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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? 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 aquatic habitats Low demanding In the hospital: Water Hospital plumbing, sinks Medical devices Antiseptic solutions Vegetables and fruits
Colonization • Oropharynx • Upper digestive tract • Trachea • Urinary tract • 10% adults, 50-60% hospitalized people • Endogeneous infection >60% • Exogeneous 30%
Infections • 10% of hospital acquired infection (National prevalence study, 2006) • Immunocompromized host: • neutropenia, HIV+
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
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
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
2 strategies in ICUs Rapid and conserted attack Acute infection Attachment Invisibility resistance Prolonged colonization Devices’ attachment
Surface factors Regulatory system Secreted factors Virulence factors Sadikot et al - AJRCCM Vol 171. pp 1209–1223, 2005
Extra-cellular secretions Lazdunski Ann Fr Anesth Réanim 2003,22,523
TTSS: a needle P aeruginosa Eukariotic cell Kubori et al. Science 1998,280,602
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
Mortality in excess with TTSS (Roy Burman et al, J Infect Dis. 2001 )
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
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)
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%
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
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
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…
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
3 QS system in PA:las, rhl, PQS Regulations of 6-10% of PA genes
Quorum sensing is more frequent in virulent strains (n=270) (n=50) Van Delden C – Personnal communication – RICAI 2007
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
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…
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
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.
Effect of clarithromycin in patients with sepsis and VAP Giamarellos-Bourboulis CID 2008:1157 (*) P=0.006
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
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))
Candida Colonization of the RespiratoryTract and Subsequent PseudomonasVentilator-Associated Pneumonia Azoulay E on behalf of the OUTCOMEREA study group Chest 2006
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
Aknowledgments Benoit Guery Benoit Misset Pierre Moine Olivier Epaulard Christian Van Delden Jean Carlet Jean Chastre Kalobios pharma
Scanning electron micrograph of a biofilm on a metal surface from an industrial water system
Clinical importance • Virulence factors • Therapeutic targets • Copathogenicity (candida-PA)