Integron-associated structures underlie the accumulation of antibiotic resistance genes on plasmids beyond cassette arrays

The emergence and spread of multidrug-resistant bacteria posing serious threat to global public health has been a focus for many studies. Coexistence of integrons and antibiotic resistance genes (ARGs) has been widely observed in various environments. It has long been considered that most correlated ARGs were carried by integron gene cassettes. In this study, we show, for the first time, evidence that integron presence is linked to the accumulation of multiple ARGs beyond their cassette arrays, resulting in the formation of multi-ARG plasmids. By analyzing 1102 plasmid sequences retrieved from PLSDB RefSeq across incompatibility groups, host genera, and plasmid sizes, we show that the increased ARGs on integron-positive plasmids included not only ARGs present in integron cassette arrays but also those located outside integron regions. These effects were consistently observed across diverse incompatibility groups, host genera, and plasmid size ranges. Aminoglycoside resistance genes were found to be the most abundant both inside and outside integron regions, while beta-lactam, tetracycline, macrolide, and quinolone resistance genes were mostly found outside integron regions on integron-positive plasmids. We also observed the same ARG accumulation on integron-positive plasmids and lack of ARG accumulation on integron-negative plasmids from the same sampling sites by analyzing 32 long-read sequenced plasmids of Escherichia coli isolates collected from human and pig wastewater. This finding can reject the alternative explanation that integrons were co-selected with multi-ARG plasmids under the same antibiotic pressure. Our findings provide insights into the structural contexts underlying the accumulation of multiple ARGs on plasmids, which may inform new directions for future antimicrobial resistance surveillance and mitigation strategies.