Evolution and adaptation of Pseudomonas aeruginosa in cystic fibrosis airways: cystic fibrosis as a model system

For centuries evolution has been investigated with an "end-point" approach,through comparisons between species or fossil records. However,to understand processes in general, including evolution, it is highly valuable to observe the dynamics as they unfold, in "realtime".This is possible through laboratory experiments, with a high degree of control and rigour. But to truly understand evolution and the complex mechanisms it deploys, it is necessary to combine the laboratory learnings with investigations of natural systems. –Though, this can be tricky. Because of the heterogeneity and constant change of natural environments, the primary obstacle is re-sampling of the samepopulation over time, especially if the population is small.Nevertheless, it has been accomplished: Chronic airway infections of cystic fibrosis (CF) patients have offered a unique view into the adaptationand evolution of Pseudomonas aeruginosa to this natural environment,spanning thousands of bacterial generations. Because of the prolonged and persistent infections, they provide a valuable modelsystem for the investigation of evolutionary mechanisms.The main focus of this thesis has been to show the link between evolutionary studies in the CF model system and general evolutionary theories, many of which have been developed from observations of other organisms.This comparison has initially been sought by showing the plausibility of using comprehensive collections of longitudinally sampled single isolates, for their use in evolutionary studies (Study 1). This was done by comparing five metagenomes with single isolates from four CF patients, and identifying significant genetic links found within the patient specific P. aeruginosa populations. This evident genetic link was even found for two populations, where a recent patient-to-patienttransmission had occurred.Secondly a comprehensive collection of 474 longitudinal single P. aeruginosa isolates from 34 young Danish CF patients was investigatedby whole genome sequencing (Study 2). This was done to reconstruct the recent evolutionary history, and identify genes targeted in the initial adaptation to the CF airways. From this analysis we found common clonal lineages among the patients, evidence of patient-to-patient transmission, historic contingencies, and convergent evolution of 52 candidate pathoadaptive genes. By further genome sequencing 26 P. aeruginosa isolates from four Italian CF patients (Study 3), and 35 P. aeruginosa isolates from 12 primary ciliary dyskinesia (PCD) patients (Study 4), we were able to find genetic and phenotypic links across countries and diseases.All three studies (not including the metagenome study) had common clonal lineages and clear overlaps of genetic adaptational patterns.However, the genetic overlap between CF and PCD isolates did not extend to a phenotypic overlap, which indicates that the mucus, which is different in CF patients compared to PCD patients, is a significant selective factor for the evolution and adaptation of P. aeruginosa to these environments.Independently and together the studies presented in this thesis provide new knowledge of adaptation and evolution in both CF and PCD airways. With further characterisation of genetic and phenotypic adaptationsit should be possible to translate these results into clinically relevant information, leading to better epidemiological predictions,valuable information with regards to treatment strategies, and perhaps extrapolation of this knowledge to other infection scenarios. OVER ALL: Through the convergence of genetic and phenotypic adaptations observed in CF studies and by linking processes of evolution to these observations, this thesis shows that collections of longitudinal P. aeruginosa isolates from CF patients provide a valuable basis for the study of adaptation and evolution in natural environments.