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New concept in Antibiotic therapy; Lisboa Sept 22nd, 2008 Why P. aeruginosa so virulent?

New concept in Antibiotic therapy; Lisboa Sept 22nd, 2008 Why P. aeruginosa so virulent?. Jean-François TIMSIT MD Ph D Grenoble, France. I have no conflict of interest to declare. Pseudomonas aeruginosa :an opportunistic pathogen. Gram-negative rod Ubiquitous : soil

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New concept in Antibiotic therapy; Lisboa Sept 22nd, 2008 Why P. aeruginosa so virulent?

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  1. New concept in Antibiotic therapy; Lisboa Sept 22nd, 2008 Why P. aeruginosa so virulent? Jean-François TIMSIT MD Ph D Grenoble, France I have no conflict of interest to declare

  2. Pseudomonas aeruginosa :an opportunistic pathogen Gram-negative rod Ubiquitous: soil aquatic habitats Low demanding In the hospital: Water Hospital plumbing, sinks Medical devices Antiseptic solutions Vegetables and fruits

  3. Colonization • Oropharynx • Upper digestive tract • Trachea • Urinary tract • 10% adults, 50-60% hospitalized people • Endogeneous infection >60% • Exogeneous  30%

  4. Infections • 10% of hospital acquired infection (National prevalence study, 2006) • Immunocompromized host: • neutropenia, HIV+

  5. National ICU database:REA-RAISIN • 2004-2006 (3 years), 56,535 patients • 7808 with at least one NI (UTI, VAP, Bacteremia) • 1875 with P. aeruginosa NI (VAP:58%, UTI:17%, BC: 15%, more than one: 15%) 24% of all infected patients 3.3% of patients • Late onset NI: 18 days (2-237d) (vs 10 days for NI due to other organisms) • TIC S: 48%, TIC R/CAZ S: 31%, CAZ R: 21% stable • High SAPS II, DS: 40 days, ICU death 35% From AG Venier - National Meeting REA-RAISIN 2008

  6. Genetic flexibility Large genome (E.coli : 4,6, M.tuberculosis : 4,4, S.aureus : 2,5) 5 500 genes (saccharomyces : 6 200) Function?? 8.4% regulatory genes Adaptability to environment Escape to innate immunity Take advantage to immunity to a concerted attack Hypermutators Transcriptional regulation

  7. Host response Inate immunity Surfactant proteins Alveolar Macrophages Defensins Cytokines Chemokines Activation Phagocytosis Alveolar space Lymphocytes Specific immunity (adaptative) Mainly chronic infections Vascular space Neutrophiles

  8. 2 strategies in ICUs Rapid and conserted attack Acute infection Attachment Invisibility resistance Prolonged colonization Devices’ attachment

  9. Surface factors Regulatory system Secreted factors Virulence factors Sadikot et al - AJRCCM Vol 171. pp 1209–1223, 2005

  10. Extra-cellular secretions Lazdunski Ann Fr Anesth Réanim 2003,22,523

  11. TTSS: a needle P aeruginosa Eukariotic cell Kubori et al. Science 1998,280,602

  12. TTSS Invasivness R to phagocytosis bacteremia • Exo S and T: • ADP rybosyl tranferase and GTPase activity domains • Cytosqueletal alterations ( DNA synth.) • Cytotoxicity • Inh. Of bacterial internalisation by both phagocytic and non phagocytic mamalian cells • Exo Y: • Adenylate cyclase ( intra cellular C-AMP) • Exo U: • necrotizing toxin with a P lipase activity • Rapid lysis of mamalian cells •  caspase 1 driven proinflammatory cytokine production ( innate response) Cytotoxicity (epith cells) Tissue damage Septic shock

  13. Mortality in excess with TTSS (Roy Burman et al, J Infect Dis. 2001 )

  14. Anti-PcrV Antibodies Protect Mice Challenged with Lethal Pa Doses Shime et al. J. Immunol 2001;167:5880-5886 Improvement of lung inflammation and damage, hemodynamic parameters of septic shock and mortality

  15. Human Fab’ with V-region sequence close to human germ-line sequence 91% sequence identity to germ-line Low likelihood of immunogenicity High affinity (0.67nM) and potent biological activity Lacks Fc-mediated effector functions Unlikely to increase inflammation in the lung PEGylation Prolongs half life to approximately 2 weeks Further reduces potential immunogenicity KB001 (Humaneered™ Anti-PcrV)

  16. Principal investigator:Prof J Chastre

  17. T III secretion system and persistence of PA after VAP El Sohl et al – AJRCCM 2008; 178:513 25 TTSS + 13 PA at Day 8 Death 68% 34 VAP MonoABx 9 TTSS - 9 eradication Death 33%

  18. T III Secretion System and persistence of PA after VAP El Sohl et al – AJRCCM 2008; 178:513 1- 71% PA-VAP TTSS+ 2- VAP-PA-TTSS+: neutrophilic Apoptosis 3- Neutro cytotox correlated with ExoU(ExoS)/Pcrv phenotypes

  19. Future prospect for anti-PCRV? • Anti PcrV in P. aeruginosa VAP patients already treated with persistent PA at Day 5-8 of antimicrobial treatment • End-point • Relapse, recurrence and mortality • Neutrophilic cytotoxicity and elastase

  20. Quorum sensing Regulation of >100 genes in a density-dependent manner  Homoserine lactones (HSL) • Important gene for the life cycle of the bacteria: DNA replication, transcription, cell division, aminoacid synthesis Persistence of the bacteria in the lung, (increase bacterial resistance, quiecent phase) • Life in community Promotion of biofilm formations. • Virulence factors Pyocyanin, siderophores, rhamnolipids…

  21. Metabolic, physiologic regulation Extra cellular product Quorum sensing system? I-gene R-gene Target-genes Binding and genes activation Transcriptional activator (R-protein) Auto-inducer synthetase PA membrane AI/R complex Freely diffusible AI (3-oxo-C12-HSL C4-HSL) AI signals to (from) other bacterias Adapted from Tateda K 2007

  22. 3 QS system in PA:las, rhl, PQS Regulations of 6-10% of PA genes

  23. Quorum sensing is more frequent in virulent strains (n=270) (n=50) Van Delden C – Personnal communication – RICAI 2007

  24. QS activity and virulence factors in clinically pathogenic isolates of P aeruginosa – Le Berre et al – CMI 2008; 14:337 Correlation las R=0.7, p=2 10-9 Correlation rhl R=0.7, p=2.2 10-9 Correlation rhl R=0.3, p=0.02

  25. Synthetic furanones inhibit QS and enhance bacterial clearance in PA lung infection in miceWu et al – JAC 2004;53:1054 • Semi-synthetic derivates from QS inhibitors from macro alga Delisea Pulchra • In a mouse model: • Supression of bacterial QS in the lung • Accelerated lung clearance • Reduced the severity of lung pathology • In a lethal PA pneumonia mouse model, it prolonged survival time…

  26. Inhibition of QS • Macrolides (azythromycin) • QS,  inflammation,  extracellular virulence factors •  the survival of mouse challenged with PA (Nicolau 1999) •  pulmonary function of cystic fibrosis (Jaffe 1998) • 70% the risk of PA infection in HIV patients (Sorvillo 2001) • Tateda et al J infect chemother 2007

  27. Impact of Macrolides on host defenses (+) TIGHT JUNCTION (-) QS (-) MOTILITY (+) PHOGOCYTOSIS (-) NF K B, AP-1 (-) TNF  IL-8 Giamerellos-Bourboulis et al - J. Antimicrob. Agents (2008), doi:10.1016/j.

  28. Effect of clarithromycin in patients with sepsis and VAP Giamarellos-Bourboulis CID 2008:1157 (*) P=0.006

  29. ANB 006/2001 Phase IIa : Pseudomonas aeruginosa prevention • Multinational multicentric study, P-o-C study • Prevention of VAP in PA colonized patient • Azithromycin 300 mg daily for 20 days • Study stopped after 92 patients/200 85 per protocol analysis • Pa VAP Acquisition and QS markers • Subgroup analysis of QS producing virulence factors strains… • Under publication, data not shown

  30. Candida-Pseudomonas copathogenicity? • Epidemiologic association between both micro-organisms (Vincent 1995) • PA infection is a risk factor of Candidaemia in burned mice (Neely 1986) • PA forms a dense biofilm on C albicans filaments and kills the fungus (Hogan, Science 2002) • Several virulence factors of PA are involved in killing C albicans filaments (Hogan 2002) • PA HSL is able to inhibit Candida filamentation (Hogan 2004) • Candida Tracheal colonization favors PA pneumonia in Rats (Roux 2006, (abstract))

  31. Candida Colonization of the RespiratoryTract and Subsequent PseudomonasVentilator-Associated Pneumonia Azoulay E on behalf of the OUTCOMEREA study group Chest 2006

  32. Impact of an antifungal treatment of tracheal candida colonization on PA VAP risk • Preliminary retrospective data • Case (19)/ Control (38) study • Decrease in the risk of PA VAP or PA colonization: OR=0.68 [0.49-0.9], p=0.046 Nseir et al – ICM 2007 • International interventional study planned

  33. Aknowledgments Benoit Guery Benoit Misset Pierre Moine Olivier Epaulard Christian Van Delden Jean Carlet Jean Chastre Kalobios pharma

  34. Scanning electron micrograph of a biofilm on a metal surface from an industrial water system

  35. Clinical importance • Virulence factors • Therapeutic targets • Copathogenicity (candida-PA)

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