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Letter
Carbapenem-Resistant Pseudomonas
aeruginosa–Carrying VIM-2 Metallo-ß-Lactamase Determinants,
Croatia
Sanda Sardelic,* Lucia Pallecchi,† Volga Punda-Polic,* and Gian Maria
Rossolini†
*University Hospital and School of Medicine Split, Split, Croatia; and
†University of Siena, Siena, Italy
Suggested citation for this article: Sardelic
S, Pallecchi L, Punda-Polic V, Rossolini GM. Carbapenem-resistant Pseudomonas
aeruginosa–carrying VIM-2 metallo-ß-lactamase determinants,
Croatia. Emerg Infect Dis [serial online] 2003 Aug [date cited].
Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no8/02-0373.htm
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-ß-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 blaIMP, blaVIM
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 ß-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 mg/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 blaIMP
and a blaVIM 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 blaVIM-2
allele located in a gene cassette inserted in the attI site of
a class 1 integron.
The metallo-ß-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 >1x10-8 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 blaVIM-2 probe generated with PCR amplification
of the entire blaVIM-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-ß-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-ß-lactamases within
the TMR program (contract no. FMRX-CT98-0232) and by grant “M.I.U.R”
(no. 2001068755_003).
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