The facts: opportunist pathogen responsible for ~30% of nosocomial - PowerPoint PPT Presentation

Pseudomonas aeruginosa pneumonia: from microbial physiopathology to treatment Christian van Delden MD Service of Infectious Diseases, University Hospital, Geneva February 2011

Pseudomonas aeruginosa The facts: • opportunist pathogen • responsible for ~30% of nosocomial infections � 47% of ventilator associated pneumonia (VAP) � leading cause of bacteremia associated with high mortality (> 40%) • therapeutic approaches are limited because of: � broad intrinsic antimicrobial resistance � its tendency to rapidly acquire resistance during antimicrobial therapies

Impact of primary infection site on mortality P-value Mortality % Primary site Cases Unknown 58 13 - Respiratory tract 24 55 0.03 Urinary tract 22 0 ND Line infections 5 0 ND Respiratory 0.21 P=0.03 Urinary/vascular 0.01 0.1 1 10 AAC 2003 ;47:2756 Adjusted hazard ratio (95% CI)

P. aeruginosa and intubated patients Colonization Infection D1 D5 D10 D18 D25 intubation extubation Risk for colonization increases with time of intubation 10-20% of colonized patients develop P. aeruginosa VAP 30 - 40% mortality due to VAP

Are there microbiological determinants that influence the outcome of P. aeruginosa infections ? Is the expression of specific virulence determinants (phenotypes) associated with a worse outcome ?

Major virulence determinants Siderophores: pyoverdine pyochelin Quorum sensing Cytotoxicity elastase phospholipase C TTSS lipase > 100 genes Type IV pili rhamnolipids pyocyanin cyanide > 100 genes flagellum

Could outcome be linked to specific strains ? _ Type III secretion system – 35 VAP isolates � 27 (77%) produced type III secreted proteins in vitro � 22 (81%): severe disease (death or relapse) � 8 strains didn’t produce type III secreted proteins � 3 (38%): severe disease (p<0.05) � 10 strains produced ExoU � 9 (90%): severe disease VAP with isolates producing type III secretion-dependent _ exoproducts, especially ExoU, in vitro are associated with worse clinical outcome. However these studies didn’t analyze whether cytotoxicity is associated with infections Crit Care Med 2002 ;30:521

QS regulation in P. aeruginosa Other regulators QS controls expression of 200-300 genes ( ∼ 5% of genome) elastase lipase rhamnolipid pyocyanin lipase cyanide rhamnolipid pyocyanin cyanide mexGHI-opmD Adapted from Wade et al. J. Bacteriol. 2005

Inter-cellular communication Quorum Allows a bacterial population to coordinate Keller and Surette, Nat Rev Microbiol. 2006

QS essential for P. aeruginosa virulence in... Plants ( Arabidopsis ) (Lettuce) Nematodes ( C. elegans ) Insects ( Drosophila ) Amoeba ( D. discoideum ) Human infections Mouse

Prospective study on P. aeruginosa colonization in the absence of antibiotic treatment Colonization Infection D1 D5 D10 D18 D25 intubation extubation 13 European ICUs: 31 patients Daily tracheal aspirate - total genomic DNA one P. aeruginosa - total RNA isolate - autoinducer

QS-proficiency and rhamnolipid production of initial colonizing isolates is associated with pneumonia in the placebo group - 57% of patients initially colonized by QS-proficient isolates versus 9% colonized by QS-deficient isolates developed VAP (P= 0.018) - Production of the QS-dependent virulence factor rhamnolipids is Thorax 2010 ; 65:703 associated with VAP (P= 0.003)

Role of rhamnolipids • Uptake of hydrophobic molecules (1992) • Surfactant for swarming motility (2000) • Lysis of amoeba ( D. discoideum ) (2002) • Maintain biofilm structure (2003) • Disrupt tight junctions in human airway epithelia (2006) • Lyse PMNs in vitro (2007)

QS-deficient isolates (LasR mutants) increase during colonization 31 placebo patients 80 LasR % patients with QS mutants 60 40 20 RhlR 0 -1 5 10 15 20 Days of colonization PNAS 2009 ;106:6339

In patient population dynamics: one genotype Isolate ( in vitro ) Δ lasR wt -1 1 3 5 7 9 11 RAPD 16101 lasR lasI primer only detects wt

In patient population dynamics: one genotype Isolate Population in vitro in patient Genomic DNA Δ lasR wt -1 1 3 5 7 9 11 10 9 70 genomic copies / g aspirate genomic copies 60 lasR wild type 10 8 % lasR wild type 50 10 7 40 30 10 6 20 10 10 5 0 RAPD 16101 10 4 -1 1 3 5 7 9 11 lasR lasI Days of colonization primer only detects wt … lasR mutants dominant in the population !!!

In patient population dynamics: two genotypes Population in patient Genomic DNA lasR wt lasR mutant genomic copies genomic copies 8 10 9 10 15108 15101 7 10 8 10 6 10 7 10 5 10 6 10 4 10 5 10 E429 239A OC2E 239A 120 120 % total population 239A ( lasR ) 239A ( lasR ) % total population 100 100 80 80 60 60 40 40 20 20 0 0 -1 2 4 6 8 10 12 14 16 18 -1 2 4 6 8 10 12 14 16 18 20 Days of colonization Days of colonization … lasR mutants dominant in the population !!! PNAS 2009 ;106:6339

Bacterial social behaviours Effect on recipient pos neg mutual benefit selfishness pos Effect on actor recipient actor neg altruism spite Signal : elicits response in recipient, induced response is beneficial for the actor Public good : resource that is costly to produce and provides benefit to all individuals in the population Cooperation : behavior that benefits another individual (recipient) and that is maintained because of its beneficial effect on the recipient Cheater : individual who does not cooperate, but gain benefit from others cooperating

Why do lasR mutants outcompete wt ? elastase Cooperator Public goods (ex: QS wild type isolate) (ex: polypeptides, produced by elastase)

Quorum sensing as a social behavior elastase Cooperator Public goods Non-cooperator or cheater (ex: QS wild type isolate) (ex: polypeptides, produced by (ex: a lasR mutant) elastase)

Quorum sensing as a social behavior elastase Cooperator Public goods Non-cooperator or cheater (ex: QS wild type isolate) (ex: polypeptides, produced by (ex: a lasR mutant) elastase) QS cheaters ( lasR mutants) have fitness advantage BUT only in the presence of QS cooperators !! PNAS 2009 ;106:6339

QS is important for development of VAP QS+ early VAP (2/6) P = 0.001 1 12 QS- late VAP (4/25) - VAP occurs earlier in patients colonized by QS-proficient isolates - Progressive accumulation of QS-deficient isolates might protect from VAP PNAS 2009 ;106:6339

Antibiotic therapy and virulence factor production Patient A Patient B 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 Imipenem Maxipim 1.5 Amikacine Ciprofloxacin 1.5 Fluconazole Diflucan 1.0 Cˇ fˇ pime Tazobactam Tobramycine M 1.0 0.5 Pipˇ racilline Gentamycine 0.5 l 0.0 t 0.0 l 24-12 28-12 01-1 05-1 09-1 13-1 17-1 21-1 25-1 29-1 02-2 06-2 10-2 14-2 18-2 22-2 26-2 01-3 05-3 09-3 13-3 17-3 21-3 25-3 20-12 24-12 28-12 01-1 05-1 09-1 13-1 17-1 21-1 25-1 29-1 02-2 06-2 10-2 14-2 18-2 22-2 26-2 01-3 05-3 09-3 13-3 17-3 21-3 25-3 l Date Date Patient C 3.2 3.0 2.8 2.6 2.4 Vancomycine 2.2 Cˇ fˇ pime Flucloxacilline 2.0 Amoxycilline Van Delden et al , unpublished results Pˇ nicilline G 1.8 1.6 1.4 1.2 14-02 16-02 18-02 20-02 22-02 24-02 26-02 28-02 01-03 03-03 05-03 07-03 09-03 11-03 Date Conclusion : 1. fluctuations of quorum ‐ sensing dependent virulence factor production appear after discontinuation of antimicrobial therapies 2. antimicrobial therapies might select quorum ‐ sensing proficient isolates

Bacterial warfare: R-pyocin mediated killing Landing Drilling Core Core contracted relaxed Killing

R-pyocin warfare in vivo ? 8 genomic copies 10 15101 7 10 6 10 10 5 4 10 L (wt) G 120 L ( lasR ) % of total population 100 80 60 40 20 0 -1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Days of colonization D-1, clone G D6, clone L Initial clone G is -1 +1 -1 +1 killed by clone L R1 by R2 pyocin 6B 6A 6B 6A R2 O6 O16 J Bact 2010 ;192:1921

Working model for R-pyocin – LPS interaction R-pyocin Shield B-band LPS Receptor Core region and A-band LPS Other serotypes: receptors may be the same, but shielding differs according to B-band charge and packaging

Summary Phenotype and NOT genotype associated with P. aeruginosa VAP � � Rhamnolipid production ( rhlR QS system) high risk factor for VAP � P. aeruginosa adapts to lung environment by mutation of lasR → Many patients co-colonized by wt and lasR mutants → lasR mutants: social « cheaters » or part of cooperative strategy ? → one genotype: lasR mutant out-competes wild-type population → multiple genotypes: other factors such as bacterial warfare determine population dynamics

How should we treat Pseudomonas infections ?

Resistance of P. aeruginosa can be predicted CID 2001 ;33:1859 Conclusion : preceding ceftazidime and imipenem exposure, especially as monotherapy, was associated with resistant P. aeruginosa bacteremic isolates

Evolution of antibiotic resistance Patient A , VAP First detection VAP VAP 6 days 6 days 6 days Tob Tobramycin Pip-Taz Imipenem Pip-Taz Pip-Taz 0 5 6 7 20 23 26 30 36 43 47 50 54 57 68 71 76 78 92 Pip. Pip. + Taz Cefta Cefep. Imi. Mero. Aztreo. Amika. Genta. Netil. Tobra. Norflo. Cipro. Susceptible Intermediate Resistant Reinhardt et al., AAC 2007