Pseudomonas aeruginosa

About Pseudomonas aeruginosagram-negative aerobe bacteriaCommonly found in the environmentAt any moist locationCommon cause of nosocomial infections

P. aeruginosa is an opportunistic pathogenExtremely broad host spectrumHardly any infections in the normal human hostSevere immunodeficiencies and medical devices predispose the patients to P. aeruginosa infectionsBroad spectrum of clinical symptomsUrinary tract infections Pulmonary infectionsSoft tissue infectionsSepsisBone and joint infectionsEndocarditis

P. aeruginosa infectionsP. aeruginosa infections are of particular concern for Cystic fibrosis patientsBurn patientsHospitalised patientsCase mortality rate for patients infected with P. aeruginosa approaches 50%

Nosocomial infectionsFourth most common isolated nosocomial pathogen accounting for approx. 10 % of all hospital acquired infections.Patient-to-patient spread and direct patient contact with environmental reservoirsdisinfectants, respiratory equipment, food, sinks, taps

Diagnosis of P. aeruginosa Isolation and lab identification of the pathogenP. 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 infectionsP. 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

PathomechanismsAdhesionPili, flagella and fimbriaeInvasionExtracellular enzymes and toxins (proteases, elastase, phospholipases, rhamnolipids, Exotoxin A)DisseminationLeukocidin inhibits neutrophils und leukocytesLPS (Endotoxin)ProtectionCapsule (Alginate)

Interbacterial Communication

McKnight et al, 2000Quorum Sensing in P. 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 Cystic 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 diseasesSlow progressing infectionsFunctional loss of the affected organHighly resistant to host defences and antimicrobial therapy Recovery is rareRole in the development of cancer, peptic ulcer and possibly atherosclerosis

Why is traditional antimicrobial therapy ineffective 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)

SCVs of P. aeruginosa in CFSlow 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 BraunschweigCupA encoded fimbria expression in P. aeruginosa

Molecular Mechanisms controlling the conversion to a SCV biofilm phenotype

Biofilms, City of microbes

Cooperative traits and coordinated behavior of bacteria

McKnight et al, 2000Quorum Sensing in P. aeruginosa

Pseudomonas Quinolone Signal (PQS)

Hydroxy Alkyl QuinolonesPQSHHQ

PQS directly interacts with bacterial DNA in a computed model

PQS enhances iron dependent DNA fragmentationDNA+ PQSDNA+ HHQDNA+ PQS+ Fe(II)DNA+ Fe(II)DNADNAControlDNA+ HHQ+ Fe(II)

PQS enhances the fragmentation of DNA in vivo

PQS production is linked to the release of DNA

PQS non-producing mutantPAO1 wild-type

Regulated DNA release in response to environmental stresses

DNA breakdown products enhance PQS production

Autoinductive PQS production cyclePQSDNApqsA-E transcriptionCell deathPyocyaninPQSLectinRhl Quorum SensingVirulence factorsBiofilm formation

P. aeruginosa WTP. aeruginosa PQS negative MutantePhenotypic Diversity is PQS dependent

Establishment of a chronic state of infection

Chronic InfectionsBiofilmFormationBacterial DiversityInterbacterial Communication

Molecular mechanisms underlying genetic diversityHorizontal gene transfer

Hypermutation

Adaptive mutations

Tiling ArraysMicroarray 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).

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Systems BiologyPrediction of the impact of cellular metabolism on the P. aeruginosa quorum sensing mediated virulence 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