a UMR 175, CNRS-MNHN, 6 Rue de l'Université, 29000 Quimper b Laboratoire de Bactériologie, Faculté de Médecine, 28 Place Henri-Dunant, 63001 Clermont-Ferrand Cedex c CHU Cochin, Laboratoire de Bactériologie, 75014 Paris, France
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Abstract |
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Introduction |
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This review attempts to summarize and to discuss the many available data concerning the IRT ß-lactamases.
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Phenotypic characteristics |
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The IRT phenotype was characterized by resistance to ß-lactam-clavulanate combinations with susceptibility to cephalosporins, which is not observed in the overproduced penicillinase phenotype. A number of studies have tried to determine the resistance pattern which allows a reliable detection of IRT-producing strains.22,23,24,25 When susceptibilities to amoxycillin, amoxycillin plus clavulanate, ticarcillin, ticarcillin plus clavulanate, piperacillin, piperacillin plus tazobactam and cephalothin were evaluated by a disc diffusion method with the critical diameters interpreted according to French guidelines,26 the phenotype amoxycillin-resistant, ticarcillin-resistant, amoxycillin or ticarcillin plus clavulanate-intermediate or -resistant and cephalothin-susceptible allowed the detection of about 87% of E. coli strains producing an IRT ß-lactamase alone or in association with a parental TEM ß-lactamase.24 However, this phenotype did not allow the discrimination of OXA-producing strains, which appeared indistinguishable from IRT strains (Table II). Libert et al.27 proposed the measurement of the inhibition diameters to cefepime, mecillinam and ceftazidime for the routine differentiation of strains producing IRT and OXA enzymes.
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ß-Lactam susceptibility of IRT-producing E. coli strains
The susceptibility of 98 IRT-producing isolates of E. coli, collected in 1993 in the teaching hospital of Clermont Ferrand (France), was assessed by determination of ß-lactam MICs by the agar dilution method (Table III). The isolates selected produced nine different IRT enzymes: TEM-30/IRT-2 (n = 19), TEM-32/IRT-3 (n = 4), TEM-33/IRT-5 (n = 16), TEM-34/IRT-6 (n = 13), TEM-35/IRT-4 (n = 13), TEM-36/IRT-7 (n = 11) TEM-37/IRT-8 (n = 19), TEM-38/IRT-9 (n = 1), and TEM-39/IRT-10 (n = 2).
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It is noteworthy that the piperacillintazobactam combination showed the
best bacteriostatic effect against the isolates producing IRT enzymes. However, the cidal effect
of
this combination was not obtained (1% of survivors at 6 h) with concentrations of 1
x MIC, 2 x MIC, and 4 x MIC of piperacillin with tazobactam (4 mg/L), and
regrowth was observed at 24 h (data not shown).
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Genetic characteristics |
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These data are clear evidence for the convergent evolution of IRT enzymes because mutations have occurred independently on different gene frameworks (ancestor sequence), but all confer an identical IRT phenotype in response to selective pressure imposed by the clinical use of ß-lactamase inhibitors.13
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Biochemical data |
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Kinetic parameters (kcat and Km) and catalytic efficiency (kcat/Km) of TEM-1 and IRTs are shown in Table VI. Generally, most IRT ß-lactamases have lower catalytic efficiency values for all substrates than those of TEM-1. This results from a decrease of kcat values and an increase of Km values. The mutants which have one amino acid substitution at position 69 show catalytic efficiency values higher than those of other IRT mutants. This indicates the important contribution of residues 244, 275 and 276 in the enzymesubstrate interaction. It is noteworthy that all IRTs have high Km values for ticarcillin (a carboxypenicillin). Similar results are obtained with carbenicillin, another carboxypenicillin (data not shown). For all IRTs this characteristic may be related to electrostatic interactions, as they have low Km values for carfecillin (a phenyl ester of carbenicillin) (data not shown). The structures of ticarcillin, carbenicillin and carfecillin are shown inFigure 1. For the mutants at position 69, modelling suggests repulsion between the carboxylate of the side chain of ticarcillin (or carbenicillin) and a carboxylate of side chains of Glu-104 and Glu-240.33 Other residue(s) required for these electrostatic interactions are still to be found.
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The structures of ß-lactamase inhibitors are shown inFigure 1. All the IRTs have IC50 and Ki values for ß-lactamase inhibitors higher than those of TEM-1 (Table VII). Those with mutations at position 69 exhibit lower IC50 and Ki values than those of other mutants. Sulbactam is a poor inhibitor of all the IRT ß-lactamases (high IC50 and Ki values), whereas tazobactam was the most active inhibitor (low IC50 and Ki values), except against those mutations at position 69, indicating a more favourable interaction with the triazole ring-substituted penicillanic acid sulphone than with the naked sulphone. This finding is consistent with work published recently by Bonomo et al.34 This study complements and extends previous investigations in which clavulanic acid and tazobactam have been shown to be more effective ß-lactamase inhibitors than sulbactam against extended-spectrum and conventional-spectrum enzymes and that clavulanic acid had activities equivalent to those of tazobactam.35,36
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Relationship between structure and function |
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Residue 69 is rather variable in size and character among class A ß-lactamases
but is always in high conformational energy.39,40,43,44 The importance of this amino acid is not its closeness to the reactive Ser-70, but
rather the position of its side chain behind the ß-3 and ß-4 strands. It is adjacent to the
oxyanion binding pocket formed by the amides of Ser-70 and Ala-237. The molecular modelling
showed that the methyl of Val-69 (C1) and Ile-69 (C
2) produced steric constraints
with the side chain of Ser-70 and Asn-170.33 The
hydrophobicity could be the main factor responsible for the kinetic properties of the
variant Met-69
Leu (TEM-33/IRT-5), as no steric effects could be detected by molecular
modelling.33 Thus, hydrophobicity and steric constraints
could be combined in the variants Met-69
Val (TEM-34/IRT-6) and Met-69
Ile
(TEM-40/IRT-11). In addition, we speculate that residue 69 could interfere with the guanidinium
group of Arg-244, as previously suggested for the Met-69
Ile mutant of the SHV-type
OHIO-1
ß-lactamase.45
Residue 165
Located at the beginning of the -loop (position 161 to 179), the side chain of
this residue is solvent-oriented. A change of Trp-165
Arg is found in the TEM-39/IRT-10
ß-lactamase, but associated with two other substitutions at positions 69 and 276. The
TEM-type variant Trp-165
Arg made by site-directed mutagenesis exhibited a slight
decrease to
the inhibitory effect of clavulanic acid.46 Molecular
modelling suggests that the side chain of Arg-165 is able to form a salt bond with the
-loop
Glu-168 (unpublished data).
Residue 182
Located just before the -8 helix (position 183 to 195), this residue is rather
far from the binding site. A threonine is present in the TEM-32/IRT-3 ß-lactamase.
However, the enzyme contains a second change at position 69 that was shown to be the dominant
factor in the resistance to ß-lactamase inhibitors.16
Molecular modelling showed a novel hydrogen bond between the hydroxyl of Thr-182 and the
carbonyl of the amide bond of Glu-64.47 That strengthens
the dense hydrogen bond network that stabilizes the active site, and therefore was expected to be
responsible for the increase in the catalytic activity of the TEM-32/IRT-3 ß-lactamase
compared with that of TEM-40/IRT-11. Moreover, Huang & Palzkill48 have recently demonstrated that the addition of the Met-182
Thr substitution to
the TEM-1 variant Met-69RIle increased the stability of the Met-69
Ile enzyme. The
Met-182
Thr substitution may have been selected in natural isolates as a suppressor of
folding or stability defects resulting from mutations associated with drug resistance.48 It is noteworthy that with the sequences of 28 class A
ß-lactamases previously aligned, TEM-1 was the sole protein exhibiting a Met at position
182, a position that generally has hydrogen bond-forming residues such as threonine, serine or
cysteine.49
Residue 244
Arg-244 is a relatively conserved residue on the ß-4 strand of class A ß-lactamases, but when absent a basic residue (Arg or Lys) is found at position 220 or at position 276.38,49,50 It is anchored in place by two hydrogen bonds to Asn-276. Via a well-ordered, structurally conserved water molecule, it may interact with the C-3 (C-4) carboxylic acid group of ß-lactams.51,52,53,54 However, Delaire et al.55 believe there are no direct interactions with the acid group and that the role of Arg-244 is to destabilize the enzyme product complex and optimize the turnover rate.
When Arg-244 is replaced by an amino acid with a short side chain such as
cysteine, serine or histidine, the enzymesubstrate interaction is modified and affinity for
the substrate decreases (Table VI). Moreover, the shorter side chains of
these residues would be unable to activate the water molecule involved in the inactivation
process of clavulanate.51 Sulbactam and
tazobactam are thought to use a different mechanism and are not dependent on the structurally
conserved water molecule.56 An unexpected finding that
the doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics
of the Ser-164 mutant enzyme, e.g. enhanced activity against ceftazidime and sensitivity to
inactivation by clavulanate, is perhaps due in part to structural changes resulting from the
disruption of the -loop.57 Arg-244 or a water
molecule co ordinated to its side chain also plays an essential role in the carbapenem
tautomerization in the ß-lactamase TEM-1 active site.58,59
Residue 261
Located at the ß-5 strand, its side chain is buried at the hydrophobic region far
from the active site. The amino acid substitution Val-261Ile is found in TEM-58,15 but is associated with the change Arg-244
Ser
which is involved in the resistance of TEM-30/IRT-2 to ß-lactamase inhibitors.
Residue 275
Located at the C-terminal of the -11 helix, its side chain is in close vicinity to the
guanidinium group of Arg-244. Substitution of Arg-275 by leucine or glutamine is found in the
ß-lactamases TEM-38/IRT-9 and TEM-45/IRT-14, respectively. However, these enzymes
contain a second change at position 69 (Val or Leu). Kinetic study of the Arg-275
Leu
variant of the TEM-type ß-lactamase has shown the involvement of this change in the
resistance to inactivation by clavulanic acid.60
This could be related to electrostatic interactions with Arg-244 and/or to a possible displacement
of the water molecule involved in the inactivation.
Residue 276
The partially exposed side chain at residue 276 is on the C-terminal -11
helix. In the TEM-1 ß-lactamase the carbonyl group of Asn-276 accepts two hydrogen
bonds
from Arg-244 that orient the guanidinium group. The amino acid substitution Asn-276
Asp
is found in the natural variants TEM-35/IRT-4, TEM-37/IRT-8 and TEM-39/IRT-10, but
associated with another change at position 69. Brun et al.,10 by comparing the kinetic properties of the TEM-35/IRT-4 enzyme and the
Met-69
Leu variant of the TEM-type enzyme, have suggested a direct or an indirect role of
Asp-276 in the catalytic mechanism. Thus, the TEM-type variant Asn-276
Asp made
by site-directed mutagenesis exhibited decreased affinity and catalytic efficiency for
ß-lactam substrates, as well as a 20-fold higher Ki for clavulanate.61 The resistance to the inactivation process of clavulanic
acid could be linked to electrostatic interactions with Arg-244 and/or to a
possible displacement of the water molecule involved in the inactivation. From an evolutionary
point of view, it is interesting to note that the Staphylococcus aureus enzyme PC1 is
highly sensitive to clavulanic acid, although it has an aspartic acid at position 276 as in Streptomyces albus G and other Gram-positive enzymes.38,49,50
Nevertheless, a full clavulanic acid molecule is bound to the ß-lactamase PC1 active site,62 whereas only a part of the inhibitor molecule is bound to
the ß-lactamase TEM-1 active site.63
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Conclusions |
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Genetic studies argue in favour of the convergent evolution of the blaIRT genes. It seems that such evolution of the parent TEM ß-lactamase to resistance to ß-lactamase inhibitors involves both forward and backward mutations,66 as previously suggested for TEM- and SHV-derived extended-spectrum ß-lactamases.67 Recently the extended-spectrum ß-lactamases TEM-AQ and TEM-50 (CMT-1) derived from TEM-1, which also have reduced susceptibility to clavulanic acid, provided a new example of convergence in this evolution process.68,69 On the other hand, inhibitor-resistant ß-lactamases have also been reported in the SHV family.70,71
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Notes |
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References |
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Received 30 June 1998; returned 17 September 1998; revised 26 October 1998; accepted 30 November 1998