Complete Sequence of a bla _NDM-1 -Harboring Plasmid in an Acinetobacter bereziniae Clinical Strain Isolated in Argentina

Abstract

b 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, bla NDM-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 bla NDM-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 bla IMP , bla VIM , bla SPM , bla NDM , bla OXA-23-like , bla OXA-24/40-like , and bla OXA-58-like genes (11-13) and sequencing analysis revealed the presence of bla NDM-1 . Evidence that bla NDM-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 Pseudo...

The plasmid diversity ofAcinetobacter bereziniaeHPC229 provides clues on the ability of the species to thrive on both clinical and environmental habitats

“…The different contigs obtained after HPC229 genomic pyrosequencing were extensively analyzed to search for plasmid sequences other than pNDM229 characterized in detail previously [20]. Among them we identified 5 putative plasmid sequences, which were thoughtfully assembled in silico and the resulting inferred final structures subsequently validated by PCR using specifically designed primer pairs (S1 Table, Figs 1, 2 and 4).…”

“…We have recently characterized by WGS a carbapenem-resistant Acinetobacter bereziniae strain isolated in Argentina designated HPC229 [19]. HPC229 carries a bla NDM-1 -bearing carbapenem-resistance plasmid of 44 kbp designated pNDM229 [20], which displays a similar structure than plasmid pNDM-BJ01 carried by an A. lwoffii strain isolated in China [21]. This strongly suggested that pNDM229 was acquired by HPC229 as the result of HGT from other member of the genus, and selected due to the antimicrobial pressure common to the clinical setting [20].…”

“…HPC229 carries a bla NDM-1 -bearing carbapenem-resistance plasmid of 44 kbp designated pNDM229 [20], which displays a similar structure than plasmid pNDM-BJ01 carried by an A. lwoffii strain isolated in China [21]. This strongly suggested that pNDM229 was acquired by HPC229 as the result of HGT from other member of the genus, and selected due to the antimicrobial pressure common to the clinical setting [20]. As a species, A. bereziniae has a wide distribution and has been isolated from various sources including soils, vegetables, animals, and human-associated environments such as wastewaters [22-25].…”

“…More recently, A. bereziniae has been also increasingly associated to healthcare-associated infections in humans, and is currently considered an emerging Acinetobacter opportunistic pathogen [24,26]. In fact, although A. bereziniae isolates were in the past generally susceptible to most antimicrobials of clinical use [23], since 2010 a number of carbapenem-resistant clinical strains bearing IMP-, SIM-, VIM- or NDM-type metallo-β-lactamases (MβL) have been reported [20,24,27,28]. MβL genes are generally carried in these strains by plasmids, suggesting both the mechanism of their acquisition and potential spread to other Gram-negative species [20,24].…”

“…In fact, although A. bereziniae isolates were in the past generally susceptible to most antimicrobials of clinical use [23], since 2010 a number of carbapenem-resistant clinical strains bearing IMP-, SIM-, VIM- or NDM-type metallo-β-lactamases (MβL) have been reported [20,24,27,28]. MβL genes are generally carried in these strains by plasmids, suggesting both the mechanism of their acquisition and potential spread to other Gram-negative species [20,24]. A. bereziniae might easily acquire plasmids, and has been proposed recently as a reservoir of clinically-relevant antimicrobial resistance genes [24].…”