Eco-evolutionary analysis of persistence in P. aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that is able to cause serious complications in patients with an impaired immune system or in some chronic infections, e.g. in cystic fibrosis patients (CF). In fact,P. aeruginosa is recognized as the most abundant bacterium in pulmonary infections of CF patients. Successfully treating chronic P. aeruginosa infections, however, can be hard due to the occurrence of persistence, which is the phenomenon in which a small subpopulation of bacterial cells makes a phenotypic switch to a non-growing but antibiotic tolerant state. Persistence is increasingly being recognized as one of the main reasons for the recalcitrance of chronic infections. Hence, there is an urgent need to develop new strategies for the eradication of these tolerant cells. Despite intense research in this field, many aspects of persistence still remain largely unexplored, particularly,nbsp;and evolutionary aspects of persistence. Previously, several theoretical models have been developed in order to unravel the evolutionary forces that generate variation in persistence levels. The predictions of these models, however, remain largely untested. In this work, naturally occurring levels of persistence in P. aeruginosa were compared against those predicted by previous models. It was found that levels of persistence in natural populations of P. aeruginosa were much lower than predicted and that this was likely caused by the occurrence of several fitness costs and trade-offs of persistence. For example, we found that increased persistence was linked to longer lag times upon dilution and regrowth innbsp;medium as well as increased mortality in stationary phase. The existence of a small subpopulation of non-growing but antibiotic tolerant persister cells provides an example of a risk spreading or rdquo; strategy in which instantaneous growth is traded for long-term survival, and resembles dormancy in plant and Crustacean seed banks. In order to further check whether persistence conforms to bet-hedging and dormancy theory, we experimentally tested some of the predictions in P. aeruginosa. Observations consistent with theory, for example, were that following each antibiotic strike the same percentage of cells woke up from the dormant persister stage, and that the hatching percentage was directly dependent upon the quality of thenbsp;environment. Addition of unconditioned medium, indicative of good growth conditions, for example, was shown to increase resuscitation of persisters, thereby making them sensitive to killing by antibiotic – a finding that may havenbsp;implications with respect to possible treatment strategies of bacterial infections. By contrast, addition of spent medium from stationary phase cultures of P. aeruginosa, and indicative of bad growth conditions, helped maintain the bacteria in the persister state. In addition, we found evidence for N-acyl-homoserine lactone quorum sensing moleculesnbsp;important in maintaining cells in the persister state. These molecules could be possible future targets to interfere with persister cell maintenance.nbsp;the molecules and cues used to maintain cells in the persister state were recognized and were generic across different P. aeruginosa strains, but induced increased mortality in E. coli. Finally, natural variation of persister levels were verified in populations with various backgrounds, including longitudinal isolates originating from pneumonia patients and CF patients with a known history of antibiotic treatment. The work presented in this thesis contributes to a better understanding of the ecological and evolutionary drivers affecting bacterial persistence in the opportunistic human pathogen P. aeruginosa. Information obtained from fitness measurements and various experiments in this work can help to set-up a more realistic model fornbsp;persistence, as well as to design better treatment regimens for anti-persistence therapy in targeted groups of patients infected with P. aeruginosa.Table of contents Scope of the thesis…………………………………………………………………………...........1 CHAPTER 1. Bacterial persistence in Escherichia coli and Pseudomonas aeruginosa………………………………………………………..3 1.1 Defining persistence……………………………………………………………………..3 1.2 Formation of persisters………………………………………………………………….4 1.3 Mathematical modeling of persistence………………………………………………..7 1.4 Persistence in P. aeruginosa……………………………………………………………9 CHAPTER 2. The opportunistic pathogen Pseudomonas aeruginosa...………………....11 2.1 General characteristics of P. aeruginosa…………………………………………….11 2.2 Development of P. aeruginosa biofilms and clinical importance…………………13 2.3 Quorum sensing in P. aeruginosa………………………………………………........15 2.4 QS regulation by sRNA in P. aeruginosa…………………………………………….17 2.5 Virulence and infection of P. aeruginosa…………………………………………….18 2.6 Clinical importance of P. aeruginosa infections…………………………………….20 CHAPTER 3. Sociobiology of microbial systems……………………………..……..……….21 3.1 Dormancy as a survival strategy……………………………………..……………….21 3.2 Evolution of dormancy………………………………………………………………….23 3.3 Bet-hedging as an evolutionary stable strategy……………………………………..24 3.3.1 Types of bet-hedging and predictions driven from this strategy……………………………………………………………24 3.4 Quorum sensing as a social behavior………………………………………………..25 3.5 Persistence as a form of dormancy and bet-hedging……………………………….26 CHAPTER 4. Fitness trade-offs explain low levels of persister cells in the opportunistic pathogen Pseudomonas aeruginosa…………………..29 4.1 Introduction……………………………………………………………………………...29 4.2 Materials and Methods……………………………………………………………….. 31 4.2.1 Strains and culture conditions………………………………………………31 4.2.2 Competition assays between high- and low-persister strains………..…32 4.2.3 Variation in persistence levels among natural isolates…………….……33 4.2.4 Costs and trade-offs of persistence………………………………...……..34 4.3 Results……………………………………..…………………………………………….35 4.3.1 Low-persister strains have a competitive benefit in the absence of antibiotic treatment…………………………………………...35 4.3.2 Low levels of persistence among natural P. aeruginosa isolates………………………………………………….…38 4.3.3 Complex fitness trade-offs explain low levels of persistence…………..41 4.4 Conclusion……………………………………..……………………………..…………44 CHAPTER 5. Awakening from persistence - dormancy characteristics determine susceptibility to antibiotics in Pseudomonas aeruginosa…….……..47 5.1 Introduction……………………………………..……………………………………….47 5.2 Materials and Methods…………………………………………………………………49 5.2.1 Strains and culture conditions……………………………………...………49 5.2.2 Quantifying background awakening from persister cells………………..49 5.2.3 Response to relief from antibiotics………………………………………..50 5.2.4 Response to unconditioned medium………………………………………51 5.2.5 Quantifying the dose-response relationship to unconditioned medium…………………………………………………………51 5.2.6 Bet-hedging: awakening fractions across favorable periods…………..52 5.2.7 Preparation of supernatant from stationary phase cultures……………..52 5.2.8 Estimation of the decline of persisters through time……………………..53 5.3 Results.………………..…………………………………………………………...……53 5.3.1 The biphasic killing curve and background mortality …………..………..53 5.3.2 Enhanced awakening rates upon incubation in unconditioned medium …………………………………………..…………….53 5.3.3 Persister decline is independent of the presence of antibiotics in spent medium……………………………………………..…55 5.3.4 Awakening rates scale with dilution of spent medium…………...……..57 5.3.5 Persistence as a bet-hedging mechanism…………………………..……58 5.4 Conclusion……………………………………………………………………………….59 CHAPTER 6. On the role of the quorum-sensing system for the maintenance of persistence in Pseudomonas aeruginosa…………….………….61 6.1 Introduction……………………………………………………………………………...61 6.2 Materials and Methods…………………………………………………………………62 6.2.1 Bacterial strains and growth conditions…………………………………..62 6.2.2 Manipulations during washing steps ………………………………..….…63 6.2.3 Preparation of supernatant from stationary phase cultures …………...63 6.2.4 Addition of synthetic molecules to the medium………………………….64 6.2.5 Estimation of the decline of persisters throughout time…………………64 6.3 Results and Discussion…………………………………………………...……………64 6.3.1 Manipulations during washing step have no effect on resuscitation of persisters…………………………...………….64 6.3.2 Addition of nutrients cannot promote resuscitation of persister cells…..65 6.3.3 A QS double mutant does not produce molecules that keep the persistent state……………………………………………..…66 6.3.4 Complementation of spent medium from QS mutants by specialized molecules could only partially rescue the persister state in P. aeruginosa……………………………………………..67 6.4 Conclusion…………………………………………………………………………….…69 CHAPTER 7. Inter- and intra-species complementation of maintenance of persister dormancy…………………………………………..71 7.1 Introduction……………………………………………………………………………...71 7.2 Materials and Methods…………………………………………………………………72 7.2.1 Bacterial strains and growth conditions……………………………………72 7.2.2 Preparation of supernatant from stationary phase cultures…………..…72 7.2.3 Estimation of the decline of persisters throughout time………………….72 7.3 Results and Discussion………………………………………………………………...73 7.3.1 Intra-species complementation maintains the persister state………..…73 7.3.2 Inter-species complementation recovers persister levels partially……………………………………………………….……74 7.4 Conclusion……………………………………………………………………………....76 CHAPTER 8. Natural variation of persistence levels in isolates of Pseudomonas aeruginosa from environmental samples and acute and chronic infections…………………………………..77 8.1 Introduction………………………………………………………………………………77 8.2 Materials and Methods…………………………………………………………………78 8.2.1 Bacterial strains and growth conditions………………………………..…78 8.2.2 Persistence assay……………………………………………………………78 8.3 Results and Discussion………………………………………………………………..78 8.3.1 Natural variation of persistence level among P. aeruginosa isolates……………………………………………………………..78 8.3.2 Longitudinal isolates from pneumonia and CF patients have elevated levels of persistence……………………………………81 8.4 Conclusion……………………………………………………………………………….85 CHAPTER 9. Discussion and perspectives……………………………………………………87 List of publications………………………………………………………………………………...95 Bibliography…………………………………………………………………………………………97 nrpages: 108status: publishe