The R plasmid pRAS2 in the fish pathogen Aeromonas salmonicida is a global mediator of antibiotic resistance in aquaculture and can be used by pRAS3 for mobilization.

Horizontal gene transfer (HGT) mediated by conjugative plasmids is a major driver of antimicrobial resistance (AMR) dissemination in aquatic environments. In Aeromonas salmonicida, plasmid-mediated HGT is largely driven by the pRAS plasmid family. Here we present genomic and experimental characterization of pRAS2 from A. salmonicida strain 1682/92 and demonstrate its role in helper-dependent mobilization of the co-resident pRAS3. Whole genome sequencing (WGS) divided the pRAS2 backbone into three modules. Module A encodes replication, maintenance and inheritance stability functions, including transposase, resolvase, partitioning and antitoxin-associated genes. Module B encodes a canonical type IV secretion system (T4SS) conjugation module containing virB2-virB6, virB8-virB11 and virD4 with virB2 and virB5 forming the transfer pilus. Module C encodes the resistance determinants sul2, aph(6)-Id, neo and Tet 31. Modules A and B were organized as in plasmids of Vibrio alginolyticus, Edwardsiella piscicida and Piscirickettsia salmonis, indicating that pRAS2 shares a conserved backbone with plasmids of other aquatic bacteria, including fish pathogens isolated on different continents. Module C was the most variable region: sul2 and aph(6)-Id were shared with pEIB202 of E. piscicida, whereas the tetracycline resistance determinant remained the same, being Tet 31 in pRAS2 as well as in pEIB202, p3PS10 and p3PS11, but absent from pVb1978 (Vibrio alginolyticus), pVIM-20710 (Enterobacter hormaechei) and p4PS9. The pRAS3 plasmids of strains 1682/92 and 2131/90, used as donors in conjugation experiments with an Escherichia coli DH5α recipient, carried the same mobilization-associated (MOB) genes and the tetracycline resistance determinant Tet C. Transfer of pRAS3 occurred only from strain 1682/92, which carried both pRAS2 and pRAS3; no transconjugant colonies were recovered from strain 2131/90, which carried pRAS3 alone. WGS showed an identical organization of the MOB, tetA(C) and tetR(C) genes in the 1682/92 donor and its transconjugant, providing experimental evidence that pRAS2 functions as a conjugative helper plasmid mediating mobilization of pRAS3. Conjugation trials performed with these strains between 1993 and 1995 further show that pRAS2 is self-transmissible, transferring autonomously at a frequency approximately three orders of magnitude higher than that of its co-mobilization of pRAS3. This demonstrates plasmid-plasmid cooperation within the pRAS plasmid family as a mechanism supporting the spread of AMR genes in aquatic bacterial communities.