THE MOLECULAR GENETIC ANALYSIS OF

Transcription

1 THE MOLECULAR GENETIC ANALYSIS OF DIABETIC FOOT WOUNDS ERIN E KLEIN, DPM, MS SARAH E HALLER, DPM; BRETT J WAVERLY, DPM; LOWELL WEIL, DPM; ADAM E FLEISCHER, DPM, MPH WEIL FOOT & ANKLE INSTITUTE DES PLAINES, ILLINOIS, USA

2 Consultant for BESPA DISCLOSURES Primary author on three ACFAS granted studies Nothing related to the current presentation

3 TENANTS OF GOOD WOUND CARE Evaluate and manage blood flow to the extremity Evaluate and manage deep infection (i.e. osteomyelitis) Offload Medical management of systemic conditions CONTROL THE BIOBURDEN OF THE WOUND

4 BIOFILM AN INTRODUCTION What is biofilm? A thin, slimy film of bacteria that adheres to a surface. Can include bacteria, yeast, algae and mold. Tooth plaque Why are we so interested in this? Community of bacteria

5 TRADITIONAL CULTURES VS MOLECULAR ANALYSIS Aim to identify organisms in a wound Organisms must be ALIVE when they arrive at the lab Amenable to growth on a plate/agar May favor the hardiest micro-organisms 16s amplicon sequencing (next gen sequencing) Organisms can be alive or dead when they arrive at the lab Can identify ALL organisms in a biofilm or in a wound

6 PURPOSE The purpose of this study is to: Analyze the content of biofilm utilizing 16s amplicon technology Identify any common factors between wounds

7 METHODS Consecutive patients were included History of diabetes Foot ulceration that has been present for 4 weeks (or longer) that has NOT responded standard wound care treatment Excluded Any other type of wound (i.e. venous wounds, pressure ulcers, arterial wounds in patients without diabetes, traumatic wounds, post operative wounds)

8 SPECIMEN COLLECTION Wound debridement Sterile instrumentation Biofilm sent to the lab Level 1 Analysis 10 most common organisms Level 2 Analysis Biofilm compared to an extensive national database 2% threshold to report Results compiled in data base Included chart review of potentially connected problems

9 SPECIMEN COLLECTION Wound debridement Sterile instrumentation Biofilm sent to the lab Level 1 Analysis 10 most common organisms Level 2 Analysis Biofilm compared to an extensive national database 2% threshold to report Results compiled in data base Included chart review of potentially connected problems

10 SPECIMEN COLLECTION Wound debridement Sterile instrumentation Biofilm sent to the lab Level 1 Analysis 10 most common organisms Level 2 Analysis Biofilm compared to an extensive national database 2% threshold to report Results compiled in data base Included chart review of potentially connected problems

11 SPECIMEN COLLECTION Wound debridement Sterile instrumentation Biofilm sent to the lab Level 1 Analysis 10 most common organisms Level 2 Analysis Biofilm compared to an extensive national database 2% threshold to report Results compiled in data base Included chart review of potentially connected problems

12 RESULTS 74 wounds in 53 patients 30 males; 23 females 38 left feet; 33 right feet 40 patients with one wound 34 patients with multiple wounds Average age: (range: 33 89) HbA1C 7.5% + 1.9% (range %)

13 RESULTS Previous amputations 3 BKA; 2 TMA; 2 partial 1 st ray Charcot 4 cases all in quiescent stage Expiration 3 patients (4 wounds) were deceased at the time of analysis

14 RESULTS SMOKING STATUS 11% 13% Current Smoker 76% Former Smoker Never Smoked

15 RESULTS BLOOD FLOW AND INFECTION Dichotomous vascular disease All patient had some evidence of vascular disease ABI were either >1.1 or <0.4 All patients were referred to vascular surgery/interventional radiology as part of their treatment Underlying osteomyelitis 41 positive 33 negative No differences noted in bacterial species, quantity of microorganisms, type of organisms or resistance patterns

16 LEVEL 1 ANALYSIS Bacterial load Bacterial Load Antibiotic Resistance 39% 18% Gene markers for antibiotic resistance 43% High (>10^7) Medium (10^5-10^7) Low (<10^5) 47% 3% 1% 33% 8% 5% 3% Methicillin Vancomycin Aminoglycosides Beta-lacta Tetracycline Macrolides None

17 LEVEL 1 ANALYSIS S aureus S agalactiae E faecalis E faecum P aeruginosa K pneumoniae C albicans

18 LEVEL 1 ANALYSIS % 24.7% 11.1% 6.0% 35.8% 6.4% S aureus S agalactiae E faecalis E faecum P aeruginosa K pneumoniae C albicans

19 LEVEL 1 ANALYSIS % 8.0% 1.0% 6.0% 12.0% 8.5% S aureus S agalactiae E faecalis E faecum P aeruginosa K pneumoniae C albicans

20 LEVEL 2 ANALYSIS 56 genera 124 species Average number of species per wound: (range 1 15) Median: 3

21 LEVEL 2 ANALYSIS Oxygen Status of Isolated Organisms 67% 2% 31% Aerobes Anaerobic Unknown

22 LEVEL 2 ANALYSIS 5% 1% 3% 1% 1% 31% 58% Bacillus Cocci Coccibacillus Curved/bent Not reported Variable clusters of 8

23 LEVEL 2 ANALYSIS Top performers Isolated Oxygen Status Gram Stain Shape Staphylococcus 43 Facultative anaerobes Positive Cocci Anaercoccus 37 Anaerobic Positive Cocci Corynebacterium 36 Aerobic Positive Bacillus Peptoniphilus 22 Anaerobic Positive Cocci

24 LEVEL 2 ANALYSIS Isolated Oxygen Status Gram Stain Shape Staphylococcus 43 Facultative anaerobes Positive Cocci Anaercoccus 37 Anaerobic Positive Cocci Corynebacterium 36 Aerobic Positive Bacillus Peptoniphilus 22 Anaerobic Positive Cocci Isolated Oxygen Status Gram Stain Shape Finegoldia 16 Anaerobic Positive Cocci Streptococcus 16 Facultative anaerobic Positive Cocci Porphyromonas 15 Obligate anaerobe Netative Bacillus Escheria 14 Facultative anaerobic Negative Bacillus Psuedomonas 10 Facultative anaerobic Negative Bacillus

25 WHY SO MANY ANAEROBES? LET S TAKE A WALK BACK TO THAT BIOFILM THING

26 BIOCIDES VS BIOFILM After 60 minutes of exposure to bleach Dying cells (green) Alive cells (red)

27 COAGGREGATION AND SYMBIOTIC RELATIONSHIPS Is it possible that bacteria live in communities where certain bacteria have symbiotic relationships with others? Aerobes live at surface Anaerobes live a bit deeper Oxygen penetrates biofilm 4 microns.

28 WHAT DOES ALL OF THIS MEAN? In many ways, the clinical significance of this data is currently unclear. Identified that there is a higher than previously thought level of anaerobic bacteria that is present in wound beds. Variability of bacteria in biofilms and wound beds. There may be a role for topical antibiotics that are aimed at all of the microbial species in the wound bed, rather than just one or two.

29 FUTURE STUDIES IN THIS AREA Personalized/customized medicine based on genomics may be the way of the future. 16S amplicon vs PCR vs plated culture study Prospective study on customized topical medication and wound outcomes

30 THANK YOU! ERIN E KLEIN, DPM, MS EEK@WEIL4FEET.COM LOWELL WEIL, JR, DPM LWJ@WEIL4FEET.COM ADAM E FLEISCHER, DPM AEF@WEIL4FEET.COM