The worldwide spread of metallo-␤-lactamase (MBL)-producing gram-negative bacilli represents a great concern nowadays. Sensitive assays for their specific detection are increasingly demanded to aid infection control and to prevent their dissemination. We have developed a novel microbiological assay employing crude bacterial extracts, designated EDTA-imipenem microbiological assay (EIM), to identify MBLs in nonfermentative gram-negative clinical strains. We also evaluated the ability of EIM to detect MBLs in comparison to those of other currently employed screening methods, such as the EDTA disk synergy test (EDS) with imipenem as a substrate and the Etest method. The sensitivities of EIM and Etest were similar (1 versus 0.92, respectively) and much higher than that of EDS (0.67). Moreover, both EIM and Etest displayed the maximum specificity. Modifications were introduced to EDS, including the simultaneous testing of three different ␤-lactams (imipenem, meropenem, and ceftazidime) and two different EDTA concentrations. This resulted in a sensitivity improvement (0.92), albeit at a cost to its specificity. A simple strategy to accurately detect MBL producers is proposed; this strategy combines (i) an initial screening of the isolates by the extended EDS assay to select the potential candidates and (ii) confirmation of the true presence of MBL activity by EIM.

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...

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