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Antimicrobial Agents and Chemotherapy, June 1998, p. 1459-1462, Vol. 42, No. 6
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Ceftazidime and Aztreonam Resistance in
Providencia stuartii: Characterization of a Natural
TEM-Derived ExtendedSpectrum 
-Lactamase, TEM-60
Nicola
Franceschini,1
Mariagrazia
Perilli,1
Bernardetta
Segatore,1
Domenico
Setacci,1
Gianfranco
Amicosante,1,*
Annarita
Mazzariol,2 and
Giuseppe
Cornaglia2
Dipartimento di Scienze e Tecnologie
Biomediche, Università degli Studi dell'Aquila, Loc. Coppito
I-67010, L'Aquila,1 and
Istituto di
Microbiologia, Università degli Studi di Verona, I-37134,
Verona,2 Italy
Received 11 August 1997/Returned for modification 1 December
1997/Accepted 30 March 1998
 |
ABSTRACT |
A plasmid-encoded 
-lactamase produced from a clinical strain of
Providencia stuartii has been purified and characterized. The gene coding for the -lactamase was cloned and sequenced. It
appears to be a new natural TEM-derived enzyme, named TEM-60. Point
mutations (Q39K, L51P, E104K, and R164S) are present with respect to
the TEM-1 enzyme; the mutation L51P has never been previously reported,
with the exception of the chromosomally encoded extended-spectrum
-lactamase PER-1. Kinetic parameters relative to penicillins,
cephalosporins, and monobactams other than mechanism-based inactivators
were related to the in vitro susceptibility phenotype.
| |
INTRODUCTION |
The use of stable -lactams to
treat bacterial infectious diseases in intensive care units or in
immunocompromised patients has resulted in the large spread of
resistant strains. These isolates are often characterized by the
presence of mutant -lactamases belonging to the TEM and/or SHV
families, which are diffused worldwide in clinically relevant strains.
The presence of these extended-spectrum -lactamases (ESBL)
usually confers a phenotype of resistance to ureido-penicillins and to
newer cephalosporins. Occasionally these enzymes appear to be resistant
to mechanism-based inactivators used in therapy, such as clavulanic
acid and penicillanic acid sulphones (14).
Besides the presence of natural -lactamase mutants,
to better understand the molecular basis of the extension
substrate specificity, a large number of laboratory mutants have been
selected in vitro (18). A detailed knowledge of
extended-spectrum activity allows us to anticipate the future;
presumably, plasmid or chromosomally encoded -lactamases, not
necessarily belonging to the TEM or SHV families, could acquire
favorable mutations to inactivate all antibiotics actually used in
clinical therapy (11). The aim of the present study was to
characterize a new TEM-derived enzyme showing an ESBL behavior found in
a clinical isolate of Providencia stuartii. Usually, strains
belonging to the Providencia genus are most frequently
isolated from urinary tract infections in hospitalized patients
(7). Moreover, P. stuartii may be the causative
agent of fatal bacteriemia not related to the urinary tract. The
increasing role of P. stuartii as a nosocomial pathogen in
the dissemination of plasmid-mediated resistance is confirmed, although
this species usually preserves its susceptibility to extended-spectrum
cephalosporins such as cefotaxime and ceftazidime.
| |
MATERIALS AND METHODS |
Bacterial strain.
P. stuartii VR-1 was a clinical
strain isolated in Milan, Italy, from the urinary tract of a
hospitalized elderly patient. It was identified with API-20 E
(bioMerieux, Marcy l'Etoile, France). On the basis of agar disk
diffusion assay, the strain was resistant to extended-spectrum
-lactams other than aztreonam. P. stuartii VR-1 was
also resistant to gentamicin, tobramycin, netilmycin, ciprofloxacin,
and polymixin B.
Antibiotics and determination of MICs.
The following
antibiotics were obtained from the respective manufacturers:
piperacillin (Lederle Wyeth, Catania, Italy); cefotaxime and
desacetyl-cefotaxime (Hoechst AG, Frankfurt, Germany); imipenem (Merck
Sharp & Dohme, Pomezia, Italy); aztreonam (Squibb Institute for Medical
Research, Princeton, N.J.); ceftazidime (Glaxo Wellcome, Verona,
Italy); clavulanic acid (SmithKline Beecham Research Laboratories, Brentford, United Kingdom); 6--iodopenicillanic acid
(-IP) and sulbactam (Pfizer Central Research, Sandwich, United
Kingdom); carumonam (Roche, Basel, Switzerland); and benzylpenicillin,
ampicillin, cephalothin, and cephaloridine (Sigma Chemical Co., St.
Louis, Mo.). Nitrocefin was purchased from Unipath (Milan, Italy). MICs were determined by the conventional macrodilution broth procedure, according to the National Committee for Clinical Laboratory Standards. Tazobactam and clavulanic acid were used at fixed concentrations of 8 and 4 µg/ml, respectively. Two different inocula (5 × 104 and 5 × 107 CFU) were used for
testing.
Plasmid content and transformation.
Lysates of P. stuartii VR-1 cultures were prepared as reported by Kado and Liu
(13). The plasmid content was analyzed running the DNA
preparation on an 0.8% agarose gel electrophoresis (120 V, 2 h).
An approximative plasmid size was established by comparison with a
standard mixture of known DNA fragments (MW-XV Boehringer, Mannheim,
Germany). Escherichia coli HB101 was used as the recipient for the plasmid. Transformation experiments were performed as reported
by Sambrook et al. (20). Transformants were selected on
Luria-Bertani (LB) agar plates containing ampicillin (100 µg/ml).
Recombinant DNA technology.
PCR was applied to amplify the
gene coding the -lactamase, by using the purified plasmid as DNA
template. Two to 5 ng of DNA were subjected to 30 cycles of
amplification by using 2 U of Taq polymerase (Perkin-Elmer,
Roche Molecular Systems Inc., Branchburg, N.J.). Oligonucleotides
designed on the basis of a published TEM-1 -lactamase nucleotide
sequence were used for the fragment amplification as reported also in
Mabilat et al. (16). Direct sequencing of PCR amplimers was
performed according to the dideoxy-chain termination method by using a
Sequenase 2.0 DNA sequencing kit (USB, Amersham, Little Chalfont,
United Kingdom), according to the manufacturer's specifications
(2).
-Lactamase preparation and purification.
Two liters
of Luria-Bertani medium was inoculated with 100 ml of an overnight
preculture of P. stuartii VR-1 at 37°C in a rotary
incubator (120 rpm). The bacterial suspension was recovered by
centrifugation and the pellet resuspended in 50 mM sodium phosphate buffer (pH 7). Crude enzyme was obtained by sonication. The membrane debris was removed by high-speed centrifugation. Forty milliliters of
the clarified supernatant containing the -lactamase activity was
extensively dialyzed against 50 mM sodium phosphate buffer (pH 7) and
loaded onto a Sepharose Q FF column (2.5 by 30 cm; Pharmacia Biotech,
Milan, Italy) equilibrated with the same buffer. The unbound material
was eliminated from the column by washing with the same buffer
(A280 < 0.05). The enzyme was eluted by a linear gradient (0 to 1 M NaCl) in the same buffer. Five fractions containing the -lactamase activity were pooled and dialyzed
against 25 mM bis-tris buffer (pH 7.4). The protein was purified to
homogeneity by the fast-chromatofocusing technique with a Mono P HR5/20
column equilibrated in the above buffer. The protein was eluted with 2.5% Ampholine solution (1:10 diluted) (pH 4). The pH of the single fractions was immediately adjusted to 7 by the addition of 1 M potassium phosphate buffer (pH 7) (1/10 of the fraction volume).
-Lactamase activity.
The -lactamase
activity was measured in a spectrophotometer by following the
hydrolysis of a 100 µM cephaloridine solution (
M260 = 
10.000 M
1
cm1). One enzyme unit was defined as the amount of enzyme
hydrolyzing 1 µmole of cephaloridine/min at maximum rate, at 30°C
in 50 mM sodium phosphate buffer (pH 7).
Mr and isoelectric focusing
analysis.
The relative -lactamase molecular mass was
estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis with 15% acrylamide (15). Proteins were
boiled 5 min at 100°C in a solution containing 1% SDS and 4%
mercaptoethanol before being loaded on the gel. The low-range marker
proteins (Bio-Rad, Richmond, N.J.) were subjected to the same treatment
and used as the standard reference.
Crude -lactamase extract and purified enzyme were analyzed on
precast polyacrylamide gel (5% wt/vol) containing Ampholines (pH
range, 3.5 to 9.5) on a Multiphor II flatbed apparatus (Pharmacia Biotech) at 6°C. Enzyme activity was revealed by overlaying the gel
with a paper strip soaked with 250 µM nitrocefin.
Kinetic parameter determinations.
The complete time course
of the substrate hydrolysis was recorded with a lambda2
spectrophotometer connected to an IBM-compatible microcomputer via
an RS232 interface. The Km and
Vmax values were derived as reported by De
Meester et al. (9). When the Km was quite low it was calculated as Ki by using 100 µM nitrocefin as a reporter substrate. The interaction with
-IP was studied with the substrate reporter method as described
by De Meester et al. (8). The reported values are the means
of three different experiments, with errors below 10%.
Determination of N-terminal amino acid sequence.
The mature
TEM-60 was used to characterize its N-terminal amino acid sequence.
N-terminal amino acid derivatives were analyzed with the help of a
gas-phase Sequenator (Applied Biosystems, Foster City, Calif.).
Nucleotide sequence accession number.
The nucleotide
sequence data appear in the GenBank database under accession no. AF
047171.
| |
RESULTS |
-Lactamase purification.
A typical purification
procedure is reported in Table 1. A
117-fold purification of the enzyme was obtained with a yield of 29%.
The enzyme used for the kinetic measurements was 95% pure.
Isolation of the P. stuartii VR-1 plasmid.
A
plasmid (pVR-1) estimated to be 20 kb was isolated from the P. stuartii VR-1 strain. It was transferred into the E. coli HB101 strain by transformation
(20). The size of the plasmid purified from the
transformed E. coli HB101 was identical to that found in
P. stuartii VR-1 and conferred the same phenotypic
pattern.
MICs.
The reported MICs showed that the presence of the VR-1
-lactamase in P. stuartii is able to confer
resistance to a broad spectrum of -lactam antibiotics, including
monobactams. Susceptibility to penicillins is restored by the
combination piperacillin-tazobactam at two inocula but not by
amoxicillin-clavulanate or by ampicillin-sulbactam. However, the
amoxicillin-clavulanate combination is able to lower the MIC (Table
2). The susceptibility data suggested the
presence of an extended-spectrum -lactamase responsible for the
resistance phenotype in P. stuartii VR-1.
Recombinant DNA technology and N-terminal amino acid sequence.
PCR experiments performed on the purified plasmid gave a single
1,100-bp fragment including an open reading frame of 865 bp potentially
encoding a 286-amino-acid polypeptide. The nucleotide sequence
determined on both DNA strands showed the main characteristics of a
class A -lactamase, such as the four conserved elements involved
in the active site delimitation, i.e., S*XXK (where S* is the
active-site Ser residue), SDN, EXXLN, and KSG. Sequence comparison with
other TEM enzymes revealed the highest sequence homology with TEM-8 and
TEM-7 enzymes, even if other amino acid substitutions related to
TEM-2, TEM-3, and TEM-18 were present (Table
3).
The N-terminal amino acid sequence (HPETLVKVKDAEDKL...)
determined by microsequencing is in complete agreement with that
deduced
from the gene sequence. By comparison with TEM-1 enzyme, four
mutated residues were found (Table
3).
Mr and isoelectric point.
The
SDS-polyacrylamide gel electrophoresis analysis showed the purified
enzyme to have a relative mass of 27,500 Da (data not shown). An
isoelectric point value of 6.4 was also determined.
Kinetic parameters.
The values of Km
and kcat determined for the purified enzyme are
reported in Table 4 in comparison with
the values calculated for TEM-1 (19). Penicillins were the
best substrates; moreover, cephalosporins had relatively high
kcat values compared to penicillins. Surprisingly, aztreonam is one of the best substrates, showing a
catalytic efficiency higher than that of most of the cephalosporins tested in the present study. A comparative analysis of catalytic efficiency between P. stuartii VR-1 -lactamase,
TEM-1, TEM-3, and TEM-18 is reported in Table
5.
View this table:
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|
TABLE 4.
Kinetic parameters determined for the TEM-60
-lactamase purified from P. stuartii VR-1
compared to TEM-1 enzymea
|
|
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|
TABLE 5.
Comparative analysis of catalytic efficiency between
TEM-60 from P. stuartii VR-1 and some related
TEM enzymes
|
|
Interaction with -iodopenicillanate, clavulanic acid,
tazobactam, and sulbactam.
The P. stuartii
VR-1 -lactamase was rapidly inactivated after incubation with
-IP. The complete inactivation of the enzyme was reached at
a 1:1 ratio with the inhibitor. No reactivation was observed by
dilution of the complex in the presence of a good substrate such as 100 µM nitrocefin at a pH of 7. The inactivation rate constants were
computed according to the following
scheme: K k+2 k+3
E + I 
EC 
EC* E + P 
k+4
ECi
where EC* is the acylenzyme and EC
i is the
irreversibly inactivated complex.
The variation of the pseudo-first order inactivation rate constant
(
ki) versus the inhibitor concentration allowed
the calculation
of the acylation constant (
k+2 = 0.021 s
1), the dissociation constant (
K = 0.08 µM), and the acylation
efficiency
(
k+2/
K = 260,000 M
1 s
1) of the process.
After incubating clavulanic acid (0.48 to 1.6 µM) as a competitive
inhibitor with 100 µM nitrocefin and measuring the initial
reaction
rate, a
Ki = 0.7 µM was computed. The values
of the first-order
rate constant were also estimated by longer
incubation and by
monitoring the relative hydrolysis of the same
substrate as reported
for
-IP interaction. The deacylation
process was measured by
the direct hydrolysis of clavulanic acid at 265 nm (
M265 = + 2,000 M
1
cm
1) and a
k+3 of 8.7 10
3 s
1 was calculated. The acylation
efficiency reported as
k+2/
K was
estimated to be 12,000 M
1 s
1. These values
were estimated as reported by Galleni et al. (
10).
Tazobactam (0.1 to 5 µM) was used as a competitive inhibitor to
calculate a
Ki of 0.018 µM. The deacylation
process of the
enzyme-inhibitor complex was estimated by incubating the
enzyme
and the inhibitor (1:5,000 ratio) at 30°C. After 10 min, a
small
aliquot of the complex (5 µl) was removed and added to 500 µl
of 100 µM nitrocefin. The hydrolysis of nitrocefin was continuously
monitored at 30°C until the rate remained constant. The
k+3 computed for the deacylation process was 4.2 10
4 s
1 and a
k+2/
K of 21,000 M
1
s
1.
No direct hydrolysis was detected with sulbactam by using a large
quantity of enzyme (5 to 10 µg). A
Ki value of
0.075 µM
was estimated in competitive inhibition assays when a range
of
0.5 to 5 µM inhibitor was used with 100 µM nitrocefin as the
reporter
substrate. The results of interactions with inhibitors are
summarized
in Table
6.
| |
DISCUSSION |
The contribution of TEM-derived or chromosome-encoded ESBLs
in the resistance mechanism to -lactam-stable
antibiotics has been reported worldwide (4, 5,
12). Nevertheless P. stuartii spp. usually have
not shown a loss of sensitivity to extended-spectrum cephalosporins. In
this study a P. stuartii -lactamase-producing strain has been isolated during a survey from institutionalized elderly
patients with urinary tract infections, whose phenotypic resistance
pattern toward expanded-spectrum cephalosporins was evident
(7). The -lactamase encoded by a 20-kb plasmid had a
molecular mass and reactivity parameters relative to the interaction with -IP consistent with a class A -lactamase as proposed
by Ambler (1). This finding was confirmed by gene
sequencing, which revealed the typical conserved motifs of class A
-lactamases (1). Moreover, our data confirmed that
the P. stuartii VR-1 enzyme is a natural
TEM-1-derived -lactamase with the following mutations: G39K, L51P, E104K, and R164S. The mutation L51P
is a unique characteristic of the P. stuartii
VR-1 enzyme compared to TEM-2, TEM-3, TEM-7, and TEM-18 enzymes that
have the other mutations. This mutation is also present on the
chromosomally encoded -lactamase PER-1 (17); but at
the present its role is not yet explored.
The Leu-51 residue lies on the beta sheet S1, just a few residues
before beta sheet S2. The L51 side chain is in the vicinity of R191,
L194, and T195 belonging to helix H8 and I260 (beta sheet S5). Thus a
substitution of P for L51 could contribute to the -lactamase
hydrolytic properties. Site-directed mutagenesis could help clarify how
this residue influences -lactamase activity.
Comparison of the catalytic efficiency with TEM-1
-lactamase showed a marked diminution of the
kcat/Km ratio relatively
to some oldest -lactams, while the same ratio was relatively
higher for the P. stuartii enzyme, especially for
cefuroxime, cefotaxime, ceftazidime and aztreonam. The higher
ratio of kcat/Km is due to the higher affinity for cefotaxime and cefuroxime, and the same
behavior was found for aztreonam and ceftazidime. The high hydrolytic
activity against ceftazidime and a greater degree of resistance to
ceftazidime than cefotaxime confirm that, as reported for TEM-8
(21), the mutation R164S leads to an increase of resistance to ceftazidime. Hydrolysis of desacetyl-cefotaxime, a cephalosporin lacking a good leaving group in the C-3 position, was no different from
that for cefotaxime with TEM-60. A completely different behavior was found for carumonam; for this monobactam a 35-fold reduction of the
kcat was measured with respect to the aztreonam.
This finding could prompt the investigation of structural analogs of
monobactams that could be used with ESBL producing strains.
The comparative analysis of mechanism-based serine-active
-lactamase inhibitors confirmed the partial sensitivity of this -lactamase to these compounds. -IP leads to the complete
inactivation of the enzyme; no enzyme activity recovery occurred after
dilution of the complex in a good substrate such as nitrocefin. The
high acylation efficiency value
(k+2/K = 260,000 M1 s1) was similar to that reported for
other class A -lactamases (8). Clavulanic acid,
although showing a high k+2/K value and a low Ki value (0.8 µM), was hydrolyzed by
the enzyme. This could explain the MICs reported for the combination
ampicillin-clavulanic acid; clavulanic acid reduces at least 10-fold
the MIC but is not able to completely restore susceptibility of the
strain. Tazobactam was able to restore piperacillin sensitivity in the
P. stuartii VR-1 wild type and also in E. coli HB101 harboring the pVR-1 plasmid. The P. stuartii TEM-60 -lactamase, as suggested by Bush and Jacoby (6), confirms that additional combinations of mutations
could play a key role in the future dissemination of antibiotic
resistance.
| |
ACKNOWLEDGMENTS |
This work was in part supported by the Consiglio Nazionale delle
Ricerche (CNR, Italy) and by the Ministero dell'Università e
della Ricerca Scientifica e Tecnologica (MURST, Italy).
We thank Moreno Galleni (CIP, University of Liège, Liege,
Belgium) for N-terminal amino acid sequence determination. We also thank C. Bianchi, Wyeth-Lederle, Italy, for his interest in this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dipartimento di
Scienze e Tecnologie Biomediche Univ. degli Studi dell'Aquila
Loc. Coppito 1-67010, L'Aquila, Italy. Phone: 39 862 433455. Fax: 39 862 433433. E-mail: Amicosante{at}aquila.infn.it.
| |
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Antimicrobial Agents and Chemotherapy, June 1998, p. 1459-1462, Vol. 42, No. 6
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
