The role of biofilms in driving AMR-hypervirulence convergence and pathogenicity in K. pneumoniae

Antimicrobial resistance (AMR) is a significant global health threat, making bacterial infections harder to treat, resulting in longer illnesses, and ultimately untreatable infections. Particularly, bacteria such as Klebsiella pneumoniae with resistance to carbapenem antibiotics are of critical concern. Resistance to carbapenems is predominantly carried on mobile DNA called plasmids which transfer accessory genes between different bacteria. Klebsiella are notorious for carrying multiple unique plasmids. Importantly, AMR is not the only accessory genes carried on plasmids. Concerningly, K. pneumoniae plasmids carrying hyper-virulence (HV) genes are arising. Historically there was a divide between AMR and HV K. pneumoniae. However, since both AMR and HV are conferred by plasmids, increasingly these plasmid types are observed converging in the same K. pneumoniae cell. This results in bacteria that have both HV and AMR. After convergence these plasmids can join together, rearrange, or integrate into the bacterial chromosome. We are at the relatively early stages of HV-AMR K. pneumoniae emergence. Understanding the fundamental biology that drives multi-plasmid convergence and subsequent evolution and adaptation to multiple plasmids will improve our ability to predict and prevent convergence and understand the implications for bacterial physiology. Research, including our own, has shown that plasmid transfer occurs at much higher levels in bacterial biofilms. Biofilms are complex structured communities in which bacteria demonstrate unique characteristics. Furthermore, biofilms are the context in which bacteria exist in the "real-world", e.g. in the environment and within recalcitrant infections. Together, this means convergence of AMR and HV plasmids may be much more prevalent in biofilm settings than expected from experiments using liquid cultures. Our aim is to use AMR and HV plasmids to investigate how the biofilm lifestyle drives unique plasmid dynamics and evolutionary trajectories for convergent cells, and the impact of this evolution on bacterial virulence. We will accomplish these aims by generating distinct fluorescently tagged AMR and HV plasmids. These will be used to compare AMR and/or HV plasmid dynamics including convergence rates (conjugation frequency, fitness impact and rate of plasmid loss) in biofilm and planktonic culture using experimental biology and mathematical modelling. In parallel, we will use eco-evolutionary experiments to compare multi-plasmid convergence dynamics in biofilm versus planktonic populations and track the longer-term evolutionary pathways of convergent populations, including monitoring how multiple plasmids may rearrange. We will identify and recapitulate the causal mutations enabling stable plasmid convergence. The strains generated during these evolution experiments will be used to characterise the impact of evolution on plasmid dynamics and the convergence of HV-AMR K. pneumoniae. Finally, using a combination of microbiological techniques, in vitro and in vivo virulence assays we will determine how convergence and biofilm evolution impact upon virulence. We will do this by investigating convergent, ancestral, and evolved populations virulence attributes, macrophage survival and immune response. This will be followed by testing select strains in mouse pneumonia models to assess immune interactions and pathogenic potential. From this interdisciplinary programme of work we will address unanswered questions about the drivers and dynamics of plasmid convergence in K. pneumoniae, their contribution to HV-AMR emergence, and impact on pathogenicity. Over the long term this information will help us devise strategies to predict and mitigate the emergence of HV-AMR K. pneumoniae, and the principles discovered here could be applied to other bacterial systems.