Transcript Pseudomonas aeruginosa tolerance totobramycin

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.
• H2O2 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.
2.
3.
Tobramycin: 10 µg ml-1 and 20µg ml-1, 48h.
H2O2: 100mM, 15min.
PMNs: the oxidative burst of PMNs (Furanone C30)
• 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
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
•Increased sensitivity towards H2O2 is QS
dependent
untreated
Wild-type
mutant
H2O2 treated
Increased sensitivity of QS mutants to PMNs
activity
Wild-type
(a)
Mutant (d)
(g)
(b)
(c)
(e)
(f)
(h)
Syto-62
The oxidative burst of PMNs activation is controlled by QS
signal molecules.
(b)
10µM Furanone C-30
Wild-type
mutant
• 123dihydrorhodami
ne (123-DHR) is
oxidized (H2O2)
to 123rhodamine
(d) 3-Oxo-C12-HSL (1 mM)
and-C4-HSL (2 mM) were
Added before inculating with
the PMNs.
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~