pseudomonas aeruginosa (pao1) after phagocytosis by …intracellular pseudomonas aeruginosa. april...
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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
April 13, 2010Intracellular Pseudomonas aeruginosa 2April 13, 2010Intracellular Pseudomonas aeruginosa 2
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)
April 13, 2010Intracellular Pseudomonas aeruginosa 3April 13, 2010Intracellular Pseudomonas aeruginosa 3
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
April 13, 2010Intracellular Pseudomonas aeruginosa 4April 13, 2010Intracellular Pseudomonas aeruginosa 4
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
April 13, 2010Intracellular Pseudomonas aeruginosa 6April 13, 2010Intracellular Pseudomonas aeruginosa 6
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
April 13, 2010Intracellular Pseudomonas aeruginosa 13April 13, 2010Intracellular Pseudomonas aeruginosa 13
Acknowledgments
Thank you for your kind attention