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Antimicrobial Agents and Chemotherapy, April 1998, p. 879-884, Vol. 42, No. 4
Microbiology Department,
Received 4 August 1997/Returned for modification 16 December
1997/Accepted 31 January 1998
Plasmid-mediated mechanisms, comprising TEM hyperproduction, TEM
derivative production, and OXA production, lead to
amoxicillin-clavulanic acid resistance in enterobacteria. The ability
of the single-strand conformation polymorphism (SSCP)-PCR method to
differentiate the genes encoding inhibitor-resistant Resistance to amoxicillin-clavulanic
acid appeared first in Escherichia coli isolates, then in
other species of enterobacteria, and most recently in Haemophilus
influenzae (7, 12, 13, 21, 22, 32). Four enzymatic
mechanisms for this resistance have been described in E. coli: hyperproduction of class C chromosomal However, because only cephalosporinase hyperproduction can be
indisputably detected by the classical antibiogram (resistance to both
cephalothin and cefoxitin, in addition to amoxicillin-clavulanic acid
resistance), other methods must be used to differentiate the mechanisms
involving plasmid-encoded amoxicillin-clavulanic acid resistance. Such
methods include the determination of the Reference genes.
The genes blaTEM-1A,
blaTEM-1B, and blaTEM-2,
which were expressed by E. coli C600, were used as wild-type
reference genes (15, 34). Previously described IRT-encoding
genes were also included as reference genes in the study. These genes
have been either sequenced (blaTEM-30 from
E. coli E-GUER, blaTEM-32 from E. coli 1408, and blaTEM-35 from
E. coli CF0042) or defined by oligotyping from clinical
E. coli isolates (blaTEM-33,
blaTEM-34, and from
blaTEM-36 to blaTEM-39)
(4, 6, 16, 31). The nucleotide differences which have been
defined by the gene sequence and those which have been determined only
by oligotyping are indicated in Table 1.
Primers.
Three pairs of primers were designed from the
nucleotide sequence of the blaTEM-1A gene and
were used to amplify three overlapping fragments (Table
2). The first pair of primers allowed the
amplification of a fragment including the whole-gene promoter. The
positioning of each fragment in relation to the
blaTEM gene is included in Table 1. One pair of
primers was selected to amplify an internal 609-bp fragment of
blaOXA-1 (Table 2).
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Discriminatory Detection of Inhibitor-Resistant
-Lactamases in
Escherichia coli by Single-Strand Conformation
Polymorphism-PCR
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-lactamases was
evaluated with three blaTEM primer pairs. The
blaTEM genes, which were known to be different
on the basis of their nucleotide sequences
(blaTEM-1A, blaTEM-1B,
blaTEM-2, blaTEM-30,
blaTEM-32, and
blaTEM-35), were identified as different by
their electrophoretic mobilities. The blaTEM-33, blaTEM-34,
blaTEM-36, blaTEM-37,
blaTEM-38, and
blaTEM-39 genes, whose sequence differences
have been established by oligotyping, displayed different SSCP profiles
for different fragments, suggesting genetic differences in addition to
those defined by oligotyping. Confirmed by sequencing, these additional
genetic events concerned silent mutations at certain positions and,
notably, a G
T transversion at position 1 of the 
10 consensus
sequence in blaTEM-34,
blaTEM-36, blaTEM-37,
and blaTEM-39. Applied to eight clinical
isolates of Escherichia coli resistant to
amoxicillin-clavulanic acid, the SSCP method detected TEM-1 in three
strains and TEM-30, TEM-32, and TEM-35 in three other strains,
respectively. A novel TEM derivative (TEM-58) was detected in another
strain, and the deduced amino acid sequence showed two substitutions:
Arg244Ser, which is known to confer amoxicillin-clavulanic acid
resistance in TEM-30, and Val261Ile, which has not been described
previously. The eighth strain produced an OXA -lactamase. Given the
discriminatory power and the applicability of SSCP-PCR, this method can
be proposed as a means of following the evolution of the
frequencies of the different inhibitor-resistant -lactamases.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-lactamase
(cephalosporinase), hyperproduction of plasmid-mediated TEM-1 or TEM-2,
production of inhibitor-resistant TEM (IRT), and production of a
relatively inhibitor-resistant OXA-type -lactamase (5, 8, 9,
29, 36). Epidemiological studies carried out in Europe have shown
that the frequency of each mechanism varies in different countries
(17, 33). The frequency of cephalosporinase hyperproduction
was similar in France and England, but IRT production seemed to be much
more frequent in France, whereas OXA production seemed to be more
frequent in England. Such differences may be related to the different
use of -lactam antibiotics in the two countries, and the evolution
of the incidence of the different mechanisms may influence the future
use of different -lactams in the hospital as well as in the
community. Thus, it is important to define a strategy which allows
continued observation of the frequency of the different mechanisms.
-lactamase isoelectric
point, determination of -lactamase kinetic parameters, and/or
oligotyping, but these methods are fastidious procedures and are too
time-consuming for a national epidemiological survey of
amoxicillin-clavulanic acid resistance. In the present study, the
single-strand conformation polymorphism (SSCP)-PCR technique was
evaluated to differentiate the plasmid-encoded enzymatic mechanisms of
amoxicillin-clavulanic acid resistance (25). This technique,
which is able to detect any genetic modification, i.e., a point
mutation, deletion, or insertion, should be able to differentiate wild-type blaTEM genes from IRT-encoding genes
derived from blaTEM genes by point mutations.
Moreover, the use of specific primers should allow differentiation
between blaTEM and blaOXA
genes.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Nucleotide substitutions in
blaTEM-1B, blaTEM-2, and
IRT-encoding genes in comparison with the nucleotides of
blaTEM-1A
TABLE 2.
Nucleotide sequences of the oligonucleotides used for
blaTEM and
blaOXA-1 amplification
SSCP-PCR.
Samples were prepared by suspending a freshly
grown colony in 0.5 ml of lysis buffer (20 mM Tris HCl [pH 8.3], 50 mM KCl, 0.1% Tween 20) which was heated at 94°C for 10 min. Five
microliters of this preparation was submitted to PCR in a final volume
of 25 µl. To the PCR master mixture containing 20 mM Tris HCl (pH 8.0), 100 mM KCl, 3 mM MgCl2, 400 µM (each)
deoxynucleotide triphosphate, 2.5 U of Taq DNA polymerase
(Boehringer Mannheim GmbH, Mannheim, Germany), and 25 pmol of each
primer, 0.25 µl (2.5 µCi) of radioactive [
-32P]dCTP was added. The amplification reaction
consisted of 36 cycles of 30 s of denaturation at 94°C, 30 s of hybridization at 42°C, and 60 s of extension at 72°C,
with a final extension step at 72°C for 10 min. The radioactive PCR
product was diluted 1:2 with SSCP dilution buffer (2 mM EDTA, 0.1%
sodium dodecyl sulfate), and 5 µl of the diluted product was mixed
with 5 µl of loading buffer (95% formamide, 0.05% bromophenol blue,
0.05% xylene cyanole, 50 mM EDTA). Immediately prior to loading of the
SSCP gel the samples were denatured for 15 min at 94°C, cooled on
ice, and loaded onto a nondenaturating polyacrylamide gel. The
nondenaturating polyacrylamide gel was prepared by mixing 20 ml of
acrylamide-bisacrylamide (29:1) with 80 ml of 1× TBE
(Tris-borate-EDTA). The gel was run for 4 h at 65 W with constant
cooling. After termination of the run, the gel was transferred to a
filter paper, dried, and exposed for 2 h to an X-ray film at
70°C with an intensifying screen.
Sequencing. The PCR products for sequencing were prepared as indicated above but the radioactive nucleotide was omitted. The PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN, Courtaboeuf, France) following the manufacturer's recommendations. The nucleotide sequences of the purified PCR fragments were determined with the Sequenase PCR Product Sequencing Kit (Amersham, Les Ulis, France) and by following the manufacturer's indications exactly. The sequencing reactions were run on a standard denaturating sequencing gel.
Clinical isolates. Eight clinical isolates of E. coli (isolates AP1 to AP8), obtained from Ambroise-Paré Hospital between 1993 and 1994, were studied because they were resistant to amoxicillin and amoxicillin-clavulanic acid and susceptible to cefoxitin and broad-spectrum cephalosporins by the disk diffusion test according to the recommendations of the Antibiogram Committee of the French Microbiology Society (1).
Characterization of the amoxicillin-clavulanic acid resistance in the eight clinical isolates. The MICs of amoxicillin (SmithKline Beecham, Nanterre, France), alone and associated with a fixed concentration of 2 µg of clavulanic acid (SmithKline Beecham) per ml, and piperacillin (Léderlé, St. Cloud, France), alone and associated with a fixed concentration of 4 µg of tazobactam (Léderlé) per ml, for the eight clinical isolates were measured by a dilution method on Mueller-Hinton agar with a Steers replicator device and an inoculum of 104 CFU per spot.
For each clinical E. coli isolate, crude extracts were submitted to isoelectric focusing as described previously (3), and their pIs were compared with the pI values of the following enzymes: RP4/TEM-1, pI 5.4; R111/TEM-2, pI 5.6; pUD101/TEM-30, pI 5.2 (4); and RGN238/OXA-1, pI 7.4 (26). The kinetic parameters for the enzymes, the-lactamase specific activity (in milliunits per milligram of total
protein), and Km values (in micromolar) were determined with crude extracts by computerized microacidimetry as
described previously (20). All extracts were first studied at pH 7 and 37°C in the presence of NaCl. When an OXA-type
-lactamase was suspected, a complementary set of experiments was
performed at pH 7 and 20°C and in the presence of
Na2SO4 instead of the NaCl solution, since OXA
enzymes are inhibited by chloride ions. After preincubation of the
crude extracts for 10 min at 37°C with 100 µg of clavulanic acid
per ml, the residual activity of the -lactamases was
determined in order to differentiate TEM enzymes which display a
residual activity of 
10% from IRT enzymes which display a residual
activity of >20% (11). Under these experimental conditions, it has been previously observed that OXA enzymes are highly
unstable (11).
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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SSCP-PCR of the wild-type reference genes.
In accordance with
the nucleotide sequences (Table 1), we obtained three different SSCP
profiles for fragment 1 covering 389 bp starting at position 7
(34) for the three wild-type reference genes
blaTEM-1A, blaTEM-1B, and
blaTEM-2 (Fig.
1A). This was also the case for fragment
2, which covers 426 bp starting at position 301 (Fig. 1B). For fragment
3 we obtained, as expected (Table 1), two different SSCP migration
profiles, one for both blaTEM-1 genes and one
for blaTEM-2 (Fig. 1C).
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SSCP-PCR of sequenced IRT-encoding genes. According to the nucleotide sequences of blaTEM-30, blaTEM-32, and blaTEM-35, we observed two different and specific SSCP profiles of fragments 1 of blaTEM-30 and blaTEM-35, whereas the profile for fragment 1 of blaTEM-32 was identical to that of fragment 1 of blaTEM-1A. For fragments 2 (Fig. 2) and 3, each of the IRT-encoding genes displayed a specific profile that was in accordance with the previously determined nucleotide sequence.
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SSCP-PCR of oligotyped IRT-encoding genes. No data were available for the nucleotide sequences corresponding to fragment 1 of the oligotyped IRT encoding genes. By the SSCP method, we found that fragment 1 of blaTEM-33 was clearly different from the fragments 1 of all reference genes included in this study. For blaTEM-34, blaTEM-36, blaTEM-37, and blaTEM-39, the SSCP migration profile of fragment 1 differed slightly from that of fragment 1 of blaTEM-30, whereas the profile of fragment 1 of blaTEM-38 differed slightly from that of fragment 1 of blaTEM-1B.
According to oligotyping analysis, at least two SSCP profiles different from those of the three blaTEM genes and the three sequenced IRT-encoding genes could be expected for fragment 2: one for blaTEM-34, blaTEM-36, and blaTEM-38 and one for blaTEM-39. On the other hand, the SSCP profile of fragment 2 of blaTEM-33 could be expected to be identical to that of fragment 2 of blaTEM-35, and that of fragment 2 of blaTEM-37 could be expected to be identical to that of fragment 2 of blaTEM-32. In fact, we obtained specific fragment 2 profiles for five of the six oligotyped IRT-encoding genes because it was shown that blaTEM-33 differed from blaTEM-35, blaTEM-37 differed from blaTEM-32, and blaTEM-38 differed from both blaTEM-34 and blaTEM-36. The fifth specific profile corresponded to blaTEM-39, as expected (Fig. 3A).
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Characterization of the amoxicillin-clavulanic acid resistance in
the eight clinical isolates. (i) Kinetic parameters of the
-lactamases.
The amoxicillin-clavulanic acid resistance was
evaluated first by the disk diffusion test for the eight clinical
isolates and was confirmed, as indicated in Table
3, by the MICs (MICs, >16 µg/ml).
Compared to the MICs of amoxicillin, those of piperacillin were lower,
and in association with 4 µg of tazobactam per ml, piperacillin was
active against six of the eight clinical isolates (MIC, <8 µg/ml)
(Table 3). The -lactamases of strains AP1, AP2, AP3, and AP5
comigrated with TEM-1 (pI 5.4) and that of strain AP4 comigrated with
OXA-1 (pI 7.4). The three remaining strains, AP6 to AP8, produced a
-lactamase with a pI of 5.2 (Table 3). According to the
Km values for penicillin G, the clinical
isolates could be separated into three groups:
Km of <10 for strains AP4 and AP5,
Km of ~25 for strains AP1 to AP3, and
Km of >100 for strains AP6 to AP8. Among the
-lactamases which displayed a pI of 5.4, three (those from strains
AP1 to AP3) showed a very low residual activity (<10%) in the
presence of clavulanic acid, suggesting the production of TEM enzymes,
whereas one (that from strain AP5) kept a relatively high residual
activity (56%), similar to those which were measured for the three
-lactamases having pIs of 5.2 (Table 3). These findings suggested
that the clinical isolates AP6 to AP8 and AP5 produced IRT
-lactamases. The necessity of using Na2SO4
instead of NaCl solution to measure the -lactamase activity of the
enzyme produced by strain AP4 and the fact that this enzyme was highly
unstable under the experimental conditions established for measurement
of the residual activity suggested the presence of an OXA -lactamase
in this strain.
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(ii) SSCP-PCR.
Strain AP4, which was expected to produce an
OXA-1 -lactamase, did not yield an amplification product with the
TEM-derived primers in repetitive experiments but yielded products with
the specific primers of blaOXA-1 (data not
shown). As indicated in Table 4, strains
AP1 to AP3, whose -lactamases displayed a very low residual activity
in the presence of clavulanic acid, were shown to produce TEM enzymes
by the SSCP method. The bla gene of strain AP1 could be
identified as blaTEM-1B, and that of strain AP3
could be identified as blaTEM-1A. An unusual
situation was observed with the bla gene of strain AP2
because the SSCP profiles of its fragments 1 and 2 corresponded
to those of blaTEM-1A and blaTEM-1B, respectively. The four clinical
strains which showed an important residual -lactamase activity in
the presence of clavulanic acid could be expected to
produce IRT enzymes according to the SSCP results, namely, TEM-32 for
strain AP5, TEM-35 for strain AP6, and TEM-30 for strain AP8. The
amplification product obtained for fragment 3 of the
blaTEM gene expressed by strain AP7 showed a
particular migration profile that did not correspond to the profiles of
any of the reference genes, while those of fragments 1 and 2 showed the
same electrophoretic mobility as fragment 1 of
blaTEM-33 and fragment 2 of
blaTEM-1B, respectively.
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Nucleotide sequencing.
Given the great diversity of SSCP
profiles of oligotyped IRT-encoding genes, sequencing of amplified
fragments 1 and 2 and some of fragment 3 was carried out and showed
that different silent point mutations occurred. As indicated in Table
5 and in comparison with the
blaTEM-1A gene sequence, these mutations
consisted of a CT transition at position 32 for
blaTEM-38, a GT transversion at
position 162 for blaTEM-34,
blaTEM-36, blaTEM-37, and
blaTEM-39, an AG transition at position 175 for blaTEM-33 and
blaTEM-38, a CT transition at
position 226 for blaTEM-38, an AG transition at position 346 for blaTEM-34,
blaTEM-36, blaTEM-37, and
blaTEM-39, a CT transition at
position 436 for all the blaTEM-derived genes, a
GT transversion at position 604 for
blaTEM-33, a TC transition at position 682 for blaTEM-34, blaTEM-36,
and blaTEM-37, and a GA transition at
position 925 for blaTEM-36. By sequencing the blaTEM gene of clinical isolate AP7, for which
the SSCP analysis was not able to define the type of IRT produced,
three nucleotide changes were observed (Table 5). The first one
consisted of an AG transition at position 175, as was found in
blaTEM-33 and blaTEM-38.
The second one concerned a CA transversion at position 929, leading
to the amino acid substitution arginineserine at position 244, while
the third one was a GA transition at position 980, leading to the
amino acid replacement valineisoleucine at position 261 (numbering
of Ambler et al. [2]).
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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The amoxicillin-clavulanic acid resistance which appeared for the
first time about 10 years ago in E. coli has now been
observed in Klebsiella pneumoniae, Proteus
mirabilis, Salmonella typhimurium, Shigella
flexneri, and H. influenzae (7, 12, 13, 21, 22,
32). Contrary to E. coli, the recently involved
species do not possess a chromosomal cephalosporinase whose
hyperproduction results in amoxicillin-clavulanic acid resistance.
Subsequently, strains of these species have acquired plasmid-encoded
mechanisms, comprising TEM hyperproduction and IRT production, which
were first identified in E. coli (7, 13, 21, 32).
OXA production, which is the third plasmid-encoded mechanism, seems to
be limited to E. coli. The reasons why bacteria,
particularly E. coli, have developed so many mechanisms are
unknown. However, we can observe differences in the incidences of the
mechanisms in individual countries and differences in the spectrum of
-lactam resistance according to the amoxicillin-clavulanic acid
resistance mechanism (17, 28, 30, 33).
Because plasmid-encoded mechanisms involved either completely different
genes (blaTEM, blaOXA) or
blaTEM-derived genes (IRT-encoding genes), the
PCR-based SSCP method seemed suitable to us for the rapid
identification of the genes responsible for amoxicillin-clavulanic acid-resistant phenotypes. In fact, using this method we were able to
differentiate blaTEM genes which were known to
be different on the basis of their nucleotide sequences. SSCP-PCR was
able to indicate the occurrence of further genetic events in the genes for which only short stretches were oligotyped, and these were confirmed by sequencing. Thus, a GT transversion at position 1 of the 10 consensus sequence was identified in
blaTEM-34,
blaTEM-36, blaTEM-37,
and blaTEM-39. Such a transversion, which is
known to be at the origin of a higher level of enzyme production, was recently described in the blaTEM-1 gene
expressed by an S. flexneri isolate resistant to
amoxicillin-clavulanic acid and in IRT-encoding genes, notably,
blaTEM-45 (10, 32). Our study shows,
as has already been described by Caniça et al. (10),
that there is a great molecular diversity in the
blaTEM genes encoding IRT but that some
mutations are very frequent in such genes, notably, A346G, C436T, and
T682C transitions. Although we have observed the G925A transition only
in blaTEM-36, this mutation also seems to be
frequent in IRT-encoding genes according to the study of Caniça
et al. (10). Inversely, the C32T and G604T transitions which
we identified in blaTEM-38 and
blaTEM-33, respectively, seem to be rare in
IRT-encoding genes (10).
The eight amoxicillin-clavulanic acid-resistant clinical isolates were
studied by both enzymatic methods and SSCP-PCR. The results obtained by
each method were in perfect concordance. Enzymes determined by the
SSCP-PCR technique to be TEM enzymes showed by enzymatic analysis the
typical Km values and residual activity of TEM
enzymes (9, 11). Those enzymes determined by the SSCP-PCR method to be IRT enzymes displayed kinetic parameters which have previously been published for such enzymes (10).
Nevertheless, the SSCP-PCR method, which is more rapid and whose
performance is less fastidious, allowed us to characterize precisely
the type of the IRT enzymes. The presence of TEM-32 in strain AP5,
suggested by the Km value, which is known to be
particularly low for this enzyme, was definitely proven by SSCP-PCR
(11). Moreover, SSCP-PCR allowed us to detect a novel
blaTEM gene (blaTEM-58)
in clinical isolate AP7. The deduced amino acid sequence showed a
previously unknown amino acid substitution, Val261Ile, in addition to
the already known amino acid substitution Arg244Ser, which confers amoxicillin-clavulanic acid resistance in TEM-30 (8). By the two crystal structures of TEM-1 -lactamase which are available (entries 1BTL and 1XPB; Protein Data Bank, Brookhaven National Laboratory, Brookhaven, N.Y. [14, 19]), Val261 was
shown to be located on sheet s-5 in a group of four hydrophobic
residues: Ile, Val, Val, and Ile, starting at position 260. Moreover,
the side chain of Val261 is in the close vicinity of at least Leu221 (helix h10), Leu250, and Leu286 (helix h11), which form a highly hydrophobic pocket. The role of the substitution Val261Ile on the
enzymatic activity of this TEM-derived enzyme should be determined in
further studies.
We can also note that the blaTEM gene of strain AP7 showed the same migration profile as fragment 1 of blaTEM-33, for which we demonstrated a guanine at position 175. Such a mutation is extremely rare, because it is apparently present only in blaTEM-1B, blaTEM-5, blaTEM-6b, and blaTEM-38; however, it is present in association with a thymine at position 216 in these four genes (15, 18, 27).
Three clinical isolates (isolates AP1 to AP3) were shown by the
SSCP-PCR method and kinetic parameters to produce TEM enzymes. Three
molecular mechanisms have previously been described to explain the
amoxicillin-clavulanic acid-resistant phenotype related to wild-type
blaTEM genes: up-mutation in the regulatory
region of the gene, the presence of multiple copies of the plasmid, and a gene dose effect (23, 29, 32, 35). Because we did not observe for the TEM enzymes produced by strains AP1 to AP3 specific SSCP-PCR profiles of fragment 1 covering the promoter region, an
up-mutation cannot be assumed to be responsible for the
amoxicillin-clavulanic acid resistance. However, AP2 has been shown by
determination of kinetic parameters and SSCP-PCR to yield a higher
level of -lactamase activity, and its -lactamase gene has been
shown to have an unusual structure.
In conclusion, this study shows that the SSCP-PCR method is a suitable tool for rapidly screening for the different plasmid-encoded mechanisms which are known so far to confer amoxicillin-clavulanic acid resistance in enterobacteria. We have shown that this method can also be used to identify the different IRT enzymes and detect novel enzymes of this type. However, because it is a comparative technique and because IRT-encoding genes have a great molecular diversity, a large number of reference genes must be used. To decrease the number of reference genes the amplified fragments could be shortened and concentrated on mutations leading to relevant amino acid substitutions, as was shown for SHV derivatives (24).
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ACKNOWLEDGMENTS |
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This work received financial support from the Beecham Institute, Paris, France.
We thank Danielle Sirot for providing us with strains producing the oligotyped IRT enzymes and Patrice Courvalin for providing us with blaTEM-1b and blaTEM-2.
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FOOTNOTES |
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* Corresponding author. Mailing address: Microbiology Department, Hôpital Ambroise-Paré, Université Paris V, 9 avenue Charles de Gaulle, 92100 Boulogne-Billancourt, France. Phone: 33-1-49 09 55 40. Fax: 33-1-49 09 59 21. E-mail: marie-helene.nicolas-chanoine{at}aph.ap-hop-paris.fr.
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Abstract
Introduction
Materials & Methods
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Discussion
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Antimicrobial Agents and Chemotherapy, April 1998, p. 879-884, Vol. 42, No. 4
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