Characterization of the diverse plasmid pool harbored by the blaNDM-1-containing Acinetobacter bereziniae HPC229 clinical strain

Abstract

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes, and functional analysis of their transfer regions revealed the mobilizable nature of them. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Fig 1. Schematic representation of pAbe229-114 plasmid.

ORFs are shown as arrows indicating the direction of transcription. Disrupted/incomplete genes are indicated with a “Δ” symbol preceding the gene denomination. The six consecutive blue light arrows upstream of the repB gene denote the predicted iteron sequences linked to the oriV region (see Table 1 for details). The putative oriT located within the conjugal/transfer region is indicated with a closed circle. The Tn6637 transposon bracketed by ISAba1 elements (one of them truncated, ΔISAba1) is highlighted in light yellow. The 9-bp (AATAAAGAT) direct repeats found at the insertion target site (DR) are indicated next to the external inverted repeats. From the outer circle inward, the circles display: i) the predicted ORFs. The colored arrows describe the location, identification, and direction of transcription of genes with described functions in databases. ORFs with undescribed functions are indicated by open arrows, ii) in grey with different designs, homologous regions described in plasmids/chromosomes of Acinetobacter non-bereziniae strains (see Table 2 for details), iii) GC content relative to the mean GC content of the plasmid, iv) GC skew, where green and purple represent positive and negative skew, respectively, v) scale in kbp. The hybridation sites of the PCR primer pairs used to verify the structure of pAbe229-114 as well as those used to amplify the transfer region (see Materials and Methods) are also indicated (S1 Table).

Fig 2

Schematic representation of plasmids pAbe229-15 (A), pAbe229-9 (B), pAbe229-4 (C) and pAbe229-1 (D). The regions encoding RNAI- and RNAII-homologous sequences in pAbe229-4 are highlighted in light blue, and the direction of transcription is also indicated in each case. In pAbe229-1, the dark triangle denotes a high-AT region (putative oriV) predicted on the basis of the cumulative GCskew (see the text). Plasmids are not drawn to scale. For details, see the legend to Fig 1.

Fig 3. Phylogenetic analysis of HPC229 plasmid relaxases.

A ML tree was inferred from the alignments of the first 300 amino acids of the N-terminal domains of MOB_Q relaxases from A. bereziniae and the Acinetobacter local database [3,30]. The MOB_Q1, MOB_Q2, MOB_Q3 and MOB_QAci sub-families are indicated. Relaxases encoded in A. bereziniae genomes are specified with black arrows, and those corresponding to HPC229 plasmids are additionally highlighted in bold. In the case of the CHI-40-1 draft genome (GenBank accession number CDEL01000000.1), the contig number in which a given relaxase gene was identified is additionally indicated. The evolutionary scale (number of amino acid substitution per site) is indicated at the botton left. Bootstrap values higher than 50% (100 replications) are indicated at the branching nodes of the ML tree.

Fig 4. Identification of homologous sequences to HPC229 plasmids in other Acinetobacter genomes.

The colored arrows in the outer ring describe the location, identification, and direction of transcription of genes in the corresponding plasmids with described functions in databases. ORFs encoding for unknown functions are indicated by open arrows. In the subsequent rings, the regions of homology between the corresponding plasmid sequences and genome sequences of other A. bereziniae strains (indicated by arcs of different colors, see key near the upper right margin) as detected by BlastN searches are shown. The strains have been located from the highest (outer rings) to the lowest (inner rings) sequence coverage. The height of the colored arcs in each case is proportional to the percentage of nucleotide identity obtained in the BlastN search. BlastN hits with ≥70% nucleotide identity and a minimum alignment length of 1,000 bp (pAbe229-114) or 300 bp (pAbe229-15, pAbe229-9 and pAbe229-4) are detailed in Table 2. The darker lines in the arcs mark overlapping hits. The external Abe1-Abe12 grey regions indicate homologous sequences found in Acinetobacter non-bereziniae genomes (For details see Figs 1 and 2; and also Table 2).