Abstract Carbapenem resistance and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter , but many plasmid types in this genus have not been well-characterised. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains bla NDM-1 and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by p dif sites recognised by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1086 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin-antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus. Impact statement Acinetobacter species are particularly well-adapted for persistence in hospital environments where they pose a life-threatening infection risk to the most clinically-vulnerable patients. Plasmids with the potential to transfer multiple antibiotic resistance determinants between Acinetobacter species are therefore concerning, but most are not well-characterised. This work sheds further light on the poorly-understood mobile gene pool associated with Acinetobacter . We show here that GR13 plasmids carry a small set of core genes but have access to a highly diverse set of accessory segments that might provide fitness advantages under certain conditions. The complex evolutionary dynamics of GR13 plasmids appear to be driven by the exchange of dif modules and by the actions of a diverse population of insertion sequences. The novel dif modules characterised here emphasise the broader importance of these elements to the dissemination of accessory genes in Acinetobacter . This study has improved our understanding of the diversity and distribution of dif modules, plasmids that carry them, and how both disseminate in the continuum of Acinetobacter populations that link hospitals and the wider environment.

GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes