Gain and loss of plasmid-borne antibiotic resistance genes are associated with their chromosomal counterparts and the broader plasmid gene repertoire in Enterobacteriaceae

Plasmids are central vehicles for the dissemination of antibiotic resistance genes (ARGs). They are among the most mobile and evolvable genetic elements, with broad host ranges and high rates of gene turnover, making them especially effective in spreading antibiotic resistance across bacterial lineages. Using the phylogeny-aware gene gain and loss model applied to 6,895 Enterobacteriaceae genomes, we quantified four evolutionary processes--gene gain, loss, expansion, and reduction-- for plasmid-borne genes. We found that, overall, plasmid-borne ARGs (pARGs) exhibit similar gain and loss rates compared with other plasmid genes, but significantly higher expansion and reduction rates. All four processes were strongly species dependent, with only a minor influence of antibiotic class. Further, pARGs were co-transferred with a broad range of plasmid functional genes, including defense, metabolic, and mobilome modules throughout evolutionary history. Moreover, bacterial clades harboring chromosomal ARGs (cARGs) showed significantly higher acquisition of plasmid-borne resistance than did their sister clades lacking corresponding cARGs. Our results demonstrate that pARGs in Enterobacteriaceae are co-transferred with the broader plasmid gene repertoire on an evolutionary timescale, whereas copy-number dynamics are distinct from plasmids. Moreover, cARGs can serve as markers of lineages predisposed to accumulate plasmid-mediated resistance. ImportancePlasmids play a central role in the spread of antibiotic resistance genes (ARGs), and the long-term evolutionary behavior of plasmid-borne ARGs (pARGs) could provide insights into the emergence of novel multidrug resistance. We studied nearly 7,000 Enterobacteriaceae genomes and show that pARGs evolve through the same gain and loss processes as other plasmid genes but exhibit markedly higher and species-dependent copy number changes. Moreover, bacterial lineages carrying chromosomal ARGs are significantly more likely to acquire additional plasmid-mediated resistance. This association suggests that plasmid and chromosomal ARGs could act synergistically to accelerate resistance accumulation within specific lineages. Identifying such evolutionarily high-risk lineages may provide a basis for predicting and monitoring the emergence of multidrug resistance.