Characterisation of a new megaplasmid family associated with the spread of multidrug resistance in Pseudomonas aeruginosa

Unlike in other important pathogens, the role of plasmids in the emergence of antimicrobial resistance (AMR) in Pseudomonas aeruginosa (Pa) has remained largely unaddressed. Previous work on AMR in Pa has mostly used genome sequencing methods that are limited because of the difficulty of using short-read data to detect and reconstruct complex plasmids. Here, using superior long-read sequencing, comprehensive bioinformatics analyses, and experimental characterization, we uncover an emerging family of important Pseudomonas megaplasmids and report its contribution to dissemination of multidrug resistance (MDR) on a global scale. Firstly, we identified large plasmids with a key role in the spread of MDR in a hospital in Thailand, and characterised their resistance regions revealing evidence of duplication, recombination and shared repeats, indicative of dynamic adaptation. Applying phylogenomics and pangenomics approaches we linked related megaplasmids and defined a core and pangenome for the family, exposing wide variations in AMR genes carriage. We then surveyed thousands of publicly available genomes, leading to discovery of dozens of megaplasmid relatives overlooked in multiple datasets, including already published studies. By integrating all this information and looking beyond the pathogenic species we gained valuable insights into the evolution of the megaplasmid family and revealed its widespread and multispecies distribution. We also showed that members of this family are stable in the absence of antibiotic pressure, bear no fitness cost to their host, and can be readily transferred between different Pseudomonas species. Our findings expand the bacterial plasmidome and provide insights on how MDR plasmids emerge from environmental reservoirs.