Abstract

Among the 443 clinical isolates of Escherichia coli and Klebsiella spp. collected between June and November 2003 from 3 university hospitals in Korea, 62 isolates were confirmed as extended-spectrum β-lactamase (ESBL)- or plasmid-mediated AmpC β-lactamase-producers by double disk synergy test, PCR and sequencing for β-lactamase genes. The most frequently identified ESBL gene among E. coli and K. pneumoniae isolates was blaSHV-12 and blaCTX-M (blaCTX-M-9, blaCTX-M-14, blaCTX-M-3, and blaCTX-M-15). Four kinds of plasmid-mediated AmpC β-lactamases, ACT-1, CMY-1, CMY-2, and DHA-1, were detected. ESBL production was associated with high levels of resistance to tetracycline, sulfisoxazole, streptomycin, kanamycin, gentamicin and tobramycin when compared to non-ESBL producing isolates. Conclusively, this study suggests that the CTX-M β-lactamases are prevalent and various kinds of plasmid-mediated AmpC enzymes are distributed in clinical isolates of E. coli and Klebsiella spp. in Korea.

Introduction

The evolution and dissemination of extended-spectrum β-lactamases (ESBL) have compromised the clinical use of third-generation cephalosporins worldwide and caused the crisis of antimicrobial drug resistance. ESBLs are usually described as acquired β-lactamases that are encoded mostly by plasmid-located genes, leading to the complex and dynamic evolution and epidemiology of ESBLs. The number of ESBL variants identified has been constantly growing and more than 100 different ESBL variants are known at present (http://www.lahey.org/studies/webt.htm). Most ESBLs are derivatives of TEM-1, TEM-2, or SHV-1 enzymes; however, reports describing the emergence of β-lactamases belong to other families, such as PER, VEB, CTX-M and/or OXA derivatives, are increasing worldwide [1–5].

Among gram-negative pathogens in Korea, the incidence of resistance to extended-spectrum β-lactam antibiotics is becoming an ever-increasing problem and rapid increase in ESBL-producing Escherichia coli and Klebsiella pneumoniae has been reported [6–9]. Although, the most commonly identified ESBLs in Korea are TEM-52, SHV-2a and SHV-12 [6–9], recent report suggests dissemination of CTX-M-14 in Korea [10]. Indeed, we here report the dissemination of CTX-M enzymes, such as CTX-M-3, CTX-M-9, CTX-M-15, and CTX-M-14, among the isolates of E. coli and Klebsiella spp. collected in 2003 from 3 university hospitals located in different cities of Korea. We also report on the first identification of ACT-1 and CMY-2, plasmid-mediated AmpC β-lactamases, from the isolates of E. coli in Korea.

Materials and methods

Clinical isolates

Consecutive non-duplicate nosocomial isolates of E. coli, K. oxytoca, and K. pneumoniae were collected between June and November 2003 from 3 university hospitals located in 3 different cities in Korea. Species identification was carried out with VITEK-GNI CARDS (bioMérieux Vitek Inc., Hazelwood, Mo.) by standard methods [11]. For selection of ESBL-producing isolates, strains for which the cefotaxime MIC was ?2 mg l−1 were subjected to the double-disk synergy test with cefotaxime, ceftazidime, aztreonam, cefepime, and amoxicillin-clavulanic acid disks (Oxoid, Basingstoke, United Kingdom) as described previously [8]. The disks were placed 20 mm apart (from center to center); for selected isolates the test was repeated with the distance reduced to 15 mm.

Antimicrobial susceptibility tests

Minimal inhibitory concentrations (MICs) were measured using a standard agar dilution method according to the approved method of the National Committee for Clinical Laboratory Standards [12]. E. coli ATCC 25922 was used as a quality reference strain. The antimicrobial agents included were ampicillin, cefoxitin, cefotaxime, kanamycin, tobramycin, gentamicin, amikacin, sulfisoxazole, chloramphenicol, streptomycin, tetracycline, and trimethoprim (all from Sigma).

PCR amplification and nucleotide sequencing

PCR was performed to detect β-lactamase genes with primers shown in Table 1. PCRs were performed with 30 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 50 °C for TEM, CTX-M-9 and DHA-1, and 55 °C for SHV, CTX-M-3 and OXA, respectively, and 1 min at 72 °C for polymerization. Final products were extended by incubation for 10 min at 72 °C. Amplified PCR products were sequenced on both strands. Sequencing was carried out with the Taq DyeDeoxyTerminal cycle-sequencing kit using primers used for PCR and the sequence was analyzed in an automatic DNA sequencer (377 ABI Prism, Perkin Elmer). DNA sequence analysis was performed with DNASIS for Windows (Hitachi Software Engineering America Ltd., San Bruno, CA). Database similarity searches for both the nucleotide sequences and deduced protein sequences were performed with BLAST at the National Center for Biotechnology Information website.

1

Oligonucleotide primers used for detection of β-lactamase genes

Primers Tma (°C) Nucleotide sequences References (GenBank No.) Expected amplicon size (bp) 
CTX-M-9-S 50 5′-TAT TGG GAG TTT GAG ATG GT-3′ AF4546633.2; 742–761 932 
CTX-M-9-AS  5′-TCC TTC AAC TCA GCA AAA GT-3′ AF4546633.2; 1655–1674  
CTX-M-3-S 55 5′-CGT CAC GCT GTT GTT AGG AA-3′ AJ632119.1; 180–209 780 
CTX-M-3-AS  5′-ACG GCT TTC TGC CTT AGG TT-3′ AJ632119.1; 941–960  
TEM-S 50 5′-ATA AAA TTC TTG AAG ACG AAA-3′ AB103506; 166–186 1080 
TEM-AS  5′-GAC AGT TAC CAA TGC TTA ATC-3′ AB103506; 1225–1245  
SHV-S 55 5′-TGG TTA TGC GTT ATA TTC GCC-3′ AY223863; 166–186 865 
SHV-AS  5′-GGT TAG CGT TGC CAG TGC T-3′ AY223863; 1015–1031  
OXA-1-S 55 5′-AGC CGT TAA AAT TAA GCC C-3′ AV162283.2; 1052–1070 908 
OXA-1-AS  5′-CTT GAT TGA AGG GTT GGG CG-3′ AV162283.2; 1941–1960  
CMY-1-S 60 5′-GAG CAG ACC CTG TTC GAG AT-3′ X92508; 570–589 846 
CMY-1-AS  5′-GAT TGG CCA GCA TGA CGA TG-3′ X92508; 1397–1416  
CMY-2-S 60 5′-TGGCCAGAACTGACAGGCAAA-3′ X78117; 478–498 462 
CMY-2-AS  5′-TTTCTCCTGAACGTGGCTGGC-3′ X78117; 919–939  
ACT-1-S 50 5′-AAACCTGTCACTCCACAAAC-3′ U58495; 244–264 887 
ACT-1-AS  5′-GGGTTCGGATAGCTTTTATT-3′ U58495; 1111–1130  
DHA-1-S 50 5′-GTT ACT CAC ACA CGG AAG GT-3′ AY205600; 75–94 869 
DHA-1-AS  5′-TTT TAT AGT AGC GGG TCT GG-3′ AY205600; 925–944  
Primers Tma (°C) Nucleotide sequences References (GenBank No.) Expected amplicon size (bp) 
CTX-M-9-S 50 5′-TAT TGG GAG TTT GAG ATG GT-3′ AF4546633.2; 742–761 932 
CTX-M-9-AS  5′-TCC TTC AAC TCA GCA AAA GT-3′ AF4546633.2; 1655–1674  
CTX-M-3-S 55 5′-CGT CAC GCT GTT GTT AGG AA-3′ AJ632119.1; 180–209 780 
CTX-M-3-AS  5′-ACG GCT TTC TGC CTT AGG TT-3′ AJ632119.1; 941–960  
TEM-S 50 5′-ATA AAA TTC TTG AAG ACG AAA-3′ AB103506; 166–186 1080 
TEM-AS  5′-GAC AGT TAC CAA TGC TTA ATC-3′ AB103506; 1225–1245  
SHV-S 55 5′-TGG TTA TGC GTT ATA TTC GCC-3′ AY223863; 166–186 865 
SHV-AS  5′-GGT TAG CGT TGC CAG TGC T-3′ AY223863; 1015–1031  
OXA-1-S 55 5′-AGC CGT TAA AAT TAA GCC C-3′ AV162283.2; 1052–1070 908 
OXA-1-AS  5′-CTT GAT TGA AGG GTT GGG CG-3′ AV162283.2; 1941–1960  
CMY-1-S 60 5′-GAG CAG ACC CTG TTC GAG AT-3′ X92508; 570–589 846 
CMY-1-AS  5′-GAT TGG CCA GCA TGA CGA TG-3′ X92508; 1397–1416  
CMY-2-S 60 5′-TGGCCAGAACTGACAGGCAAA-3′ X78117; 478–498 462 
CMY-2-AS  5′-TTTCTCCTGAACGTGGCTGGC-3′ X78117; 919–939  
ACT-1-S 50 5′-AAACCTGTCACTCCACAAAC-3′ U58495; 244–264 887 
ACT-1-AS  5′-GGGTTCGGATAGCTTTTATT-3′ U58495; 1111–1130  
DHA-1-S 50 5′-GTT ACT CAC ACA CGG AAG GT-3′ AY205600; 75–94 869 
DHA-1-AS  5′-TTT TAT AGT AGC GGG TCT GG-3′ AY205600; 925–944  

aAnnealing temperature used for PCR.

Results

Between June and November 2003, 443 isolates including 272 of E. coli, 13 of K. oxytoca, and 158 of K. pneumoniae were collected from 3 university hospitals in Korea. Among them, 87 isolates revealed ?2 mg l−1 of MIC against cefotaxime and they were subjected to the double disk synergy test (DDST), PCR of β-lactamase genes, and subsequent sequencing of amplified β-lactamase genes. Of 87 isolates revealed ?2 mg l−1 of MIC against cefotaxime, 45 isolates (35 of E. coli and 10 of K. pneumoniae) showed resistance to cefoxitin. They were screened for plasmid-mediated AmpC β-lactamases with the primers shown in Table 1 and amplified PCR products were analyzed by nucleotide sequencing.

From the results, 62 strains were identified as ESBL- or plasmid-mediated AmpCs-producers, including 32 isolates of E. coli, 2 isolates of K. oxytoca, and 28 isolates of K. pneumoniae (Table 2). The most frequently identified ESBL gene among E. coli and K. pneumoniae isolates was blaSHV-12 and blaCTX-M which identified from 18 (29%) and 18 (29%) isolates, respectively. Among 32 isolates of E. coli, 8 isolates carried blaTEM- and SHV-derived genes (blaTEM-52 in 2 isolates and blaSHV-12 6 isolates); 15 isolates carried blaCTX-M (blaCTX-M-9 in 1 isolate, blaCTX-M-14 in 4, blaCTX-M-3 in 2, and blaCTX-M-15 in 8); blaCMY-2 and blaACT-1, PABL-coding genes, were identified in one and six isolates, respectively; and remaining 2 isolates carried several blaESBL genes (blaSHV-12 and blaCTX-M-9 in 1 isolate and blaCTX-M-3, blaOXA-30, and blaCMY-1 in 1 isolate). Among 28 isolates of K. pneumoniae, 10 isolates carried blaSHV-12 and 3 isolates carried blaCTX-M (blaCTX-M-14 in one and blaCTX-M-3 in two); blaCMY-1 and blaDHA-1 were identified in one and one isolate, respectively; and remaining 13 isolates carried several blaESBL genes. Seven of eight isolates carrying blaDHA-1 also carried blaSHV-12, blaTEM, blaCTX-M-3, and/or blaOXA-30. One K. pneumoniae isolate carried five different kinds of bla genes, such as blaTEM-52, blaSHV-12, blaCTX-M-3, blaDHA-1, and blaOXA-30.

2

Distribution of ESBLs among the clinical isolates of E. coli and Klebsiella spp. collected from 3 university hospitals in Korea

Species Number of isolates Type of ESBL (number of isolates) 
  TEM SHV CTX-M AmpC Mixed ESBLs 
E. coli 32 TEM-52 (2) TEM-1/SHV-12 (3) CTX-M-9 (1) ACT-1 (1) SHV-12/CTX-M-9 (1) 
   SHV-12 (1) CTX-M-14 (1) TEM-1/ CMY-2 (6) CTX-M-3/OXA-30/CMY-1(1) 
   TEM-1/SHV-12/OXA-30 (2) TEM-1/CTX-M-14 (2)   
    TEM-1/CTX-M-14/OXA-30 (1)   
    TEM-1/CTX-M-3 (1)   
    TEM-1/CTX-M-3/OXA-30 (1)   
    TEM-54/CTX-M-15/OXA-30 (3)   
    TEM-1/CTX-M-15/OXA-30 (5)   
K. oxytoca  TEM-1/SHV-12 (1)    
   TEM-54/SHV-12 (1)    
K. pneumoniae 28  TEM-1/SHV-12/OXA-30 (1) SHV-11/CTX-M-3 (2) TEM-1/SHV-1/OXA-30/CMY-1(1) SHV-12/CTX-M-3/DHA-1/OXA-30 (1) 
   SHV-12 (3) SHV-11/CTX-M-14(1) TEM-1/DHA-1(1) TEM-54/SHV-12/DHA-1(2) 
   TEM-54/SHV-12 (2)   TEM-52/SHV-12 (1) 
   TEM-1/SHV-12 (4)   SHV-12/DHA-1(2) 
      SHV-12/CTX-M-14 (4) 
      TEM-52/SHV-12 (1) 
      TEM-52/SHV-12/OXA-30 (1) 
      TEM-1/SHV-12/CTX-M-3/DHA-1/OXA-30 (1) 
Number of strains (%) 62 2 (3.3) 18 (29.0) 18 (29.0) 9 (14.5) 15 (24.2) 
Species Number of isolates Type of ESBL (number of isolates) 
  TEM SHV CTX-M AmpC Mixed ESBLs 
E. coli 32 TEM-52 (2) TEM-1/SHV-12 (3) CTX-M-9 (1) ACT-1 (1) SHV-12/CTX-M-9 (1) 
   SHV-12 (1) CTX-M-14 (1) TEM-1/ CMY-2 (6) CTX-M-3/OXA-30/CMY-1(1) 
   TEM-1/SHV-12/OXA-30 (2) TEM-1/CTX-M-14 (2)   
    TEM-1/CTX-M-14/OXA-30 (1)   
    TEM-1/CTX-M-3 (1)   
    TEM-1/CTX-M-3/OXA-30 (1)   
    TEM-54/CTX-M-15/OXA-30 (3)   
    TEM-1/CTX-M-15/OXA-30 (5)   
K. oxytoca  TEM-1/SHV-12 (1)    
   TEM-54/SHV-12 (1)    
K. pneumoniae 28  TEM-1/SHV-12/OXA-30 (1) SHV-11/CTX-M-3 (2) TEM-1/SHV-1/OXA-30/CMY-1(1) SHV-12/CTX-M-3/DHA-1/OXA-30 (1) 
   SHV-12 (3) SHV-11/CTX-M-14(1) TEM-1/DHA-1(1) TEM-54/SHV-12/DHA-1(2) 
   TEM-54/SHV-12 (2)   TEM-52/SHV-12 (1) 
   TEM-1/SHV-12 (4)   SHV-12/DHA-1(2) 
      SHV-12/CTX-M-14 (4) 
      TEM-52/SHV-12 (1) 
      TEM-52/SHV-12/OXA-30 (1) 
      TEM-1/SHV-12/CTX-M-3/DHA-1/OXA-30 (1) 
Number of strains (%) 62 2 (3.3) 18 (29.0) 18 (29.0) 9 (14.5) 15 (24.2) 

Resistance rates of the ESBL- or plasmid-mediated AmpCs-producing E. coli and Klebsiella spp. against non-β-lactam antibiotics are given in Table 3. Production of ESBL or plasmid-mediated AmpCs among E. coli and Klebsiella spp. was associated with high levels of resistance to tetracycline, sulfisoxazole, streptomycin, kanamycin, gentamicin and tobramycin when compared to non-ESBL producing isolates. Especially, resistance rates of ESBL-producers to aminoglycosides except amikacin were very high as 63% to streptomycin, 81% to kanamycin, 60% to gentamicin, and 92% to tobramycin. But the resistance rates to ampicillin, chloramphenicol, trimethoprim, and amikacin were the same or lower in ESBL-producing isolates than in non-ESBL producing isolates.

3

Antimicrobial susceptibilities (%) of ESBL-producing isolates of E. coli and Klebsiella spp.

Antimicrobials Total (87)a E. coli (51) Klebsiella spp. (36) 
 ESBL (62) Non-ESBL (25) ESBL (32) Non-ESBL (19) ESBL (30) Non-ESBL (6) 
 
Ampicillin 89 11 96 84 16 95 85 15 100 
Chloramphenicol 32 68 32 68 41 59 37 63 21 79 17 83 
Tetracycline 40 60 28 72 69 31 37 63 91 100 
Sulfisoxazole 73 27 56 44 81 19 58 42 58 42 50 50 
Trimethoprim 47 53 52 48 59 41 53 47 30 70 50 50 
Streptomycin 63 37 40 60 69 31 47 53 52 48 17 83 
Kanamycin 81 19 52 48 75 25 53 47 79 21 50 50 
Gentamicin 60 40 48 52 66 34 47 53 48 52 50 50 
Amikacin 29 71 28 72 25 75 26 74 30 70 33 67 
Tobtramycin 92 48 52 91 47 53 85 15 50 50 
Antimicrobials Total (87)a E. coli (51) Klebsiella spp. (36) 
 ESBL (62) Non-ESBL (25) ESBL (32) Non-ESBL (19) ESBL (30) Non-ESBL (6) 
 
Ampicillin 89 11 96 84 16 95 85 15 100 
Chloramphenicol 32 68 32 68 41 59 37 63 21 79 17 83 
Tetracycline 40 60 28 72 69 31 37 63 91 100 
Sulfisoxazole 73 27 56 44 81 19 58 42 58 42 50 50 
Trimethoprim 47 53 52 48 59 41 53 47 30 70 50 50 
Streptomycin 63 37 40 60 69 31 47 53 52 48 17 83 
Kanamycin 81 19 52 48 75 25 53 47 79 21 50 50 
Gentamicin 60 40 48 52 66 34 47 53 48 52 50 50 
Amikacin 29 71 28 72 25 75 26 74 30 70 33 67 
Tobtramycin 92 48 52 91 47 53 85 15 50 50 

aParenthesis indicates the number of isolates.

Discussion

In this study, the overall prevalence rates of ESBL-producers in E. coli and K. pneumoniae isolates were 11.8% (32 of 272 isolates) and 17.7% (28 of 158 isolates), respectively. The result was very similar to the ESBL survey in 1999 in 28 hospitals in Korea which revealed a frequency of 8.3% and 18.1% ESBL producers among E. coli and K. pneumoniae, respectively [13]. Data from the SENTRY Antimicrobial Surveillance Program, performed on K. pneumoniae, E. coli, P. mirabilis and Salmonella spp. isolates collected in 1997–1999 from all over the world, showed that ESBL frequency in K. pneumoniae may account for about 45% in Latin America, 25% in the Western Pacific, 23% in Europe and 8% in the USA [14]. Therefore, it seems that the frequency of ESBL among K. pneumoniae in Korea may be higher than in USA but lower than in Latin America and Europe.

So far, TEM-52 and SHV-12 are the most common types of ESBL in Korea and CTX-M enzyme has been rarely found in Korea [6–10]. Although CTX-M-14 has been identified from only 4 isolates in Korea [10], identification of this enzyme in three different genera, i.e., Shigella sonnei, E. coli, and K. pneumoniae, and from different parts of Korea suggests dissemination of this enzyme in Korea. Indeed, CTX-M ESBL, with SHV-12, was the most frequently found ESBL among E. coli and K. pneumoniae isolates in this study. Moreover, four kinds of CTX-M ESBLs, CTX-M-3, CTX-M-9, CTX-M-15, and CTX-M-14, were found, suggesting that CTX-M enzymes have been persisted for longer periods and evolved in Korean hospital environments. Therefore, more attention and evaluation for CTX-M enzymes are needed to catch the precise situation of ESBLs in Korea.

Since first CTX-M-β-lactamase, FEC-1 was discovered in a cefotaxime-resistant E. coli strain in 1986 [14], CTX-M β-lactamases become the most widespread non-TEM, non-SHV plasmid-mediated class A ESBLs. In Far East, there have been reports of CTX-M-2, CTX-M-3, CTX-M-15, and CTX-M-14 in Japan [15–17] and of CTX-M-3, CTX-M-9, CTX-M-13, and CTX-M-14 from isolates of E. coli, K. pneumoniae and S. marcescens in China and Taiwan [18–21]. CTX-M-14 and CTX-M-17 have commonly been observed in E. coli and K. pneumoniae strains in Vietnam [22,23] and CTX-M-15 was reported in India [24]. Therefore, it seems that CTX-M-3 and CTX-M-14 types are the most prevalent type of CTX-M enzymes in Far East.

Since 1989, over 20 plasmid-mediated AmpC β-lactamases have been reported worldwide [25,26]. Whereas ESBLs confer resistance to the oxyimino-cephalosporins such as ceftazidime, cefotaxime, and aztreonam, plasmid-mediated AmpCs confer resistance to cephamycins such as cefoxitin and cefotetan. From this study, 8 of 272 (2.9%) E. coli isolates and 8 of 158 (5.1%) K. pneumoniae isolates demonstrated plasmid-mediated AmpCs-producers. Similarly, a survey performed in 2002 in 12 university hospitals in Korea showed that the prevalence rate of plasmid-mediated AmpCs-producers was 1.5% (8 of 544 isolates) in E. coli and 5.4% (20 of 367 isolates) in K. pneumoniae[27].

To date in Korea, CMY-1, CMY-11, and DHA-1 plasmid-mediated AmpCs has been identified from cefoxitin-resistant E. coli and K. pneumoniae isolates [8,27–29]. However, from this study, CMY-2 and ACT-1, in addition to CMY-1 and DHA-1, were firstly identified in six E. coli isolates and one E. coli isolate, respectively. Although this may be a first report on the blaCMY-2-carrying E. coli isolates in Korea, the spread of blaCMY-2 among E. coli and Salmonella isolates from food animals has recently been reported in North America and has raised a public health concern [15,30–34]. Moreover, a connection between CMY-2-producing E. coli and Salmonella isolates from humans and those from food animals has been established in the United States [32–35] and blaCMY-2-carrying Salmonella and E. coli isolates associated with community-acquired infections in Taiwan [36]. To prevent further spread of blaCMY-2-carrying isolates in Korea, constant and consistent surveillance is needed.

Since ESBL producers express their β-lactamase genes from plasmids, these organisms may also have genes coding for resistance to additional classes of antibiotics. This study showed association of ESBL production with high levels of resistance to tetracycline, sulfisoxazole, and aminoglycosides such as streptomycin, kanamycin, gentamicin and tobramycin when compared to non-ESBL producing isolates. This finding suggests that genes coding for ESBLs and genes coding for resistance to these antibiotics may reside within the same plasmids and therefore be spread together. This means that resistance to two different kinds of drugs may be co-selected by the use of either one.

It should be also emphasized that most of ESBL-producers from this study carried more than two kinds of bla genes. Of 62 ESBL-producers, 30 isolates (48.4%) and 18 isolates (29.0%) carried blaTEM-1 or blaOxa-30, respectively, besides to blaESBL. Even more, 15 (24.2%) of 62 ESBL-producers carried more than two kinds of blaESBL. Surprisingly, one isolate of K. pneumoniae carried three kinds of blaESBL, such as blaSHV-12, blaCTX-M, and blaDHA-1, in addition to blaTEM-1 and blaOXA-30. These findings indicate that very complex and dynamic evolution and dissemination of bla genes have been progressed in hospital environments for long periods.

In conclusion, the overall prevalence rates of ESBL-producers were relatively high as 11.8% and 17.7% in E. coli and K. pneumoniae, respectively, and the most common types of ESBLs were CTX-M and SHV-12. To our knowledge, CTX-M-3, CTX-M-9, CTX-M-15, ACT-1, and CMY-2 were firstly identified in E. coli and K. pneumoniae isolates from Korea. The relatively high prevalence of CTX-M enzyme and the presence of diverse plasmid-mediated AmpCs underline the need for routine surveillance for these β-lactamases.

Acknowledgments

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).

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Author notes

1
Present address: Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Republic of Korea.