Derivatization of pBACpAK Entrapment Vectors for Enhanced Mobile Genetic Elements Transposition Detection in Multidrug-Resistant Escherichia coli

Aim: Antimicrobial resistance (AMR) poses a critical global health threat, driven by the dissemination of resistance genes via mobile genetic elements (MGEs). This study aims to enhance the detection of MGEs insertions in multidrug-resistant Escherichia coli by derivatizing the pBACpAK entrapment vector. Methods and results: Three derivatives were constructed with additional nucleotides upstream of the cI repressor gene, resembling experimentally proven insertion sites of transposable elements. The derivatives showed increased MGE capture rates (10.7–73.1%) compared to the wild-type vector (3.75%), leading to the identification of multiple MGEs, including the novel composite transposon Tn7824. Tn7824 harbors the blaOXA-181 carbapenem resistance gene and the qnrS1 quinolone resistance gene, highlighting the clinical relevance of these findings. Long-read sequencing of transposants confirmed the accuracy of MGE identification and structural characterization, which also revealed chromosomal integration events of the pBACpAK derivatives mediated by flanking insertion sequences. Conclusions: The modifications introduced in the pBACpAK derivatives could increase the detection of transposition events by alleviating spatial constraints, allowing for more robust MGE detection. Impact statement: These findings highlight the utility of entrapment vectors for studying MGE-associated AMR dissemination in clinical and environmental settings. By improving the detection of novel and clinically relevant MGEs, such as Tn7824, this approach contributes to a better understanding of resistance gene mobility and may aid future AMR surveillance efforts.