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Antimicrobial Agents and Chemotherapy, February 1999, p. 297-301, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization and Nucleotide Sequence of CARB-6,
a New Carbenicillin-Hydrolyzing 
-Lactamase from Vibrio
cholerae
Danièle
Choury,1,*
Gérald
Aubert,2
Marie-France
Szajnert,3
Kemal
Azibi,3,4
Marc
Delpech,1 and
Gérard
Paul5
Laboratoire de Biologie Moléculaire des
Cellules Eucaryotes,1
INSERM
U129,3 and
Laboratoire de Recherche en
Microbiologie,5 UFR Cochin Port-Royal, 75014 Paris, and
Laboratoire de Bactériologie, CHU,
Saint-Etienne,2 France, and
CHU
Alger-Ouest, Algiers, Algeria4
Received 28 May 1998/Returned for modification 21 September
1998/Accepted 20 November 1998
 |
ABSTRACT |
A clinical strain of Vibrio cholerae non-O1 non-O139
isolated in France produced a new 
-lactamase with a pI of 5.35. The purified enzyme, with a molecular mass of 33,000 Da, was characterized. Its kinetic constants show it to be a carbenicillin-hydrolyzing enzyme
comparable to the five previously reported CARB -lactamases and to
SAR-1, another carbenicillin-hydrolyzing -lactamase that has a pI of
4.9 and that is produced by a V. cholerae strain from Tanzania. This -lactamase is designated CARB-6, and the gene for
CARB-6 could not be transferred to Escherichia coli K-12 by conjugation. The nucleotide sequence of the structural gene was determined by direct sequencing of PCR-generated fragments from plasmid
DNA with four pairs of primers covering the whole sequence of the
reference CARB-3 gene. The gene encodes a 288-amino-acid protein that
shares 94% homology with the CARB-1, CARB-2, and CARB-3 enzymes, 93%
homology with the Proteus mirabilis N29 enzyme, and 86.5%
homology with the CARB-4 enzyme. The sequence of CARB-6 differs from
those of CARB-3, CARB-2, CARB-1, N29, and CARB-4 at 15, 16, 17, 19, and
37 amino acid positions, respectively. All these mutations are located
in the C-terminal region of the sequence and at the surface of the
molecule, according to the crystal structure of the
Staphylococcus aureus PC-1 -lactamase.
| |
INTRODUCTION |
Clinical strains of Vibrio
cholerae are naturally susceptible in vitro to broad-spectrum
penicillins and cephalosporins. Some isolates that exhibit
plasmid-mediated resistance to -lactams that are susceptible to the
effects of -lactamase inhibitors produce TEM-1 (9, 11,
35) or SAR-1, a carbenicillin-hydrolyzing -lactamase with an
isoelectric point of 4.9 (29).
Until 1992, V. cholerae of serogroup O1 was the only known
causative agent of pandemic cholera. Strains termed non-O1 had never
been shown to generate epidemic cholera, although some were responsible
for sporadic gastrointestinal or extraintestinal diseases, such as
pyogenic infection and septicemia in susceptible hosts (24,
30). However, a non-O1 strain is causing the current cholera
pandemic described in Bangladesh. The strain is V. cholerae O139, which was initially isolated in southern Asia (34).
We report here the biochemical and structural properties of a new
-lactamase produced by a clinical strain of V. cholerae non-O1, non-O139 that is resistant to -lactams and that was
responsible for the death of a cirrhotic patient after a lower-limb
infection (3). The strain was isolated at Bellevue Hospital
(CHU, Saint-Etienne, France).
In preliminary studies, the -lactamase was shown to have a pI of
5.35 and to be inhibited by an anti-CARB-3 serum (3) that
inactivates the five previously described enzymes (27). We
report here the physical and biochemical properties of the purified
enzyme. The nucleotide sequence of the gene and the deduced amino acid
composition of this -lactamase were determined and compared to those
of other -lactamases.
(This work was presented, in part, at the 16th and 17th
Interdisciplinary Meetings of Anti-Infectious Chemotherapy, Paris, France, in 1996 [5] and 1997 [6],
respectively.)
| |
MATERIALS AND METHODS |
Clinical strain.
The clinical strain of V. cholerae non-O1, non-O139 used in this study was resistant to
aminopenicillins and carboxypenicillins and was isolated from a
cirrhotic French farmer at the Bellevue Hospital (CHU, Saint-Etienne,
France). This strain was responsible for a fatal septic shock as a
consequence of a severe infection of the right leg that developed
within 2 weeks after a stay on the Mediterranean coast (3).
-Lactamase purification and analytical IEF.
The
-lactamase extract was prepared from 24 liters (92 g [wet weight])
of an overnight culture grown in nutrient broth (Difco) with
amoxicillin (50 µg/ml). The cells were collected by centrifugation and broken by sonication at 4°C (100 W at 20 kHz) in 50 mM Tris HCl
buffer (pH 7) containing 2 mM EDTA, 7 mM -mercaptoethanol, and 10%
sucrose. The sample was centrifuged at 20,000 rpm (Sorvall RC2B) for 20 min, and the supernatant containing the -lactamase was collected.
The enzyme was purified by ammonium sulfate precipitation to between 40 and 80% saturation, ion-exchange chromatography (DE 52), and gel
filtration on 5% acrylamide and 4% agarose (Ultrogel AcA54;
IBF-BIOSEPRA). The purified enzyme was concentrated by ultrafiltration
(26). Purity was verified by sodium dodecyl sulfate
(SDS)-electrophoresis (19). Analytical isoelectric focusing (IEF) was carried out in a polyacrylamide gel with an Ampholine (Pharmacia) gradient (pH 3.5 to 9.5) (20). The -lactamase
in the gel was revealed by the iodine procedure in the presence of benzylpenicillin (17). The pI of the enzyme was determined
by using TEM-1 (R111) and PSE-4 (16, 23) as reference
-lactamases.
Molecular weight.
The molecular weight was determined by
SDS-gel electrophoresis (19).
Substrate and inhibitor profiles of the -lactamase.
The
following antibiotics and -lactamase inhibitors were used to
determine kinetic constants: benzylpenicillin (Sarbach); amoxicillin,
ticarcillin, clavulanic acid, and cloxacillin (Smith Kline Beecham);
piperacillin and tazobactam (Lederlé); methicillin and oxacillin
(Bristol-Myers); cephaloridine (Glaxo); sulbactam (Pfizer); cephalothin
(Panpharma); and mezlocillin (Bayer).
Kinetic parameters (Km and relative
Vmax) were determined and inhibitor studies were
performed at pH 7 and 37°C with a pH Stat apparatus by the
microacidimetric method (15). One -lactamase unit is
defined as the amount of enzyme that hydrolyzes 1 µmol of
benzylpenicillin per min at pH 7 and 37°C.
Preparation of DNA for PCR.
Plasmid DNA was extracted and
purified by the X-Trax procedure (Medgene Science).
PCR amplification and nucleotide sequencing.
The nucleotide
sequence was determined by direct sequencing of PCR-generated DNA
fragments with four pairs of synthetic primers (Table
1) covering the whole reference sequence
of CARB-3 gene (18). The first fragment (V2-V2') was
obtained by the nested PCR method (Fig.
1).
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FIG. 1.
Sequencing strategy for CARB-6 -lactamase gene. The
CARB-6 gene is represented by the black box. The arrows indicate the
oligonucleotide positions.
|
|
Amplification by PCR with specific primers was performed with 10 µl
of the plasmid DNA preparation and 2 µl of each primer
solution (100 ng/µl) in a reaction mixture with a total volume
of 100 µl. The
reaction mixture contained 10 µl of PCR buffer
(GIBCO BRL), each
deoxynucleoside triphosphate at a concentration
of 0.2 mM, 4.5 mM
MgCl
2, and 2.5 U of
Taq DNA polymerase (GIBCO
BRL). The PCR program used was an initial denaturation step at
96°C
for 5 min, followed by 30 cycles (50 s of denaturation at
96°C,
50 s of annealing at 55°C, and 50 s of extension at 72°C)
and a final extension at 72°C for 7 min. Nested PCR amplification
was
performed with V1 and V1' as the outer primer pair and V2
and V2' as
the inner primer pair (Table
1). One microliter of
the PCR product
obtained with the outer primer pair was used for
the PCR with the inner
primer pair under the conditions described
above. Amplifications were
carried out on a Perkin-Elmer Cetus
apparatus, and the products were
analyzed on 2% agarose
gels.
The PCR products were sequenced with an automatic sequencer (ABI 377)
by using a dye terminator cycle sequencing kit (Ready
Reaction 402080)
with Ampli Taq polymerase (Fluorescent
Sequencing).
Sequence analysis.
The BLASTN program for nucleic acid
databases (GenBank, EMBL) and the BLASTP program for amino acid
database (SwissProt) were used to search for related -lactamases
with sequences homologous to that of the purified -lactamase through
the World Wide Web BLAST server of the National Center for
Biotechnology Information (1). Multiple sequence alignments
were performed with the CLUSTALW facilities of the BISANCE software
package in the INFOBIOGEN server (8, 33).
The InsightII program was used for molecular modeling of the
Staphylococcus aureus PC-1
-lactamase
molecule.
Nucleotide sequence accession number.
The CARB-6
-lactamase sequence has been submitted to GenBank. Its accession no.
is AF 030945.
| |
RESULTS |
Purification and molecular weight determination.
The enzyme
was purified 200-fold, and the yield was 10%. Its molecular weight was
estimated to be 33,000 (Table 2).
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TABLE 2.
Physicochemical properties of the CARB-6 -lactamase of
V. cholerae and comparison with TEM-1 and other
carbenicillin-hydrolyzing enzymes
|
|
pI.
The -lactamase, both as a crude extract and after
purification, was subjected to IEF. In both cases a single band was
obtained at pI 5.35, which is between those of TEM-1 (pI 5.4) and PSE-4 (CARB-1) (pI 5.3) (Table 2).
Substrate and inhibition profiles.
The substrate and
inhibition profiles of the purified enzyme were determined (Tables
3 and 4).
They indicate that it is a penicillinase which hydrolyzes ticarcillin
and mezlocillin (59 and 100% hydrolysis relative to that for
penicillin, respectively). The Km values
indicate that the purified enzyme has a higher affinity for
carboxypenicillins such as ticarcillin (55 µM) and ureidopenicillins such as mezlocillin (58.5 µM) than for benzylpenicillin (96 µM) and
amoxicillin (157 µM) (Table 3).
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TABLE 3.
Kinetic parameters for V. cholerae CARB-6
-lactamase in comparison with the published values for
-lactamases TEM-1, SAR-1, and CARB-1
to CARB-3a
|
|
The inhibition profile is compatible with that of a class A
-lactamase (Table
4).
Sequence analysis.
The nucleotide sequence of the PCR product
obtained with consecutive primers (967 nucleotides) contains an open
reading frame of 962 nucleotides. The coding region (CDS) of 867 nucleotides (positions 96 to 962) encodes a protein of 288 amino acids
(Fig. 2 and
3).
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FIG. 2.
Gene sequence and deduced amino acid sequence of the
V. cholerae -lactamase (CARB-6). The deduced amino acid
sequence is designated by the one-letter code. The active site,
STFK, is boxed, and the differences relative to CARB-1 to
CARB-3 are underlined.
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FIG. 3.
Multiple sequence alignment of the amino acid sequences
of CARB-1, CARB-2, CARB-3, CARB-4, P. mirabilis N29,
P. mirabilis GN79, and CARB-6 -lactamases. The shadowed
boxes (I to VII) correspond to amino acid boxes conserved in all
penicillin-recognizing enzymes, as identified by Joris et al.
(14). Alpha-helix and beta-barrel motifs are indicated from
the PC-1 crystal structure (4, 12). Asterisks indicate the
conserved residues specific for class A -lactamases. Amino acid
changes are written as black letters in white boxes. Sequences are
numbered as described by Ambler (2).
|
|
The deduced amino acid sequence is very similar to those of class A
-lactamases: a
bla active-site (STFK) tetrad at positions
65 to 68 (positions 70 to 73 according to the standard numbering
scheme
of Ambler [
2]), cysteine residues at positions 72 and
118 (Ambler positions 77 and 123), and all seven conserved amino
acid
boxes (
4,
14) and specific conserved residues (Fig.
3).
Nucleic and amino acid analyses with the BLAST and FASTA programs
showed the purified enzyme has substantial homology (94%)
with the
CARB-1, CARB-2, and CARB-3
-lactamases of the same group.
The
starting codon and the length of the coding region (288 amino
acids)
are strictly conserved (Fig.
2).
Multiple sequence alignment of the CARB-6 amino acid sequence with the
sequences of the previously described CARB-1, CARB-2,
and CARB-3
(
16,
18), CARB-4 (
28,
32), and
Proteus
mirabilis N29 (
13)
-lactamases confirms that it is a
carbenicillinase.
Moreover, CARB-6 possess an RSG box VII that is
encountered only
in this class of
-lactamases. It appears to be more
closely related
to CARB-3, CARB-2, CARB-1, and N29 than to CARB-4 (Fig.
3). The
nucleotide and amino acid sequences of CARB-6 and CARB-3 are
very
similar. CARB-6 differs from CARB-3 at 15 amino acid positions
(Table
5) located in the downstream third
of the sequence (Fig.
2). By molecular modeling with the known crystal
coordinates of
the
S. aureus PC-1 enzyme (
12),
these mutations were localized
on the surface of the molecule.
| |
DISCUSSION |
V. cholerae strains belonging to antigenic group O1
were the etiologic agents of the first seven cholera pandemics, and
O139 strains are the etiologic agents of the current pandemic. Some non-O1, non-O139 isolates of V. cholerae are responsible for
sporadic intestinal or extraintestinal noncholera infections.
Generally, wild-type strains of V. cholerae are naturally
susceptible to antibiotics that are active against gram-negative
bacteria, but biotype El Tor is resistant to polymyxins. However,
acquired multiple-drug resistance, presumably plasmid mediated, may
occur (9, 11, 25, 29, 35). Moreover,
blaCARB and blaPSE genes
are known to be plasmid mediated (22) and to be part of
transposons and integrons (4).
The phenotypic expression of plasmid-mediated -lactamase production
by strains is resistance to aminopenicillins and carboxypenicillins, but no interference with cephalosporin activities occurs. -Lactamase inhibitors such as clavulanic acid can restore susceptibility to
inactive penicillins. These characteristics are consistent with the
presence of TEM-1 or SAR-1, as reported previously for resistant
strains. However, a pyogenic strain of V. cholerae non-O1, non-O139 expressed the same resistance phenotype, but its phenotype was
associated with the production of a new -lactamase of pI 5.35. Indeed, the pI was clearly different from that of SAR-1 (pI 4.9) but
was similar to that of TEM-1 (pI 5.4) (29). Conversely, its
kinetic constants identify this enzyme as a carbenicillin-hydrolyzing enzyme similar to SAR-1 but different from TEM-1 (16, 21). Finally, the same substrates are similarly hydrolyzed by CARB-6, CARB-1
to CARB-3, and SAR-1, but the affinity of CARB-6 for most substrates
except for piperacillin (50% inhibition) is slightly lower than those
of the CARB -lactamases. The inhibition profile reveals that
clavulanic acid, an inhibitor of class A -lactamases, inhibits
CARB-6, as well as SAR-1 and TEM-1. If CARB-6 is compared to
-lactamases not yet found in V. cholerae strains, the
enzyme with the nearest pI (pI 5.3), molecular mass (33,000 Da) (Table 2), and substrate profile is CARB-1 (PSE-4) (16, 21).
The resistance gene could not be transferred by conjugation to the
recipient Escherichia coli K-12 strain, despite several attempted mating experiments. This inability to be transferred by
conjugation is a character often reported for the V. cholerae and other CARB or CARB-like genes (35).
Initially, only the TEM-1 enzyme was found in ampicillin-resistant
V. cholerae (9, 10). Later, a novel -lactamase
designated SAR-1 was reported in a strain of V. cholerae
(29). Twenty-nine strains isolated in Africa have been
studied in order to assess the distribution of resistance genes in
V. cholerae (25). None of the
-lactam-resistant isolates studied cross-hybridized with oligonucleotide probes specific for TEM-1 or OXA-1 -lactamases. This
confirms the presence of -lactamases other than TEM-1 in V. cholerae.
Most CARB -lactamases were described as being
Pseudomonas-specific enzymes, hence the designation PSE.
This indicates that they were originally identified in
Pseudomonas aeruginosa strains (28). Indeed,
these enzymes have rarely been reported in members of the family
Enterobacteriaceae (22) and have been reported only once in Achromobacter xylosoxidans (7). No
structural information is available for SAR-1, which is found in
V. cholerae, or for CARB-5, which is found in members of the
Acinetobacter genus (27). The two
carbenicillin-hydrolyzing enzymes described in Japan from P. mirabilis are antigenically and structurally unrelated (Fig. 3):
that from strain N29 is a variant of the CARB group of enzymes
(13), while that from strain GN79 has a distant relationship
with these enzymes but contains the important Arg-234 CARB signature
(31). The AER-1 -lactamase produced by a strain of
Aeromonas hydrophila is also a structurally unique
carbenicillin-hydrolyzing enzyme (32). A feature common to
all bacterial species that produce these -lactamases is their
distribution in an aquatic environment.
Nucleotide sequencing, analysis, and comparison of the amino acid
sequence of CARB-6 with those of other members of the -lactamase family showed the presence of the seven boxes described by Joris et al.
(14) and the active-site tetrad characteristic of class A
-lactamases, according to the classification scheme of Ambler (2). Multiple sequence alignment with class A -lactamase
amino acid sequences confirmed the relatedness of CARB-6 to
carbenicillinases. The amino acid substitutions that characterize the
CARB-6 sequence make this enzyme more similar to P. mirabilis N29, CARB-1, CARB-2, and CARB-3 -lactamases (19, 17, 16, and 15 amino acid differences, respectively) than to the CARB-4
-lactamase (38 amino acid differences) (Fig. 3 and Table 5).
Identification of these mutated positions on the 2-Å crystal structure
of the S. aureus penicillinase showed that all of them are
located on the surface of the protein. These changes would therefore be
expected to modify the function of the enzyme rather than its structure
and are consistent with faster amoxicillin hydrolysis (higher
Vmax) and a lower affinity for all -lactam
substrates (higher Km) (Table 3). Further
analysis may elucidate the mechanisms by which these mutations affect
enzyme function.
In conclusion, CARB-6, described for the first time in V. cholerae, appears to be a new carbenicillin-hydrolyzing enzyme
with a unique combination of enzymatic and physicochemical properties.
| |
ACKNOWLEDGMENTS |
We thank I. Siebert for efficient technical assistance, F. Letourneur (ICGM) for the nucleotide sequencing, and L. Camoin (UPR
415, ICGM) for fruitful help with and discussions about the modeling experiments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Biologie Moléculaire des Cellules Eucaryotes, UFR Cochin
Port-Royal, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France.
Phone: 00 33 1 44 41 23 47. Fax: 00 33 1 44 41 23 42. E-mail:
paul{at}citi2.fr.
| |
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Antimicrobial Agents and Chemotherapy, February 1999, p. 297-301, Vol. 43, No. 2
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
