Carbapenem-resistant Pseudomonas aeruginosa--carrying VIM-2 metallo-[beta]-lactamase determinants, Croatia. (Letters).

To the Editor: Carbapenem-hydrolyzing enzymes of the VIM-type (six different variants are known: VIM-1, VIM-2, VIM-3, VIM-4, VIM-5, and VIM-6) are new molecular class B metallo-[beta]-lactamases. These enzymes have recently been identified in carbapenem-resistant isolates of Pseudomonas aeruginosa and other gram-negative nonfermenters from European countries in the Mediterranean basin (Italy, France, Greece, Spain, Portugal, and Turkey), as well as in Far East countries (Korea, Taiwan, and Singapore) and the United States (1-3, Midilli et al., GenBank accession no. AY144612, Koh et al., GenBank accession no. AY165025). Similar to [bla.sub.IMP], [bla.sub.VIM] genes are located on mobile gene cassettes inserted in the variable regions of integrons (1), a condition that provides a wide potential for expression and dissemination in gram-negative pathogens. VIM enzymes possess the broadest range of substrate hydrolysis and can degrade virtually all [beta]-lactams, except monobactams (4).

According to a recent report, the overall resistance rate to imipenem in P. aeruginosa isolated from 17 representative laboratories in Croatia was 11% (range 0%-20%) (5). However, molecular basis of carbapenem resistance was not investigated.

In October 2000, two P aeruginosa isolates with an unusual resistance profile were isolated from two Croatian patients (66 and 74 years of age, respectively) who underwent hysterectomies at the Split University Hospital. Both isolates were cultured from urine a week after surgery; a urinary catheter had been used for both patients who had become febrile and had signs and symptoms of urinary tract infection. Analysis of the macrorestriction profiles of chromosomal DNA of the two isolates by pulsed-field gel electrophoresis, carried out as described previously (6), indicated that the two isolates were clonally related (the two profiles were apparently identical). In routine antibiotic susceptibility testing, done by disk diffusion, both isolates showed a multidrug-resistant phenotype, including ureidopenicillins, piperacillin, piperacillin-tazobactam, ceftazidime, cefoperazone, cefepime, aztreonam, ciprofloxacin, gentamicin, netilmicin, imipenem, and meropenem.

MICs to imipenem and meropenem were high (>128 [micro]g/mL). These findings suggested production of an acquired carbapenemase. In fact, crude extracts of the two isolates exhibited carbapenemase activity in a spectrophotometric assay (7) (imipenem hydrolyzing-specific activity was, in either case, >170 nmol/min/mg protein).

A colony blot hybridization, carried out as described with a [bla.sub.IMP] and a [bla.sub.VIM] probe (6), yielded a positive result with the latter probe. Polymerase chain reaction (PCR) amplification of the variable region of class 1 integrons, carried out as described previously by using primers designed on the 5'- and 3'-conserved segments of the integron (8), yielded a 4-kb amplification product from either isolate. Direct sequencing of these amplification products showed, in both cases, the presence of a [bla.sub.VIM-2]. allele located in a gene cassette inserted in the attI site of a class 1 integron.

The metallo-[beta]-lactamase determinant was not transferred to Escherichia coli MKD135 or P aeruginosa 10145/3 (9) in diparental mating experiments conducted on solid medium (the sensitivity of the assay was [greater than or equal to] 1 x [10.sup.-8] transconjugants per donor). Plasmid extraction was performed with several techniques, including lysis with sodium dodecyl sulfate (10) and alkaline lysis conducted with a conventional method (10) or with the Nucleobond BAC100 system (Macherey-Nagel, Duren, Germany). Extraction of whole genomic DNA was also performed, as described (8). Plasmid DNA was not detected in any of these preparations, either when analyzed by agarose gel electrophoresis or after Southern blot hybridization analysis with a [bla.sub.VIM-2] probe generated with PCR amplification of the entire [bla.sub.VIM-2] gene. In Southern blots, a hybridization signal was only detectable in correspondence of the band of chromosomal DNA.

To our knowledge, this isolation is the first one of clinical strains producing acquired metallo-[beta]-lactamase in Croatia. A similar finding underscores the progressive emergence of these determinants in different geographic areas and emphasizes the need for an early recognition of these strains. In fact, monitoring dissemination of new antibiotic resistance determinants is essential to enforce adequate control measures and adjust guidelines for antimicrobial chemotherapy in different hospital settings.

This work was supported by the European research network on metallo-[beta]lactamases within the TMR program (contract no. FMRX-CT98-0232) and by grant "M.I.U.R" (no. 2001068755_003).

Sanda Sardelic, * Lucia Pallecchi, ([dagger]) Volga Punda-Polic, * and Gian Maria Rossolinil ([dagger])

* University Hospital and School of Medicine Split, Split, Croatia; and ([dagger]) University of Siena, Siena, Italy

References

(1.) Nordmann P, Poirel L. Emerging carbapenemases in gram-negative aerobes. Clin Microbiol Infect 2002;8:321-31.

(2.) Pournaras S, Tsakris A, Maniati M, Tzouvelekis LS, Maniatis AN. Novel variant (bla(VIM-4)) of the metallo-[beta]-lactamase gene bla(VIM-1) in a clinical strain of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2002;46:4026-8.

(3.) Toleman MA, Rolston K, Jones RN, Walsh TR. Molecular characterization of VIM-4, a novel metallo-[beta]-lactamase isolated from Texas: report from the CANCER surveillance program. In: 42nd ICAAC Abstracts, San Diego, CA, 2002 Sep 27-30; Washington: American Society for Microbiology; 2002.

(4.) Docquier JD, Lamotte-Brasseur J, Galleni M, Amicosante G, Frere JM, Rossolini GM. On functional and structural heterogeneity of VIM-type metallo-[beta]-lactamases. J Antimicrob Chemother 2003;51:257-66.

(5.) Tambic Andrasevic A, Tambic T, Kalenic S, Jankovic V, Working Group of the Croatian Committee for Antibiotic Resistance Surveillance. Surveillance for antimicrobial resistance in Croatia. Emerg Infect Dis 2002;8:14-8.

(6.) Cornaglia G, Mazzariol A, Lauretti L, Rossolini GM, Fontana R. Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM1, a novel transferable metallo-[beta]-lactamase. Clin Infect Dis 2000;31:1119-25.

(7.) Lauretti L, Riccio L, Mazzariol G, Cornaglia G, Amicosante G, Fontana R, et al. Cloning and characterization of [bla.sub.VIM], a new integron-borne metallo-[beta]-lactamase gene from Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother 1999 ;43:1584-90.

(8.) Riccio ML, Franceschini N, Boschi L, Caravelli B, Cornaglia G, Fontana R, et al. Characterization of the metallo-[beta]-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of bla(IMP) allelic variants carried by gene cassettes of different phylogeny. Antimicrob Agents Chemother 2000;44:1229-35.

(9.) Riccio M, Pallecchi L, Fontana R, Rossolini GM. In 70 of plasmid pAX22, a [bla.sub.VIM-1]-containing integron carrying a new aminoglycoside phosphotransferase gene cassette. Antimicrob Agents Chemother 2001 ;45:1249-53.

(10.) Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1989.

Address for correspondence: Gian Maria Rossolini, Dipartimento di Biologia Molecolare, Sez. Microbiologia, Universita di Siena, Policlinico Le Scotte, 53100, Siena, Italy; fax: [phone]; email: [e-mail]