VIM-2–producing Pseudomonas putida, Buenos Aires

To the Editor: Pseudomonas putida infections (0.03% of isolates from the culture collection of inpa-tients, SIR Program 2003–2004, www.aam.org.ar) are mainly reported in immunocompromised patients, such as newborns, neutropenic patients, and cancer patients. They are usually susceptible to extended-spectrum cephalosporins, aminoglyco-sides, fluoroquinolones, and car-bapenems. However, isolates have been identified that produce acquired metallo-β-lactamases (MBLs) and are resistant to most β-lactams, including carbapenems. Two multidrug-resistant P. puti-da isolates were obtained from clinical samples at the Sanatorio Mater Dei in Buenos Aires. One isolate was obtained in March 2005 from a urine specimen of a 76-year-old woman with a urinary tract infection who was using a urethral catheter. The second isolate was obtained in May 2005 from a tracheal aspirate of a 67-year-old man with nosocomial pneumonia. Bacteria were identified by using conventional biochemical tests and the API 20NE System (API, bioMérieux, Lyon, France). Susceptibility tests were performed according to standard procedures. Both isolates were resistant to imi-penem and meropenem (MICs >32 µg/mL) but were susceptible to amikacin and colistin. Susceptibility data are shown in the Table. Screening for MBLs was performed by using a double-disk diffusion method. Disks containing 1 µmol EDTA (metal chelator) were placed on Mueller-Hinton agar plates containing the 2 isolates. Disks containing carbapenem were placed 15 mm from disks containing EDTA. An increase in the inhibition zone of the disk containing drug near the disk containing EDTA was observed for both isolates, which suggested the presence of MBLs. PCR amplification of imp and vim genes was conducted by using primers based on conserved regions of the imp and vim genes (blaIMP-F: 5′-GAAG-G C G T T TAT G T T C ATA C T T-3 ′ , blaIMP-R: 5′-GTTTGCCTTACCAT ATTTGGA-3′, blaVIMG-F: 5′-GGT-GTTTGGTCGCATATC-3′, and bla VIMG-R 5′-TGGGCCATTCAGC CAGATC-3′) and heat-extracted DNA as template. Reactions were performed in a T-gradient instrument (Biometra, Göttingen, Germany) with the following reaction conditions: 1 cycle at 95°C for 5 min, 52°C for 15 min, and 72°C for 6 min, followed by 30 cycles at 95°C for 1 min, 52°C for 1 min, and 72°C for 1 min, and a final reaction at 72°C for 20 min. Amplified fragments were sequenced on both strands by using an ABI Prism DNA 3700 (Applied Biosystems, Foster City, CA, USA), and nucleotide sequences were compared by using BLAST Tools/). Nucleotide sequences were completely homologous to the vim-2 coding gene. were used to characterize isolates. PCR conditions were …