Relationship between blaSHV-12 and blaSHV-2a in Korea

Jungmin Kima, Haeng-Seop Shinb, Sung-Yong Seolb and Dong-Taek Chob,*

a Department of Microbiology, College of Medicine, Dankook University, Cheonan; b Department of Microbiology, Kyungpook National University School of Medicine, 101, DongIn-2Ga, Taegu 700-422, South Korea


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
In contrast to the USA and Europe, where SHV-2, SHV-4 and SHV-5 are the prevalent extended-spectrum SHV enzymes, in Korea SHV-2a and SHV-12 are the most frequently identified extended-spectrum SHV enzymes. A 6.6 kb BamHI fragment containing the blaSHV-12 gene of strain K7746 isolated from one university hospital in Korea was cloned into the pCRScriptCAM vector. Sequencing of the constructed recombinant plasmid pK7746-C1 revealed that the immediate upstream sequence of the blaSHV-12 gene showed little similarity to the part of the prototype blaSHV-1 gene due to the insertion of an IS26 element next to the –10 region. Instead, the upstream sequences of blaSHV-12 retained 100% DNA identity with the part of plasmid pMPA2a from Klebsiella pneumoniae KPZU-3 carrying blaSHV-2a. The restriction map of the inserted 6.6 kb DNA fragment of plasmid pK7746-C1 was also homologous to that of plasmid pMPA2a, suggesting a common lineage of blaSHV-12 and blaSHV-2a. We also studied, using PCR, the upstream non-coding region of several SHV ß-lactamase genes for the presence of IS26 sequence. The flanking IS26 sequence in the immediate upstream region of the blaSHV gene was not detected in five standard strains producing SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. However, IS26 was detected in all 69 clinical strains producing SHV-2a or SHV-12 isolated from three university hospitals in Korea during 1993–1999. The above findings suggest a direct evolution of SHV-12 from SHV-2a, not from SHV-2 to -5, and it is considered to be one of the reasons for the absolute predominance of SHV-2a and SHV-12 in Korea.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Extended-spectrum ß-lactamases (ESBLs), such as the plasmid-mediated class A TEM- and SHV-type enzymes, have developed by stepwise mutations in their structural genes, resulting in either single or multiple amino acid changes in the encoded enzymes, and these changes sufficiently remodel the active site to allow attack on aminothiazolyl compounds.1

Since they were first identified at the beginning of the 1980s, ESBL-producing microorganisms, belonging mostly to the Enterobacteriaceae, have spread by nosocomial routes throughout the world. The incidence of ESBL producers in Korean isolates of Escherichia coli and Klebsiella pneumoniae were in the range 4.8–22.5% and 13.2–22.4%, respectively.2

In contrast to the USA and Europe, where SHV-2, SHV-4 and SHV-5 are the most prevalent extendedspectrum SHV enzymes, SHV-12 and SHV-2a are the most frequently identified extended-spectrum SHV enzymes among K. pneumoniae strains3 in Korea. They are also found in E. coli4 and even in Enterobacter cloacae, in which chromosomal AmpC cephalosporinases predominate.5 SHV-2a first appeared in Germany in 1991.6 It has since been seen around the world and is particularly common in countries that border the Mediterranean, where it often occurs in Salmonella isolates. Recently, SHV-2a has been reported to be produced by Pseudomonas aeruginosa,7 indicating that the ESBL gene is no longer limited to Enterobacteriaceae. SHV-12 was first described in a survey of SHV ß-lactamases in Switzerland in 1997.8 Although the prevalence of SHV-12 in Enterobacteriaceae in Europe and the USA is not known, it is widespread among K. pneumoniae and E. coli strains in Korea.3,5 In addition, no other SHV-ESBLs besides SHV-2a and SHV-12 have been isolated in Korea. The reason for the prevalence of SHV-2a and SHV-12 in Korea is not clear, but our study provides some clues.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains and plasmids

K. pneumoniae K7746 was isolated from a urine specimen of a patient hospitalized in Kyungpook National University Hospital of Taegu, Korea, in 1997. Strain K7746 was resistant to ampicillin and cephalosporins, including extended-spectrum cephalosporins, but susceptible to cefoxitin and imipenem. It also conferred resistance to chloramphenicol, sulfisoxazole, trimethoprim, kanamycin, gentamicin and tobramycin. The MIC of ceftazidime was 256 mg/L, the MICs of ceftriaxone and cefotaxime were 128 and 512 mg/L, respectively, and the MIC of aztreonam was 64 mg/L. E. coli RG488 and E. coli XL1-Blue were used as recipients in conjugation and transformation experiments, respectively.

Antibiotic susceptibility testing and analytical isoelectric focusing

MICs were determined by the agar dilution method according to NCCLS guidelines.9 The drugs tested were chloramphenicol, sulfisoxazole, trimethoprim, kanamycin, gentamicin, tobramycin, ampicillin, ticarcillin, ceftriaxone, cefotaxime, cefoxitin, ceftazidime, aztreonam and imipenem. Isoelectric focusing of ß-lactamase was performed as described previously.10

Transfer of resistance and plasmid analysis

To test the transmissibility of the ceftazidime and aztreonam resistance of the isolate K7746, a conjugation experiment was performed with E. coli RG488 as the recipient. Logarithmic phase cells of K7746 were mated with similar cultures of E. coli RG488 on trypticase soy agar plates (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). After overnight incubation, transconjugants were selected on Mueller–Hinton agar plates containing 50 mg/L of rifampicin (Sigma, St Louis, MO, USA) and 10 mg/L of aztreonam (Dong-A Biotech Co., Seoul, Korea). To confirm the presence of plasmids and to estimate their sizes, plasmids from clinical isolates and transconjugants were extracted by the method of Kado & Liu.11

Cloning and sequencing of the ß-lactamase gene

Plasmid DNA was prepared with the Qiagen plasmid kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer' instructions and then digested with BamHI (Boehringer Mannheim, Mannheim, Germany). T4 DNA ligase, ligation buffer and calf intestinal phosphatase were purchased from Gibco-BRL, Tsuen Wan, Hong Kong, and cloning of the BamHI fragments into pCRScriptCAM SK+ cloning vector (Stratagene, La Jolla, CA, USA) followed by transformation of E. coli strain XL1-Blue was performed according to Maniatis et al.12 Clones were initially selected on Luria–Bertani agar plates containing 100 mg/L of chloramphenicol, X-Gal and IPTG. The ß-lactamases of the selected clones were tested by isoelectric focusing. A clone showing the ß-lactamase with a pI of 8.2 was finally selected for further studies. DNA sequencing was performed by the dideoxy chain-termination method13 using the OmniBase sequencing kit (Promega, Madison, WI, USA) and [32P]dATP (DuPont, North Billerica, MA, USA) according to the manufacturer' instructions. DNA sequence homology search was carried out with the GenBank BLAST program.

Restriction endonuclease mapping of recombinant clone and Southern blot hybridization

Restriction enzymes were purchased from Boehringer Mannheim. Restriction enzyme digests, 1% agarose gel electrophoresis and Southern blotting by vacuum on to nylon membranes (Boehringer Mannheim) were carried out using conventional methods.12 The location of the blaSHV gene was studied by Southern blot hybridization with the blaSHV gene probe. The probe was prepared by PCR with the primers S1 (5'-CTACTCGCCGGTCAGCG-3') and S2 (5'-GACCCGATCGTCCACCAT-3'), corresponding to nucleotides 486–502 and 805–822 of the blaSHV-2 gene, respectively.14 The probe hybridized to the IS26 element was prepared by PCR with the primers IS26-1 (5'-TTACATTTCAAAAACTCTGC-3') and IS26-2 (5'-ATGAACCCATTCAAAGGCCGG-3'), corresponding to nucleotides 681–700 and 1365–1385 of pMPA2a clone, respectively.15 Probe labelling and hybridization were performed with the digoxigenin labelling and detection kit (Boehringer Mannheim), according to the manufacturer' instructions.

PCR mapping of IS26-blaSHV region

The presence of IS26 in the promoter region of seven SHV ß-lactamase genes, including blaSHV-1, blaSHV-2, blaSHV-3, blaSHV-4, blaSHV-5, blaSHV-2a and blaSHV-12, was examined using a PCR mapping method. Five strains (kindly provided by G. A. Jacoby), each carrying one of blaSHV-1 (R1010), blaSHV-2 (pMG229), blaSHV-3 (pUD18), blaSHV-4 (pUD21) or blaSHV-5 (pAFF2), and 69 clinical isolates carrying blaSHV-2a or blaSHV-12 (55 strains of K. pneumoniae, four strains of E. coli and 10 strains of E. cloacae)3,5 were used. Primers used in the PCR mapping experiment were IS (5'-GCGTTCAGCCAGCATC-3') and S3 (5'-GGCCAGATCCATTTCTATCA-3'), corresponding to nucleotides 1272–1287 and 1646–1665 of the pMPA2a clone,15 respectively. The primer set was designed to detect both sequences of IS26 and blaSHV. PCR amplification was performed in 25 µL reaction mixtures containing 1 µL of crude cellular lysate, 50 mM KCl, 10 mM Tris–HCl pH 8.3, 1 mM MgCl2, 0.1 µM oligonucleotide primers, 200 µM deoxynucleoside triphosphate mix and 2.5 U of Taq DNA polymerase (Promega). PCR assay was performed in a Gene Cycler thermal cycler (Bio-Rad, Hercules, CA, USA) with the following cycling parameters: denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 30 s; and a final extension period of 72°C for 10 min.

Nucleotide sequence accession number

The nucleotide sequence of upstream non-coding and coding region of blaSHV-12 reported in this study will appear under the GenBank accession number AY008838.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
ß-Lactam resistance of the strain K7746 was transferred to E. coli RG488 in conjugation experiments. Resistance to chloramphenicol, sulfisoxazole, trimethoprim, kanamycin and tobramycin was not cotransferred with ceftazidime resistance; however, gentamicin resistance was transferred. Isoelectric focusing of crude lysates of K7746 and E. coli RG488 transconjugant T7746 revealed a single ß-lactamase in each with a pI of 8.2, presumed to be SHV-12 or SHV-5. Plasmid DNA from K7746 and transconjugant T7746 was extracted and analysed by gel electrophoresis. A 126 kb plasmid was detected in both strains.

Plasmid DNA (126 kb) from the transconjugant T7746 was digested with BamHI and ligated to the BamHI-digested vector plasmid pCRScriptCAM, a chloramphenicol resistance conferring cloning vector. By further isoelectric focusing of crude lysates of pre-selected clones, one clone of the ß-lactamase with a pI of 8.2, K7746-C1, was selected and analysed. Its plasmid, pK7746-C1 (10 kb), consisted of pCRScriptCAM (3.4 kb) and a 6.6 kb BamHI fragment containing the blaSHV gene. Locations of sites for restriction endonucleases and the location of the blaSHV gene on pK7746-C1 were determined by several digestions and subsequent Southern blot hybridization with the blaSHV gene probe (Figure 1Go). The restriction map of pK7746-C1 obtained is shown in Figure 2Go.



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Figure 1. (a) Restriction endonuclease analysis of pK7746-C1. Lanes 1 and 15: Lambda/HindIII; lanes 2–14, no cut, ApaI, BamHI, BglI, ClaI, EcoRI, HincII, PvuI, SalI, SmaI, pCRScriptCAM/SalI, TEM-1, SHV-2. (b) Southern hybridization with SHV probe. Lanes 1 and 15: Lambda/HindIII; lanes 2–14 [size (kb) of fragment hybridized with SHV probe): no cut, ApaI (6.9), BamHI (3.6), BglI (0.9), ClaI (6.3), EcoRI (11), HincII (2.4), PvuI (1.6, 0.9), SalI (4.8), SmaI (5.9), pCRScriptCAM/SalI, TEM-1, SHV-2.

 


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Figure 2. Physical map of the BamHI fragment (6.6 kb) of pK7746-C1 including blaSHV-12 and IS26.

 
The 1134 bp nucleotide sequence, including the blaSHV gene, within pK7746-C1 was determined (GenBank accession number AY008838). It revealed that blaSHV-12 was the gene responsible for extended-spectrum cephalosporin resistance of strain K7746. Notably, the nucleotide sequence upstream of the –10 region of blaSHV-12 showed little similarity to that of the prototype blaSHV-1 gene, but it corresponded exactly to the sequence from IS26, including the 14 bp terminal inverted repeat sequence (IRS).16 In addition, sequences homologous to blaSHV-1 begin immediately past the outside base of the IS26 IRS, suggesting an insertion of IS26 next to the –10 sequence of blaSHV-12. The IS26-insertion generated a hybrid promoter in which the TTGTGA –35 region of the blaSHV-12 promoter was replaced by the –35 sequence TTGCAA provided by the left inverted repeat of IS26.

The immediate upstream sequences of blaSHV-12 (Figure 3Go) retained 100% DNA identity with the part of plasmid pMPA2a from K. pneumoniae KPZU-3 producing SHV-2a15 and pMK105 from Shigella dysenteriae PB-10 producing SHV-11.17 Comparison of the sequence of the coding region also revealed that the bla genes shared the same substitution of glutamine for leucine at position 35, and the same silent mutations in the coding triplets for Leu-138 (CTG) and for Thr-268 (ACG). The restriction map of the inserted 6.6 kb DNA fragment of pK7746-C1 was also homologous to plasmid pMPA2a.15 This high degree of identity between blaSHV-12, blaSHV-2a and blaSHV-11 in both non-coding and coding regions of the genes suggests a common lineage.



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Figure 3. Nucleotide sequences of the immediate upstream region of blaSHV-12, blaSHV-2a,15 blaSHV-1117 and blaSHV-1. The boxed sequence corresponds to the left inverted repeat (IRL) of IS26;16 the –35 and –10 promoter sequences are those described by Podbielski et al.25

 
SHV-11 (or SHV-1a), SHV-2a and SHV-12 (or SHV-5-2a) differ from SHV-1, SHV-2 and SHV-5, respectively, by one amino acid change from leucine to glutamine at position 35, which is far from the active site and known not to alter the isoelectric point. They are thus considered as variants of SHV-1, SHV-2 and SHV-5, respectively. However, the above data indicate that the upstream non-coding region of SHV-11, SHV-2a and SHV-12 might be different from that of SHV-1, SHV-2 and SHV-5 by the presence of the IS26 sequence inserted next to the –10 sequence. To confirm this possibility, we studied the presence of the IS26 element in the immediate upstream regions of the following SHV ß-lactamase genes: blaSHV-1, blaSHV-2, blaSHV-3, blaSHV-4, blaSHV-5, blaSHV-2a and blaSHV-12 using PCR mapping. First, we performed PCRs on 69 clinical isolates carrying blaSHV-2a or blaSHV-12 isolated from three university hospitals during 1993–1999 (55 strains of K. pneumoniae, four E. coli and 10 E. cloacae).3,5 The flanking IS26 sequence was detected in all of the 69 strains. However, we could not find the flanking IS26 sequence in the five standard strains, each producing one of SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. This suggests the separate evolutionary development of SHV-2a and SHV-2. In a further plasmid analysis and Southern blot hybridization study with a blaSHV or IS26 probe among 39 strains of K. pneumoniae carrying blaSHV-2a or blaSHV-12,3 both blaSHV and IS26 probes were hybridized to the same plasmid and the sizes of the plasmids carrying both genes were in the range 30–121 kb.

The above findings indicate that SHV-12 may have evolved directly from SHV-2a, not from SHV-2 to -5. Therefore, we present a diagram of the possible evolutionary relationship of eight members of the SHV family (Figure 4Go). SHV-11 is a narrow-spectrum ß-lactamase with activity against ampicillin, piperacillin and to some extent early cephalosporins (e.g. cefalothin), and it has been considered to be a variant of SHV-1. However, our recent finding implies that SHV-11 may be another chromosomal ß-lactamase of K. pneumoniae carried by >90% of clinical isolates of K. pneumoniae. Our recent study determining the nucleotide sequences of the blaSHV genes of the strains shown by a ligase chain reaction5 to have SHV-2a- or SHV-12-specific mutations confirmed the results from the ligase chain reaction and revealed that the majority of clinical isolates of K. pneumoniae with SHV-2a or SHV-12 sequence also had SHV-1 (three of 13 strains) or SHV-11 (five of 13 strains) sequence. The gene encoding SHV-2a or SHV-12 was transferred by conjugation, but the gene encoding SHV-11 was not transferred. From these results, we suspect that chromosomal SHV-11 might be a kind of chromosomal ß-lactamase of K. pneumoniae, like SHV-1 or LEN-1. It is also possible that chromosomal SHV-11 has evolved from chromosomal SHV-1 by one amino acid substitution, L35Q. In any case, chromosomal SHV-11 is thought to be an ancestor of the plasmid-mediated SHV-11 found in an isolate of S. dysenteriae17 and extendedspectrum ß-lactamases such as SHV-2a and SHV-12.



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Figure 4. Diagram of the possible evolutionary relationship between members of the ß-lactamases. SHV-11, SHV-2a and SHV-12 have the same amino acid substitution, L35Q, and an identical upstream non-coding region in which IS26 is inserted; IS26 sequence was not detected in the upstream non-coding region of SHV-2, SHV-3, SHV-4 and SHV-5, indicating separate evolutionary development of SHV-2 and SHV-2a. This finding strongly indicates direct evolution of SHV-12 from SHV-2a, not from SHV-2 to -5.

 
The presence of the IS26 element in the upstream noncoding regions of blaSHV-11, blaSHV-2a and blaSHV-12 led to the question of the role of the sequence in the evolution of these genes. The close association of insertion sequences (ISs) with antibiotic resistance genes strongly suggests an active role for these sequences in the evolution or dissemination of antibiotic resistance genes. IS26 has been associated with several antibiotic resistance genes, including aphA1 in Tn2680 and other transposons,18 a blaTaac5 operon in plasmid pUZ3644,19 an IAB operon in plasmid pBWH77,20 dhfrVIII in plasmid pLMO22621 and dfr13, aadA4, blaTEM-1 and sul2 gene in plasmid pUK2381.22 It has been assumed from sequencing data that IS26 provides part of a hybrid promoter for aacC genes in plasmids pWP7b and pWP14a.23 In addition, Prenki et al.24 suggested that IS26 is a portable –35 promoter site. Insertion of the IS26 sequence immediately upstream of blaSHV-11, blaSHV-2a and blaSHV-12 genes generates the hybrid promoter consisting of the –35 region derived from IS26 and the –10 region derived from the blaSHV promoter itself. This hybrid promoter has been reported to increase ß-lactam resistance when coupled to blaSHV-2,25 indicating that the IS26 insertion produces a more efficient promoter. This more efficient promoter could be adaptively significant, producing more gene product. It therefore appears that acquisition of both a strong promoter and point mutations in the structural genes, which contribute to the ability of the host to resist the lethal effect of third-generation cephalosporins, was selected for by extensive use of these drugs in clinical settings. In Korea, cefotaxime and ceftazidime have been in use since the early 1980s and early 1990s, respectively, and usage of these agents has been increasing. The amount of ceftazidime and cefotaxime produced in Korea was 3.5 tons (5.3% of total cephalosporins produced) and 1.2 tons (1.8%), respectively, in 1993, but had increased to 9.2 tons (8.6%) and 4.1 tons (3.8%), respectively, in 1998.26

In conclusion, we suggest three possible explanations for the unusual predominance of SHV-2a and SHV-12 in Korea: the first is direct evolution of SHV-12 from SHV-2a; the second is a separate evolutionary development of SHV-2a and SHV-2; the third is the acquisition of a strong hybrid promoter of SHV-2a and SHV-12, created by IS26 insertion. The latter may contribute to the survival and dissemination of SHV-2a- and SHV-12-producing bacteria in the presence of third-generation cephalosporins. The presence of the IS26 sequence immediately upstream of SHV-2a and SHV-12 may also discriminate between strains producing these enzymes from the strains producing SHV-2 and SHV-5.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We are grateful to Dr G. Jacoby, for providing strains that produce the standard SHV ß-lactamases. This work was supported in part by a grant from Dankook University in Korea to J. K. (2001). This work was presented in part at the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, on 26 September 1999.


    Notes
 
* Corresponding author. Tel: +82-53-420-6951; Fax: +82-53-427-5664; E-mail: dtcho{at}knu.ac.kr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
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Received 31 May 2001; returned 3 July 2001; revised 26 October 2001; accepted 31 October 2001