Pseudomonas aeruginosa

1. Pseudomonas aeruginosa

2. About Pseudomonas aeruginosa • gram-negative aerobe bacteria • Commonly found in the environment • At any moist location • Common cause of nosocomial infections

3. P. aeruginosa is an opportunistic pathogen • Extremely broad host spectrum • Hardly any infections in the normal human host • Severe immunodeficiencies and medical devices predispose the patients to P. aeruginosa infections • Broad spectrum of clinical symptoms • Urinary tract infections • Pulmonary infections • Soft tissue infections • Sepsis • Bone and joint infections • Endocarditis

4. P. aeruginosa infections • P. aeruginosa infections are of particular concern for Cystic fibrosis patients • Burn patients • Hospitalised patients • Case mortality rate for patients infected with P. aeruginosa approaches 50%

5. Nosocomial infections • Fourth most common isolated nosocomial pathogen accounting for approx. • 10 % of all hospital acquired infections. • Patient-to-patient spread and direct patient contact with environmental reservoirs • disinfectants, • respiratory equipment, • food, • sinks, taps

6. Diagnosis of P. aeruginosa • Isolation and lab identification of the pathogen • P. aeruginosa grows well on most laboratory media • Identified on the basis of its: • Gram morphology, • inability to ferment lactose, • a positive oxidase reaction, • its characteristic odor, • its ability to grow at 42° C. • Fluorescence is helpful in early identification of P. aeruginosa colonies and may also help identify its presence in wounds.

7. Treatment of P. aeruginosa infections • P. aeruginosa is frequently resistant to many commonly used antibiotics. • To archive synergy a combination of e.g. gentamicin and carbenicillin is frequently used. • No vaccines so far

8. Pathomechanisms • Adhesion • Pili, flagella and fimbriae • Invasion • Extracellular enzymes and toxins (proteases, elastase, phospholipases, rhamnolipids, Exotoxin A) • Dissemination • Leukocidin inhibits neutrophils und leukocytes • LPS (Endotoxin) • Protection • Capsule (Alginate)

9. Interbacterial Communication

10. Quorum Sensing in P. aeruginosa McKnight et al, 2000

11. „....It is time to close the book on infectious disease….“ William Stewart, Surgeon in a message to the United State Congress in 1969

12. Bacterial Biofilms

13. Biofilms in the environment

14. Catheter associated biofilms

15. Chronic biofilm infections

16. Despite even intensified antibiotic therapy, no eradication of chronic P. aeruginosa infections of the cystic fibrosis lung

17. Cystic fibrosis • Most common life-threatening inherited genetic disorder in the Caucasian population • Mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene • one in every 25 carry the mutated recessive gene and more than 1 in 4000 live births suffer from CF. • Life expectancy: • Until the 1930s: the life expectancy of a baby with CF was only a few months, • in the 1980s, most deaths from CF occurred in children and teenagers. Today with improved treatments, nearly 40 percent of the CF population is aged 18 and older, • for a person with CF the median age of survival is nearly 37 years. • Cystic Fibrosis affects a number of organs in the body, cycles of infection and inflammation lead to a progressive deterioration of lung function.

18. Chronic infection of the Cystic Fibrosis lung

19. CF and Transplantation Centre • 360 patients regularyly attend the CF outpatient clinic at the Medical School Hannover, Germany • Informations of more than 500 CF patients in the local CF register • 2000 – 2005: 100 lung transplantation / year at the MHH

20. Indications for lung transplantation

21. Lung Transplantation in Germany

22. Post lung transplant mortality

23. Chronic infectious diseases • Slow progressing infections • Functional loss of the affected organ • Highly resistant to host defences and antimicrobial therapy • Recovery is rare • Role in the development of cancer, peptic ulcer and possibly atherosclerosis

24. Why is traditional antimicrobial therapy ineffective against biofilm bacteria?

25. Biofilms, City of microbes

26. Adaptation and survival is facilitated by diversity

27. Emergence of morphological distinct niche-specific phenotypes From: Rainey R, Travisano M. Nature, 1998; 39: 69-72

28. Phenotypic Diversity via the selection of niche-specialists

29. „Small colony variants“ (SCV)

30. SCVs of P. aeruginosa in CF • Slow growing subpopulations (3% of the P. aeruginosa positive sputum specimens) • SCVs exhibit an increased resistance towards a broad spectrum of antimicrobial agents • The recovery of SCV correlates with parameters revealing poor lung function and an inhalative antimicrobial therapy • Fast growing revertants can be isolated from the SCV population

31. Auto-aggregation in liquid cultures

32. CupA encoded fimbria expression in P. aeruginosa M. Rohde, GBF Braunschweig

33. Molecular Mechanisms controlling the conversion to a SCV biofilm phenotype

34. Biofilms, City of microbes

35. Cooperative traits and coordinated behavior of bacteria

36. Quorum Sensing in P. aeruginosa McKnight et al, 2000

37. Pseudomonas Quinolone Signal (PQS)

38. Hydroxy Alkyl Quinolones HHQ PQS

39. PQS directly interacts with bacterial DNA in a computed model

40. PQS enhances iron dependent DNA fragmentation DNA + PQS + Fe(II) DNA + HHQ + Fe(II) DNA + HHQ DNA + Fe(II) DNA + PQS Control DNA DNA

41. PQS enhances the fragmentation of DNA in vivo

42. PQS production is linked to the release of DNA PAO1 wild-type PQS non-producing mutant

43. Regulated DNA release in response to environmental stresses

45. DNA breakdown products enhance PQS production

46. Autoinductive PQS production cycle PQS Rhl Quorum Sensing PQS pqsA-E transcription Lectin Pyocyanin Cell death DNA Virulence factors Biofilm formation

47. Phenotypic Diversity is PQS dependent P. aeruginosa WT P. aeruginosa PQS negative Mutante

48. Establishment of a chronic state of infection Biofilm Formation Bacterial Diversity Chronic Infections Interbacterial Communication

49. Molecular mechanisms underlying genetic diversity • Horizontal gene transfer • Hypermutation • Adaptive mutations

50. Tiling Arrays • Microarray hybridisation-based method technique to find mutations in bacterial genomes was used to studymetronidazole resistance in Helicobacter pylori TJ Albert et al. Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori. Nature Methods 2, 951 - 953 (2005).