Lead


Lay summary
Pseudomonas aeruginosa is an important opportunistic pathogen responsible for severe nosocomial respiratory tract infections in mechanically ventilated patients. These infections are associated with major morbidity and mortality, and generate important costs. The patho-physiology of these infections involves an initial phase of colonization which in 10-20% of patients leads to an acute ventilator associated pneumonia (VAP). P. aeruginosa is also the main pathogen responsible for chronic colonization and acute exacerbations leading to the progressive loss of lung function in cystic fibrosis (CF) patients. Whereas adaptive responses of P. aeruginosa to the specific lung environment during chronic colonization (over years) of CF patients have been described, no such studies have been performed so far in intubated patients which follow a more acute (over days) clinical course. We have now gathered two probably unique prospective collections of P. aeruginosa isolates and tracheal aspirates collected longitudinally from intubated patients who were either treated (collection A) or not treated (collection B) with anti-pseudomonal drugs. In vitro and preliminary in vivo analyses of isolates from both collections, performed in the frame of our current FN-grant, suggest that the virulence phenotype of P. aeruginosa evolves rapidly (within 3 to 4 days) during the colonization of intubated patients. The most prominent genetic adaptation we identified in both collections is the emergence of mutations in the lasR gene, encoding one of the quorum-sensing regulators in P. aeruginosa. While this adaptive change occurred in 70% of intubated patients in collection B, we observed that VAP occurred in patients colonized predominantly by lasR wild-type isolates. This strongly suggests that quorum-sensing proficiency represents a major risk factor for progression from colonization towards pneumonia. We also identified lasR mutants among isolates from antibiotic treated patients (collection A), however these emerged once antibiotic treatment had been discontinued. In PartA of this project we propose to analyze in vitro and in vivo this important adaptation as part of a social behavior of P. aeruginosa and to identify further adaptive responses by global in vivo gene expression studies.A second project (PartB) addresses the issue of P. aeruginosa persistence observed in both CF and intubated patients. Unlike classical antibiotic resistances that arise due to target mutations or to acquisition of resistance determinants, persister cells are a non-growing, ”dormant” subpopulation insensitive to conventional antibiotics and present also in bacterial biofilms. Genetic determinants required for the formation of these “specialized” persister cells have not been investigated in P. aeruginosa and are the subject of this part of the grant proposal. Both projects address essential issues for the development of novel alternative strategies to challenge the problem of antibiotic resistance. Raising interest in such strategies is illustrated by a recent review entitled: “Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaptation” 3.