1. Invitro Testing of Anti-Biofilm Burn and Wound Care Formulations Scott Tufts, Vice President Cardinal Health, Infection Prevention Cardinal Health, Infection Prevention

2. Acknowledgements: Dr. Paul S. Attar, President, Bridge PTS (Preclincal Testing Services), San Antonio, TX Mr. Kan Lam, Vice President, Bridge PTS (Preclincal Testing Services), San Antonio, TX Miss Yanira Hernandez, Scientist, Enturia, Inc.

3. The Way We Used to Think About Bacteria

4. The Real World of Chemotactic Communication

5. Bean Sprouts with E. coli O157:H7

6. E coli on Spinach

7. Biofilms on Fresh Vegetables According to statistics from the Centers for Disease Control and Prevention (CDC) in Atlanta, only 0.6 percent of disease outbreaks from food in the 1970s could be traced to fresh produce. However, since 1998, the percentage has jumped up to 14%. According to statistics from the Centers for Disease Control and Prevention (CDC) in Atlanta, only 0.6 percent of disease outbreaks from food in the 1970s could be traced to fresh produce. However, since 1998, the percentage has jumped up to 14%.

8. Now We Know About the EPS

9. The Biofilm Process

10. Biofilms and Human Disease It is estimated that over 80% of all microbial infections of the human body are associated with biofilm structure for example: It is estimated that over 80% of all microbial infections of the human body are associated with biofilm structure for example: Acne Acne Tuberculosis Tuberculosis Periodontal Disease Periodontal Disease Pneumonia Pneumonia Osteomyelitis Osteomyelitis Ear, Nose, and Throat Infections Ear, Nose, and Throat Infections All Catheter and Percutaneous Device Infections All Catheter and Percutaneous Device Infections All Wound and Burn Infections All Wound and Burn Infections

11. Problems Presented by Biofilms It is difficult to deliver antimicrobial agents through the exopolysaccharide matrix at effective concentrations. Concentrations required to penetrate the biofilm are thousands of times greater than necessary to treat planktonic bacteria. Often higher than the toxicity threshold of the antibiotic! It is difficult to deliver antimicrobial agents through the exopolysaccharide matrix at effective concentrations. Concentrations required to penetrate the biofilm are thousands of times greater than necessary to treat planktonic bacteria. Often higher than the toxicity threshold of the antibiotic! The cells at the base of the biofilm are operating at a very low metabolic rate, they are hibernating, waiting for a nutrient source to revive them. This low metabolic rate of the sessile organisms, drastically reduces the effectiveness of antibiotics and promotes antibiotic resistance. The cells at the base of the biofilm are operating at a very low metabolic rate, they are hibernating, waiting for a nutrient source to revive them. This low metabolic rate of the sessile organisms, drastically reduces the effectiveness of antibiotics and promotes antibiotic resistance.

12. Infected Burns and Wounds

13. Testing Objective / Challenge: To utilize laboratory methods to screen and evaluate trial formulations for their potential to eliminate biofilms found in burns and wounds. Methods Development: Laboratory methods must be validated to produce reliable and reproducible results. Methods must be cost effective at screening large numbers of samples (dose ranging) Testing Objectives

14. Basic MRD Experimental Setup

15. Biofilm Structure The Biofilms tested were “complex biofilms” containing approximately equal portions of: The Biofilms tested were “complex biofilms” containing approximately equal portions of: Pseudomonas aeruginosa (ATCC 27317) Pseudomonas aeruginosa (ATCC 27317) Staphylococcus aureus (ATCC 25923) Staphylococcus aureus (ATCC 25923) Escherichria coli (ATCC # 47022) Escherichria coli (ATCC # 47022)

16. Actual Biofilm Device Set Up Note- In actual operation media flask would be immersed in a heated water bath

17. Actual Biofilm Device Set Up Note- MRD sits in a heated Aluminum block

18. Secondary Device for Antimicrobial Gel Testing

19. Experimental Logic Flow Determine the effective concentrations for the disruption agent in the vehicle Determine the effective concentrations for the disruption agent in the vehicle Determine the effectiveness of the antimicrobial at various levels in the vehicle Determine the effectiveness of the antimicrobial at various levels in the vehicle Verify that in combination with the disruption agent, the antimicrobial agent exhibits effectiveness at lower concentrations Verify that in combination with the disruption agent, the antimicrobial agent exhibits effectiveness at lower concentrations

20. END