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Antimicrobial Agents and Chemotherapy, April 2001, p. 1278-1280, Vol. 45, No. 4
Laboratoire de Bactériologie,
Faculté de Médecine, 63001 Clermont-Ferrand
Cedex,1 and Laboratoire de
Bactériologie-Hygiène, Faculté de Médecine
Pitié-Salpêtrière, 75634 Paris Cedex
13,2 France
Received 27 July 2000/Returned for modification 28 October
2000/Accepted 1 January 2001
The sequences of the blaTEM genes encoding
TEM-92 in Proteus mirabilis and Providencia
stuartii isolates were determined and were found to be identical.
Except for positions 218 (Lys-6) and 512 (Lys-104), the nucleotide
sequence of blaTEM-92 was identical to that of
blaTEM-20, including the sequence of the
promoter region harboring a 135-bp deletion combined with a G-162 The extended-spectrum
beta-lactamases (ESBLs) observed in Proteus mirabilis
and rarely in Providencia stuartii are often difficult to
detect. Their detection requires a modified synergy test
(16) because they are usually produced at low levels
(5).
We report here on two strains, P. mirabilis CF 529 and
P. stuartii 1606 (Table 1),
isolated in 1998 from the urinary tracts of two patients hospitalized
at Clermont-Ferrand Hospital and Pitié-Salpêtrière
Hospital in Paris, France, respectively. These isolates, in particular,
P. mirabilis CF 529, were noticed because of their high
level of resistance to cefotaxime and the results of the synergy test,
which was unequivocally positive with oxyiminocephalosporins and
clavulanic acid.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1278-1280.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
New TEM Variant (TEM-92) Produced by Proteus
mirabilis and Providencia stuartii
Isolates
ABSTRACT
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substitution. The deduced amino acid sequence of TEM-92 differed from
that of TEM-52 by the presence of a substitution (Gln-6Lys) in the
peptide signal.
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TABLE 1.
ESBL-producing clinical strains used in this study
MICs were determined by a dilution method on Mueller-Hinton agar with an inoculum of 104 CFU per spot (6). Antibiotics were provided as powders by SmithKline Beecham Pharmaceuticals (amoxicillin, ticarcillin, and clavulanate), Eli Lilly, Paris, France (cephalothin, moxalactam), Roussel-Uclaf (cefotaxime, cefpirome), Glaxo-Wellcome Research and Development (ceftazidime), Bristol-Myers Squibb (aztreonam, cefepime), and Merck Sharp & Dohme (cefoxitin).
Table 2 lists the MICs of aztreonam,
cefoxitin, cefotaxime alone and combined with clavulanate at a fixed
concentration of 2 µg/ml, ceftazidime, cefepime, cefpirome, and
moxalactam for the two strains producing TEM-92, P. mirabilis CF 529 and P. stuartii 1606. They were
compared with those for P. mirabilis CF 39 and P. mirabilis CF 669, two strains of the same species that produce TEM-3 and TEM-66, respectively (3). These three ESBLs,
TEM-92, TEM-3, and TEM-66, have the same mutations, Glu-104Lys and
Gly-238Ser, implicated in the extension of the spectrum of activity.
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P. mirabilis CF 529 and P. stuartii 1606 differed from P. mirabilis strains CF 39 (TEM-3) and CF 669 (TEM-66) in that the first two strains had higher levels of resistance to cefotaxime (MICs for CF 529 and 1606, 64 and 32 µg/ml, respectively; MICs for CF 39 and CF 669, 2 and 8 µg/ml, respectively). The ceftazidime MIC was 8 µg/ml for all strains except P. mirabilis CF 39 (TEM-3), for which it was 0.5 µg/ml.
Aztreonam MICs (
2 µg/ml) remained low. The four strains were
susceptible to moxalactam (MICs, 0.25 µg/ml) and cefoxitin (MICs,
8 µg/ml), unlike Klebsiella pneumoniae NEM 865 producing TEM-52 (14). Clavulanate restored the impaired activity of
cefotaxime. Analytical isoelectric focusing was performed with crude
lysates from polyacrylamide gels containing ampholines with a pH range of 3.5 to 10.0, as described previously (15). Both of the
clinical strains (P. mirabilis CF 529 and P. stuartii 1606) produced a beta-lactamase with a pI of 6.0.
Several transfer experiments were tried with mutants of Escherichia coli HB 101, E. coli C600, and P. mirabilis ATCC 29906 as recipient strains. Only one transconjugant strain was obtained by mating P. mirabilis CF 529 with rifampin-resistant P. mirabilis ATCC 29906. The phenotype of resistance to aminoglycosides (kanamycin, tobramycin, and gentamicin) observed by the diffusion method was cotransferred with the ESBL phenotype.
Plasmids from clinical isolates and the transconjugant were extracted by the method of Kado and Liu and electrophoresed at 250 V for 4 h in a 0.7% agarose gel. They were blotted onto Nytran filters.
Hybridization with a TEM probe obtained by PCR with primers TEM-A
(5'-TAAAATTCTTGAAGACG-3') and TEM-B
(5'-TTACCAATGCTTAATCA-3') (3) and labeled by
random priming (DNP-DNA labeling kit; Appligen Oncor, Illkirch, France)
showed that the TEM gene resided on a 50-kb plasmid of P. mirabilis CF 529 and P. stuartii 1606 (data not shown).
In K. pneumoniae NEM 865 (TEM-52), 
-lactam resistance was
transferred alone, without aminoglycoside resistance genes, and was
located on a 13.5-kb plasmid. Great variability in plasmid size in
TEM-52-producing E. coli and K. pneumoniae
strains (between 71 and 100 kb) was also observed in Korea
(12). PCR amplification and DNA sequencing of the promoter
and coding regions of the blaTEM-92 gene were
performed as described previously (3).
The nucleotide sequences of the two blaTEM genes
from P. mirabilis CF 529 and P. stuartii 1606 were identical. Analysis of the deduced protein sequence compared to
that of blaTEM-1 showed four amino acid
substitutions, Gln-6Lys, Glu-104Lys, Met-182Thr, and
Gly-238Ser. This protein sequence is identical to that of TEM-52
reported previously (12, 14) except for the substitution Gln-6Lys in the signal peptide. We suggest that the enzyme be designated TEM-92.
In Table 3, which shows the positions
known to allow discrimination of blaTEM genes
(10), the sequence of blaTEM-92 is compared with those of blaTEM-52 genes reported
previously (12, 14) and with that of
blaTEM-20 (2).
blaTEM-92 and blaTEM-20 have the same promoter with a 135-bp deletion between nucleotides 22 and 158 combined with the mutation G-162T. This combination of the
deletion and the mutation resulted in a promoter sequence that
contained TTGAA for the 
35 region and TACAAT for the 10 region and that is thereby of closer similarity to the consensus promoter sequence, which conferred a strong promoter (2).
No deletion was observed in the promoters of
blaTEM-52 genes reported previously, in which
strong promoters, P4 (G-162T) for K. pneumoniae NEM 865 (14) and Pa + Pb (C-32T) for K. pneumoniae KMK 107 (BLAST program, National Center for
Biotechnology Information, accession number AF 027 199 [http://www.ncbi.nlm.nih.gov/BLAST/]), were observed.
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If we consider the silent mutations at positions 226, 346, 436, 604, 682, and 925 known to allow the discrimination of
blaTEM genes, blaTEM-92
is related to blaTEM-2 (10). In
comparison, blaTEM-52 from K. pneumoniae NEM 865 was identical to
blaTEM-1a, and
blaTEM-52 from K. pneumoniae KMK 107 was identical to blaTEM-1b. Compared to the
blaTEM-15 gene (10), we could
designate the gene from K. pneumoniae NEM 865 blaTEM-52a and the gene from K. pneumoniae KMK 107 blaTEM-52b. If
we consider the sequences of the promoter and coding regions,
blaTEM-92 was identical to
blaTEM-20 (2) except at two
positions, 218 and 512 (Glu-6Lys and Glu-104Lys, respectively),
and both genes belong to the
blaTEM-2-like group (4).
Among ESBLs, TEM-52 was hitherto not frequent except in Korea, where it was observed in an epidemic (12). It was first reported in a K. pneumoniae strain isolated from a girl originating from Athens, Greece (14), and a strain was observed in France (8). These TEM-52 and TEM-92 enzymes, which harbor the same critical substitutions involved in the extension of the beta-lactamase spectrum at positions 104, 182, and 238, differed by their geographical locations, the species implicated, the sizes of the plasmids carrying the blaTEM gene, and their nucleotide sequences. The occurrence of these enzymes could be due to a convergent evolution from different blaTEM genes (11).
A wide variety of TEM-type ESBLs were observed in P. mirabilis. Some of them (TEM-8, TEM-10, TEM-24, TEM-26
[3], and TEM-87) confer a ceftazidimase resistance
phenotype, whereas others (TEM-3, TEM-21, TEM-66 [3] and
TEM-72 [13]) confer a cefotaximase resistance phenotype.
The latest, TEM-87, has the same mutation (Gln-6Lys) in the peptide
signal as TEM-92. This diversity is perhaps related to the variety of
the ecological niches of this species, which is rarely implicated in
nosocomial infections (7). In P. stuartii,
unlike in P. mirabilis, ESBLs were rarely reported (TEM-24
in our hospital [unpublished data] and TEM-60 [9]). That could be due to the level of expression in this species. A lower
level of expression of TEM-92 was observed in P. stuartii 1606 than in P. mirabilis CF 529. For both species we could
suspect the existence of a factor that leads to weak expression of
-lactam resistance, despite the presence of a strong promoter
(3).
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ACKNOWLEDGMENTS |
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We thank Rolande Perroux, Marlène Jan, and Dominique Rubio for technical assistance. We are grateful to C. Poyart, who kindly provided K. pneumoniae NEM 865.
This work was supported in part by a grant from the Direction de la Recherche et des Etudes Doctorales, Ministère de l'Education Nationale de la Recherche et de la Technologie, Paris, France.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Bactériologie, Faculté de Médecine, 28, place Henri Dunant, 63001 Clermont-Ferrand Cedex, France. Phone: 33.(0)4.73.60.80.18. Fax: 33.(0)4.73.27.74.94. E-mail: Christophe.DECHAMPS{at}u-clermont1.fr.
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Antimicrobial Agents and Chemotherapy, April 2001, p. 1278-1280, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1278-1280.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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