Derivatization of pBACpAK entrapment vectors for enhanced mobile genetic element transposition detection in multidrug-resistant Escherichia coli

Abstract

Aim. Antimicrobial resistance 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 MGE 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, based on conserved regions identified from GenBank sequences containing known IS26 and IS1 insertions. Using colony PCR, intracellular transposition screening was performed on 194 tetracycline-resistant colonies from four E. coli ESI123 strains carrying different pBACpAK constructs. The derivatives showed increased MGE capture rates (10.7–73.1%) compared to the WT vector (3.75%), identifying multiple MGEs, including the novel composite transposon Tn7824. Tn7824 harbours the bla<inf>OXA-181</inf> 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.