Institute of Medical Microbiology, University of Zürich, PO Box, CH-8028 Zürich, Switzerland
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Abstract |
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Introduction |
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During the 1980s, ß-lactamase inhibitor-resistant (IR) strains began to emerge at low to moderate frequency. In most cases, they were found to overproduce an intrinsically inhibitor-susceptible ß-lactamase (e.g. TEM-1), in sufficient quantity to overwhelm the inhibitor, leaving excess enzyme capable of destroying the accompanying ß-lactam.8,9 Since 1992, when TEM-30, the first mechanism-based IR variant emerged by a single amino acid substitution from TEM-1,10 a whole new subgroup, designated 2br,1 evolved. By 1999, this subgroup contained 18 variants derived from TEM-1 and one, TEM-50, derived from TEM-15, as well as one member of the SHV family, SHV-10.11 The 18 TEM variants contain one or two of the three following single amino acid substitutions: Met-69(Ile/Leu/Val), Arg-244
(Ser/Thr/Cys) or Asn-276
Asp. TEM-5012 and SHV-1013 are of great concern since they combine amino acid substitutions of ESBL and IR variants. Nevertheless, characterization of TEM-50 and SHV-10 implied that both enzymes appeared to be unable to confer a full ESBL phenotype.
In order to investigate the effects of amino acid substitutions mediating IR in TEM ß-lactamases on the resistance phenotypes conferred by SHV ß-lactamases, we introduced the substitutions at homologous positions within the SHV sequence.
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Materials and methods |
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The low copy plasmid vector pCCR914 harbouring a tetracycline selection marker was used for cloning of various blaSHV genes. Escherichia coli DH515 was used as a recipient for transformation with recombinant plasmids. The strains MPB-1, MPB-2 and MPB-5, producing the parental ß-lactamases SHV-1, SHV-2 and SHV-5, respectively, were taken from a panel of isogenic SHV producers described previously.16
Antibiotics
Tetracycline was obtained from Pfizer (Groton, CT, USA).
Oligonucleotides
The sequences of the oligonucleotides used for site-directed mutagenesis were the following: for Met-69 Ile (ATG
ATT), 5'-GAACGCTTTCCCATGATTAGC ACCTTTAAAGTA-3'/3'-CTTGCGAAAGGGTACTA ATCGTGGAAATTTCAT-5'; for Arg-244
Ser (CGC
AGC), 5'-CGGGGTGCGAGCGGGATTGTCGCCCT GCTTGGC-3'/3'-GCCCCACGCTCGCCCTAACAGC GGGACGAACCG-5'; and for Asn-276
Asp (AAT
GAT); 5'-AGCATGGCCGAGCGAGATCAGCAAAT CGCCGGG-3'/3'-TCGTACCGGCTCGCTCTAGTCGTTTAGCGGCCC-5'. All oligonucleotides for site-directed mutagenesis (33-mers) and sequencing (20-mers) were custom synthesized by Microsynth (Balgach, Switzerland).
Antibiotic susceptibility testing
Disc agar diffusion testing was performed according to the guidelines of the NCCLS.17 Etests (AB Biodisk, Solna, Sweden) were performed on plates containing a 4 mm layer of MuellerHinton agar (Difco, Detroit, MI, USA), according to the manufacturer's protocols.
DNA preparation
DNA of recombinant plasmids was prepared using the Qiagen plasmid kit (Qiagen, Hilden, Germany). The manufacturer's instructions were followed. Standard protocols were applied for the extraction of total DNA.18
Mutagenesis
The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to introduce single nucleotide exchanges into blaSHV genes located on recombinant plasmids. These alterations were confirmed by sequencing as were the entire open reading frames and the 400 bp upstream and 300 bp downstream flanking regions. Genes that had received the correct modifications were re-cloned on 3.6 kb fragments into pCCR9 vector to prevent aberrations from complete isogenicity through possible undetected mistakes within the non-sequenced part occurring upon mutagenesis. Re-cloning was carried out using Asp718SphI restriction sites and the ligation products were transformed into E. coli DH5 for evaluation of resistance.
Re-cloning of blaSHV genes and confirmation of sequence
Ten micrograms of vector or recombinant plasmid DNA were digested with appropriate restriction enzymes following the supplier's protocols (Hoffmann La Roche, Basel, Switzerland). After agarose gel electrophoresis, the linearized vector band and selected insert fragments were cut from the gel, extracted and purified by using the NucleoSpin Extrakt Kit (Machery-Nagel, Düren, Germany). Recircularization of vector was prevented by pre-treatment with calf intestinal phosphatase (Hoffmann La Roche), and ligation was with T4 DNA ligase (Hoffmann La Roche) for 18 h at 4°C, according to the manufacturer's instructions. The ligation mixtures were used to transform competent E. coli DH5 cells according to the standard protocols of Sambrook et al.18 Recombinants were picked and purified on LB agar plates (Difco) containing 10 mg/L tetracycline. The recombinant plasmids were checked for correct size and orientation of the insert by restriction mapping.18
DNA sequences were determined by the dideoxy nucleotide chain termination method19 using an ABI Prism 310 Genetic Analyzer. The ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA, USA) was used according to the supplier's recommendations. Sequences were processed with the Auto Assembler, version 1.4.0 (Perkin-Elmer) and analysed with the GCG sequence analysis software package, version 9.0 (Genetics Computer Group, Madison, WI, USA).
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Results |
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Three amino acid substitutions, Met-69Ile, Arg-244
Ser and Asn-276
Asp, were introduced by site-directed mutagenesis of the bla genes within three parental isogenic strains carrying either blaSHV-1, blaSHV-2 or blaSHV-5. They were introduced alone and in all possible combinations involving two or three mutations, until seven variants of each parental strain were established. Designation of the resulting clones was in accordance with the following examples: the strain expressing SHV-1 and carrying the substitutions Met-69
Ile and Arg-244
Ser was called ICB-1(Ile-69, Ser-244).
Decrease of resistance against ß-lactam antibiotics
Etests were performed to obtain a resistance pattern for each mutant (Table I). The results obtained with the disc diffusion method were in good agreement with the Etest (data not shown). Comparison of the resistance of parental and mutant strains revealed that the new strain constructions generally expressed reduced resistance against non-combined ß-lactam antibiotics. The ESBL phenotypes (defined by the MICs of expanded-spectrum cephalosporins and aztreonam) of the mutants derived from SHV-2 and SHV-5 were drastically reduced, resulting in resistance levels similar to those of the producer of the non-ESBL SHV-1 (Table I
). This effect was particularly pronounced in mutants that contained two or three amino acid substitutions (Table I
). As an exception, the substitution Asn-276
Asp exerted little or no impairment of resistance to these compounds in mutants derived from either SHV-2 or SHV-5, and cefepime resistance was even increased two- to three-fold. Single substitution at position 69 caused moderate reduction of resistance against the ß-lactams tested, and the greatest reduction by a single change was observed through the substitution at position 244. Of the mutants with combinations of IR-specifying substitutions, those carrying Met-69
Ile+Asn-276
Asp clearly had the least diminishing effect. Their cephalosporin resistance, apart from a few exceptions, remained higher than those expressed by carriers of the single substitution at position 244. Combinations of the other substitutions, Arg-244
Ser+Asn-276
Asp, Met-69
Ile+Arg-244
Ser and Met-69
Ile+Arg-244
Ser+Asn-276
Asp, abolished resistance almost totally. The MICs of expanded-spectrum cephalosporins were hardly, if at all, above those reached by the control carrying the unaltered vector.
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Increase of resistance against ß-lactamase inhibitors
Etests were performed to assess the IR phenotypes of the mutants (Table II). As expected, MICs of inhibitor/ß- lactam combinations for the carriers of IR-specifying mutations were generally higher than those for the producers of the respective parental enzymes. This effect, however, was barely noticeable with ceftazidime/clavulanic acid (Table II
). No effect was observed with cefotaxime/clavulanic acid (not shown), because all MICs were below the range covered by the respective Etest strips. In the non-ESBL SHV-1, introduction of Met-69
Ile alone or together with Asn-276
Asp, caused the strongest increase in resistance, two- to 43-fold (Table II
), followed by the derivatives carrying the single substitutions Asn-276
Asp or Arg-244
Ser. Other double or triple substitutions barely lowered the MICs of inhibitor combinations (Table II
).
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Only a few derivatives of the non-ESBL SHV-1, e.g. ICB-1(Ile-69), ICB-1(Ile-69, Asp-276) and, to a lesser extent, ICB-1(Asp-276), were able to mediate inhibitor resistance significantly above the lower NCCLS breakpoints for clinical relevance.20 None of the combined IRESBL mutants reached MICs of inhibitors that were >32-fold above background (E. coli DH5/pCCR9; Table II
), and none of the IRESBL mutants, except ICB-5(Asp-276) and ICB-5(Ile-69, Asp-276), led to MICs of expanded-spectrum cephalosporins that were above the lower NCCLS breakpoint of 8 mg/L or even above the NCCLS ESBL screening breakpoint of 1 mg/L,20 the vast majority remaining below 0.25 mg/L (Table I
).
Absence of combined IR-ESBL phenotypes
The fold-increases of resistance to inhibitor combinations conferred by the IRESBL constructions, derived from SHV-2 and SHV-5, were compared with the corresponding fold-decreases of resistance to expanded-spectrum cephalosporins (Tables I and II). Some of the constructions, predominantly among those harbouring two or three substitutions, even suffered loss of activity within both phenotypes. However, the relative loss of expanded-spectrum cephalosporin resistance was greater than that of inhibitor resistance. Cumulative ranking of the data on cefotaxime, ceftriaxone, ceftazidime and cefepime for the IRESBL derivatives of SHV-2 and SHV-5 revealed that the substitution Asn-276
Asp caused the least impairment of resistance, followed by Met-69
Ile, Met-69
Ile+Asn-276
Asp, Arg-244
Ser, Met-69
Ile+Arg-244
Ser, Arg-244
Ser+Asn-276
Asp and Met-69
Ile+Arg-244
Ser +Asn-276
Asp in increasing order. The corresponding ranking based on the data for co-amoxiclav, ampicillin/ sulbactam, piperacillin/tazobactam and cefoperazone/ sulbactam, showed that Arg-244
Ser led to the strongest increase of inhibitor resistance, followed by Met-69
Ile +Arg-244
Ser, Asn-276
Asp, Met-69
Ile+Asn276
Asp, Arg-244
Ser+Asn-276
Asp, Met-69
Ile+Arg-244
Ser+Asn-276
Asp and Met-69
Ile in decreasing order.
As a general trend, we noted that a gain in inhibitor resistance came at the expense of expanded-spectrum cephalosporin resistance. Consequently, none of the hybrid IRESBL SHV enzymes alone (Tables I and II) was able to mediate simultaneous resistance to both expanded-spectrum cephalosporins and inhibitor/ß-lactam combinations. Moreover, even the highest MICs of inhibitor combinations mediated by these hybrid enzymes were only at moderate levels. This was probably owing to the general loss of hydrolytic activity towards all ß-lactams including penicillins (Table I
). Such an effect of mutual compensation by the two types of substitution were not seen with the mutants derived from non-ESBL SHV-1. Consequently, these mutants showed the highest levels of resistance to inhibitor combinations (Table II
).
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Discussion |
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We present the results of a systematic study on the phenotypic effect of amino acid substitutions typical for IR-TEM enzymes, when introduced at the homologous and otherwise conserved positions of selected non-ESBL and ESBL SHV ß-lactamases in an isogenic genetic background. The isogenic background crucial for accurate evaluation of mutational influences is warranted through a system established previously16 and refined recently.14
The most important finding of the present study is that none of the 14 SHV-IRESBL derivatives was able to confer high-level resistance to both expanded-spectrum cephalosporins and inhibitor/ß-lactam combinations. This was found even when all seven possible combinations of the three TEM-type inhibitor-determining substitutions were systematically introduced into the two most important types of SHV ESBL: (i) SHV-2 carrying the most abundant alteration, Gly-238Ser; and (ii) SHV-5 featuring Gly-238
Ser and Glu-240
Lys, the latter being responsible for a boost of ceftazidime resistance.14,35 These results confirm and extend the findings of others, who worked with IRESBL constructions.2830,33,34,36 Moreover, they reveal that the levels of resistance to both expanded-spectrum cephalosporins and inhibitor combinations of the SHV-IRESBL derivatives were generally lower than those of the respective TEM derivatives. This may partly explain why the three TEM-specific IR substitutions have not been found in clinical SHVs so far, and why far fewer SHV than TEM variants have been discovered to date.
A second important finding is that the SHV derivatives generally did not benefit from accumulation of multiple IR-type substitutions, since in most cases both the ESBL and the IR phenotypes conferred were drastically reduced. This observation, which is consistent with an earlier report on TEM derivatives,37 may reflect a destabilizing effect of mutations on the enzyme structure. This may result in reduced activity and/or a reduction in the amount of functional protein in the periplasm, leading to reduced resistance. The only partial exceptions were Met-69Ile +Asn-276
Asp, which caused only moderate impairment of the ESBL phenotype, and Met-69
Ile+Arg-244
Ser, which resulted in the second highest overall inhibitor resistance. Despite these partial exceptions, it is reasonable to conclude that no IRESBL synergy between any of the three IR substitutions will occur through homologous recombination.
The fact that resistance to inhibitors and extendedspectrum cephalosporins are indirectly proportional when the MICs for all SHV-IRESBL derivatives are analysed suggests that the two phenotypes are mutually exclusive. This hypothesis is supported by the observation that TEM-ESBLs challenged with inhibitor combinations have been shown to revert back to TEM-1 rather than to evolve to IR-TEMESBLs.38 Similarly, a strain expressing an IR-TEM enzyme exposed to a single ß-lactam rather than IR-TEMESBLs produced TEM-1 revertants.39 This has prompted some workers to call for more formulations combining later-generation cephalosporins with inhibitors.7 Our data strongly support this strategy, since none of our SHV-IRESBL constructions showed reduced susceptibility to cefoperazone/sulbactam or ceftazidime/clavulanate (Table II). Indeed, in the latter case, the MICs for most derivatives remained below the limit of detection of the respective Etest strips.
Further interesting new aspects were noted. In our system, modification of position 244 had the most profound effect on augmentation of inhibitor resistance in both SHV-ESBLs used. In contrast, TEM-ESBLs gained the greatest lR increase when positions 69 and 276 were altered.34 Two factors could potentially explain this difference: (i) distinct structural properties at the active site of the TEM and the SHV structure; or (ii) the introduction of Arg-244Ser as opposed to Arg-244
Cys replacements realized in the TEM system. Although both effects may be important, the intrinsic structural differences between TEM- and SHV-ESBLs seem to have a predominant effect, as in our SHV system, the non-ESBL variants behaved exactly as the non-ESBL IR-TEM variants, with Met-69
Ile and Met-69
Ile plus Asn-276
Asp increasing inhibitor resistance most and Arg-244
Ser ranking only fourth out of seven (Table II
). This interpretation is also supported by Bret and co-workers,27 who found only minor differences when they compared the effects of either Ser, Cys or His as replacements for Arg-244. In this context, it is also worth mentioning that although Arg-244
Ser alone or in combination with Met-69
Ile had the greatest impact on the increase in inhibitor resistance in SHV-IRESBL variants, this rise was only one- to eight-fold, and was accompanied by an up to 2000-fold decrease of expanded-spectrum cephalosporin resistance (Tables I and II
). Interestingly, and consistent with our results, OHIO-1, an non-ESBL SHV enzyme very closely related to SHV-1, also gained little inhibitor resistance by Arg-244
Ser, or became even more susceptible to certain combinations including ampicillin/sulbactam and piperacillin/tazobactam while Met-69
Ile led to broad-spectrum inhibitor resistance.31,32 The reason Arg-244
Ser behaves so differently within the SHV-IRESBLs compared with within the SHV-IRnon-ESBL is not known.
Considering that substitution Asn-276Asp alone caused almost no weakening of resistance to single ß-lactams on the one hand, and an only 1.5- to five-fold increase of inhibitor resistance on the other (Tables I and II
), it is clearly the alteration with the least influence. This observation is not surprising since it reflects the relatively unimportant role that this change plays in the TEM background, where it has never been found alone but always in concert with a change at position 69.11 Comparing our data with those reported by Stapleton and co-workers,34 it is obvious that the TEM derivatives confer increased co-amoxiclav resistance and remain fully susceptible to piperacillin/tazobactam, while the opposite is true for the hybrid SHV variants. This suggests that the active sites of hybrid TEM and SHV enzymes may be differentially accessible for clavulanate and tazobactam.
In conclusion, SHV ß-lactamases will not necessarily benefit from recombination events that unify substitutions leading to the IR and the ESBL phenotype. If a hybrid IRESBL phenotype does occur, it is likely to be even weaker than that mediated by an analogous IR-TEM ESBL derivative. In the case of the inhibitor tazobactam only, hybrid SHVs may be superior. These findings are reassuring for clinicians and clinical microbiologists who are concerned about the possible evolution of hybrid IRESBL enzymes. However, some caution is justified because compensatory substitutions away from the critical positions identified thus far, might lead to a ß-lactamase that is able simultaneously to confer high-level resistance to both expanded-spectrum cephalosporins and inhibitor/ ß-lactam combinations. A structure of this kind has been presented at a recent meeting but has not as yet been described in full.40
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Acknowledgments |
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Notes |
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References |
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2 . MacKenzie, F. M. & Gould, I. M. (1998). Extended spectrum ß-lactamases. Journal of Infection 36, 2558.[ISI][Medline]
3
.
Fierer, J. & Guiney, D. (1999). Extended-spectrum ß-lactamases a plague of plasmids. Journal of the American Medical Association 281, 5634.
4 . Philippon, A., Labia, R. & Jacoby, G. (1989). Extended-spectrum ß-lactamases. Antimicrobial Agents and Chemotherapy 33, 11316.[ISI][Medline]
5 . Jacoby, G. A. & Medeiros, A. A. (1991). More extended-spectrum ß-lactamases. Antimicrobial Agents and Chemotherapy 35, 1697704.[ISI][Medline]
6 . Piroth, L., Aube, H., Doise, J. M. & Vincent-Martin, M. (1998). Spread of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae: are ß-lactamase inhibitors of therapeutic value? Clinical Infectious Diseases 27, 7680.[ISI][Medline]
7
.
Amyes, S. G. B. & Miles, R. S. (1998). Extended-spectrum ß-lactamases: the role of inhibitors in therapy. Journal of Antimicrobial Chemotherapy 42, 4157.
8 . Page, J. W., Farmer, T. H. & Elson, S. W. (1989). Hyperproduction of TEM-1 ß-lactamase by Escherichia coli strains. Journal of Antimicrobial Chemotherapy 23, 1601.[ISI][Medline]
9 . Wu, P. J., Shannon, K. & Phillips, I. (1995). Mechanisms of hyperproduction of TEM-1 ß-lactamase by clinical isolates of Escherichia coli. Journal of Antimicrobial Chemotherapy 36, 92739.[Abstract]
10 . Vedel, G., Belaaouaj, A., Gilly, L., Labia, R., Philippon, A., Nevot, P. et al. (1992). Clinical isolates of Escherichia coli producing TRI ß-lactamases: novel TEM-enzymes conferring resistance to ß-lactamase inhibitors. Journal of Antimicrobial Chemotherapy 30, 44962.[Abstract]
11 . Jacoby, G. & Bush, K. (2000). Amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant ß-lactamases. Lahey Clinic. [On-line.] http://www.lahey.org/studies/ webt.htm (16 October 2000, date last accessed).
12 . Sirot, D., Recule, C., Chaibi, E. B., Bret, L., Croize, J., Chanal-Claris, C. et al. (1997). A complex mutant of TEM-1 ß-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate. Antimicrobial Agents and Chemotherapy 41, 13225.[Abstract]
13 . Prinarakis, E. E., Miriagou, V., Tzelepi, E., Gazouli, M. & Tzouvelekis, L. S. (1997). Emergence of an inhibitor-resistant ß-lactamase (SHV-10) derived from an SHV-5 variant. Antimicrobial Agents and Chemotherapy 41, 83840.[Abstract]
14
.
Randegger, C. C., Keller, A., Irla, M., Wada, A. & Hächler, H. (2000). Contribution of natural amino acid substitutions in SHV extended-spectrum ß-lactamases to resistance against various ß-lactams. Antimicrobial Agents and Chemotherapy 44, 275963.
15 . Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166, 55780.[ISI][Medline]
16 . Nüesch-Inderbinen, M. T., Hächler, H. & Kayser, F. H. (1995). New system based on site-directed mutagenesis for highly accurate comparison of resistance levels conferred by SHV ß-lactamases. Antimicrobial Agents and Chemotherapy 39, 172630.[Abstract]
17 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Disk Susceptibility Tests: Approved Standard M2-A6. NCCLS, Villanova, PA.
18 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
19 . Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA 74, 54637.[Abstract]
20 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility TestingNinth Informational Supplement: M100-S9, Vol. 19, no. 1. NCCLS, Villanova, PA.
21
.
Chaibi, E. B., Sirot, D., Paul, G. & Labia, R. (1999). Inhibitor-resistant TEM ß-lactamases: phenotypic, genetic and biochemical characteristics. Journal of Antimicrobial Chemotherapy 43, 44758.
22 . Nüesch-Inderbinen, M. T., Hächler, H. & Kayser, F. H. (1996). Detection of genes coding for extended-spectrum SHV betalactamases in clinical isolates by a molecular genetic method, and comparison with the E test. European Journal of Clinical Microbiology and Infectious Diseases 15, 398402.[ISI][Medline]
23 . Maiti, S. N., Phillips, O. A., Micetich, R. G. & Livermore, D. M. (1998). Beta-lactamase inhibitors: agents to overcome bacterial resistance. Current Medicinal Chemistry 5, 44156.[ISI][Medline]
24 . Rice, L. B., Carias, L. L. & Shlaes, D. M. (1994). In vivo efficacies of ß-lactamß-lactamase inhibitor combinations against a TEM-26-producing strain of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 38, 26634.[Abstract]
25 . Bradford, P. A., Cherubin, C. E., Idemyor, V., Rasmussen, B. A. & Bush, K. (1994). Multiply resistant Klebsiella pneumoniae strains from two Chicago hospitals identification of the extended-spectrum TEM-12 and TEM-10 ceftazidime-hydrolyzing ß-lactamases in a single isolate. Antimicrobial Agents and Chemotherapy 38, 7616.[Abstract]
26 . Rice, L. B., Carias, L. L., Bonomo, R. A. & Shlaes, D. M. (1996). Molecular genetics of resistance to both ceftazidime and ß-lactamß-lactamase inhibitor combinations in Klebsiella pneumoniae and in vivo response to ß-lactam therapy. Journal of Infectious Diseases 173, 1518.[ISI][Medline]
27 . Bret, L., Chaibi, E. B., Chanal-Claris, C., Sirot, D., Labia, R. & Sirot, J. (1997). Inhibitor-resistant TEM (IRT) beta-lactamases with different substitutions at position 244. Antimicrobial Agents and Chemotherapy 41, 25479.[Abstract]
28
.
Giakkoupi, P., Tzelepi, E., Legakis, N. J. & Tzouvelekis, L. S. (1999). Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 ß-lactamases. Journal of Antimicrobial Chemotherapy 43, 239.
29 . Giakkoupi, P., Tzelepi, E., Legakis, N. J. & Tzouvelekis, L. S. (1998). Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 ß-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes. FEMS Microbiology Letters 160, 4954.[ISI][Medline]
30
.
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, 12813.
31 . Lin, S., Thomas, M., Mark, S., Anderson, V. & Bonomo, R. A. (1999). OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors. Biochimica et Biophysica Acta Protein Structure and Molecular Enzymology 1432, 12536.
32 . Lin, S., Thomas, M., Shlaes, D. M., Rudin, S. D., Knox, J. R., Anderson, V. et al. (1998). Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 ß-lactamase, an SHV-family class A enzyme. Biochemical Journal 333, 395400.[ISI][Medline]
33 . Bonomo, R. A., Knox, J. R., Rudin, S. D. & Shlaes, D. M. (1997). Construction and characterization of an ohio-1 beta-lactamase bearing Met69Ile and Gly238Ser mutations. Antimicrobial Agents and Chemotherapy 41, 19403.[Abstract]
34
.
Stapleton, P. D., Shannon, K. P. & French, G. L. (1999). Construction and characterization of mutants of the TEM-1 ß-lactamase containing amino acid substitutions associated with both extended-spectrum resistance and resistance to ß-lactamase inhibitors. Antimicrobial Agents and Chemotherapy 43, 18817.
35
.
Huletsky, A., Knox, J. R. & Levesque, R. C. (1993). Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type ß-lactamases probed by site-directed mutagenesis and 3-dimensional modeling. Journal of Biological Chemistry 268, 36907.
36 . Imtiaz, U., Manavathu, E. K., Mobashery, S. & Lerner, S. A. (1994). Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 ß-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime. Antimicrobial Agents and Chemotherapy 38, 11349.[Abstract]
37
.
Vakulenko, S. B., Geryk, B., Kotra, L. P., Mobashery, S. & Lerner, S. A. (1998). Selection and characterization of ß-lactamß-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 ß-lactamase gene. Antimicrobial Agents and Chemotherapy 42, 15428.
38 . Du Bois, S. K., Marriott, M. S. & Amyes, S. G. B. (1995). TEM- and SHV-derived extended-spectrum ß-lactamases: relationship between selection, structure and function. Journal of Antimicrobial Chemotherapy 35, 722.[Abstract]
39 . Thomson, C. J. & Amyes, S. G. (1995). Back mutations to the TEM-1 ß-lactamase from TRC-1 lead to restored sensitivity to clavulanic acid. Journal of Medical Microbiology 42, 42932.[Abstract]
40 . Vakulenko, S. B., Mobashery, S. & Lerner, S. A. (1999). Extended-spectrum (E-S), inhibitor-resistant mutants of the TEM-1 ß-lactamase. In Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy. Abstract 2048, p. 137. American Society for Microbiology, Washington, DC.
Received 28 July 2000; returned 26 September 2000; revised 7 November 2000; accepted 19 January 2001