Plasmid diversity in an Acinetobacter bereziniae clinical strain, HPC229: antimicrobial and heavy metal resistance genes, different replicases, and XerC/D-flanked modules contribute to the adaptability to different habitats and to the Acinetobacter plasmi

REPIZO GUILLERMO; VIALE ALEJANDRO M.; MARCHIARO PATRICIA; BROVEDAN, MARCO A.; LIMANSKY ADRIANA S.

Seattle

Simposio; Plasmid Biology 2018; 2018

Acinetobacter bereziniae (Abe, formerly Acinetobacter genomic species 10), primarily isolated from clinical specimens and healthcare associated environments, is generally susceptible to antimicrobials. However, Abe clinical strains bearing metallo-β-lactamase genes have recently been described. We have previously reported the whole-genome sequence of the carbapenem-resistant A. bereziniae strain HPC229 (1), which contains a 44 kbp plasmid, pNDM229, carrying a blaNDM-1 gene (2). We characterized here the whole HPC229 plasmidic content, and conducted a comparative analysis against the Acinetobacter spp database. DNA sequencing was conducted using a 454 pyrosequencing platform, following by assembly of contigs into five novel plasmid structures which were validated by PCR/primer walking. Genes encoding replicases of the Rep-3 superfamily (pfam1051) were identified among them, and classified according to a recently-described A. baumannii Rep classification scheme (3). These plasmids were designated pAbe229-114 (114 kbp; 42.1% GC), pAbe229-15 (15 kbp; 35.0%), pAbe229-9 (9 kbp; 35.4%), pAbe229-4 (4 kbp; 36.4%) and pAbe229-1 (1 kbp; 36.9%). pAbe229-114, pAbe229-15 and pAbe229-9 encode proteins exhibiting replication, stability, transfer, and adaptability functions. The Rep proteins from pAbe229-114 and pAbe229-15 could be confidentially assigned to GR13 and GR12, respectively, while Rep from pAbe229-9 represents a novel GR. The stability modules found among HPC229 plasmids include several toxin-antitoxin (TA) systems such as RelE/B (pAbe229-114 and pAbe229-15); HipA/B and ParE/D (pAbe229-114); and BrnT/A and Doc (pAbe229-9). Concerning transfer functions, mobA genes were identified in pAbe229-15 and pAbe229-9, whereas a truncated conjugative module was located in pAbe229-114. Adaptability genes included heavy metal ions (copper, arsenic and nickel) resistance loci in pAbe229-114. Comparative analyses of pAbe229-114, pAbe229-15 and pAbe229-4 plasmid sequences against Abe and other Acinetobacter nucleotide databases revealed regions displaying high nucleotide identity (>85% and >80%, respectively), suggesting both intra- and inter-species recombinatorial exchange of particular plasmid regions. Shorts sequences recognized by the XerC/D recombinases were searched by using a consensus motif defined in A. baumannii (4) as a query, and 13 total XerC/D-like sites were recognized: 2 in pAbe229-114; 5 in pAbe229-15; 4 in pAbe229-9; and 2 in pAbe229-4. Notably, transfer, stability and replication regions from pAbe229-9 and pAbe229-15 were flanked by these sites, suggesting a possible mechanism of mobilization mediated by site-specific recombination and co-integrate formation (4).In summary, our analysis identified 6 plasmids in Abe HPC229 strain from which only some carried genes involved in resistance to antimicrobials (pNDM229, blaNDM-1, aphA6, 2), and heavy metals (pAbe229-114). From them, pAbe229-114, pAbe229-15 and pAbe229-9 carried Rep-3 replicases, while no replicases were detected in pAbe229-4 and pAbe229-1. Moreover, four HPC229 plasmids harbored each more than one XerC/D recognition sites bracketing specific regions, suggesting that they could mediate their dissemination through cointegrate formation (4). The number and diversity of plasmids harbored by HPC229 strongly suggests that this species acts as a plasmid reservoir contributing to the plasmidome of the Acinetobacter genus and the adaption of its members to clinical and environmental habitats.1- Brovedan et al., Gen Announc. 4(2): e00117-16, 2016. 2- Brovedan et al., AAC 59:6667?69, 2015. 3- Cameranesi et al., J Infect Dis Epidemiol 3:046, 2017. 4- Cameranesi et al., Front Microbiol. 9: 66, 2018.