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Antimicrobial Agents and Chemotherapy, November 2005, p. 4443-4447, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4443-4447.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

TEM-109 (CMT-5), a Natural Complex Mutant of TEM-1 ß-Lactamase Combining the Amino Acid Substitutions of TEM-6 and TEM-33 (IRT-5)

Faculté de Médecine, Centre Hospitalo-Universitaire, Clermont-Ferrand, France

Received 30 March 2005/ Returned for modification 6 June 2005/ Accepted 3 August 2005

	ABSTRACT

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Escherichia coli CF349 exhibited a complex ß-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a ß-lactamase of pI 5.9. The corresponding bla gene was identified by PCR and sequencing as a bla_TEM gene. The deduced protein sequence revealed a new complex mutant of TEM-1 ß-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum ß-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 (k_cat, 56 s^–1 and 105 s^–1, respectively; K_m values, 226 and 247 µM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 µM and 0.27 µM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 µM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.

	INTRODUCTION

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The most prevalent mechanism of resistance to ß-lactam antibiotics in members of the family Enterobacteriaceae is the production of ß-lactamases belonging to Bush group 2b (6, 21). These enzymes are able to inactivate penicillins and narrow-spectrum cephalosporins before they can reach their target.

Extended-spectrum ß-lactamases (ESBLs) were isolated first in Europe and then worldwide shortly after the introduction of oxyimino cephalosporins (28, 30). According to the structural classification of Ambler et al. (1) and the functional classification of Bush and Jacoby (6), these first ESBLs were class A enzymes of the 2be group that arose subsequent to a few number of amino acid substitutions from the common plasmid-mediated TEM and SHV-1 ß-lactamases. These enzymes confer resistance to penicillins, oxyimino cephalosporins, and aztreonam and are susceptible to ß-lactam inhibitors.

The use of ß-lactamase inhibitors has also been followed by the emergence of resistant clinical isolates, which overproduce TEM-type ß-lactamases (18) or which produce inhibitor-resistant TEM variants (IRTs) (3). As was the case for the ESBLs, IRTs arose from the common plasmid-mediated TEM and SHV-1 penicillinases subsequent to a few amino acid substitutions. These substitutions conferred resistance to inhibitors but not the ability to hydrolyze oxyimino ß-lactams.

A new subgroup of TEM- and SHV-type ß-lactamases which harbors both mutations conferring extended-spectrum activity and resistance to inhibitors has emerged since the end of the 1990s in different species of the family Enterobacteriaceae: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Enterobacter aerogenes. Four enzymes are derived from TEM-type enzymes and were designated complex mutant TEM (CMT-1 to CMT-4) (10, 23, 24, 29). Another complex mutant derived from SHV-1 has been identified: SHV-10 (25). In 2001, we isolated E. coli CF349, a clinical isolate resistant to amoxicillin and ticarcillin alone and in combination with clavulanate and also to some extended-spectrum cephalosporins. The aim of this work was to characterize the ß-lactamases involved in this resistance phenotype.

	MATERIALS AND METHODS

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Strains and plasmids. The strains used in this work included E. coli CF349, E. coli CF0051 producing TEM-33 (12), E. coli HB251 producing TEM-6 (2, 11), and E. coli CF001 producing TEM-1 (12). E. coli DH5 (Novagen, Darmstadt, Germany) and E. coli BL21(DE3) (Novagen, Darmstadt, Germany) were used for the cloning experiments, and E. coli C600 was used for the mating-out assays. The plasmid pBK-CMV (Stratagene, Amsterdam, The Netherlands) was used for the initial cloning experiments, and a modified pET9a plasmid (20) was used for the overexpression of the ß-lactamase-encoding genes.

Susceptibility to ß-lactams. Antibiotic-containing disks were used for antibiotic susceptibility testing by the disk diffusion assay (Sanofi-Diagnostics Pasteur, Marnes la Coquette, France). The double-disk synergy test was performed as described previously (13). MICs were determined by a microdilution method on Mueller-Hinton agar (Sanofi Diagnostics Pasteur) with an inoculum of 10⁴ CFU per spot. The antibiotics were provided as powders by GlaxoSmithKline (amoxicillin, ticarcillin, cefuroxime, ceftazidime, and clavulanic acid), Lederle Laboratories (piperacillin and tazobactam), Eli Lilly (cephalothin), Roussel-Uclaf (cefotaxime and cefpirome), Bristol-Myers Squibb (aztreonam and cefepime), and Merck Sharp & Dohme-Chibret (cefoxitin and imipenem).

Isoelectric focusing. Isoelectric focusing was performed with polyacrylamide gels containing ampholines with a pH range of 3.5 to 10.0, as described previously (29). ß-Lactamases with known pIs were used as standards: TEM-33 (pI 5.2), TEM-1 (pI 5.4), TEM-2 (pI 5.6), and TEM-6 (pI 5.9).

Mating-out experiment. Direct transfer of plasmids coding for resistance genes was performed by mating donor strains with in vitro-obtained rifampin-resistant mutants of E. coli C600 as the recipient strain at 37°C in solid Mueller-Hinton medium (26). Transconjugants were selected on agar containing rifampin (300 µg/ml) and ticarcillin (32 µg/ml).

Plasmid content analysis. Plasmid DNAs were extracted from the transconjugants by the method of Kado and Liu (14). The plasmid size was determined by comparison with those of plasmids Rsa (39 kb), TP114 (61 kb), pCFF04 (85 kb), and pCFF14 (180 kb).

Genotyping. The clinical isolates of E. coli CF349 were compared by enterobacterial repetitive intergenic consensus sequence PCR (ERIC2-PCR) and ribotyping, as described previously (9, 33).

Cloning experiments. Recombinant DNA manipulation and transformations were performed as described by Sambrook et al. (26). T4 DNA ligase and proofreading Taq polymerase were purchased from Appligène (Oncor, Illkirch, France). The TEM-encoding genes, including their promoters, were amplified by PCR with primers TEM-A (5'-TAA AAT TCT TGA AGA CG-3') and TEM-B2 (5'-TCT GAC AGT TAC CAA TGC-3') and cloned into SmaI restriction site of pBK-CMV plasmid. The correct orientation of the insert was checked by PCR with the primers TEM-A and pBK-CMV2' (5'-AAT TGG GTA CAC TTA CCT GGT ACC C-3'). The TEM-encoding genes were also amplified with the primers NdeI-TEM-A (5'-GGA ATT CCA TAT GAG TAT TCA ACA TTT CCG-3') and NotI-TEM-B (5'-ATA GTT TAG CGG CCG CTT AAT GCT TAA TCA GTG AG-3'), which included restriction sites for the enzymes NdeI and NotI, respectively. The PCR products were digested by these enzymes and ligated into the corresponding restriction sites of a modified pET9a plasmid. The plasmids derived from pBK-CMV and pET9a (pBK-TEM-1, pET9-TEM-1, pBK-TEM-33, pET9-TEM-33, pBK-TEM-6, pET9-TEM-6, pBK-TEM-109, and pET9-TEM-109) were transformed after sequencing of the control into E. coli strains DH5 and BL21(DE3), respectively. E. coli transformants were selected on Mueller-Hinton agar supplemented with 30 µg kanamycin and 0.5 µg ceftazidime.

Sequencing of DNA amplified by PCR. Direct sequencing of both strands of the PCR products was performed by the dideoxy chain termination with an Applied Biosystems sequencer (ABI 377) (27). The nucleotide and deduced protein sequences were analyzed with software available at the website of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). The ClustalW program (http://infobiogen.fr) was used to align the multiple protein sequences (32).

Overexpression and purification of ß-lactamases. TEM-producing E. coli BL21(DE3) were used to overproduce the ß-lactamases, as described previously (8). The strains were cultured in 2x YT broth (Qbiogene, Irvine, Calif.) containing kanamycin (30 µg/ml) and 0.1 mM isopropyl-ß-D-thiogalactopyranoside (Sigma Chemical Co., St. Louis, Mo.) The bacteria were disrupted by sonication. The supernatant was loaded onto a Q Sepharose column (10 ml; Amersham Pharmacia Biotech.) equilibrated with 20 mM Tris-HCl (pH 7.0). The bound proteins were eluted with a linear NaCl gradient (0 to 500 mM). The ß-lactamase-containing elution peak was loaded onto a Superose 12 column (3.2 by 30 cm; Amersham Pharmacia Biotech.) and eluted with the buffer 20 mM Tris-HCl—100 mM NaCl (pH 7.0). The ß-lactamase-containing elution peak was dialyzed against 100 mM NaCl, concentrated by ultrafiltration, and stored at –20°C until use. The total protein concentration was estimated by the Bio-Rad (Richmond, Calif.) protein assay, with bovine serum albumin (Sigma Chemical Co.) used as a standard. The level of purity was estimated to be >97% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4, 17).

Determination of ß-lactamase kinetic parameters k_cat and K_m and IC₅₀s. The Michaelis constant (K_m) and catalytic activity (k_cat) were determined with purified extracts by a computerized microacidimetric method (16). The 50% inhibitory concentrations (IC₅₀s) were determined for clavulanic acid and tazobactam, as described previously, with 100 µM benzylpenicillin as the reporter substrate (4).

Nucleotide sequence accession number. The nucleotide sequence of the bla_TEM-109 gene has been assigned accession number AY628175 in the GenBank database.

	RESULTS AND DISCUSSION

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Clinical isolates. Two E. coli isolates were collected from stool specimens of patients admitted in the intensive care unit of the teaching hospital of Clermont-Ferrand in 2001 and were selected on the basis of their ability to grow on agar plates containing 4 µg/ml ceftazidime. The two E. coli isolates had the same resistance phenotype and were genotypically identical by ribotyping and ERIC2-PCR (data not shown). These isolates, designated CF349, presented high levels of resistance to penicillins and ceftazidime and low levels of resistance to cephalothin and aztreonam. MICs of methoxyimino cephalosporins were in the susceptible range but were significantly higher than those obtained for E. coli DH5, which did not produce a TEM-type ß-lactamase. In contrast, the MICs of cefoxitin and imipenem were identical for CF349 and DH5. If tazobactam restored the activity of piperacillin, clavulanic acid was less effective in decreasing the MICs of amoxicillin, ticarcillin, ceftazidime, and aztreonam (Table 1). However, the synergy test was positive with ceftazidime.

View this table: [in this window] [in a new window]   TABLE 1. MICs of ß-lactams for E. coli CF349; transconjugant E. coli C600(pCF349); and TEM-109-, TEM-6-, TEM-33-, and TEM-1-producing E. coli DH5 and E. coli DH5(pBK-CMV)

PCR experiments and DNA sequencing. Analysis of the bla_TEM nucleic acid sequences from the transconjugants revealed a TEM-1-encoding gene corresponding to the ß-lactamase of pI 5.4 and a new bla_TEM-type gene called bla_TEM-109 corresponding to the ß-lactamase of pI 5.9. In its promoter zone, bla_TEM-109 differed from bla_TEM-1a by the substitution CT at position 32, according to the numbering of Sutcliffe (31), which leads to the strong overlapping promoter P_a/P_b (18, 19). The sequence of bla_TEM-109 showed a pattern of silent mutations identical to those of bla_TEM-1b: A175G, C226T, C436T, and G604T. In the coding region, bla_TEM-109 differed from bla_TEM-1b by three point mutations: the nucleotide change AC at position 407, which led to the amino acid substitution MetLeu at position 69, and the nucleotide change GA at positions 512 and 693, which led to the amino acid substitutions GluLys at position 104 and ArgHis at position 164 (1). This enzyme harbors the substitutions described in the IRT-5/TEM-33 (Leu-69) and in the ESBL enzyme TEM-6 (Lys-104 and the His-164). This is the fifth member of the complex mutant TEM group (and therefore is also indicated CMT-5) and the second example of the production of such an enzyme by E. coli.

MICs for TEM-6-, TEM-33-, and TEM-109-producing transformants. TEM-109-, TEM-6-, and TEM-33-producing E. coli DH5 transformants were used to compare these related TEM-encoding genes (Table 1). TEM-109- and TEM-6-producing E. coli DH5 exhibited the same high level of resistance to penicillins and carboxypropyl oxyimino ß-lactams. The MICs of cephalothin, cefotaxime, cefepime, and cefpirome were also identical for these two strains and increased slightly in comparison with those for E. coli DH5. In contrast, the MICs of the combinations of ß-lactams and clavulanic acid were significantly higher for TEM-109-producing E. coli DH5 than for TEM-6-producing E. coli DH5.

The MICs of the combinations amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, and piperacillin-tazobactam were comparable for TEM-109- and TEM-33-producing E. coli DH5.

Enzymatic parameters. The kinetic parameters for TEM-1, TEM-6, TEM-33, and TEM-109 were determined under the same experimental conditions (Table 2). The kinetic constants obtained for TEM-1 were similar to those previously determined in another study (29).

The kinetic constants of TEM-109 and TEM-6 against cephalosporins clearly differed from those of TEM-1 and TEM-33. TEM-33 was 10- to 70-fold less efficient against cephalothin than TEM-109, TEM-6, and TEM-1. In addition, only the TEM-109 and TEM-6 enzymes exhibited significant hydrolytic activity against oxyimino ß-lactams (k_cat, 4 to 105 versus <0.1 s^–1). Only slight differences in k_cat and K_m values were observed for these substrates between these two enzymes. The catalytic efficiencies of TEM-109 and TEM-6 against oxyimino ß-lactams were therefore globally similar (k_cat/K_m, 0.1 to 0.4 µM^–1 s^–1). This activity of TEM-109 and TEM-6 against oxyimino ß-lactams is explained by the substitutions Glu104Lys and Arg164His.

The presence of lysine at position 104 slightly enhances oxyimino cephalosporin hydrolysis (15) but seems insufficient alone to confer a high level of resistance to extended-spectrum cephalosporins. This substitution is often associated with an additional amino acid substitution at position 238 or 164 (15). TEM-109 harbors the substitution Arg164His, which confers improved catalytic efficiency against oxyimino ß-lactams such as ceftazidime and aztreonam. Position 164 is located in a wall of the binding site, the loop, which limits the accommodation of large ß-lactams such as oxyimino ß-lactams. The substitution at position 164 leads to the flexibility of the loop (15, 34), which can favor the accommodation of the acyl-amide substitutes of ceftazidime and aztreonam, as observed in the crystal structure of TEM-64 (34).

The IC₅₀ of clavulanic acid (0.13 ± 0.01 µM) for TEM-109 was 10-fold higher than that of TEM-6 (0.01 ± 0.001 µM), slightly higher than that of TEM-1 (0.08 ± 0.01 µM), and 10-fold lower than that of TEM-33 (1.9 ± 0.1 µM). Similarly, tazobactam was less efficient against TEM-109 (0.27 ± 0.02 µM) than against TEM-6 (0.06 ± 0.004 µM) and TEM-1 (0.13 ± 0.01 µM) and more efficient than against TEM-33 (2.3 ± 0.1 µM). Compared to other CMT enzymes, TEM-109 had lower resistance to clavulanic acid than CMT-1 (29) or CMT-4 (24). CMT-1 and CMT-4 harbor highly efficient IRT-type mutations or associations of mutations. TEM-109 harbors the substitution Met69Leu of TEM-33. TEM-33, which harbors only this substitution, is one of the IRT-type enzymes that is the least resistant to clavulanic acid (5, 7, 22). Residue Leu at position 69 enhances clavulanic acid resistance by increasing the turnover against inhibitors (22). However, no structural evidence explains the behavior induced by Leu69 (22). The weak efficiency of residue Leu69 partially explains the level of resistance of TEM-109 to inhibitors. Despite the slight inhibitor resistance of TEM-109, TEM-109-producing E. coli DH5 harbored MICs of penicillins and clavulanic acid in association similar to those of TEM-33-producing E. coli DH5. Its resistance to ß-lactamase inhibitors was probably enhanced by the presence of the overlapping promoter Pa/Pb, which is responsible for the overproduction of TEM enzymes (18, 19), as assessed by their specific activities (Table 1).

The kinetic constants for previous CMT enzymes suggest that the combination of ESBL and IRT mutations have antagonist effects, which conferred to the CMT enzymes kinetic parameters against expanded-spectrum generation cephalosporins lower than those observed with the parental ESBL enzyme (10, 23-25, 29). TEM-109 differed from previously reported CMT enzymes by kinetic parameters almost identical to those of its parental ESBL, TEM-6. The presence of a leucine at position 69 may be responsible for the persistence of the catalytic efficiency of its parental ESBL, TEM-6. Different work suggests that the slight modification of the active site due to the Met69Leu substitution modified only the hydrolysis of small ß-lactams, such as clavulanic acid or tazobactam (22). If ESBL mutations Glu104Lys and Arg164His decrease the stability of the enzyme (–1.94 kcal/mol) (34), Wang et al. have also shown that residue Leu69 enhances enzyme stability (1 kcal/mol), unlike residues Ile69 and Val69 (35). Residue Leu69 may partially restore the stability and therefore contribute to the low discrepancies of oxyimino ß-lactam MICs between TEM-109 and TEM-6.

In conclusion, we observed E. coli isolates resistant to oxyimino ß-lactams and penicillins and clavulanic acid in association. The resistance was due to a bla_TEM gene containing a strong promoter P_a/P_b and producing the novel enzyme TEM-109 (CMT-5). This enzyme harbored ESBL mutations Glu104Lys and Arg164His in association with the weak and stabilizing IRT mutation Met69Leu. The association of these mutations with a strong promoter may constitute a good compromise that allows extended-spectrum activity, resistance to inhibitors, and stability.

	ACKNOWLEDGMENTS

 
We thank Marlene Jan, Rolande Perroux, and Dominique Rubio for technical assistance and Sophie Quevillon-Cheruel for providing us the modified pET9a plasmid.

This work was supported in part by a grant from Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, Paris, France, and a grant from the Centre Hospitalier Régional Universitaire de Clermont-Ferrand, France, and the Ministère de la Santé, de la Famille et des Personnes Handicapées, France (Projet Hospitalier de Recherche Clinique).

	FOOTNOTES

 
* Corresponding author. Mailing address: Laboratoire de Bactériologie, Faculté de Médecine, 28 place H. Dunant, 63 001 Clermont-Ferrand, France. Phone: (33) 4 73 17 81 50. Fax: (33) 4 73 27 74 94. E-mail: frobin{at}chu-clermontferrand.fr.

This paper is devoted to the memory of Catherine Chanal.

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