Carbapenem-Resistant Pseudomonas aeruginosa–Carrying VIM-2 Metallo-β-Lactamase Determinants, Croatia

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-β-lactamases. These enzymes have recently been identified in carbapenem-resistant isolates of Pseudomonas aeruginosa and other gram-negative nonfermenters from European countries in the Koh et al., GenBank accession no. AY165025). Similar to bla IMP , bla 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 β-lac-tams, 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. aerugi-nosa 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 uri-nary 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 chromoso-mal 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, includ-MICs to imipenem and meropen-em were high (>128 µg/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) (imipen-em hydrolyzing–specific activity was, in either case, >170 nmol/min/mg protein). A colony blot hybridization, carried out as described with a bla IMP and a bla 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 VIM-2 allele located in a gene cassette inserted in the attI site of a class 1 integron. The …