JAC Advance Access originally published online on June 17, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):315-319; doi:10.1093/jac/dkl252
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A novel ceftazidime-hydrolysing extended-spectrum ß-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop
1 Department of Laboratory Medicine, Kosin University College of Medicine 602-030, 34 Amnam-Dong, Suh-Gu, Busan, Korea 2 Department of Laboratory Medicine, Pochon CHA University College of Medicine 463-712, 351 Yatap-Dong, Bundang-Gu, Sungnam, Korea 3 Department of Laboratory Medicine, Pusan National University School of Medicine 602-739, Suh-Gu, Ami-Dong 1-10, Busan, Korea 4 Center for Food Safety Evaluation, Korea Food and Drug Administration 122-704, 231 Jinheung-Ro, Eunpyung-Gu, Seoul, Korea 5 R&D Park, LG Life Sciences Ltd 305-380, 104-1 Moonji-Dong, Yuseong-Gu, Daejeon, Korea
*Corresponding author. Tel: +82-51-990-6373; Fax: +82-51-990-3034; E-mail: kscpjsh{at}ns.kosinmed.or.kr
Received 25 April 2006; returned 16 May 2006; revised 19 May 2006; accepted 24 May 2006
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Abstract |
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Objectives: To characterize a novel ceftazidime-hydrolysing CTX-M mutant, designated CTX-M-54, produced by Klebsiella pneumoniae clinical isolate BDK0419 and to investigate its genetic environment.
Methods: Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A ß-lactamases was performed by PCR amplification, and the genetic organization of the blaCTX-M-54 gene was investigated by PCR and sequencing of the regions surrounding this gene. Kinetic parameters were determined from purified CTX-M-54.
Results: The strain BDK0419 contained a transferable plasmid with a molecular size of 
21 kbp that carries both blaSHV-2a and bla CTX-M-54 ß-lactamase genes, along with two other plasmids. The blaCTX-M-54 gene was flanked upstream by an ISEcp1 insertion sequence and downstream by an IS903-like element. CTX-M-54 had a P167Q substitution within the omega loop region of class A ß-lactamases compared with the sequence of CTX-M-3. The MIC of ceftazidime for K. pneumoniae BDK0419 was 16-fold higher than that of cefotaxime; however, the kinetic parameter of CTX-M-54 against ceftazidime revealed a low catalytic efficiency.
Conclusions: This work shows once again that novel CTX-M enzymes with an expanded activity towards ceftazidime through a single amino acid substitution can be identified from clinical isolates. Thus, detection of CTX-M enzymes can no longer be based solely on the resistance phenotypes of clinical isolates towards ceftazidime and cefotaxime.
Keywords: cefotaximase , ISEcp1 , IS903
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Introduction |
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CTX-M-type extended-spectrum ß-lactamases (ESBLs), the most widespread enzymes among non-TEM and non-SHV plasmid-mediated ESBLs, were initially reported in the second half of the 1980s in Europe.1 As the designation ‘CTX’ indicates, these enzymes preferentially hydrolyse cefotaxime but not ceftazidime. However, there have been recent reports of CTX-M mutants exhibiting a significant hydrolytic activity against ceftazidime. Amino acid substitutions at positions 167 and 240 (Ambler's numbering scheme) in CTX-M-type enzymes have been associated with expansion of activity towards ceftazidime. CTX-M-15, CTX-M-16 and CTX-M-27 harbour an Asp
Gly substitution at position 240.2–4 CTX-M-19 (derived from CTX-M-18) and CTX-M-23 (derived from CTX-M-19) harbour a ProSer and a ProThr substitution at position 167 in the omega loop, respectively.5,6
This report identifies another ceftazidime-hydrolysing CTX-M mutant, designated CTX-M-54. CTX-M-54 differed from CTX-M-3 only by the substitution Pro-167Gln and is the third CTX-M enzyme harbouring an amino acid substitution at position 167 after CTX-M-19 and CTX-M-23.
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Materials and methods |
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Bacterial strains
Klebsiella pneumoniae clinical isolate BDK0419 was identified with the API 20E system (bioMérieux, Marcy l'Etoile, France). Escherichia coli BL21(DE3) was the host for cloning experiments, and E. coli J53 AzideR was used as a recipient strain for conjugative transfer. E. coli ATCC 25933 was used as an MIC reference strain.
Antimicrobial susceptibility testing
Antibiotic-containing discs (BBL, Cockeysville, MD, USA) were used for routine antibiograms by disc diffusion assay according to the recommendations of the CLSI.7 The double-disc synergy test was carried out on Mueller–Hinton agar (Difco Laboratories, Detroit, MI, USA) with discs of ceftazidime, cefotaxime and aztreonam, each containing 30 µg of the drug, placed at distances of 20 mm (centre to centre) from a disc containing amoxicillin–clavulanic acid (20 µg/10 µg) in the centre of the plate.8 MICs were determined by the agar dilution method with Mueller–Hinton agar with an inoculum of 104 cfu.9 MICs of ß-lactams were determined alone or in combination with a fixed concentration of clavulanic acid (4 mg/L).
PCR experiments
Plasmid DNA of K. pneumoniae BDK0419 was extracted as described previously,10 and this DNA was used as a template in PCR experiments with a series of primers designed for the detection of class A ß-lactamase genes and their extended-spectrum derivatives: blaTEM, blaSHV, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, blaPER-1, blaVEB-1, blaIBC/GES and blaTLA (Table 1). The PCR products were subjected to direct sequencing by the dideoxynucleotide chain-termination method with an automatic DNA sequencer (ABI 3700, Perkin-Elmer, Foster City, CA, USA). Both strands of the PCR products were sequenced twice.
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The genetic organization of the blaCTX-M-54 gene was investigated by PCR and sequencing of the regions surrounding this gene. The internal ISEcp1 forward primers (TN1-F and BTN-F) and CTX-M-1 reverse primers (CTX-M-1R and FM-1R) were used to investigate the promoter regions of the blaCTX-M-54 gene. PCR primers corresponding to sequences upstream (pemK and ISEcp1) and downstream (IS903) of the blaCTX-M-54 gene were also used (Table 1).
Mating-out assays
Conjugation experiments were carried out between K. pneumoniae BDK0419 (donor) and azide-resistant recipient strain E. coli J53 on Mueller–Hinton agar plates.11 Transconjugants were selected on Mueller–Hinton agar plates supplemented with ceftazidime (2 mg/L) and sodium azide (100 mg/L).
Purification of CTX-M-54 ß-lactamase
The PCR product obtained with primers CTX-M-54 pcrF (5'-GG GAA TTC CAT ATG GTT AAA AAA TCA CTG CG-3'; NdeI site underlined) and CTX-M-54 pcrR (5'-CCG CTC GAG CAA ACC GTC GGT GAC GAT TTT-3'; XhoI site underlined) was purified with a QIAquick column (Qiagen, Courtaboeuf, France) and ligated in the NdeI and XhoI sites of plasmid pET30a (Novagen, Milan, Italy). It was then subjected to confirmatory sequencing. The expression vector constructed, named pET30a-CTX-M-54, was introduced into E. coli BL21(DE3) competent cells. E. coli BL21(DE3) carrying plasmid pET30a-CTX-M-54 was cultured at 37°C in 1 L of LB broth supplemented with kanamycin (50 mg/L). Isopropyl-ß-D-thiogalactopyranoside (final concentration, 0.4 mM) was added when the culture reached an OD600 of 0.6, and the culture was incubated overnight at 20°C. The cells were harvested by centrifugation and were resuspended in 50 mL of buffer A [50 mM Tris (pH 7.0), 500 mM NaCl, 10 mM imidazole] and were then disrupted by microfluidizer at 15 000 psi. The cell lysate was finally centrifuged at 15 000 g for 40 min to remove cell debris. The cleared supernatant was loaded onto an Ni-NTA column (XK16, Amersham-Pharmacia-Biosciences, Milan, Italy) pre-equilibrated with buffer A. After loading, buffer B [50 mM Tris (pH 7.0), 500 mM NaCl, 25 mM imidazole] was used to wash the column. The ß-lactamase was eluted with a linear gradient of imidazole (25–300 mM in 1 h). The fractions containing nitrocefin-hydrolysing activity and purity-confirmed by SDS–PAGE were pooled and dialysed with buffer C [50 mM Tris (pH 7.0), 300 mM NaCl without imidazole]. The final protein concentration was 0.315 mg/mL (purity >>90%).
Isoelectric focusing (IEF)
To determine the isoelectric point (pI), 5 µL of the condensed supernatant containing ß-lactamase was loaded onto a Novex IEF Gel (pH 3-10; Invitrogen, Carlsbad, CA, USA) with an Xcell surelock Mini-Cell system (Invitrogen). Running conditions were 100 V constant for 1 h, 200 V constant for 1 h and 500 V for 30 min.11 The pI of the ß-lactamase was measured by staining the gel with a 0.05% solution of nitrocefin (Oxoid, Basingstoke, UK).
Kinetic measurements
Purified ß-lactamase was used for kinetic measurements performed at 30°C with 100 mM sodium phosphate buffer (pH 7.0) with a Cary 300 Bio UV-visible spectrophotometer (Varian Inc., Palo Alto, CA, USA). Wavelengths of 235 nm for benzylpenicillin (Sigma, St Louis, MO, USA); 236 nm for ampicillin (Sigma); 318 nm for aztreonam (Sigma); 482 nm for nitrocefin (Oxoid); 260 nm for ceftazidime (Sigma), cefotaxime and cefuroxime (Sigma); 265 nm for cefalothin (Sigma); and 260 nm for cefaloridine (Sigma) were used. Extinction coefficients of each antibiotic substrate used in the spectrophotometric assays were same as described previously.4 The steady-state kinetic parameters (Km and kcat) were determined under initial-rate conditions using Lineweaver–Burk plot.
Nucleotide sequence accession number
The nucleotide sequence data reported in this paper are available in the GenBank nucleotide database under accession number DQ303459.
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Description of the clinical isolate
K. pneumoniae BDK0419 was isolated from a sputum specimen of a female patient hospitalized at an intensive care unit in a tertiary-care hospital in Sungnam, Korea, in May 2004, for hypertensive intracranial haemorrhage and pulmonary oedema. Strain BDK0419 exhibited resistance to ampicillin, ampicillin–sulbactam, piperacillin, ceftazidime, amikacin, gentamicin and tobramycin and susceptibility to piperacillin-tazobactam, cefoxitin, cefotaxime, cefepime, aztreonam, imipenem, ciprofloxacin and trimethoprim/sulfamethoxazole. The strain exhibited a positive double-disc synergy test, thus indicating the production of ESBLs.
PCR and sequencing of the ß-lactamase genes
PCR amplifications using primers specific for ESBL-encoding genes revealed that K. pneumoniae BDK0419 possessed both blaSHV and blaCTX-M-1-type genes. Sequence of the blaSHV PCR amplicon with K. pneumoniae BDK0419 was 100% identical to the blaSHV-2a sequence. Sequence data of the blaCTX-M-1-type gene indicate an open reading frame of 873 bp, corresponding to a putative protein of 291 amino acids. The blaCTX-M-1-type gene differed from blaCTX-M-3, the nearest CTX-M neighbour, by only a Pro (CCG) to Gln (CAG) substitution at Ambler position 167. As this substitution has not previously been described in the CTX-M-type ß-lactamases, the enzyme from K. pneumoniae BDK0419 appears to be a novel ß-lactamase and has been designated CTX-M-54.
Genetic environment of blaCTX-M-54
An ISEcp1 insertion sequence, comprising an intact tnpA gene, was located 128 bp upstream of blaCTX-M-54. ISEcp1 possesses two imperfect inverted repeats (IRs), the left IR (IRL, CCTAGATTCTACGTCAGT) and the right IR (IRR, ACACACGTGGAATTTAGG), made of 18 bp with 14 of these 18 bp being complementary. A putative promoter consisting of the –10 (TACAAT) and –35 (TTGAA) regions, which drives blaCTX-M-54 transcription, was observed within the 3' non-coding sequence of ISEcp1. To identify further DNA sequences surrounding the blaCTX-M-54 gene, several long-range PCR experiments were performed using a series of primers. The upstream region of the ISEcp1 insertion sequence contained the pemK gene. The pemK protein, the pemK gene product, is supposed to inhibit the growth of host cells that have lost the pem+ plasmid.12 One-hundred and twenty-four nucleotides from the stop codon, the blaCTX-M-54 gene was flanked by a IS903-like element (Figure 1).
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Transfer of resistance to ceftazidime
K. pneumoniae BDK0419 contained three plasmids with molecular sizes of 21 (pBDK0419), 3.5 and 3 kbp. The strain transferred pBDK0419 containing both blaSHV-2a and blaCTX-M-54 genes to the E. coli J53 AzideR recipient by mating experiments.
ß-Lactam susceptibility
Agar dilution MIC testing confirmed that the K. pneumoniae BDK0419 was resistant to ampicillin and ceftazidime, intermediate to aztreonam, and susceptible to cefoxitin, cefotaxime, cefepime and imipenem (Table 2). Notably, the MIC of ceftazidime for this strain was 16-fold higher than that of cefotaxime. The ß-lactam resistance phenotypes of the transconjugant (E. coli trcBDK0419) and E. coli BL21(DE3) carrying plasmid pET30a-CTX-M-54 were almost identical to those of the K. pneumoniae BDK0419 (i.e. the MICs of ceftazidime were 16-fold higher than those of cefotaxime). Clavulanic acid restored the activities of ceftazidime in all three strains.
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IEF analysis
IEF of the sonic extract of E. coli trcBDK0419 showed two bands with pI values of 7.6 and 8.0. IEF of the partially purified ß-lactamase of E. coli BL21(DE3) carrying plasmid pET30a-CTX-M-54 revealed a band with a pI value of 8.0. The relative molecular mass of CTX-M-54, determined by SDS–PAGE analysis, was 28 kDa (data not shown).
Kinetic studies
The kinetic parameters for the CTX-M-54 ß-lactamase showed that it had activity against most ß-lactams including benzylpenicillin, cefaloridine, cefuroxime and cefotaxime (Table 3). There was a discrepancy between MIC results and the kinetic parameters of the enzyme against ceftazidime and cefotaxime. The catalytic efficiency (kcat/Km) of CTX-M-54 against cefotaxime (0.2 µM–1 s–1) was higher than that against ceftazidime (0.003 µM–1 s–1).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
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The strain BDK0419 contained a transferable plasmid with a molecular size of
21 kbp (pBDK0419) that carries both blaSHV-2a and blaCTX-M-54 ß-lactamase genes, along with two other plasmids. CTX-M-54 had a P167Q substitution within the omega loop region of class A ß-lactamases compared with the sequence of CTX-M-3. Amino acid substitutions at this position of CTX-M-type ß-lactamases have been associated with expansion of activity towards ceftazidime.13 MICs of ceftazidime for all K. pneumoniae BDK0419, the transconjugant (E. coli trcBDK0419) and E. coli BL21(DE3) carrying plasmid pET30a-CTX-M-54 were 16-fold higher than those of cefotaxime. These results indicate that CTX-M-54 preferentially hydrolyses ceftazidime but not cefotaxime. However, the kinetic parameters of CTX-M-54 against ceftazidime revealed a low catalytic efficiency. A similar discrepancy between MIC results and kinetic data has been reported in CTX-M-19, ascribed to a rapid loss of the activity of the enzyme caused by instability of the protein.5
The blaCTX-M-54 gene was flanked upstream by an ISEcp1 insertion sequence and downstream by an IS903-like element. The blaCTX-M genes belonging to the CTX-M-1, CTX-M-2 and CTX-M-9 clusters are associated with ISEcp1-like insertion sequences.14 The blaCTX-M-17 and blaCTX-M-19 genes are also associated with IS903-like elements.14,15 ISEcp1-like insertion sequences may play a role in the mobilization of the blaCTX-M genes by a transcriptional mechanism by recognizing a variety of DNA sequences as right IRs. However, the role of IS903-like elements in the mobilization process of the blaCTX-M genes has not yet been demonstrated.
The present work shows once again that novel CTX-M enzymes with an expanded activity towards ceftazidime through a single amino acid substitution can be identified from clinical isolates. Thus, detection of CTX-M enzymes can no longer be based solely on the resistance phenotypes of clinical isolates towards ceftazidime and cefotaxime.
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None to declare.
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Acknowledgements |
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This work was supported by a research grant from the Korea Food and Drug Administration.
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References |
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1 Bonnet R. (2004) Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 48:1–14.
2
Poirel L, Gniadkowski M, Nordmann P. (2002) Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum ß-lactamase CTX-M-15 and of its structurally related ß-lactamase CTX-M-3. J Antimicrob Chemother 50:1031–4.
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Bonnet R, Dutour C, Sampaio JL, et al. (2001) Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240Gly. Antimicrob Agents Chemother 45:2269–75.
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Bonnet R, Recule C, Baraduc R, et al. (2003) Effect of D240G substitution in a novel ESBL CTX-M-27. J Antimicrob Chemother 52:29–35.
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Poirel L, Naas T, Le Thomas I, et al. (2001) CTX-M-type extended-spectrum ß-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrob Agents Chemother 45:3355–61.
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Sturenburg E, Kuhn A, Mack D, et al. (2004) A novel extended-spectrum ß-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance. J Antimicrob Chemother 54:406–9.
7 Clinical and Laboratory Standards Institute. (2006) Performance Standards for Antimicrobial Disk Susceptibility Tests—Ninth Edition: Approved Standard M2-A9 (CLSI, Wayne, PA, USA).
8 Jarlier V, Nicolas MH, Fournier G, et al. (1988) Extended broad-spectrum ß-lactamases conferring transferable resistance to newer ß-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis 10:867–78.[Web of Science][Medline]
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Jeong SH, Lee K, Chong Y, et al. (2003) Characterization of a new integron containing VIM-2, a metallo-ß-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae. J Antimicrob Chemother 51:397–400.
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Ryoo NH, Kim E-C, Hong SG, et al. (2005) Dissemination of SHV-12 and CTX-M-type extended-spectrum ß-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES-3 in Korea. J Antimicrob Chemother 56:698–702.
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13
Kimura S, Ishiguro M, Ishii Y, et al. (2004) Role of a mutation at position 167 of CTX-M-19 in ceftazidime hydrolysis. Antimicrob Agents Chemother 48:1454–60.
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Poirel L, Lartigue M-F, Decousser J-W, et al. (2005) ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli. Antimicrob Agents Chemother 49:447–50.
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Cao V, Lambert T, Courvalin P. (2002) ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum ß-lactamase CTX-M-17. Antimicrob Agents Chemother 46:1212–7.
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