Multi-replicon Architecture Drives the Global Accumulation of Resistance to Antimicrobials, Biocides, and Metals in IncF and IncH Plasmids

Plasmids are major vectors driving the environmental dissemination of antimicrobial resistance (AMR) and other stress-resistance traits. The convergence between AMR and tolerance to multiple environmental stressors has become increasingly concerning, as these interactions intensify horizontal gene transfer and enhance plasmid conjugation. In this study, we investigated whether the co-occurrence of resistance determinants against different stressors results from random aggregation or statistically meaningful associations. We analyzed 25,116 complete plasmids from PLSDB and applied multivariate correspondence analysis to examine relationships between incompatibility groups and resistance categories. Pairwise co-occurrence patterns among resistance genes were assessed using Fisher's exact test to determine whether their distribution deviated from randomness. IncF and IncH plasmids emerged as the incompatibility groups most strongly enriched in multidrug resistance, showing a marked tendency to co-carry genes conferring tolerance to antimicrobials, biocides, and metals-traits highly relevant under environmental co-selection. While pairwise tests did not reveal significant associations between specific gene pairs, the broader patterns of resistance accumulation highlight the structural evolution of plasmids via multireplicon cointegration as a primary mechanism for multi-stressor resistance. Our findings underscore the ecological importance of multireplicon plasmids, particularly those involving IncF and IncH, as high-risk vectors that sustain multi-stressor resistance in microbial communities.