a Department of Bacteriology, Hellenic Pasteur Institute; b Laboratory of Antimicrobial Agents, Department of Microbiology, Medical School, University of Athens, M. Asias 75, 11527, Athens, Greece
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Abstract |
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Introduction |
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Characterization of an in-vitro constructed mutant of TEM-1 ß-lactamase showed that
Asn276Asp substitution conferred resistance to clavulanate and reduced hydrolytic
activity against penicillins and cephalosporins.17 In
addition, Asn276
Gly replacement in OHIO-1 ß- lactamase (an
enzyme of the SHV family) modified inhibitor binding specificity and altered affinity for
penicillins.18 In this work, we examined the effect of
Asn276
Asp substitution in
SHV-1 ß-lactamase and its extended-spectrum derivative SHV-5.
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Materials and methods |
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The Escherichia coli DH5 strain (deoR, endA1, gyrA96, hsdR17 (rk-mk+), recA1, relA1, supE44, thi-1
(lacIZYA-argFV169),
80
lac
M15,
F-,
-) was used to express the wild-type and mutant ß-lactamases. The
plasmids
used in the study are described in Table I. To achieve isogenic
conditions, 1.4 kb SmaIClaI fragments, encompassing the coding and
promoter regions of blaSHV-1 and blaSHV-5, were
purified from low-melting point agarose and ligated into the multicloning
site of pBCSK(+). The resulting plasmids were used to transform E. coli DH5
competent cells. ß-Lactam-resistant clones were selected in Luria Bertani agar
(Unipath Ltd, Basingstoke, UK) supplemented with ampicillin (50 mg/L) plus chloramphenicol
(20 mg/L).
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Mutant ß-lactamases were constructed by the `megaprimer' PCR-based site-directed mutagenesis method essentially as described previously.19 A mutagenic and an external primer were used in the first round of PCR to create the `megaprimer'. In the second round of PCR, the `megaprimer' and an external primer were used. The resulting amplicons were digested with SmaI and ClaI and subcloned into pBCSK(+). The mutagenic primer (5'-TGGCCGAGCGAGATCAGCAAAT-3') was 22 nucleotides long and contained a single base mismatch close to the centre of the sequence in order to direct mutagenesis at codon 276 of the mature peptide (with GAT instead of AAT at codon 276). Oligonucleotide primers were prepared in an Applied Biosystems DNA synthesizer according to the instructions of the manufacturer (Applied Biosystems, Foster City, CA, USA). DNA sequencing was performed by the dideoxy chain termination method using a Sequenase 2.0 kit (United States Biochemical Corp., Cleveland, OH, USA). To confirm the lack of unwanted changes, the complete nucleotide sequences of both strands of the mutant genes were determined.
Susceptibility testing
Susceptibility to various ß-lactam antibiotics and penicillin-inhibitor combinations was determined by an agar dilution assay according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).20 Mueller Hinton agar (Unipath) containing the appropriate antibiotic concentrations was inoculated with 104 cfu/spot and incubated for 18 h at 37°C. The initial screening for clones producing mutant ß-lactamases was performed by a disc diffusion method. Antibiotic discs, ß-lactam antibiotics, clavulanate and sulbactam were purchased from commercial sources. Tazobactam was a gift from Wyeth Hellas S.A. Etest strips detecting the extended-spectrum ß-lactamases (ESBLs) (ceftazidime and ceftazidime clavulanate) were also used according to the instructions of the manufacturer (Biodisk, Solna, Sweden).
ß-Lactamase assays
To obtain enzyme preparations containing the wild-type and mutant ß-lactamases, the
respective E. coli DH5 clones were grown exponentially at 37°C for 18 h in
tryptone soya broth (Unipath). Bacterial cells were harvested and washed twice in
phosphate-buffered saline (pH 7.0). ß-Lactamases were released after mild ultrasonic
treatment of cells suspended in phosphate buffer (PB, 100 mM, pH 7.0). The extracts were
clarified by ultracentrifugation and dialysed overnight against PB. The protein content of the
extracts was determined with a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA,
USA). Isoelectric focusing (IEF) was performed in polyacrylamide gels containing ampholytes
which covered a pH range from 3.5 to 9.5 (Pharmacia-LKB Biotechnology, Uppsala, Sweden).
ß-Lactamase bands were visualized with nitrocefin (Unipath). The ß-lactamase activity
of the extracts was quantified using nitrocefin as substrate. Results were expressed as units of
activity. One unit was the amount of enzyme hydrolysing 1 mmol of substrate/ min/mg of protein
at pH 7.0 and 37°C. Inhibition profiles were determined using clavulanate, tazobactam and
sulbactam as described previously.21 Nitrocefin was used
as the reporter substrate at a concentration of 100 mM. The
amount of each ß-lactamase preparation was normalized to give 150 mM nitrocefin
hydrolysed per minute. The IC50 values were determined by incubating the enzyme
preparations with various
concentrations of inhibitor for 5 min before the addition of nitrocefin. The maximum rates of
hydrolysis of various ß-lactam substrates were determined by UV spectrophotometry
(37°C, pH 7.0) as described previously
15 and expressed relative to that of nitrocefin, which was set at 100 (relative Vmax).
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Results |
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The clone producing SHV-1(Asn276Asp) was slightly less resistant to amoxycillin,
ticarcillin and piperacillin than the clone producing the SHV-1 ß-lactamase. In contrast, the
mutant enzyme, compared with SHV-1, conferred higher levels of resistance to
penicillin-inhibitor combinations. Cephalothin and cefprozil were four-fold more active against
the isogenic clone that expressed the SHV-1 (Asn276
Asp) mutant ß-lactamase.
Compared with the SHV-1-producing E. coli clone, its
SHV-1(Asn276
Asp)-producing counterpart was found to be more susceptible to all
`third-generation' oxyimino-ß-lactams tested, except ceftazidime. The MICs
of the latter antibiotic remained unaltered. Notably, the MICs of cefpirome and cefepime were
consistently one- to two-dilutions higher in the E. coli clone producing the
SHV-1(Asn276
Asp) mutant ß- lactamase (Table II).
As is shown in Table III, Asn276Asp substitution rendered the
SHV-1
ß-lactamase less susceptible to mechanism-based inhibitors, increasing the IC50
values of clavulanic acid and tazobactam by a factor of 8.8 and 8.0 respectively.
The IC50 of sulbactam, when tested against SHV-1(Asn276
Asp), was >20
µM and higher concentrations of this inhibitor were not used.
The relative maximum hydrolysis rates are presented in Table IV. The
results for cephalothin
and cefepime were in line with the differences observed in the isogenic MIC determinations; the
Asn276Asp mutant enzyme hydrolysed cefepime more rapidly and cephalothin more
slowly than the SHV-1 ß-lactamase. Despite the evident decrease in efficiency against
penicillins (Table II), the mutant ß-lactamase hydrolysed penicillin
G more quickly than the
parental enzyme.
Effect of Asn276Asp on SHV-5
Like the pair of clones described above, the E. coli strain expressing the
SHV-5(Asn276Asp) mutant enzyme was less resistant to penicillins, cephalothin and
cefprozil than its SHV-5-producing isogenic counterpart. The MICs of penicillin-inhibitor
combinations were higher for the SHV-5(Asn276
Asp)-producing clone than for the
strain expressing the SHV-5 ESBL. The most noticeable effect of the Asn276
Asp
replacement on the resistance phenotype conferred by SHV-5 was the drastic reduction of
activity against cefotaxime and ceftriaxone. The MICs of ceftazidime and aztreonam were
virtually unaltered. The combination of ceftazidime with clavulanate was active against the
SHV-5-producing E. coli strain. The clone expressing the
SHV-5(Asn276
Asp) mutant ß-lactamase was less susceptible to the latter
combination (Table II and Figure 2). As can also
be seen inFigure 2, the
SHV-5(Asn276
Asp) mutant did not seem to be an ESBL according to the
requirements of this particular Etest-based method. Cefpirome and cefepime were slightly less
active against the clone that produced the mutant SHV-5 (Table II).
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SHV-5(Asn276Asp) hydrolysed penicillin G and cefepime more quickly and
cefotaxime more slowly than the SHV-5 ß-lactamase. Although the mutant ß-lactamase
conferred a lower level of resistance to cephalothin than did SHV-5 (Table II), the antibiotic was
hydrolysed more quickly by the former enzyme (Table IV).
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Discussion |
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ß-Lactam susceptibility testing using isogenic systems can demonstrate differences in
hydrolytic efficiencies of related ß-lactamases.
24,25 The MICs of E. coli clones producing SHV-1, SHV-5 and their
respective Asn27Asp mutants were determined under isogenic conditions. Therefore,
the observed changes in the MICs of the ß-lactams and ß-lactam-inhibitor combinations
suggested that replacement of Asn276 by aspartic acid conferred resistance to ß-lactamase
inhibitors and influenced the hydrolytic efficiencies of SHV-1 and SHV-5 ß-lactamases.
Asn276 is on the C-terminal -helix and its side-chain lies far from the active site of TEM
and
SHV enzymes. The carbonyl group of Asn276 accepts two hydrogen bonds from Arg244 and
this bonding contributes to the proper orientation of the guanidium group of Arg244.23 The latter positively charged group is critical for ß-lactam
binding, and for
inactivation by `suicide' inhibitors: one of its NH2 groups forms a
hydrogen bond with the C-3 (C-4) ß-lactam carboxylate, and
the second more exposed amino group holds in place a water molecule (W673) which
participates in the process of irreversible inactivation by clavulanate.22 In TEM-1(Asn276
Asp), Asp276 may form a salt bridge with Arg244,17 leading to a
slight change in the orientation of the
guanidium group and altering
the position of W673. Such alterations in the active site cavity may explain why
SHV-1(Asn276
Asp) and SHV-5(Asn276
Asp) were less susceptible to
inhibitors than were the parental ß-lactamases.
As shown by the MIC determinations, the net result of the Asn276Asp substitution in
SHV-1 and SHV-5 ß-lactamases was to reduce hydrolytic activity against
mostß-lactams. As mentioned above, Arg244 is involved mainly in substrate binding.
Assuming that the consequence of (Asn276
Asp mutation is to alter the position of the
Arg244 side-chain, this reduction resulted from lower enzyme substrate affinity.
Replacement of Asn276 by Asp caused diverse changes in the substrate profiles of SHV-1 and
SHV-5 (Table IV). An increase in the hydrolysis rates of some
ß-lactams has been observed
with the analogous mutant ß-lactamases TEM-1(Asn276
Asp)17 and OHIO-1 (Asn276
Gly),18
while the respective catalytic efficiencies were lower than those of the parental
ß-lactamases. Such `discrepancies' underline the different interactions of
each particular ß-lactamase with different ß-lactams, and the differences in the active
site of an ESBL with that of its parental penicillinase. Interestingly, the replacement of Asn276
by Asp improved the ability of SHV-1 and SHV-5 to inactivate the
`fourth-generation' cephalosporins cefpirome and cefepime. The presence of
aspartic acid
at position 276 leads to a decrease in the positive potential of the active site,22 and thus may facilitate electrostatically the docking of the latter antibiotics,
which possess a positively charged quaternary ammonium group at C-3.
Several recent studies have reported the emergence of inhibitor-resistant TEM-1/TEM-2
ß-lactamase variants. In some areas (e.g. Clermont-Ferrand, France), these enzymes appear
at a relatively high frequency among enterobacteria.6,7,8 Analogous
inhibitor-resistant SHV variants have not been described.
The inhibitor-resistant SHV-10 has been derived from an SHV-5 variant by replacement of
Ser130 by Gly.26 TEM-1/TEM-2 ß-lactamases
occur more frequently than SHV-1
among enterobacteria (reviewed in references 27 and 28). Therefore the possibility of selection of
inhibitor-resistant SHV variants is expected to be lower. Furthermore, the emergence of some of
these mutants, e.g. SHV-1(Asn276Asp), could pass unnoticed in routine susceptibility
tests. The above hypotheses may explain partly the apparent absence of inhibitor-resistant SHV
ß-lactamases.
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Acknowledgments |
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Notes |
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References |
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Received 20 May 1998; returned 9 July 1998; revised 3 August 1998; accepted 17 August 1998