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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, May 1998, p. 1181-1186, Vol. 42, No. 5
Department of Microbiology,
Received 9 October 1997/Returned for modification 15 January
1998/Accepted 9 March 1998
Escherichia coli TUM1083, which is resistant to
ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin,
cefuzonam, and aztreonam while being sensitive to cefoxitin,
moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from
the urine of a patient treated with The expanded-spectrum In this report, we discuss a correlation between the mutation and the
substrate specificity of the Bacterial strains and plasmids.
Table
1 shows the bacterial strains and
plasmids used in this study. E. coli TUM1083 was isolated in
March 1995 from the urine of a 69-year-old male who suffered from colon
cancer. Before strain isolation, the patient had received an empiric
antibiotic treatment consisting of a combination of piperacillin,
cefuzonam, cefotiam, imipenem, levofloxacin, and tosulfloxacin. The
strain did not ferment lactose but was identified as E. coli
by tests with API 20E (Asuka, Tokyo, Japan) and the Vitek system
(bioMerieux Vitek, Inc., Hazelwood, Mo.). E. coli ML4909,
used for plasmid conjugation, was provided by Matsuhisa Inoue of
Kitasato University. E. coli AS226 (13) was used
for Conjugation.
Conjugation was performed by the broth method
(7). E. coli TUM1083 and E. coli
ML4909 (recipient) were incubated for 30 min at 35°C before selection
of transconjugant.
Antibacterial agents.
Penicillin G and ampicillin (Meiji
Seika, Ltd., Tokyo, Japan); oxacillin and imipenem (Banyu
Pharmaceutical Co., Ltd., Tokyo, Japan); carbenicillin and ceftizoxime
(Fujisawa Pharmaceutical Co., Ltd., Tokyo, Japan); piperacillin (Toyama
Chemical Co., Ltd., Tokyo, Japan); cefuzonam (Lederle Japan Ltd.,
Tokyo, Japan); cephalothin, cephaloridine, and moxalactam (Shionogi & Co., Ltd., Osaka, Japan); ceftazidime (Nippon Glaxo Ltd., Tokyo,
Japan); cefotaxime (Hoechst Marion Roussel, Ltd., Tokyo, Japan);
aztreonam (Sankyo Co., Ltd., Tokyo, Japan); and tazobactam and YP-14, a
combination of tazobactam and piperacillin at a ratio of 1 to 4, respectively (Taiho Pharmaceutical Co., Ltd., Tokyo, Japan), all with
known potencies, were used.
Drug sensitivity tests.
MICs were determined by the broth
microdilution method with Mueller-Hinton broth (Difco, Detroit, Mich.).
The organisms were inoculated at about 5 × 105
cells/well with MIC2000 (Dynatech, McLean, Va.).
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cloning and Sequencing of the Gene Encoding Toho-2, a Class A
-Lactamase Preferentially Inhibited by Tazobactam
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-lactam antibiotics. The
-lactamase (Toho-2) purified from the bacteria hydrolyzed -lactam
antibiotics such as penicillin G, carbenicillin, cephaloridine,
cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had
increased relative hydrolysis rates for cephalothin, cephaloridine,
cefotaxime, and ceftizoxime. Different from other extended-spectrum
-lactamases, Toho-2 was inhibited 16-fold better by the
-lactamase inhibitor tazobactam than by clavulanic acid. Resistance
to -lactams was transferred by conjugation from E. coli
TUM1083 to E. coli ML4909, and the transferred plasmid was
about 54.4 kbp, belonging to the incompatibility group IncFII. The
cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp
plasmid. The sequence of the gene was determined, and the open reading
frame of the gene was found to consist of 981 bases. The nucleotide
sequence of the gene (DDBJ accession no. D89862) designated as
blatoho was found to have 76.3% identity to
class A -lactamase CTX-M-2 and 76.2% identity to Toho-1. It has
55.9% identity to SHV-1 -lactamase and 47.5% identity to TEM-1
-lactamase. Therefore, the newly isolated -lactamase designated
as Toho-2 produced by E. coli TUM1083 is categorized as an
enzyme similar to Toho-1 group -lactamases rather than to mutants of
TEM or SHV enzymes. According to the amino acid sequence deduced from
the DNA sequence, the precursor consisted of 327 amino acid residues.
Comparison of Toho-2 with other -lactamase (non-Toho-1 group)
suggests that the substitutions of threonine for Arg-244 and arginine
for Asn-276 are important for the extension of the substrate
specificity.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-Lactam antibiotics are widely
used as front line agents in the clinical field. In the early 1980s,
expanded-spectrum -lactams, with stability for -lactamase and
good activity against gram-negative bacteria, were first used in the
clinical setting. Not long after the beginning of wide use of the
expanded-spectrum -lactams, extended-spectrum -lactamases were
isolated in Europe and the United States and now have become a serious
problem in the clinical field (29). In the late 1980s and
early 1990s, those enzymes hydrolyzing the expanded-spectrum
-lactams were generally derived from TEM- or SHV-type -lactamases
through several mutations (6, 24). The mutations of Glu-104,
Arg-164, and Glu-240 have been suggested to be important for the
spectrum expansion (16, 24). In more recent years, non-TEM-
or non-SHV-type -lactamases such as Toho-1 (13), CTX-M-2
(5), and MEN-1 (3) have been identified. Those
-lactamases have high homology to the chromosomally encoded -lactamase of Proteus vulgaris or Klebsiella
oxytoca (2, 9, 23). In most cases, the
-lactamase-producing organisms show resistance to expanded-spectrum
-lactams such as cefotaxime and ceftazidime (6, 24). On
the other hand, they are susceptible to carbapenems such as
imipenem (6, 24). The main characteristic of those class
A -lactamases, except TEM-30 to TEM-40, is that they are sensitive
to -lactamase inhibitors such as clavulanic acid, sulbactam, and
tazobactam (6). The reaction mechanism and the amino acid
residues associated with the spectrum expansion of -lactamases are
still under investigation. Ishii et al. (13) proposed that
mutations at positions 244 and 276 are important for the substrate
extension after performing sequence alignment of Toho-1 and other
-lactamases.
-lactam-resistant strains isolated from
several hospitals were surveyed and collected. We investigated those
strains by enzymological and molecular biological methods and focused
upon Escherichia coli TUM1083, a cefotaxime-resistant clinical isolate.
-lactamase from E. coli TUM1083 based on the sequence alignment and a three-dimensional structure of a related -lactamase of Bacillus
licheniformis (17).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-lactamase purification, and E. coli MV1184
(31) was used for transformation.
TABLE 1.
Bacterial strains and plasmids used
Incompatibility tests and -lactamase assay.
Incompatibility tests were carried out according to the method
described in a previous report (7). The plasmids and strains used in this study were described previously (13).
Purification of -lactamase.
The -lactamase was
purified from E. coli TUM1103, in which pMTY036 was
transformed. The organisms were incubated for 5 h in 2 liters of
Luria-Bertani broth and centrifuged at 7,000 × g for
10 min at 4°C. The supernatant was discarded, and MES
(morpholineethanesulfonic acid)-NaOH buffer (20 mM, pH 6.5) was added
to the tube to suspend the sediments. The suspended solution was
disrupted by sonication (100 W, 30 min) in a volume of 15 ml of
MES-NaOH buffer (20 mM, pH 6.5). The lysates were centrifuged again at
45,000 × g for 30 min. The supernatant was dialyzed
for 24 h against 10 mM MES-NaOH buffer (pH 6.5), was then applied
to carboxymethyl-Bio-Gel A (column size, 2.5 by 10 cm; Bio-Rad,
Richmond, Calif.), and washed overnight with 10 mM MES-NaOH buffer (pH
6.5). Elution was performed with 10 mM MES-NaOH buffer (pH 6.5)
containing 0.05 M NaCl. The activities of the eluted fractions were
checked with nitrocefin (Oxoid, Basingstoke, England), and the active
fractions were pooled and concentrated with a Centriprep-10
concentrator (Amicon, Beverly, Mass.) and purified again by fast
protein liquid chromatography (column; Mono S 5/5 [Pharmacia Biotech,
Uppsala, Sweden]; binding buffer, 10 mM MES-NaOH [pH 6.5]; elution
buffer, 10 mM MES-NaOH [pH 6.5]-25 mM NaCl; flow rate, 0.5 ml/min;
detector; UV, 280 nm). The purified -lactamase was concentrated with
a Centriprep-10 concentrator (Amicon). Isoelectric focusing was carried
out with a Multiphor II electrophoresis system (Pharmacia Biotech) and
a gel plate containing 2% Ampholine (pH 6.0 to 8.0). The enzyme
protein on the gel plate was detected by staining with Coomassie
brilliant blue R-250. The molecular weight was determined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (18).
Assay of -lactamase activity.
The activity of the highly
purified -lactamase was measured by spectrophotometric assay
(32) with a spectrophotometer (DU640; Beckman, Fullerton,
Calif.) with a thermoregulator. The peak wavelength for each antibiotic
used for the measurement was set according to that described in
previous reports (13). Km and
kcat values were derived by the linear
regression analysis of Lineweaver-Burk plots (19) of initial
velocity data that were obtained at different substrate concentrations
ranging from 10 to 100 µM. The apparent Ki
value was determined with nitrocefin as substrate at concentrations from 10 to 100 µM, after preincubation for 1 min with -lactamase inhibitor concentrations from 1 to 25 µM, and data were analyzed by a
Dixon plot (8).
Cloning and analysis of recombinant plasmids.
Plasmid DNA
was purified by extracting plasmid DNA by the large-scale alkaline
method and the ethidium bromide-CsCl linear gradient method
(26) at 80,000 rpm for 16 h with a Beckman TLA centrifuge (Beckman). Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo Co., Ltd. (Shiga, Japan). The plasmid size
was calculated from the size of the fragments obtained by cleaving the
plasmid with restriction enzymes with cleaving 
phage DNA cleaved
with StyI as a molecular marker. The cefotaxime resistance
gene was cloned as follows. After plasmid DNA was cleaved partially by
Sau3AI, the resultant fragments were ligated into the
BamHI site of pHSG397 (30). E. coli
MV1184 (31) was transformed with the ligated DNA, and
cefotaxime-resistant colonies were selected on an L agar plate
(26) supplemented with 10 µg of cefotaxime per ml.
DNA sequencing analysis. After pMTY036 was double digested with KpnI and XhoI, SphI and SalI deletion mutants were prepared by using a Takara deletion kit (Takara Shuzo). From these deletion mutants, five subclones were sequenced with the universal primer M13 pUC sequencing primer (Takara Shuzo), the Takara Bca BEST Dideoxy sequencing kit (Takara Shuzo), and a DSQ-1000 DNA sequencer (Shimadzu, Tokyo, Japan). Then, a 17-mer oligonucleotide reversed primer was prepared on the basis of the results obtained with the universal primer. The sequence was determined according to the scheme shown in Fig. 1.
Computer analysis. The DNA sequence data were analyzed primarily by using a UNIX computer and software from the DNA Data Bank of Japan (National Institute of Genetics, Mishima, Japan). The alignments of the DNA and peptide sequences were examined by using the Fasta mail server (22), and the multiple sequence alignment was examined by using the ODEN and Karashi programs (12). The sequences extracted from the database and used for examination of the multiple alignment were Toho-1 from E. coli TUH12191 (13), MEN-1 from E. coli MEN-1 (3), KLEOX from K. oxytoca E23004 (2), CITDI from Citrobacter diversus ULA 27 (25), STRAL from Streptomyces albus G (20), TEM-1 from E. coli TEM-1 (14), SHV-1 from E. coli SHV-1 (4), PC-1 from Staphylococcus aureus PC-1 (11), PROVU from Proteus vulgaris RO104 (23), and YEREN from Yersinia enterocolitica (28).
Nucleotide sequence accession number. The nucleotide sequence data for the Toho-2 gene appear in the EMBL-GenBank-DDBJ data libraries under accession no. D89862.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
One hundred fifty strains of E. coli or
Klebsiella pneumoniae which show resistance to
expanded-spectrum -lactams were examined by PCR with the specific
primer sets of the TEM (14), SHV (4), KOXY
(2), Sme-1 (21), and Toho-1 (13) types
of -lactamases. No positive strain was detected by PCR with all the
primer sets. The cefotaxime-resistant E. coli TUM1083
strain, which produces -lactamase Toho-2, was identified from those
strains. E. coli TUM1083 was isolated in March 1995 from the
urine of a 69-year-old male patient. The patient suffered from colon
cancer and was administered piperacillin, cefuzonam, cefotiam,
imipenem, levofloxacin, and tosulfloxacin. The strain produced a new
-lactamase with an isoelectric point of 7.7 named Toho-2.
Plasmid profile.
Transconjugants which acquired cefotaxime
resistance by conjugation appeared at a frequency of 10
4
to 106. A plasmid profile of 20 transconjugants revealed
the presence in each of a single 54.4-kbp plasmid. The plasmid, which
is called pMTY002 (pMTY; registered with the Plasmid Reference Center), was cleaved into seven segments by EcoRI or
HindIII. From the size of the fragments obtained, the
size of pMTY002 was estimated to be about 54.4 kbp. The incompatibility
of pMTY002 was examined by conjugation with E. coli C600
strains containing various different Inc plasmids listed in Table 1. In
this way, pMTY002 was shown to be within incompatibility group FII.
Cloning of the -lactamase gene.
The fragments of pMTY002
generated by partial digestion with Sau3AI were inserted
into pHSG397 and were transformed to E. coli MV1184. One
plasmid, pMTY036, of about 4.3 kbp was isolated from a transformant
selected on an L agar plate containing 10 µg of cefotaxime per ml. A
restriction map and the position of the -lactamase structural gene
blatoho are shown in Fig.
1.
|
Susceptibility to antibiotics.
Table
2 shows MICs of -lactam antibiotics
against E. coli TUM1083 and E. coli ML4909 with
and without pMTY002. Susceptibility tests were conducted with 10 clones
of transconjugants. MICs of all penicillins, cephalothin, cefotaxime,
and cefuzonam against E. coli TUM1083 and E. coli
ML4909 (pMTY002) were 
256 µg/ml. The MICs of inhibitors against
E. coli TUM1083 and E. coli ML4909(pMTY002) were
512 µg/ml or more. However, MICs of piperacillin to these strains
were markedly decreased to 16 and 2µg/ml in the presence of 4 and 0.5 µg/ml of tazobactam per ml. The MICs of aminoglycoside antibiotics
against E. coli ML4909 were not changed in the presence of
pMTY002.
|
Kinetic parameters.
-Lactamase was purified from E. coli cells harboring pMTY036, as described under Materials and
Methods. The purified enzyme gave a single band on isoelectric focusing
(Coomassie blue staining) and on sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, with an estimated molecular mass of 28,000 Da. As
shown in Table 3, purified Toho-2 had
high catalytic activity toward cephalothin, cephaloridine, cefotaxime,
and piperacillin. the catalytic efficiency (kcat/Km) for those drugs
was also much higher than for the other substrates tested. However,
Toho-2 did not have high kcat values toward
ampicillin, oxacillin, cefoxitin, ceftazidime, and imipenem. Relatively
high Km values of these substrates reduced the
catalytic efficiency (relative
kcat/Km). The
-lactamase inhibitors, tazobactam and clavulanic acid, appeared to
have the highest affinities of all the agents.
|
DNA sequencing.
The nucleotide sequence of 981-bp
blatoho was determined by the strategy shown in
Fig. 1. An 870-nucleotide open reading frame with a GC content of
70.2% was present in this sequence (Fig. 2). The sequence initiation codon (ATG)
was preceded by a possible 
10 region (TGGAAT) and a
35 region (TTGAAG) of a putative promoter. The termination
codon was TAA. From the putative open reading frame, the precursor form
of Toho-2 seemed to consist of 289 amino acid residues with a molecular
mass of 30,725 Da. The processing site was determined by the comparison
of the hypothetical amino acid sequence predicted by the DNA sequence
with the last 10 amino acids from the N-terminal sequence determined by
a peptide sequencer. The mature form consisted of 261 amino acid
residues with a molecular mass of 27,752 Da. The consensus sequences
such as STSK, SDN, and KTG in class A -lactamases were found in the
amino acid sequence of Toho-2 -lactamase. Thus, Toho-2 is a class A
-lactamase.
|
Homology with other -lactamases.
The DNA sequence of the
gene for Toho-2 showed high homology (76% or higher) with those for
plasmid-mediated -lactamases of CTX-M-2 (5) and Toho-1.
Amino acid sequence identities of Toho-2 with E. coli MEN-1
(3), K. oxytoca -lactamase (2), and
Toho-1 are 72, 72, and 70%, respectively, while Toho-2 showed less
than 30% amino acid sequence identity with TEM and SHV. The multiple
sequence alignment of Toho-2 and other -lactamases is shown in Fig.
3. The consensus sequences S70XXK73,
S130DN132, and K234TG236 (Ambler numbering) and the highly conserved
E166 residue, which are essential for the catalysis of class A
-lactamases, were found in Toho-2.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Generally, most of the class A -lactamases are strongly
inhibited by -lactamase inhibitors (6). Clavulanic acid
is often the most potent inhibitor for these enzymes (6).
For Toho-1, Ki values for sulbactam and
tazobactam were 5.8 and 5.3 µM, respectively; on the other hand, the
apparent Ki value of Toho-1 with clavulanic acid
was 0.6 µM (13). However, the extended-spectrum
-lactamase Toho-2 was more strongly inhibited by tazobactam than by
clavulanic acid or sulbactam. It is very different from the
-lactamase reported heretofore.
Toho-2 has three major sequences distinct from those of other class A
-lactamases. Two of those are found in the N- and C-terminal sequences, though Toho-1 has a sequence similar to that of Toho-2. The
other distinct sequence is located at Ala-185 to Ala-219 (Fig. 3). This
sequence has almost no sequence homology with the other class A
-lactamases. The sequence may constitute a loop structure at the
substrate-binding site and has a deletion of 2 amino acid residues at
the binding site. This deletion may well correlate with the observation
that tazobactam, a -lactamase inhibitor, showed a higher affinity
for Toho-2 than for Toho-1, whereas sulbactam binds to both enzymes
with similar affinities. Tazobactam has the 1,2,3-triazolylmethyl group
at C-2 instead of methyl groups for sulbactam. Examination of the
location of the triazolylmethyl moiety at the binding site through
three-dimensional structure modeling of the acyl enzyme suggests that
the triazole moiety interacts with the loop where the deletion of 2 amino acid residues occurred. Thus, the triazole moiety would find
suitable room to reside in a larger substrate-binding pocket.
Ishii et al. (13) have pointed out that in Toho-1 the
replacement of Asn-276 by Arg with the concomitant substitution of Thr
for Arg-244 is important for the extension of the substrate specificity
and that Arg-276 may function as Arg-244. The Arg-274 of Toho-1 is also
a characteristic residue since this residue could also be located at a
site similar to Arg-276. Toho-2 had the same substitutions as Toho-1 at
positions 244 and 276. However, the Ser-274 residue was found in
Toho-2, instead of the Arg-274 residue found in Toho-1. Since the
Ser-274 residue is found also in the -lactamases of E. coli MEN-1 (3) and K. oxytoca E23004 (2), the basic residue at position 274 in Toho-1 may not
play an important role for the cefotaxime hydrolysis, while it would contribute to the higher affinity of aztreonam, which has the carboxylate in the oxyimino side chain.
The substitution of serine for Ala-237 is also a common mutation
observed in substrate-extended class A -lactamases. We assume that
this mutation brings about an enlarged binding site for the bulky
oxyimino moiety of the expanded-spectrum cephalosporins such as
cefotaxime, and thus the mutation should be very important for the
substrate extension.
In conclusion, Toho-2 has two characteristic mutation sites for the substrate extension. The mutations at 237, 244, and 276, which are aligned at a peripheral site of the substrate-binding site, may contribute to the specificity for oxyiminocephalosporins such as cefotaxime and ceftizoxime. The other mutation-deletion at 185 to 219, particularly at the triazole-binding loop, may be responsible for the higher affinity for tazobactam.
| |
ACKNOWLEDGMENTS |
|---|
We thank Hirosuke Matsuo for providing us with E. coli TUM1083. We express our thanks to Teru Ogura and Akira Inoue for their valuable advice.
This study was supported in part by a grant from the Ministry of Health and Welfare of Japan in 1996 (Scientific Research Foundation on Drug Resistant Bacteria) and by Project Research grant 8-25 from the Toho University School of Medicine. Ling Ma was supported by a grant from the Suntory Institute for Bioorganic Research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 1438540, Japan. Phone: 81-3-3762-4151. Fax: 81-3-5493-5415. E-mail: yoishii{at}sirius.med.toho-u.ac.jp.
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