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, ecological 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 in new 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 “bet-hedging” 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 the new 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 have important 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 molecules being important in maintaining cells in the persister state. These molecules could be possible future targets to interfere with persister cell maintenance. Moreover, 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 for bacterial persistence, as well as to design better treatment regimens for anti-persistence therapy in targeted groups of patients infected with P. aeruginosa.