A novel extended-spectrum ß-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance

Enno Stürenburg*, Alexandra Kühn, Dietrich Mack and Rainer Laufs

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objective: In recent years, cefotaximases of the CTX-M type have become a predominant cause of resistance to extended-spectrum cephalosporins in Gram-negative bacteria. Although most enzymes provide higher levels of resistance to cefotaxime than to ceftazidime, mutants with enhanced catalytic efficiency against ceftazidime have recently been described. This report identifies another ceftazidime-resistant mutant of the CTX-M class of enzymes.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In recent years, the frequency of isolation of CTX-M-type extended-spectrum ß-lactamases (ESBLs) has increased sharply worldwide.13 The family currently comprises at least 40 enzymes, most of which have been described in the last 5 years. Generally, these enzymes are characterized by a considerable imbalance in their hydrolytic activities against oxyimino cephalosporins, having high activity against cefotaxime but not ceftazidime. Accordingly, CTX-M producers appear, on laboratory testing, to be highly resistant to cefotaxime but susceptible to ceftazidime.1,3 Exceptions to this rule are four recently described enzymes, CTX-M-15, CTX-M-16, CTX-M-19 and CTX-M-27, which have significant hydrolytic activity against ceftazidime.4,5 Among them, CTX-M-15, CTX-M-16 and CTX-M-27 harbour an Asp-to-Gly substitution at position 240 (Ambler's numbering scheme).6 From molecular modelling studies of TEM/SHV-type ESBLs, amino acid substitutions at residue 240 have been associated with expansion of activity towards ceftazidime, most probably due to its position in a key ß-strand of the catalytic site of class A ß-lactamases.79 A key role in expansion of activity towards ceftazidime has also been attributed to Pro-167, which is located in the omega loop of Ambler class A enzymes. One recent report identified a CTX-M producing Klebsiella pneumoniae strain that produces a novel Pro-167->Ser variant, designated CTX-M-19 and derived from CTX-M-18. The Ser-167-harbouring CTX-M-19 conferred higher MICs of ceftazidime than did the Pro-167-harbouring CTX-M-18 enzyme (MIC, 512 versus 1 mg/L), indicating that though residue 167 is not a direct part of the active site, it must be considered a crucial determinant of substrate specificity.5

This report identifies another ceftazidime-resistant CTX-M mutant, designated CTX-M-23.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

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 Mueller–Hinton (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 3892–3912 in the sequence published under GenBank accession number J01749); TEM-R, 5'-TTGGTCTGACAGTTACCAATGC-3' (nucleotides 4822–4801 in the same sequence); SHV-F, 5'-TTATCTCCCTGTTAGCCACC-3' (nucleotides 28–47 in the sequence listed under GenBank accession number AF148850); SHV-R, 5'-GATTTGCTGATTTCGCTCGG-3' (nucleotides 824–805 in the same sequence); CTX-M-F, 5'-TCTTCCAGAATAAGGAATCCC-3' (nucleotides 42–62 in the sequence listed under GenBank accession number X92506); and CTX-M-R, 5'-CCGTTTCCGCTATTACAAAC-3' (nucleotides 950–930 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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
E. coli IFI-1 and K. pneumoniae IFI-2 were both found to be resistant to oxyimino cephalosporins and exhibited a positive double-disc synergy test, thus indicating the presence of an extended-spectrum ß-lactamase. Broth microdilution MIC testing confirmed that both isolates were highly resistant to penicillins, cefuroxime and ceftazidime; susceptible or intermediate to cefotaxime; and susceptible to cefoxitin, cefepime and imipenem (Table 1). Notably, both clinical strains had a 32-fold higher level of resistance to ceftazidime than to cefotaxime (Table 1). Both clinical isolates were mated with E. coli C600 (nalidixic acid-resistant) and yielded E. coli transconjugants T/IFI-1 and T/IFI-2. The ß-lactam resistance phenotypes of the transconjugants were almost identical to those of the donor strains (i.e. the MICs of ceftazidime were up to 32-fold higher than those of cefotaxime). In all four strains, clavulanate and tazobactam partially or totally restored the activities of the oxyimino ß-lactams (Table 1). E. coli IFI-1 was also resistant to gentamicin, tobramycin and tetracycline yielding a multiple resistance phenotype, which could be co-transferred with ceftazidime resistance into E. coli C600 (data not shown). Isoelectric point determination with penicillin G as substrate revealed the presence of two to three ß-lactamases per strain (with pIs of 5.4, 7.6 and 8.9), but using ceftazidime as substrate, only one enzyme with a pI of approximately 8.9 showed hydrolytic activity.


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Table 1. MIC values of antimicrobials against E. coli IFI-1 and K. pneumoniae IFI-2, and their E. coli C600 transconjugants T/IFI-1 and T/IFI-2

 
When a PCR assay for CTX-M-type genes was used, amplicons were detected in the parents E. coli IFI-1 and K. pneumoniae IFI-2 and in their E. coli transconjugants T/IFI-1 and T/IFI-2. Sequence data indicate an open reading frame of 873 bp, corresponding to 291 amino acid residues (Figure 1). From homology with previously characterized CTX-M-type ß-lactamases, the most likely signal peptide would consist of 29 amino acid residues, whereas the mature ß-lactamase could comprise 262 amino acids with a predicted molecular weight of 28 884 Da. Compared to blaCTX-M-1, the nearest CTX-M neighbour, three silent point mutations were found at positions Arg-43 (AGA to CGA), Arg-191 (CGT to CGG) and Gly-200 (GGT to GGC) and three amino acid substitutions were found at positions: 114, Asp (GAT) to Asn (AAT); 140, Ser (TCT) to Ala (GCT); and 167, Pro (CCG) to Thr (ACG). As these substitutions are not shared by other recorded CTX-M-type ß-lactamases, the enzyme from our strains appears to be a novel extended-spectrum ß-lactamase and has been designated CTX-M-23.



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Figure 1. Alignments of the CTX-M-23 amino acid sequence with those of CTX-M-1 and CTX-M-3. Dashes indicate amino acids identical to those of CTX-M-1. Italic letters represent the likely signal peptide of CTX-M-1, as determined by homology with previously characterized CTX-M ß-lactamases. The amino acids are numbered according to the standard numbering scheme for the class A ß-lactamases of Ambler et al.6The boxed area represents the active-site omega loop.

 
Since PCR experiments also gave positive results for blaTEM with the parents and their transconjugants, and for blaSHV with K. pneumoniae IFI-2, these amplicons were sequenced too. The amino acid sequence encoded by blaTEM was identical to the sequence encoded by blaTEM-1, and the blaSHV sequence was 100% identical to the blaSHV-1 sequence. These results correlate the isoelectric focusing findings of pIs 5.4 and 7.6 corresponding to blaTEM-1 and blaSHV-1, respectively.

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-167->Thr change in the active-site omega loop (residues 161–179), 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 Ser->Thr 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.


    Footnotes
 
* Corresponding author. Tel: +49-40-42803-3147; Fax: +49-40-42803-4881; Email: e.stuerenburg{at}uke.uni-hamburg.de


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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16 . Ho, P. L., Cheung, T. K. M., Yam, W. C. et al. (2002). Characterization of a laboratory-generated variant of BPS ß-lactamase from Burkholderia pseudomallei that hydrolyses ceftazidime. Journal of Antimicrobial Chemotherapy 50, 723–6.[Abstract/Free Full Text]

17 . Poirel, L., Naas, T., Le Thomas, I. et al. (2001). CTX-M-type extended-spectrum ß-lactamase that hydrolyses ceftazidime through a single amino acid substitution in the omega loop. Antimicrobial Agents and Chemotherapy 45, 3355–61.[Abstract/Free Full Text]