Pseudomonas aeruginosa tolePseudomonas aeruginosa tolerance to tobramycin, hydrogen rance to tobramycin, hydrogen

peroxide andperoxide andpolymorphonuclear leukocytepolymorphonuclear leukocyte

s is s is quorum-sensing dependentquorum-sensing dependent

銘傳大學生科四甲銘傳大學生科四甲邱熒珊邱熒珊

2005,12,062005,12,06

IntroductionIntroduction• The opportunistic human pathogen Pse

udomonas 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.

BiofilmBiofilm

• 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.

• H2O2 has a bactericidal effect.

Materials and MethodsMaterials and Methods• Bacteria strains: The wild-type P. aeruginosa PAO1. The ΔlasR rhlR mutant: knock-out systems. A st

able green fluorescent protein (GFP) constitutively expressed on plasmid pMRP9 was used to tag the bacteria.

treatment:1. Tobramycin: 10 µg ml-1 and 20µg ml-1, 48h.2. H2O2: 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 DiscussionResults and Discussion

• both expressing GFP as a tag• Add propidium iodide• Increased sensitivity towards tobramycin is QS dependent

Untreated 10µg ml-1 tobrmycin 20µg ml-1 tobrmycin

Wild-type

Mutant

•Increased sensitivity towards HIncreased sensitivity towards H22OO22 is QS dependent is QS dependent

Wild-type

mutant

untreated H2O2 treated

Increased sensitivity of QS mutants to PMNs activityIncreased sensitivity of QS mutants to PMNs activity

Wild-type

Mutant

(a)

(d)

(b) (c)

(e)

( f )

(g) (h)

Syto-62

• 123-dihydrorhodamine (123-DHR) is oxidized (H2O2) 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 A ∆∆lasR rhlRlasR rhlR mutant is cleared rapidly in viv mutant is cleared rapidly in vivoo

• The 0.04ml either wild-type P. aeruginosa or the ∆lasR rhlR mutant wer

e 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.

ConclusionConclusion• Experiments performed in vivo support

our in vitro data.• the immune system is activated to a hig

her 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 endThe end~Thank You~~Thank You~