JAC Advance Access originally published online on September 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1257-1259; doi:10.1093/jac/dkl397
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Emergence of CTX-M-12 extended-spectrum ß-lactamase-producing Escherichia coli in Korea
1 Department of Laboratory Medicine, Kosin University College of Medicine 602-030, 34 Amnam-Dong, Suh-Gu, Busan, Korea 2 Department of Quality Improvement, Pusan National University Hospital 602-739, 1-10 Ami-Dong, Suh-Gu, Busan, Korea 3 Center for Food Safety Evaluation, Korea Food and Drug Administration 122-704, 231 Jinheung-Ro, Eunpyung-Gu, Seoul, Korea 4 Department of Laboratory Medicine, Hallym University College of Medicine 150-950, 948-1 Daerim 1-Dong, Yongdeungpo-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 31 May 2006; returned 5 July 2006; revised 25 August 2006; accepted 11 September 2006
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Abstract |
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Objectives: To characterize CTX-M-12 extended-spectrum ß-lactamase (ESBL) produced by clinical Escherichia coli isolates 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 environments of the blaCTX-M-12 genes were investigated by PCR and sequencing of the regions surrounding the genes. Kinetic parameters were determined from purified CTX-M-12.
Results: Sequence data for the CTX-M-1 cluster from three clinical E. coli isolates indicated the presence of CTX-M-12. An ISEcp1 insertion sequence was located 49 bp upstream of blaCTX-M-12 in all three E. coli isolates. CTX-M-12 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and was encoded on a self-transferable 
18 kbp plasmid.
Conclusions: This work shows that CTX-M-12, which confers high-level resistance to cefotaxime but not to ceftazidime, has emerged in Korea. The blaCTX-M-12 gene was associated with an upstream ISEcp1 insertion sequence.
Keywords: ISEcp1 , horizontal transfer , ERIC-PCR
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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 At present, the CTX-M family comprises more than 50 enzymes that have greater hydrolytic activity against cefotaxime than ceftazidime. In Korea, CTX-M-15 and CTX-M-3 were reported to be the most prevalent ESBLs in clinical Escherichia coli isolates in a survey of 12 Korean hospitals in 2003.2
CTX-M-12 ESBL was first detected from Klebsiella pneumoniae isolates from an outbreak among six newborn babies in Kenya in 2001.3 And then, the ESBL was also detected from a clinical K. pneumoniae isolate from Colombia and from a clinical E. coli isolate from China.4,5 CTX-M-12 differs from CTX-M-3, the nearest CTX-M neighbour, by three amino acid substitutions along with five silent point changes.
The ISEcp1 element is able to achieve the transfer of the downstream DNA sequence by a one-ended transposition process.1 This element has repeatedly been observed upstream of ORFs encoding the CTX-M enzymes. In the present study, we report the first isolation of CTX-M-12 ESBL from three clinical E. coli isolates from Korea. Additionally, we have characterized the genetic environment of the blaCTX-M-12 genes and have measured kinetic parameters of CTX-M-12.
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Materials and methods |
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Bacterial strains
Clinical isolates of E. coli were identified with the VITEK system (bioMérieux, Marcy l'Etoile, France). E. coli BL21(DE3) was the host for cloning experiments. E. coli J53 AzideR and E. coli ATCC 25933 were used as a recipient strain for conjugative transfer and an MIC reference strain, respectively.
Antimicrobial susceptibility testing and mating-out assays
Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods according to the recommendations of the CLSI.6,7 MICs of ß-lactams were determined alone or in combination with a fixed concentration of clavulanic acid (4 mg/L). ESBL-production was detected by the double-disc synergy (DDS) test.8 Conjugation experiments were carried out as described previously.2
PCR experiments
Searches for genes coding for class A ESBLs were performed by PCR amplification.2 The templates for PCR amplification in clinical isolates were a whole-cell lysate. The PCR products were subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 373A; Applied Biosystems, Weiterstadt, Germany). The genetic organization of the blaCTX-M-12 gene was investigated by PCR and sequencing of the regions surrounding this gene.
Purification of CTX-M-12 ß-lactamase
The PCR product obtained with the primers CTX-M-12 pcrF (5'-GG GAA TTC CAT ATG GTT AAA AAA TCA CTG CG-3'; NdeI site underlined) and CTX-M-12 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 pET30a (Novagen, Milan, Italy). The resultant pET30a-CTX-M-12 expression construct was transformed into E. coli BL21(DE3). LB broth (1 L) supplemented with kanamycin (50 mg/L) was cultured at 37°C. Isopropyl-ß-D-thiogalactopyranoside (final concentration 0.4 mM) was added when the culture reached an A600 of 0.6, and the culture was incubated overnight at 20°C. The cells were harvested by centrifugation and resuspended in 50 mL of buffer A [50 mM Tris (pH 7.0), 500 mM NaCl, 10 mM imidazole]. Cells were disrupted in a microfluidizer (at 15 000 psi) and the lysate centrifuged at 15 000 g for 40 min. The supernatant was loaded onto a Ni-NTA column (XK16, Amersham-Pharmacia-Biosciences, Milan, Italy) pre-equilibrated with buffer A, and washed with buffer B [50 mM Tris (pH 7.0), 500 mM NaCl, 25 mM imidazole]. The ß-lactamase was eluted with a linear gradient of imidazole (25–300 mM in 1 h). The fractions containing nitrocefin-hydrolysing activity (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 3 mg/mL (purity >>98%).
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 a 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.2 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). Extinction coefficients of each antibiotic substrate used in the spectrophotometric assays were the same as described previously.9 The steady-state kinetic parameters (Km and kcat) were determined under initial-rate conditions using Lineweaver–Burk plot.
Enterobacterial repetitive consensus (ERIC)-PCR
ERIC-PCRs were performed in 50 µL volumes containing 10 ng of genomic DNA from three clinical E. coli isolates containing the blaCTX-M-12 gene, 4 mM MgCl2, 50 pM of each primer [ERIC-1R (5'-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC-2 (5'-AAGTAAGTGACTGGGGTGAGCG-3')], 1.25 U of TaKaRa Ex Taq polymerase (TaKaRa, Otsa, Shiga, Japan), 0.2 mM each of dATP, dCTP, dGTP and dTTP in 25 mM TAPS [N-Tris(hydroxy)methyl-3-amino-propane sulphonic acid, pH 9.3], 50 mM KCl and 1 mM 2-mercaptoethanol. Amplification was carried out as described previously10 and amplicons were analysed by gel electrophoresis.
Nucleotide sequence accession numbers
The nucleotide sequence data reported in this paper are available in the GenBank nucleotide database under accession numbers DQ658220 [GenBank] , DQ658221 [GenBank] and DQ658222 [GenBank] .
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Results and discussion |
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E. coli SME3, SSE5 and SCE4 were isolated from urine specimens of three patients hospitalized at three different hospitals in Seoul and Gumi, Korea, in 2004. The isolates were resistant to cefotaxime, but susceptible to ceftazidime and all exhibited positive results in the DDS test, indicating ESBL production.
PCR amplifications using primers specific for ESBL-encoding genes revealed that all three E. coli isolates possessed both blaTEM and blaCTX-M-1-type genes. Sequences of the blaTEM PCR amplicons were 100% identical to the blaTEM-1 sequence. Sequence data from the amplicons of the CTX-M-1 cluster indicated the presence of CTX-M-12 (GenBank accession no. AF305837 [GenBank] ). An ISEcp1 insertion sequence, which 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 inverted repeats (IRs),11 was located 49 bp upstream of blaCTX-M-12 in all three E. coli isolates. ISEcp1 possessed two imperfect IRs, the left IR (CCTAGATTCTACGTCAGT) and the right IR (ACACACGTGGAATTTAGG), made of 18 bp with 14 of these 18 bp being complementary. A putative promoter consisting of the –10 (TACAAT) and –35 (TTGAAA) regions was observed within the 3' non-coding sequence of ISEcp1.
IEF of the partially purified ß-lactamase of E. coli BL21(DE3) carrying plasmid pET30a-CTX-M-12 revealed a band with a pI value of 9.0. The kinetic parameters for the CTX-M-12 ß-lactamase showed that it had activity against benzylpenicillin, cefaloridine and cefotaxime (Table 1). The catalytic efficiency (kcat/Km) of CTX-M-12 against cefotaxime (3.130 µM–1 s–1) was much higher than that against ceftazidime (0.004 µM–1 s–1).
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All three E. coli isolates contained a plasmid with molecular sizes of
18 kbp containing blaCTX-M-12 and blaTEM-1 genes. But ERIC-PCR of the three E. coli isolates proved that these strains were not clonal (data not shown), indicating that horizontal transfer of the blaCTX-M-12 gene had occurred and suggesting the possibility of further spread of this gene in the future. Despite repeated attempts, only one (SCE4) among three E. coli isolates transferred the plasmid (pSCE4) to the E. coli J53 AzideR recipient by mating experiments. Agar dilution MIC testing confirmed that all three E. coli isolates were highly resistant to ampicillin, intermediate or resistant to aztreonam, cefoxitin, cefotaxime and cefepime, and susceptible to ceftazidime and imipenem (Table 2). The ß-lactam resistance phenotypes of the transconjugant (E. coli trcSCE4) were almost identical to those of the parent strain. In the Kenya study, the CTX-M-12-producing K. pneumoniae isolates were resistant to cefotaxime (MIC 24 mg/L by Etest), but the presence of clavulanic acid lowered the MIC of the drug 750 times to 0.032 mg/L.3 In our cases, however, clavulanic acid restored the activities of cefotaxime in E. coli BL21(DE3) carrying plasmid pET30a-CTX-M-12 only, but not in both the clinical E. coli isolates and the E. coli transconjugant.
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In summary, this work shows that CTX-M-12 has now emerged in Korea, in addition to its recent description in China.5 Our kinetic characterizations show that CTX-M-12 was more active against cefotaxime than against ceftazidime, and we have also demonstrated the association of the blaCTX-M-12 with an upstream ISEcp1 element.
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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 (06042HangNaeMae129).
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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
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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Kariuki S, Corkill JE, Revathi G, et al. (2001) Molecular characterization of a novel plasmid-encoded cefotaximase (CTX-M-12) found in clinical Klebsiella pneumoniae isolates from Kenya. Antimicrob Agents Chemother 45:2141–3.
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Villegas MV, Correa A, Perez F, et al. (2004) CTX-M-12 ß-lactamase in a Klebsiella pneumoniae clinical isolate in Colombia. Antimicrob Agents Chemother 48:629–31.
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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.[ISI][Medline]
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