Pseudomonas aeruginosa

About Pseudomonas aeruginosa

• gram-negative aerobe bacteria

• Commonly found in the environment– At any moist location

• Common cause of nosocomial infections

P. aeruginosa is an opportunistic pathogen

• Extremely broad host spectrum• Hardly any infections in the normal human host• Severe immunodeficiencies and medical devices

predispose the patients to P. aeruginosa infections• Broad spectrum of clinical symptoms

– Urinary tract infections – Pulmonary infections– Soft tissue infections– Sepsis– Bone and joint infections– Endocarditis

P. aeruginosa infections

• P. aeruginosa infections are of particular concern for Cystic fibrosis patients

• Burn patients

• Hospitalised patients– Case mortality rate for patients infected with P.

aeruginosa approaches 50%

Nosocomial infections

• Fourth most common isolated nosocomial pathogen accounting for approx.

• 10 % of all hospital acquired infections.• Patient-to-patient spread and direct patient contact

with environmental reservoirs– disinfectants, – respiratory equipment, – food, – sinks, taps

Diagnosis of P. aeruginosa

• Isolation and lab identification of the pathogen• P. aeruginosa grows well on most laboratory media • Identified on the basis of its:

– Gram morphology, – inability to ferment lactose, – a positive oxidase reaction, – its characteristic odor, – its ability to grow at 42° C. – Fluorescence is helpful in early identification of P.

aeruginosa colonies and may also help identify its presence in wounds.

Treatment of P. aeruginosa infections

• P. aeruginosa is frequently resistant to many commonly used antibiotics.

• To archive synergy a combination of e.g. gentamicin and carbenicillin is frequently used.

• No vaccines so far

Pathomechanisms

• Adhesion– Pili, flagella and fimbriae

• Invasion– Extracellular enzymes and toxins (proteases, elastase,

phospholipases, rhamnolipids, Exotoxin A)

• Dissemination– Leukocidin inhibits neutrophils und leukocytes– LPS (Endotoxin)

• Protection– Capsule (Alginate)

Interbacterial Communication

McKnight et al, 2000

Quorum Sensing in Quorum Sensing in P. aeruginosaP. aeruginosa

„....It is time to close the book on infectious disease….“

William Stewart, Surgeon in a message to the United State Congress in 1969

Bacterial Biofilms

Biofilms in the environment

Catheter associated biofilms

Chronic biofilm infections

Despite even intensified antibiotic therapy,

no eradication of chronic P. aeruginosa infections of the cystic fibrosis lung

Cystic fibrosis

• Most common life-threatening inherited genetic disorder in the Caucasian population

• Mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

• one in every 25 carry the mutated recessive gene and more than 1 in 4000 live births suffer from CF.

• Life expectancy: – Until the 1930s: the life expectancy of a baby with CF was only a few months, – in the 1980s, most deaths from CF occurred in children and teenagers. Today with

improved treatments, nearly 40 percent of the CF population is aged 18 and older, – for a person with CF the median age of survival is nearly 37 years.  

• Cystic Fibrosis affects a number of organs in the body, cycles of infection and inflammation lead to a progressive deterioration of lung function.  

Chronic infection of the Chronic infection of the Cystic Fibrosis lungCystic Fibrosis lung

CF and Transplantation Centre

• 360 patients regularyly attend the CF outpatient clinic at the Medical School Hannover, Germany

• Informations of more than 500 CF patients in the local CF register

• 2000 – 2005: 100 lung transplantation / year at the MHH

Indications for lung transplantation

Lung Transplantation in Germany

Post lung transplant mortality

Chronic infectious diseases

• Slow progressing infections

• Functional loss of the affected organ

• Highly resistant to host defences and antimicrobial therapy

• Recovery is rare

• Role in the development of cancer, peptic ulcer and possibly atherosclerosis

Why is traditional antimicrobial therapy Why is traditional antimicrobial therapy ineffective against biofilm bacteriaineffective against biofilm bacteria??

Biofilms, City of microbes

Adaptation and survival is facilitated by diversity

Emergence of morphological distinct niche-specific phenotypes

From: Rainey R, Travisano M. Nature, 1998; 39: 69-72

Phenotypic Diversity via the selection of niche-specialists

„„Small colony variants“ (SCV)Small colony variants“ (SCV)

SCVs of SCVs of P. aeruginosaP. aeruginosa in CF in CF

• Slow growing subpopulations (3% of the P. aeruginosa positive sputum specimens)

• SCVs exhibit an increased resistance towards a broad spectrum of antimicrobial agents

• The recovery of SCV correlates with parameters revealing poor lung function and an inhalative antimicrobial therapy

• Fast growing revertants can be isolated from the SCV population

Auto-aggregation in liquid cultures

M. Rohde, GBF Braunschweig

CupA encoded fimbria expression in P. aeruginosa

Molecular Mechanisms controlling the conversion to a SCV biofilm phenotype

Biofilms, City of microbesBiofilms, City of microbes

Cooperative traits and coordinated behavior of bacteria

McKnight et al, 2000

Quorum Sensing in P. aeruginosa

Pseudomonas Quinolone Signal (PQS)

Hydroxy Alkyl Quinolones

PQS HHQ

PQS directly interacts with bacterial DNA in PQS directly interacts with bacterial DNA in a computed modela computed model

PQS enhances iron dependent DNA fragmentationDNA

+ PQSDNA

+ HHQ

DNA+ PQS+ Fe(II)

DNA+ Fe(II)

DNADNADNA

ControlDNA

+ HHQ+ Fe(II)

PQS enhances the fragmentation of DNA in vivo

PQS production is linked to the release of DNA

PQS non-producing mutant

PAO1 wild-type

Regulated DNA release in response to environmental stresses

DNA breakdown products enhance PQS DNA breakdown products enhance PQS productionproduction

Autoinductive PQS production cycleAutoinductive PQS production cycle

PQS

DNA

pqsA-E transcription

Cell death Pyocyanin

PQS

Lectin

Rhl Quorum Sensing

Virulence factorsBiofilm formation

P. aeruginosa WT P. aeruginosa PQS negative Mutante

Phenotypic Diversity is PQS dependent

Establishment of a chronic state of infection

Chronic Infection

s

BiofilmFormation

Bacterial Diversity

Interbacterial Communication

Molecular mechanisms underlying genetic diversity

• Horizontal gene transfer

• Hypermutation

• Adaptive mutations

Tiling Arrays• Microarray hybridisation-based method technique to find

mutations in bacterial genomes was used to study metronidazole resistance in Helicobacter pylori TJ Albert et al. Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori. Nature Methods 2, 951 - 953 (2005).

Thank You for your attention!

Systems Biology

• Prediction of Prediction of the the impact of cellular impact of cellular metabolism on the metabolism on the P. P. aeruginosaaeruginosa quorum quorum sensing mediated sensing mediated virulence phenotypevirulence phenotype

Use a theoretical model to complete

the knowledge on the system, to plan

new experiments and to predict the

behavior of the system under changing

genetic or environmental conditions