Complete Sequence of a bla(NDM-1)-Harboring Plasmid in an Acinetobacter bereziniae Clinical Strain Isolated in Argentina.

The New Delhi metallo-lactamase (NDM-1) was initially identified in clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sweden from a patient previously hospitalized in India (1). Since then, blaNDM-1 has frequently been reported in Enterobacteriaceae and Acinetobacter spp., with a fast dissemination in the Indian subcontinent, the Balkan countries, China, and the Middle East (2). NDM-1 producers generally associated with Enterobacteriaceae species have also been reported, albeit with much lower frequency, in Latin American countries, including Guatemala, Mexico, Colombia, and Brazil (3). Moreover, production of NDM-1 in this geographic region has also been noted in Acinetobacter baumannii in Honduras and Brazil (3, 4) and in Acinetobacter pittii in Paraguay and Brazil (5, 6). Here, we report the first case of an NDM-1-producing Acinetobacter species in Argentina, an Acinetobacter bereziniae clinical isolate. We describe also the complete sequence of a blaNDM-1-containing plasmid in this strain. (Part of this work was presented at the 10th International Symposium on the Biology of Acinetobacter 2015, Athens, Greece, 3 to 6 June 2015 [7].) A. bereziniae HPC229 was isolated on June 2014 from a blood sample of a 53-year-old female patient that underwent chemotherapy due to leukemia at a hospital located in Rosario, Argentina. The patient was treated with ciprofloxacin plus tigecycline, resulting in clinical and microbiological cure as evaluated by negative blood cultures. The patient was readmitted to the hospital 3 months later with symptoms of severe sepsis and died due to septic shock, with positive blood cultures that grew Escherichia coli. HPC229, originally identified as Acinetobacter lwoffii by the Vitek 2 system (bioMérieux), was reclassified by DNA sequence comparison analyses of its 16S rRNA, gyrB (8), and rpoB (9) genes. The highest identity was found to the corresponding orthologs of the A. bereziniae ATCC 17924 type strain (99.9%, 99.7%, and 99.8%, respectively). The antibiotic susceptibility profile of HPC229 was determined by either the Vitek 2 system or the agar dilution method. The interpretation of the obtained MICs based on CLSI breakpoints (10) indicated resistance to -lactams, including carbapenems (Table 1). PCR amplification using specific primers for the blaIMP, blaVIM, blaSPM, blaNDM, blaOXA-23-like, blaOXA-24/40-like, and blaOXA-58-like genes (11–13) and sequencing analysis revealed the presence of blaNDM-1. Evidence that blaNDM-1 was located in a plasmid was first obtained by plasmid curing (14) in which HPC229 was cultured in 5 ml of LB liquid medium containing 0.2 ml of 10% SDS for 48 h at 40°C. This procedure allowed the isolation of HPC229c, which showed a marked increase in susceptibility to -lactams (Table 1). Susceptibility to aztreonam as judged by the disk diffusion assay, in contrast, showed no differences between HPC229 and HPC229c. All conjugation attempts using HPC229 as the donor and Escherichia coli DH5 , A. baumannii ATCC 17978, or Pseudomonas aeruginosa PAO1 as the recipient (11) were unsuccessful. The genomic relatedness between HPC229 and HPC229c was confirmed using previously described PCR-based procedures (15), and the loss of blaNDM-1 was confirmed by specific PCR analysis. Analysis of whole-genome sequencing performed with pyrosequencing (454 Genome Sequencer FLX system; Roche Diagnostics) demonstrated that the blaNDM-1 metallo-lactamase gene was located in HPC229 in a 44,560-bp plasmid, designated pNDM229. This gene was found located inside a composite transposon bracketed by two copies of ISAba125 and identical to that first described in A. baumannii (16) and designated Tn125 by Poirel et al. (17). The immediate genetic environment of Tn125 in