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Antimicrobial Agents and Chemotherapy, April 2007, p. 1304-1309, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01058-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evolution of TEM-Type Enzymes: Biochemical and Genetic Characterization of Two New Complex Mutant TEM Enzymes, TEM-151 and TEM-152, from a Single Patient ^,

Frédéric Robin,^1,2* Julien Delmas,^1,2 Cédric Schweitzer,^1,2 Olivier Tournilhac,³ Olivier Lesens,⁴ Catherine Chanal,^1,2 and Richard Bonnet^1,2

CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Bactériologie Clinique, Clermont-Ferrand F-63003, France,¹ Université Clermont 1, UFR Médecine, Laboratoire de Bactériologie, EA3844, Clermont-Ferrand F-63001, France,² CHU Clermont-Ferrand, Hôtel-Dieu, Service d'Hématologie Clinique, Clermont-Ferrand F-63003, France,³ CHU Clermont-Ferrand, Hôtel-Dieu, Service de Maladies Infectieuses et Tropicales, Clermont-Ferrand F-63003, France⁴

Received 22 August 2006/ Returned for modification 26 October 2006/ Accepted 2 January 2007

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two clinical isolates of Escherichia coli, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related. E. coli recombinant strains exhibiting the resistance phenotype of the clinical strains were obtained by cloning. The clones corresponding to strains CF1179 and CF1295 produced TEM-type beta-lactamases with pI values of 5.7 and 5.3, respectively. Sequencing analysis revealed two novel bla_TEM genes encoding closely related complex mutant TEM enzymes, designated TEM-151 (pI 5.3) and TEM-152 (pI 5.7). These two genes also harbored a new promoter region which presented a 9-bp deletion. The two novel ß-lactamases differed from the parental enzyme, TEM-1, by the substitution Arg164His, previously observed in extended-spectrum beta-lactamases (ESBLs), and by the substitutions Met69Val and Asn276Asp, previously observed in the inhibitor-resistant penicillinase TEM-36/IRT-7. They differed by two amino acid substitutions: TEM-152 harbored a Glu240Lys ESBL-type substitution and TEM-151 had an Ala284Gly substitution. Functional analysis of TEM-151 and TEM-152 showed that both enzymes had hydrolytic activity against ceftazidime (k_cat, 5 and 16 s^–1, respectively). TEM-152 was more resistant than TEM-151 to the inhibitor clavulanic acid (50% inhibitory concentrations, 1 versus 0.17 µM). These results confirm the evolution of TEM-type enzymes toward complex enzymes harboring the two kinds of substitutions which confer an extended spectrum of action against beta-lactam antibiotics and resistance to inhibitors.

Top Abstract Introduction Materials and Methods Results Discussion References

 
ß-Lactams are the most important antibiotic family because of their wide spectrum of efficiency, their bactericidal activity, and their low toxicity. The major resistance mechanism in Enterobacteriaceae is the production of ß-lactamases. The most prevalent enzymes (TEM-1 and SHV-1) were able to inactivate penicillins and narrow-spectrum cephalosporins but remained susceptible to ß-lactamase inhibitors, such as clavulanic acid, and inactive against oxyimino cephalosporins, such as ceftazidime.

However, the intensive use of these molecules was quickly followed by the evolution of TEM- and SHV-type ß-lactamases. Extended-spectrum ß-lactamases (ESBLs) were identified during the 1980s (14, 28, 29). They differed from the TEM and SHV penicillinases by a few amino acid substitutions which confer hydrolytic activity against oxyimino cephalosporins. However, these ESBLs remained susceptible to clavulanate and tazobactam. A synergy picture between a disk containing an oxyimino cephalosporin and a disk containing a penicillin-clavulanate combination allows their detection (8).

Starting from 1990, the clinical use of combinations of penicillins and ß-lactamase inhibitors was followed by the emergence of TEM-type enzymes resistant to inhibitors (2). These enzymes, designated inhibitor-resistant TEM (IRT) enzymes, were inactive against all cephalosporins (5).

Since the middle of the 1990s, a third subgroup of TEM enzymes has emerged that combines the IRT- and ESBL-type substitutions. These new ß-lactamases, called complex mutant TEM (CMT) enzymes, have been identified in different species of Enterobacteriaceae, including Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Enterobacter aerogenes (11, 21-24, 27). These enzymes confer different levels of resistance to clavulanic acid and to oxyimino cephalosporins, depending on the harbored mutations.

We report here two isolates of E. coli, obtained from the same patient, with combined resistance to penicillin-clavulanate combinations and to ceftazidime.

(This work was presented in part at the 25th Réunion Interdiciplinaire de Chimiothérapie Anti-Infectieuse [RICAI], Paris, France, December 2005.)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial isolates and plasmids. The strains used in this study were E. coli CF1179, E. coli CF1295, E. coli CF0072 producing TEM-36 (13), E. coli 2300 producing TEM-28 (4), E. coli CF628 producing TEM-29 (9), and E. coli CF001 producing the penicillinase TEM-1 (13). E. coli DH5 (Novagen, Darmstadt, Germany) and E. coli BL21(DE3) (Novagen) were used for cloning experiments (25), and E. coli C600 was used for mating-out assays. Plasmid pBK-CMV (Stratagene, Amsterdam, The Netherlands) was used for the initial cloning experiments, and a modified pET9a plasmid (20) was used for overexpression of the ß-lactamase-encoding genes.

Genomic typing. The clinical isolates E. coli CF1295 and CF1179 were compared by enterobacterial repetitive intergenic consensus sequence PCR (ERIC2-PCR) as previously described (10).

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). MICs were determined by a dilution method on Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Marnes la Coquette, France), with an inoculum of 10⁴ CFU per spot, and were interpreted according to the guidelines of the Clinical Laboratory Standards Institute (7). The antibiotics were provided as powders by Glaxo Smith Kline (amoxicillin, ticarcillin, cefuroxime, ceftazidime, and clavulanic acid), Wyeth Laboratories (piperacillin and tazobactam), Eli Lilly (cephalothin), Roussel-Uclaf (cefotaxime and cefpirome), Bristol-Myers-Squibb (aztreonam and cefepime), and Merck Sharp and Dohme-Chibret (cefoxitin and imipenem).

Isoelectric focusing. Isoelectric focusing of ß-lactamases was performed with polyacrylamide gels containing ampholytes with a pH range of 3.5 to 10.0, as previously described (3), with TEM-39 (pI 5.2), TEM-12 (pI 5.25), TEM-1 (pI 5.4), and TEM-2 (pI 5.6) as standards.

Transfer experiments. Plasmids carrying resistance genes were directly transferred by mating donor strains with rifampin-resistant mutants of E. coli C600 obtained in vitro as recipient strains at 37°C on solid Mueller-Hinton medium (25). Transconjugants were selected on agar containing rifampin (300 µg/ml) and ceftazidime (0.5 µg/ml).

When mating-out experiments were negative, plasmid DNA was extracted and purified by alkaline lysis according to the Qiafilter protocol (QIAGEN, Hilden, Germany). Electroporation of plasmid DNA into E. coli DH5 was performed according to the manufacturer's instructions (Bio-Rad, Richmond, CA). Transformants were selected on agar containing ceftazidime (0.5 µg/ml).

Cloning experiments. The TEM enzyme-encoding genes, including the promoter region, were cloned into pBK-CMV and a modified plasmid vector, pET-9a, as previously described (23). The recombinant plasmids obtained were transformed into E. coli strains DH5 and BL21(DE3), respectively. E. coli clones were selected on Mueller-Hinton agar supplemented with 30 µg/ml kanamycin and 0.5 µg/ml ceftazidime.

Sequencing of DNAs amplified by PCR. Direct sequencing was performed on three independent PCR products, which were obtained from the transconjugant E. coli C600 and the recombinant E. coli DH5 strains. These PCR products were sequenced by dideoxy chain termination on both strands with an Applied Biosystems sequencer (ABI 377) (26). The nucleotide and deduced protein sequences were analyzed using software available at the website of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).

Overexpression and purification of ß-lactamases. TEM-producing E. coli BL21(DE3) clones were used to overproduce the TEM-type ß-lactamases as previously described (6). Bacteria were disrupted by sonication. TEM purification was carried out, as previously described (23), by ion-exchange chromatography with a Q Sepharose column (Amersham Pharmacia Biotech, Orsay, France) and gel filtration chromatography with a Superose 12 column (Amersham Pharmacia Biotech), using a fast-protein liquid chromatography system. The total protein concentration was estimated by the Bio-Rad protein assay (Bio-Rad, Richmond, CA), 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 (3, 17).

Determination of ß-lactamase kinetic parameters k_cat and K_m and of IC₅₀s. The Michaelis constant (K_m) and catalytic activity (k_cat) were determined with purified extracts, using a computerized microacidimetric method (16). The 50% inhibitory concentrations (IC₅₀s) were determined for clavulanic acid and tazobactam, as previously described (3), with 100 µM benzylpenicillin as the reporter substrate.

Nucleotide sequence accession numbers. The nucleotide sequences of the bla_TEM-151 and bla_TEM-152 genes have been assigned the accession numbers DQ834729 and DQ834728, respectively, in the GenBank nucleotide sequence database.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clinical strains and resistance phenotype. The clinical isolates CF1295 and CF1179 were isolated during the summer of 2004 from the same patient, who was hospitalized for a myelodysplastic syndrome in the hematology unit of the teaching hospital of Clermont-Ferrand, France. Before the isolation of strains, the patient received amoxicillin, an amoxicillin-clavulanate combination, and ceftazidime. Isolate CF1295 was isolated from feces after 3 weeks of hospitalization, and CF1179 was isolated from blood after 6 weeks of hospitalization. These isolates harbored the same ERIC2-PCR profile (data not shown). The patient was successfully treated for this bacteremia by imipenem associated with amikacin.

The isolates E. coli CF1295 and CF1179 harbored high levels of resistance against the penicillins, alone or in combination with clavulanate, and were susceptible to cefuroxime, cefoxitin, aztreonam, cefotaxime, cefepime, cefpirome, and imipenem. Isolate CF1179 was resistant to ceftazidime (MIC, 64 µg/ml), whereas isolate CF1295 had a ceftazidime MIC of 4 µg/ml. The double-disk synergy test, which was performed with a 30-mm interdisk distance, was positive for E. coli CF1179 but negative for E. coli CF1295. However, low synergy was observed for the latter isolate when the interdisk distance was decreased to 20 mm.

Isoelectric focusing and transfer experiment. The clinical isolates produced two ß-lactamases each, with pI values of 5.3 and 5.4 for isolate CF1295 and 5.4 and 5.7 for isolate CF1179. These values were compatible with those of TEM-derived enzymes. However, only the enzyme with a pI of 5.4 was transferred by mating-out experiments, using 32 µg/ml of amoxicillin instead of 0.5 µg/ml of ceftazidime, and it conferred a penicillinase-type resistance phenotype to the corresponding transconjugants, TC1295 and TC1179.

Two transformants, TF1295 and TF1179, exhibited resistance phenotypes similar to those of their parental strains, CF1295 and CF1179, respectively. They produced only one beta-lactamase each, with a pI of 5.3 for transformant TF1295 and of 5.7 for transformant TF1179.

PCR, cloning, and DNA sequencing. PCR experiments and direct DNA sequencing using TEM-A and TEM-B2 primers confirmed the presence of bla_TEM-1 in the two transconjugants producing the enzyme with a pI of 5.4. Cloning experiments were performed by PCR with the pBK-CMV plasmid vector to characterize the genes encoding the two other enzymes. Two clones, designated CL1295 and CL1179, produced one ß-lactamase each, with pIs of 5.3 and 5.7, respectively, and harbored resistance phenotypes similar to those of their parental isolates, CF1295 and CF1179, respectively.

The bla_TEM nucleic acid sequences revealed two new bla_TEM genes corresponding to the ß-lactamases with pIs of 5.3 (bla_TEM-151) and 5.7 (bla_TEM-152) (Table 1). They both presented a new promoter region which had a 9-bp deletion between positions 32 and 41, a T53A substitution, and a G162T substitution according to the Sutcliffe numbering system (30) (Fig. 1). In the coding region, the two genes differed from bla_TEM-1a by the same silent mutations, namely, A346G, C436T, T682C, and G925A. This pattern of silent mutations is characteristic of bla_TEM-1f (19).

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TABLE 1. Amino acid substitutions in TEM-151, TEM-152, TEM-28, TEM-29, and TEM-36 compared with TEM-1 sequence

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FIG. 1. Nucleotide sequences of promoters Pc and Pd of blaTEM-151 and blaTEM-152 compared with those of promoters P3, Pa/Pb, and P4. The promoter sequences are numbered according to the Sutcliffe numbering system (30). The –35 and –10 sequences of each promoter are boxed. Bold letters indicate mutations, deleted nucleotides are indicated by dashes, and the start codon is underlined.

They both presented four additional mutations, which resulted in four amino acid substitutions. Three of these mutations were present in both genes, including A407G, causing a Met69Val substitution; G693A, causing an Arg164His substitution; and A1022G, causing an Asn276Asp substitution. bla_TEM-151 presented a fourth mutation, C1047G, which led to the substitution Ala284Gly, and bla_TEM-152 presented a G917A mutation, which led to the substitution Glu240Lys. The resulting enzymes combined the mutations of the IRT enzyme TEM-36 (Met69Val and Asn276Asp) for both enzymes, of the ESBL TEM-29 (Arg164His) for TEM-151, and of the ESBL TEM-28 (Arg164His and Glu240Lys) for TEM-152.

ß-Lactam MICs. The clinical strain CF1179 and the corresponding E. coli DH5 clone, CL1179, presented higher levels of resistance to a piperacillin-tazobactam combination (16 to 128 versus 2 to 4 µg/ml), ceftazidime (64 to 512 versus 4 to 8 µg/ml), aztreonam (8 to 64 versus 2 µg/ml), and cefepime (2 to 16 versus 0.5 µg/ml) than did the clinical strain CF1295 and its corresponding clone, CL1295.

E. coli DH5 clones which produced the enzymes TEM-151 and TEM-152 and their parental enzymes (the ESBLs TEM-28 and TEM-29 and the IRT enzyme TEM-36) from the same genetic background were used to microbiologically compare the related TEM-encoding genes. The MICs of ß-lactams alone were similar for the TEM-151- and TEM-152-producing clones and the clones producing the corresponding parental ESBLs (Table 2), with the exception of the MICs of cefotaxime for the TEM-152- and TEM-28-producing clones (0.25 versus 4 µg/ml). The MICs of penicillin-clavulanate combinations were higher for the TEM-151- and TEM-152-producing clones than for those producing the corresponding ESBLs. These MICs were closely related to those for the clone producing the IRT enzyme TEM-36 (512 to 2,048 versus >2,048 µg/ml). In contrast, the TEM-151-producing clone exhibited a lower resistance level to piperacillin-tazobactam than those for clones producing TEM-152 and TEM-36 (2 versus 128 and >2,048 µg/ml).

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TABLE 2. MICs of ß-lactam antibiotics for E. coli CF1295, E. coli CF1179, and the recombinants E. coli DH5(pBK-TEM-151), E. coli DH5(pBK-TEM-152), E. coli DH5(pBK-TEM-29), E. coli DH5(pBK-TEM-28), E. coli DH5(pBK-TEM-36), E. coli DH5(pBK-TEM-1), and E. coli DH5(pBK-CMV)

Enzymatic parameters. TEM-151 and TEM-152 had lower hydrolytic activities (k_cat) against penicillins than did TEM-1 (k_cat, 51 to 306 versus 176 to 1,216 s^–1), as did their corresponding parental ESBLs, TEM-28 and TEM-29 (Table 3). Their K_m values were similar to those of TEM-1 (15 to 50 versus 15 to 55 µM). The K_m values and hydrolytic activities of TEM-151 and TEM-152 against oxyimino-ß-lactams were closer to those of their corresponding ESBLs, TEM-29 and TEM-28, than to those of TEM-1 and TEM-36. Overall, the catalytic efficiencies of TEM-152 and TEM-28 against oxyimino-ß-lactams were greater than those of TEM-151 and TEM-29.

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TABLE 3. Kinetic parameters of ß-lactamases TEM-151, TEM-152, TEM-29, TEM-28, TEM-36, and TEM-1a

In contrast, the hydrolytic activities of TEM-151 and TEM-152 against cephalothin were closer to that of the IRT enzyme TEM-36 (k_cat, 4 to 15 versus 18.4 s^–1) than to those of the ESBLs TEM-29 and TEM-28 (k_cat, 4 to 15 versus 126 to 194 s^–1). K_m values were similar for the five enzymes (205 to 335 µM).

TEM-152 was 2- to 50-fold more resistant to ß-lactamase inhibitors than was TEM-151 (IC₅₀, 0.4 to 1 versus 0.17 to 0.27 µM) (Table 4). The IC₅₀s of clavulanic acid for TEM-151 and TEM-152 were 2- to 50-fold higher than those for TEM-1, TEM-28, and TEM-29 (0.17 to 1 versus 0.02 to 0.08 µM) but 20- to 120-fold lower than that for TEM-36 (0.17 to 1 versus 20.5 µM). Similar results were obtained with tazobactam, which was 2- to 20-fold more efficient against TEM-1, TEM-28, and TEM-29 and 6- to 10-fold more efficient against TEM-36 than against TEM-151 and TEM-152.

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TABLE 4. IC50s of clavulanic acid and tazobactam for TEM-151, TEM-152, TEM-28, TEM-29, TEM-36, and TEM-1

Top Abstract Introduction Materials and Methods Results Discussion References

 
The clinical isolates CF1295 and CF1179 produced two novel ß-lactamases, TEM-151 and TEM-152, respectively, belonging to the CMT-type ESBL subgroup. They harbored the same ERIC2-PCR profile. These results suggest an evolution of these bla_TEM genes in the same strain from a common bla_TEM gene. This hypothesis is strengthened by the presence in both the TEM-151- and TEM-152-encoding genes of a novel and atypical TEM promoter and of the same pattern of silent mutations at positions 346, 436, 682, and 925. A classical P₃ promoter region was observed for the bla_TEM-1 gene observed in the two isolates, excluding a direct genetic linkage with the CMT-encoding genes.

The new promoter region presents a 9-bp deletion between positions 32 and 41 (Fig. 1). This deletion allows the association of the –35 sequence of the Pa promoter with a new –10 promoter sequence, 5'-TACGAT-3', which seems closer to the consensus sequence 5'-TATAAT-3' than to the classical –10 sequence of the Pa promoter (5'-TACGCT-3') (12). We suggest calling this new promoter Pc. This deletion also leads to a modification of the distance between the –35 and –10 sequences of the Pb promoter, which are separated by only 9 bp, which is incompatible with a functional promoter (12). However, the sequence 5'-GATAAT-3', which is close to the consensus sequence 5'-TATAAT-3' and separated from the –35 sequence of promoter Pb by the ideal distance of 17 bp, may be a functional –10 sequence. The –35 sequence of the Pb promoter and this novel –10 sequence may form a new promoter, Pd. Lastly, this promoter region also presents the substitution G162T, which leads to the promoter P₄ (18), and a new substitution, T53A.

Overall, the 5' regions of the two new bla_TEM genes may contain three promoters, namely, Pc, Pd, and P₄, and thereby confer a high level of transcription.

The gene bla_TEM-151 also harbored the mutation C1047G, which led to the substitution Ala284Gly. TEM-151 is the first described TEM-type ß-lactamase which harbors this substitution. Ala284 is located at a distance from the active site, on helix h11, and its side chain is oriented toward the solvent. Thus, the Ala284Gly mutation probably does not affect the hydrolytic activity.

As previously observed for other complex mutants belonging to the TEM subgroup (11, 21-24, 27), TEM-151 and TEM-152 were slightly less efficient against oxyimino-ß-lactams than the corresponding ESBLs TEM-28 and TEM-29 (k_cat, 1 to 16 versus 2.1 to 64 s^–1). However, they both significantly hydrolyzed ceftazidime, especially TEM-152, as assessed by their hydrolytic activities (k_cat, 5 to 16 s^–1). These two ß-lactamases behaved like ESBLs with regard to their hydrolytic activities. The presence of the additional Glu240Lys substitution in TEM-152 may explain the higher level of activity observed for ceftazidime and the lower K_m values observed for aztreonam, cefotaxime, and cefepime (15).

TEM-151 and TEM-152 were also less resistant to inhibitors than TEM-36, which presented the same IRT substitutions at positions 69 and 276. Such a loss of resistance to inhibitors was previously observed for all CMT-type ß-lactamases that presented ESBL hydrolytic activity (11, 22-24, 27). However, they were both more resistant to clavulanic acid and tazobactam than TEM-28 and TEM-29 and even the parental enzyme TEM-1. Their diminished susceptibility to inhibitors could explain the small decreases in the MICs of ß-lactam-clavulanic acid compared to those of the ß-lactams alone.

We report here the emergence in a single patient of two new TEM-type ß-lactamases belonging to the CMT subgroup of ESBLs. The two corresponding genes harbored a novel promoter region characterized by a 9-bp deletion which appeared to modify the classical Pa/Pb overlapping promoter. This work supports the currently observed evolution of the TEM-type ß-lactamases toward enzymes combining both hydrolysis of oxyimino cephalosporins and resistance to ß-lactamase inhibitors.

 
We thank Patricia Bradford for kindly providing us with E. coli strain 2300. We also thank Marlene Jan, Rolande Perroux, and Dominique Rubio for technical assistance and Sophie Quevillon-Cheruel for providing us with 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 by 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).

 
* Corresponding author. Mailing address: CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Bactériologie Clinique, Clermont-Ferrand F-63003, France. Phone: 33-473178150. Fax: 33-473277494. E-mail: frobin{at}chu-clermontferrand.fr

Published ahead of print on 12 January 2007.

This work is dedicated to the memory of Catherine Chanal.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, April 2007, p. 1304-1309, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01058-06
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