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Antimicrobial Agents and Chemotherapy, April 2005, p. 1572-1575, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1572-1575.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Occurrence of CTX-M-3, CTX-M-15, CTX-M-14, and CTX-M-9 Extended-Spectrum ß-Lactamases in Enterobacteriaceae Clinical Isolates in Korea

Jungmin Kim,^1* Yu-Mi Lim,¹ Young-Sook Jeong,² and Sung-Yong Seol²

Department of Microbiology, Dankook University College of Medicine, Chonan,¹ Department of Microbiology, School of Medicine, Kyungpook National University, Daegu, Korea²

Received 9 August 2004/ Returned for modification 11 September 2004/ Accepted 22 November 2004

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Among 603 isolates of Enterobacteriaceae collected between June and November 2003 from three university hospitals within Korea, bla_CTX-M-3, bla_CTX-M-15, bla_CTX-M-14, and bla_CTX-M-9 were detected in 41 isolates of species from five different genera of Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter spp., and Serratia marcescens.

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Although most extended-spectrum ß-lactamases (ESBL) belong to the TEM- and SHV-type ESBL families, the members of a novel ESBL family, CTX-M, are increasingly being reported in gram-negative bacilli (1). Here, we examined the presence of CTX-M enzymes and the predominant type of CTX-M enzyme in Korea.

Between June and November 2003, 603 consecutive nonduplicate nosocomial isolates of Enterobacteriaceae were collected from three university hospitals located in three different cities—Daegu, Daejun, and Cheonan—in Korea. Among the 603 isolates collected, 163 (27%) were grown on Mueller-Hinton agar plates containing 2 µg of cefotaxime (Sigma)/ml, and they were subjected to PCR for detecting bla_CTX-M with primers listed in Table 1, designed for detection of enzymes from the CTX-M-1, CTX-M-2, and CTX-M-9 groups. As a result of the PCR experiment, 41 of 163 isolates (25.2%) have been shown to carry bla_CTX-M: 28 strains were positive for the PCR of the CTX-M-1 group, and 13 strains were positive for the PCR of the CTX-M-9 group. Further determination of bla_CTX-M alleles was performed by nucleotide sequencing of PCR products on both strands with primers used for PCR. Sequencing was carried out with the Taq DyeDeoxyTerminal cycle-sequencing kit using primers used for PCR, and the sequence was analyzed by using an automatic DNA sequencer (377 ABI Prism; Perkin Elmer). Of the 28 strains positive for the CTX-M-1 group, 17 were confirmed to carry bla_CTX-M-3, and the remaining 11 strains carried bla_CTX-M-15. Of the 13 strains positive for the CTX-M-9 group, 9 were confirmed to carry bla_CTX-M-14, and the remaining 4 strains carried bla_CTX-M-9 (Table 2). In Escherichia coli isolates, all four kinds of bla_CTX-M were demonstrated. bla_CTX-M-3 was identified in species from four different genera of Enterobacteriaceae, Citrobacter freundii (one isolate), E. coli (three isolates), Klebsiella pneumoniae (four isolates), and Serratia marcescens (nine isolates), indicating horizontal transfer and wide dissemination of bla_CTX-M-3 among the family Enterobacteriaceae. bla_CTX-M-14 and bla_CTX-M-15 were detected in all three university hospitals located in three different cities. bla_CTX-M-3 and bla_CTX-M-9 were not detected in the hospital located in Daejun and in the hospital located in Cheonan, respectively.

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TABLE 1. Oligonucleotide primers used for detection of ß-lactamase genes

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TABLE 2. Phenotypic and genotypic characterization of 41 isolates carrying the blaCTX-M gene

Characterization of 41 isolates carrying bla_CTX-M was performed via antimicrobial susceptibility testing, an isoelectric focusing (IEF) assay (2), PCR, and nucleotide sequencing for ß-lactamase genes. MICs were measured using a standard agar dilution method according to the approved method of the National Committee for Clinical Laboratory Standards (3). E. coli ATCC 25922 was used as a quality reference strain. Isoelectric focusing and inhibition assays with 0.3 mM clavulanic acid or cloxacillin were performed as described previously (2, 4).

As shown in Table 2, most isolates expressing CTX-M enzyme were found to produce additional ß-lactamases. The ß-lactamase with a pI of 5.4 was confirmed as TEM-1 or TEM-54, inhibitor-resistant TEM, by TEM-specific PCR and sequencing. The ß-lactamases with pIs of 7.6 and 8.2 and whose activity was inhibited by 0.3 mM clavulanic acid were SHV-1 and SHV-12, respectively. The ß-lactamase with a pI of 7.4 whose activity was not inhibited by either 0.3 mM clavulanic acid or 0.3 mM cloxacillin was OXA-30, confirmed by OXA-1-specific PCR and subsequent sequencing. The ß-lactamases with pIs of 8.0 and 7.8 whose activity was inhibited by 0.3 mM cloxacillin were CMY-1 and DHA-1, respectively.

For almost all strains expressing CTX-M enzyme, except five strains which coexpressed SHV-12 or CMY-1, the MICs of cefotaxime were higher than those of ceftazidime (Table 2). The cefotaxime MICs for such strains were two- to sevenfold higher dilutions than those of ceftazidime. Ratios of cefotaxime MIC to ceftazidime MIC for isolates expressing CTX-M-15 were lower than those for isolates expressing CTX-M-3, as demonstrated by other reports (6, 7). Although there is only one amino acid difference between CTX-M-3 and CTX-M-15 (Asp²⁴⁰Gly), Poirel et al. (6) demonstrated that the amino acid difference in the omega loop region of CTX-M-15 results in increased ceftazidime hydrolysis and antibiotic resistance compared to those for CTX-M-3. For four strains expressing CTX-M-9 (two E. coli strains and two Enterobacter cloacae strains), the MICs of cefotaxime were lower, ranging from 8 to 32 µg/ml, than those for strains expressing CTX-M-3, CTX-M-15, or CTX-M-14.

Some strains demonstrated high levels of resistance to cefoxitin, and these strains were found to produce additional chromosomal AmpC enzyme or plasmid-mediated AmpC enzymes, such as CMY-1 and DHA-1.

Transferability of cefotaxime resistance was determined by conjugation experimentation using E. coli J53 Azide^R (confers resistance to sodium azide) as a recipient. Donor and recipient strains at logarithmic phase were grown in 4 ml of Trypticase soy broth (Difco Laboratories) and were mixed at a ratio of 4 (recipient) to 1 (donor) at 37°C for 20 h. Transconjugants were selected on Mueller-Hinton agar plates (Difco Laboratories) supplemented with sodium azide (150 µg/ml) and cefotaxime (4 µg/ml). By conjugation, cefotaxime resistance was transferred in 29 isolates, and the bla_CTX-M gene was confirmed in all 29 transconjugants by PCR (Table 3). Some other bla genes, such as bla_OXA-30, bla_TEM, bla_DHA-1, and bla_SHV-12, were cotransferred with bla_CTX-M to transconjugants. Especially, bla_OXA-30 was cotransferred with bla_CTX-M-3 in almost all strains, indicating that bla_CTX-M-3 and bla_OXA-30 might be located on the same transferable plasmid.

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TABLE 3. Transfer of resistance for cefotaxime and other antimicrobial agents of clinical isolates carrying blaCTX-M

Resistance to chloramphenicol, tetracycline, aminoglycosides, and co-trimoxazole was found in most strains carrying bla_CTX-M, and the resistance was also found in most transconjugants (Table 3). Interestingly, a high level of amikacin resistance (MIC, 512 µg/ml) was demonstrated in all 17 isolates carrying bla_CTX-M-3 but not in isolates carrying another subtype of bla_CTX-M, and the amikacin resistance was transferred to transconjugants.

In conclusion, the occurrence of CTX-M-3, CTX-M-15, CTX-M-9, and CTX-M-14 in species from five different genera of Enterobacteriaceae, C. freundii, E. coli, Enterobacter spp., K. pneumoniae, and S. marcescens was demonstrated. This finding indicates horizontal transfer and wide dissemination of these enzymes in Korea and would suggest that CTX-M enzymes have existed for several years and have evolved in Korean hospital environments. Although CTX-M-14 was identified in one isolate of Shigella sonnei, two of K. pneumoniae, and one of E. coli in Korea in 2001 (5), to our knowledge this study represents the first identification of CTX-M-3, CTX-9, and CTX-M-15 in Korea.

 
We are grateful to the following people who supplied the clinical isolates used in this study: Je-Chul Lee, Kyung-Pook National University School of Medicine, and Insoo Rheem, Dankook University College of Medicine.

This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ1-PG1-CH03-0002).

 
* Corresponding author. Present address: Department of Microbiology, Kyungpook National University School of Medicine, Daegu, South Korea. Phone: 82-53-420-4845. Fax: 82-53-427-5664. E-mail: minkim{at}dankook.ac.kr.

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Antimicrobial Agents and Chemotherapy, April 2005, p. 1572-1575, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1572-1575.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, J. Articles by Seol, S.-Y. Search for Related Content PubMed PubMed Citation Articles by Kim, J. Articles by Seol, S.-Y.