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Antimicrobial Agents and Chemotherapy, December 2001, p. 3591-3594, Vol. 45, No. 12
Laboratoire de Bactériologie,
Hôpital Universitaire du Bocage, 21034 Dijon
Cedex,1 and Unité FRE 2125, CNRS,
UBO, MNHN, 29000 Quimper,2 France
Received 20 February 2001/Returned for modification 29 May
2001/Accepted 28 August 2001
TEM-89 (CMT-3) is the first complex mutant Inhibitor-resistant TEM (IRT)
Two strains of P. mirabilis were isolated from the same
blood culture of a paraplegic and diabetic patient who had chronically infected wounds and who was hospitalized in the Nephrology Unit of the
University Hospital in Dijon, France. The first one, strain Pm 631, was
resistant to amoxicillin-clavulanate, whereas the second one, strain Pm
631SE, was susceptible to amoxicillin-clavulanate. The production of an
ESBL was easily detected in Pm 631SE by a double-disk synergy test
(13), but Pm 631was negative by this test. Analysis of
chromosomal DNA by pulsed-field gel electrophoresis after digestion
with SmaI as described previously (17) revealed that the two strains were identical (data not shown). Analytical isoelectric focusing (14) revealed that Pm 631 produced a
Conjugation experiments were performed with E. coli
K-12 C600, which is resistant to sodium azide, and transconjugants were selected at a frequency of 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3591-3594.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
TEM-89
-Lactamase Produced by a Proteus
mirabilis Clinical Isolate: New Complex Mutant (CMT 3) with
Mutations in both TEM-59 (IRT-17) and TEM-3
ABSTRACT
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Abstract
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References
-lactamase produced
by a clinical strain of Proteus mirabilis (strain Pm
631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM -lactamase IRT-17): Ser-130 to Gly. TEM-89
hydrolyzed penicillins to the same extent that TEM-3 did but
lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.
TEXT
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Abstract
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References
-lactamases are mostly produced by strains of Escherichia
coli (12, 21), but some of them have been described
in Klebsiella spp. and Proteus mirabilis clinical strains (2, 3, 6, 15). Until now, 23 enzymes of this type
have been reported (G. Jacoby and K. Bush, Amino acid sequences for
TEM, SHV, and OXA extended-spectrum -lactamases,
http://www.lahey.org//studies/webt.htm, 2000), and they are widely
described in strains responsible for community-acquired infections
(11). The chimera enzymes which associate two types of
mutations, one set of which is encountered in extended-spectrum
-lactamases (ESBLs) and the other set of which is encountered in
inhibitor-resistant -lactamases, are more unusual. Two such
so-called complex mutants have been reported in the TEM family: TEM-50
(CMT-1) in a strain of E. coli and TEM-68 (CMT-2) in
Klebsiella pneumoniae (8, 22). Complex mutant SHV-10 has been isolated from a strain of K. pneumoniae
(19). In the present work we describe a novel one: TEM-89
produced by a clinical isolate of P. mirabilis.
-lactamase of pI 6.28 and that Pm 631SE produced a -lactamase of
pI 6.3 (data not shown). A plasmid of about 25 kb was extracted
from both strains by the method of Birnboim and Doly (4).
2 on agar
containing sodium azide (128 mg/liter) and netilmicin (8 mg/liter). The
restriction patterns obtained after digestion of the plasmid with
EcoRI were very similar in Pm 631, Pm 631SE, and the
transconjugants. The MICs of the -lactams tested for the
clinical strains and their transconjugants (Tc), E. coli Tc 443 producing TEM-59 (IRT-17), and E. coli K-12 C600, which
is resistant to sodium azide, are reported in Table
1. Pm 631 was more resistant to
piperacillin than to ticarcillin, which is somewhat unusual for
IRT -lactamase-producing strains, but inhibitors were more efficient
when they were combined with piperacillin (lowering the MICs by 3 or
more dilutions). The synergy was much weaker in E. coli Tc
631SE. Permeability and binding to the beta-lactam targets were perhaps
different in the original host and the transconjugants; this could
explain the differences in the MICs.
TABLE 1.
MICs for P. mirabilis clinical isolates,
E. coli K-12 C600, and the transconjugants producing TEM-89,
TEM-3, and TEM-59
PCR analysis was performed as described previously with plasmid
DNA extracted from strain Pm 631, strain Pm 631SE, and their transconjugants with primers GOU1 (forward primer;
5'-ATAAAATTCTTGAAGACGAAA-3'), SIE2 (reverse primer;
5'-AAAACTCTCAAGGATCTTACC-3'), J (forward primer;
5'-CTTATTCCCTTTTTTGCGGC-3'), and E (reverse primer;
5'-GGTCTGACAGTTACCAATGC-3') (7) at positions
5, 380, 236, and 1079, respectively, of the blaTEM gene, according to Sutcliffe
(25). Both strands of the PCR products were sequenced with
an Applied Biosystems 373A sequencer according to the manufacturer's
instruction. The coding region of the
blaTEM-89 gene differed from that of the
blaTEM-2 gene (10) by three
mutations: a G-to-A change at position 512, an A-to-G change at
position 590, and a G-to-A change at position 914. Thus, the
blaTEM-89 gene differed from the
blaTEM-3 gene by a single mutation at
position 590. The amino acid sequences of the enzymes deduced as
described by Ambler et al. (1) revealed that Pm
631SE produced TEM-3, whereas Pm 631 produced the new enzyme, TEM-89
(Lys-39, Lys-104, Gly-130, and Ser-238). TEM-89 represents a new
variant of complex mutant -lactamase which combines mutations
encountered in the TEM-3 ESBL (Lys-39, Lys-104, Ser-238) with mutations
encountered in TEM-59 (Lys-39, Gly-130), which belongs to the IRT
-lactamase family (3, 20, 24). The promoter sequence of
blaTEM-89 was identical to that of
blaTEM-2 except at position 147, where
there is a T residue for blaTEM-2 and an A
residue for blaTEM-89.
The TEM-2, TEM-3, TEM-59, and TEM-89 -lactamases were
purified to homogeneity by previously described methods
(3). After precipitation with ammonium sulfate the enzyme
was submitted to gel filtration chromatography on Toyopearl HW-50 resin
(fractionation range, 500 to 80,000; Sigma). Then, the active fractions
were pooled, dialyzed, concentrated, and purified by ion-exchange
chromatography (fast-performance liquid chromatography) with
MonoQ HR 5/5 resin (Pharmacia) (8, 14, 16). The purified
enzymes were homogeneous, as shown by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (purity, 
97%). Kinetic
constants (Table 2) were determined by computerized microacidimetry (14) at pH 7 and 37°C in
0.1 M NaCl solution.
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TEM-89 was characterized by low kcat
values for all antibiotics, and these values were comparable to those
of TEM-3 for penicillins. Km values for
penicillins remained low. TEM-89 had low levels of hydrolytic activity
for cephalothin and cephaloridine and had lost all detectable
hydrolytic activity for cefuroxime, cefotaxime, ceftriaxone, and
ceftazidime. In terms of
kcat/Km,
TEM-89 had an efficiency comparable to that of TEM-59. The
concentrations of -lactamase inhibitors required to attain
50% enzyme inhibition (IC50s) were determined
after incubation of the enzyme and the inhibitor for 10 min at
37°C. The reporter substrate was benzylpenicillin, which was used at
a concentration of 1 mM. The values determined for TEM-89 were very
high and were very similar to those for TEM-59, whereas TEM-3 was
extremely susceptible (Table 3).
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Two enzymes which bear the amino acid substitutions of an ESBL
associated with substitutions of an IRT -lactamase have been described previously. The first one, TEM-50 (CMT-1), associates mutations from the TEM-17 ESBL (Lys-104) plus those from TEM-35, IRT-4
(Leu-69, Asp-276) (22). The second one is TEM-68, which combines mutations from the TEM-47 ESBL (Ser-238, Lys-240, Met-265) and
an IRT (Leu-275) (9). Surprisingly, TEM-50 and TEM-68 have conserved a capacity to hydrolyze cephalosporins associated with decreased inhibitor efficacy. In the case of TEM-89, the enzyme lost
almost all activity against cephalosporins and was highly resistant to
inhibitors, as was the case for TEM-59. From this point of view, TEM-89
can be compared with SHV-10, the first inhibitor-resistant ESBL and the
single complex mutant from the SHV family (19). SHV-10
bears the mutations of the SHV-9 ESBL associated with the Ser-to-Gly
change at position 130. A similar situation was also observed for
IRKO-1, an inhibitor-resistant OXY-2-derived -lactamase produced by Klebsiella oxytoca, which also had Gly-130
(23). SHV-10, IRKO-1, and TEM-89 have kept their abilities
to hydrolyze penicillins but have lost nearly all activity against
cephalosporins compared with the activities of their parental
-lactamases (SHV-9, OXY-2, and TEM-3, respectively). It
therefore appears that the mutation Ser-130 to Gly in class A
-lactamases can confer a high level of resistance to -lactamase
inhibitors and can lead to a reduction in the activity of the enzyme.
A broad diversity of TEM mutants has been described in P. mirabilis species, but the frequency of occurrence of TEM-2 is
high (5). The only two IRT -lactamases which have been
characterized until now in P. mirabilis (TEM-44 [IRT-12]
and TEM-65 [IRT-16]) were derived from TEM-2
(18). Our results are consistent with these observations:
TEM-89 is also derived from TEM-2. This new enzyme has probably evolved
from TEM-3 by a single amino acid substitution: Ser-130 to Gly.
The patient from whom strain Pm 631 was isolated received amoxicillin-clavulanate 2 weeks before he developed septicemia and had received cefotaxime 2 months earlier. The concentrations of the antibiotics in the large, chronically infected wounds were probably too low, thereby allowing the selection of Pm 631SE producing TEM-3 and of Pm 631 producing TEM-89.
TEM-89 conferred resistance to -lactamase inhibitors, but the
TEM-89-producing strain remained susceptible to the
broad-spectrum cephalosporins widely used in our hospital. That could
explain why the strain did not spread. Nevertheless, such complex
mutants of clinical strains could reflect a new direction in the
evolution of the enzyme. The benefit for the TEM-89-producing
strains remains unclear, but the phenomenon should be taken into account.
Nucleotide sequence accession number. The GenBank accession number for blaTEM-89 is AY039040.
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ACKNOWLEDGMENTS |
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Claudine Quentin (Bordeaux, France) is acknowledged for the gift of strain E. coli Tc 443 producing TEM-59.
C.N. and S.M. have contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Bactériologie, Hôpital Universitaire du Bocage, BP 1542, 21034 Dijon Cedex, France. Phone: 33-3 80 29 32 60. Fax: 33-3 80 29 36 67. E-mail: catherine.neuwirth{at}chu-dijon.fr.
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Antimicrobial Agents and Chemotherapy, December 2001, p. 3591-3594, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3591-3594.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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