Next Generation of Tn 7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii

Kaleigh Ducas-Mowchun; P Malaka De Silva; Leandro Crisostomo; Dinesh M Fernando; Tzu-Chiao Chao; Peter Pelka; Herbert P Schweizer; Ayush Kumar

doi:10.1128/AEM.00066-19

Next Generation of Tn 7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii

Appl Environ Microbiol. 2019 May 16;85(11):e00066-19. doi: 10.1128/AEM.00066-19. Print 2019 Jun 1.

Authors

Kaleigh Ducas-Mowchun^#¹, P Malaka De Silva^#¹, Leandro Crisostomo¹, Dinesh M Fernando¹, Tzu-Chiao Chao², Peter Pelka¹, Herbert P Schweizer³, Ayush Kumar^{4

5}

Affiliations

¹ Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
² Department of Biology, University of Regina, Regina, Saskatchewan, Canada.
³ University of Florida, College of Medicine, Department of Molecular Genetics and Microbiology, Emerging Pathogens Institute, Gainesville, Florida, USA.
⁴ Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada Ayush.Kumar@umanitoba.ca.
⁵ Manitoba Chemosensory Biology Group, University of Manitoba, Winnipeg, Manitoba, Canada.

^# Contributed equally.

Abstract

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 Acinetobacter baumannii In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the ble and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 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-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application.IMPORTANCEAcinetobacter baumannii 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 A. baumannii 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-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.

Keywords: apramycin; cloning; gene expression; single copy; zeocin.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acinetobacter baumannii / drug effects*
Acinetobacter baumannii / genetics*
Acinetobacter baumannii / isolation & purification
Anti-Bacterial Agents / pharmacology
Bacterial Proteins / genetics
Bleomycin / pharmacology
Cloning, Molecular
DNA Transposable Elements / genetics*
Drug Resistance, Multiple, Bacterial / genetics*
Escherichia coli / genetics
Gene Expression Regulation, Bacterial
Genes, Bacterial / genetics
Genetic Vectors
Humans
Microbial Sensitivity Tests
Plasmids / genetics
Promoter Regions, Genetic
Sequence Alignment
Transformation, Bacterial

Substances

Anti-Bacterial Agents
Bacterial Proteins
DNA Transposable Elements
Bleomycin
Zeocin