銘傳大學生科四甲 邱熒珊 2005,12,06

1. Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide andpolymorphonuclear leukocytes is quorum-sensing dependent 銘傳大學生科四甲 邱熒珊 2005,12,06

2. Introduction • The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) (纖維性囊腫)patients. • The bacteria form biofilmsin the host, which makes the bacteria tolerant to antibiotic treatment and the action of the host defence system.

3. Biofilm

4. quorum-sensing: a cell–cell communication system. In P. aeruginosa the QS communication apparatus is composed of the Las and the Rhl systems. • 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL) for the las system. • Butyryl homoserine lactone (C4-HSL) for the rhl system.

6. PMNs react to intruding foreign organisms either by phagocytosis or secretion; in both cases the PMNs launch a cocktail of antimicrobial agents, in particular free oxygen radicals. • H2O2 has a bactericidal effect.

8. Materials and Methods • Bacteria strains: The wild-type P. aeruginosa PAO1. The ΔlasR rhlR mutant: knock-out systems. A stable green fluorescent protein (GFP) constitutively expressed on plasmid pMRP9 was used to tag the bacteria. treatment: • Tobramycin: 10 µg ml-1 and 20µg ml-1, 48h. • H2O2: 100mM, 15min. • PMNs: the oxidative burst of PMNs (Furanone C-30)

9. Experimental animals: BALB/c mice female, at 10~11 weeks.  The 0.04ml 菌液 were instilled in the left lung of each mouse.

10. Results and Discussion Untreated 10µg ml-1 tobrmycin 20µg ml-1 tobrmycin Wild-type • both expressing GFP as a tag • Add propidium iodide • Increased sensitivity towards tobramycin is QS dependent Mutant

12. Increased sensitivity towards H2O2 is QS dependent untreated H2O2 treated Wild-type mutant

13. Syto-62 Increased sensitivity of QS mutants to PMNs activity (b) Wild-type (a) (c) (d) (e) ( f ) Mutant (g) (h)

14. The oxidative burst of PMNs activation is controlled by QS signal molecules. (b) 10µM Furanone C-30 • 123-dihydrorhodamine (123-DHR) is oxidized (H2O2) to 123-rhodamine Wild-type (d) 3-Oxo-C12-HSL (1 mM) and-C4-HSL (2 mM) were Added before inculating with the PMNs. mutant

15. A ∆lasR rhlR mutant is cleared rapidly in vivo • The 0.04ml either wild-type P. aeruginosa or the ∆lasR rhlR mutant were instilled in two groups(72,72) of mice. 1. For 5 days,deaths within 24 h were rejected. 2. wild-type:73% died; ∆lasR rhlR mutant :46% died.

16. On day 1, the ∆lasR rhlR group being cleared fastest. • On day 5, ∆lasR rhlR were sterile, whereas for the wild-type, five out of seven contained P. aeruginosa.

17. Conclusion • Experiments performed in vivo support our in vitro data. • the immune system is activated to a higher level when are infected with the ∆lasR rhlR mutant compared to the wild-type.

18. A combination of the action of PMNs and a QS inhibitors along with conventional antibiotics would then eliminate the biofilm-forming bacteria before a chronic infection is established.

19. The end ~Thank You~