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
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Abstract |
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Introduction |
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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 4.822.5% and 13.222.4%, 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.
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Materials and methods |
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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 MuellerHinton 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 LuriaBertani 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 486502 and 805822 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 681700 and 13651385 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 12721287 and 16461665 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 TrisHCl 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.
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Results and discussion |
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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 1). The restriction map of pK7746-C1 obtained is shown in Figure 2
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The immediate upstream sequences of blaSHV-12 (Figure 3) 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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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 4). 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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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.
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Acknowledgements |
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Notes |
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References |
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Received 31 May 2001; returned 3 July 2001; revised 26 October 2001; accepted 31 October 2001