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Adaptation and persistence of Pseudomonas aeruginosa during colonization and infection of ventilated patients

English title Adaptation and persistence of Pseudomonas aeruginosa during colonization and infection of ventilated patients
Applicant Van Delden Christian
Number 120011
Funding scheme Project funding (Div. I-III)
Research institution Dépt Microbiologie et Médecine Moléculaire Faculté de Médecine Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Medical Microbiology
Start/End 01.05.2008 - 30.04.2011
Approved amount 318'000.00
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All Disciplines (2)

Discipline
Medical Microbiology
Experimental Microbiology

Keywords (4)

Pseudomonas aeruginosa; Bacterial infections; quorum-sensing; persistance

Lay Summary (English)

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.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
108106 Adaptation of Pseudomonas aeruginosa during respiratory tract colonization and infection in mechanically ventilated patients: in vitro and in situ studies 01.04.2005 Project funding (Div. I-III)
140929 Pseudomonas aeruginosa in lung transplant patients: adaptation, bacterial competition and novel therapeutic approaches 01.06.2012 Project funding (special)

Abstract

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 are almost always preceded by colonization of the upper respiratory tract. Classical antimicrobial treatments are unable to eradicate and/or prevent the progression from colonization to infection. 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. Recently evidence from CF patients has supported progressive loss of virulence determinants, including motility, cytotoxicity and quorum-sensing (QS) dependent virulence, during chronic colonization (over years). This adaptive response of P. aeruginosa to the specific CF lung environment results from accumulation of individual mutations in different genetic loci, including the lasR gene encoding one of the quorum-sensing regulators in P. aeruginosa. So far no similar investigations have been reported in intubated patients, which follow a more acute (over days) clinical course. Over the last 5 years we have gathered two prospective respiratory tract collections. The first collection includes more than 4000 isolates consecutively obtained from tracheal aspirates of 13 intubated patients, exposed to a variety of antimicrobial treatments. The second collection consists of more than 500 P. aeruginosa isolates, as well as corresponding genomic DNA and total RNA extracts from consecutively collected tracheal aspirates obtained from 62 intubated, non-treated patients. In vitro analysis of isolates from both collections suggested 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. These adaptive changes occur in 70% of patients and lead in many patients to co-colonization with wild type and lasR mutants. Considering quorum-sensing as a cooperative behavior, the lasR mutants could be considered as “selfish cheaters”, since they profit from “public goods” produced by the wild type without paying their cost. So far it remains unknown whether these cheaters take over by directly eliminating the wild type population or simply have a growth advantage, and how this “asocial” behavior affects the long-term survival of P. aeruginosa populations in intubated patients? Strikingly we observed that antimicrobial therapies influence these adaptive modifications; indeed whereas lasR wild type isolates predominate during antimicrobial therapies, lasR mutants rapidly appear after treatment discontinuation. So far unpublished results obtained by our laboratory during the current FN research grant suggest that colonized patients in whom P. aeruginosa lasR wild type strains disappear progressively, as a consequence of adaptation to the lung environment, are less likely to evolve towards infection. In contrast, patients colonized predominantly by lasR wild type isolates, due to insufficient adaptive changes, are at high risk to develop infection.In PartA of this project we propose to further verify this hypothesis, to dissect these adaptive modifications, to characterize how they are modulated by antimicrobial treatments, and to investigate their role during pathogenesis. We suggest to analyse the competition between wild type strains and lasR mutants both in vitro and during the colonization of intubated patients using qRT-PCR to follow the proportion of wild type and mutant populations. We further plan to determine whether specific clones might take an advantage by killing competing cells by toxins such as pyocins, or whether particular nutrients (such as certain amino-acids) might confer a growth advantage to lasR mutants. Comparative transcriptome analysis will be used to obtain a global picture of gene expression alterations due to lasR mutations in clinical isolates. In vitro co-cultures studies, and co-infection of mice using in vivo bioluminescence will allow determining how co-colonization of wild type and mutant strains affects persistence and transition to infection. Taking advantage of our collections, a global transcriptome analysis performed on RNA extracts on tracheal aspirates from patients during their evolution from colonization to infection is planned. In a fourth part, we suggest to investigate the role of antibiotic pressure on population dynamics. These studies will provide valuable information concerning the pathogenesis of such infections, which could lead to the identification of new targets for anti-virulence strategies.The second part of this project (PartB) relates to a different aspect of the social behavior of P. aeruginosa. Failure to eradicate P. aeruginosa from both CF and intubated patients has been well documented. Relapse of P. aeruginosa pneumonia after antimicrobial therapy is a frequent event. While classical antibiotic resistance and biofilm formation have been incriminated, alternative explanations including the formation of “specialized” persister cells have been suggested but never verified experimentally in P. aeruginosa. Persisters, which represent a non-growing, ”dormant” subpopulation insensitive to conventional antibiotics can be considered as a “risk-insurance” for the cell population in case of catastrophes (antibiotic exposure). One patient presented such a persistent colonization for more than one month. The tested isolates, although susceptible did not respond to antimicrobial treatments. The project proposes to identify genetic determinants involved in persister cell formation in reference strains and clinical isolates from our collections. Persisters will be enriched by exposure of cells during early stationary phase to bactericidal concentrations of ß-lactam antibiotics. The proportion of surviving cells that regrow as susceptible cells will be defined as persister population. Using classical genetic screens (transposon mutagenesis, expression libraries), we will seek for genetic determinants which increase or drastically reduce the proportion of persister cells. A second approach proposes to test the involvement of specific regulatory and signaling pathways (stringent response, quorum sensing, toxin/antitoxin modules) in persister formation by generating mutants (toxin/antitoxin) or using available mutants (stringent response, quorum sensing) derived from a reference strain. Understanding the mechanisms governing the dynamics of bacterial populations (adaptation and persistence) should permit to develop novel strategies manipulating adaptive processes and/or interfering with persistence in order to prevent the progression from colonization to infection. Both projects therefore address essential issues for the development of novel alternative strategies to challenge the problem of antibiotic resistance.
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