MOLECULAR DIAGNOSIS OF BACTERIA AND CUSTOMIZED ANTIBIOTIC TREATMENT Where the Future of Wound Care is Heading? Caroline E. Fife, MD CMO, Intellicure Medical Director, St. Luke’s Wound Care Center, The Woodlands, TX

WHY SHOULD YOU NEED AIR FRESHENER TO WORK IN A WOUND CENTER? •

For 15 years one of the most important supplies we carried was a wide variety of air fresheners.

•

Even the ID doctors told us that if the cultures were negative, the odor was not something to worry about.

•

The patients found it a problem, and it was not fun for the staff.

•

I began to ask some basic questions: • Why did some wounds really stink but have negative cultures? • Did odor matter other than for social reasons?

30 YEAR SURVIVOR OF 80% BODY BURN

Multiple chronically draining leg ulcers with foul odor that cultured only Pseudomonas.

MR. AND MRS. CELLULITIS •

Morbidly obese husband and wife couple with cellulitis and draining wounds

•

Constantly re-infecting each other despite all the antibiotics we gave them.

wife

husband

WHAT IS THIS STUFF AND WHAT SHOULD I DO ABOUT IT?

BIOBURDEN 101 VS. 102 Bioburden 101: Planktonic (the world is flat) Historical Paradigm (predatory-acute)

Key word: “Planktonic” or free mobile microbes like we learned in biology.

BIOBURDEN 101 VS. 102

Bioburden 102: Biofilm

Medical Reality (parasitic-chronic)

Key word: “Biofilm” or microbes ATTACHED to a surface.

THE TRANSITION ILLUSTRATED

THE INFECTION CONTINUUM • The “infection continuum” has been described as contamination followed by colonization, “critical colonization” and finally “infection.” • This continuum does not reflect recent advances in understanding of the microbial/host relationship. • A more current description is: contamination => attachment => proliferation => critical concentration (of signaling molecules rather than bacterial counts) => adoption of a biofilm phenotype => progressively polymicrobial biofilm

WOUND CARE: CHANGING MEDICAL PRACTICE AS WE KNOW IT? • Medical biofilms ARE chronic infection (e.g an infection that does not respond to antibiotics) • Chronic infections are treated the same in all specialties: • Use antibiotics and if that fails cut it out. • Examples: Sinus = sinusectomy, tonsils = tonsillectomy, hardware = revision surgery, heart valve = remove valve & tissue, osteomyelitis = cut the bone out • If we consider chronic infection as one disease, it impacts 3-4 times more patients than heart disease and kills >500,000 per year (on par ~ heart disease and cancer).

Del Pozo and Patel Clin Pharm Ther Vol 82, 2007

BIOFILMS IN NATURE

PERIVASCULAR CUFF

FLUORESCENT IN SITU HYBRIDIZATION

host nuclei

red blood cells

P. aeruginosa biofilm

Schaber et al, 2007, I&I

WOUND BIOFILM

WBC ACTIVITY

When I say resistant… I mean RESISTANT!

Note: Biofilms provide PHYSICAL resistance, not genetic resistance!

Why can ’t we heal this? can’t

LET THE BALANCE GUIDE YOU

(All Host Defenses) (All Treatments)

HOS T

(Hypoxia / CLI) (Systemic Disease / DM) (Malnutrition) (Trauma / Pressure) (Bioburden)

PATHOLOG Y

WHAT IS WRONG WITH THIS PICTURE? ACUTE WOUND

CHRONIC WOUND

How do we diagnose these bacteria?

James Wound Repair and Regeneration Vol 16, 2008

PROBLEM 1: BIOFILMS ARE POLYMICROBIAL In chronic infection, we see a community of bacteria, acting as a group -wounds / cystic fibrosis / sinus / endocarditis… All chronic infections are polymicrobial.

MY NAME IS LEGION, FOR WE ARE MANY!

PROBLEM 2: RESISTANCE & SENSITIVITY TESTING MRSA, MDRPA, VRE Biofilm difference: (10 Staph isolates of 3 species) Vancomycin & Linezolid Kills took 100 – 1000 fold > conc than planktonic MIC90 If biofilm disrupted: 25 - 50% > potency per ABX

Whatever susceptibility data you have are based on planktonic sensitivities and SYSTEMIC CONCENTRATIONS.

Khardori et al, Ann Clin Microbiol Antimicrob. 2005; 4:2.

PROBLEM 3: PLANKTONIC VS. BIOFILM

Same Genotype !

Phenotype II

Phenotype I

Same Genotype !

Phenotype I

Phenotype II

PROBLEM 4: Limitations Of Culture Dx •

DYK? In the most advanced laboratories in the world specializing in culturing hard to grow bacteria… • Only 5% of known bacteria can be grown using traditional culturing techniques?

•

Staph, I want you!

Only ~ 1% of bacteria are grown quickly and easily

• Staphylococcus aureus, • Enterococcus spp, • Streptococcus spp, • Pseudomonas aeruginosa, • E. coli, etc. •

When we send off to culture, why is X is reported?

Why? Cause I can’t grow in a lab???

Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing. Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA, Wolcott RD. BMC Microbiol. 2008 Mar 6;8:43.

SOLUTION: SAME GENOTYPE!

Same Genotype !

Phenotype I

Phenotype II

You may not be able to get these organisms to grow like this in culture but their DNA is the same

Same Genotype !

Phenotype I

Phenotype II

1000’S OF SAMPLES = REALITY • DFU “top ten”: • 6 of 10 highly fastidious (HARD to grow) • 50% anaerobes • 2 of 10 easy to grow

• PU’s:

5 of top 10; > 60%; 2 of 10

I can grow in 24hrs on agar! Can I be your pathogen?

• VLU’s: 4 of top 10; >40%; 3 of 10

• 97% of chronic wounds contain easy to grow bacteria at < 1% of total population. Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and titanium amplicon pyrosequencing and metagenomic approaches. Wolcott RD, Gontcharova V, Sun Y, Dowd SE. BMC Microbiol. 2009 Oct 27;9:226.

POLYMERASE CHAIN REACTION (PCR): THE XEROX MACHINE FOR DNA

HOW DO YOU OBTAIN PCRS?

PATHOGENIUS LABORATORY REPORT

1ST: SHARP DEBRIDEMENT

First line to remove biofilm in any chronic wound! Frequently q 7-14 days New Wound Bed Biofilms removal

EVIDENCE: Will it be worth it? DNA Guided Personalized Medicine

Control Group A:

Culture Directed Therapy

Treatment Group B: DNA Directed Systemic ABX  Quantification & DNA level certainty. Treatment Group C: DNA Directed Topical ABX  Topical antibiofilm agents & ABX.  NO systemic antibiotics at all.

Class II-B Evidence: >1400 patients

DNA Directed Systemic

DNA Directed Topical

%  healing =

%  healing =

%  days to healing =

%  days to healing =

How much % improvement in outcomes (patients healed) would it take to make you adopt either DNA guided approach? How much of a reduction in days to healing would it take for you to adopt?

% HEALED ~Doubled healing in every period

IMPACT ON RECALCITRANT WOUNDS

~3 to 4 X faster

BIOFILM MANAGEMENT ILLUSTRATED Add DNA & ABX

Add ABF

BF RTN

Debridement

A TYPICAL CHRONIC ULCER PATIENT

Bilateral leg ulcers present for 5 years

PCR FINDINGS (NOT ATYPICAL) • • • • • • • • • • • •

Parvimonas micra Fusobacterium nucleatum Peptostreptococcus anaerobius Bacteroides fragilis Streptococcus angionosus Prevotella timonensis Anaerococcus vaginalis Dialister pneumosintes Coryebacterium urealyticum Prophyomonas bennonis Dialister propionicifaciens Prevotella ruminicola

32% 25% 6% 5% 4% 4% 2% 2% 2% 1% 1% 1%

Her customized gel for these bacteria

TOPICAL ANTIBIOTIC GEL

Collagenase was used to help debride the icthyotic skin.

TOPICAL ANTIBIOTIC GEL

Right lateral leg

WOUND HEALING WITH TOPICAL ANTIBIOTIC GEL AND COMPRESSION

Left medial leg

HOW ABOUT AN ISCHEMIC PATIENT?

58 yo WF with PVD and connective tissue disease on steroids, progressive heart failure and low PtcO2. Confirmed osteomyelitis, in palliative wound care with exposed 1st MT head.

YES, THAT IS EPITHELIUM

The distal toe lesions has closed, the joint space and bone are covered and there is epithelium over the surface

ISCHEMIA WITH EXPOSED ANKLE JOINT

74 y.o. WF diabetic with poor perfusion and exposed ankle joint. No good surgical options.

TOPICAL GEL USED BEFORE AND AFTER BIOSYNTHETIC SKIN

REMEMBER HIM?

Multiple chronically draining leg ulcers for >10 years with overpowering foul odor that cultured only Pseudomonas.

WITH REGULAR USE OF ANTIBIOTIC GEL

Odor is gone. Ulcerations are still an issue, but wounds are improved and is quality of life is much better.

WHAT IS THE TAKE HOME MESSAGE? • Chronic wounds are colonized, often with bacteria which cannot be identified using traditional culture techniques • Evidence is accumulating that polymicrobial biofilms may contribute to non-healing • PCR is a way to identify these bacteria • In addition to debridement, topical antibiotics directed at specific bacteria may be a useful therapeutic option. • The cost of PCR analysis is not significantly greater than wound culture and is covered by Medicare. • Topical antibiotics may be associated with fewer significant side effects than systemic therapy (issues relating to resistance not known)