# Impact of IS26 mobilization on genetic manipulation of multidrug-resistant Acinetobacter baumannii

## TL;DR

The study explores how IS26 transposition complicates genetic manipulation in multidrug-resistant Acinetobacter baumannii, offering insights into overcoming these challenges.

## Contribution

The study reveals how IS26 transposition interferes with gene editing in Acinetobacter baumannii and proposes methods to circumvent this issue.

## Key findings

- IS26 transposition disrupts gene knockout attempts in Acinetobacter baumannii by inserting into target regions.
- Suicide plasmids can evade IS26 interference through natural transformation and conjugation methods.
- IS26 is highly prevalent in Acinetobacter baumannii genomes, especially in IC2 strains, suggesting a role in their evolutionary success.

## Abstract

Multidrug-resistant Acinetobacter baumannii poses significant challenges for genetic manipulation, historically hindering research on this organism.

To elucidate the factors contributing to these difficulties, comA and xcpW knockouts were performed on a multidrug-resistant (MDR) clinical isolate of international clone 2 (IC2), designated HN85.

Through electroporation, both constructed telR-marked suicide plasmids were recruited via active IS26 transposition into adjacent genomic regions, complicating attempts to delete the target genes through homologous recombination. Transferred by natural transformation and conjugation methods, the suicide plasmids successfully evaded targeting by IS26 and ultimately achieved comA and xcpW knockouts. During mutant screening following transformation, false positive colonies consistently emerged on tellurite plates without undergoing plasmid integration. Genomic sequencing revealed that this tellurite resistance resulted from the interruption of pitA caused by IS26 transposition. To investigate whether the high transposition activity of IS26 was attributable to its high copy number in HN85, a single IS26 copy was introduced into a susceptible clinical A. baumannii isolate W068. Although W068 possesses a higher density of insertion sequence (IS) elements, IS26 remained preferentially mobilized and exhibited similar active transposition behavior in the new host cell.

IS26 is prevalent in A. baumannii genomes (78.1%, 698/931), particularly among strains belonging to IC2 (99.8%, 509/510), implying its significant role in the evolution and success of IC2. The potential implications of active IS26 transposition for gene editing and screening warrant careful consideration beyond just A. baumannii.