Journal of Antimicrobial Chemotherapy (2000) 45, 101-104
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Detrimental effect of the combination of R164S with G238S in TEM-1 ß-lactamase on the extended-spectrum activity conferred by each single mutation
a Laboratory of Antimicrobial Agents, Department of Microbiology, Medical School, University of Athens, M. Asias 75, Athens 11527; b Department of Bacteriology, Hellenic Pasteur Institute, Athens, Greece
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Abstract |
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The non-naturally occurring TEM-1 ß-lactamase mutant R164S:G238S, as well as the R164S (TEM-12) and G238S (TEM-19) ß-lactamases, were constructed and expressed in Escherichia coli under isogenic conditions. Comparison of susceptibilities to ß-lactam antibiotics and substrate profiles showed that the combination of R164S with G238S drastically reduced the extended-spectrum activity of the respective single mutants.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The emergence of extended-spectrum ß-lactamases (ESBLs) was observed soon after the introduction of broad-spectrum cephalosporins in the clinical setting. Most ESBLs are derivatives of the ubiquitous plasmid-mediated penicillinases TEM-1/2 and SHV-1.1 In TEM-1/2-derived ESBLs, one or more substitutions at critical amino acid residues are responsible for the expansion of hydrolytic activity to oximino ß-lactams.2 Substitution of Ser or His for Arg-164 is the most common change observed in these TEM variants. In addition, replacement of Gly-238 by Ser appears in various TEM-derived ESBLs. An R164S:G238S TEM ß-lactamase has not been detected in the clinic to date. Only in TEM-8, a descendant of TEM-2, do the substitutions Arg-164
Ser and Gly-238Ser co-exist along with a replacement of Glu-104 by a lysine.3 This triple mutant possesses high extended-spectrum ß-lactamase activity.4 In the present study, we compared the ß-lactam resistance levels conferred on Escherichia coli by the naturally occurring TEM-1 derivatives TEM-12 (R164S) and TEM-19 (G238S), with those of the mutant R164S:G238S constructed in vitro. The substrate and inhibition profiles of the latter enzyme were also evaluated and compared with those of the respective single mutant ß-lactamases.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Bacterial strains and plasmids
E. coli XL1 Blue (F'::Tn10 proA+B+ lacIq 
(lacZ)M15/ recA1 endA1 gyrA96 (Nalr) thi hsdR17 (rk–mk+) supE44 relA1 lac –1) (Stratagene, La Jolla, CA, USA) was used as host for plasmid DNA manipulations. E. coli C600 (F– e14-(McrA–) thr-1 leuB6 thi-1 lacY1 supE44 rfbD1 fhuA21) was used in susceptibility testing and ß-lactamase studies. Plasmid pBCSK(+) (Stratagene) was used for cloning and site-directed mutagenesis. Plasmid pBGTEM-1 was used as the source of blaTEM-1.5
Site-directed mutagenesis
An EcoRI–SalI fragment (2.2 kb) from plasmid pBGTEM-1, containing the blaTEM-1 gene, was cloned into the multicloning site of pBCSK(+). The resulting plasmid was used for the construction of mutants. The genes encoding the three mutant ß-lactamases (R164S, G238S and R164S:G238S) were obtained using a PCR-based sitespecific mutagenesis technique as described previously.6 Two pairs of primers were used for each substitution. One primer for each pair contained a single base mismatch to direct mutagenesis. The mutagenic primers were 20–22 bases long and the mismatch was close to the centre of the sequence. The primers were prepared in an Applied Biosystems (Foster City, CA, USA) DNA synthesizer. The complete nucleotide sequences of the mutant genes were determined using a Sequenase 2.0 kit (United States Biochemical Corp., Cleveland, OH, USA) and a set of universal and custom synthesized oligonucleotides. The plasmids encoding TEM-1 and mutant ß-lactamases were used to transform E. coli competent cells according to standard techniques.
Susceptibility testing
Susceptibility to ß-lactam antibiotics was evaluated using an agar dilution technique in accordance with the guidelines of the National Committee for Clinical Laboratory Standards.7 E. coli strain ATCC 25922 was included as control. Cefepime and cefprozil were provided by Bristol-Myers-Squibb, Athens, Greece. Tazobactam was a gift from Wyeth Hellas S.A., Athens, Greece. The other ß-lactams were purchased from commercial sources.
ß-Lactamase studies
Bacterial mid-log phase cultures in tryptone-soy broth (Unipath Ltd, Basingstoke, UK) were harvested and washed twice in phosphate-buffered saline (pH 7.0). ß- Lactamases were released by mild ultrasonic treatment of cells suspended in phosphate buffer (PB, 100 mM, pH 7.0). The extracts were clarified by ultracentrifugation and dialysed overnight against PB. The protein content was measured with a Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The ß-lactamase activity of the extracts was quantified using nitrocefin (Unipath) as substrate. Results were expressed as units of activity. One unit was the amount of enzyme hydrolysing 1 µmol of substrate/min/mg of protein at pH 7.0 and 37°C. The maximum rates of hydrolysis of various ß-lactams were estimated by UV spectrophotometry as described previously8 and expressed relative to that of penicillin G, which was set at 100. Inhibitory activities of clavulanate and tazobactam were also assessed as described in a previous study,9 using nitrocefin as the reporter substrate. The 50% inhibitory concentrations (IC50s) were determined from plots of the inhibitor concentration versus percentage inhibition. Isoelectric focusing (IEF) was performed in polyacrylamide gels containing ampholytes (pH range 3.5–9.5) (Pharmacia-LKB Biotechnology, Uppsala, Sweden).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Upon IEF, the mutants R164S and R164S:G238S displayed a lower isoelectric point (pI 5.25) than the TEM-1 and G238S ß-lactamases (pI 5.4), due to replacement of the positively charged Arg by the weakly polar Ser (data not shown). Quantification of enzymic activity using nitrocefin gave the following results: TEM-1, 18.5 U; R164S, 24.1 U; G238S, 25.0 U and R164S:G238S, 17.7 U, indicating that the enzymes were expressed in comparable amounts. Since blaTEM-1 and its mutant derivatives had all been introduced into the same genetic background, MIC values allowed an acceptable comparison of the activities of the respective enzymes. This approach has also been used in previous studies.5,8
MICs of ß-lactams are presented in Table I
. All three mutant ß-lactamases conferred similar levels of resistance to the penicillins and penicillin–inhibitor combinations tested. Compared with TEM-1, the mutant enzymes were less active against penicillin–inhibitor combinations. The most marked reduction was observed with the MICs of ampicillin–sulbactam and piperacillin–tazobactam. Since the MICs of penicillins remained largely unaffected, this effect must have been due to increased susceptibility of mutant ß-lactamases to inhibitors. Indeed, IC50 values of inhibitors were clearly reduced for the mutant enzymes with respect to TEM-1 (Table II).
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The less active ß-lactamase against narrow-spectrum cephalosporins cephalothin and cefprozil, was the double mutant R164S:G238S (Table I
). As is also shown in Table II, R164S:G238S displayed the lowest relative rate of hydrolysis of cephalothin among the three ß-lactamases tested.
Substitution of Ser for Arg-164 in TEM-1 results in an ESBL (TEM-12) which confers high-level resistance to ceftazidime. Introduction of the G238S mutation in TEM-12 reversed this effect. Both the MIC of ceftazidime (Table I) and its relative hydrolysis rate (Table II) were reduced to low levels, comparable to those observed with the single mutant G238S. On the other hand, the ability of the double mutant to hydrolyse cefotaxime was even lower than that of G238S. In fact, the resistance to cefotaxime conferred by R164S:G238S was reduced to the level conferred by TEM-1 (Table I) and hydrolysis of the antibiotic was hardly detectable (Table II). The MICs of aztreonam, cefepime and cefpirome did not differ significantly for the strains producing the three TEM mutant ß-lactamases (Table I).
These results show that the combination of substitutions R164S and G238S is, in general, neutral or detrimental with respect to the single mutants. In TEM-1, Arg-164 interacts strongly with the conserved Asp-179 forming the neck of the 
loop. Weakening of this linkage by substitution of Ser for Arg-164 may render this structure more flexible thus allowing accommodation of ß-lactams with bulky substituents such as ceftazidime. Also, replacement of Gly-238 by Ser may expand the active site cavity owing to development of steric conflicts with adjacent residues (reviewed in references 2 and 10). It could be hypothesized that a combination of these effects distorts the active site in such a way as to impede hydrolysis of oximino cephalosporins. On the other hand, R164S:G238S retains hypersusceptibility to mechanism-based inhibitors, which is characteristic of most ESBLs.
These properties of the R164S:G238S TEM ß-lactamase may explain its apparent absence in vivo. Nevertheless, R164S:G238S combined with E104K results in a ß- lactamase (TEM-8) with high extended-spectrum activity. According to a scheme proposed by Du Bois et al.,4 the most likely sequence of mutational events leading to emergence of TEM-8 is R164SE104KG238S. The low hydrolytic efficiency of the double TEM mutant R64S:G238S against the most commonly used broadspectrum cephalosporins, as observed here, would therefore seem to be in agreement with the above mentioned scheme.
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Notes |
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* Corresponding author. Tel: +350-1-778-5638; Fax: +350-1-770-9180; E-mail: tzouvel{at}cc.uoa.gz
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Amyes, S. G. B. (1998). Genes and spectrum: the theoretical limits. Clinical Infectious Diseases 27, Suppl. 1, 21–8.
2 . Knox, J. R. (1995). Extended-spectrum and inhibitor-resistant TEM-type ß-lactamases: mutations, specificity, and threedimensional structure. Antimicrobial Agents and Chemotherapy 39, 2593–601.[Web of Science][Medline]
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Chanal, C., Poupart, M.-C., Sirot, D., Labia, R., Sirot, J. & Cluzel, R. (1992). Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 ß-lactamase genes. Antimicrobial Agents and Chemotherapy 36, 1817–20.
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Du Bois, S. K., Marriott, M. S. & Amyes, S. G. (1995). TEMand SHV-derived extended-spectrum ß-lactamases: relationship between selection, structure and function. Journal of Antimicrobial Chemotherapy 35, 7–22.
5 . Blazquez, J., Morosini, M. I., Negri, M. C., Gonzalez-Leiza, M. & Baquero, F. (1995). Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM ß-lactamases. Antimicrobial Agents and Chemotherapy 39, 145–9.[Abstract]
6 . Nelson, R. M. & Long, G. L. (1989). A general method of sitespecific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Analytical Biochemistry 180, 147–51.[Web of Science][Medline]
7 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A3. NCCLS, Villanova, PA.
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Giakkoupi, P., Miriagou, V., Gazouli, M., Tzelepi, E., Legakis, N. J. & Tzouvelekis, L. S. (1998). Properties of mutant SHV-5 ß-lactamases constructed by substitution of isoleucine or valine for methionine at position 69. Antimicrobial Agents and Chemotherapy 42, 1281–3.
9 . Tzouvelekis, L. S., Gazouli, M., Prinarakis, E. E., Tzelepi, E. & Legakis, N. J. (1997). Comparative evaluation of the inhibitory activities of the novel penicillanic acid sulfone Ro 48-1220 against ß-lactamases that belong to groups 1, 2b and 2be. Antimicrobial Agents and Chemotherapy 41, 475–7.[Abstract]
10 . Matagne, A., Lamotte-Brasseur, J. & Frere, J.-M. (1998). Catalytic properties of class A ß-lactamases: efficiency and diversity. Biochemical Journal 330, 581–98.
Received 14 December 1998; returned 26 March 1999; revised 5 April 1999; accepted 16 June 1999
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