Reviews. Inhibitor-resistant TEM -lactamases: phenotypic, genetic and biochemical characteristics

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1 Journal of Antimicrobial Chemotherapy (1999) 43, Reviews JAC Inhibitor-resistant TEM -lactamases: phenotypic, genetic and biochemical characteristics E. B. Chaïbi a, D. Sirot b, G. Paul c and R. Labia a * a UMR 175, CNRS MNHN, 6 Rue de l Université, Quimper; b Laboratoire de Bactériologie, Faculté de Médecine, 28 Place Henri-Dunant, Clermont-Ferrand Cedex; c CHU Cochin, Laboratoire de Bactériologie, Paris, France -Lactamases represent the main mechanism of bacterial resistance to -lactam antibiotics. The recent emergence of bacterial strains producing inhibitor-resistant TEM (IRT) enzymes could be related to the frequent use of -lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam in hospitals and in general practice. The IRT -lactamases differ from the parental enzymes TEM-1 or TEM-2 by one, two or three amino acid substitutions at different locations. This paper reviews the phenotypic, genetic and biochemical characteristics of IRT -lactamases in an attempt to shed light on the pressures that have contributed to their emergence. Introduction Increased and repeated use of -lactam antibiotics (e.g. penicillins and cephalosporins) leads to them becoming ineffective, principally as a result of the onset and worldwide spread of enzymatic resistance via -lactamase production by bacteria. Two strategies have been employed to counter this resistance problem. Firstly, new -lactam drugs have been developed that are inherently less susceptible to -lactamases. A second approach utilizing combinations of a mechanism-based inactivator for -lactamases (e.g. clavulanic acid, sulbactam and tazobactam) and a penicillin has also been used. The rationale for such combined therapy is based on a synergic effect of the two molecules: the inactivator destroys the -lactamase activity, whereby the penicillin is protected from inactivation. Combinations of hydrolysable penicillins with a -lactamase inhibitor are a successful strategy to overcome TEM-type mediated resistance. Reports have established that the susceptibility of Escherichia coli isolates to -lactamase inhibitors can be affected by hyperproduction of unmodified TEM-type - l a c t a m a s e, 1 3 or by the modification of the outer membrane proteins, or by both. 4 Resistance may also be attributable to production of OXA-type enzymes, or to hyperproduction of cephalosporinases. 5 Since 1990, the effect of -lactamase inhibitors has also been compromised by the emergence of mutant TEM-type -lactamases, 6,7 collectively designated inhibitor-resistant TEM or IRT -lactamases (Table I). Until now, the IRT -lactamases have been described only in Europe (France, 7 15 Spain 16 and the UK 6,17 ). It is probable that bacterial strains producing IRT enzymes are also widespread on other continents, but the lack of reports may result from inadequate techniques for detection of the IRT phenotype. The production of IRT has been detected only in strains of Enterobacteriacae, particularly in E. coli, but also in three clinical strains of Klebsiella pneumoniae 18,19 and ten strains of Proteus mirabilis. 12 The presence of IRT was also reported in 1993 in strains of E. coli and of Citrobacter freundii, isolated from calf faeces. 20 IRT enzymes have never been observed in Haemophilus influ e n z a e although co-amoxiclav is widely prescribed to treat infections caused by -lactamase-producing strains of this bacterium. As suggested by Nicolas-Chanoine, 2 1 this could be related to a high intrinsic activity of penicillins against such strains or to an inadequate number of bacteria in the natural reservoir (the oropharynx) to allow for the spontaneous point mutation of the plasmidic TEM gene. Moreover, this species makes only a small amount of -lactamase. This review attempts to summarize and to discuss the many available data concerning the IRT -lactamases. *Corresponding author. Tel: ; Fax: ; roger.labia@univ-brest.fr 1999 The British Society for Antimicrobial Chemotherapy 447

2 E. B. Chaïbi et al. Table I. Amino acid substitutions in the IRT -lactamases Amino acid a at sequence position b -Lactamase c Alternative name pi Reference(s) TEM-1 Gln Met Trp Met Arg Val Arg Asn TEM-30 TEM-41 d IRT-2 Ser 5.2 8, 9, 11, 17 TEM-31 IRT-1 Cys 5.2 8, 9 TEM-32 IRT-3 Ile Thr TEM-33 IRT-5 Leu 5.4 9, 11 TEM-34 IRT-6 Val 5.4 9, 11, 17 TEM-35 IRT-4 Leu Asp 5.2 9, 10, 11 TEM-36 IRT-7 Val Asp 5.2 9, 11 TEM-37 IRT-8 Ile Asp TEM-38 IRT-9 Val Leu TEM-39 IRT-10 Leu Arg Asp TEM-40 IRT-I67, IRT-11 Ile , 17 TEM-44 IRT 2 2, IRT-13 Lys Ser TEM-45 IRT-14 Leu Gln TEM-51 IRT-15 His TEM-58 Ser Ile 15 a Ala, alanine; Asn, asparagine; Arg, arginine; Asp, aspartic acid; Cys, cysteine; Gln, glutamine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Ser, serine; Thr, threonine; Trp, tryptophan; Val, valine. b According to the ABL numbering system of Ambler et al. 50 c TEM -lactamase classification. 72 d Data from references 73 and 74. Phenotypic characteristics Detection of IRT-producing strains The IRT phenotype was characterized by resistance to -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 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 cephalothinsusceptible allowed the detection of about 87% of E. coli strains producing an IRT -lactamase alone or in association with a parental TEM -lactamase. 24 However, this phenotype did not allow the discrimination of OXAproducing 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. In a study using an automated and rapid (4 5 h) ATB method (biomérieux, La Balme-les-Grottes, France) linked to a knowledge-based expert system (ATB Plus System, Golo V.1.5, biomérieux) detecting IRT on the basis of growth indices obtained with -lactams, it was demonstrated that this system was able to detect 86.7% of IRT-producing E. coli strains. 28 By routine susceptibility tests, IRT production was inconstantly detected when it was present with additional -lactamases such as AmpC cephalosporinase. 24 -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 -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). For all the IRT-producing isolates high-level resistance to amoxycillin and ticarcillin (90% inhibitory concentration 4096 mg/l) was observed. Addition of clavulanic acid (2 mg/l) reduced the MICs of amoxycillin and ticar- 448

3 Inhibitor-resistant TEM -lactamases Table II. -Lactam resistance phenotypes of amoxycillin clavulanate-intermediate (I) and -resistant (R) E. coli strains a Amoxycillin Ticarcillin -Lactamase Amoxycillin clavulanate Ticarcillin clavulanate Cephalothin Cefoxitin IRT R I/R I/R I/R S/I S OXA R I/R R I/R S S TEM-type penicillinase R I/R b R S/I b /R b S/I b S Cephalosporinase c I/R I/R S/I S/I I/R I/R a Data obtained from references 23 and 24. b Overproduced penicillinase. c Enhanced production of chromosomal cephalosporinase. Table III. Distribution of -lactam MICs (mg/l) for 98 IRT-producing E. coli isolates No. of isolates inhibited at following concentration (mg/l) -Lactams MIC 50 MIC 90 Amoxycillin clavulanic acid a Ticarcillin clavulanic acid a Piperacillin tazobactam b a Clavulanic acid at fixed concentration of 2 mg/l. b Tazobactam at fixed concentration of 4 mg/l. cillin only by two dilutions, this reduction being insufficient to render any of the isolates susceptible to these combinations. A lower degree of resistance to piperacillin than to amoxycillin and ticarcillin was observed for all IRTproducing isolates. These isolates had intermediate susceptibility to piperacillin (8 MIC 64 mg/l) and only 39% of isolates were resistant (MIC 64 mg/l). Addition of tazobactam (4 mg/l) reduced substantially the piperacillin MIC, as 84% of isolates were classified as susceptible to a piperacillin tazobactam combination (MIC 8 mg/l). This could be due to the greater inherent activity of piperacillin alone against IRT-producing isolates. In general, no particular differences were observed among MIC values obtained for isolates producing the various IRT types. It is noteworthy that the piperacillin tazobactam 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 MIC, 2 MIC, and 4 M I C of piperacillin with tazobactam (4 mg/l), and regrowth was observed at 24 h (data not shown). Genetic characteristics Three bla gene sequences designated TEM-1A, TEM-1B and TEM-2 encode two different -lactamases, namely TEM-1 and TEM-2 (Table IV). There are a total of nine nucleotide sequence differences between these genes at positions 32, 175, 226, 317, 346, 436, 604, 682 and 925 (numbering according to Sutcliffe 29 ). Caniça et al. 13 have explored the molecular diversity of IRT enzymes by using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP) and direct nucleotide sequencing. Study of the primary structure of the genes encoding these enzymes with altered phenotype is expected to provide insight into the molecular basis of the phenotype and to help in tracing their evolution. Table V shows that the IRT -lactamase genes can be grouped in three sequence linkage groups: TEM-1A like, TEM-1B like and TEM-2 like. It should be noted that a given mutation conferring an IRT phenotype can be found in two different gene frameworks: e.g. TEM-30/IRT-2, TEM-31/IRT-1 and TEM- 34/IRT-6 are found independently in both TEM-1B like and TEM-2 like gene sequences. As suggested by Caniça 449

4 E. B. Chaïbi et al. Table IV. Sequence differences between bla TEM 1A, bla TEM 1B and bla TEM 2 genes Nucleotide a (amino acid) b in: promoter region coding region bla gene Enzyme (6) 317 (39) 346 (48) 436(78) 604 (134) 682 (160) 925 (242) Reference(s) bla TEM 1A TEM-1 C A C C (Gln) A C G T G 29 bla TEM 1B TEM-1 C G T C A T T T G 30, 75 bla TEM 2 TEM-2 T A C A (Lys) G T G C A 30, 75 a Nucleotide positions are numbered according to Sutcliffe. 29 b The amino acid is indicated when a point mutation leads to amino acid substitution compared with the sequence of TEM-1. Numbering is according to the ABL numbering system of Ambler et al. 50 Table V. Isolated or associated amino acid substitutions present in the IRT -lactamases; distribution among sequence linkage groups Amino acid TEM -lactamase position a Mutation a Linkage group b classification c (IRT) Reference(s) Met-69 Leu 1B TEM-33 (IRT-5) 9 (ATG) (CTG) Val 1B TEM-34 (IRT-6) 9 (GTG) 2 Ile 2 TEM-40 (IRT-11) 11 (ATT) Ile Thr-182 1A TEM-32 (IRT-3) 16 (ATA) (ACG) Arg-244 Ser 1B TEM-30 (IRT-2) 8, 18, 19 (CGC) (AGC) 2 Cys 1B TEM-31 (IRT-1) 8, 18 (TGC) 2 His 1A TEM-51 (IRT-15) 14 (CAC) Arg-275 Leu Val-69 1B TEM-38 (IRT-9) 11 (CGA) (CTA) (GTG) Gln Leu-69 2 TEM-45 (IRT-14) 13 (CAA) (TTG) Asn-276 Asp Leu-69 2 TEM-35 (IRT-4) 9 (AAT) (GAT) (CTG) Asp Leu-69 2 TEM-35 (IRT-4) 11 (GAT) (TTG) Asp Leu-69 Arg TEM-39 (IRT-10) 11 (GAT) (CTG) (CGG) Asp Val-69 2 TEM-36 (IRT-7) 11 (GAT) (GTG) Asp Ile-69 2 TEM-37 (IRT-8) 11 (GAT) (ATA) a ABL numbering system of Ambler et al. 50 The nucleotide triplet codons are indicated in parentheses. b The bla gene framework(s) in which the mutation is found. c TEM -lactamase classification

5 Inhibitor-resistant TEM -lactamases et al. 13 these mutations can be considered as either recurrent (and hence at hot spots for mutations), or much more ancient than others, allowing their occurrence on two different gene matrices. Contrasting with this situation, -lactamases other than IRT-1, IRT-2 and IRT-6 are associated only with one of the sequence linkage groups, TEM-1A like, TEM-1B like or TEM-2 like. It is significant to note that the IRT -lactamases can be hyperproduced by mutation at the level of their promoter genes. Indeed, Caniça et al. 13 have described two nucleotide substitutions in the promoter region of the bla IRT genes: C32 T and G162 T. The first substitution, C32 T, characteristic of the gene promoter of TEM-2, 30 was found in the promoter region of the TEM-1 gene of 15 clinical isolates of E. coli. 3 The second substitution, G162 T, was found in the -lactamase genes of TEM-30/IRT-2, TEM- 35/IRT-4, TEM-34/IRT-6, TEM-36/IRT-7, TEM-37/IRT- 8, TEM-39/IRT-10 and TEM-45/IRT ,15 This G162 T transversion falls within the functional 10 Pribnow box and consequently renders the 10 consensus region of the IRT -lactamase genes more similar to the optimal promoter of E. coli 5 -TATAAT The G162 T transversion has been also described as the mechanism of TEM-1 hyperproduction in two ampicillin sulbactam-resistant Shigella flexneri isolates from Hong Kong. 32 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 -lactamase inhibitors. 13 Biochemical data Kinetic parameters Kinetic parameters (k cat and K m ) and catalytic efficiency (k cat /K m ) of TEM-1 and IRTs are shown in Table VI. Generally, most IRT -lactamases have lower catalytic efficiency values for all substrates than those of TEM-1. This results from a decrease of k cat values and an increase of K m 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 enzyme substrate interaction. It is noteworthy that all IRTs have high K m 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 K m values for carfecillin (a phenyl ester of carbenicillin) (data not shown). The structures of ticarcillin, carbenicillin and carfecillin are shown in Figure 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 Other residue(s) required for these electrostatic interactions are still to be found. Interaction with clavulanic acid, sulbactam and tazobactam The structures of -lactamase inhibitors are shown in Figure 1. All the IRTs have IC 50 and K i values for Figure 1. Structures of some substrates and -lactamase inhibitors. 451

6 E. B. Chaïbi et al. 452

7 Inhibitor-resistant TEM -lactamases Table VII. IC 50 and K i values of -lactamase inhibitors Clavulanic acid Sulbactam Tazobactam -Lactamase IC 50 a K i a IC 50 K i IC 50 K i TEM TEM-30 IRT b b b 0.6 TEM-31 IRT-1 31 b b b 2 TEM-32 IRT (12) c (160) c (5) c 0.58 TEM-33 IRT TEM-34 IRT TEM-35 IRT TEM-36 IRT TEM-37 IRT TEM-38 IRT TEM-39 IRT TEM-40 IRT (13) c (150) c (5) c 0.14 TEM-45 d IRT TEM-51 e IRT a IC 50 and K i values, expressed in micromolar units, were determined by micro-acidimetry with penicillin as substrate. b Data are from reference 7. c In parentheses are the IC 50 values which were determined spectrophotometrically with nitrocefin as substrate by Blazquez et al. 16 d Data are from reference 77. e Data are from reference 14. -lactamase inhibitors higher than those of TEM-1 (Table VII). Those with mutations at position 69 exhibit lower IC 50 and K i values than those of other mutants. Sulbactam is a poor inhibitor of all the IRT -lactamases (high IC 50 and K i values), whereas tazobactam was the most active inhibitor (low IC 50 and K i 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 -lactamase inhibitors than sulbactam against extended-spectrum and conventionalspectrum enzymes and that clavulanic acid had activities equivalent to those of tazobactam. 35,36 Relationship between structure and function In the absence of crystal structures, most structure function relationships of the IRT -lactamases have been studied by molecular modelling. Two excellent reviews have recently discussed these relationships. 37,38 Figure 2 shows the ribbon representation of the three-dimensional structure of a class A TEM-type -lactamase, established at the atomic level by X-ray crystallography, 39,40 together with the location of each of the point mutations. All the IRT variants arise from point mutations in the gene encoding either TEM-1 or TEM-2. At amino acid position 39, located at the end of the N-terminal -2 helix, TEM-1 enzymes have a glutamine and TEM-2 a lysine. The catalytic properties of these parental enzymes are slightly, but significantly, different. 41,42 Residue 69 Residue 69 is rather variable in size and character among class A -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 -3 and -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 (C 1) and Ile-69 (C 2) produced steric constraints with the side chain of Ser-70 and Asn 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 -lactamase

8 E. B. Chaïbi et al. Figure 2. Ribbon representation of a class A TEM-type -lactamase. In the -lactam binding site, the reactive Ser-70 is located at the N-terminus of the -2 helix. 39,40 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 -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 -lactamase. However, the enzyme contains a second change at position 69 that was shown to be the dominant factor in the resistance to -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 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 -lactamase compared with that of TEM-40/IRT-11. Moreover, Huang & Palzkill 48 have recently demonstrated that the addition of the Met-182 Thr substitution to the TEM-1 variant Met-69 Ile increased the stability of the Met-69 I l e 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 -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 -4 strand of class A -lactamases, but when absent a basic residue (Arg or Lys) is found at position 220 or at position ,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 -lactams 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 enzyme substrate 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 454

9 Inhibitor-resistant TEM -lactamases 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 coordinated to its side chain also plays an essential role in the carbapenem tautomerization in the -lactamase TEM-1 active site. 58,59 Residue 261 Located at the -5 strand, its side chain is buried at the hydrophobic region far from the active site. The amino acid substitution Val-261 Ile 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 -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 -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 -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 -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 -lactam substrates, as well as a 20-fold higher K i 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 -lactamase PC1 active site, 62 whereas only a part of the inhibitor molecule is bound to the -lactamase TEM-1 active site. 63 Conclusions The emergence of IRT-producing strains might be related to the frequent use of clavulanate-containing formulations in hospitals and in general practice. The IRT-producing strains cannot, however, be detected reliably by routine susceptibility tests. Thus, this characterization must be completed by iso-electric points of -lactamases, determination of kinetic parameters, 6 4, 6 5 and the use of molecular biology techniques. Genetic studies argue in favour of the convergent evolution of the bla IRT genes. It seems that such evolution of the parent TEM -lactamase to resistance to -lactamase inhibitors involves both forward and backward mutations, 66 as previously suggested for TEM- and SHV-derived extended-spectrum -lactamases. 67 Recently the extendedspectrum -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 -lactamases have also been reported in the SHV family. 70,71 References 1. Martinez, J. L., Vicente, M. F., Delgado-Iribarren, A., Perez-Diaz, J. C. & Baquero, F. (1989). Small plasmids are involved in amoxycillin clavulanate resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy 33, Wu, P.-J., Shannon, K. & Phillips, I. (1994). Effect of hyperproduction of TEM-1 -lactamase on in vitro susceptibility of Escherichia coli to -lactam antibiotics. Antimicrobial Agents and Chemotherapy 38, Wu, P.-J., Shannon, K. & Phillips, I. (1995). Mechanisms of hyperproduction of TEM-1 -lactamase by clinical isolates of Escherichia coli. Journal of Antimicrobial Chemotherapy 36, Reguera, J. A., Baquero, F., Pérez-Diaz, J. C. & Martinez, J. L. (1991). Factors determining resistance to -lactam combined with -lactamase inhibitors in Escherichia coli. Journal of Antimicrobial Chemotherapy 27, Bergström, S. & Normark, S. (1979). -Lactam resistance in clinical isolates of Escherichia coli caused by elevated production of the ampc-mediated chromosomal -lactamase. Antimicrobial Agents and Chemotherapy 16, Thomson, C. J. & Amyes, S. G. B. (1992). TRC-1: emergence of a clavulanic acid TEM -lactamase in a clinical strain. FEMS Microbiology Letters 70,

10 E. B. Chaïbi et al. 7. Vedel, G., Belaaouaj, A., Gilly, L., Labia, R., Philippon, A., Névot, P. et al. (1992). Clinical isolates of Escherichia coli producing TRI -lactamases: novel TEM-enzymes conferring resistance to -lactamase inhibitors. Journal of Antimicrobial Chemotherapy 30, Belaaouaj, A., Lapoumeroulie, C., Caniça, M. M., Vedel, G., Névot, P., Krishnamoorthy, R. et al.(1994). Nucleotide sequences of the genes coding for the TEM-like -lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiology Letters 120, Zhou, X. Y., Bordon, F., Sirot, D., Kitzis, M.-D. & Gutmann, L. (1994). Emergence of clinical isolates Escherichia coli producing TEM-1 derivatives or an OXA-1 -lactamase conferring resistance to -lactamase-inhibitors. Antimicrobial Agents and Chemotherapy 38, Brun, T., Péduzzi, J., Canica, M. M., Paul, G., Névot, P., Barthélémy, M. et al. (1994). 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