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Volume 13, Number 4—April 2007

Letter

VIM-2–producing Pseudomonas putida, Buenos Aires

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To the Editor: Pseudomonas putida (0.03% of isolates from the culture collection of the Argentina Association of Microbiology, www.aam.org.ar) infections are mainly reported in immunocompromised patients, such as newborns, neutropenic patients, and cancer patients. They are usually susceptible to extended-spectrum cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems. 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. putida 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 imipenem 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/L 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′-GAAGGCGTTTATGTTCATACTT-3′, blaIMP-R: 5′-GTTTGCCTTACCATATTTGGA-3′, blaVIMG-F: 5′-GGTGTTTGGTCGCATATC-3′, and blaVIMG-R 5′-TGGGCCATTCAGCCAGATC-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 (National Center for Biotechnology Information, Bethesda, MD, USA, http://www.ncbi.nlm.nih.gov/Tools/). Nucleotide sequences were completely homologous to the vim-2 coding gene.

Two repetitive-element–based PCR (rep-PCR) assays (ERIC-PCR and REP-PCR) with primers REP-1 (5′-IGCGCCGICATCAGGC-3′), REP-2 (5′-CGTCTTATCAGGCCTAC-3′), ERIC-1 (5′-CACTTAGGGGTCCTCAATGTA-3′), and ERIC-2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) were used to characterize isolates. PCR conditions were 94°C for 2 min, 30 cycles at 94°C for 30 s, 50°C for 1 min, and 72°C for 4 min, and a final reaction at 72°C for 7 min. Banding patterns were visually analyzed after electrophoresis of samples. Variations in band intensity were not considered to indicate genetic differences. Banding patterns obtained by REP-PCR and ERIC-PCR assays were identical in both isolates (data not shown).

Among the MBLs acquired by P. putida, IMP-1 was reported by Senda et al. in Japan in 1996 (1) and later reported in Taiwan and Japan (2). IMP-12 was the first IMP MBL described in P. putida in Europe (3). VIM-1 in P. putida was first reported in Europe (4), and VIM-2 in P. putida was first reported in Taiwan, Republic of Korea, Japan, and France (5,6). Our isolates were resistant to aztreonam (MIC 64 μg/mL). However, carbapenem-susceptible P. putida had low levels of susceptibility because the MIC50 was only 1 dilution below the current breakpoint (7,8). Aztreonam resistance could not be transferred by conjugation between IMP-1–producing (aztreonam-resistant) P. putida and P. aeruginosa (2) and is not associated with a transposon carrying blaVIM-2 (6). No evidence of extended-spectrum β-lactamases was detected in our isolates by classic synergy assays with clavulanate plus aztreonam, ceftazidime, or cefotaxime. VIM-6–producing P. putida isolates from Singapore (9) were more resistant to aztreonam (MIC >128 μg/mL), ceftazidime, and cefepime (MIC >256 μg/mL).

Detection of blaVIM-2 in Pseudomonas in South America was initially reported by the SENTRY Antimicrobial Surveillance Program (10) and included 1 P. fluorescens isolate in Chile and 3 P. aeruginosa isolates in Venezuela. To the best of our knowledge, our report is the first of VIM-2 in P. putida in Latin America. VIM-2–producing P. putida, which were originally restricted to East Asia and only very recently found in France, may represent an emerging pathogen or function as reservoirs for resistance because of their widespread presence in the hospital environment.

Marisa Almuzara*, Marcela Radice*, Natalia de Gárate*, Alejandra Kossman*, Arabela Cuirolo*, Gisela Santella*, Angela Famiglietti*, Gabriel Gutkind*Comments to Author , and Varolos Vay*
Author affiliations: *Universidad de Buenos Aires, Buenos Aires, Argentina;

Acknowledgment

This work was partially supported by grants from the Secretaría de Ciencia y Técnica de la Universidad de Buenos Aires (UBACyT) and the Agencia Nacional de Promoción Científica y Tecnológica to G.G. and the UBACyT to C.V. G.G. is a member of Carrera del Investigador, Consejo Nacional de Investigaciones Científicas y Técnicas.

References

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  2. Yomoda S, Okubo T, Takahashi A, Murakami M, Iyobe S. Presence of Pseudomonas putida strains harboring plasmids bearing the metallo-β-lactamase gene bla IMP in a hospital in Japan. J Clin Microbiol. 2003;41:424651. DOIPubMed
  3. Docquier JD, Riccio ML, Mugnaioli C, Luzzaro F, Endimiani A, Toniolo A, IMP-12, a new plasmid-encoded metallo-β-lactamase from a Pseudomonas putida clinical isolate. Antimicrob Agents Chemother. 2003;47:15228. DOIPubMed
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  5. Lee K, Lim JB, Yum JH, Yong D, Chong Y, Kim JM, bla VIM-2 Cassette-containing novel integrons in metallo-β-lactamase- producing Pseudomonas aeruginosa and Pseudomonas putida isolated disseminated in a Korean hospital. Antimicrob Agents Chemother. 2002;46:10538. DOIPubMed
  6. Poirel L, Cabanne L, Collet L, Nordman P. Class II transposon-borne structure harboring metallo-β-lactamase gene blaVIM-2 in Pseudomonas putida. Antimicrob Agents Chemother. 2006;50:288991. DOIPubMed
  7. Vay CA, Almuzara M, Rodríguez C, Pugliese M, Lorenzo Barba F, Mattera J, ‘In vitro’ activity of different antimicrobial agents on gram-negative nonfermentative bacilli, excluding Pseudomonas aeruginosa and Acinetobacter spp. [in Spanish]. Rev Argent Microbiol. 2005;37:3445.PubMed
  8. Sader HS, Jones RN. Antimicrobial susceptibility of uncommonly isolated non-enteric gram-negative bacilli. Int J Antimicrob Agents. 2005;25:95109. DOIPubMed
  9. Koh TH, Wang GCY, Song LH. IMP-1 and a novel metallo-β-lactamase, VIM-6, in fluorescent pseudomonads isolated in Singapore. Antimicrob Agents Chemother. 2004;48:23346. DOIPubMed
  10. Mendes RE, Castanheira M, Garcia P, Guzman M, Toleman MA, Walsh TR, First isolation of blaVIM-2 in Latin America: report from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother. 2004;48:14334. DOIPubMed

Table

Suggested citation for this article: Almuzara M, Radice M, de Gárate N, Kossman A, Cuirolo A, Santella G, et al. VIM-2–producing Pseudomonas putida, Buenos Aires [letter]. Emerg Infect Dis [serial on the Internet]. 2007 Apr [date cited]. Available from http://wwwnc.cdc.gov/eid/article/13/4/06-1083

DOI: 10.3201/eid1304.061083

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Table of Contents – Volume 13, Number 4—April 2007

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Gabriel Gutkind, Facultad de Famacia y Bioquímica, Catedra de Microbiologia, Universidad de Buenos Aires, Junin 954, Buenos Aires 1113, Argentina;

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