Next Generation of Tn-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii.

The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of In this study, mini-Tn vectors with zeocin and apramycin selection markers were created by cloning the and genes, respectively, enabling either inducible gene expression (pUC18T-mini-TnT-Zeo-LAC and pUC18T-mini-TnT-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-TnT-Zeo and pUC18T-mini-TnT-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn vectors described above. Combinations of these novel mini-Tn plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of via these apramycin and zeocin mini-Tn constructs to demonstrate their application. is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of displaying MDR and XDR phenotypes.