Drivers of antimicrobial resistance gene clustering in plasmids: distribution, host clinical relevance, and plasmid mobility.

Plasmids play a central role in the dissemination of antimicrobial resistance genes (ARGs) through horizontal gene transfer. However, the extent to which different resistance determinants are associated and how these associations vary with plasmid mobility and bacterial host context remains poorly understood. Here, we analysed more than 52,000 complete bacterial plasmids to investigate pairwise co-occurrence patterns among ARGs conferring resistance to 28 antibiotic classes. We found that non-random ARG associations are widespread, with more than half of all detected resistance-class combinations occurring more frequently than expected. Conjugative plasmids exhibited substantially more co-occurrences than mobilizable or non-transmissible plasmids, consistent with their prominent role in horizontal gene transfer. Although most ARG combinations are present in plasmids from both ESKAPEE pathogens and non-ESKAPEE species, several associations differed significantly in prevalence between the two groups. Among all resistance classes, tetracycline resistance genes showed the broadest spectrum of associations, whereas nitroimidazole resistance genes occurred exclusively in isolation. Together, these findings reveal that ARG co-occurrence in plasmids is structured rather than random and is strongly associated with both plasmid mobility and host context, providing new insights into the evolutionary organization of multidrug resistance.