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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2000, p. 3061-3068, Vol. 44, No. 11
Laboratoire de
Bactériologie,1 and Laboratoire de
Biochimie,3 Faculté de Médecine,
63001 Clermont-Ferrand Cedex, and CNRS-UBO-MNHN, FRE 2125,
29000 Quimper,4 France, and Setor de
Bacteriologia, Laboratório Lâmina LTDA, 71-Botafogo,
Rio de Janeiro, RJ, Brazil 22280-0302
Received 13 April 2000/Returned for modification 19 July
2000/Accepted 24 August 2000
Serratia marcescens Rio-5, one of 18 extended-spectrum
Shortly after the introduction of
extended-spectrum Since the first report of MEN-1 (CTX-M-1) at the beginning of the 1990s
(3), non-TEM, non-SHV, class A ESBLs have been observed
for strains of the family Enterobacteriaceae and in
Pseudomonas aeruginosa. Except for the ESBL SFO-1,
which is closely related to the chromosomal enzyme of Serratia
fonticola (28), these ESBLs, especially the enzyme
GES-1 (39), are distantly related not only to one another
but also to chromosome-borne enzymes. However, two groups can be
considered; on the one hand, the ESBLs of the growing family
CTX-M (7), which cluster with the class A chromosomally
encoded To estimate the diversity of ESBLs in Brazil, clinical strains that
exhibited ESBL phenotypes in different species were collected in
hospitals in Rio de Janeiro in 1996 and 1997. In this report, we
describe a novel type of non-TEM, non-SHV, class A ESBL from a
Serratia marcescens clinical isolate, designated
BES-1 (Brazil extended spectrum).
Strains and plasmids.
Table 1
shows the strains and plasmids used in this study. S. marcescens Rio-5 was isolated from the blood of a newborn
hospitalized in the intensive care unit of a hospital in Rio de
Janeiro, Brazil, in 1996. pRio-5 was the natural BES-1-encoding plasmid
of S. marcescens Rio-5, and pClRio-5 was the
recombinant plasmid, obtained by cloning the gene of BES-1 in phagemid
vector pBK-CMV.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Novel Class A Extended-Spectrum
-Lactamase
(BES-1) in Serratia marcescens Isolated in Brazil
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase (ESBL)-producing strains isolated in several
hospitals in Rio de Janeiro (Brazil) in 1996 and 1997, exhibited a high level of resistance to aztreonam (MIC, 512 µg/ml) and a distinctly higher level of resistance to cefotaxime (MIC, 64 µg/ml) than to
ceftazidime (MIC, 8 µg/ml). The strain produced a plasmid-encoded ESBL with a pI of 7.5 whose bla gene was not related to
those of other plasmid-mediated Ambler class A ESBLs. Cloning and
sequencing revealed a bla gene encoding a novel class
A -lactamase in functional group 2be, designated BES-1 (Brazil
extended-spectrum -lactamase). This enzyme had 51% identity
with chromosomal class A penicillinase of Yersinia
enterocolitica Y56, which was the most closely related enzyme and
47 to 48% identity with CTX-M-type -lactamases, which were the most
closely related ESBLs. In common with CTX-M enzymes, BES-1
exhibited high cefotaxime-hydrolyzing activity
(kcat, 425 s
1). However, BES-1
differed from CTX-M enzymes by its significant ceftazidime-hydrolyzing activity (kcat, 25 s1), high affinity for aztreonam
(Ki, 1 µM), and lower susceptibility to
tazobactam (50% inhibitory concentration [IC50], 0.820 µM) than to clavulanate (IC50, 0.045 µM). Likewise,
certain characteristic structural features of CTX-M enzymes, such as
Phe-160, Ser-237, and Arg-276, were observed for BES-1, which, in
addition, harbored different residues (Ala-104, Ser-171, Arg-220,
Gly-240) and six additional residues at the end of the sequence. BES-1,
therefore, may be an interesting model for further investigations of
the structure-function relationships of class A ESBLs.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactams such as cefotaxime, aztreonam, and
ceftazidime, extended-spectrum -lactamases (ESBLs) were
characterized for members of the family Enterobacteriaceae, firstly in Europe (23, 47)
and then worldwide. These enzymes hydrolyze extended-spectrum
cephalosporins and aztreonam to varying extents but usually neither
cephamycins (cefoxitin and moxalactam) nor carbapenems (imipenem and
meropenem). A common feature of these enzymes is inhibition of their
activity by clavulanic acid. According to the structural classification
of Ambler et al. (1) and the latest functional scheme of
Bush et al. (11), these ESBLs are generally class A
enzymes of the 2be group, which arise as the result of a few amino acid
substitutions from the common plasmid-mediated TEM and SHV-1
-lactamases.
-lactamases of Proteus vulgaris (36), S. fonticola (35), Citrobacter
diversus (37), Klebsiella oxytoca (2), Burkholderia cepacia (52), and
Yersinia enterocolitica (45), and on the other
hand, the ESBLs of the PER type (5, 32), TLA-1
(46), and VEB-1 (40), which cluster with
Bacteroides class A chromosomal -lactamases (42,
48) and the -lactamase CME-1 of Chryseobacterium
(Flavobacterium) meningosepticum (43).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in the study
Plasmid study.
Conjugations of plasmids carrying the
-lactamase gene were performed by mating donor strains with in
vitro-obtained rifampin- or nalidixic acid-resistant mutants of
Escherichia coli HB101 (44) as recipient strains
at 37 or 30°C in solid or liquid Mueller-Hinton medium.
Transconjugants were selected on Mueller-Hinton agar containing rifampin (300 µg/ml) or nalidixic acid (150 µg/ml) and cefotaxime (2 µg/ml).
was
performed according to the manufacturer's instructions (Bio-Rad,
Richmond, Calif.). Transformants were selected on Mueller-Hinton agar
containing cefotaxime (2 µg/ml).
Plasmid DNA was extracted and purified by alkaline lysis according to
the Quiafilter protocol (Qiagen, Hilden, Germany). The plasmid size was
determined after digestion with restriction endonucleases EcoRI and SalI (Boehringer Mannheim, Mannheim,
Germany). Restriction fragments were visualized after electrophoresis
in 0.8% agarose gels with a 1-kb DNA ladder (Eurogentec).
Susceptibilities to -lactams.
MICs were determined by a
dilution method on Mueller-Hinton agar (Sanofi Diagnostics Pasteur,
Marnes la Coquette, France) with an inoculum of 104 CFU per
spot. Antibiotics were provided as powders by SmithKline Beecham
Pharmaceuticals (amoxicillin, ticarcillin, and clavulanate), Lederle
Laboratories (piperacillin and tazobactam), Eli Lilly (Paris, France)
(cephalothin), Roussel-Uclaf (cefotaxime and cefpirome), Glaxo Wellcome
Research and Development (ceftazidime), and Bristol-Myers Squibb (cefepime).
Isoelectric focusing.
Isoelectric focusing was performed
with 6% polyacrylamide gels containing ampholines (Pharmacia Biotech,
Uppsala, Sweden) with a pH range of 3.5 to 10. Proteins were focused at
a constant temperature (6°C) for 3 h at 1 W of constant power
per cm with a Multiphor II flatbed apparatus (Pharmacia Biotech). After
focusing, the -lactamase activity was revealed with iodine agar by
overlaying the polyacrylamide gel with an agar gel containing 0.6%
(wt/vol) penicillin G, 6% (wt/vol) potassium iodide, and 0.6%
(wt/vol) iodine. -Lactamases with known pIs were used as standards:
TEM-1 (pI 5.4), SHV-1 (pI 7.6), and MEN-1 (pI 8.6).
-Lactamase preparation.
The ESBL-producing strain was
grown in 6 liters of brain heart infusion broth containing cefotaxime
at 2 µg/ml for 16 h at 37°C. The bacteria collected by
centrifugation were suspended in morpholineethanesulfonic acid
(MES)-NaOH (20 mM; pH 6.0; 20 ml/5 g of cells) and disrupted by
ultrasonic treatment (four times for 30 s, each time at 20 W).
After centrifugation (48,000 × g for 10 min at 4°C),
the clarified supernatant was loaded onto an SP Sepharose column (10 ml; Amersham Pharmacia Biotech) equilibrated with MES-NaOH (50 mM; pH
6.0). After washing of the column with the same buffer, the bound
proteins were eluted with a linear NaCl gradient (0 to 500 mM). After
ultrafiltration-concentration (Centriprep YM-10; Amicon; Millipore
Corporation, Bedford, Mass.), the -lactamase-containing elution peak
(spot test with nitrocefin as substrate) was loaded onto a Superose 12 (3.2 by 30 cm; Amersham Pharmacia Biotech) column that had been
equilibrated and eluted with the buffer MES-NaOH (20 mM)-NaCl (100 mM;
pH 6.0). The -lactamase-containing elution peak was extensively
dialyzed and concentrated by ultrafiltration against NaCl (100 mM) and
stored at 
20°C until use. The total protein concentration was
estimated by the Bio-Rad protein assay (Bio-Rad) with bovine serum
albumin (Sigma Chemical Co., St. Louis, Mo.) used as a standard. The
purity of BES-1 extract was estimated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was performed
as described by Laemmli (26) with final acrylamide concentrations of 12 and 5% (wt/vol) for the separating and the stacking gels, respectively. Proteins were stained with Coomassie blue
R-250 (Sigma Chemical Co.). After SDS-PAGE, renaturation of
-lactamase was performed as previously described (27),
using the renaturation buffer Tris-HCl (100 mM)-Triton X-100 (2%
[wt/vol]; pH 7.0). -Lactamase activity was detected by overlaying
the polyacrylamide gel with 0.5 mM nitrocefin (Oxoid, Paris, France) in
100 mM phosphate buffer (pH 7.0).
N-terminal protein sequence. The N-terminal sequence of enzyme BES-1 was determined with a gas-phase sequencer as recommended by the manufacturer (Applied Biosystems, Foster City, Calif.) after loading the purified extract of BES-1 onto a polyvinylidene difluoride membrane (Millipore Corp.).
Determination of -lactamase kinetic constants.
The
Km and Kcat constants of
the -lactamase were obtained by a computerized microacidimetric
method (25) with a purified extract. The enzyme BES-1 was
purified from crude lysates of E. coli DH5(pClRio-5). The
concentrations of the inhibitors (clavulanate and tazobactam) required
to inhibit enzyme activity by 50% (IC50s) were determined
as described previously (7). IC50 and
Ki were monitored with penicillin G (225 mM) as
the reporter substrate.
-Lactamase gene cloning.
Recombinant DNA manipulation and
transformations were performed as described by Sambrook et al.
(44). The T4 DNA ligase used was purchased from Boehringer
Mannheim. The BES-1-encoding gene was cloned as follows. Natural
plasmid pRio-5 was partially cleaved by Sau3A, and the
resultant fragments were ligated into the BamHI site of
pBK-CMV phagemid (Stratagene, La Jolla, Calif.). E. coli
DH5 (44) was transformed by electroporation. The
transformant harboring the recombinant BES-1-encoding plasmid pClRio-5
was selected on Mueller-Hinton agar supplemented with 2 µg of
cefotaxime per ml.
DNA sequencing. The insert of recombinant plasmid pClRio-5 was sequenced by the dideoxy chain termination procedure on both complementary strands using an Applied Biosystems sequencer (ABI 1377), as previously described (7).
Computer analysis.
The nucleotide sequence and the deduced
protein sequence were analyzed with the software available on the
Internet at the National Center for Biotechnology Information
site (http://www.ncbi.nlm.nih.gov/, BLAST and ORF finder). A
hydrophobic blot was obtained with the method of Nielsen et al.
(31). The multiple sequence alignment and the phylogenic
analysis, performed by the parsimony method, were carried out as
previously described (7). BES-1 was compared with 23 class A
-lactamases: TEM-3 (49), SHV-2 (15), PSE-4 (6), GES-1 (39), VEB-1 (40), PER-1
(32), CEPA (42), CBLA (48), NMC-A
(30), SME-1 (29), YENT (45), BCEP
(52), PVUL (36), CDIV (37), SFO-1
(28), OXY-1 (2), OXY-2 (41), CTX-M-1
(MEN-1) (3, 4), CTX-M-2 (4), TOHO-1
(19), CTX-M-4 (16, 17), CTX-M-5 (10),
and CTX-M-8 (7).
Nucleotide sequence accession number. The blaBES gene nucleotide sequence data appear in the GenBank nucleotide sequence database under accession no. AF234999.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
S. marcescens Rio-5 (Table
2) exhibited a resistance to
broad-spectrum cephalosporins and a positive double-disk synergy test and produced three -lactamases with pIs of 5.4, 7.5, and >8.6.
PCR and DNA sequencing identified the -lactamase with a pI of
5.4 as TEM-1. The -lactamase with a pI of >8.6 is likely the
chromosomal cephalosporinase of the S. marcescens isolate. No hybridizations were obtained
with the probes of CTX-M and SHV types.
|
Transfer of -lactam resistance.
No transconjugant was
obtained by a mating-out assay in the conditions used. However, a
transformant which exhibited an ESBL phenotype and produced two
-lactamases with pIs of 5.4 and 7.5 was obtained by electroporating
the plasmid DNA content of S. marcescens Rio-5 into
E. coli DH5. The analysis of the plasmid content of this
transformant revealed a 15.5-kb plasmid, pRio-5 (data not shown). No
associated resistance markers were encoded by this plasmid.
Cloning and sequencing of the -lactamase gene.
The ESBL
gene encoded by pRio-5 was cloned in plasmid vector pBK-CMV. Different
transformants which exhibited an ESBL phenotype and produced only
the -lactamases with a pI of 7.5 were obtained. One of these
transformants contained a recombinant plasmid, pClRio-5, which harbored
an insert of about 2 kb.
35
and 10 consensus sequences and a putative ribosome-binding site. A
terminator hairpin loop was detected 12 nucleotides from the stop codon
(Fig. 1).
|
-lactamases: S-X-X-K
at positions 70 to 73, S-D-N at positions 130 to 132, E at position
166, and K-T-G at positions 234 to 236 (Fig. 1).
Homology with other -lactamases.
A phylogenic study was
performed to relate BES-1 to the most closely related enzymes and to
the representatives of major lineages of class A -lactamases (Fig.
2). BES-1 exhibited the highest percentage of identity (51 to 45%) with the following group of gram-negative bacterial -lactamases: the chromosomal class A -lactamases of Y. enterocolitica Y56, B. cepacia 249, K. oxytoca, P. vulgaris R0104,
and C. diversus CUV and the plasmid-borne -lactamases of
the CTX-M type and SFO-1. The phylogenic study located the enzyme BES-1
on a distinct branch between the cluster containing these class A
-lactamases and class A imipenemases NMC-A and SME-1 (Fig. 2).
|
-lactamase BES-1 and the most
closely related enzymes is shown in Fig.
3. BES-1 possessed the highly
conserved amino acid residues of class A -lactamase, i.e., boxes I
to VII (22), which interact with -lactam compounds.
|
loop contained Arg-164 and Glu-179. Arg was observed at
positions 220 and 276. Six more amino acid residues than in CTX-M-type
ESBLs were observed for BES-1 at the end of the sequence.
These residues were encoded by duplicated nucleotides (nucleotides 1052 to 1071) of the terminal hairpin loop (nucleotides 1080 to 1099) (Fig.
1).
-Lactam susceptibility.
MICs of -lactams for S. marcescens Rio-5 and for E. coli
DH5(pClRio-5) are listed in Table 2 and compared with those for CTX-M-8-producing E. coli DH5(pClRio-2) and E. coli DH5. The three -lactamase-producing strains exhibited a
high level of resistance to amoxicillin (MICs, >2,048 µg/ml),
ticarcillin (MICs, >2,048 µg/ml), and cephalothin (MICs, 
1,024
µg/ml).
-lactams (MICs, 4 to 64 µg/ml). For BES-1- and CTX-M-8-producing strains, MICs of cefotaxime
(MICs, 32 to 64 µg/ml) were 2- to 16-fold higher than those of
ceftazidime (MICs, 2 to 16 µg/ml), cefpirome (MICs, 4 to 16 µg/ml), and cefepime (MICs, 4 to 8 µg/ml).
Clavulanate partially or totally restored the activity of the
-lactams against BES-1- and CTX-M-8-producing strains (Table 2). In
contrast to clavulanate, tazobactam was much less effective in
decreasing the MICs of extended-spectrum -lactams for
BES-1-producing strains: the MICs of cefotaxime, ceftazidime, and
aztreonam associated with tazobactam were 16, 2 to 8, and 128 µg/ml, respectively.
Biochemical properties of -lactamase BES-1.
The purified
proteins appeared on SDS-polyacrylamide gels as a band of
28.5 kDa for BES-1 (28.2 kDa for CTX-M-8), which exhibited -lactamase activity (Fig. 4). The
overall recovery of enzyme from clarified extract was around 65%. The
specific activity of purified -lactamase (97% pure) BES-1
was 85 µmol · min1 · mg of
protein1, determined with 225 µM benzylpenicillin as
the substrate.
|
1) and cephalothin
(kcat, 173 s1) than did CTX-M-8
(kcat, 17 to 150 s1 and 1,600 s1, respectively) but had slightly better affinity
(Km, 3 to 18 µM) for these substrates than did
CTX-M-8 (Km, 11 to 87 µM).
|
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-lactams (ceftazidime
and aztreonam) and hence are cefotaximase-type ESBLs.
For BES-1, ceftazidime was a better substrate
(kcat, 25 s1) than was aztreonam
(kcat, 1 s1). Surprisingly,
however, BES-1 had high affinity to this monobactam (aztreonam
Ki, 1 µM) and thus higher catalytic
efficiency against aztreonam
(kcat/Km, 1 µM1 s1) than against ceftazidime
(kcat/Km, 0.025 µM1 s1). BES-1 exhibited at least
10-fold-higher catalytic activity than did CTX-M-8 against ceftazidime.
BES-1 and CTX-M-8, like TEM-1, were susceptible to the -lactam
inhibitors clavulanate and sulbactam (Table 4). Sulbactam was
approximately 2 orders of magnitude less effective than was clavulanate. However, tazobactam, which is as efficient as clavulanate against TEM-1 (IC50, 0.090 versus 0.085 µM) and slightly
better than clavulanate against CTX-M-8 (IC50, 0.010 versus
0.036 µM), was 20-fold less efficient than clavulanate against BES-1
(IC50, 0.820 versus 0.045 µM).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The starting point of this work was the observation of the
clinical strain S. marcescens Rio-5, which exhibited
resistance to aztreonam and cefotaxime and, to a lesser extent, to
ceftazidime and had a positive double-disk synergy test. In the course
of cloning, a novel bla gene was characterized, which
induced this ESBL phenotype. The deduced protein was a novel class
A -lactamase, designated BES-1, which had only 51% identity with
the most closely related -lactamase (chromosomal penicillinase of
Y. enterocolitica). BES-1 is distantly related to either the
CTX-M or GES-1 enzyme that has been isolated from South American
isolates (7, 39). The origin of BES-1 thus remains unknown.
The SHV- and TEM-type ESBLs, first observed in 1985 (23, 47) shortly after the introduction of broad-spectrum cephalosporins such as cefotaxime, aztreonam, and ceftazidime, resulted from point mutations of widespread class A penicillinases. Non-SHV- and non-TEM-type ESBLs, MEN-1, PER-1, VEB-1, TLA-1, GES-1, and CTX-M enzymes, have been characterized since 1992 (3, 4, 5, 7, 10, 16, 32, 39, 40, 46). Some of these ESBL-encoding genes were mobile, since blaVEB-1 and blaGES-1 are located in mobile cassettes of class 1 integrons (39, 40), blaPER genes are chromosomal and plasmidic (5, 13, 32), and blaCTX-M-3 and blaCTX-M-8 genes are observed in different plasmids (7, 33). The type 1 integrase gene and associated resistance markers were not detected in the natural BES-1-encoding plasmid. In the same way, amplification performed with primers specific for 5' and 3' conserved regions of type 1 integrons did not reveal their presence (data not shown).
In view of its susceptibility to clavulanate and its enzymatic properties against extended-spectrum cephalosporins, BES-1 could be classified in the 2be group of the functional classification (11). Like CTX-M enzymes, BES-1 is an ESBL with a strong cefotaxime-hydrolyzing activity inducing a distinctly higher level of resistance to cefotaxime than to ceftazidime. However, BES-1 exhibited a catalytic activity against ceftazidime and an affinity for aztreonam 10- and 1,000-fold higher, respectively, than those of CTX-M enzymes (3, 10). The activity of BES-1 against ceftazidime and aztreonam has been observed to a lesser extent with SHV-4 (34).
The residue Thr-237, observed for some TEM-type -lactamases and
implicated in cefotaxime-hydrolyzing activities (24), is observed for BES-1. This residue, Thr-237, which has the same hydrogen-bonding capacity as Ser, could be involved, in association with Arg-276, in the cefotaximase activity of BES-1, as previously suggested for residues Ser-237 and Arg-276 of CTX-M enzymes (3, 16, 17, 18, 19). Four glycine residues, including Gly-232, which
is frequently observed for ESBL enzymes (18), were
present in BES-1, as they were in CTX-M enzymes. They could increase
the flexibility of strand 3, one wall of the active-site
cavity. BES-1, like CTX-M enzymes, contains residue Phe-160. This
residue, conserved in non-ESBL -lactamases, is
responsible for the lack of a hydrogen bond between the N and C termini
of the loop (18). In addition, Ser-171 was observed for
BES-1, as in PER-1 and in the Streptomyces albus G
-lactamase (9, 14). It eliminates the hydrogen bond
between residues 171 and 164 (14). Thus, Ser-171 and Phe-160
of BES-1 could increase movement of the loop (positions 162 to
179), thereby facilitating the hydrolysis of bulky -lactams. The glycine residues of strand 3 and Phe-160 and Ser-171 of BES-1 could facilitate binding to and/or hydrolysis of
expanded-spectrum cephems.
BES-1, which exhibited ceftazidime-hydrolyzing activity,
harbored neither the residues in positions 164 and 179 of the loop (positions 162 to 179) nor residues Lys-104 and Lys-240 of SHV- and
TEM-type ESBLs (24). Residue 104 could contribute to the precise positioning of the SDN loop (residues 130 to 132), which is involved in substrate binding and catalysis (38). In
addition, Lys-104, frequently observed for TEM-type ESBLs, is
thought to interact with the carboxylic acid group in the alkyloximino
substituent of ceftazidime and aztreonam (24). The polar
residue Asn-104 of CTX-M-type ESBLs is replaced in BES-1 by
the small nonpolar residue Ala-104, which may contribute to the
particular properties of BES-1.
The mutation Glu-240
Lys plays a major role in the
extended-spectrum activity of TEM- and SHV-type ESBLs
against ceftazidime and aztreonam. CTX-M enzymes contain the acid
residue Asp in position 240, which could repel the propyloximino
carboxylic acid group of ceftazidime and aztreonam and impair their
binding. Residue Gly-240, observed for both BES-1 and PER-1
(8), may enhance the binding of aztreonam but not correct
positioning. This could explain the poor catalytic activity and the
high affinity of BES-1 against this substrate.
Arg-220 in the S. albus G -lactamase, Arg-244 in some
class A enzymes, and Arg-276 in CTX-M enzymes play an important role in
enzyme catalysis (18-20). BES-1 is the first enzyme to
harbor two of these residues: residue Arg-220, located at the end of helix H10, and residue Arg-276, located at the beginning of helix H11.
Given the structure of Toho-1 (18), it is likely that the side chain of Arg-220 plays a role in the catalytic process, whereas Arg-276 is more probably involved in the extension of substrate specificity. The association of the two residues Arg-220 and Arg-276, which extend their side chains close to one another, and the six additional amino acids observed in helix H11 of BES-1 could lead to
changes in positioning of Arg-276 and could be responsible for the
enzymatic properties of BES-1.
In addition, BES-1 exhibited resistance to the inhibitor
tazobactam, whereas CTX-M enzymes are susceptible to all
-lactam inhibitors. Surprisingly, the respective IC50s
of sulbactam and clavulanate for BES-1 are close to those observed for
CTX-M-8 and TEM-1. Thus, this is the first report of a -lactamase
that exhibits selective resistance to tazobactam. The residues in
positions 69, 244, 275, and 276 responsible for the resistance of
TEM-type enzymes to the -lactam inhibitors were not observed for
BES-1. For TEM-1 -lactamase, molecular modeling suggests the
existence of one hydrogen bond between the triazole ring of tazobactam
and the side chain of Arg-244 via a water molecule (12). In
BES-1, the absence of Arg-244 associated with the environment created by Arg-220 and Arg-276 could suppress interaction or produce
unfavorable interactions between the triazole ring of tazobactam and
BES-1.
In conclusion, the functional activity of BES-1 against cefotaxime
resembles that of CTX-M-type ESBLs. However, BES-1 exhibits particular enzymatic properties against alkyloximinocephems,
monobactams, and -lactam inhibitors. Thus, BES-1 could be an
interesting model for further investigations of the structure-function
relationships of class A ESBLs. S. marcescens
Rio-5 was the sole BES-1-producing strain from among 18 ESBL-producing Enterobacteriaceae strains, which
produced mainly SHV-type ESBLs (10 strains out of 18) and CTX-M-type enzymes (5 strains out of 18). The recent emergence of
non-SHV- and non-TEM-type ESBLs could be the result of mobilization of genes from environmental strains to clinical strains as observed with the blaSFO-1 gene (28). The G+C
content of blaBES-1 (60%), not typical of
Enterobacteriaceae genes (around 50%), is compatible with
this hypothesis. It will be interesting to investigate the spread of
BES-1 or to detect BES-1-like enzymes.
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ACKNOWLEDGMENTS |
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We thank Angeline Serre, Rolande Perroux, Marlène Jan, and Dominique Rubio for technical assistance. We are also grateful to Ekkehard Collatz, Laboratoire de Recherche Moleculaire sur les Antibiotiques (LRMA), INSERM E0004, Universite Paris VI, for his help in N-terminal protein sequencing.
This work was supported in part by a grant from M.E.N.R.T. (Ministère de l'Education Nationale, de la Recherche et de la Technologie).
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FOOTNOTES |
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* Corresponding author. Mailing address: Faculté de Médecine, Service de Bactériologie-Virologie, 28, Place Henri Dunant, 63001 Clermont-Ferrand Cedex, France. Phone: 33 (0)4 73 60 80 18. Fax: 33 (0)4 73 27 74 94. E-mail: Richard.Bonnet{at}u-clermont1.fr.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. |
Ambler, R. P.,
A. F. W. Coulson,
J.-M. Frère,
J. M. Ghuysen,
B. Joris,
M. Forsman,
R. C. Levesque,
G. Tiraby, and S. G. Waley.
1991.
A standard numbering scheme for the class A -lactamases.
Biochem. J.
276:269-272.
|
| 2. |
Arakawa, Y.,
M. Ohta,
N. Kido,
M. Mori,
H. Ito,
T. Komatsu,
Y. Fujii, and N. Kato.
1989.
Chromosomal -lactamase of Klebsiella oxytoca, a new class A enzyme that hydrolyzes broad-spectrum -lactam antibiotics.
Antimicrob. Agents Chemother.
33:63-70 |
| 3. |
Barthélémy, M.,
J. Péduzzi,
H. Bernard,
C. Tancrède, and R. Labia.
1992.
Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum -lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca.
Biochim. Biophys. Acta
1122:15-22[CrossRef][Medline].
|
| 4. |
Bauernfeind, A.,
I. Stemplinger,
R. Jungwirth,
S. Ernst, and J. M. Casellas.
1996.
Sequences of -lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other -lactamases.
Antimicrob. Agents Chemother.
40:509-513[Abstract].
|
| 5. |
Bauernfeind, A.,
I. Stemplinger,
R. Jungwirth,
P. Mangold,
S. Amann,
E. Akalin,
O. Ang,
C. Bal, and J. M. Casellas.
1996.
Characterization of -lactamase gene blaPER-2, which encodes an extended-spectrum class A -lactamase.
Antimicrob. Agents Chemother.
40:616-620[Abstract].
|
| 6. |
Boissinot, M., and R. C. Levesque.
1990.
Nucleotide sequence of the PSE-4 carbenicillinase gene and correlations with the Staphylococcus aureus PC1 beta-lactamase crystal structure.
J. Biol. Chem.
265:1225-1230 |
| 7. |
Bonnet, R.,
J. L. M. Sampaio,
R. Labia,
C. de Champs,
D. Sirot,
C. Chanal, and J. Sirot.
2000.
A novel CTX-M -lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae isolated in Brazil.
Antimicrob. Agents Chemother.
44:1936-1942 |
| 8. | Bouthors, A. T., N. Dagoneau-Blanchard, T. Naas, P. Nordmann, V. Jarlier, and W. Sougakoff. 1998. Role of residues 104, 164, 166, 238 and 240 in the substrate profile of PER-1 beta-lactamase hydrolysing third-generation cephalosporins. Biochem. J. 15:1443-1449. |
| 9. |
Bouthors, A. T.,
J. Delettré,
P. Mugnier,
V. Jarlier, and W. Sougakoff.
1999.
Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 beta-lactamase hydrolysing expanded-spectrum cephalosporins.
Protein Eng.
12:313-318 |
| 10. |
Bradford, P. A.,
Y. Yang,
D. Sahm,
I. Grope,
D. Gardovska, and G. Storch.
1998.
CTX-M-5, a novel cefotaxime-hydrolyzing -lactamase from an outbreak of Salmonella typhimurium in Latvia.
Antimicrob. Agents Chemother.
42:1980-1984 |
| 11. |
Bush, K.,
G. A. Jacoby, and A. A. Medeiros.
1995.
A functional classification scheme for -lactamases and its correlation with molecular structure.
Antimicrob. Agents Chemother.
39:1211-1213[Medline].
|
| 12. | Chaibi, E. B., J. Péduzzi, S. Farzaneh, M. Barthélémy, D. Sirot, and R. Labia. 1998. Clinical inhibitor-resistant mutants of the beta-lactamase TEM-1 at amino-acid position 69: kinetic analysis and molecular modeling. Biochim. Biophys. Acta 1382:38-46[CrossRef][Medline]. |
| 13. |
Danel, F.,
L. M. Hall,
D. Gur,
H. E. Akalin, and D. M. Livermore.
1995.
Transferable production of PER-1 -lactamase in Pseudomonas aeruginosa.
J. Antimicrob. Chemother.
35:281-294 |
| 14. | Dideberg, O., P. Charlier, J.-P. Wery, P. Dehottay, J. Dusart, T. Erpicum, J.-M. Frère, and J.-M. Ghuysen. 1987. The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution. Biochem. J. 245:911-913[Medline]. |
| 15. |
Garbarg-Chenon, A.,
V. Godard,
R. Labia, and J. C. Nicolas.
1990.
Nucleotide sequence of SHV-2 beta-lactamase gene.
Antimicrob. Agents Chemother.
34:1444-1446 |
| 16. |
Gazouli, M.,
N. J. Legakis, and L. S. Tzouvelekis.
1998.
Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A -lactamase CTX-M-4.
FEMS Microbiol. Lett.
169:289-293[Medline].
|
| 17. |
Gazouli, M.,
E. Tzelepi,
S. V. Sidorenko, and L. S. Tzouvelekis.
1998.
Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A -lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis.
Antimicrob. Agents Chemother.
42:1259-1262 |
| 18. |
Ibuka, A.,
A. Taguchi,
M. Ishiguro,
S. Fushinobu,
Y. Ishii,
S. Kamitori,
K. Okuyama,
K. Yamaguchi,
M. Konno, and H. Matsuzawa.
1999.
Crystal structure of the E166A mutant of extended-spectrum -lactamase Toho-1 at 1.8 Å resolution.
J. Mol. Biol.
285:2079-2087[CrossRef][Medline].
|
| 19. |
Ishii, Y.,
A. Ohno,
H. Taguchi,
S. Imajo,
M. Ishiguro, and H. Matsuzawa.
1995.
Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A -lactamase isolated from Escherichia coli.
Antimicrob. Agents Chemother.
39:2269-2275[Abstract].
|
| 20. |
Jacob-Dubuisson, F.,
J. Lamotte-Brasseur,
O. Dideberg,
B. Joris, and J.-M. Frère.
1991.
Arginine 220 is a critical residue for the catalytic mechanism of the Streptomyces albus G beta-lactamase.
Protein Eng.
4:811-819 |
| 21. |
Jarlier, V.,
M. H. Nicolas,
G. Fournier, and A. Philippon.
1988.
Extended broad-spectrum -lactamases conferring transferable resistance to newer -lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns.
Rev. Infect. Dis.
10:867-878[Medline].
|
| 22. |
Joris, B.,
P. Ledent,
O. Dideberg,
E. Fonze,
J. Lamotte-Brasseur,
J. A. Kelly,
J. M. Ghuysen, and J. M. Frère.
1991.
Comparison of the sequences of class A -lactamases and of the secondary structure elements of penicillin-recognizing proteins.
Antimicrob. Agents Chemother.
35:2294-2301 |
| 23. |
Kliebe, C.,
B. A. Nies,
S. F. Meyer,
R. M. Tolxdorff-Neutzling, and B. Wiedeman.
1985.
Evolution of plasmid-coded resistance to broad-spectrum cephalosporins.
Antimicrob. Agents Chemother.
28:302-307 |
| 24. |
Knox, J. R.
1995.
Extended-spectrum and inhibitor-resistant TEM-type -lactamases: mutations, specificity, and three-dimensional structure.
Antimicrob. Agents Chemother.
39:2593-2601[Medline].
|
| 25. |
Labia, R.,
J. Andrillon, and F. Le Goffic.
1973.
Computerized microacidimetric determination of -lactamase Michaelis-Menten constants.
FEBS Lett.
33:42-44[CrossRef][Medline].
|
| 26. | Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[CrossRef][Medline]. |
| 27. |
Massidda, O.,
G. M. Rossolini, and G. Satta.
1991.
The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo--lactamases.
J. Bacteriol.
173:4611-4617 |
| 28. |
Matsumoto, Y., and M. Inoue.
1999.
Characterization of SFO-1, a plasmid-mediated inducible class A beta-lactamase from Enterobacter cloacae.
Antimicrob. Agents Chemother.
43:307-313 |
| 29. |
Naas, T.,
D. M. Livermore, and P. Nordmann.
1995.
Characterization of an LysR family protein, SmeR from Serratia marcescens S6, its effect on expression of the carbapenem-hydrolyzing -lactamase Sme-1, and comparison of this regulator with other -lactamase regulators.
Antimicrob. Agents Chemother.
39:629-637[Abstract].
|
| 30. |
Naas, T., and P. Nordmann.
1994.
Analysis of a carbapenem-hydrolyzing class A -lactamase from Enterobacter cloacae and of its LysR-type regulatory protein.
Proc. Natl. Acad. Sci. USA
91:7693-7697 |
| 31. |
Nielsen, H.,
J. Engelbrecht,
S. Brunak, and G. von Heijne.
1997.
Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.
Protein Eng.
10:1-6 |
| 32. |
Nordmann, P., and T. Naas.
1994.
Sequence analysis of PER-1 extended-spectrum -lactamase from Pseudomonas aeruginosa and comparison with class A -lactamases.
Antimicrob. Agents Chemother.
38:104-114 |
| 33. |
Palucha, A.,
B. Mikiewicz,
W. Hryniewicz, and M. Gniadkowski.
1999.
Concurrent outbreaks of extended-spectrum -lactamase-producing organisms of the family Enterobacteriaceae in a Warsaw hospital.
J. Antimicrob. Chemother.
44:489-499 |
| 34. |
Péduzzi, J.,
M. Barthélémy,
K. Tiwari,
D. Mattioni, and R. Labia.
1989.
Structural features related to hydrolytic activity against ceftazidime of plasmid-mediated SHV-type CAZ-5 -lactamase.
Antimicrob. Agents Chemother.
33:2160-2163 |
| 35. | Péduzzi, J., S. Farzaneh, A. Reynaud, M. Barthélémy, and R. Labia. 1997. Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolysing class A beta-lactamase from Serratia fonticola CUV. Biochim. Biophys. Acta 1341:58-70[CrossRef][Medline]. |
| 36. |
Péduzzi, J.,
A. Reynaud,
P. Baron,
M. Barthélémy, and R. Labia.
1994.
Chromosomally encoded cephalosporin-hydrolysing -lactamase of Proteus vulgaris R0104 belongs to Ambler's class A.
Biochim. Biophys. Acta
1207:31-39[CrossRef][Medline].
|
| 37. | Perilli, M. G., N. Franceschini, B. Segatore, G. Amicosante, A. Oratore, C. Duez, B. Joris, and J. M. Frère. 1991. Cloning and nucleotide sequencing of the gene encoding the beta-lactamase from Citrobacter diversus. FEMS Microbiol. Lett. 67:79-84[CrossRef][Medline]. |
| 38. |
Petit, A.,
L. Maveyraud,
F. Lenfant,
J. P. Samama,
R. Labia, and J. M. Masson.
1995.
Multiple substitutions at position 104 of -lactamase TEM-1: assessing the role of this residue in substrate specificity.
Biochem. J.
305:33-40.
|
| 39. |
Poirel, L.,
I. Le Thomas,
T. Naas,
A. Karim, and P. Nordmann.
2000.
Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae.
Antimicrob. Agents Chemother.
44:622-632 |
| 40. |
Poirel, L.,
T. Naas,
M. Guibert,
E. B. Chaibi,
R. Labia, and P. Nordmann.
1999.
Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum -lactamase encoded by an Escherichia coli integron gene.
Antimicrob. Agents Chemother.
43:573-581 |
| 41. |
Reynaud, A.,
J. Péduzzi,
M. Barthélémy, and R. Labia.
1991.
Cefotaxime-hydrolysing activity of the -lactamase of Klebsiella oxytoca D488 could be related to a threonine residue at position 140.
FEMS Microbiol. Lett.
81:185-192[CrossRef].
|
| 42. |
Rogers, M. B.,
A. C. Parker, and C. J. Smith.
1993.
Cloning and characterization of the endogenous cephalosporinase gene, cepA, from Bacteroides fragilis reveals a new subgroup of Ambler class A -lactamases.
Antimicrob. Agents Chemother.
37:2391-2400 |
| 43. |
Rossolini, G. M.,
N. Franceschini,
L. Lauretti,
B. Caravelli,
M. L. Riccio,
M. Galleni,
J.-M. Frère, and G. Amicosante.
1999.
Cloning of a Chryseobacterium (Flavobacterium) meningosepticum chromosomal gene (blaCME) encoding an extended-spectrum class A -lactamase related to the Bacteroides cephalosporinases and the VEB-1 and PER -lactamases.
Antimicrob. Agents Chemother.
43:2193-2199 |
| 44. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed., vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 45. |
Seoane, A.,
M. V. Francia, and J.-M. Garcia Lobo.
1992.
Nucleotide sequence of the ampC-ampR region from the chromosome of Yersinia enterocolitica.
Antimicrob. Agents Chemother.
36:1049-1052 |
| 46. |
Silva, J.,
C. Aguilard,
G. Ayala,
M. A. Estrada,
U. Garza-Ramos,
R. Lara-Lemus, and L. Ledezma.
2000.
TLA-1: a new plasmid-mediated extended-spectrum -lactamase from Escherichia coli.
Antimicrob. Agents Chemother.
44:997-1003 |
| 47. |
Sirot, D.,
J. Sirot,
R. Labia,
A. Morand,
P. Courvalin,
A. Darfeuille-Michaud,
R. Perroux, and R. Cluzel.
1987.
Transferable resistance to third-generation cephalosporins in clinical isolates of Klebsiella pneumoniae: identification of CTX-1, a novel -lactamase.
J. Antimicrob. Chemother.
20:323-334 |
| 48. |
Smith, C. J.,
T. K. Bennett, and A. C. Parker.
1994.
Molecular and genetic analysis of the Bacteroides uniformis cephalosporinase gene, cblA, encoding the species-specific -lactamase.
Antimicrob. Agents Chemother.
38:1711-1715 |
| 49. |
Sougakoff, W.,
S. Goussard, and P. Courvalin.
1988.
The TEM-3 -lactamase, which hydrolyzes broad-spectrum cephalosporins, is derived from the TEM-2 penicillinase by two amino acid substitutions.
FEMS Microbiol. Lett.
56:343-348[CrossRef].
|
| 50. |
Swaren, P.,
L. Maveyraud,
X. Raquet,
S. Cabantous,
C. Duez,
J. D. Pedelacq,
S. Mariotte-Boyer,
L. Mourey,
R. Labia,
M. H. Nicolas-Chanoine,
P. Nordmann,
J.-M. Frère, and J. P. Samama.
1998.
X-ray analysis of the NMC-A -lactamase at 1.64-Å resolution, a class A carbapenemase with broad substrate specificity.
J. Biol. Chem.
273:26714-26721 |
| 51. |
Tassios, P. T.,
M. Gazouli,
E. Tzelepi,
H. Milch,
N. Kozlova,
S. Sidorenko,
N. J. Legakis, and L. S. Tzouvelekis.
1999.
Spread of a Salmonella typhimurium clone resistant to expanded-spectrum cephalosporins in three European countries.
J. Clin. Microbiol.
37:3774-3777 |
| 52. | Trepanier, S., A. Prince, and A. Huletsky. 1997. Characterization of the penA and penR genes of Burkholderia cepacia 249 which encode the chromosomal class A penicillinase and its LysR-type transcriptional regulator. Antimicrob. Agents Chemother. 41:2399-2405[Abstract]. |
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