Dipartimento di Biologia Molecolare, Sezione
di Microbiologia, Università di Siena,
I-53100-Siena,1 Dipartimento di
Scienze e Tecnologie Biomediche, Università di L'Aquila,
I-67100-L'Aquila,2 and Dipartimento
di Patologia, Sezione di Microbiologia, Università di Verona,
I-37134-Verona,3 Italy
Received 9 August 1999/Returned for modification 13 December
1999/Accepted 1 February 2000
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INTRODUCTION |
The integron-based recombination
system is a powerful mechanism of discrete genetic rearrangement that
operates in procaryotic genomes and that plays a major role in the
spread of antibiotic resistance determinants (see references
13 and 30 for reviews). Although
the most widespread secondary 
-lactamases are not integron associated (see references 6 and
25 and references therein), various -lactamase
determinants are carried by integron-borne mobile cassettes
(30). The repertoire of integron-associated -lactamases
has been shown to include also metallo--lactamases (2, 16, 20,
22), which is a most worrying development in the field of
microbial drug resistance. In fact, these enzymes are able to hydrolyze
virtually all -lactam compounds (including carbapenems and
expanded-spectrum cephalosporins) and are not susceptible to the
mechanism-based serine--lactamase inhibitors (3, 5, 21, 22, 29,
32).
The blaIMP gene, which encodes the IMP-1 enzyme,
was the first metallo--lactamase determinant to be identified as
part of gene cassettes inserted into chromosomal or plasmid-borne
integrons carried by nosocomial isolates of Serratia
marcescens, Klebsiella pneumoniae, and
Pseudomonas aeruginosa from Japan (2, 16, 20,
28; EMBL/GenBank database entry D29636). Spread of the
blaIMP determinant has essentially remained
limited to Japan, with single reports from Korea (K. Lee, Y. Chong,
H. B. Shin, and D. Yong, Abstr. 38th Intersci. Conf. Antimicrob.
Agents Chemother. abstr. E85, p. 193, 1998) and Singapore (T. H. Koh, G. S. Babini, N. Woodford, L. H. Sng, L. M. Hall,
and D. M. Livermore, Letter, Lancet 353:2162, 1999),
until recently, when a multidrug-resistant Acinetobacter
baumannii strain (strain AC-54/97) isolated from an Italian
patient in Verona, Italy, was found to produce a metallo--lactamase and to carry blaIMP-related sequences (G. Cornaglia, M. L. Riccio, A. Mazzariol, P. Piccoli, L. Lauretti,
R. Fontana, and G. M. Rossolini, Letter, Lancet
353:899-900, 1999). The unusual nature of this host
(blaIMP has never been reported in
Acinetobacter isolates in the Far East) and its different
geographical origin raised a question about the nature and origin of
this determinant.
In the work described here we cloned and characterized the
blaIMP-related determinant acquired by A. baumannii AC-54/97. Since it was found to be an allelic variant
(named blaIMP-2) of
blaIMP found in Japanese isolates, we also
purified the IMP-2 enzyme and analyzed its kinetic properties with
various -lactam substrates.
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MATERIALS AND METHODS |
Bacterial strains and genetic vectors.
A. baumannii
AC-54/97 was isolated in 1997 from an Italian inpatient at the
Intensive Care Unit of the Verona University Hospital. The properties
of this strain and the clinical history of the patient have been
described previously (Cornaglia et al., Letter, Lancet
353:899-900, 1999). Escherichia coli DH5
(GIBCO-BRL, Gaithersburg, Md.) was used as the host for recombinant
plasmids. Bacterial strains were always grown aerobically at 37°C.
Plasmid pBC-SK (Stratagene, La Jolla, Calif.) was used as a cloning vector.
Antibiotics.
Antibiotics were obtained from Sigma Chemical
Co. (St. Louis, Mo.) unless otherwise specified. Nitrocefin was from
Unipath (Milan, Italy), imipenem was from Merck Research Laboratories (Rahway, N.J.), meropenem was from Zeneca Pharmaceuticals (Cheshire, United Kingdom), ceftazidime was from Glaxo-Wellcome (Verona, Italy),
and cefepime and aztreonam were from Bristol-Myers Squibb (Wallingford,
Conn.). All antibiotic solutions were prepared immediately before use.
Recombinant DNA methodology.
Basic recombinant DNA
procedures were performed as described by Sambrook et al.
(33). Genomic DNA was extracted from A. baumannii
as described previously (17). PCR for amplification of gene
cassette arrays inserted into class 1 integrons (ICA-PCR) was performed
with primers INT/5CS and INT/3CS designed on the 5' conserved segment
(5'-CS) and the 3' conserved segment (3'-CS) of class 1 integrons,
respectively (Fig. 1). Reactions were performed in a 100-µl volume
with the Expand High-Fidelity PCR System (Boehringer Mannheim,
Mannheim, Germany; 2.5 U of the enzyme per reaction) in the reaction
buffer provided by the manufacturer, which contained 1.5 mM
MgCl2, 200 µM deoxynucleoside triphosphates, 50 pmol of each primer, and 10 ng of bacterial genomic DNA as the template. Reaction parameters were as follows: annealing at 51°C for 60 s,
extension at 70°C for 180 s (with an increment of 3 s for
each cycle), and denaturation at 93°C for 40 s, which were
repeated for 15 cycles, and then annealing at 55°C for 60 s,
extension at 70°C for 180 s (with an increment of 3 s for
each cycle), and denaturation at 93°C for 40 s, which were
repeated for 20 cycles. Reactions were performed in 0.2-ml tubes with a
Gene Amp PCR system 2400 (Perkin-Elmer, Rahway, N.J.).
DNA sequencing and computer analysis of sequence data.
DNA
sequences were determined either with crude PCR products or with
plasmid templates by the dideoxy-chain termination method with an
automatic DNA sequencer (model 4000; LI-COR Inc., Lincoln, Nebr.), the
Thermosequenase DNA sequencing kit (Amersham Pharmacia Biotech, Milan,
Italy), and IRD 800-labeled custom sequencing primers (MWG-Biotech,
Munich, Germany). Similarity searches against sequence databases were
performed with an updated version of the BLAST program (1).
Computer analysis of sequence data was performed with the Wisconsin
package (version 8.1; Genetics Computer Group Inc., Madison, Wis.) at
the Italian EMBL Node of Bari. The cleavage site of the IMP-2 signal
peptide was predicted with the SIGCLEAVE program of the EGCG extension
of the Wisconsin package. Codon usage tables were compared as described
by Grantham et al. (12) by using the CORRESPOND program of
the Wisconsin package.
In vitro susceptibility testing.
MICs were determined by a
broth macrodilution method (26) with cation-supplemented
Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) and a
bacterial inoculum of 5 × 105 CFU per tube. Results
were recorded after incubation for 18 h at 37°C. MIC
determinations were performed in triplicate. The results of the
susceptibility tests were interpreted according to the guidelines of
the National Committee for Clinical Laboratory Standards
(27).
Protein analysis techniques.
Metallo--lactamase activity
in crude cell extracts and in fractions during the purification
procedure was assayed as described previously, with imipenem as the
substrate (22). The protein concentration in the solution
was assayed by the method of Bradford (4) with a commercial
kit (Bio-Rad Protein Assay; Bio-Rad, Richmond, Calif.), with bovine
serum albumin used as a standard. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) was performed as described by Laemmli
(18), with final acrylamide concentrations of 15 and 5%
(wt/vol) for the separating and the stacking gels, respectively. After
electrophoresis the protein bands were stained with Coomassie brilliant
blue R-250. Analytical isoelectric focusing was performed in precast
5% polyacrylamide gels containing ampholytes (pH range, 3.5 to 9.5;
Ampholine PAGplate; Pharmacia) with a Multiphor II Apparatus
(Pharmacia). Gels were focused at 0.1 W/cm2 for 2 h at
10°C. -Lactamase activity was detected as purple bands after
overlaying the gel with filter paper soaked with a 0.25 mM nitrocefin
solution in 50 mM HEPES (pH 7.5) supplemented with 2 mM
ZnCl2.
Purification of IMP-2 enzyme.
The IMP-2 enzyme was purified
from E. coli DH5(pBAUX-30) as follows. The strain was
grown in 1 liter of brain heart infusion broth (Difco) containing
chloramphenicol (60 µg/ml) for 16 h at 37°C. The cells were
harvested by centrifugation, washed twice with 50 mM Tris-HCl (pH 8.7)
(Tris buffer [TB]), resuspended in the same buffer, and disrupted by
sonication (five times for 30 s each time at 60 W). Cell debris
was removed by high-speed centrifugation (105,000 × g
for 60 min at 4°C), and the clarified supernatant was loaded onto an
S-Sepharose FF column (2.5 by 30 cm; Pharmacia) equilibrated with TB.
After washing of the column with the same buffer, the bound proteins
were eluted with a linear NaCl gradient (0 to 1 M) in TB (300 ml). The
fractions showing metallo--lactamase activity were pooled, dialyzed
against 50 mM HEPES (pH 7.5), concentrated 10-fold by ultrafiltration,
and loaded onto a Superdex-75 column (1.6 by 75 cm; Pharmacia) that had
been equilibrated and eluted with the same buffer. The
-lactamase-containing elution peak was concentrated at 0.5 mg/ml and
was stored at 
80°C until use.
Determination of kinetic parameters.
Kinetic parameters of
the IMP-2 enzyme were determined by essentially the same methodology
previously adopted for characterization of IMP-1 (21).
Hydrolysis of -lactams was monitored with a lambda 2 spectrophotometer (Perkin-Elmer) equipped with thermostatically controlled cells. The enzyme concentration in the reaction mixture was
in the range 8 to 60 nM.
Nucleotide sequence accession number.
The nucleotide
sequence reported in this paper has been submitted to the
EMBL/GenBank/DDBJ sequence databases and assigned the accession no.
AJ243491.
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RESULTS AND DISCUSSION |
Cloning of the blaIMP-related determinant
of A. baumannii AC-54/97.
A. baumannii AC-54/97
is a clinical isolate from Europe that was previously shown to produce
a metallo--lactamase and to carry a chromosomal copy of a
determinant related to blaIMP by means of
Southern blot hybridization with a
blaIMP-specific probe (Cornaglia et al., Letter,
Lancet 353:899-900, 1999). AC-54/97 exhibits high-level
resistance to carbapenems and is also resistant to several other
-lactam and aminoglycoside antibiotics (Cornaglia et al., Letter,
Lancet 353:899-900, 1999) (Table
1).
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TABLE 1.
MICs of various -lactams and aminoglycosides for
A. baumannii AC-54/97 and for E. coli
DH5(pBAUX-30), which carries the cloned cassette array of In42
in pBC-SKa
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Since in members of the family Enterobacteriaceae and the
genus Pseudomonas blaIMP was found to be carried
on gene cassettes inserted into integrons (2, 16, 20,
28; EMBL/GenBank database entry D29636), a PCR methodology
designed for amplification of integron cassette arrays (ICA-PCR; Fig.
1), modified after that previously
developed by Lévesque et al. (23), was adopted to
isolate the blaIMP-related determinant of
AC-54/97. With the genomic DNA of AC-54/97 as the template, ICA-PCR
yielded a 3-kb amplification product which was recognized by a
blaIMP-specific probe that comprised the 0.5-kb
HindIII fragment internal to the blaIMP gene (28) in a Southern blot
hybridization (data not shown). Direct sequencing of this product with
the primer ATT/SEQ, designed from the sequence upstream of the
attI site of class 1 integrons (Fig. 1), yielded a sequence
identical to that of the attI site of In1 from plasmid R46
(14) until the recombination core site of the first inserted
cassette, from which a double sequence ladder began, preventing further
reading (data not shown). This indicated that the 3-kb amplification
product was actually contributed by a mixed population of two different
amplicons of approximately the same size. Digestion of the
amplification product with XbaI yielded a partial
restriction pattern, suggesting that only one of the two amplicons
contained an internal XbaI site that yielded two fragments
of 1.75 and 1.25 kb. Southern blot hybridization with the
blaIMP-specific probe showed that the
blaIMP-related determinant was carried by the
amplicon that did not contain the internal XbaI site (data
not shown). Therefore, after digestion of the amplification product
with XbaI, the 3-kb band was purified by agarose gel
electrophoresis and was cloned into the pBC-SK vector to obtain
recombinant plasmid pBAUX-30. Metallo--lactamase activity was
detected in crude extracts of E. coli DH5(pBAUX-30) (data
not shown), confirming that the cloned gene encoded a functional product.
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FIG. 1.
Design of primers INT/5CS and INT/3CS used in the
ICA-PCR methodology to target amplification of the cassette arrays
inserted into class 1 integrons and of primer ATT/SEQ used for direct
sequencing of the attI site and downstream region of the
amplification products. To facilitate cloning of the amplicons, an
XbaI site (underlined) was engineered near the 5' end of the
amplification primers by introducing a point mutation in each of them
(indicated by lowercase letters). Gene cassettes are represented by
black arrows (coding sequences) followed by empty circles (59-base
elements).
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The ICA-PCR methodology developed for amplification of integron-borne
cassette arrays, therefore, was successful in rapid isolation of the
new metallo--lactamase determinant and could be used for rapid
characterization of new class 1 integrons carried by
antibiotic-resistant clinical isolates. If the amplification product
obtained is unique, direct sequencing can be used for characterization
of the entire cassette array. On the other hand, if two or more class 1 integrons are present in the strain studied, yielding multiple
amplicons (as it was the case with A. baumannii AC-54/97),
it may be necessary to separate them by size or by cloning to achieve
their characterization. The introduction of a restriction site in the
primers used for amplification in ICA-PCR facilitates cloning of the
amplification products. Another difference of ICA-PCR compared to the
approach previously developed by Lévesque et al. (23)
is that the amplified region also contains the Pant promoter, whose configuration may be variable in different integrons (24, 34) and is relevant to the expression of the genes
carried by the integrated cassettes (9, 24).
Sequence of the integron-borne metallo--lactamase determinant
and flanking regions.
The nucleotide sequence of the cloned
amplicon carried by pBAUX-30 was determined, and its fidelity was
subsequently verified by direct sequencing of PCR products obtained
from the genomic DNA of AC-54/97 with custom primers designed from the
sequences of unique regions.
Analysis of the sequence data revealed the presence of three gene
cassettes, which contained a blaIMP-related
allele (named blaIMP-2), an aacA4
allele, and an aadA1 allele, respectively, inserted between
the 5'-CS and the 3'-CS of a class 1 integron which was named In42
(Fig. 2).
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FIG. 2.
Structure of the cloned amplicon carried by recombinant
plasmid pBAUX-30, which contains the cassette array of In42 along with
part of the 5'-CS and 3'-CS of the integron. Gene cassettes are
indicated as described in the legend to Fig. 1.
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The partial 5'-CS of In42 contained in the cloned amplicon was
identical to that of In1 (14) except for two differences located within the 35 hexamer of the Pant promoter (a T
in place of a G in the second position and a G in place of an A in the fourth position; Fig. 3). The
configuration of the Pant promoter found in this integron,
with a TTGGCA 35 hexamer and a TAAACT 10
hexamer, is different from those present in other integrons (24, 34; results of a BLAST search on updated
EMBL/GenBank sequence databases), with a 10 hexamer identical to that
found in the strong and hybrid versions of Pant
(24) and a 35 hexamer which is unique for the presence of
a G in the fourth position. Since the Pant configuration of
In42 is original, it will be interesting to investigate its functional
behavior in comparison with that of Pant versions found in
other integrons (9, 24).
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FIG. 3.
Nucleotide sequence of the
blaIMP-2 gene cassette of In42 and flanking
regions. Initiation codons of the various ORFs are indicated, and
protein translations are reported below the sequence. The nucleotide
sequence of the blaIMP gene and the amino acid
sequence of the IMP-1 protein (28) are also reported above
that of blaIMP-2 and below that of IMP-2,
respectively, with dots representing identical residues. The putative
signal peptide of IMP-2, deduced with the SIGCLEAVE program, and the
signal peptide of IMP-1 (21) are underlined. The
blaIMP-2 cassette boundaries are indicated by
vertical arrows. The conserved recombination core sites located at the
cassette boundaries and the inverse core site are boxed. The internal
2L and 2R core sites of the 59-base element (35) are
overlined with arrows. The 35 and 10 hexamers of Pant
are underlined, and the two mutations in the 35 hexamer of the
Pant promoter compared to that of In1 (14) are
in boldface.
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The partial 3'-CS of In42 contained in the cloned amplicon was
identical to that of In1 (34).
The gene cassette containing blaIMP-2 is 831 bp
long. The blaIMP-2 open reading frame (ORF)
exhibits 88% nucleotide sequence identity to
blaIMP found in Japanese isolates (Fig. 3). The
deduced amino acid sequence of the IMP-2 enzyme is 85% identical to
that of IMP-1, with several differences (10 of 36) being clustered within the signal peptide region (Fig. 3). Of the differences found in
the mature protein, none falls in the highly conserved residues
(His-86, His-88, Asp-90, His-149, Cys-168, and His-210, by use of the
numbering for -lactamase II [Bc-II] of Bacillus cereus
569/H [8]) that are known to be involved in the
binding of zinc ions in the B. cereus and Bacteroides
fragilis metallo--lactamases (7, 8, 10) and that are
also conserved in IMP-1 (Fig. 4) and in
the other enzymes of subclass B1 (3, 22, 29, 32). Only two
of the differences between IMP-2 and IMP-1 (Gln-142 and Asp-164, by use
of the numbering for IMP-2; Fig. 4) are found at positions that, in
Bc-II, are known to be in or close to the active site (8).
The G+C content of blaIMP-2 (39.0%) is similar to that of blaIMP (39.1%), and the codon usage
of the two genes is not significantly different
(D2 value = 1.26). The 59-base element of
the blaIMP-2 cassette is 78 bp long and exhibits
the features typical of these elements, with putative IntI1-binding
domains at the left and right ends (Fig. 3). The 59-base element of the
blaIMP-2 cassette is unrelated to those of the
blaIMP cassettes found in Japanese isolates
(Fig. 5).
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FIG. 4.
Sequence comparison between the IMP-1 (28)
and IMP-2 proteins and additional comparison with Bc-II of B. cereus 569/H (15). Identical residues are indicated by
an asterisk; conserved amino acid substitutions are indicated by a
colon. Identity and similarity symbols reported below the Bc-II
sequence refer to the comparison among the three sequences, while those
reported below the IMP-2 sequence refer to the comparison between IMP-1
and IMP-2. The highly conserved residues known to be involved in the
binding of zinc ions in the B. cereus and B. fragilis metallo--lactamases (7, 8, 10) are boxed.
Secondary structure elements of Bc-II (8) are also indicated
above the sequence.
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FIG. 5.
Comparison of the structure of the 59-base elements of
the blaIMP-2 and blaIMP
gene cassettes. The trinucleotides in boldface at the beginning of each
sequence correspond to the stop codons of the
blaIMP-2 and blaIMP ORFs.
The 59-base elements are in uppercase letters and are those present in
the circular forms of the cassettes. The inverse core sites and the
recombination core sites are boxed, and the internal 2L and 2R core
sites of the 59-base elements (35) are overlined with
arrows. Identical residues are indicated by asterisks. The
blaIMP 59-base element reported in the figure is
that of the blaIMP cassette inserted in In31
(20). The minor differences observed between this sequence
and those of previously sequenced blaIMP
cassettes (2, 28) are indicated above (for comparison with
data from reference 28) or below (for comparison
with data from reference 2) the sequence and are
italicized.  , a deletion from that position;  , an insertion at
that position. It should be noted that sequence data from reference
28 are available for comparison only until
nucleotide 101 (in the numbering of the blaIMP
59-base element).
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The aacA4 gene cassette inserted in In42 (Fig. 2) contains
an aacA4 allele that encodes an aminoglycoside
acetyltransferase identical to that encoded by plasmid pIP1855 of
Pseudomonas fluorescens BM2687 (19).
The aadA1 cassette inserted in In42 (Fig. 2) contains an
aadA1 allele that encodes a protein which is nearly
identical (except for the substitution Ala 240
Ser) to the
aminoglycoside adenylyltransferase encoded by In1 of plasmid R46
(14).
Characterization of the metallo--lactamase determinant carried by
A. baumannii AC-54/97 therefore revealed that it is an allelic variant of the blaIMP gene that is
circulating in Japan (2, 20, 28; EMBL/GenBank
database entry D29636) and, evidently, had been acquired independently.
A similar finding demonstrates that different allelic variants of this
resistance determinant exist in nature and can be acquired by
clinically relevant species. It also suggests that the environmental
reservoir of blaIMP alleles could be more
widespread than was originally believed. In fact, finding of different
blaIMP alleles in isolates from different
epidemiological settings raises a question concerning their
geographical distribution and their degree of genetic variability, which could be relevant when screening for similar resistance determinants by PCR-based assays. Recently, two A. baumannii
clinical isolates that produce a metallo--lactamase of alkaline pI
have been reported in Portugal (11). It would be interesting
to investigate the nature of the acquired metallo--lactamase
determinants carried by those isolates to ascertain their potential
relationships with blaIMP or
blaIMP-2.
Similar to blaIMP (2, 16, 20,
28; EMBL/GenBank database entry D29636)
blaIMP-2 was also found to be carried on an
integron-borne gene cassette. Interestingly, however, the sequence similarity between the cassettes containing the two
blaIMP allelic variants was essentially limited
to the coding sequences, while their 59-base elements were unrelated to
each other, revealing different phylogenies of the two cassettes. A
similar situation, which has also been observed with gene cassettes
containing different alleles of class A and class D -lactamases
(31), is consistent with the hypothesis that gene cassettes
could be assembled from individual pools of genes and 59-base elements
or that 59-base elements can be shuffled during cassette evolution
(31). Identification of the environmental source(s) of
blaIMP alleles not only would be relevant from
the epidemiological standpoint but also could provide an interesting
model for study of the evolution of similar cassette-borne resistance
determinants. The strong similarity of base composition and of codon
usage pattern that exists between blaIMP-2 and
blaIMP suggests that their original hosts could
belong to closely related, although not identical, taxa, considering the consistent divergence between the signal peptides of IMP-1 and
IMP-2.
In vitro susceptibility of E. coli carrying the cloned
cassette array of In42.
The susceptibility of E. coli
DH5(pBAUX-30), which carries the cloned cassette array of In42, to
various -lactams and aminoglycosides was determined and compared to
that of A. baumannii AC-54/97 and to that of the E. coli host carrying an empty vector.
The presence of the resistance genes carried by In42 was associated
with a decrease in the in vitro susceptibility to several -lactams
(ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime,
cefepime, imipenem, and meropenem) and aminoglycosides (gentamicin,
tobramycin, and netilmicin), while the susceptibility to aztreonam and
amikacin was apparently unaffected (Table 1).
The pattern of decreased aminoglycoside susceptibility exhibited by
E. coli DH5(pBAUX-30) was consistent with the pattern for the aacA4 allelic variant carried by In42, which encodes
an AAC(6')-II aminoglycoside acetyltransferase active on gentamicin but
not on amikacin (19). The pattern of decreased -lactam susceptibility exhibited by E. coli DH5(pBAUX-30)
indicated that IMP-2 has a broad substrate specificity and was overall
consistent with the kinetic parameters determined with the purified
enzyme (see below). The higher MICs of some antibiotics for A. baumannii AC-54/97 compared to those for E. coli
DH5(pBAUX-30) (Table 1) are likely due to the contribution of
additional resistance mechanisms in the former strain. We are
investigating the nature of the gene cassettes carried by the other
integron detected by ICA-PCR in AC-54/97 (see above).
Purification and characterization of IMP-2 enzyme.
The IMP-2
enzyme was purified from E. coli DH5(pBAUX-30) by
means of an anion-exchange chromatography step, followed by a gel
permeation chromatography step. By SDS-PAGE the purified protein appeared as a single 26-kDa band. The pI of the purified protein, determined by analytical isoelectric focusing, was 8.1 (data not shown). This value is in agreement with the theoretical pI calculated for mature IMP-2 (7.96), assuming the presence of a signal peptide of
17 amino acids (Fig. 3), and is similar to the alkaline pI value
(9 ± 0.2) previously reported for IMP-1 (21).
The purified IMP-2 protein hydrolyzed many -lactam substrates
including penicillins, narrow- to expanded-spectrum cephalosporins including cephamycins, and carbapenems. No hydrolysis of aztreonam was
detected (Table 2). Compared to IMP-1,
the kinetic parameters of IMP-2 were similar to those of some
-lactam substrates but were remarkably different from those of
others (Table 2). Hydrolysis of ampicillin and carbenicillin by IMP-2
showed differences in both Km values and
turnover rates for the two substrates but resulted in similar
kcat/Km ratios, whereas
IMP-1 exhibits a much higher (240-fold)
kcat/Km ratio with
ampicillin than with carbenicillin. Moreover, IMP-2 exhibited
Km and kcat values
considerably lower than those of IMP-1 with cephaloridine and
meropenem. With the former substrate these variations resulted in a
nearly 10-fold lower
kcat/Km ratio for IMP-2,
while with meropenem the much higher affinity of IMP-2 actually
resulted in a higher value for the kcat/Km ratio (Table 2).
The structural polymorphism that exists between IMP-1 and IMP-2
therefore appears to be relevant to their functional properties, as
shown by the notable differences in their kinetic parameters with some
-lactam substrates. For this reason, the two IMP variants could be
an interesting model for study of the structure-function relationships
of these clinically important enzymes.
We acknowledge the excellent technical support of Tiziana di Maggio and
Michela Cappelli and the secretarial assistance of Francesco Lissi and
Elena Sestini.
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-Lactamase
Determinant of Acinetobacter baumannii AC-54/97 Reveals the
Existence of blaIMP Allelic Variants
Carried by Gene Cassettes of Different Phylogeny
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Abstract
Introduction
