1 Department of Laboratory Medicine, Keimyung University College of Medicine, Daegu; 2 Seoul National University College of Medicine, Seoul; 3 Pochon CHA University College of Medicine, Sungnam; 4 The Catholic University of Korea College of Medicine, Seoul; 5 Department of Laboratory Medicine and 6 Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul; and 7 Department of Laboratory Medicine, Kosin University College of Medicine, Busan, Korea
Received 26 April 2005; returned 5 June 2005; revised 15 August 2005; accepted 15 August 2005
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Abstract |
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Methods: Clinical isolates of Escherichia coli and Klebsiella pneumoniae collected from 12 Korean hospitals during FebruaryJuly 2003 were evaluated. Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the putative ESBL-producing strains were tested by the double-disc synergy method. Detection of genes encoding class A ß-lactamases was performed by PCR amplification, and the PCR products were subjected to direct sequencing.
Results: The double-disc synergy test showed positive results in 9.3% (23/246) of E. coli and 23.0% (55/239) of K. pneumoniae isolates. The most prevalent types of Ambler class A ESBLs in E. coli isolates were CTX-M-15 (n = 4) and CTX-M-3 (n = 3), and those in K. pneumoniae isolates were SHV-12 (n = 30) and CTX-M-3 (n = 13). Two isolates produced both SHV-12 and GES-3, simultaneously.
Conclusions: CTX-M-type and/or SHV-12 ESBL-producing E. coli and K. pneumoniae isolates are spreading, and a GES-type ESBL has emerged in Korea.
Keywords: ESBLs , resistance , prevalence
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Introduction |
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The aim of the present study was to describe the prevalence and shift of the ESBLs including non-TEM or -SHV ESBLs in E. coli and K. pneumoniae in recent years in Korea.
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Methods |
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Consecutive non-duplicate nosocomial isolates of E. coli (n = 246) and K. pneumoniae (n = 239) were collected during FebruaryJuly 2003 from 12 hospitals in Korea (Figure 1). E. coli J53 AzideR was used as the recipient strain for conjugation. E. coli ATCC 25933 was used as the reference strain for antimicrobial susceptibility testing.
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Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods according to the recommendations of the NCCLS.7,8 ESBL production was detected by the double-disc synergy (DDS) method. Mating experiments were performed as described previously.1 Transconjugants were selected on MacConkey agar supplemented with ceftazidime (2 mg/L) and sodium azide (150 mg/L; Sigma, St Louis, MO, USA).
Isoelectric focusing
Crude bacterial extracts from clinical isolates were prepared as described previously.1 Sonic extracts and sample buffer (TEFCO corporation, Tokyo, Japan) were mixed in equal amounts and separated by electrophoresis on precast polyacrylamide gels (pH 310, TEFCO corporation, Tokyo, Japan) for 1 h at 100 V, 1 h at 200 V and 40 min at 300 V. ß-Lactamase activity was detected with 0.5 mM nitrocefin (Oxoid, Basingstoke, UK).
Molecular analysis
Primers used in this study are listed in Table 1. The templates for PCR amplification in clinical isolates were a whole-cell lysate or a plasmid preparation. The PCR products were then subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 373A; Applied Biosystems, Weiterstadt, Germany).
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Results |
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Among 23 and 55 DDS-positive E. coli and K. pneumoniae isolates, genes encoding TEM-type ß-lactamases were detected in 78.3% (18/23) of E. coli and 60% (33/55) of K. pneumoniae. Most of them were TEM-1 except for one isolate each of E. coli and K. pneumoniae that had TEM-52. The most common types of class A ESBLs identified were SHV-12 and CTX-M-3 in K. pneumoniae, and CTX-M-15 and CTX-M-3 in E. coli. GES-3, which was detected for the first time in Korea, was detected in two isolates of K. pneumoniae from Bundang city. Six isolates (12.2%) of K. pneumoniae and one E. coli carried multiple ESBL genes (Table 2). Non-TEM- and non-SHV-type ESBLs including PER-1, VEB, IBC and TLA-type ESBLs and members of CTX-M-2 and -8 groups were not detected in this survey. For 14 of 23 (60.9%) DDS-positive E. coli and six of 55 (10.9%) DDS-positive K. pneumoniae isolates no ESBL was detected. These isolates may have produced another ESBL, which was not determined in this study or might have given positive results for ESBL activity.
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Discussion |
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We have previously shown that only 1.7% (9/520) of clinical E. coli and K. pneumoniae isolates produced CTX-M-type ESBLs in 2002,3 but the prevalence of these enzymes was increased to 4.4% (26/585) in the present study. CTX-M-3 was the most common enzyme among CTX-M-type ESBLs, and was isolated from five hospitals (Figure 1). CTX-M-15 differs from CTX-M-3 by one amino acid substitution from glycine to aspartate at position 240, and this amino acid change results in increased enzymatic activity against ceftazidime.2 It was also observed in this study that MICs of ceftazidime for four isolates of E. coli that produced CTX-M-15 were all 128 mg/L, which was higher than that of isolates producing other CTX-M-type ESBLs. Another noteworthy finding was that five CTX-M-type ESBL-producing isolates also produced SHV-12, simultaneously, and they were highly resistant to both ceftazidime and cefotaxime. Simultaneous production of both cefotaximase and ceftazidimase may confer a higher level of resistance against these oxyimino-cephalosporins in clinical isolates.
GES-3 was originally found in an E. coli isolate from a hospital in Greece.5 GES-type ESBLs have not been reported before in Korea. However, in the present study, we found that two isolates of K. pneumoniae from a hospital in Bundang city produced both GES-3 and SHV-12 ESBLs. The MIC of ceftazidime (MIC 256 mg/L) was more than fourfold higher than that of cefotaxime (MIC 64 mg/L) in both of these isolates, and showed little change when clavulanic acid was added.
The present data suggest that the incidence of isolation of SHV-12 and CTX-M-type ESBLs has increased in E. coli and K. pneumoniae isolates in Korea. In conclusion, 9.3% of E. coli and 23.0% of K. pneumoniae isolates from Korea have produced Ambler class A ESBLs. The most common ESBLs in E. coli isolates were CTX-M-15 and CTX-M-3, and in K. pneumoniae were SHV-12 and CTX-M-3. In addition, a GES-type ESBL has emerged in Korea.
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Transparency declarations |
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Acknowledgements |
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References |
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