Journal List > Korean J Clin Microbiol > v.11(2) > 1038157

TOOLS
Kim, Yum, Yong, Jeong, Lee, and Chong: Detection of CTX-M-Type Extended-Spectrum β-Lactamase in Clinical Isolates of Chromosomal AmpC β-Lactamase-Producing Enterobacteriaceae from Korea and Their Molecular Characteristics
Original Article

Korean J Clin Microbiol 2008;11(2):90-97.

Published online: 20 October 2008

DOI: https://doi.org/10.5145/kjcm.2008.11.2.90

Detection of CTX-M-Type Extended-Spectrum β-Lactamase in Clinical Isolates of Chromosomal AmpC β-Lactamase-Producing Enterobacteriaceae from Korea and Their Molecular Characteristics

Chang-Ki Kim1, Jong Hwa Yum2, Dongeun Yong3, 4, Seok Hoon Jeong3, 4, Kyungwon Lee3, 4, , Yunsop Chong3, 4

1Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul, Korea

2Department of Biomedical Laboratory Science, Dong-Eui University College of Natural Science, Busan, Korea

3Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea

4Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea

Correspondence: Kyungwon Lee, Department of Laboratory Medicine, Yonsei University College of Medicine, 250, Seongsan-ro, Seodae-mun-gu, Seoul 120-752, Korea. (Tel) 82-2-2228-2446, (Fax) 82-2-313-0908, (E-mail) leekcp@yuhs.ac

Received 4 February 2008       Accepted 20 August 2008

Copyright © 2008 Korean Journal of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Clinical isolates of AmpC β-lactamase-producing Enterobacteriaceae were evaluated to determine the prevalence of CTX-M extended-spectrum β-lactamases (ESBLs) and their genetic environments.

Methods

A total of 250 non-duplicate isolates of Eneterobacter aerogenes, E. cloacae, Citrobacter freundii, Serratia marcescens and Morganella morganii were collected at a Korean hospital. ESBL production was determined by double disk synergy test. For ESBL producers, bla genes were sequenced and blaCTX-M environment was characterized by PCR mapping and sequencing.

Results

Among the 250 isolates 29 (11.6%) produced ESBL, and 14 of the 29 isolates produced CTX-M ESBLs, including CTX-M-9 by 8 isolates, CTX-M-3 by 4 isolates, CTX-M-12 by 1 isolate, and CTX-M-14 by 1 isolate. ISEcp1 was present upstream of blaCTX-M-3, 12, and 14. Three of the four CTX-M-3 producers had the same genetic environment (pemK-ISEcp1-blaCTX-M-3-orf477-mucA). An IS903-like element was found downstream of blaCTX-M-14. ISCR1 was identified upstream of blaCTX-M-9 and ISCR1 and blaCTX-M-9 were located on sul1-type class 1 integron. The variable region between the 5'-CS and the first 3'-CS contained dfrA16 and aadA2. Its structure was similar to that of In60, but our isolates did not have IS3000 or second 3'-CS.

Conclusion

CXT-M type ESBL was prevalent in AmpC β-lactamase-producing Enterobacteriaceae, particularly E. cloacae. blaCTX-M genes were associated with ISEcp1 or ISCR1. This is the first report on the genetic environment of blaCTX-M in Korean isolates.

Keywords: Extended-spectrum β-lactamase, CTX-M, Genetic characteristics

References

1. Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C, Ould-Hocine Z, et al. Dissemination of CTX-M-type β-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother. 2004; 48:1249–55.
2. Perez F, Endimiani A, Hujer KM, Bonomo RA. The continuing challenge of ESBLs. Curr Opin Pharmacol. 2007; 7:459–69.
crossref
3. Ryoo NH, Kim EC, Hong SG, Park YJ, Lee K, Bae IK, et al. Dissemination of SHV-12 and CTX-M-type extended-spectrum β-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES-3 in Korea. J Antimicrob Chemother. 2005; 56:698–702.
crossref
4. Kim JY, Park YJ, Kim SI, Kang MW, Lee SO, Lee KY. Nosocomial outbreak by Proteus mirabilis producing extended-spectrum β-lactamase VEB-1 in a Korean university hospital. J Antimicrob Chemother. 2004; 54:1144–7.
crossref
5. Walther-Rasmussen J and H⊘iby N. Cefotaximases (CTX-M-ases), an expanding family of extended-spectrum β-lactamases. Can J Microbiol. 2004; 50:137–65.
6. Bonnet R. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004; 48:1–14.
crossref
7. Eckert C, Gautier V, Arlet G. DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother. 2006; 57:14–23.
8. Brizio A, Vasco S, Goncalves AR, Lito LM, Cristino JM, Salgado MJ, et al. Survey of extended-spectrum β-lactamases in Escherichia coli isolates from a Portuguese hospital and characterisation of a novel class 1 integron (In60A) carrying the blaCTX-M-9 gene. Int J Antimicrob Agents. 2006; 28:320–4.
9. Canton R and Coque TM. The CTX-M β-lactamase pandemic. Curr Opin Microbiol. 2006; 9:466–75.
10. Shibata N, Kurokawa H, Doi Y, Yagi T, Yamane K, Wachino J, et al. PCR classification of CTX-M-type β-lactamase genes identified in clinically isolated gram-negative bacilli in Japan. Antimicrob Agents Chemother. 2006; 50:791–5.
crossref
11. Pai H, Choi EH, Lee HJ, Hong JY, Jacoby GA. Identification of CTX-M-14 extended-spectrum β-lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J Clin Microbiol. 2001; 39:3747–9.
12. Bae IK, Jeong SH, Lee K, Yong D, Lee J, Hong SG, et al. Emergence of CTX-M-12 and a novel CTX-M type extended-spectrum β-lactamaseproducing Klebsiella pneumoniae. Korean J Lab Med. 2006; 26:21–6.
13. Kim J, Lim YM, Jeong YS, Seol SY. Occurrence of CTX-M-3, CTX-M-15, CTX-M-14, and CTX-M-9 extended-spectrum β-lactamases in Enterobacteriaceae clinical isolates in Korea. Antimicrob Agents Chemother. 2005; 49:1572–5.
14. Kim YR, Kim SI, Lee JY, Park YJ, Lee KY, Kang MW. Nosocomial transmission of CTX-M-15 and OXA-30 β-lactamase-producing Escherichia coli in a neurosurgical intensive care unit. Ann Clin Lab Sci. 2005; 35:297–301.
15. Oh CE, Hong JS, Bae IK, Song EH, Jeong SH, Lee KW, et al. Dissemination of CTX-M type extended-spectrum β-lactamases and emergence of CTX-M-12 in Escherichia coli. Korean J Lab Med. 2005; 25:252–8.
16. Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests-Ninth Edition: Approved Standard M2-A9. Wayne, PA; CLSI. 2006.
17. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995; 33:2233–9.
crossref
18. Winokur PL, Canton R, Casellas JM, Legakis N. Variations in the prevalence of strains expressing an extended-spectrum β-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin Infect Dis. 2001; 32(Suppl 2):S94–103.
crossref
19. Lee JH, Bae IK, Kwon SB, Jeong SH, Woo GJ, Lee J, et al. Prevalence of CTX-M-type extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae Isolates in Korea, 2003. Korean J Clin Microbiol. 2004; 7:111–8.
20. Park JH, Lee SH, Jeong SH, Kim BN, Kim KB, Yoon JD, et al. Characterization and prevalence of Escherichia coli and Klebsiella pneumoniae isolates producing an extended-spectrum β-lactamase from Korean hospitals. Korean J Lab Med. 2003; 23:18–24.
21. Tzelepi E, Giakkoupi P, Sofianou D, Loukova V, Kemeroglou A, Tsakris A. Detection of extended-spectrum β-lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes. J Clin Microbiol. 2000; 38:542–6.
22. Park YJ, Park SY, Oh EJ, Park JJ, Lee KY, Woo GJ, et al. Occurrence of extended-spectrum β-lactamases among chromosomal AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens in Korea and investigation of screening criteria. Diagn Microbiol Infect Dis. 2005; 51:265–9.
crossref
23. Hoffmann H, Sturenburg E, Heesemann J, Roggenkamp A. Prevalence of extended-spectrum β-lactamases in isolates of the Enterobacter cloacae complex from German hospitals. Clin Microbiol Infect. 2006; 12:322–30.
crossref
24. Ho PL, Shek RH, Chow KH, Duan RS, Mak GC, Lai EL, et al. Detection and characterization of extended-spectrum β-lactamases among bloodstream isolates of Enterobacter spp. in Hong Kong, 2000–2002. J Antimicrob Chemother. 2005; 55:326–32.
crossref
25. Yu WL, Cheng KC, Chi CJ, Chen HE, Chuang YC, Wu LT. Characterisation and molecular epidemiology of extended-spectrum β-lactamase-producing Enterobacter cloacae isolated from a district teaching hospital in Taiwan. Clin Microbiol Infect. 2006; 12:579–82.
crossref
26. Pitout JD, Reisbig MD, Venter EC, Church DL, Hanson ND. Modification of the double-disk test for detection of Enterobacteriaceae producing extended-spectrum and AmpC β-lactamases. J Clin Microbiol. 2003; 41:3933–5.
27. Bae IK, Lee BH, Hwang HY, Jeong SH, Hong SG, Chang CL, et al. A novel ceftazidime-hydrolysing extended-spectrum β-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop. J Antimicrob Chemother. 2006; 58:315–9.
crossref
28. Jeong SH, Bae IK, Kwon SB, Lee JH, Song JS, Jung HI, et al. Dissemination of transferable CTX-M-type extended-spectrum β-lactamase-producing Escherichia coli in Korea. J Appl Microbiol. 2005; 98:921–7.
29. Jeong SH, Bae IK, Lee JH, Sohn SG, Kang GH, Jeon GJ, et al. Molecular characterization of extended-spectrum β-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey. J Clin Microbiol. 2004; 42:2902–6.
30. Pai H. The characteristics of extended-spectrum β-lactamases in Korean isolates of Enterobacteriaceae. Yonsei Med J. 1998; 39:514–9.
31. Pai H, Lyu S, Lee JH, Kim J, Kwon Y, Kim JW, et al. Survey of extended-spectrum β-lactamases in clinical isolates of Escherichia coli and Klebsiella pneumoniae: prevalence of TEM-52 in Korea. J Clin Microbiol. 1999; 37:1758–63.
32. Kariuki S, Corkill JE, Revathi G, Musoke R, Hart CA. Molecular characterization of a novel plasmid-encoded cefotaximase (CTX-M-12) found in clinical Klebsiella pneumoniae isolates from Kenya. Antimicrob Agents Chemother. 2001; 45:2141–3.
33. Lartigue MF, Poirel L, Nordmann P. Diversity of genetic environment of blaCTX-M genes. FEMS Microbiol Lett. 2004; 234:201–7.
34. Golebiewski M, Kern-Zdanowicz I, Zienkiewicz M, Adamczyk M, Zylinska J, Baraniak A, et al. Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum β-lactamase gene blaCTX-M-3. Antimicrob Agents Chemother. 2007; 51:3789–95.
35. Poirel L, Lartigue MF, Decousser JW, Nordmann P. ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli. Antimicrob Agents Chemother. 2005; 49:447–50.
36. Novais A, Canton R, Valverde A, Machado E, Galan JC, Peixe L, et al. Dissemination and persistence of blaCTX-M-9 are linked to class 1 integrons containing CR1 associated with defective transposon derivatives from Tn402 located in early antibiotic resistance plasmids of IncHI2, IncP1-α, and IncFI groups. Antimicrob Agents Chemother. 2006; 50:2741–50.
37. Boyd DA, Tyler S, Christianson S, McGeer A, Muller MP, Willey BM, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum β-lactamase involved in an outbreak in longterm-care facilities in Toronto, Canada. Antimicrob Agents Chemother. 2004; 48:3758–64.
38. Morosini MI, Garcia-Castillo M, Coque TM, Valverde A, Novais A, Loza E, et al. Antibiotic coresistance in extended-spectrum-β-lactamase-producing Enterobacteriaceae and in vitro activity of tigecycline. Antimicrob Agents Chemother. 2006; 50:2695–9.
39. Jacoby GA, Walsh KE, Mills DM, Walker VJ, Oh H, Robicsek A, et al. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother. 2006; 50:1178–82.
40. Nordmann P and Poirel L. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother. 2005; 56:463–9.
41. Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D, Park CH, et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med. 2006; 12:83–8.
crossref

Fig. 1.
Schematic representation of the genetic environment of blaCTX-M genes. (A) C. freundii 06–5-U1734 and E. cloacae 06–2-R1363 (CTX-M-9), (B) C. freundii 06–5-P448, S. marcescens 06-6-R1088 and 06-7-B4288 (CTX-M-3), (C) M. morganii 06-8-P334 (CTX-M-14).
kjcm-11-90f1.tif
Fig. 2.
PFGE analysis of CTX-M-9 producing E. cloacae isolates. Lane 1, 06-2-R1363; Lane 2, 06-5-R1137; Lane 3, 06-5-R468; Lane 4, 06–5-C242; Lane 5, 06–5-R1186; Lane 6, 06–5-R1124. Lane; M, DNA molecular marker.
kjcm-11-90f2.tif
Table 1.
Sequences of the primers for bla genes and blaCTX-M genetic environment
Target Primer name Primer sequence Reference
blaCTX-M CTX-M 1F 5'-CCGTCACGCTGTTGTTAGG-3' [27]
CTX-M 1R 5'-GACGATTTTAGCCGCCGAC-3' [27]
CTX-M 2F 5'-CGGTGCTTAAACAGAGCGAG-3' [27]
CTX-M 2R 5'-CCATGAATAAGCAGCTGATTGCCC-3' [27]
CTX-M 8F 5'-ACGCTCAACACCGCGATC-3' [27]
CTX-M 8R 5'-CGTGGGTTCTCGGGGATAA-3' [27]
CTX-M 9F 5'-GATTGACCGTATTGGGAGTTT-3' [27]
CTX-M 9R 5'-CGGCTGGGTAAAATAGGTCA-3' [27]
CTXM-3A 5'-CCCATGGTTAAAAAATCACT-3' This study
CTXM-3B 5'-CCGTTTCCGCTATTACAAAC-3' This study
blaTEM TEM F 5'-ATGAGTATTCAACATTTCCGT-3' [27]
TEM R 5'-TTACCAATGCTTAATCAGTGA-3' [27]
blaSHV SHV F 5'-CCGGGTTATTCTTATTTGTCGCT-3' [27]
SHV R 5'-TAGCGTTGCCAGTGCTCG-3' [27]
blaGES GES/IBC F 5'-GTTAGACGGGCGTACAAAGATAAT-3' [27]
GES/IBC R 5'-TGTCCGTGCTCAGGATGAGT-3' [27]
blaVEB VEB1-F 5'-ACGAAGAACAAATGCACAAGG-3' This study
VEB1-R 5'-GAACAGAATCAGTTCCTCCG-3' This study
orf477 orf477 reverse 5'-ACTTCAAAAATTATGCCACC-3' [7]
mucA mucA lower 5'-GGCATCAGGCAGGGGTAAGG-3' [7]
orf513 orf513 5'-TGGAAGAGGGCGAAGACGAT-3' [7]
orf513 rev 5'-GCGTTTTATCGGTAGTCGTC-3' [7]
3'CS qacED1 5'-TCGCAACATCCGCATTAAAA-3' [7]
qacED1 reverse 5'-TTTTAATGCGGATGTTGCGA-3' [7]
sulI rev 5'-GCTCAAGAAAAATCCCATCCCC-3' [7]
ISEcp1 tnpA1 ISEcp1 5'-AATACTACCTTGCTTTCTGA-3' [7]
ISEcp1 5 5'-TTCAAAAAGCATAATCAAAGCC-3' [7]
ISEcp1 reverse 5'-CAACCACCTTTCAATCATTTTT-3' [7]
IS903 IS903 Rev 5'-CATCATCCAGCCAGAAAGTT-3' [7]
tnpA1 IS903 Rev 5'-CGGTTGTAATCTGTTGTCCA-3' [7]
pemK pemK F 5'-AGGGGAAATCTGGCTTGTCT-3' [27]
pemK R 5'-TCTGTCGAAACAATGGGTCA-3' [27]
Table 2.
Types of ESBL produced by species
Species (No. tested) No. (%) of DDS positive ESBL (No. of isolates)
CTX-M type SHV type TEM type Mixed ESBLs
E. aerogenes (50) 6 (12) – SHV-12 (3) TEM-52 (2) CTX-M-9+SHV-12 (1)
E. cloacae (50) 10 (20) CTX-M-3 (1), −9 (1) SHV-12 (3) – CTX-M-9+SHV-12 (5)
S. marcescens (50) 8 (16) CTX-M-3 (2) SHV-12 (3) TEM-52 (3) –
C. freundii (50) 4 (8) CTX-M-3 (1), −9 (1), −12 (1) SHV-2a (1) – –
M. morganii (50) 1 (2) CTX-M-14 (1) – – –
Total (250) 29 (12) 8 10 5 6

Abbreviation: DDS, double disk synergy.

Table 3.
Characteristics of CTX-M type ESBLs in clinical isolates
Isolate Specimen Ward CTX-M type Other β-lactamases Upstream of blaCTX-M Associated resistances Associated resistances 1
ISEcp1 ISCR1
C. freundii 06–5-U664 Urine Hematology CTX-M-12 TEM-1 + – GEN TOB
C. freundii 06–5-U1734 Urine Medical ICU CTX-M-9 – – + AMK GEN NET TOB COT
C. freundii 06–5-P448 Wound Oncology CTX-M-3 TEM-1 + – AMK GEN NET TOB COT
E. aerogenes 06–6-R756 Sputum Neurolgogy CTX-M-9 TEM-1, SHV-12 - + GEN NET TOB LEV COT TET
E. cloacae 06–2-R1363 Sputum Cardiosurgery CTX-M-9 SHV-12 – + AMK NET TOB LEV COT TET
E. cloacae 06–5-R1137 Sputum Neurology ICU CTX-M-9 SHV-12 – + NET TOB COT TET
E. cloacae 06–5-R468 Sputum GI CTX-M-9 SHV-12 – + NET TOB COT TET
E. cloacae 06–5-R1186 Sputum GI CTX-M-9 SHV-12 – + NET TOB COT TET
E. cloacae 06–5-R1124 Sputum GI CTX-M-9 – – + AMK GEN NET TOB LEV COT TET
E. cloacae 06–5-C242 Pleural fluid CCU CTX-M-9 SHV-12 – + AMK GEN TOB LEV COT TET
E. cloacae 06–4-P445 Wound Neurosurgery CTX-M-3 – + – AMK NET TOB LEV COT TET
M. morganii 06–8-P334 Bile Oncology CTX-M-14 TEM-1 + –  
S. marscecens 06–6-R1088 8 Sputum Pediatrics CTX-M-3 – + – AMK GEN NET TOB LEV COT TET
S. marscecens 06–7-B4288 8 Discharge ENT CTX-M-3 – + – AMK GEN NET TOB LEV COT TET

Abbreviations: GEN, gentamicin; TOB, tobramycin; AMK, amikacin; NET, netilmicin; COT, cotrimoxazole; LEV, levofloxacin; TET, tetracycline.

TOOLS
Similar articles