INTRODUCTION

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Gram-negative bacteria may exhibit reduced susceptibility to -lactam antibiotics by a number of mechanisms including reduced outer membrane permeability, target-site modification, and efflux of the -lactam out of the cell (20, 23). However, by far the most common mechanism of resistance is the enzymatic inactivation of the -lactam by a -lactamase (18). There are many types of -lactamases, which have been classified by their amino acid sequences and corresponding substrate profiles (6). The TEM-1 -lactamase belongs to a functional group of broad-spectrum enzymes that are inhibited by clavulanate (6). This group includes enzymes such as the SHV-1 and OHIO-1 -lactamases. Although the TEM-1 -lactamase does not usually provide protection against extended-spectrum cephalosporins such as ceftazidime and cefotaxime or -lactamase inhibitors like clavulanate and tazobactam (except in the case of TEM-1 overproduction), amino acid substitutions can alter the hydrolytic spectrum of the -lactamase to encompass these compounds.

Extended-spectrum TEM -lactamases (ESBLs) do not usually confer resistance to -lactamase inhibitors, suggesting that the two phenotypes may be incompatible. In support of this suggestion, Imtiaz et al. (15) have shown that introduction of an amino acid substitution (Arg164Ser) that confers on the TEM-1 -lactamase the ability to efficiently hydrolyze ceftazidime leads to the loss of clavulanate resistance when introduced into the inhibitor-resistant -lactamase TEM-31. However, recently a clinical Escherichia coli isolate that expressed a -lactamase, TEM-50 (CMT-1), that conferred low-level resistance both to -lactamase inhibitors and to extended-spectrum cephalosporins has been reported (22).

In order to investigate this phenomenon further we used site-directed mutagenesis of the TEM -lactamase encoding gene to introduce into ESBLs amino acid substitutions known to confer inhibitor resistance. We found that the different amino acid substitutions gave rise to enzymes that conferred different resistance phenotypes. None of the substitutions conferred high-level resistance to both -lactamase inhibitors and extended-spectrum cephalosporins, although the double amino acid substitution (Met69Leu, Asn276Asp) in the TEM-12 -lactamase did give rise to an ESBL with a moderate level of clavulanate resistance.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. E. coli CJ236 [dut-1 ung-1 thi-1 relA1; pCJ105 (Cm^r)] and E. coli MV1190 [(lac-proAB) thi supE (srl-recA)306::Tn10(Tet^r); (F':traD36 proAB lacI^qZM15)] were used in this study. The plasmid vector pTZ18U was used as the initial source of the bla_TEM gene. All bacteria were grown in Luria-Bertani (LB) broth or on LB agar (Oxoid, Basingstoke, United Kingdom) containing the appropriate antibiotic (chloramphenicol, 20 µg/ml; amoxicillin, 100 µg/ml; or tetracycline, 10 µg/ml).

Antibiotics and reagents. The following companies kindly supplied antibiotic powders of known potencies: Bristol Meyers Squibb (cefepime and aztreonam); American Cyanamid (piperacillin and tetracycline); Glaxo Group Research Ltd. (ceftazidime and cephaloridine); Roussel Laboratories Ltd. (cefotaxime and chloramphenicol); and SmithKline Beecham (amoxicillin, clavulanate, temocillin, and ticarcillin). Nitrocefin was obtained from Oxoid.

Susceptibility testing. MICs were determined by agar dilution on Diagnostic Sensitivity Test Agar (CM261; Oxoid) with an inoculum of about 10⁴ organisms per spot as described previously (24). E. coli NCTC 10418 was used as the control strain.

Site-directed mutagenesis. Site-directed mutagenesis was performed with the reagents contained within the Muta-Gene Phagemid In Vitro Mutagenesis kit (version 2) from Bio-Rad (Hemel Hempstead, United Kingdom). The procedures used in this kit are based on the method originally described by Kunkel et al. (17). Oligonucleotides were designed with the aid of oligonucleotide design software (PrimerSelect; DNAStar) and were based on the sequence of the bla_TEM-1 gene reported by Sutcliffe (27). The oligonucleotides were custom made by Pharmacia Biotech (St. Albans, United Kingdom) (Table 1).

DNA sequencing. In order to confirm that mutations had been introduced, plasmid DNA was extracted with a Qiagen QIAprep kit (Qiagen Ltd., Crawley, United Kingdom) and was sequenced in both directions with fluorescein-labelled primers (Table 1). DNA sequencing was performed with the reagents contained in a cycle sequencing kit (RPN 2438) from Amersham Life Sciences (Little Chalfont, United Kingdom) by following the manufacturer's instructions. The annealing temperature for the cycle sequencing reactions was 60°C, and the DNA sequence was determined with an automated DNA sequencer (Pharmacia Biotech).

Determination of IC₅₀s. Each strain was grown at 37°C in brain heart infusion broth (Oxoid) for 16 h, with shaking (200 rpm). The cells were harvested by centrifugation and were resuspended in 0.5 ml of sterile distilled water, and the -lactamase was released by sonication. Sonication was performed for 20 s with a W-385 sonicator (Heat Systems; Ultrasonics, Inc., Farmingdale, N.Y.) with the following settings: 5-s cycle time, 50% duty cycle, and a 1.5 output control setting. -Lactamase activity was measured by monitoring the rate of nitrocefin hydrolysis (10 µM) at 482 nm in a Biochrom 4060 spectrophotometer (Pharmacia Biotech). All assays were performed in 0.1 M phosphate buffer (pH 7.0) and at 37°C. In order to take into account the different levels of -lactamase activities within the samples the activity of each sample was standardized to give an absorbance change of 0.15 per min. Samples were preincubated for 10 min at 37°C with various concentrations (0.01 to 50 µM) of the -lactamase inhibitor before the -lactamase activity was determined with nitrocefin (10 µM) as the reporter substrate. The concentration of -lactamase inhibitor required to inhibit 50% of the -lactamase activity (IC₅₀) was then determined graphically.

	RESULTS

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Mutagenesis. Phagemid pTZ18U conveniently encodes a bla_TEM gene, and use of pTZ18U thus negates the need to subclone the bla_TEM gene from another source. However, the bla_TEM gene from pTZ18U is not identical to bla_TEM-1 as the result of two nucleotide changes, G²⁴⁴A and C⁵⁴⁵T, that were introduced to remove PstI and HincII restriction sites, respectively. While the resulting amino acid substitutions, Ile84Val and Ala184Val, have been regarded as neutral (22a), Chaibi et al. (8) have demonstrated that the catalytic efficiency of the "artificial" TEM -lactamase was one-half to one-third lower than that of the TEM-1 -lactamase. Consequently, in this study we initially converted the artificial bla_TEM into bla_TEM-1 and subsequently used this gene as the template for the construction of the TEM mutants. Four ESBL enzymes (TEM-12, TEM-15, TEM-19, and TEM-26) were constructed together with three -lactamase-inhibitor-resistant mutants (TEM-31, TEM-33, and TEM-35) (Table 2). In order to investigate whether the amino acid substitutions found in -lactamase inhibitor-resistant mutants could confer inhibitor resistance if introduced into ESBL enzymes, the three sets of amino acid substitutions that confer inhibitor resistance were engineered into the extended-spectrum antibiotic-resistant TEM -lactamases by altering the gene-coding sequence. The amino acid substitutions corresponded to those found in the TEM-31 (Arg244Cys), TEM-33 (Met69Leu), and TEM-35 (Met69Leu and Asn276Asp) -lactamases. In all cases the introduced nucleotide changes in the bla_TEM gene were confirmed by DNA sequencing.

View this table: [in this window] [in a new window]   TABLE 2.   MICs of -lactams and -lactamase inhibitor combinations for E. coli MV1190 producing the TEM-1 and mutant TEM -lactamases

Phenotypic characterization of TEM-1 -lactamase and mutant derivatives. (i) TEM-1 and ESBL enzymes. The MICs of ampicillin and ticarcillin in the presence of clavulanate (2 µg/ml) and piperacillin in the presence of tazobactam (4 µg/ml) for E. coli MV1190 expressing the TEM-1 -lactamase were relatively high (Table 2). This could be accounted for by the large quantity of the TEM-1 -lactamase expressed as a result of the high copy number of the pTZ18U plasmid carrying the bla_TEM-1 gene (Table 3). Despite this, because the TEM-1 -lactamase and the mutant enzymes in this study shared the same genetic background, comparisons between the mutant enzymes and the TEM-1 -lactamase could still be made.

View this table: [in this window] [in a new window]   TABLE 3.   -Lactamase activities and clavulanate and tazobactam IC50s for TEM-1 -lactamase and TEM mutant enzymes

(ii) Inhibitor-resistant enzymes. The substitutions Arg244Cys, Met69Leu, and Met69Leu plus Asn276Asp in the TEM-1 -lactamase gave rise to enzymes with resistance to clavulanate combined with resistance to amoxicillin or ticarcillin. Substitution of a Cys residue at position 244 of the TEM-1 -lactamase also resulted in an enzyme (TEM-31) that conferred lower levels of resistance to penicillins and cephaloridine than the levels conferred by TEM-1 (Table 2). In contrast, a single Met69Leu substitution and a double substitution, Met69Leu plus Asn276Asp, in the TEM-1 -lactamase did not greatly affect the MICs of piperacillin, although a fourfold reduction in resistance to cephaloridine was observed (Table 2). None of the inhibitor-resistant enzymes conferred resistance to extended-spectrum cephalosporins, aztreonam, or temocillin.

(iii) Substitution of Cys for Arg at position 244 in ESBLs. Introduction of the Arg244Cys substitution into the ESBL enzymes had an effect similar to that in TEM-1. Like the TEM-31 -lactamase, the resulting hybrid enzymes conferred lower levels of resistance to penicillins and cephaloridine than their respective parent enzymes did. However, the MICs of penicillin-inhibitor combinations were elevated for the strain with TEM-26 plus the Arg244Cys substitution. In addition, the amino acid substitution resulted in enzymes that either had lost (TEM-12, TEM-15, and TEM-19) or had a reduced ability (TEM-26) to confer resistance to ceftazidime. The MICs of cefepime and aztreonam were reduced 4- and 16-fold, respectively, for E. coli MV1190 expressing the hybrid TEM-26 -lactamase compared to the MICs for the strain expressing the TEM-1 -lactamase. All the hybrid enzymes with the Arg244Cys substitution were found to be more resistant to clavulanate and tazobactam inhibition than the TEM-1 -lactamase, with the IC₅₀s for the hybrid enzymes being comparable to those for the naturally occurring inhibitor-resistant TEM -lactamases (Table 3).

(iv) Substitution of Leu for Met at position 69 in ESBLs. The IC₅₀s of clavulanate for the parental ESBL enzymes were lower than those of the TEM-1 -lactamase, indicating that the ESBL enzymes were more susceptible to clavulanate inhibition than the TEM-1 -lactamase. Introduction of a Leu residue at position 69 in the ESBLs resulted in hybrid enzymes that conferred increased levels of resistance to both clavulanate and tazobactam compared to the level of resistance conferred by their respective parental ESBL enzymes (Table 3). In the case of the TEM-12 -lactamase, the amino acid substitution gave rise to a hybrid enzyme that was less susceptible to clavulanate inhibition than the TEM-1 -lactamase. For the other ESBLs, however, the substitution resulted in hybrid enzymes for which clavulanate IC₅₀s were similar to those for the TEM-1 -lactamase. Tazobactam was found to be equally effective against the TEM-1 -lactamase and the hybrid ESBLs with a Gly238Ser substitution (TEM-15 and TEM-19). However, tazobactam was less effective against hybrid ESBLs with the Arg164Ser substitution (TEM-12 and TEM-26) (Table 3).

(v) Substitution of Leu for Met at position 69 and Asp for Asn at position 276 in ESBLs. Introduction of the double amino acid substitution Leu-69 and Asp-276 in the ESBLs gave rise to hybrid enzymes that were more resistant to clavulanate inhibition than hybrid ESBL enzymes with a single Leu-69 substitution. In the case of the TEM-12 -lactamase, this gave rise to an enzyme for which the IC₅₀ of clavulanate was similar to that for the inhibitor-resistant enzyme TEM-33. For the other hybrid ESBLs, however, the IC₅₀s of clavulanate were intermediate between that for the TEM-1 -lactamase and those for the inhibitor-resistant enzymes. The IC₅₀s of tazobactam were similar for the hybrid enzymes with single or double amino acid substitutions. The double substitution did not greatly affect the ability of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was apparent with E. coli MV1190 expressing the hybrid derivatives of the TEM-19 and TEM-26 -lactamases. Two of the hybrid enzymes, TEM-12 and TEM-26, showed a markedly reduced susceptibility to ceftazidime combined with their susceptibility to clavulanate. As a consequence of the double amino acid substitution, extended-spectrum resistant variants of TEM-12, TEM-15, and TEM-26 -lactamases that also conferred increased levels of resistance to -lactamase inhibitors were constructed. However, the levels of clavulanate resistance conferred by the hybrid ESBLs were not as high as that conferred by the corresponding inhibitor-resistant TEM -lactamase TEM-35.

	DISCUSSION

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Substitution of Cys for Arg at position 244. In this study we replaced the Arg at position 244 in the TEM-12, TEM-15, TEM-19, and TEM-26 -lactamases with a Cys residue in order to investigate whether the amino acid substitution would give rise to inhibitor-resistant ESBLs. In each case the substitution conferred increased levels of resistance to -lactamase inhibitors, but the substitution also gave rise to enzymes that conferred lower degrees of resistance to penicillin and cephalosporins. Both these observations are consistent with a disruption of the Arg244 hydrogen-bonding arrangement predicted to occur in the TEM-1 -lactamase (26, 30). Since the Cys residue at position 244 would be unable to form a hydrogen bond to the common carboxylate group of -lactam antibiotics, this probably explains why the MICs of both penicillins and cephalosporins were affected by the amino acid substitution. This would be especially pertinent if, as suggested by Zafaralla et al. (30), the binding energy of Arg244 is used to lower the activation energy of the hydrolytic reaction.

Substitution of Leu for Met at position 69. Unlike the TEM-1 -lactamase, in which substitutions of Leu, Val, or Ile for the Met at position 69 have all given rise to inhibitor-resistant enzymes (9, 11, 25, 31), substitution of a Leu residue for the Met residue at this position in the four ESBLs did not give rise to clavulanate-resistant enzymes. This probably can be explained by a different binding arrangement of the clavulanate molecule in the active site of the hybrid ESBL enzymes compared to that in the TEM-1 -lactamase. As noted previously the parental ESBLs were more sensitive to clavulanate inhibition than the TEM-1 -lactamase, suggesting that alterations within the active site enhanced the inhibitory action of clavulanate. Although the hybrid ESBLs were not resistant to clavulanate, they were less sensitive to clavulanate inhibition than their respective parent enzymes were. The substitutions at position 69 are thought to cause slight alterations to the active-site structure of the TEM-1 -lactamase, resulting in deformation of the oxyanion hole and a less favorable binding orientation of the clavulanate molecule (10). This suggests that the clavulanate molecule still interacts with the oxyanion hole but possibly in a different manner.

Substitution of Asp for Asn at position 276 plus Leu for Met at position 69. Amino acid substitutions at position 276 have been found naturally only in combination with changes at position 69 in the TEM -lactamase (5, 13, 22, 31), although the change can confer inhibitor resistance in the absence of a substitution at position 69 (7, 21, 28). Recently, Sirot et al. (22) have reported on a natural variant of the TEM-15 -lactamase, designated TEM-50, with amino acid substitutions, Met69Leu and Asn276Asp, found in the inhibitor-resistant -lactamase TEM-35. An E. coli strain expressing the TEM-50 -lactamase displayed susceptibilities to -lactams, including ceftazidime and cefotaxime, that were between those for strains expressing the TEM-15 or TEM-35 -lactamases. In our study we artificially constructed the TEM-50 -lactamase together with mutants of the TEM-12, TEM-19, and TEM-26 -lactamases. In contrast to Sirot et al. (22), we found that the MIC of ceftazidime for E. coli MV1190 expressing the TEM-50 -lactamase was only twofold lower than the MIC of ceftazidime for the same strain expressing the TEM-15 -lactamase. A possible explanation for this difference may have been the exceptionally high level of -lactamase expressed from the high-copy-number plasmid pTZ18U harboring the TEM-coding gene used in this study. Such high levels of -lactamase expression may have masked small differences in hydrolytic activities between the enzymes.