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

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 biofilms in the host, which makes the bacteria tolerant to antibiotic treatment and the action of the host defence system.

Biofilm

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.

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. H 2 O 2 has a bactericidal effect.

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: 1.Tobramycin: 10 µ g ml -1 and 20 µ g ml -1, 48h. 2.H 2 O 2 : 100mM, 15min. 3.PMNs: the oxidative burst of PMNs (Furanone C-30)

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

Results and Discussion both expressing GFP as a tag Add propidium iodide Increased sensitivity towards tobramycin is QS dependent Untreated10µg ml -1 tobrmycin20µg ml -1 tobrmycin Wild-type Mutant

Increased sensitivity towards H 2 O 2 is QS dependentIncreased sensitivity towards H 2 O 2 is QS dependent Wild-type mutant untreated H 2 O 2 treated

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

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

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.

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.

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.

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.

The end ~Thank You~