Rapid evolution of Klebsiella pneumoniae biofilms in vitro delineates adaptive changes selected during infection

Biofilm formation facilitates infection by the opportunistic pathogen Klebsiella pneumoniae, primarily via indwelling medical devices. Here, we explore the adaptive evolution of classical pathotype K. pneumoniae in surface-attached biofilms through experimental evolution that mimics catheter-associated infections. We observe rapid convergent evolution that alters or abolishes capsule production, modifies the fimbrial adhesin MrkD, or increases the production of fimbriae and cellulose via upregulated c-di-GMP-dependent pathways. We show that multiple aspects of biofilm formation, including early attachment, topology, surface preference, and extracellular matrix composition, are affected in a mutation-specific manner. However, evolutionary trajectories and resulting phenotypes show strain-specific differences, illustrating the importance of genetic background on biofilm adaptation. Additionally, changes in acute virulence are linked to the underlying genetic change rather than the overall biofilm capacity. Identified adaptive changes conferring hypermucoviscosity or affecting c-di-GMP-related regulatory pathways overlap extensively with those previously identified in clinical UTI and wound isolates, confirming biofilm as an important selective trait in vivo.