• The Journal of the Korean Society for Microbiology
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• v.35 no.3
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• pp.263-271
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• 2000

A clinical isolate of Klebsiella pneumoniae K7746 produced the extended-spectrum ${\beta}$-lactamase (ESBL) SHV-12. A 6.6 kb BamHI fragment containing the $bla_{SHV-12}$ gene of K7746 strain was cloned into pCRScriptCAM vector resulting in the recombinant plasmid p7746-Cl. The restriction map of 3.6 kb inserted DNA and sequences immediately surrounding $bla_{SHV-12}$ of p7746-C1 were homologous to plasmid pMPA2a carrying $bla_{SHV-2a}$. In addition, both $bla_{SHV-12}$ and $bla_{SHV-2a}$ were expressed from a common hybrid promoter made of the -35 region derived from the left inverted repeat of IS26 and the -10 region from the $bla_{SHV}$ promoter itself. The results indicate that $bla_{SHV-12}$ and $bla_{SHV-2a}$ may have evolved from a common ancestor in the sequential order of $bla_{SHV-2a}$ first, followed by $bla_{SHV-12}$. Furthermore, by the PCR mapping method using primers corresponding to the IS26 and $bla_{SHV}$, the association between IS26 and $bla_{SHV}$ was studied in 12 clinical isolates carrying $bla_{SHV-2a}$, 27 clinical isolates carrying $bla_{SHV-12}$, and 5 reference strains carrying $bla_{SHV-1}$ to $bla_{SHV-5}$. All 39 strains carrying $bla_{SHV-2a}$ or $bla_{SHV-12}$ were positive by the PCR, providing confirmative evidence that IS26 has been involved in the evolution and dissemination of $bla_{SHV-2a}$ and $bla_{SHV-12}$. But 5 reference strains carrying $bla_{SHV-1}$ to $bla_{SHV-5}$ were negative by the PCR. Therefore, we concluded that the molecular evolutionary pathway of $bla_{SHV-2a}$ and $bla_{SHV-12}$ may be different from that of other $bla_{SHV-ESBL}$, e.g., $bla_{SHV-2}$, $bla_{SHV-3}$, $bla_{SHV-4}$, and $bla_{SHV-5}$.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.221-227
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• 2000

This study was performed to investigate the biological characteristics of twenty isolates of extended spectnlm $\beta$-lactamase (ESBL) producing Klebsiellapnezm~onia collected kom the various clinical specimens of three hospitals in Pusan. Isoelectric focusing (IEF) and PCR were used to determine the types of $\beta$-lactamase gene in this study. Twenty isolates of ESBL producing Klebsiellnp~ieun~or~iae could be divided by PCR, such as TEM type (I1 strains), SHV type (8 strains); non TEM non SHV type (1 strain). In the isoelechic focusing test, the PI of TEM type was 5.2-6.0 and that of SHV type was 6.9-7.4. According to the pI value and PCR bands, twenty strains of ESBL Klebsiellapneumoniae were divided into 5 types: TEM type @I 5.2-6.0; 1080 bp on PCR band), TEM + SHV type (pI 5.2-6.0; andpI 7.0-7.4; 1080 bp and 599 bp on PCR band), SHV type (pl7.0-7.4; 599 bp on PCR band), non TEM non SHV type, and otber type (PCR result was SHV type but pI was not detected).

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.1065-1069
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• 2009

Of 143 clinical isolates of Klebsiella pneumoniae collected from Korean non-tertiary hospitals, 24 (16.8%) showed an extended-spectrum ${\beta}$-lactamase-positive phenotype. PCR and sequence analysis revealed the presence of TEM-116 (n=13), CTX-M-3 (n=5), CTX-M-14 (n=2), CTX-M-15 (n=3), and SHV-12 (n=16). Each of the 24 isolates encoded more than one ${\beta}$-lactamase, and seven isolates (29%) harbored two different SHV-type ${\beta}$-lactamase genes ($bla_{SHV-11}$ and $bla_{SHV-12}$) bounded by insertion sequence IS26 in a single transferable plasmid.

• Journal of Life Science
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• v.19 no.12
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• pp.1718-1723
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• 2009

In this study, we characterized extended-spectrum $\beta$-lactamase (ESBL)-producing Enterobacteriaceae isolated from clinical specimens in Korea and found two strains harboring plasmid-mediated $bla_{SHV-11}$, Klebsiella pneumoniae. First, the isolates were detected using the Vitek system and confirmed by the double-disk synergy test. The classification of gene coding for ESBL was also performed by polymerase chain reactions and followed by DNA sequencing. The transmission of genes was confirmed by transconjugation and transformation. Resistant expression of transformants was determined by broth microdilution minimal inhibitory concentration test. Genotypic analysis revealed that one strain harbored the $bla_{TEM-1}$, $bla_{SHV-11}$ and $bla_{CTX-M-15}$ and the other strain harbored the $bla_{SHV-11}$ and $bla_{CTX-M-15}$. They showed high resistance to oxyiminocephalosphorins (3rd-generation cephalosporins), while the transformant containing only $bla_{SHV-11}$ did not show any resistance to the antibiotics.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.4
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• pp.153-157
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• 2009

The production of extended-spectrum ${\beta}$-lactamases ($ESBL_S$) of the TEM or SHV type by bacterial pathogens is a major threat to the use of the clinically important expanded-spectrum cephalosporins. The characterization of the SHV ESBLs producing Klebsiella pneumoniae strains present in clinical isolates is time-consuming processes. We describe here in the development of a novel system, which consists of a real time PCR. We found 11 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disk synergy test showed 8 ESBL positive and conventional PCR showed 10 SHV ESBL positive, which were K. pneumoniae strains isolates. By real time PCR analysis, SHV gene in 11 of 11 strains were identified. When sequencing analysis was compared with real time PCR, both analysis were presented 99% similarity. In this study, we used a rapid, sensitive, and specific real-time PCR (RT-PCR) method for detection of the assay SHV ESBL producing K. pneumoniae strains in clinical isolates.

• Journal of Life Science
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• v.25 no.12
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• pp.1399-1407
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• 2015

Klebsiella pneumoniae is found in the normal flora of the skin, mouth, respiratory tract, urinary tract, and intestines in human. However, the stain is opportunistic pathogen, which is the causative agent of community acquired pneumonia. Corn silk has been known to be effective for antimicrobial activity against pathogenic bacteria, including K. pneumoniae, Staphylococcus aureus, Bacillus subtilis, Shigella spp., Salmonella spp., Escherichia coli, Pseudomonas aeruginosa, et al. In this study we focused on the antimicrobial properties of con silk water extract of K. penumoniae. K. pneumoniae isolates K. pneumoniae ATCC 13883 and broad-spectrum β-lactamase (BSBL), exteded-spectrum β-lactamase (ESBL), carbapenemase-producers. Antimicrobial susceptibilities were determined by the disk diffusion method. Searches for bla genes were performed by PCR amplication and direct sequencing. MacConkey agar plate medium was prepared using the corn silk extracts (50% or 100%) instead of distilled water for antimicrobial activity test. The microbial growth inhibitory potential of K. pneumoniae was determined by using the MacConkey agar plate spreading method, and the plate was incubated 18 hr at 37℃. Genes encoding β-lactamases including SHV-1 (n=8), SHV-2a (n=8), SHV-5 (n=2), SHV-11 (n=2), SHV-12 (n=18), TEM-1 (n=10), CTX-M-3 (n=2), CTX-M-14 (n=2), CTX-M-15 (n=1), GES-5 (n=5), KPC-2 (n=6), KPC-3 (n=4), and NDM-1 (n=2) were detected. The corn silk extract showed significantly antimicrobial activity against K. pneumoniae ATCC 13883, but BSBLs, ESBLs, and carbapenemase producers were not. Therefore, corn silk extract is thought to be able to assist in the prevention and rapid recovery of infectious disease caused by K. pneumoniae.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.889-895
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• 2006

To investigate the antibiotic-resistant patterns and the gene types of extended-spectrum $\beta$-lactamase (ESBL)-producing Klebsiella pneumoniae, we collected 226 Klebsiella pneumoniae strains from three general hospitals with more than 500 beds in Busan, Korea from September 2004 to October 2005, The minimum inhibitory concentration (MIC) of antibiotics was measured using the Gram-negative susceptibility (GNS) cards of Vitek (Vitek system, Hazelwood Inc., MO, U.S.A.). Of the 226 K, pneumoniae isolates, 65 ESBL-producing K. pneumoniae strains were detected by the Vitek system and confirmed by the double-disk synergy test. TEM (Temoniera) type, SHV (sulfhydryl variable) type, and CTX-M (cefotaxime) type genes were detected by polymerase chain reaction. All 65 K. pneumoniae strains were resistant to ampicillin, cefazolin, cefepime, ceftriaxone, and aztreonam, and 83.0% of the organisms were resistant to ampicillin/sulbactam, 66.1% to tobramycin, 67.6% to piperacillin/tazobactam, 61.5% to ciprofloxacin, and 47.6% to trimethoprim/sulfamethoxazole, and 43.0% to gentamicin. TEM-type ESBLs (TEM-1 type, -52 type) were found in 64.6% (42 of 65) of the isolates, SHV-type ESBLs (SHV-2a type, -12 type, -28 type) in 70.7% (46 of 65) of isolates, and CTX-M-type ESBLS (CTX-M-15 type) in 45% (29 of 65) of isolates. Of the 65 ESBL-producing K. pneumoniae strains, two strains were found to harbor blaSHV-28, which were detected in Korea for the first time. Therefore, more investigation and research on SHV-28 are needed in order to prevent the ESBL type-producing K. pneumoniae from spreading resistance to oxyimino cephalosporin antibiotics.

• Journal of Life Science
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• v.17 no.10
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• pp.1426-1433
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• 2007

To investigate the antibiotic resistant patterns of the bacteria producing ESBL, we isolated one organism of Klebsiella pneumoniae from a clinical laboratory in Busan. The organism that produces ESBL gene was detected by double disk synergy test and the presence of three ESBL genes (TEM-1, SHV-12, CTX-M-15) was confirmed by polymerase chain reaction and DNA sequencing analysis. To analyse the characteristics of three ESBL genes, we performed transconjugation, transformation and cloning experiment with the organism. The MIC of Klebsiella pneumoniae was revealed that ceftazidime, cefotaxime and ceftriaxone were $256\;{\mu}g/ml,\;128\;{\mu}g/ml\;and\;128\;{\mu}g/ml$ respectively. The MIC of conjugant (E. coli $RG176^{Na(r)}$) af was revealed that ceftazidime, cefotaxime and ceftriaxone were $256\;{\mu}g/ml,\;64\;{\mu}g/ml\;and\;128\;{\mu}g/ml$ respectively. The MIC of transformant (E. cofi $DH5{\alpha}$) was revealed that ceftazidime, cefotaxime and ceftriaxone were $128\;{\mu}g/ml,\;32\;{\mu}g/ml,\;and\;32\;{\mu}g/ml$ respectively, The MIC of cloned organism of SHV-12 gene (E. coli $DH5{\alpha}$) was revealed that ceftazidime, cefotaxime and ceftriaxone were $128\;{\mu}g/ml,\;8\;{\mu}g/ml,\;and\;32\;{\mu}g/ml$ respectively. The results indicated that MIC of conjugant was higher than MIC of transformant and also SHV-12 gene were not resistant against cefotaxime antibiotic.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.1
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• pp.26-33
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• 2006

Recently, the rapid increase in extended-spectrum ${\beta}$-lactamase (ESBL) producing clinical isolates has become a serious problem. In this study, the epidemiologic features and molecular characteristics of ESBL among clinical isolates of Escherichia coli and Klebsiella pneumoniae, antibiotic susceptibility testing, genotype of the ESBL and patterns of chromosomal DNA from PFGE (pulsed field gel electrophoresis) were observed. A total of 53 ESBL-producing clinical isolates (30 of E. coli and 23 of Klebsiella pneumoniae) were collected from two university hospitals in the period of June to July in 2002 and 2003 respectively. The antibiotic resistance frequency of those 53 strains was tested by the disk agar diffusion method with the result that all the strains were resistant to cephalothin. To other antibiotics, the resistance rates of E. coli (30 isolates) were in order of ceftazidime (90.0%), cefotaxime and aztreonam (respectively 83.3%). Also, the resistance rates of K. pneumoniae (23 isolates) were in order of aztreonam (78.3%), ceftazidime (73.9%) and cefotaxime (65.3%). Also the sensitivity of ceftazidime-clavulanic acid were 100% in E. coli and 95.7% in K. pneumoniae. And the sensitivity of cefotaxime-clavulanic acid was 96.7% in E. coli and 91.3% in K. pneumoniae. The types of the ESBL genes were determined by using polymerase chain reaction (PCR). Among the 30 isolates of ESBL-producing E. coli, 6 (20.0%) have SHV only, 5 (16.7%) have TEM only and, 18 (60.0%) have both of TEM and SHV. Among the 23 isolates of ESBL-producing K. pneumoniae, 7 (30.4%) have SHV only, 2 (8.7%) have TEM only, and 14 (60.9%) have both of TEM and SHV. These results show that 52 strains, with only one exception, were confirmed as either TEM or SHV. The patterns of Xba I-digested chromosomal DNA of ESBL-producing E. coli and K. pneumoniae isolates were analyzed by PFGE. PFGE patterns of E. coli and K. pneumoniae were multiclonal, but many strains were grouped into a few types. Therefore, it seems that there were clonal outbreaks or possible horizontal spread. In conclusion, the TEM and SHV ${\beta}$-lactamase are most widely spread in E. coli and K. pneumoniae in Korea. As these types are usually carried by plasmids, the spread of these ${\beta}$-lactamase genes could compromise the future usefulness of third generation cephalosporins for the treatment of infections caused by E. coli and K. pneumoniae.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.116-123
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• 2007

The purpose of this study was typing the plasmid mediated extended spectrum ${\beta}-lactamases$ produced by enteric bacteria isolated from rivers in Pusan. Six strains of Eschericha coli and fifteen strains of Klebsiella pneumoniae transferred their plasmid mediated extended spectrum ${\beta}-lactamase$ genes to the recipient strain Eschericha coli J53 $Azid^{R}$. The plasmid mediated extended spectrum ${\beta}-lactamase$ genes were sequenced directly after PCR and the types were determined by the BCM Search Launcher and GenBank nucleotid database. Determined types of the plasmid mediated extended spectrum ${\beta}-lactamases$ were TEM-52 and SHV-12. TEM-52 was isolated from both Escherichia coli and Klebsiella pneumoniae. However SHV-12 was isolated from Klebsiella pneumoniae only. The results indicated that the plasmid mediated extended spectrum ${\beta}-lactamase$ producing bacteria spreded over the area of clinical to the nature in Korea.