A β-lactamase with an M(r) of 29,000 and a pI of 7.6 was partially purified from a clinical isolate of Shigella dysenteriae. The bla gene encoded the SHV-11 enzyme carrying the substitution Leu→Gln at position 35 and was linked to a strong promoter. This variant, unlike the prototype SHV-1 enzyme, hydrolyzed oxacillin, cloxacillin, and oxyiminocephalosporins such as cefotaxime

Ahamed, Jasimuddin

Kundu, Manikuntala

PubMed

A β-lactamase with an Mr of 29,000 and a pI of 7.6 was partially purified from a clinical isolate of Shigella dysenteriae. The bla gene encoded the SHV-11 enzyme carrying the substitution Leu→Gln at position 35 and was linked to a strong promoter. This variant, unlike the prototype SHV-1 enzyme, hydrolyzed oxacillin, cloxacillin, and oxyiminocephalosporins such as cefotaxime

Publications of the IAS Fellows

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
0066-4804/99/$04.0010
Aug. 1999, p. 2081–2083 Vol. 43, No. 8
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Molecular Characterization of the SHV-11 b-Lactamase of
Shigella dysenteriae
JASIMUDDIN AHAMED AND MANIKUNTALA KUNDU*
Department of Chemistry, Bose Institute, Calcutta 700 009, India
Received 16 November 1998/Returned for modification 3 March 1999/Accepted 6 June 1999
A b-lactamase with an Mr of 29,000 and a pI of 7.6 was partially purified from a clinical isolate of Shigella
dysenteriae. The bla gene encoded the SHV-11 enzyme carrying the substitution Leu3Gln at position 35 and
was linked to a strong promoter. This variant, unlike the prototype SHV-1 enzyme, hydrolyzed oxacillin,
cloxacillin, and oxyiminocephalosporins such as cefotaxime.
Shigella species are a major cause of diarrheal diseases and
mortality in developing countries (4), and the frequency of
strains multiply resistant to ampicillin, trimethoprim-sulfameth-
oxazole, and streptomycin is causing growing concern. b-Lac-
tamases are primarily responsible for b-lactam resistance in
gram-negative bacteria. The presence of TEM-, OXA-1, and
OXA-3 b-lactamases in S. flexneri and S. sonnei has been pre-
viously reported (21). However, detailed characterization of
these enzymes in S. dysenteriae is lacking. We report a clinical
strain of S. dysenteriae PB-10 (obtained from the National
Institute of Cholera and Enteric Diseases [NICED], Calcutta,
India) harboring the SHV-11 b-lactamase capable of hydrolyz-
ing oxacillin, cloxcillin, and oxyimnocephalosporins.
Bacteria were routinely grown in tryptic soy broth on a
rotary shaker at 37°C. The b-lactamase was partially purified
from sonic extracts of cells by size-exclusion chromatography
on Sephadex G-75, followed by successive chromatography on
a Q-Sepharose anion exchanger at pH 7 (at which time b-lac-
tamase activity remained in the flowthrough) and then at pH
10 (where b-lactamase activity eluted at a salt concentration of
0.1 M NaCl in 20 mM ethanolamine containing 5% glycerol
and 5% ethylene glycol). During purification of the enzyme,
b-lactamase activity was routinely determined at 30°C by mon-
itoring the hydrolysis of nitrocefin spectrophotometrically at
482 nm (17). One unit of b-lactamase activity is the amount of
enzyme hydrolyzing 1 mmol of nitrocefin per min.
Hydrolysis of b-lactams was monitored at 30°C in 100 mM
sodium phosphate buffer, pH 7.2, at wavelengths that gave a
maximum in the difference spectrum of the hydrolyzed antibi-
otic against the unhydrolyzed antibiotic. The relative Vmax val-
ues of the substrates were obtained after absorbance data were
fit to the integrated Michaelis-Menten equation (7). The rate
of hydrolysis of benzylpenicillin was set at 100.
Conjugative transfer of S. dysenteriae PB-10 antibiotic resis-
tance plasmid into Escherichia coli HB-101 was performed as
described previously by Philippon et al. (18). Transconjugants
were selected by plating on medium containing 25 mg of am-
picillin/ml and 10 mg of rifampin/ml.
For cloning of the b-lactamase gene, plasmid DNA from the
transconjugant was partially digested with BamHI and ligated
to the BamHI-digested vector pK19 (20). Transformants were
selected on Luria-Bertani agar supplemented with 1 mM iso-
propyl-b-D-thiogalactopyranoside (IPTG), 50 mg of ampicillin,
and 50 mg of kanamycin per ml. Plasmid DNA containing an
’9-kb insert was subsequently subcloned in several steps by
using successively the enzymes SstI, SaII, and AvaI and select-
ing each time for Amp Kanr recombinants. The plasmid ob-
tained in the final step, pMK105, contained a 1.8-kb insert. A
library of nested deletions of the clone pMK105 was finally
generated in the vector pK19 to sequence the entire cloned
DNA, by using the double-stranded nested deletion kit from
Amersham Pharmacia Biotech. DNA sequencing was done by
using the Thermosequenase cycle sequencing kit (Amersham
Pharmacia Biotech). Both strands of DNA were sequenced
from two clones. The nucleotide sequence and the deduced
protein sequence were analyzed by using the Genetics Com-
puter Group software package. Similarity searches were per-
formed by using the Gap BLAST algorithm (1).
The b-lactam resistance of strain PB10 could be transferred
to a rifampin-resistant strain of E. coli, HB101, by transconju-
gation. MICs were determined on Mueller-Hinton agar plates
containing serial dilutions of antibiotics by inoculating with 104
CFU per spot and reading after 18 h of growth at 37°C. The
clinical isolate PB-10 was resistant toward all the penicillins
and some of the cephalosporins tested (Table 1). Cefoxitin and
imipenem appeared to be the most effective antibiotics. The
MICs of selected antibiotics were determined in combination
with that of clavulanic acid (MIC, 32 mg/ml) at a concentration
of 4 mg/ml. The decreased MICs indicated the involvement of
a transferable b-lactamase resistant to strain PB-10. The cefo-
taxime and ceftazidime MICs are high compared to data re-
ported earlier for Shigella species (6, 21). For cefotaxime, this
could be due, at least in part, to the presence of the b-lacta-
mase reported here, since a decrease in the MIC was observed
in the presence of clavulanic acid for strain PB-10. The higher
ceftazidime MIC could not be correlated with b-lactamase
activity, particularly since there was no decrease in the MIC in
the presence of clavulanate.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
of the cell lysate and the partially purified b-lactamase of strain
PB-10, followed by renaturation and overlay of gels with ni-
trocefin (12), showed the presence of a single band of b-lac-
tamase activity, with an approximate Mr of 29,000 6 2,000
(mean 6 standard deviation). Isoelectric focusing of the ref-
erence SHV-1 enzyme as well (3) as crude and partially puri-
fied b-lactamase of PB-10, followed by overlay with nitrocefin,
showed in each case a single band with b-lactamase activity at
an isoelectric point of 7.6 6 0.2, which corresponded to that
reported for SHV enzymes (2, 5).
The hydrolysis of cephaloridine and of almost all other ceph-
alosporins tested was much slower than that of benzylpenicil-
* Corresponding author. Mailing address: Department of Chemis-
try, Bose Institute, 93/1 Acharya Prafulla Chandra Rd., Calcutta 700
009, India. Phone: 91 33 350 6619. Fax: 91 33 3506790. E-mail: mani
@boseinst.ernet.in.
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lin, nitrocefin being the only exception (Table 2). The oxyimi-
nocephalosporins cefotaxime, cefuroxime, and ceftizoxime
were hydrolyzed at measurable rates. No detectable hydrolytic
activity was observed for ceftazidime, cefoxitin, cefsulodin, az-
treonam, or imipenem. Also, these antibiotics failed to inhibit
the enzyme. Unlike other SHV enzymes, the PB-10 enzyme
hydrolyzed oxacillin and cloxacillin at appreciable rates. Com-
parison of relative rates of hydrolysis of b-lactams by a refer-
ence SHV-1 enzyme and the b-lactamase harbored by
pMK105 demonstrate that, unlike the reference SHV-1 en-
zyme, the b-lactamase produced by pMK105 hydrolyzed ox-
acillin. Cefotaxime was hydrolyzed by this enzyme, whereas its
hydrolysis could not be measured with the reference SHV-1
enzyme. Sensitivity to clavulanic acid is characteristic of TEM
and SHV enzymes. The 50% inhibitory concentrations (IC50)
of clavulanic acid and sulbactam were determined by incubat-
ing the enzyme with either inhibitor for 10 min prior to the
addition of benzylpenicillin (1 mM). The IC50 was determined
graphically. For clavulanic acid, the values were 2 and 40 nM,
while for sulbactam, the values were 17,000 and 30,000 nM for
the reference strain SHV-1 and the S. dysenteriae enzyme,
respectively. The S. dysenteriae enzyme was therefore less sus-
ceptible to clavulanic acid and sulbactam than the SHV-1 en-
zyme.
The nucleotide sequence (EMBL nucleotide sequence da-
tabase, accession no. Y18299) of the plasmid pMK105 revealed
a 286-amino-acid open reading frame. The nucleotide se-
quence corresponding to positions 1 through 286 of the de-
duced amino acid sequence was identical to that reported for
the Klebsiella pneumoniae SHV-1a (or SHV-11) (GenBank
accession no. X98101), except for a silent CAC-to-CAT muta-
tion encoding histidine 108. Mutations of the plasmid-deter-
mined TEM, SHV, and OXA b-lactamases that enhance their
affinity for expanded-spectrum cephalosporins usually occur
between positions 104 and 240. These mutations have resulted
in roomier active-site cavities that permit expanded-spectrum
b-lactams with bulky side chains to enter and bind to the active
site serine-70 (10, 13). With site-directed mutants it has been
reported that the leu3gln substitution at position 35 on the
protruding NH2 terminus (16) increases resistance to ceftazi-
dime but reduces the MICs of all other cephalosporins tested
compared to SHV-2. In our case, the situation was not com-
parable, since the SHV-11 enzyme has glycine at position 238
rather than serine, as in SHV-2. (8) A survey of SHV b-lacta-
mases in Switzerland previously led to the identification of the
variant enzyme SHV-11 (15) harboring the Leu3Gln substi-
tution at position 35. In this report, K. pneumoniae KPZU-12,
harboring the SHV-11 b-lactamase, showed b-lactam suscep-
tibilities virtually identical to K. pneumoniae KPAA-I harbor-
ing the SHV-1 b-lactamase. We demonstrate that the SHV-11
enzyme from S. dysenteriae hydrolyzes oxyiminocephalospor-
ins. The hydrolysis of oxacillin and cloxacillin was intriguing.
There was little similarity of the nucleotide sequence up-
stream of the 210 with that of the prototype SHV-1 bla gene
(14). Changes in the 235 and 210 regions are a powerful
influence on promoter strength (11). In E. coli, the closer the
sequence is to the consensus, the stronger the promoter (235
consensus sequence, TTGACA; 210 consensus sequence,
TATAAT) (9). The promoter comprising 59TTGCAA939 (235
box) and 59TATTCT39 (210 box) identified in the pMK105 bla
gene has been reported to increase b-lactam resistance when
coupled to the SHV-2 gene. (19). The association of the bla
gene with a strong promoter likely influenced susceptibilities to
b-lactams in the present study as well.
The continued emergence of b-lactam-resistant S. dysente-
riae is of particular concern to developing countries. Surpris-
ingly, reports of noteworthy efforts to characterize the b-lac-
tamases most likely to be associated with this resistance
phenomenon are sparse in the literature, particularly in terms
of nucleotide sequence information. Our report describes per-
haps the first serious attempt both to characterize the struc-
tural bla gene of a b-lactamase from a clinical isolate of S.
dysenteriae and to biochemically characterize the elaborated
enzyme. Although the leu-353gln substitution had been re-
ported while this work was in progress (15), its effect on sub-
strate profile had not been investigated. Rather, conclusions
had been drawn merely from an evaluation of MIC data, which
may be influenced by an interplay of factors, including the
permeability of the outer membrane of the hosts, the amount
TABLE 1. In vitro susceptibilities of S. dysenteriae and
transconjugant to b-lactam antibiotics
Antibiotic(s)
MIC (mg/ml) for:
E. coli
HB-101
S. dysenteriae
C152a
S. dysenteriae
PB-10 Transconjugant
Ampicillin 8 8 .1000 .1,000
Ampicillin 1 CAc 8 8 40 52
Ticarcillin 8 NDb .1000 .1,000
Ticarcillin 1 CA 8 ND 100 100
Cefotaxime 0.25 0.25 16 16
Cefotaxime 1 CA 0.25 0.25 4 4
Ceftazidime 0.16 1 16 16
Ceftazidime 1 CA 0.16 1 16 16
Cefuroxime ND ND 250 ND
Piperacillin ND 8 500 ND
Carbenicillin ND ND 1,000 ND
Cefoxitin ND 4 4 ND
Imipenem ND 2 2 ND
a S. dysenteriae C152 was a susceptible clinical isolate obtained from NICED,
Calcutta, India.
b ND, not determined.
c CA, clavulanate.
TABLE 2. Hydrolysis parameters for SHV-1 and SHV-11 b-
lactamases for selected b-lactamsc
Antibiotic Wavelength De (M21cm21)
SHV-1
J53R1010
(reference)a
SHV-11
(S. dysenteriae)
Benzylpenicillin 232 800 100 100
Ampicillin 235 670 150 300
Oxacillin 260 400 ,0.1 96
Cloxacillin 260 140 ,0.1 45
Carbenicillin 235 830 20 45
Nitrocefin 482 15,000 NDc 150
Cephaloridine 255 9,000 50 35
Cefazolin 273 6,600 10 10
Cefuroxime 273 7,500 ,0.1 6
Cefotaxime 264 6,700 ,0.1 8
Cefacetrile 260 7,400 5 4
Cefoperazone 260 5,700 ND 4
Cefaclor 260 7,200 8 15
Ceftizoxime 255 5,000 ,0.1 18
Ceftazidime 260 8,600 ,0.1 ,0.1
Cefsulodin 265 5,800 ,0.1 ,0.1
Cefoxitin 265 7,380 ,0.1 ,0.1
Imipenem 297 9,000 ,0.1 ,0.1
a SHV-1 J53R1010 was a gift from Antone A. Medeiros.
b ND, not determined.
c Results are the means of three determinations (standard deviation, #10%).
Rates of hydrolysis (Vmax) of b-lactams are expressed relative to the rate of
hydrolysis of benzylpenicillin, which was set at 100.
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of b-lactamase produced, the affinities of the target penicillin-
binding proteins for the tested b-lactam, and the involvement
of efflux pumps. We demonstrate, using the SHV-1 J35R1010
as a reference enzyme, that the SHV-11 enzyme hydrolyzes
oxacillin, cloxacillin, and oxyiminocephalosporins such as ce-
fotaxime and is less sensitive to clavulanic acid. The results
from our biochemical characterization of this enzyme suggest
that an amino acid substitution in the protruding amino ter-
minus of the b-lactamase likely alters the overall conformation
of the molecule and, consequently, its substrate profile. The
structural basis for this needs to be investigated further.
This work was supported in part by grants from the Department of
Science and Technology and the Council of Scientific and Industrial
Research, government of India.
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English

Molecular characterization of the SHV-11 β-lactamase of Shigella dysenteriae

http://repository.ias.ac.in/102776/1/Antimicrob.%20Agents%20Chemother.-1999-Ahamed-2081-3.pdf

Molecular Characterization of the SHV-11 β-Lactamase of Shigella dysenteriae

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