Service de Bactériologie-Virologie,
Hôpital de Bicêtre, Assistance Publique/Hôpitaux de
Paris, Faculté de Médecine Paris-Sud, 94275 Le
Kremlin-Bicêtre,1 Laboratoire de
Biologie, Institut Paoli-Calmettes, 13273 Marseille,2 and Laboratoire de Biologie
Médicale, Hôpital d'Instruction des Armées
Bégin, 94160 Saint Mandé,3 France
Received 13 October 1999/Returned for modification 28 December
1999/Accepted 18 January 2000
 |
INTRODUCTION |
Among the class B metalloenzymes,
two carbapenem-hydrolyzing 
-lactamases have been genetically
characterized in Pseudomonas aeruginosa: IMP-1 and
VIM-1 (7, 10, 15). Both enzymes possess the broadest
substrate of hydrolysis range among P. aeruginosa -lactamases, including penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems, but not monobactams. Their activity is
zinc dependent and is inhibited by EDTA. Since 1991, IMP-1 has spread
among gram-negative rods, including P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens,
Burkholderia cepacia, Alcaligenes xylosoxidans,
and members of the family Enterobacteriaceae in Japan
(7). According to the results of a 1996 to 1997 survey of
IMP-1-producing gram-negative bacteria in Japan, 1.3% of P. aeruginosa isolates and 4.4% of Serratia marcescens
isolates produced IMP-1 through acquisition of plasmids (H. Kurokawa,
T. Yagi, N. Shibata, K. Shibayama, and Y. Arakawa, Letter, Lancet
354:955, 1999). Other uncharacterized
carbapenem-hydrolyzing -lactamases have been reported in P. aeruginosa and Acinetobacter baumannii isolates
in Europe (5; N. M. Woodford,
M.-F. I. Palepou, G. S. Babini, J. Bates, and
D. M. Livermore, Letter, Lancet 352:546-547, 1998).
VIM-1 has been described recently from several P. aeruginosa Italian isolates and shares 28% amino acid identity with IMP-1 (10; G. Cornaglia, Abstr. 39th Intersci.
Conf. Antimicrob. Agents Chemother., abstr. 1482, 1999).
blaIMP-1 is plasmid or chromosome located, while
blaVIM-1 was identified as chromosome
borne only (7, 10).
blaVIM-1 and blaIMP-1 are
encoded within the variable region of class 1 integrons (1, 9,
10). Integrons are genetic structures capable of capturing gene
cassettes. Class 1 integrons, which are most commonly isolates from
antibiotic-resistant clinical isolates, possess two conserved segments
(5'-CS and 3'-CS) located on either side of the integrated genes
(20). Gene cassettes are discrete mobile units comprising a
gene, usually an antibiotic resistance gene, and a recombination site
that is recognized by the integrase (20). The
cassette-associated recombination sites, known as 59-base elements, are
located downstream of inserted genes and are of variable length
(23). Integron-located resistance genes provide them with a
wide potential for expression and dissemination.
In this work, we report analysis of the -lactamase content and
genetic support of P. aeruginosa COL-1 isolated in France in
1996, which hydrolyzed imipenem but remained susceptible to monobactams.
| |
MATERIALS AND METHODS |
Bacterial strains.
The bacterial strains and plasmids used
in this work are listed in Table 1. The
P. aeruginosa COL-1 isolate was identified with the API-20
NE system (bioMérieux, Marcy l'Etoile, France).
Antimicrobial agents and susceptibility testing.
The
antimicrobial agents and the agar dilution technique for MIC
determination have been described elsewhere (16).
Antibiotic-containing disks were used for routine antibiograms by the
disk diffusion assay (Sanofi-Diagnostics Pasteur, Marnes-La-Coquette, France).
Molecular techniques.
A search for
blaVIM-1- or
blaIMP-1-like genes in P. aeruginosa was performed by PCR amplification with the
following sets of primers: for blaVIM-1,
VIM-1A (5'-TCTACATGACCGCGTCTGTC-3') and VIM-1B
(5'-TGTGCTTTGACAACGTTCGC-3'; and for
blaIMP-1, IMP-1A (5'-CTACCGCAGCAGAGTCTTTGC-3') and IMP-1B
(5'-GAACAACCAGTTTTGCCTTACC-3') (10, 15).
Whole-cell DNA of P. aeruginosa COL-1 or of P. aeruginosa MKAM 12, which produced IMP-1 (Table 1), was extracted
as described previously (16) and used as a template in these
PCR experiments (22). BamHI- or
HindIII-restricted genomic DNA of P. aeruginosa COL-1 was ligated into either BamHI or
HindIII-restricted pBK-CMV phagemid as described
previously (16). Selection (amoxicillin [30 µg/ml] or
imipenem (2 µg/ml) and kanamycin [30 µg/ml]) and analysis
of recombinant plasmids and the electroporation technique used have
been described previously (16).
Transfer of resistance genes into in vitro-obtained rifampin-resistant
Escherichia coli JM109 or ciprofloxacin-resistant P. aeruginosa PU21 was attempted by liquid and solid conjugation assays at 30 and 37°C (17). Transconjugant selection was
performed on Trypticase soy (TS) agar plates containing rifampin (200 µg/ml), ciprofloxacin (4 µg/ml) and amoxicillin (30 µg/ml),
or imipenem (2 µg/ml). Plasmid DNA extraction of P. aeruginosa COL-1 was attempted by different methods as described
previously (17). The plasmid extract from P. aeruginosa COL-1 culture was electroporated into E. coli DH10B with selection on amoxicillin- or
imipenem-containing TS plates. Conjugations were repeated with
an E. coli DH10B electrotransformant as the donor and
rifampin-resistant E. coli strain JM109 as the recipient.
To identify the location of the -lactamase gene, whole-cell DNA of
P. aeruginosa and unrestricted and restricted plasmid DNAs
of P. aeruginosa COL-1 and of a corresponding E. coli electrotransformant were run on a 0.7% agarose gel,
transferred onto a Hybond N+ membrane (Amersham Pharmacia
Biotech, Orsay, France), and hybridized with a PCR-obtained 801-bp
internal probe for blaVIM-2 (VIM-2A; 5'-ATGTTCAAACTTTTGAGTAGTAAG-3' and VIM-2B;
CTACTCAACGACTGAGCG-3'). The nonradioactive ECL (enhanced
chemiluminescence) random prime system was used (Amersham Pharmacia
Biotech). Briefly, it includes a nucleic acid labeling, hybridization,
and detection system based on a combination of enhanced
chemiluminescence detection and random primer labeling of DNA.
DNA sequencing and protein analysis.
The cloned DNA fragment
inserted into recombinant plasmid pNOR-2001 was sequenced on both
strands with an Applied Biosystems sequencer (ABI 373). The nucleotide
and deduced amino acid sequences were analyzed and compared to
sequences available over the internet as described previously
(16).
-Lactamase extraction and purification.
-Lactamase
extraction was obtained from 6 liters of TS broth culture of E. coli DH10B(pNOR-2001) as described previously (16).
Similar unpurified -lactamase extract was obtained from a 10-ml
culture of P. aeruginosa COL-1 subsequently resuspended in
0.5 ml of sodium phosphate buffer.
The -lactamase extract of E. coli DH10B(pNOR-2001) was
dialyzed overnight in 50 mM Bis-Tris buffer (pH 6.5). The -lactamase extract was loaded onto a preequilibrated Q-Sepharose column (Amersham Pharmacia Biotech). The -lactamase was eluted in 200 mM NaCl and
subsequently dialyzed overnight against 50 mM phosphate buffer containing 150 mM NaCl (pH 7.0). This prepurified extract was loaded
onto a 1.6- by 47-cm gel filtration column packed with Superdex 75 (Amersham Pharmacia Biotech) equilibrated with 50 mM phosphate buffer
(pH 7.0) containing 150 mM NaCl. The fraction containing the
-lactamase activity was dialyzed overnight against 30 mM cacodylate
buffer (pH 6.5) containing 50 µM ZnCl2 prior to a 10-fold
concentration with Centrisart-C30 columns (Sartorius, Goettingen,
Germany). At each purification step, the -lactamase activity was
determined qualitatively by nitrocefin hydrolysis (Oxoid, Dardilly,
France) or quantitatively in a spectrophotometer with 100 µM
imipenem (297 nm, 
= 9,210 M
1
cm1) as the substrate in the dialysis buffer. One unit of
enzyme activity was defined as the amount of enzyme that hydrolyzed 1 µmol of substrate per min at 30°C. The protein content was measured by using the Biorad DC protein assay (Bio-Rad), and the specific activities of the crude extract and of the purified -lactamase from
E. coli DH10B(pNOR-2001) were compared.
Analytical IEF.
The -lactamase extract from P. aeruginosa COL-1 and the purified -lactamase from E. coli DH10B(pNOR-2001) were subjected to analytical isoelectric
focusing (IEF) as described previously (16). The focused
-lactamases were detected by overlaying the gel with 1 mM nitrocefin
(Oxoid) or with an iodine starch gel containing 0.5% (wt/vol)
imipenem in 100 mM phosphate buffer (pH 7.0).
Kinetic measurements and Mr
determination.
Purified -lactamase from a culture of E. coli DH10B(pNOR-2001) was used for determination of kinetic
parameters (kcat, Km) performed at 30°C in 30 mM sodium
cacodylate buffer (pH 6.5) supplemented with 50 µM ZnCl2
as described previously (16). Inactivation by
Zn2+ removal was studied at 30°C in cacodylate buffer in
the presence of different concentrations of EDTA, with 100 µM
imipenem as the reporter substrate. The 50% inhibitory
concentration (IC50) was determined for EDTA. Reactivation
by Zn2+ (2 mM) was assayed by measuring activity after
incubation with EDTA-treated (2 mM) enzyme for 15 min at 30°C.
The relative molecular mass (Mr) of the purified
-lactamase from E. coli DH10B(pNOR-2001) was determined
by gel filtration with a 1.6- by 47-cm column packed with Superdex 75 (Amersham Pharmacia Biotech) equilibrated and eluted with 50 mM
phosphate buffer (pH 7.0) containing 150 mM NaCl. Each elution peak was tested for -lactamase activity by using nitrocefin as a substrate. The peak that showed the highest -lactamase activity was linearly plotted against the logarithm of the molecular masses of standard proteins (Amersham Pharmacia Biotech) to determine the
Mr of the purified -lactamase.
Nucleotide sequence accession number.
The nucleotide
sequence data reported in this paper have been assigned to the
EMBL/GenBank nucleotide sequence database under accession no. AF 191564.
| |
RESULTS |
Origin of the P. aeruginosa COL-1 isolate and
preliminary susceptibility testing.
P. aeruginosa COL-1 was
isolated in 1996 at the Institut Paoli-Calmettes in Marseilles, France.
A 39-year-old-French woman was hospitalized for chronic myelogenous
leukemia and pancytopenia before the performance of an allogeneic bone
marrow transplantation. She had not travelled recently to Italy or
Japan, and no information is available on any patient transfer from
Italian hospitals concomitant with her hospital stay. The patient had
fever and received a course of imipenem and amikacin. Despite
this treatment, she died of septic shock 5 days later. The day after
her death, blood cultures inoculated 3 days earlier grew a
carbapenem-resistant P. aeruginosa isolate,
COL-1. Antibiotic susceptibility testing by disk diffusion suggested an
uncommon mechanism of resistance, since the isolate was resistant to
most -lactams, including ureidopenicillins, ureidopenicillins--lactamase inhibitors, narrow-spectrum
cephalosporins, cefepime, ceftazidime, imipenem, and meropenem,
but remained fully susceptible to aztreonam (data not shown). These
results were confirmed by MIC analysis (Table
2). Disk diffusion testing revealed that
P. aeruginosa COL-1 was also resistant to kanamycin,
tobramycin, streptomycin, spectinomycin, tetracycline, and
chloramphenicol; of intermediate susceptibility to fluoroquinolones and
rifampin; and susceptible to fosfomycin.
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TABLE 2.
MICs of -lactams for P. aeruginosa COL-1,
E. coli DH10B harboring recombinant plasmid pNOR-2001,
and E. coli reference strain DH10B
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Cloning, sequencing, and analysis of the genetic support of the
-lactamase gene.
Preliminary PCR-based experiments failed to
detect blaIMP-1 or a
blaVIM-1-like gene in the P. aeruginosa COL-1 isolate, although no
blaVIM-1-containing strain was used as a
positive control. Ten recombinant E. coli clones were
obtained after cloning experiments and selection on
amoxicillin-containing plates. One of them, recombinant plasmid
pNOR-2001 (Fig. 1), produced a
-lactamase as assessed by a positive nitrocefin test. Analysis of
the nucleotide sequence from the 3,843-bp insert in pNOR-2001 revealed
an 801-bp-long open reading frame (ORF) encoding a 266-amino-acid
protein, named VIM-2 (Fig. 2). Amino acid
sequence analysis of this protein revealed a putative cleavage site
between the alanine and serine residues at positions 20 and 21, respectively (14) (Fig. 2). The G+C content of this ORF was
56%, a value that did not lie within the expected range of the
G+C content of P. aeruginosa genes (ranging from 60.1 to 69.5%). The codon usage differed as well from that of P. aeruginosa genes (28). The deduced amino acid sequence of this ORF showed low amino acid identity with most of the
Ambler class B carbapenem-hydrolyzing -lactamases, ranging from 32% to 4% for B-II from Bacillus cereus to GOB-1 from
Chryseobacterium meningosepticum, respectively (Table
3). It was most closely related to VIM-1
(90% amino acid identity), a recently identified metallo--lactamase isolated from an Italian P. aeruginosa
clinical isolate (10). VIM-1 and VIM-2 clustered
within a subgroup of carbapenem-hydrolyzing -lactamases (Fig.
3). The conserved amino acids among
carbapenem-hydrolyzing -lactamases that may bind either to
Zn2+ ions or a water molecule near or within their putative
active site were found in VIM-2 (3, 18, 24, 27): His-86,
His-88, Asp-90, His-149, His-225, Cys-168, and His-210 (Fig.
4). These amino acids were
identical for VIM-2 and VIM-1 (Fig. 4). Amino acid changes in VIM-2
compared to the sequence of VIM-1 occurred mostly within the
NH2- or COOH-terminal regions (Fig. 4).
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FIG. 1.
(A) Schematic map of the recombinant plasmid pNOR-2001
encoding blaVIM-2 (arrows indicate its
translational orientation) and (B) comparison with the
blaVIM-1-containing integron as cloned into
recombinant plasmid pBCLL/39H (10). For pNOR-2001,
the solid line represents the cloned insert from P. aeruginosa COL-1 with the ORFs that are boxed, and the dotted
lines indicate the vector pBK-CMV.
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FIG. 2.
Nucleotide sequence of the 3,843-bp fragment of the
cloned BamHI-fragment of pNOR-2001 containing the
blaVIM-2 coding region and its integron. The
deduced amino acid sequence is designated in the single-letter code
below the nucleotide sequence. The start codons of IntI1,
blaVIM-2, qacE 1, and
sul1 genes are indicated by horizontal arrows, and their
stop codons are indicated by asterisks. Only the start and the end of
the integrase, qacE1, sul1, and
orf5 genes are represented. The 35 and 10 sequences of
the promoters Pc, P2, and Pint are
underlined; RBS indicates the putative ribosome binding site for
blaVIM-2. The conserved core and inverse core
sites located at the blaVIM-2 cassette
boundaries are boxed, and the composite 59-base element is italicized.
The cassette boundaries are indicated by vertical arrows. The left part
of the attI1 site is underlined with a dotted line.
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FIG. 3.
Dendrogram obtained for 10 representative Ambler class B
carbapenem-hydrolyzing -lactamases by parsimony analysis
(16). The alignment used for tree calculation was performed
with Clustal W (16) followed by minor adjustments in order
to reduce the number of gaps and to maintain the alignment of the amino
acid residues identified as critical for activity of some class B
carbapenem-hydrolyzing -lactamases. Branch lengths are drawn to
scale and are proportional to the number of amino acid changes. The
percentage values at branching points (underlined) refer to the number
of times a particular node was found in 100 bootstrap replications (the
star indicates uncertainty about nodes with bootstrap values of less
than 50%). The distance along the vertical axis has no significance.
The origins of the -lactamases are given in Table 3.
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FIG. 4.
Comparison of the amino acid sequence of VIM-2 with that
of VIM-1. Identical amino acid residues are indicated by asterisks, and
functionally equivalent amino acid substitutions are indicated by
colons. Boldface amino acids are those of the putative active sites of
VIM-1 and of VIM-2. The numbering is according to the B-II sequence of
B. cereus (18).
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Further sequencing of the cloned fragment in pNOR-2001 revealed key
signatures of the class 1 integron, such as (i) a 5'-CS containing an
intI1 integrase gene with its own promoter region, (ii) an
attI1 recombination site, and (iii) a 3'-CS containing qacE1 and sulI1 (Fig. 2). The initiation codon
(ATG) of blaVIM-2 was preceeded by two putative
promoter regions named Pc (regions 35 [TGGACA]
and 10 [TAAGCT]) and P2 (regions 35
[TTGTTA] and 10 [TACAGT]), which lie within
the integrase structural gene (Fig. 2). The secondary promoter
P2 identified in some class 1 integrons was in its active
form, since the insertion of three guanosine molecules 119 bases
downstream of the promoter Pc between the 35 and 10
regions of P2 brought the spacing to 17 bp (11). The blaVIM-2 gene cassette, which was inserted
in the attI1 recombination site, has a core site
(GTTATGC) and an inverse core site (GCATAAC) (Fig. 2 and 5). The 59-base element
was 72 bp long. This class 1 integron, named In56, contained only the
blaVIM-2 gene cassette (Fig. 2). The G+C content
of this 59-base element was 58%. The 59-base elements for
blaVIM-1 and blaVIM-2
cassettes clearly differed in size and structure (Fig. 5). Only the
right and left ends of the 59-base element shared significant homology,
while the center part required three gaps to be introduced in the
blaVIM-2 59-base element in order to obtain an
optimal alignment (Fig. 5).
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FIG. 5.
Comparison of the sequences of the 72 bp of the
blaVIM-2 59-base element (59-be) and the 81 bp
of the blaVIM-1 59-base element present in the
circular form of the -lactamase gene cassettes to a 59-base element
consensus sequence given below. The inverse core and core sequences are
double underlined. L1, L2, R1, and R2 are four regions found to be
highly conserved within 59-base elements of class 1 integrons (20,
23). Consensus bases (Cons) in uppercase letters are present in
two-thirds or more of the 59-base elements, and bases in lowercase
letters are present in half or more of the 59-base elements. Stars
indicate bases of the 59-base element of
blaVIM-2 that fit the consensus. R, purine; Y,
pyrimidine; S, C or G; N, undetermined base.
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Conjugation experiments failed to transfer -lactam resistance from
P. aeruginosa COL-1 to rifampin-resistant E. coli
DH10B or ciprofloxacin-resistant P. aeruginosa strains.
However, plasmid extraction from P. aeruginosa COL-1
followed by electroporation into E. coli gave a ca. 45-kb
natural plasmid, pNOR-2000 (data not shown). This plasmid was not
self-transferable from E. coli to E. coli. This
plasmid conferred a similar resistance profile to -lactams, as was
found for recombinant plasmid pNOR-2001, as well as resistance to
sulfamides. Hybridization experiments confirmed the presence of a
plasmid of similar size in P. aeruginosa COL-1 as in
the E. coli DH10B electroporant (data not shown).
Biochemical properties of VIM-2 and resistance pattern conferred by
VIM-2.
IEF analysis of the -lactamase preparation revealed that
E. coli DH10B(pNOR-2001) produced only a single
-lactamase with a pI of 5.6. For P. aeruginosa COL-1, an
additional band of -lactamase activity with a pI of 9.0 was found
that likely corresponded to the chromosomal P. aeruginosa
AmpC cephalosporinase (26), the pI of 5.6 being revealed
only after imipenem hydrolysis detection.
VIM-2 was purified 400-fold from a culture of E. coli
DH10B(pNOR-2001) with a specific activity of 17.8 U · mg of
protein1 with imipenem as the substrate. The
Mr of the mature -lactamase was 29.7 kDa.
Kinetic parameters revealed that VIM-2 has a broad hydrolysis profile,
including most -lactams, except monobactams (aztreonam), cefsulodin,
cefepime, and cefpirome (Table 4). VIM-2
activity was higher against imipenem than against meropenem.
Its activity was inhibited by EDTA (IC50, 50 µM) and was
restored in the presence of 2 mM ZnCl2. Thus, VIM-2 could
be included in the functional group 3a of the Bush -lactamase
classification that includes most metalloenzymes, except those from
Aeromonas sp., Myroides odoratus, and
Legionella gormanii, which show a restricted hydrolysis spectrum to carbapenems (3, 18).
The natural plasmid pNOR-2000 (data not shown) or the recombinant
plasmid pNOR-2001 conferred resistance to aminopenicillins and narrow-
and extended-spectrum cephalosporins and a reduced susceptibility to
piperacillin, cefepime, and carbapenems in E. coli DH10B.
However, the MIC of aztreonam for E. coli DH10B(pNOR-2001) remained unchanged compared to those for the parental E. coli DH10B strain (Table 2).
| |
DISCUSSION |
The carbapenem-hydrolyzing -lactamase VIM-2 shared 90% amino
acid identity with VIM-1. It has been obtained from an isolate from the
French Riviera region (Marseilles) that is only 300 km from Verona,
where VIM-1 had been isolated (10). Moreover, patient transfers were common between Italian hospitals and Marseilles hospitals until 1994, thus underlining a possible regional outbreak of
organisms producing related enzymes.
Both VIM-1 and VIM-2 can be classified in the protein sequence-based
subclass B1 of metallo--lactamases (3). The amino acids
that may be involved in the catalytic site of these enzymes were
identical (18, 24, 27). Once cloned onto a plasmid vector
and expressed in E. coli, both enzymes provided a similar pattern of decreased susceptibility to -lactams, except aztreonam. However, their level of resistance to carbapenems remained low. As
suggested from results of experiments performed with another carbapenem-hydrolyzing -lactamase (an IMP-1-like enzyme), the permeability coefficient of each -lactam may play a major role in
explaining the level of resistance to each -lactam in gram-negative bacteria that produce metalloenzymes (13), thus explaining
the low level of resistance to ureidopenicillins in P. aeruginosa COL-1 (Table 1). VIM-2 did not significantly hydrolyze
either cefsulodin, cefepime, or cefpirome. The high MIC of cefepime for P. aeruginosa COL-1 could be due to its low permeability
coefficient (13). Interestingly, P. aeruginosa
isolates that expressed either VIM-1 or VIM-2 -lactamases were fully
susceptible to aztreonam only and resistant to most aminoglycosides,
thus limiting the choice of active drugs in clinical use.
Taking into account the structural similarity between VIM-1 and VIM-2
and the similar MIC data, it is likely that the biochemical properties
of VIM-1 are close to those of VIM-2. Although related to B-II from
B. cereus, VIM-2 did not share its peculiar property of
better hydrolyzing meropenem than imipenem (3, 6,
18). The extended hydrolysis profile of VIM-2 was different from
the restricted hydrolysis profile found for CphA-1 and ImiS from
Aeromonas species (3, 18). Therefore, VIM-2 could
be included in biochemical group 3a (3).
While the G+C content of blaVIM-1 is not typical
of P. aeruginosa genes, it could correspond to that
of genes found in members of the family
Enterobacteriaceae. Upstream of
blaVIM-2, two putative promoters,
Pc and P2, were found (Fig. 2). Compared to
other Pc sequences, the Pc promoter for
blaVIM-2 is a weak promoter (4, 11).
P2 expression may be responsible for up to 90% of
blaVIM-2 transcription, as described for other
integron-located genes (4, 11).
blaVIM-1 and blaVIM-2 are
located on different class 1 integrons not related to
blaIMP-1 integrons, and the corresponding 59-base elements were different in size and structure (Fig. 5). A
similar situation was observed for dfrA1, dfrA5,
and dfrA7 genes, which share 70% amino acid identity, but
have unrelated 59-base elements, with the first and last 20 bp of these
59-base elements showing similarity to the consensus (20,
23). The catB2 and catB3 cassettes also
contain quite different 59-base elements (20). The fact that
closely related genes such as the VIM-1 and VIM-2 genes have different
59-base elements supports the hypothesis of a separate origin of the
genes and of the 59-base element in each cassette. The class 1 integron
for blaVIM-2 contained only this gene cassette,
as opposed to the class 1 integron that contained blaVIM-1 together with at least another
gene cassette (Fig. 1) (10).
The dendrogram analysis revealed that VIM-2 and VIM-1 clustered
in the same carbapenem-hydrolyzing -lactamase
subgroup and that neither of them is related to the
chromosome-borne class B carbapenem-hydrolyzing -lactamases (Fig.
3). It may now be time to detect gram-negative rods that produce these
novel expanded-spectrum -lactamases to prevent their spread. Their
detection should be performed with a PCR technique using, for example,
the following consensus primer sequences: VIMB,
5'-ATGGTGTTTGGTCGCATATC-3'; and VIMF,
5'-TGGGCCATTCAGCCAGATC-3'.
This work was funded by the Ministère de l'Education
Nationale et de la Recherche, Université Paris XI, Faculté
de Médecine Paris Sud (UPRES, JE-2227); and the French network on
-lactamase research "Les -lactamases: de l'observation
clinique à la structure," France.
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-Lactamase and Its Plasmid- and Integron-Borne Gene from
a Pseudomonas aeruginosa Clinical Isolate in
France
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Abstract
Introduction
