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 Stop Your Curiosity

INFECTIONS COST THE HEALTHCARE SYSTEM

3

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.

Medical BiofilmsMedical 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

Gen

era

l In

fect

ion

s

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

No

soco

mia

l

Central venous catheters 250,000 $296 M-$2.3 B, 30-62.5 K

deaths

Total 20 Million $100 B, >500k deaths

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 BiofilmsContext

For Comparison

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

Biofilm Development

Masako,K Journal of Dermatologic Science June 2005

Biofilm Detachment

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

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

Host Defenses

Leid, JG Infect Immun Vol 70, 2002

Current Antimicrobial Wound Solutions are Ineffective Against Microbial Biofilms - in-vitro

testing against biofilms1

0

2

4

6

8

Log

10

Via

ble

Bac

teri

a (c

fu/m

L)

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

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).

0

2

4

6

8

Total Bacteria Biofilm NPWT SalineInstallatoin

MicrocynInstallation

10

Via

ble

Bac

teri

a (c

fu/m

L)

Treatment of Porcine Explants, 108 cfu of P. aeruginosa inoculation, 3 days growth before or after 12 cycles of 10

min installation

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 log₁₀ 16S

copies/mg of tissue values for all 10 patients

Slow Penetration

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)

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 Medical Education, TTUHSC, Lubbock, TX; 5Dept of Immunology and Molecular Microbiology, TTUHSC Lubbock, TX

AbstractChronic 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

numerous wound healing and immune response cytokines.

HypothesisS. 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

or 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

of 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 FunctionFold Change

Injured / Uninjured

Fold Change

Injured infected / Injured

uninfected

Adipoq Metabolism (fat cells) 1.58 -3.23

Bmp2 Bone mineralization 1.50 -4.00

Bmp4 Bone formation 2.00 -3.23

Bmp6 Response to injury 1.59 -4.00

Spp1 Osteoclast Function 31.96 -4.00

Cntf Central Nervous system 3.14 -4.00

TgfbWound repair, matrix maintenance,

fibrosis1.00 -4.00

Ccl-20 Wound healing 63.70 -1.27

Protein FunctionFold Change

Injured / Uninjured

Fold Change

Injured infected / Injured

uninfected

Csf3 G-CSF: differentiation of granulocytes 18.45 1.25

Osm Megakaryocyte development 20.27 1.97

Il7 B, T cell development -2.50 1.25

Il21 T, B cell development 1.00 5.89

Il23 Th 17 cell development 5.69 -1.59

Protein FunctionFold Change

Injured / Uninjured

Fold Change

Injured infected / Injured

uninfected

Csf3 G-Csf: activation of granulocytes 18.45 1.25

Cxcl1 PMN chemotaxis 43.91 -1.59

Cxcl3 PMN chemotaxis 193.55 2.50

Cxcl5 PMN chemotaxis 2198.70 1.58

Ppbp CXCL-7: PMN chemotaxis 20.43 -3.23

Ccl2MCP-1: Monocyte chemotaxis,

differentiation12.60 1.26

Ccl3MIP-1a:Monocyte chemotaxis, macrophage

diff.133.56 1.59

Ccl4MIP-1b: Monocyte chemotaxis, macrophage

diff.100.17 1.26

Ccl7MCP-3: Monocyte chemotaxis, macrophage

diff.6.32 1.25

LtbTNFb: :Lymphocyte development in

periphery-2.00 1.99

Cxcl10 Th1, CD8, NK cell movement -1.28 6.35

Ccl17 Th2, Treg trafficking 1.57 5.05

Cxcl13 B cell movement 10.02 -2.56

Protein FunctionFold Change

Injured / Uninjured

Fold Change

Injured infected / Injured

uninfected

HcC5, C5a, Proinflammatory,

antibacterial13.18 -2.04

IfnaProinflammatory, macrophage

activation; increased MHC expression-1.27 4.15

Il1bMonocyte release of cytokines, ROl,

prostaglandin80.49 1.58

Il1rn Inhibitor of IL-1a and IL-1b 80.10 -1.59

Il6 Hepatic and pituitary acute phase 33.73 2.00

OsmRegulation of endothelial cell cytokine

production20.27 1.97

Il24 Cytokine release by immune cells 11.80 -4.00

Lta Activation and cytotoxicity of T cells -1.05 -3.23

Ltb Proinflammatory -2.00 1.99

IfngPromotes activation of antigen-

presenting cell-1.28 3.17

Ccl3 T cell/dendritic cell interaction 133.56 1.56

Ccl4 T cell/dendritic cell interaction 100.17 1.26

TNfsf11Promotes cytokine production by

many cells1.71 2.48

Il23 Th17 expanded 5.69 -1.59

Il21 B cell activation -1.27 5.89

31

Compared to Americans, Okinawan eldersget 80% fewer breast and prostate cancersget 50% fewer ovarian and colon cancershave 50% fewer hip fractureshave 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

"To lengthen thy life, lessen thy meals." Benjamin Franklin

THANK YOU

MREGULSKI@COMCAST.NET