Latent evolution of biofilm formation depends on life-history and genetic background

www.nature.com/npjbiofilms ARTICLE OPEN Latent evolution of biofilm formation depends on life-history and genetic background Amandine Nucci1, Eduardo P. C. Rocha1 and Olaya Rendueles 1✉ 1234567890():,; Adaptation to one environment can often generate phenotypic and genotypic changes which impact the future ability of an organism to thrive in other environmental conditions. In the context of host-microbe interactions, biofilm formation can increase survival rates in vivo upon exposure to stresses, like the host’s immune system or antibiotic therapy. However, how the generic process of adaptation impacts the ability to form biofilm and how it may change through time has seldomly been studied. To do so, we used a previous evolution experiment with three strains of the Klebsiella pneumoniae species complex, in which we specifically did not select for biofilm formation. We observed that changes in the ability to form biofilm happened very fast at first and afterwards reverted to ancestral levels in many populations. Biofilm changes were associated to changes in population yield and surface polysaccharide production. Genotypically, mutations in the tip adhesin of type III fimbriae (mrkD) or the fim switch of type I fimbriae were shaped by nutrient availability during evolution, and their impact on biofilm formation was dependent on capsule production. Analyses of natural isolates revealed similar mutations in mrkD, suggesting that such mutations also play an important role in adaptation outside the laboratory. Our work reveals that the latent evolution of biofilm formation, and its temporal dynamics, depend on nutrient availability, the genetic background and other intertwined phenotypic and genotypic changes. Ultimately, it suggests that small differences in the environment can alter an organism’s fate in more complex niches like the host. npj Biofilms and Microbiomes (2023)9:53 ; https://doi.org/10.1038/s41522-023-00422-3 INTRODUCTION One of the central questions in microbial evolutionary biology is understanding the mechanisms by which bacteria expand their ecological breadth. The niche shift hypothesis postulates that the process of adaptation to a different environment can result from rapid adaptive changes via mutation or horizontal gene transfer1,2, leading to diversification and opening the possibility of exploiting novel niches3. Bacteria may have to contend with novel stresses to adapt. This may often involve forming a biofilm, which generically increases tolerance to a broad range of stresses4. Such resilient surface-attached multicellular structures are ubiquitous and the prevalent prokaryotic lifestyle5. In the context of host-microbe interactions, it has been shown that increased ability to form biofilm correlates with the capacity to replicate and colonize multiple hosts, whereas bacteria with narrow host ranges are usually poor biofilm-formers6,7. Within a host, biofilm formation offers numerous specific advantages, such as higher resistance to antimicrobials8 and to antibody-mediated killing and phagocytosis9. During competition with other members of the microbiome, it can also lead to niche exclusion of direct competitors10. The Klebsiella pneumoniae species complex (KpSC) is a metabolically versatile group of seven distinct and closely related taxa of Klebsiella belonging to the Enterobacteriaceae family. KpSC includes the best-studied K. pneumoniae sensu stricto but also other species like K. variicola and K. africana11. KpSC are characterised by a very large carbon and nitrogen core metabolism12. This may partly explain its ubiquity and ecological breadth13–15. These bacteria can adopt a free-living lifestyle in the soil or in the water, but they are mostly studied in its hostassociated form, colonizing plants, insects and mammals, including humans, where they can be a found as gut commensals. Hypervirulent strains of K. pneumoniae cause community-acquired infections which may result in pyogenic liver abscesses, but most of K. pneumoniae infections are opportunistic and health-care associated. They typically require a precolonization of the gastrointestinal epithelia prior to infecting other body sites11. Several factors impact the ability of K. pneumoniae to form biofilm and colonise host tissue, most notably two chaperon-usher systems16: the type I fimbriae encoded by the fimA-K operon and the type III fimbriae encoded by the mrkA-I operon. The former has been shown to preferentially bind to mannose residues in E. coli but not in K. pneumoniae17, whereas the latter has high affinity to collagen18,19 and mediates adhesion to abiotic surfaces20. In silico studies have predicted the existence of many other chaperon–usher systems that could have specific tropism or be expressed in response to specific environmental cues21. In addition to surface adhesins, another important factor determining biofilm formation in K. pneumoniae is the extracellular capsule22–25 produced by most isolates26. On the one side, some studies revealed that the capsule can strongly inhibit biofilm formation by masking surface adhesins24 or by altering surface physico-chemical properties and thus limiting surface attachment and inter-cellular interactions27,28. On the other side, presence of some Klebsiella capsules has been shown to increase the formation of biofilm and be required for its maturation22. Thus, the role of the capsule in biofilm formation is convoluted and depends both on the physical interactions between the capsule and the environment25, and the genetic interactions between the capsule locus and the rest of the genome23. Numerous studies have focused on how different microbes increase biofilm formation by positively selecting for this trait29–31. Yet, how biofilm formation evolves when it is not under strong selection, or just as a mere by-product of the generic processes of adaptation is not currently understood. Indeed, adaptation of a given population to different novel environments may impact the Institut Pasteur, Université de Paris Cité, CNRS, UMR3525, Microbial Evolutionary Genomics, F-75015 Paris, France. ✉email: 1 Published in partnership with Nanyang Technological University A. Nucci et al. 2 1234567890():,; ability of the population to adhere and form a biofilm. This can have important consequences, for instance, in host colonisation or increased tolerance to antibiotics. Here, we measured the evolution of biofilm formation to determine whether it latently changes when it is not specifically selected. If it does, we sought to enquire if this evolutionary process takes place in a progressive manner or evolves by leaps. We hypothesise that changes in biofilm could be the result of alterations in other phenotypic traits that were under strong selection in our evolution experiment, and which are known to affect biofilm formation. We thus specifically tested for correlation in changes in population yield or surfaceattached polysaccharide production (capsule or others), and changes (...truncated)