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Journal of Clinical Microbiology, June 1999, p. 2109-2110, Vol. 37, No. 6
Emergence of Carbapenem-Hydrolyzing Enzymes in
Acinetobacter baumannii Clinical Isolates
Received 3 March 1999/Accepted 10 March 1999
In recent years, the number of nosocomial infections caused by
Acinetobacter baumannii has increased significantly
(4). Many outbreaks have been reported, especially among
patients confined to hospital intensive care units, where the
widespread use of antibiotics may select multidrug-resistant
strains. The difficulty of treating A. baumannii nosocomial
infection is associated with the high resistance to a wide range
of antimicrobial agents frequently observed in this species
(8). Often, imipenem remains one of the few therapeutic
alternatives. Fortunately, imipenem resistance is relatively rare
among Acinetobacter clinical isolates. Carbapenem resistance can arise by a decrease in expression of an outer membrane protein (3) or by alteration in penicillin-binding proteins (5). In general, the emergence of carbapenem-hydrolyzing
enzymes has been limited compared to the prevalence of other
This communication reports the production of imipenem-hydrolyzing
enzymes in two A. baumannii isolates obtained from urine cultures in 1998 in a Portuguese teaching hospital.
MICs were determined by the E-test method according to the
manufacturer's instructions, and resistance was defined according to
National Committee for Clinical Laboratory Standards guidelines (6). Crude sonicates of cell suspensions were assayed by
spectrophotometry (UV/Vis Perkin-Elmer Lambda 6 spectrophotometer) with
0.1 mM imipenem and 0.1 mM nitrocefin solutions. One unit of
The results are shown in Table 1.
The isolates (strains 122FFC and 65FFC) presented similar
resistance patterns, with high-level resistance to imipenem and
meropenem (MICs of both compounds, >32 mg/liter) and cephalosporins,
including a new cephalosporin, cefepime (MICs, >256 mg/liter). They
were susceptible to ciprofloxacin (MICs of 1.5 and 0.5 mg/liter,
respectively) and to aminoglycosides (data not shown).
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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-lactamases (1). However, in 1985 in Scotland, an
A. baumannii strain that produced a plasmid-mediated
carbapenemase, ARI-I, was isolated (7), and recently,
imipenem-hydrolyzing enzymes have been noted in some
Acinetobacter isolates in a United Kingdom burns unit (10).
-lactamase activity was defined as the amount of enzyme hydrolyzing
1 nmol of the substrate per min per mg of protein at 30°C in 0.1 M phosphate buffer, pH 7.0. Isoelectric focusing (IEF) was performed in
precast polyacrylamide gels, pI 3 to 9 (Pharmacia), by using a
PhastSystem apparatus according to the manufacturer's instructions.
Prior to staining with nitrocefin, one of the gels was overlaid with 0.1 mM cloxacillin solution, which is known to inhibit Bush group 1 -lactamases (2), enzymes that are usually produced by
Acinetobacter spp. (9). Two control
strains were used in the experiments: an A. baumannii
clinical isolate susceptible to imipenem and A. baumannii
ATCC 19606. In an attempt to detect transfer of imipenem resistance,
filter mating experiments were performed at 37°C by using
Escherichia coli K802N as a recipient cell.
TABLE 1.
Characteristics of the isolates of A. baumannii studied
IEF revealed a large -lactamase band with a pI of >8,
presumably a chromosomal Bush group 1 enzyme. A sharp -lactamase
band with a pI of >8 was observed after the treatment with
cloxacillin, suggesting the production of another -lactamase
not inhibited by cloxacillin.
Imipenem was readily hydrolyzed by crude extracts of imipenem-resistant
isolates, but not by extracts of the controls, confirming the presence
of an imipenem-hydrolyzing enzyme. The observed carbapenem resistance
correlated with hydrolytic activity. A slight improvement of the
-lactamase activity was observed during the measurement of imipenem
hydrolysis in the presence of 1 mM ZnCl2 solution. Preincubation of the extract with 1 mM EDTA solution for 10 min at
30°C resulted in a decrease in -lactamase activity (between 70 and
80% inhibition). All attempts to transfer imipenem resistance to
E. coli K802N were unsuccessful.
The results obtained suggest the possibility of a metalloenzyme.
Neither of the A. baumannii carbapenemases reported in the literature are zinc-dependent enzymes (7, 10). This is the first report of an imipenem-hydrolyzing enzyme in this species found in
Portugal. The increase in carbapenem therapy might be associated with
the emergence of A. baumannii strains that produce imipenem-hydrolyzing enzymes, which is a serious concern due to the
large spectrum of these enzymes. Therefore, it is crucial to
rationalize the use of this class of compounds in an attempt to
minimize the selection of these new -lactamases.
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FOOTNOTES |
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* Phone: (351) 39 852567
Fax: (351) 39 852569
E-mail: gjsilva{at}cygnus.ci.uc.pt
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Gabriela J. Da Silva* Rui Leitão Laboratório de Microbiologia Faculdade de Farmácia Universidade de Coimbra Couraça dos Apóstolos, 51, r/c E 3030 Coimbra, Portugal | |||||
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Luísa Peixe Laboratório de Microbiologia Faculdade de Farmácia Universidade do Porto R. Aníbal da Cunha, 164 4050 Porto, Portugal |
Journal of Clinical Microbiology, June 1999, p. 2109-2110, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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