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

Hypermutators Transcriptional regulation Adaptability to environment Escape to innate immunity Take advantage to  immunity to a concerted attack

Host response

Alveolar Macrophages Surfactant proteins Defensins

Inate immunity

Cytokines Chemokines Alveolar space

Activation Phagocytosis

Vascular space Neutrophiles Lymphocytes

Specific immunity (adaptative)

Mainly chronic infections

2 strategies in ICUs

Rapid and conserted attack

Acute infection

Attachment Invisibility resistance

Prolonged colonization Devices’ attachment

Surface factors

Virulence factors

Secreted factors Regulatory system

Sadikot et al - AJRCCM Vol 171. pp 1209 –1223, 2005

Extra-cellular secretions

Lazdunski Ann Fr Anesth Réanim 2003,22,523

TTSS: a needle

Type I

P. aeruginosa

AprA Type II ExoY Type III ExoT ExoU ExoS Membrane interne PcrV Membrane externe ExoS Membrane cytoplasmique Eukariotic cell Cellule eucaryote Kubori et al. Science 1998,280,602 ExoY ExoU ExoT ExoS

TTSS

• •

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) Invasivness R to phagocytosis bacteremia •

Exo U:

– necrotizing toxin with a P lipase activity – Rapid lysis of mamalian cells –  caspase 1 driven proinflammatory cytokine production (  response) Cytotoxicity (epith cells) Tissue damage Septic shock innate

Mortality in excess with TTSS

Acute infection SSTT [+] Mortality TTSS [+] 21% 89% Chronic Infection 41% TTSS [-] 3% PcrV alone PcrV + toxin (s) RR death 7,4 8,7 (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

KB001 (Humaneered™ Anti-PcrV)

• 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

French MVP Study Schema 12 pts

Surveillance in MVP pts at high

Pa

risk

Pa

> 10 3 ETA > 10 2 BAL

R 12 pts 12 pts KB001 10mg/kg KB001 3mg/kg Placebo

Add antibiotics at clinical VAP

Endpoints

•

Day 1-3 Change vs placebo

• •

Pa

burden Bact diversity

•

Inflammation

•

Lung function

•

Day 28 Frequency

•

Pa VAP/sepsis

•

Pa relapse

•

Time to VAP

•

Clinical and MV endpts

•

Pharmacokinetics

•

KB001 airway penetration

•

Immunogenicity

Principal investigator:Prof J Chastre

23

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) 1.

2.

3.

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…

Quorum sensing system?

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

3 QS system in PA:las, rhl, PQS

las  PQS synth Rhl system Biofilm production Elastase Lipase Exo A Regulations of 6-10% of PA genes rhl  PQS synth Rhamnolipids Elastase Lipase Pyocyanin Exo S PQS (Pseudomonas quinolone signal) Rhl system Rhamnolipids Biofilm formation Elastase Pyocyanin

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=0.3, p=0.02

Synthetic furanones inhibit QS and enhance bacterial clearance in PA lung infection in mice

Wu 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

Tateda et al J infect chemother 2007

– – –   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)

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

Age PF ratio EOP/LOP Septic shock Placebo n=100 58 218 44/56 43 PA A. baumannii Crude mortality D28 Day 7 Sepsis related Time until VAP resolution* 12% 43 28 8 24 11.5

Clarithro n=100 58 224 41/59 42 17% 36 31 6 21 7 (*) 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  protocol analysis – Pa VAP Acquisition and QS markers 85 per • Subgroup analysis of QS producing virulence factors strains…

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 Respiratory Tract and Subsequent Pseudomonas Ventilator-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)