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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Introduction |
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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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E. coli XL1 Blue (F'::Tn10 proA+B+ lacIq (lacZ)M15/ recA1 endA1 gyrA96 (Nalr) thi hsdR17 (rkmk+) 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 EcoRISalI 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 2022 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 3.59.5) (Pharmacia-LKB Biotechnology, Uppsala, Sweden).
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Results and discussion |
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MICs of ß-lactams are presented in Table I. All three mutant ß-lactamases conferred similar levels of resistance to the penicillins and penicillininhibitor combinations tested. Compared with TEM-1, the mutant enzymes were less active against penicillininhibitor combinations. The most marked reduction was observed with the MICs of ampicillinsulbactam and piperacillintazobactam. 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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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 R164SE104K
G238S. 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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References |
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2
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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.
8
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Received 14 December 1998; returned 26 March 1999; revised 5 April 1999; accepted 16 June 1999