Evolutionary trajectories of Klebsiella pneumoniae : From experimental biofilm evolution to a hospital outbreak

Abstract: Bacterial evolution is closely intertwined with our lives. As their hosts, we shape how bacteria evolve by imposing numerous selective pressures during the time bacteria spend in our bodies. As a result, they adapt in various ways to colonize us or infect us better. In this thesis, I present studies aimed to expand the knowledge on the pathoadaptive changes in Klebsiella pneumoniae, which is a bacterial pathogen of critical importance worldwide. In Paper I, we present a new 3D-printed device for growing and studying surface-attached bacterial biofilms. The special aim was to increase the ease of use and versatility, and we have used this biofilm device to screen for biofilm capacity, perform experimental evolution and fundamental biofilm analysis in subsequent studies.In Paper II, we study within-host evolution by analyzing 110 isolates originating from the same multidrug-resistant K. pneumoniae clone that caused an outbreak at Uppsala University Hospital between 2005 and 2010. We whole-genome sequenced these isolates and phenotypically characterized them to show that the clone has undergone extensive changes in individual patients, leading to increased biofilm formation capacity, attenuation of systemic virulence, and altered colonization potential.In Paper III, we exploit an experimental evolution approach to decipher evolutionary trajectories towards increased biofilm formation. We show how fast this trait can be acquired in different K. pneumoniae strains by a strong convergent evolution, mostly targeting genes involved in capsule, fimbriae, and c-di-GMP-related regulatory pathways. Importantly, this genetic parallelism extends beyond in vitro observations as we find an extensive overlap with clinical outbreak isolates that carry signatures from within-host evolution.The experimental evolution experiments revealed interesting genetic changes not only in the known structures or pathways but also in completely novel factors. In Paper IV, we explore a previously uncharacterized T6SS effector that is involved in biofilm formation in K. pneumoniae and strongly enhances this phenotype upon acquiring a single and specific point mutation. We demonstrate that the toxin acts as a DNase and that this mutation results in changes at multiple levels, including protein stability, toxicity, and transcriptional profiles, which collectively lead to the formation of biofilms.