Biofilms , Antibiotic Resistance and Implications for Medical Treatment

1. Biofilms, Antibiotic Resistance and Implications for Medical Treatment James M. Coticchia M.D.,F.A.C.S. Director of Pediatric Otolaryngology Associate Professor Vice Chairman Otolaryngology Head and Neck Surgery Wayne State University School of Medicine Giancarlo ZulianiMD Chief Resident Otolaryngology Head and Neck Surgery Wayne State University School of Medicine

2. Defined as an assemblage of microbial cells enclosed in a self-produced polymeric matrix that is irreversibly associated with an inert or living surface 65% of nosocomial infections whose treatment costs an estimated 1 billion dollars (CDC) Biofilms

3. Biofilm Formation • Biofilms complex microbial lifestyle initiated by multiple genetic pathways • Planktonic cells attach to a surface • Cells then go on to form an attached monolayer

4. Biofilm Formation • Micro-colonies form • Prolific EPS matrix with micro-organisms embedded in matrix forms • Planktonic Shedding from the surface of biofilms

6. Molecular Aspects of Biofilms • Initial steps in the development of biofilms rely on altered gene expression • A large number of genes are up-regulated or down-regulated as biofilm phenotypes develop • Specific gene products are expressed to provide attachment • Motility mechanisms are used to form multicellular aggregates • Synthesis of extracellular matrix components: EPS

7. Molecular Aspects of Biofilms • Multicellular biofilms communicate via quorum sensing, which may play important mechanism in antimicrobial resistance and dispersion of planktonic organisms

8. Clinical Implications of Biofilms • Bacteria in biofilms persist despite antibiotic concentration of 100 - 1000 x MLC • Antimicrobial therapy can suppress planktonic organisms shed from biofilms and suppress clinical symptoms

9. Clinical Implications of Biofilms • Organisms embedded in biofilms resist antimicrobial therapy • When antibiotic therapy ends, organisms in biofilm may reinfect the host in a recurrent and relapsing nature

10. Clinical Implications of Biofilms • Andrel & Colleagues Antimicrobial Agents Chemotherapy 2000, 44:1818-24 • Demonstrated β-lactamasenegative Klebsiellapneumoniae, MIC 2mg/ml, survived as a biofilm in ampicillin concentration of 5000 mg/ml

11. Clinical Implications of Biofilms • Andrel & Colleagues Antimicrobial Agents Chemotherapy 2000, 44:1818-24 • Dispersed planktonic organisms readily killed • Suggests that standard resistance mechanisms such as efflux pumps may not play a central role in antibiotic resistance of biofilm organisms

12. Biofilms and Antibiotic Resistance • 10-1000 times more resistant than their planktonic counterparts • Classic teaching: resistance conferred via plasmids, transposons, and mutations • Multicellular strategies

13. Biofilms and Antibiotic Resistance • Physical proximity of cells within a biofilm would be expected to favor conjugation over the same process in planktonic counterparts • Ehlers and Bouwer demonstrated the conjugation rates between different species of Pseudomonas were significantly higher in biofilms than in their free-floating phenotype

14. Putative mechanisms : antimicrobial resistance of bacterial biofilms • Slow or incomplete penetration of antibiotics into the biofilm matrix • Ampicillin readily penetrates β-lactamaseneg biofilms • Ampicillin penetration retarded by wild strain β-lactamasepos. • Aminoglycoside antibiotics : positive charge retarded by negative ions biofilm matrix

15. Putative mechanisms : antimicrobial resistance of bacterial biofilms • Altered chemical microenvironment within the biofilm • pH gradients >1 between fluid and solid phase inhibit some antibiotics • Deeper layers of biofilm are anaerobic and decrease the efficacy of aminoglycoside antibiotics • Depletion of nutritional substrate or elevation of waste products induces sessile growth phase that renders antibiotics less effective

16. Putative mechanisms : antimicrobial resistance of bacterial biofilms • Osmotic environment within biofilms may alter membrane permeability, alteration of porins and antibiotic penetration • Subpopulation within biofilms form a unique phenotype similar to spore formation • These phenotypes may be <1% of population and develop even immature biofilms • This phenotype is extremely resistant to both antimicrobial therapy and disinfectants

17. Resistance Mechanisms • Stewart et al. demonstrated the spatial physiologic heterogeneity within biofilms of Pseudomonas aeruginosa using visualization techniques that indicated protein synthesis, respiratory activity, and relative RNA content

18. Resistance Mechanisms • Quorum sensing • lasI gene encodes protein for an acyl-homoserine lactone shown to be impotant for bacteria species (gm -) to monitor its own population density • LasI mutants are arrested after micorcolony formation but before full maturation

19. Resistance Mechanisms • Antimicrobial diffusion may be affected by aggregates of micro-organisms • Osmotic gradient may affect porins

20. Resistance Mechanisms • Quorum sensing influences small population of dormant micro-organisms • Planktonic organisms revert to original sensitivity

21. Host Immune Response & Biofilms • Bacteria within biofilms may elude normal host immune response • Shiau & Wu; Microbiol & Immunol, 42: 33-40 • Demonstrated that the slime product of S. epidermidis affected phagocytosis by macrophages

22. Host Immune Response & Biofilms • Ward & Colleagues; J. Med Microbiol, 36: 406-413 • Demonstrated lack of phagocytosis of bacterial biofilm implanted device in immunized animals • Meluleni & Colleagues; J. Immunol, 155: 209-238 • Demonstrated opsonic antibody in Cystic Fibrosis patients to be ineffective in eliminating organisms within biofilms

23. Host Immune Response & Biofilms • FISH imaging has also identified intracellular pod formation that may evade normal surveillance

24. Therapeutic Options : Biofilm Infections • Mechanical Disruption • Surgical debridement • Device removal • Ultrasonic treatment • Increases efficacy gentamycin • Chemical Disruption • Saponification • Enzyme degradation • Alginate lyase

25. Therapeutic Options : Biofilm Infections • Molecular Techniques • Disruption of bacterial adherence • Disruption of Quorum sensing pathway • Inhibition of biofilm matrix synthesis • Photodynamic therapy

26. Therapeutic Options : Biofilm Infections • Antimicrobials • Multidrug treatment regimens • Clarithromycin decreases alginate and hexose biofilm matrix • May have synergistic effect with other antibiotics like ofloxacin • Multidrug regimens routinely used for treatment of H. pylori infection : a biofilm disease

27. Therapeutic Options : Biofilm Infections • Nanotechnology • Succi & Colleagues; Chem & Biology, 14: 387-388 • Described development of viral nanoplatform (protein cage) delivery system : Staphylococcus aureus biofilm bacterium • Labeling • Drug platform

28. Thank-you / Grazie Mille • Alessandro Fiocchi MD, Marcello Giovannini MD and the inviting committee • James Coticchia MD, Aaron Duberstein MD, Michael Carlisle MD • Division of Pediatric Otolaryngology, Department of Otolaryngology-Head and Neck Surgery, Wayne State University

29. References • Anderl JN. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicob Agents Chemother 2000; 44: 1818-1824. • Cochran WL, McFeters GA, Stewart PS. Reduced susceptibility of thin Pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine. J Appl Microbiol 2000; 88: 22-30. • Ehlers LJ, Bouwer EJ. RP4 plasmid transfer among species of Pseudomonas in a biofilm reactor. Water Sci Technol 1999; 7:163-171. • Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK. The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J Immunol 2005; 175(11): 7512-8. • Mah T-F, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001; 9: 34-9. • Parsek MR, Greenberg EP. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc Natl Acad Sci USA 2000; 97: 8789-93. • Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358: 135-8. • Xu KD, McFeters GA, Stewart PS. Biofilm resistance to antimicorbial agents. Microbiology 2000; 146: 547-49.