Novel Aminoglycoside Resistance Transposons and Transposon-Derived Circular Forms Detected in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

Abstract

Acinetobacter baumannii has emerged as an important opportunistic pathogen equipped with a growing number of antibiotic resistance genes. Our study investigated the molecular epidemiology and antibiotic resistance features of 28 consecutive carbapenem-resistant clinical isolates of A. baumannii collected throughout Sweden in 2012 and 2013. The isolates mainly belonged to clonal complexes (CCs) with an extensive international distribution, such as CC2 (n = 16) and CC25 (n = 7). Resistance to carbapenems was related to blaOXA-23 (20 isolates), blaOXA-24/40-like (6 isolates), blaOXA-467 (1 isolate), and ISAba1-blaOXA-69 (1 isolate). Ceftazidime resistance was associated with blaPER-7 in the CC25 isolates. Two classical point mutations were responsible for resistance to quinolones in all the isolates. Isolates with high levels of resistance to aminoglycosides carried the 16S rRNA methylase armA gene. The isolates also carried a variety of genes encoding aminoglycoside-modifying enzymes. Several novel structures involved in aminoglycoside resistance were identified, including Tn6279, ΔTn6279, Ab-ST3-aadB, and different assemblies of Tn6020 and TnaphA6. Importantly, a number of circular forms related to the IS26 or ISAba125 composite transposons were detected. The frequent occurrence of these circular forms in the populations of several isolates indicates a potential role of these circular forms in the dissemination of antibiotic resistance genes.

FIG 1

Illustrative positioning of the primers. Gray arrows, coding regions, with the arrowhead indicating the direction of transcription; blue boxes, insertion sequence elements; black double-headed arrow, a presumed genetic element carrying an antibiotic resistance gene of interest. The figure shows an inserted genetic element surrounded by two peripheral copies of an IS element (top) coexisting with a circular form with only one copy of these IS elements (bottom right) and an insertion site with the second copy (bottom left). The genetic context was verified using a number of internal primers (red arrows). Insertion of the genetic element was detected by PCR assays and sequencing using an outwards internal and an inwards external primer (one green arrow and one blue arrow). The occurrence of circular forms was detected by PCR assays and sequencing using peripheral outwards internal primers (the two green arrows). A deficiency of the genetic element in part of the population of each strain was investigated using inwards external primers (the two blue arrows).

FIG 2

Whole-genome SNP-based phylogenetic network (constructed using NeighborNet graph). The NeighborNet graph was constructed using SplitsTree (http://www.splitstree.org/) on the basis of the alignments of the whole-genome SNP concatenations of 28 Acinetobacter baumannii isolates. Strains ATCC 17978 (sporadic), AYE (CC1), ACICU (CC2), and SDF (sporadic, isolated from a human body louse) were included as reference strains. The corresponding sequence type (ST) or clonal complex (CC) was added next to the four main clades.

FIG 3

Genetic context of bla_OXA-24/40 and bla_OXA-72. (A) Genetic structures of plasmids pA105-2 and pA077. Gray arrows, coding regions, with the arrowhead indicating the direction of transcription; blue, red, and green boxes, ISAba31, iterons, and XerC/XerD-like sites, respectively. (B) Nucleotide sequence of the XerC/XerD-like sites surrounding the bla_OXA-24/40 and bla_OXA-72 genes. Blue, XerC/XerD-like sequences; red, polymorphisms.

FIG 4

Genetic context of the armA-positive elements. (A) Genetic structure and site of chromosomal acquisition of Tn6279 and ΔTn6279. (B) Co-occurrence of circular forms derived from Tn6279, Tn1548-like-1, and Tn6020b-1 in Acinetobacter baumannii isolate A071. (C) Plasmid insertion and co-occurrence of a circular form of Tn1584-like-2 in A. baumannii isolate A068. Blue labeled arrows, genes, with the arrowhead indicating the direction of transcription; blue boxes, insertion sequence elements; red arrow in panel A, the site of a deletion of 160 bp, making the only difference between Tn6020b-1 and -2; red circular arrows in panel C, the locations of internal deletions of 7,984 and 7,907 bp, resulting in the formation of circular forms derived from Tn1584-like-2-Δ1 and -Δ2, respectively.

FIG 5

Potential sources, intermediate steps of formation, genetic structure, and site of acquisition of the Ab-ST3-aadB element. (A) Partial diagrams of plasmids pTB11 (GB accession number AJ744860) and pSN254b (GB accession number KJ909290), representing potential sources for the two segments of a prospective Ab-ST3-aadB element. Black lines, the segments involved in the construction of Ab-ST3-aadB. The coding regions of these segments were labeled according to GenBank records. (B) Circular intermediate forms of Ab-ST3-aadB. The acquisition of IS26 was associated with a target site duplication, indicated by vertical lines. (C) Comparative analysis between Ab-ST3-aadB-negative Acinetobacter baumannii strain TCDC-AB0715 (GB accession number CP002522) and Ab-ST3-aadB-positive A. baumannii strains A085 (this study) and AB4857 (GB accession number AHAG01000030). The acquisition of Ab-ST3-aadB in A085 and AB4857 was not associated with a target site duplication. (D) Comparative sequence analysis between Ab-ST3-aadB-negative Acinetobacter baylyi strain ADP1 (GB accession number CR543861) and Ab-ST3-aadB-positive A. baylyi transformant Ab(II)3 (GB accession number JX041889). A. baylyi ADP1 was used as a recipient to detect the natural transformation of Ab-ST3-aadB from A. baumannii A064. The acquisition of Ab-ST3-aadB in A. baylyi Ab(II)3 was associated with a target site duplication, indicated by vertical lines. Arrows, coding regions oriented according to the direction of transcription.

FIG 6

Genetic context of aphA1. (A) Genetic structures of Tn6020a-1, Tn6020a-2, Tn6020a-3, Tn6020b-1, and Tn6020b-2. (B) Co-occurrence of transposon-derived circular forms corresponding to Tn6020a-1, Tn6020a-2, Tn6020a-3, Tn6020b-1, and Tn6020b-2. (C) Genetic context and location of Tn6020a-1 or Tn6020a-2 in isolates A084, A072, A070, and A082. Gray arrows, genes, as labeled, with the arrowhead indicating the direction of transcription; blue boxes, insertion sequence elements. In panels A and B, the labeled blue boxes were decorated with black arrows indicating the direction of transcription of the IS26 transposase gene; the lengths of the spacers upstream and downstream of aphA1b are presented in base pairs, and the structures were drawn to the indicated scale.

FIG 7

Genetic context of aphA6. (A) Genetic structure of TnaphA6 and TnaphA6-like. Gray arrows, coding regions, with the arrowhead indicating the direction of transcription; blue boxes, ISAba125 elements; black arrows, the direction of transcription of the transposase gene. Tnaph6 is highlighted. The structures were drawn to the indicated scale. (B) Genetic structure of plasmid pA105-1 (this study). Gray arrows, coding regions, with the arrowhead indicating the direction of transcription; blue and green boxes, ISAba125 and regions of repeated sequences, respectively. (C) Genetic structure of the circular form of TnaphA6. (D) Gel electrophoresis of PCR products amplified using primers surrounding the insertion site of TnaphA6 in isolates A074 and A105. The detection of three bands indicated that the population of each isolate could be divided into three parts, namely, part 1, where the site was intact (small band of 423 bp); part 2, where the site was inserted only by ISAba125 (medium-sized band of 1,513 bp); and part 3, where the site was inserted by TnaphA6 (large band of 3,498 bp).