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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


   ABSTRACT
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Among 603 isolates of Enterobacteriaceae collected between June and November 2003 from three university hospitals within Korea, blaCTX-M-3, blaCTX-M-15, blaCTX-M-14, and blaCTX-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.


   TEXT
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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 blaCTX-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 blaCTX-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 blaCTX-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 blaCTX-M-3, and the remaining 11 strains carried blaCTX-M-15. Of the 13 strains positive for the CTX-M-9 group, 9 were confirmed to carry blaCTX-M-14, and the remaining 4 strains carried blaCTX-M-9 (Table 2). In Escherichia coli isolates, all four kinds of blaCTX-M were demonstrated. blaCTX-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 blaCTX-M-3 among the family Enterobacteriaceae. blaCTX-M-14 and blaCTX-M-15 were detected in all three university hospitals located in three different cities. blaCTX-M-3 and blaCTX-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 blaCTX-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 (Asp240->Gly), 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 AzideR (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 blaCTX-M gene was confirmed in all 29 transconjugants by PCR (Table 3). Some other bla genes, such as blaOXA-30, blaTEM, blaDHA-1, and blaSHV-12, were cotransferred with blaCTX-M to transconjugants. Especially, blaOXA-30 was cotransferred with blaCTX-M-3 in almost all strains, indicating that blaCTX-M-3 and blaOXA-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 blaCTX-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 blaCTX-M-3 but not in isolates carrying another subtype of blaCTX-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.


   ACKNOWLEDGMENTS
 
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).


   FOOTNOTES
 
* 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. Back


   REFERENCES
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 Abstract
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 References
 

  1. Bonnet, R. 2004. Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48:1-14.[Free Full Text]
  2. Mathew, A., A. M. Harris, M. J. Marshall, and G. W. Ross. 1975. The use of analytical isoelectric focusing for detection and identification of ß-lactamases. J. Gen. Microbiol. 88:169-178.[Medline]
  3. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, vol. 20, p. 7-10. Approved standard, 5th ed. NCCLS, Wayne, Pa.
  4. Pai, H., S. Lyu, J. H. Lee, J. Kim, Y. Kwon, J. W. Kim, and K. W. Choe. 1999. Survey of extended-spectrum ß-lactamases in clinical isolates of Escherichia coli and Klebsiella pneumoniae: prevalence of TEM-52 in Korea. J. Clin. Microbiol. 37:1758-1763.[Abstract/Free Full Text]
  5. Pai, H., E. H. Choi, H. J. Lee, J. Y. Hong, and G. A. Jacoby. 2001. Identification of CTX-M-14 extended-spectrum ß-lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J. Clin. Microbiol. 39:3747-3749.[Abstract/Free Full Text]
  6. Poirel, L., M, Gniadkowski, and P. Nordmann. 2002. Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum beta-lactamase CTX-M-15 and of its structurally related beta-lactamase CTX-M-3. J. Antimicrob. Chemother. 50:1031-1034.[Abstract/Free Full Text]
  7. Poirel, L., T. Naas, I. Le Thomas, A. Karim, E. Bingen, and P. Nordmann. 2001. CTX-M-type extended-spectrum ß-lactamase that hydrolyses ceftazidime through a single amino acid substitution in the omega loop. Antimicrob. Agents Chemother. 45:3355-3361.[Abstract/Free Full Text]

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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