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

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.

FIG 1

Sequence alignment of the attTn7-associated sequence and the Tn7 insertion site of Acinetobacter baumannii ATCC 17978 and clinical isolates used in this study, showing the highly conserved sequence homology between the strains. The insertion site is located 24 bp downstream of the glmS2 gene in a neutral location (as shown by the arrow). The stop codon of the glmS2 gene is outlined in the red box. The consensus sequence is shown at the bottom of the alignment and displays 100% identity.

FIG 2

Construction of the pUC18T-mini-Tn7T-Apr-LAC and pUC18T-mini-Tn7T-Zeo-LAC plasmids. The plasmids were created by replacing the aacC1 gentamicin resistance gene of pUC18T-mini-Tn7T-Gm-LAC with apramycin and zeocin resistance genes, respectively. Plasmid sequences were confirmed using Illumina Hi-Seq next-generation sequencing (MGH CCIB DNA Core Facility, Cambridge, MA, USA). Maps were constructed using SnapGene software (GSL Biotech).

FIG 3

Visualization of the inserted fluorescent-protein genes in different A. baumannii strains using the ImageXpress Micro 4 high-content imaging system with a 40× objective. Images for LAC-4::Ruby Red and 17978::mCherry was acquired using a Texas Red filter, while those for AB030::sfGFP and AB030::mTurquoise were acquired using an FITC filter and a DAPI filter, respectively. The images acquired using the filters were compared to the bright-field images of the same to observe the presence and distribution of cells. Emission and excitation wavelengths are provided in Materials and Methods.

FIG 4

PCR confirmation of mini-Tn7T-Apr-LAC and mini-Tn7T-Zeo-LAC genomic insertions in the A. baumannii strains used in this study. The mini-Tn7T-Apr-LAC and mini-Tn7T-Zeo-LAC genomic insertions in A. baumannii strains were confirmed using the ABglmS_F_New and Tn7R primers, which give the expected 368-bp band. Lane M, 100-bp molecular size marker (New England Biolabs); lane 1, A. baumannii ATCC 17978::mini-Tn7T-Apr-LAC; lane 2, A. baumannii ATCC 17978::mini-Tn7T-Zeo-LAC; lane 3, negative control for A. baumannii ATCC 17978; lane 4, A. baumannii AB030::mini-Tn7T-Apr-LAC; lane 5, negative control for A. baumannii AB030; lane 6, A. baumannii AB031::mini-Tn7T-Apr-LAC; lane 7, A. baumannii AB031::mini-Tn7T-Zeo-LAC; lane 8, negative control for A. baumannii AB031; lane 9, A. baumannii LAC-4::mini-Tn7T-Apr-LAC; lane 10, A. baumannii LAC-4::mini-Tn7T-Zeo-LAC; lane 11, negative control for A. baumannii LAC-4.

FIG 5

Deletion of apramycin or zeocin resistance markers from A. baumannii using pFLP2A. (a) Photographs of the LB agar plate (left) and the LB agar plate supplemented with 100 µg/ml of apramycin (right) to screen for the loss of the Apr^r resistance gene by using pFLP2Z, encoding Flp recombinase. The absence of growth on LB plus Apr confirms the loss of aac(3)-IV (Apr^r marker). (b) Photographs of the LB agar plate (left) and the LB agar plate supplemented with 100 µg/ml of zeocin (right) to screen for the loss of Zeo^r resistance gene. The absence of growth on LB plus Zeo confirms the loss of ble (Zeo^r marker). (c) The loss of the relevant resistance markers was further confirmed with PCR. Lanes 1, 3, and 5 show the 998-bp band corresponding to the aac(3)-IV gene in ATCC 17978, LAC-4, and AB031, respectively. Lanes 2, 4, and 6 show the loss of the 998-bp band in the pFLP2Z-treated ATCC 17978, LAC-4, and AB031 strains, respectively. Lanes 7 and 8, positive and negative controls for the 998-bp aac(3)-IV band, respectively. Lanes 9, 11, and 13 show the 569-bp band corresponding to the ble gene in ATCC 17978, LAC-4, and AB031, respectively. Lanes 10, 12, and 14 show the loss of the 569-bp band in the pFLP2A-treated ATCC 17978, LAC-4, and AB031 strains, respectively. Lanes 15 and 16, positive and negative controls for the 569-bp ble band, respectively. Lanes M, 100-bp molecular size marker.