Pseudomonas aeruginosa to Pseudomonas aeruginosa to lerance to tobramycin, hy lerance to tobramycin, hy drogen peroxide and drogen peroxide and polymorphonuclear leukocy polymorphonuclear leukocy tes is tes is quorum-sensing dependent quorum-sensing dependent 銘銘銘銘銘銘銘銘 銘銘銘銘銘銘銘銘 銘銘銘 銘銘銘 2005,12,06 2005,12,06
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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.
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Pseudomonas aeruginosa tolePseudomonas aeruginosa tolerance to tobramycin, hydrogen rance to tobramycin, hydrogen
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.