VIM-2-producing Pseudomonas putida, Buenos Aires.

To the Editor: Pseudomonas putida infections (0.03% of isolates from the culture collection of inpatients, 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, aminoglycosides, fluoroquinolones, and carbapenems. However, isolates have been identified that produce acquired metallo-[beta]-lactamases (MBLs) and are resistant to most [beta]-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, bioMerieux, Lyon, France). Susceptibility tests were performed according to standard procedures. Both isolates were resistant to imipenem and meropenem (MICs >32 [micro]g/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 [micro]mol 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-GCGTTTATGTTCATACTT-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, Gottingen, Germany) with the following reaction conditions: 1 cycle at 95[degrees]C for 5 min, 52[degrees]C for 15 min, and 72[degrees]C for 6 min, followed by 30 cycles at 95[degrees]C for l min, 52[degrees]C for 1 rain, and 72[degrees]C for 1 min, and a final reaction at 72[degrees]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, 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'-CGTCTTATCAGGCC-TAC-3'), ERIC-1 (5'-CACTTAGGG GTCCTCAATGTA-3'), and ERIC-2 (5'-AAGTAAGTGACTGGGGT-GAGCG-3') were used to characterize isolates. PCR conditions were 94[degrees]C for 2 min, 30 cycles at 94[degrees]C for 30 s, 50[degrees]C for 1 min, and 72[degrees]C for 4 min, and a final reaction at 72[degrees]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 [micro]g/mL). However, carbapenem-susceptible P. putida had low levels of susceptibility because the MI[C.sub.50] 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 [beta]-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 [micro]g/mL), ceftazidime, and cefepime (MIC >256 [micro]g/mL).

Detection of [bla.sub.VIM-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.

This work was partially supported by grants from the Secretaria de Ciencia y Tecnica de la Universidad de Buenos Aires (UBACyT) and the Agencia Nacional de Promocion Cientifica y Tecnologica to G.G. and the UBACyT to C.V.G.G. is a member of Carrera del Investigador, Consejo Nacional de Investigaciones Cientificas y Tecnicas.

Marisa Almuzara, * Marcela Radice, * Natalia de Garate, * Alejandra Kossman, * Arabela Cuirolo, * Gisela Santella, * Angela Famiglietti, * Gabriel Gutkind, * and Varolos Vay *

* Universidad de Buenos Aires, Buenos Aires, Argentina

References

(1.) Senda K, Arakawa Y, Ichiyama S, Nakashima K, Ito H, Ohsuka S, et al. PCR detection of metallo-[beta]-lactamase gene ([bla.sub.IMP]) in gram-negative rods resistant to broad-spectrum [beta]-lactams. J Clin Microbiol. 1996;34:2909-13.

(2.) Yomoda S, Okubo T, Takahashi A, Murakami M, Iyobe S. Presence of Pseudomonas putida strains harboring plasmids bearing the metallo-[beta]-lactamase gene ([bla.sub.IMP]) in a hospital in Japan. J Clin Microbiol. 2003;41:424-51.

(3.) Docquier JD, Riccio ML, Mugnaioli C, Luzzaro F, Endimiani A, Toniolo A, et al. IMP-12, a new plasmid-encoded metallo-[beta]-lactamase from a Pseudomonas putida clinical isolate. Antimicrob Agents Chemother. 2003;47:1522-8.

(4.) Lombardi G, Luzzaro E Docquier JD, Riccio ML, Perilli M, Coli A, et al. Nosocomial infections caused by multidrug-resistant isolated of Pseudomonas putida producing VIM-1 metallo-[beta]-lactamase. J Clin Microbiol. 2002:40:4051-5.

(5.) Lee K, Lim JB, Yum JH, Yong D, Chong Y, Kim JM, et al. ([bla.sub.VIM-2]) Cassette-containing novel integrons in metallo-[beta]-lactamase- producing Pseudomonas aeruginosa and Pseudomonas putida isolated disseminated in a Korean hospital. Antimicrob Agents Chemother. 2002;46:1053-8.

(6.) Poirel L, Cabanne L, Collet L, Nordman P. Class II transposon-borne structure harboring metallo-[beta]-lactamase gene ([bla.sub.VIM-2]) in Pseudomonas putida. Antimicrob Agents Chemother. 2006;50:2889-91.

(7.) Vay CA, Almuzara M, Rodriguez C, Pugliese M, Lorenzo Barba F, Mattera J, et al. 'In vitro' activity of different antimicrobial agents on gram-negative nonfermentative bacilli, excluding Psettdomonas aeruginosa and Acinetobacter spp. [in Spanish]. Rev Argent Microbiol. 2005;37:34-45.

(8.) Sader HS, Jones RN. Antimicrobial susceptibility of uncommonly isolated non-enteric gram-negative bacilli. Int J Antimicrob Agents. 2005;25:95-109.

(9.) Koh TH, Wang GCY, Song LH. IMP-1 and a novel metallo-[beta]-lactamase, VIM-6, in fluorescent pseudomonads isolated in Singapore. Antimicrob Agents Chemother. 2004;48:2334-6.

(10.) Mendes RE, Castanheira M, Garcia P, Guzman M, Toleman MA, Walsh TR, et al. First isolation of ([bla.sub.VIM-2]) in Latin America: report from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother. 2004;48: 1433-4.

Address for correspondence: Gabriel Gutkind, Facultad de Famacia y Bioquimica, Catedra de Microbiologia, Universidad de Buenos Aires, Junin 954, Buenos Aires 1113, Argentina; email: [e-mail]

Table. Antimicrobial drug susceptibility profiles of 2
[bla.sub.VIM-2]-carrying Pseudomonas putida isolates, Argentina

                              MIC ([micro]g/mL)

Drug                       Isolate 1    Isolate 2

Imipenem                       32           64
Meropenem                      64           64
Ertapenem                     128          128
Piperacillin                   64           64
Piperacillin-tazobactam        32           32
Ceftazidime                   128          128
Cefepime                       32           32
Aztreonam                      64           64
Amikacin                        4            4
Gentamicin                     16           16
Ciprofloxacin                 >64          >64
Gatifloxacin                  >64          >64
Levofloxacin                  >64          >64
Moxifloxacin                  >64          >64
Doxycycline                    64           64
Colistin                        2            2