Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent

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Pseudomonas aeruginosa tolerance to tobramycin,
hydrogen peroxide andpolymorphonuclear
leukocytes is quorum-sensing dependent

• ????????
• ???
• 2005,12,06

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Introduction

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

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Biofilm
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• quorum-sensing a cellcell 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.

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• 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.

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Materials and Methods

• Bacteria strains
• The wild-type P. aeruginosa PAO1.
• The ?lasR 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)

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• Experimental animals BALB/c mice female, at
  1011 weeks.
• ? The 0.04ml ?? were instilled in the left lung
  of each mouse.

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Results and Discussion
Untreated
10µg ml-1 tobrmycin
20µg ml-1 tobrmycin
Wild-type
Mutant

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

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Increased sensitivity towards H2O2 is QS
dependent
untreated
H2O2 treated
Wild-type
mutant
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Increased sensitivity of QS mutants to PMNs
activity
Syto-62
(b)
Wild-type
(a)
(c)
(d)
(e)
( f )
Mutant?
(g)
(h)
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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
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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-type73 died
• ?lasR rhlR mutant 46 died.

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• 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.

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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.

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• 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.

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The end

• Thank You