Americans, Okinawan elders get 80% fewer breast and prostate cancers get 50% fewer ovarian and colon cancers have 50% fewer hip fractures have 80% fewer heart attacks
40% fewer calories than Americans and 17% fewer calories than the Japanese average
http://www7.nationalgeographic.com/ngm/0511/sights_n_sounds/index.html
Okinawan Elders
Greatest # centenarians
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"To lengthen thy life, lessen thy meals." Benjamin Franklin
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