Insertion sequences (ISs) promote the transmission of antimicrobial resistance genes (ARGs) across bacterial populations. However, their contributions and dynamics during the transmission of resistance remain unclear. In this study, we selected IS26 as a representative transposable element to decipher the relationship between ISs and ARGs and to investigate their transfer features and transmission trends. We retrieved 2656 translocatable IS26‐bounded units with ARGs (tIS26‐bUs‐ARGs) in complete bacterial genomes from the NCBI RefSeq database. In total, 124 ARGs spanning 12 classes of antibiotics were detected, and the average contribution rate of IS26 to these genes was 41.2%. We found that IS26‐bounded units (IS26‐bUs) mediated extensive ARG dissemination within the bacteria of the Gammaproteobacteria class, showing strong transfer potential between strains, species, and even phyla. The IS26‐bUs expanded in bacterial populations over time, and their temporal expansion trend was significantly correlated with antibiotic usage. This wide dissemination could be due to the nonspecific target site preference of IS26. Finally, we experimentally confirmed that the introduction of a single copy of IS26 could lead to the formation of a composite transposon mediating the transmission of “passenger” genes. These observations extend our knowledge of the IS26 and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.

Pseudomonas aeruginosa is a significant pathogen mainly causing healthcare-associated infections (HAIs). Newly emerging high-risk clones of P. aeruginosa with elevated virulence profiles furtherly cause severe community-acquired infections (CAIs). Usually, it is not common for P. aeruginosa to co-carry exoU and exoS genes, encoding two type Ⅲ secretion system (T3SS) effectors. The pathogenicity mechanism of exoS⁺/exoU⁺ strains of P. aeruginosa remains unclear. Here, we provide detailed evidence for a subset of hypervirulent P. aeruginosa strains, which abundantly co-express and secrete the T3SS effectors ExoS and ExoU. The exoS⁺/exoU⁺ P. aeruginosa strains were available to cause both HAIs and CAIs. The CAI-associated strains could elicit severe inflammation and hemorrhage, leading to higher death rates in a murine acute pneumonia model, and had great virulence potential in establishing chronic infections, demonstrating hypervirulence when compared to PAO1 (exoS⁺/exoU) and PA14 (exoS^-/exoU⁺). Both ExoS and ExoU were co-expressed and co-secreted in abundance in exoS⁺/exoU⁺ strains. Their abundant protein secretion could boost exoS⁺/exoU⁺ strains’ potentials for cytotoxicity in vitro and pathogenicity in vivo. Genomic evidence indicates that exoU acquisition is likely mediated by horizontal gene transfer (HGT) of the pathogenicity island PAPI-2, while deletion of exoU was sufficient to mitigate virulence in the exoS⁺/exoU⁺ strains. Furthermore, bioinformatics analysis showed that such exoS⁺/exoU⁺ P. aeruginosa strains turned out to be widely distributed across the globe. Overall, the research provide detailed evidence for the high virulence and epidemicity of exoS⁺/exoU⁺ strains of P. aeruginosa, highlighting an urgent need for surveillance against these high-risk hypervirulent strains.