Institut für Infektionsmedizin, Zentrum für Klinisch-Theoretische Medizin I, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany
Received 7 April 2004; returned 14 May 2004; revised 21 May 2004; accepted 21 May 2004
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Abstract |
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Methods: Two ceftazidime-resistant strains, Escherichia coli IFI-1 and Klebsiella pneumoniae IFI-2, were isolated from a 46-year-old man during treatment of postoperative peritonitis with ceftazidime. Susceptibility testing, mating-out assays, isoelectric focusing as well as PCR and sequencing techniques were carried out to investigate the underlying mechanism of resistance.
Results: E. coli IFI-1 and K. pneumoniae IFI-2 exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. Notably, both strains had up to a 32-fold higher level of resistance to ceftazidime than to cefotaxime. Further characterization revealed that a novel blaCTX-M gene encoding a ß-lactamase with a pI of 8.9 was implicated in this resistance: CTX-M-23. Along with the substitutions D114N and S140A, CTX-M-23 differed from CTX-M-1, the most closely related enzyme, by a P167T replacement in the active-site omega loop, which has not previously been observed in other CTX-M enzymes. By analogy with what was observed with certain TEM/PSE/BPS-type ß-lactamases, the amino acid substitution in the omega loop may explain ceftazidime resistance, which has only rarely been reported for other CTX-M enzymes.
Conclusion: The emergence of a new ceftazidime-resistant CTX-M-type mutant provides evidence that these enzymes are able to broaden their substrate spectrum towards ceftazidime, probably due to substitutions in the active-site omega loop.
Keywords: CTX-M ß-lactamases , ESBLs , cephalosporins
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Introduction |
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This report identifies another ceftazidime-resistant CTX-M mutant, designated CTX-M-23.
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Materials and methods |
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Two clinical strains, Escherichia coli IFI-1 and K. pneumoniae IFI-2, were isolated from a 46-year-old man. Two weeks before the isolation, the patient underwent liver transplantation because of a hepatitis C cirrhosis. The review of the records revealed that the patient was previously treated with cefazolin for 9 days. On the eighth postoperative day, when peritonitis developed, the antibiotic regimen was changed to ceftazidime, metronidazole and gentamicin. A swab collected from this patient during surgical revision of a subhepatic abscess grew E. coli IFI-1 and K. pneumoniae IFI-2.
Susceptibility to ß-lactams
The MICs of various ß-lactam agents against the original isolates and transconjugants were determined by using broth microdilution according to the NCCLS.10,11 The isolates were tested for the production of an ESBL using ceftazidime and cefotaxime with and without clavulanic acid (4 mg/L) or tazobactam (4 mg/L) as described by the NCCLS.10 Control strains of E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were included with each run.
Mating-out assays
Conjugation experiments were carried out between E. coli IFI-1 and K. pneumoniae IFI-2 (donors), and nalidixic acid-resistant recipient strain E. coli C600 on MuellerHinton (MH) agar plates (Oxoid, UK). Transconjugants were selected on MH agar plates containing ceftazidime (2 mg/L) and nalidixic acid (64 mg/L).
Isoelectric focusing
To determine the number and isoelectric points (pIs) of the ß-lactamase(s) present in E. coli IFI-1 and K. pneumoniae IFI-2 and the transconjugants, isoelectric focusing was conducted using pre-cast gels with a pH gradient from 5 to 9 (Servalyt Precotes; Serva, Heidelberg, Germany) and crude extracts prepared by sonication, as described previously.12 Visualization of ß-lactamase activity was carried out as previously described,4 by layering the gel with agar containing 6% (w/v) potassium iodide, 0.6% (w/v) iodine and 0.6% (w/v) ß-lactam substrate: penicillin G to show overall ß-lactamase content and ceftazidime or cefotaxime to show oxyimino cephalosporin-hydrolysing ß-lactamases. Enzyme extracts from strains expressing SHV-2 (pI 7.6), SHV-5 (pI 8.2), SHV-12 (pI 8.2), TEM-1 (pI 5.4), TEM-52 (pI 6.0) and CTX-M-1 (pI 8.4) were used for comparison.
PCR of ß-lactamase-encoding genes
The detection of ß-lactamase-encoding genes was carried out using PCR with primers that correspond to conserved regions of blaTEM-blaSHV-and blaCTX-M-type genes: TEM-F, 5'-TCCGCTCATGAGACAATAACC-3' (corresponding to nucleotides 38923912 in the sequence published under GenBank accession number J01749); TEM-R, 5'-TTGGTCTGACAGTTACCAATGC-3' (nucleotides 48224801 in the same sequence); SHV-F, 5'-TTATCTCCCTGTTAGCCACC-3' (nucleotides 2847 in the sequence listed under GenBank accession number AF148850); SHV-R, 5'-GATTTGCTGATTTCGCTCGG-3' (nucleotides 824805 in the same sequence); CTX-M-F, 5'-TCTTCCAGAATAAGGAATCCC-3' (nucleotides 4262 in the sequence listed under GenBank accession number X92506); and CTX-M-R, 5'-CCGTTTCCGCTATTACAAAC-3' (nucleotides 950930 in the same sequence). The nucleotide sequence was determined by bidirectional sequencing of PCR products, carried out by the Bigdye dideoxy chain termination method on an ABI Prism 310 DNA sequencer (Perkin-Elmer Corp., Foster City, CA, USA). The nucleotide sequence and the deduced protein sequence were analysed using commercial software (Vector NTI suite; InforMax Inc., Paisley, UK). The sequence of blaCTX-M-23 has been given GenBank accession number AF488377.
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Results and discussion |
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Unlike the majority of CTX-M enzymes, which act primarily as cefotaximases and preferentially hydrolyse cefotaxime over ceftazidime (the designation CTX refers to this feature), the new enzyme was able to confer higher levels of resistance to ceftazidime than to cefotaxime. Along with two other mutations, the amino acid sequence of CTX-M-23 differed from that of CTX-M-1 by a Pro-167Thr change in the active-site omega loop (residues 161179), which has never been observed before in naturally occurring CTX-M enzymes. However, from other Ambler class A enzymes, it is known that residues in the omega loop play an important role in the substrate profile for cephalosporins.13 Even though residue 167 is not a direct part of the catalytic mechanism, this position seems to have a pivotal influence on substrate specificity. In laboratory-derived mutants of TEM-1, PSE-4 and BPS-1, a very similar mutation, Pro-167
Ser, has been shown to be closely associated with ceftazidime resistance.1416 Actually, the same amino acid substitution has recently been observed in a naturally occurring CTX-M ESBL, converting CTX-M-18 into ceftazidime-hydrolysing ß-lactamase CTX-M-19.17
Given these observations, it was postulated that substitutions in omega loop position 167 that can expand the catalytic activity towards ceftazidime typically involve replacement of a larger amino acid, such as proline, by a small amino acid, such as serine or glycine.14 Accordingly, more space should be available in the pocket that houses the oxyimino moiety of ceftazidime in the catalytic site of the enzyme.14 For threonine, the side chain has an additional methyl moiety in its structure compared to serine and thus occupies more space. Therefore, one would expect that the binding site of CTX-M-23 is smaller, which should result in a noticeable decrease in activity against ceftazidime. However, since we have shown that position 167 tolerates a SerThr exchange while retaining significant resistance to ceftazidime, it seems that the mechanistic model delineated above may be too simple and steric interaction cannot be the only factor in modulating substrate specificity.
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Footnotes |
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References |
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