Pseudomonas aeruginosa (PAO1) after phagocytosis by …Intracellular Pseudomonas aeruginosa. April...
Transcript of Pseudomonas aeruginosa (PAO1) after phagocytosis by …Intracellular Pseudomonas aeruginosa. April...
April 13, 2010Intracellular Pseudomonas aeruginosa 1April 13, 2010Intracellular Pseudomonas aeruginosa 1
Pharmacodynamic evaluation of the intracellular activity of tobramycin,
doripenem, levofloxacin, and colistin towards Pseudomonas aeruginosa (PAO1) after
phagocytosis by human THP-1 macrophages.
J. BUYCK, P.M. TULKENS, F. VAN BAMBEKE
Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute
Université catholique de Louvain, Brussels, Belgium
www.facm.ucl.ac.be
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Introduction (1/2)• Pseudomonas aeruginosa,
an important human pathogen
– Gram-negative bacillus
– Opportunistic human pathogen • respiratory system infections • chronic infection in CF patients
– (multi)resistance to antibiotics
– Intracellular survival• ~ 50% of strains demonstrate measurable internalization(Engel, 2003)
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Introduction (1/2)• Pseudomonas aeruginosa,
an important human pathogen
– Gram-negative bacillus
– Opportunistic human pathogen • respiratory system infections • chronic infection in CF patients
– (multi)resistance to antibiotics
– Intracellular survival• ~ 50% of strains demonstrate measurable internalization(Engel, 2003)
Angus 2008
Hirikata 2002
Darling 2003
Plotkowski 1998
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Introduction (2/2)• Current view of the internalization pathway
PAInternalization Raft:
CFTRCaveolin-1
Cholesterol
PACytoskeletal and
membrane changes
Adapted from:Kannan et al., 2008Bajmoczi et al., 2009
• Potential role for intracellular reservoir ?May constitute a source for chronic infection
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Aims of the study
• To develop a model of intracellular infection by P. aeruginosa over a 24 h period to allow intracellular growth
• To study in this model the activity of antibiotics representative of the main classes currently used in the clinics
• To compare pertinent pharmacological descriptors of antibiotic activity (maximal efficacy, relative potency) against both extracellular and intracellular forms of P. aeruginosa
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Experimental procedure• Model:
– THP-1 cells: Human acute monocytic leukemia cell line
– PAO1 strain
MOI 10
Phagocytosis (2 h)
Incubation (with ATB)(T0, T5 and T24 h)
600,000 cells/mLOpsonization (45’, 37°c)9 mL RPMI +1 mL human serum
Extracellular WashGEN 100 µg/ml (1 h)
5 to 7x105 CFU/mg prot.Time 0
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• Intracellular localisation of PA:– Confocal imaging
Setting-up the model
Blue: nucleus staining by TO-PRO 3,Red: actin staining by Rhodamin-phalloïdin,Green: Pseudomonas specific staining by a FITC-labeled antibody
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• Definition of pharmacodynamic parameters:– Gentamicin as an example
Results
MIC Emax Cstatic (xMIC)
Extracellular 0.5 -3.95 ± 0.41 0.96
Intracellular - -1.62 ± 0.19 22.95
Int. Emax
Ext. Emax
Ext. Cstat
Int. Cstat
99
Tobramycin
Colistin
Cstat
Emax
Emax Emax
Cstat Cstat =
Doripenem
Levofloxacin
Cstat = Emax
Bactericidal
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Summary• Intracellular Pseudomonas aeruginosa
Emax for all antibiotics
Cstat for GEN, TOB and CST; ~ for DOR, LVX
CstatEmax
GEN
TOB
LVX
DOR
CST
GEN
TOB
LVX
DOR
CST
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Conclusion
• P. aeruginosa is able to invade and survive within human THP-1 cells
• All antibiotics tested show reduced efficacy but to different extents
• This lower activity may contribute to persistence or recurrence of infection.
• Fluoroquinolones seem of interest for further investigation
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• Financial support:
• FACM Team:
Acknowledgments
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Acknowledgments
Thank you for your kind attention