This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bermudes, H.
Right arrow Articles by Quentin, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bermudes, H.
Right arrow Articles by Quentin, C.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, July 1999, p. 1657-1661, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Molecular Characterization of TEM-59 (IRT-17), a Novel Inhibitor-Resistant TEM-Derived beta -Lactamase in a Clinical Isolate of Klebsiella oxytoca

H. Bermudes,1 F. Jude,1 E. B. Chaibi,2 C. Arpin,1 C. Bebear,3 R. Labia,2 and C. Quentin1,*

Laboratoire de Microbiologie, UFR des Sciences Pharmaceutiques, Université de Bordeaux 2,1 and Laboratoire de Bactériologie, Hôpital Pellegrin,3 33076 Bordeaux Cedex, and UMR 175 CNRS-MNHN, 29000 Quimper,2 France

Received 16 November 1998/Returned for modification 26 February 1999/Accepted 10 May 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

A clinical isolate of Klebsiella oxytoca (Kox 443) was found to have a low-level resistance to broad-spectrum penicillins (MICs of amoxicillin and ticarcillin, 256 and 32 µg/ml, respectively), without substantial potentiation by 2 µg of clavulanic acid per ml (amoxicillin- and ticarcillin-clavulanate, 128 and 8 µg/ml, respectively), while being fully susceptible to cephalosporins and other beta -lactam antibiotics. These resistances were carried by a ca. 50-kb conjugative plasmid that encodes a single beta -lactamase with a pI of 5.6. Compared to TEM-2, this enzyme exhibited a 3- to 30-fold higher Km and a decreased maximal hydrolysis rate for beta -lactams; higher concentrations of suicide inactivators (5- to 500-fold higher concentrations giving a 50% reduction in hydrolysis) were required for inhibition. Nucleotide sequence analysis revealed identity between the blaTEM gene of Kox 443 and the blaTEM-2 gene, except for a single A-to-G change at position 590, leading to the amino acid change from Ser-130 Gly. This mutation has not been reported previously in the TEM type beta -lactamases produced by clinical strains, and the novel enzyme was called TEM-59 (alternative name IRT-17). This is the first description of an inhibitor-resistant TEM-derived enzyme in the species K. oxytoca.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Klebsiella oxytoca is an opportunistic pathogen, commonly found in the environment and in the gut. During the last decade, this organism has been increasingly isolated in patients with various pathological processes (2). In a recent study, this species accounted for 26% of all klebsiellae collected in European intensive care units (32). K. oxytoca is intrinsically low level resistant to penicillins, due to the production of small amounts of a chromosomal, constitutive, and clavulanate-susceptible penicillinase called K1 or KOXY (2). Two forms of this enzyme, OXY-1 and OXY-2, have been differentiated on the basis of their isoelectric points and gene sequences (16). In addition, about 10% of K. oxytoca strains (32, 36) are resistant or have a decreased susceptibility to all beta -lactams except for cephamycins and carbapenems (2, 17). These resistances are related to the overproduction of the chromosomal beta -lactamase, caused by point mutations in the promoter sequence of the blaOXY genes (17). Extended-spectrum beta -lactamases such as TEM-3, TEM-24 (13), or the transposon-encoded TEM-12 (21) have been only occasionally reported in this species. Finally, as other enterobacteria, K. oxytoca can produce common plasmid- and/or transposon-mediated beta -lactamases; TEM-1 is the most frequently identified (13, 36). Very recently, a K. oxytoca strain harboring an inhibitor-resistant OXY-2-derived beta -lactamase was isolated (37). However, until now, no inhibitor-resistant TEM-derived enzyme (IRT) has been described in this species. We report here the molecular characterization of a novel IRT in a clinical isolate of K. oxytoca.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains. The strain of K. oxytoca Kox 443 was isolated together with a methicillin-resistant strain of Staphylococcus aureus from the foot of a diabetic and arteritic 82-year-old woman, hospitalized in a vascular surgery unit of a university hospital of Bordeaux, France. This patient had been empirically treated with amoxicillin-clavulanic acid (3 g/24 h) over the previous 16 days for a febrile abdominal syndrome. The isolate was identified by the API 20E system and also by the Biotype 100-carbon source strips (bioMérieux, Marcy-L'Étoile, France) in order to avoid any confusion with Klebsiella planticola, the other indole-positive species of klebsiella (2, 16). A spontaneous mutant of Escherichia coli K-12 resistant to nalidixic acid and rifampin (E. coli Rifr Nalr) was used as the recipient in a transfer experiment and as the source of TEM-2 encoded by plasmid RP4.

Antimicrobial susceptibility testing. Antibiotic susceptibility patterns of the K. oxytoca Kox 443 and its transconjugant (Tc 443) were determined by a disk diffusion method (15) on Mueller-Hinton agar. MICs of various beta -lactams, alone or in combination with beta -lactamase inhibitors used at fixed concentrations (2 µg/ml for clavulanic acid, 8 µg/ml for sulbactam, and 4 µg/ml for tazobactam), were determined by an agar dilution method (15) on Mueller-Hinton medium, with an inoculum of 104 to 105 CFU per spot. The following antibiotics were provided as standard powders by the indicated laboratory suppliers: ampicillin, Bristol-Myers Squibb Laboratories; amoxicillin, ticarcillin, and clavulanic acid, SmithKline Beecham Pharmaceuticals; cephalothin, Lilly France SA; piperacillin and tazobactam, Wyeth Lederle Laboratories; and sulbactam, Pfizer Inc. Results were interpreted according to French national guidelines (14).

Isoelectric focusing. Analytical isoelectric focusing was performed as previously described (34) in polyacrylamide gels containing 0.8% ampholines with a pH range of 3.5 to 10.0. beta -Lactamase activities were detected by an iodine-starch procedure in agar gel, using benzylpenicillin (75 µg/ml) as the substrate. The pIs of the beta -lactamases produced by Kox 443 and its transconjugant were determined by comparison with the pI values of standard beta -lactamases: TEM-1 (pI 5.4), TEM-2 (pI 5.6), TEM-30/IRT-2 (pI 5.2), and TEM-3 (pI 6.3).

Kinetic studies. The beta -lactamases produced by the transconjugant Tc 443 and TEM-2 were partially purified from crude extracts by ion-exchange chromatography using AGMP-1 resin (Bio-Rad) (29). The resin was first treated with 0.1 M ammonia in water and then washed extensively with distilled water. After absorption of the extracts, elution was performed with a 0.1 M NaCl solution. The active fractions were pooled, dialyzed, and lyophilized. The Michaelis constant (Km) and the maximal hydrolysis rate (Vmax) were determined by a computerized microacidimetry assay (29), at pH 7 and 37°C in distilled water containing 85 mM NaCl. Vmax values were expressed in comparison with those of benzylpenicillin, the latter being taken as 100%. The concentrations of the inhibitors giving a 50% reduction in hydrolysis (IC50) of benzylpenicillin at 1 mM were measured after 10 min of preincubation of the enzymes with the inhibitors. The inhibition constant (Ki) was determined by a competition procedure with benzylpenicillin.

Transfer experiment. Conjugation between Kox 443 and E. coli Rifr Nalr was carried out by a broth mating procedure (15) in brain heart medium. Transconjugants were selected on Mueller-Hinton agar plates containing rifampin (100 µg/ml) and ampicillin (100 µg/ml).

Plasmid DNA analysis. Plasmid DNA from Kox 443 and its resulting transconjugant was extracted and purified with the protocol and reagents of a commercial kit (Qiagen plasmid midi kit), was then analyzed by electrophoresis in a 0.9% (wt/vol) agarose gel, and was visualized with ethidium bromide under UV light. The size of the plasmid was estimated after restriction by the endonuclease EcoRI.

Sequencing of DNA amplified by PCR. The amplification was performed with 5 ng of purified DNA plasmid from Kox 443 in a final volume of 50 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mg of gelatin per ml, 200 µM (each) deoxynucleoside triphosphate, 0.5 µM primer, and 0.25 U of Taq polymerase (Oncor-Appligène). Each sample was first subjected to a cycle of denaturation at 94°C for 5 min and then was subjected to 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 30 s, elongation at 72°C for 1 min and a final elongation step at 72°C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% (wt/vol) agarose gel. Primers used for amplification of the complete blaTEM gene were as follows: forward, A (5'-GTATCCGCTCATGAGACAATA-3'), and reverse, B (5'-TCTAAAGTATATATGAGTAAACTTGGTCTG-3'), starting, respectively, at base 148 and 1103 of the blaTEM-1 gene according to the numbering of Sutcliffe (41). The PCR products were purified for sequencing the Microspin 400 purification system (Pharmacia LKB), then they were directly sequenced on both strands by automated fluorescent sequencing, the dye terminator method (Perkin-Elmer), and six primers, including (forward) A, C (5'-GGGCAAGAGCAACTCGG-3'; 5' position: 461), and D (5'-CAGCAATGGCAACAACGTTG-3'; 5' position: 753) and (reverse) B, F (5'-CAACGTTGTTGCCATTGCTGCAG-3'; 5' position: 772), and G (5'-ACCGAGTTGCTCTTGCCC-3'; 5' position: 478).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Antibiotic resistance pattern. By the disk diffusion method, Kox 443 was resistant to amoxicillin and amoxicillin-clavulanate; exhibited an intermediate susceptibility to ticarcillin; and was susceptible to piperacillin-tazobactam, cephalothin, cefoxitin, cefuroxime, cefotaxime, ceftazidime, latamoxef, aztreonam, and imipenem. In addition, strain Kox 443 was resistant to most aminoglycosides (gentamicin, tobramycin, dibekacin, and netilmicin), chloramphenicol, sulfonamides, trimethoprim, nalidixic acid and ofloxacin. The E. coli transconjugant Tc 443 exhibited the same antibiotic resistance pattern except for ofloxacin. Results of MICs are listed in Table 1. At the fixed concentrations chosen, the beta -lactamase inhibitors lowered the MICs of penicillins by twofold dilutions or less, except for piperacillin-tazobactam (MICs decreased by 3 dilutions). Tc 443 was slightly more resistant to beta -lactams than Kox 443, and clavulanate did not exhibit synergy with amoxicillin or ticarcillin.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   MICs of beta -lactam antibiotics for the clinical isolate Kox 443, its transconjugant Tc 443, and other E. coli strains

beta -Lactamase characterization. Analytical isoelectric focusing of crude beta -lactamase extracts of Kox 443 and its transconjugant gave an identical band which comigrated with the reference enzyme TEM-2, at pI 5.6. In addition, Kox 443 expressed a beta -lactamase of pI 6.3.

Kinetic parameters (Km and Vmax) of the enzyme produced by Tc 443 compared to those of TEM-2 are given in Table 2. The Tc 443 enzyme exhibited much higher Km than TEM-2 for all beta -lactams tested: the Km values were 3 to 30 times more elevated for benzylpenicillin, ampicillin, amoxicillin, and piperacillin and were even too high to be accurately and reproducibly determined for ticarcillin and the cephalosporins. Moreover, the enzyme produced by Tc 443 hydrolyzed most penicillins somewhat more slowly than TEM-2 (2- to 7-fold decrease of the relative Vmax, except for piperacillin, which was hydrolyzed two times faster) and cephalosporins (30-fold decrease of the relative Vmax for cefoperazone): Vmax values for cephalothin and cephaloridine were inferior or equal to the lowest rates that could be measured under the assay conditions used.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Kinetic parameters of TEM-2 and TEM-59/IRT-17

Inhibition parameters (Ki and IC50) for the enzyme of Tc 443 and TEM-2 are compared in Table 3. The beta -lactamase of Tc 443 exhibited a dramatic loss of affinity for the beta -lactamase inhibitors: the Ki values were increased by a factor of 150 for sulbactam and by a factor of 1,700 to 1,800 for tazobactam and clavulanic acid. Larger quantities of inhibitors were needed for inhibiting the Tc 443 enzyme than TEM-2: the IC50s were 5-fold higher for sulbactam, 35-fold higher for tazobactam, and 500-fold higher for clavulanic acid; however, tazobactam retained significant inhibitor potency (IC50, 7 µM), 5 to 14 times more than sulbactam (35 µM) or clavulanic acid (100 µM).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Inhibition parameters of TEM-2 and TEM-59/IRT-17

Gene characterization. Upon mating K. oxytoca 443 with E. coli Rifr Nalr, amoxicillin-resistant transconjugants were selected at a frequency of 10-5. The strain Kox 443 and its transconjugant contained a single plasmid of about 50 kb.

PCR amplification specific for the blaTEM genes on both the K. oxytoca strain and its transconjugant gave a fragment of the expected size, 956 bp. Direct sequencing on both strands of the complete gene revealed that it was identical to blaTEM-2 except for a single point mutation (Table 4). This mutation consisted of the nucleotide change A to G (Aright-arrowG) at position 590, (according to Sutcliffes numbering [41]), which leads to the amino acid substitution Ser to Gly (Serright-arrowGly) at position 130 (according to Ambler's numbering) (1). This substitution has not been previously described in TEM-type enzymes produced by clinical strains. Consequently, the enzyme synthesized by Kox 443 was designated TEM-59 (26), according to the TEM nomenclature (10), or IRT-17 in the IRT series.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 4.   Nucleotide differences between bla genes

Nucleotide sequence accession number. The nucleotide sequence data reported here have been submitted to GenBank and have been assigned accession no. AF062386.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Inhibitor-resistant beta -lactamases mainly derive from TEM-1 or TEM-2 enzymes by point mutations in the active site vicinity that confer a lower substrate affinity. Currently, 15 IRT enzymes have been identified (3, 5-7, 9, 11, 19, 38, 39, 40, 43, 44), excluding TEM-41/IRT-12 which was inadvertantly placed on the list (40). These beta -lactamases have been found almost exclusively in E. coli, where they are present in up to 5% of the European clinical strains (12, 20); only rare isolates producing such enzymes have been reported in Klebsiella pneumoniae (4, 30) or Proteus mirabilis (7) responsible for human infections and in Citrobacter freundii isolated from calf feces (22). Recently, an inhibitor-resistant beta -lactamase has been described in the SHV family, SHV-10 (35).

The clinical isolate of K. oxytoca Kox 443 had an antibiogram (resistance to amoxicillin-clavulanate and full susceptibility to cephalothin) suggesting the presence of an inhibitor-resistant beta -lactamase. Indeed, the determination of the MICs confirmed that Kox 443 showed low-level resistance to penicillins and susceptibility to cephalothin and that clavulanic acid did not substantially potentiate penicillin activities. However, Tc 443 exhibited a lower resistance to amoxicillin than strains of E. coli producing most other IRT-type enzymes, as reported in the literature (MICs, >512 µg/ml), and a higher resistance to piperacillin alone or combined with tazobactam (MICs, <32 and <4 µg/ml, respectively); clavulanate at 2 µg/ml is usually more efficient, lowering the MICs of amoxicillin and ticarcillin by one or two dilutions (5-7, 9, 11, 19, 39, 40, 43, 44). MICs of penicillins, slightly lower for K. oxytoca 443 than for its E. coli transconjugant, could be related to a lower plasmid copy number in the original host or differential permeability or differential binding to penicillin-binding proteins.

The transferable beta -lactamase produced by Kox 443 had an unexpected pI of 5.6. Indeed, all IRT-type beta -lactamases reported so far have had a pI of either 5.2 or 5.4 (5-7, 9, 11, 19, 39, 40, 43, 44). The additional beta -lactamase produced by Kox 443, which was nontransferable and had a pI of 6.3, likely was the chromosomal enzyme of the species, probably of an OXY-2 type (pIs 5.2 to 6.8) (13, 16, 36).

Kinetic studies demonstrated that the Tc 443 enzyme behaved like the IRT-type enzymes, i.e., reached saturation with beta -lactams at considerably higher substrate concentrations (particularly for ticarcillin) and showed a reduction of hydrolysis (particularly for the cephalosporins). However, the increase of the Km values of the Tc 443 enzyme compared to those of TEM-2 was higher for amoxicillin and lower for piperacillin than that of other IRTs (5- to 10-fold and 6- to 24-fold increase, respectively) (6, 9, 11, 43, 44); there was a decrease of the Vmax values for amoxicillin and an increase for piperacillin in contrast with other IRTs (7, 43, 44). Inhibition data indicated that the Tc 443 enzyme had a weaker affinity than TEM-2 for beta -lactamase inhibitors and that larger concentrations of suicide inhibitors were required for inhibition, with tazobactam remaining the most efficient compound. The increase of the IC50 values was in the same range as that of other IRTs: higher for clavulanate (40- to 1,200-fold) than for sulbactam (5- to 110-fold) and tazobactam (2- to 180-fold). Because tazobactam is almost as efficient as clavulanic acid in inhibiting TEM-1 or TEM-2 enzymes, i.e., about 100 times more efficient than sulbactam (31), such differential loss of affinity for IRTs left tazobactam the best inhibitor of these mutant enzymes (5, 6, 9, 11, 43).

Clavulanate resistance of Kox 443, together with resistance to aminoglycosides [likely due to an AAC(3)-II], chloramphenicol, sulfonamides, and trimethoprim, was easily transferred by conjugation to E. coli K-12. Both Kox 443 and its transconjugant harbored a single plasmid of ca. 50 kb. The genes encoding IRT beta -lactamases are usually located on conjugative plasmids of variable size (33 to >180 kb) (4, 5, 11, 30, 44) that also code for such additional resistances (7, 9, 11, 43).

Specific PCR amplification showed that Kox 443 and its transconjugant carried a blaTEM gene. Nucleotide sequence analysis revealed almost complete identity with blaTEM-2, notably at the seven positions of the coding region which discriminate this gene from blaTEM-1A and blaTEM-1B (18, 41), in contrast with the molecular diversity previously found in the blaIRT genes (3, 6, 11, 12, 30). The fact that the blaTEM gene of Kox 443 arose from a TEM-2 ancestor is somewhat surprising, since all previously described IRT enzymes were TEM-1 derivatives, except for TEM-44/IRT-13 found in P. mirabilis (7), and since TEM-1 is much more frequent than TEM-2 in K. oxytoca (13, 36). Actually, the blaIRT gene from Kox 443, named blaTEM-59, differed from the blaTEM-2 gene by a single point mutation, Aright-arrowG at position 590, leading to the amino acid modification Serright-arrowGly at position 130. This substitution agrees well with the pI of 5.6 of the enzyme: it remained that of TEM-2, since serine, which contains an uncharged group, was replaced by glycine, another uncharged amino acid.

IRT enzymes found in clinical strains differ from TEM-1 or TEM-2 by one to three amino acid substitutions, involving at least one of the three following residues: Met-69, Arg-244, or Asn-276 (28). However, crystallographic data, kinetic study of mutant enzymes obtained by site-directed mutagenesis, and molecular modelling analysis have provided evidence that the highly conserved residue Ser-130 plays a crucial role in the structure and function of class A beta -lactamases (25, 27, 33), particularly in the inactivation process by suicide inhibitors (8, 23, 24). Very recently, Vakulenko et al. (42) have demonstrated by PCR mutagenesis of TEM-1 that only four mutations were sufficient by themselves to confer beta -lactam inhibitor resistance, including the three previously described mutations and Serright-arrowGly-130. The replacement of Ser-130 by amino acids other than glycine led to almost inactive enzymes. The Serright-arrowGly-130 mutant exhibited a profound decline in ampicillin resistance (the MIC declined from 16,000 to 1,000 µg/ml), with the relatively high residual resistance probably being related to the use of a high-copy-number plasmid.

The substitution Serright-arrowGly-130 was also found to be responsible for suicide inhibitor resistance in SHV-10, the only SHV-derived inhibitor-resistant beta -lactamase reported at present (35). This enzyme, derived from the extended-spectrum beta -lactamase SHV-9, an SHV-5 variant, partially retained its ability to hydrolyze penicillins but had a drastically reduced activity against cephalosporins (35). SHV-10 contained not only Serright-arrowGly-130 but also two additional amino acid substitutions, thus complicating the analysis of the effect of the Serright-arrowGly-130 replacement alone. Finally, the inhibitor-resistant OXY-2-derived beta -lactamase (IRKO-1) produced by a strain of K. oxytoca was found to differ from the parental enzyme by four amino acid substitutions, including Serright-arrowGly-130 (37).

The molecular characterization of TEM-59/IRT-17 emphasizes the key role of Ser-130 in conferring susceptibility to beta -lactamase inhibitors and provides further insight into understanding the catalytic process of class A beta -lactamases. The discovery of an IRT enzyme in K. oxytoca confirms the spread of these enzymes in the Enterobacteriaceae family. However, the good susceptibility of the TEM-59-producing strain to all cephalosporins may limit the spread of the gene encoding this type of beta -lactamase.


    FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Microbiologie, Université de Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. Phone: (33) 5 57 57 10 75. Fax: (33) 5 56 90 90 72. E-mail: claudine.quentin{at}bacterio.u-bordeaux2.fr.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Ambler, R. P., A. F. W. Coulson, J.-M. Frère, J. M. Ghuysen, B. Joris, M. Forsman, R. C. Levesque, G. Tiraby, and S. G. Waley. 1991. A standard numbering scheme for the class A beta -lactamases. Biochem. J. 276:269-272.
2. Arakawa, Y., M. Ohta, N. Kido, M. Mori, H. Ito, T. Komatsu, Y. Fujii, and N. Kato. 1989. Chromosomal beta -lactamase of Klebsiella oxytoca, a new class A enzyme that hydrolyzes broad-spectrum beta -lactam antibiotics. Antimicrob. Agents Chemother. 33:63-70[Abstract/Free Full Text].
3. Belaaouaj, A., C. Lapoumeroulie, M. M. Caniça, G. Vedel, P. Névot, R. Krishnamoorthy, and G. Paul. 1994. Nucleotide sequences of the genes coding for the TEM-like beta -lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiol. Lett. 120:75-80[Medline].
4. Bermudes, H., F. Jude, C. Arpin, C. Quentin, A. Morand, and R. Labia. 1997. Characterization of an inhibitor-resistant TEM (IRT) beta -lactamase in a novel strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 41:222[Medline].
5. Blazquez, J., M.-R. Baquero, R. Canton, I. Alos, and F. Baquero. 1993. Characterization of a new TEM-type beta -lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli. Antimicrob. Agents Chemother. 37:2059-2063[Abstract/Free Full Text].
6. Bret, L., E. B. Chaibi, C. Chanal-Claris, D. Sirot, R. Labia, and J. Sirot. 1997. Inhibitor-resistant TEM (IRT) beta -lactamases with different substitutions at position 244. Antimicrob. Agents Chemother. 41:2547-2549[Abstract].
7. Bret, L., C. Chanal, D. Sirot, R. Labia, and J. Sirot. 1996. Characterization of an inhibitor-resistant enzyme IRT-2 derived from TEM-2 beta -lactamase produced by Proteus mirabilis strains. J. Antimicrob. Chemother. 38:183-191[Abstract/Free Full Text].
8. Brown, R. P. A., R. T. Aplin, and C. J. Schofield. 1996. Inhibition of TEM-2 beta -lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry. Biochemistry 35:12421-12432[Medline].
9. Brun, T., J. Péduzzi, M. M. Caniça, G. Paul, P. Névot, M. Barthélémy, and R. Labia. 1994. Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta -lactamase inhibitors. FEMS Microbiol. Lett. 120:111-118[Medline].
10. Bush, K., and G. Jacoby. 1997. Nomenclature of TEM beta -lactamases. J. Antimicrob. Chemother. 39:1-3[Free Full Text].
11. Caniça, M. M., M. Barthélémy, L. Gilly, R. Labia, R. Krishnamoorthy, and G. Paul. 1997. Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta -lactamases. Antimicrob. Agents Chemother. 41:374-378[Abstract].
12. Caniça, M. M., C. Y. Lu, R. Krishnamoorthy, and G. Paul. 1997. Molecular diversity and evolution of blaTEM genes encoding beta -lactamases resistant to clavulanic acid in clinical E. coli. J. Mol. Evol. 44:57-65[Medline].
13. Chardon, H., C. Pachetti, L. Collet, O. Bellon, and E. Lagier. 1993. Détermination du point isoélectrique des beta -lactamases extraites de 67 souches de Klebsiella oxytoca et comportement phénotypique vis-à-vis de huit beta -lactamines. Pathol. Biol. 41:343-348[Medline].
14. Comité de l'antibiogramme de la Société Française de Microbiologie. 1997. Communiqué 1997. Pathol. Biol. 45:I-XII.
15. Courvalin, P., F. Goldstein, A. Philippon, and J. Sirot. 1985. L'antibiogramme. MPC-Videom, Brussels, Belgium.
16. Fournier, B., P. H. Roy, P. H. Lagrange, and A. Philippon. 1996. Chromosomal beta -lactamase genes of Klebsiella oxytoca are divided into two main groups, blaoxy-1 and blaoxy-2. Antimicrob. Agents Chemother. 40:454-459[Abstract].
17. Fournier, B., C. Y. Lu, P. H. Lagrange, R. Krishnamoorthy, and A. Philippon. 1995. Point mutation in the Pribnow box, the molecular basis of beta -lactamase overproduction in Klebsiella oxytoca. Antimicrob. Agents Chemother. 39:1365-1368[Abstract].
18. Goussard, S., and P. Courvalin. 1991. Sequence of the blaT-1B and blaT-2. Gene 102:71-73[Medline].
19. Henquell, C., C. Chanal, D. Sirot, R. Labia, and J. Sirot. 1995. Molecular characterization of nine different types of mutants among 107 inhibitor-resistant beta -lactamases from clinical isolates of Escherichia coli. Antimicrob. Agents Chemother. 39:427-430[Abstract/Free Full Text].
20. Henquell, C., D. Sirot, C. Chanal, C. De Champs, P. Chatron, B. Lafeuille, P. Texier, J. Sirot, and R. Cluzel. 1994. Frequency of inhibitor-resistant TEM beta -lactamases in Escherichia coli isolates from urinary tract infections in France. J. Antimicrob. Chemother. 34:707-714[Abstract/Free Full Text].
21. Heritage, J., P. M. Hawkey, N. Todd, and I. J. Lewis. 1992. Transposition of the gene encoding a TEM-12 extended-spectrum beta -lactamase. Antimicrob. Agents Chemother. 36:1981-1986[Abstract/Free Full Text].
22. Hunter, J. E. B., J. E. Corkill, A. G. McLannan, J. N. Fletcher, and C. A. Hart. 1993. Plasmid encoded beta -lactamases resistant to inhibition by clavulanic acid produced by calf faecal coliforms. Res. Vet. Sci. 55:367-370[Medline].
23. Imtiaz, U., E. Billings, J. R. Knox, E. K. Manavathu, S. A. Lerner, and S. Mobashery. 1993. Inactivation of class A beta -lactamases by clavulanic acid: the role of arginine 244 in a proposed non-concerted sequence of events. J. Am. Chem. Soc. 115:4435-4442.
24. Imtiaz, U., E. M. Billings, J. R. Knox, and S. Mobashery. 1994. A structure-based analysis of the inhibition of class A beta -lactamases by sulbactam. Biochemistry 33:5728-5738[Medline].
25. Jacob, F., B. Joris, S. Lepage, J. Dusart, and J.-M. Frère. 1990. Role of the conserved amino acids of the "SDN" loop (Ser130, Asp131, and Asn132) in a class A beta -lactamase studied by site-directed mutagenesis. Biochem. J. 271:399-406[Medline].
26. Jacoby, G., and K. Bush. 1999. [Online.] Amino acid sequences for TEM, SHV, and OXA extended-spectrum beta -lactamases. http://www.lahey.hitchcock.org/pages/lhc/studies/webt.htm. [2 May 1999, last date accessed.]
27. Juteau, J. M., E. Billings, J. R. Knox, and R. C. Levesque. 1992. Site-saturation mutagenesis and three-dimensional modeling of ROB-1 define a substrate binding role of Ser130 in class A beta -lactamases. Protein Eng. 5:693-701[Abstract/Free Full Text].
28. Knox, J. R. 1995. Extended-spectrum and inhibitor-resistant TEM-type beta -lactamases: mutations, specificity, and three-dimensional structure. Antimicrob. Agents Chemother. 39:2593-2601[Medline].
29. Labia, R., J. Andrillon, and F. Le Goffic. 1973. Computerized microacidimetric determination of beta -lactamase Michaelis-Menten constants. FEBS Lett. 33:42-44[Medline].
30. Lemozy, J., D. Sirot, C. Chanal, C. Huc, R. Labia, H. Dabernat, and J. Sirot. 1995. First characterization of inhibitor-resistant TEM (IRT) beta -lactamases in Klebsiella pneumoniae strains. Antimicrob. Agents Chemother. 33:2580-2582.
31. Livermore, D. M. 1993. Determinants of the activity of beta -lactamase inhibitor combinations. J. Antimicrob. Chemother. 31(Suppl. A):9-21[Free Full Text].
32. Livermore, D. M., and M. Yuan. 1996. Antibiotic resistance and production of extended-spectrum beta -lactamases amongst Klebsiella spp. from intensive care units in Europe. J. Antimicrob. Chemother. 38:409-424[Abstract/Free Full Text].
33. Matagne, A., and J.-M. Frère. 1995. Contribution of mutant analysis to the understanding of enzyme catalysis: the case of class A beta -lactamases. Biochim. Biophys. Acta 1245:109-127.
34. Matthew, M., A. M. Harris, M. G. Marshall, and G. W. Ross. 1975. The use of analytical isoelectric focusing for detection and identification of beta -lactamases. J. Gen. Microbiol. 88:169-178[Medline].
35. Prinarakis, E. E., V. Miriagou, E. Tzelepi, M. Gazouli, and L. S. Tzouvelekis. 1997. Emergence of an inhibitor-resistant beta -lactamase (SHV-10) derived from an SHV-5 variant. Antimicrob. Agents Chemother. 41:838-840[Abstract].
36. Reig, R., C. Roy, M. Hermida, D. Teruel, and A. Coira. 1993. A survey of beta -lactamases from 618 isolates of Klebsiella spp. J. Antimicrob. Chemother. 31:29-35[Abstract/Free Full Text].
37. Sirot, D., R. Labia, P. Pouedras, C. Chanal-Claris, C. Cerceau, and J. Sirot. 1998. Inhibitor-resistant OXY-2-derived beta -lactamase produced by Klebsiella oxytoca. Antimicrob. Agents Chemother. 42:2184-2187[Abstract/Free Full Text].
38. Sirot, D., C. Recule, E. B. Chaibi, L. Bret, J. Croize, C. Chanal-Claris, R. Labia, and J. Sirot. 1997. A complex mutant of TEM-1 beta -lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate. Antimicrob. Agents Chemother. 41:1322-1325[Abstract].
39. Speeldoren, V., B. Heym, R. Labia, and M. H. Nicolas-Chanoine. 1998. Discriminatory detection of inhibitor-resistant beta -lactamases in Escherichia coli by single-strand conformation polymorphism-PCR. Antimicrob. Agents Chemother. 42:879-884[Abstract/Free Full Text].
40. Stapleton, P., P. J. Wu, A. King, K. Shannon, G. French, and I. Phillips. 1995. Incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli. Antimicrob. Agents Chemother. 39:2478-2483[Abstract]. (Author's correction, 42:2773, 1998.)
41. Sutcliffe, J. G. 1978. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc. Natl. Acad. Sci. USA 75:3737-3741[Abstract/Free Full Text].
42. Vakulenko, S. B., B. Geryk, L. P. Kotra, S. Mobashery, and S. A. Lerner. 1998. Selection and characterization of beta -lactam-beta -lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta -lactamase gene. Antimicrob. Agents Chemother. 42:1542-1548[Abstract/Free Full Text].
43. Vedel, G., A. Belaaouaj, L. Gilly, R. Labia, A. Philippon, P. Névot, and G. Paul. 1992. Clinical isolates of Escherichia coli producing TRI beta -lactamases: novel TEM-enzymes conferring resistance to beta -lactamase inhibitors. J. Antimicrob. Chemother. 30:449-462[Abstract/Free Full Text].
44. Zhou, X. Y., F. Bordon, D. Sirot, M.-D. Kitzis, and L. Gutmann. 1994. Emergence of clinical isolates of Escherichia coli producing TEM-1 derivatives or an OXA beta -lactamase conferring resistance to beta -lactamase inhibitors. Antimicrob. Agents Chemother. 38:1085-1089[Abstract/Free Full Text].

Antimicrobial Agents and Chemotherapy, July 1999, p. 1657-1661, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Dubois, V., Poirel, L., Demarthe, F., Arpin, C., Coulange, L., Minarini, L. A. R., Bezian, M.-C., Nordmann, P., Quentin, C. (2008). Molecular and Biochemical Characterization of SHV-56, a Novel Inhibitor-Resistant {beta}-Lactamase from Klebsiella pneumoniae. Antimicrob. Agents Chemother. 52: 3792-3794 [Abstract] [Full Text]  
  • Zarnayova, M., Siebor, E., Pechinot, A., Duez, J.-M., Bujdakova, H., Labia, R., Neuwirth, C. (2005). Survey of Enterobacteriaceae Producing Extended-Spectrum {beta}-Lactamases in a Slovak Hospital: Dominance of SHV-2a and Characterization of TEM-132. Antimicrob. Agents Chemother. 49: 3066-3069 [Abstract] [Full Text]  
  • Poirel, L., Mammeri, H., Nordmann, P. (2004). TEM-121, a Novel Complex Mutant of TEM-Type {beta}-Lactamase from Enterobacter aerogenes. Antimicrob. Agents Chemother. 48: 4528-4531 [Abstract] [Full Text]  
  • Helfand, M. S., Bethel, C. R., Hujer, A. M., Hujer, K. M., Anderson, V. E., Bonomo, R. A. (2003). Understanding Resistance to {beta}-Lactams and {beta}-Lactamase Inhibitors in the SHV {beta}-Lactamase: LESSONS FROM THE MUTAGENESIS OF SER-130. J. Biol. Chem. 278: 52724-52729 [Abstract] [Full Text]  
  • Aumeran, C., Chanal, C., Labia, R., Sirot, D., Sirot, J., Bonnet, R. (2003). Effects of Ser130Gly and Asp240Lys Substitutions in Extended-Spectrum {beta}-Lactamase CTX-M-9. Antimicrob. Agents Chemother. 47: 2958-2961 [Abstract] [Full Text]  
  • Galan, J.-C., Morosini, M.-I., Baquero, M.-R., Reig, M., Baquero, F. (2003). Haemophilus influenzae blaROB-1 Mutations in Hypermutagenic {Delta}ampC Escherichia coli Conferring Resistance to Cefotaxime and {beta}-Lactamase Inhibitors and Increased Susceptibility to Cefaclor. Antimicrob. Agents Chemother. 47: 2551-2557 [Abstract] [Full Text]  
  • Schroeder, W. A., Locke, T. R., Jensen, S. E. (2002). Resistance to {beta}-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM {beta}-Lactamase Mutants. Antimicrob. Agents Chemother. 46: 3568-3573 [Abstract] [Full Text]  
  • Alonso, R., Gerbaud, G., Galimand, M., Courvalin, P. (2002). TEM-103/IRT-28 {beta}-Lactamase, a New TEM Variant Produced by Escherichia coli BM4511. Antimicrob. Agents Chemother. 46: 3627-3629 [Abstract] [Full Text]  
  • Arpin, C., Labia, R., Dubois, V., Noury, P., Souquet, M., Quentin, C. (2002). TEM-80, a Novel Inhibitor-Resistant {beta}-Lactamase in a Clinical Isolate of Enterobacter cloacae. Antimicrob. Agents Chemother. 46: 1183-1189 [Abstract] [Full Text]  
  • Barlow, M., Hall, B. G. (2002). Origin and Evolution of the AmpC {beta}-Lactamases of Citrobacter freundii. Antimicrob. Agents Chemother. 46: 1190-1198 [Abstract] [Full Text]  
  • Granier, S. A., Van, J.-C. N., Kitzis, M.-D., Goldstein, F. W., Leflon-Guibout, V., Nicolas-Chanoine, M.-H. (2002). First Description of a TEM-30 (IRT-2)-Producing Klebsiella oxytoca Isolate. Antimicrob. Agents Chemother. 46: 1158-1159 [Full Text]  
  • Neuwirth, C., Madec, S., Siebor, E., Pechinot, A., Duez, J.-M., Pruneaux, M., Fouchereau-Peron, M., Kazmierczak, A., Labia, R. (2001). TEM-89 beta -Lactamase Produced by a Proteus mirabilis Clinical Isolate: New Complex Mutant (CMT 3) with Mutations in both TEM-59 (IRT-17) and TEM-3. Antimicrob. Agents Chemother. 45: 3591-3594 [Abstract] [Full Text]  
  • Leflon-Guibout, V., Heym, B., Nicolas-Chanoine, M.-H. (2000). Updated Sequence Information and Proposed Nomenclature for blaTEM Genes and Their Promoters. Antimicrob. Agents Chemother. 44: 3232-3234 [Abstract] [Full Text]  
  • Martínez-Martínez, L., Conejo, M. C., Pascual, A., Hernández-Allés, S., Ballesta, S., Ramírez de Arellano-Ramos, E., Benedí, V. J., Perea, E. J. (2000). Activities of Imipenem and Cephalosporins against Clonally Related Strains of Escherichia coli Hyperproducing Chromosomal beta -Lactamase and Showing Altered Porin Profiles. Antimicrob. Agents Chemother. 44: 2534-2536 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bermudes, H.
Right arrow Articles by Quentin, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bermudes, H.
Right arrow Articles by Quentin, C.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»