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

Abstract

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.