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By transferring ecologically important traits between species, plasmids drive genomic divergence and evolutionary innovation in their bacterial hosts. Bacterial communities are often diverse and contain multiple coexisting plasmids, but the dynamics of plasmids in multispecies communities are poorly understood. Here, we show, using experimental multispecies communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to ‘the dilution effect’; an epidemiological phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species. In addition, plasmid horizontal transmission was also affected by plasmid diversity, such that the rate of plasmid conjugation was reduced from coinfected host cells carrying both plasmids. In diverse microbial communities, plasmid spread may be limited by the dilution effect and plasmid-plasmid interactions reducing the rate of horizontal transmission.

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.

Bacteria exist in complex communities that include not only many other bacterial species but also diverse mobile genetic elements which accelerate bacterial evolution via horizontal gene transfer. How multiple mobile genetic elements interact in bacterial populations and how this affects plasmid dynamics remains poorly understood. We experimentally evolved populations of Pseudomonas fluorescens SBW25 either singly-infected or co-infected with two conjugative mercury resistance plasmids either with or without positive selection (i.e. addition of Hg(II)). We show that co-infection led to higher levels of mercury resistance in the bacterial population in the absence of positive selection. Consistent with this, in the absence of positive selection the plasmids could stably coexist within bacterial cells resulting in the maintenance co-infection. By contrast, with positive selection, plasmid coexistence was destabilised, leading to the dominance of a single plasmid in several replicate bacterial populations. Plasmid co-infection appears to alter the trajectory of compensatory evolution to ameliorate the cost of plasmid carriage and may have selected for alternative mechanisms compared to singly-infected populations. Stable plasmid co-infection without positive selection for plasmid-encoded traits suggests that environments where plasmids are useless may be hot-spots for genomic innovation via plasmid-plasmid recombination.