• Journal of Plant Biotechnology
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• 제2권2호
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• pp.89-96
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• 2000

For large DNA fragment transformation in dicots and monocots, BIBAC2 vector system was applied to Arabidopsis thaliana and Oryza sativa L. cv. Jinmi as a model plant, respectively. For Arabidopsis, the Th1 gene in T23L3 BAC clone whose size is about 90 kb was used as the target gene source for transformation. Because T23L3 BAC clone was originally constructed in pBelloBAC11, the target gene was reconstructed into BIBAC2. As the results of reconstruction, 476 colonies were survived in selection medium containing 40 mg/L kanamycin. In colony hybridization analysis, 24 out of 476 colonies exhibited positive signals. In the pulsed-field gel electrophoresis analysis, 11 out of 24 positive clones exhibited the band at the location of 90 kb. In Southern hybridization, positive signal band at the location of 90 kb was observed in all 11 transformants. Using these verified clones, Agrobacterium-mediated transformation was applied to Arabidopsis thaliana th1-201 mutant for genetic complementation test. Twelve thousands T$_1$ seeds were harvested, and antibiotic selection test is being analyzed to verify whether these seeds were transformed. for rice, COR356 that contains 150 kb human genomic DNA in a BIBAC2 vector was used as the target gene. As the results of transformation, 151 out of 210 co-cultivated calli were survived in selection medium containing 5 mg/L hygromycin, and 45 out of 151 survived calli were regenerated into plants. Transformation efficiency was 21.6%. Progeny test using 71 seeds is being analyzed now. These results provide the potential that large DNA fragments can be transferred into both dicots and monocot by Agrobacterium-mediate d transformation system.

• 대한수의학회지
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• 제55권3호
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• pp.191-197
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• 2015

Escherichia (E.) coli is commensal bacteria found in the intestine; however, some pathogenic strains cause diseases in animals and humans. Although E. coli does not typically produce hydrogen sulfide ($H_2S$), $H_2S$-producing strains of E. coli have been identified worldwide. The relationship between virulence and $H_2S$ production has not yet been determined. Therefore, characteristics of $H_2S$-producing isolates obtained from swine feces were evaluated including antibiotic resistance patterns, virulence gene expression, and genetic relatedness. Rates of antibiotic resistance of the $H_2S$-producing E. coli varied according to antibiotic. Only the EAST1 gene was detected as a virulence gene in five $H_2S$-producing E. coli strains. Genes conferring $H_2S$ production were not transmissible although the sseA gene encoding 3-mercaptopyruvate sulfurtransferase was detected in all $H_2S$-producing E. coli strains. Sequences of the sseA gene motif CGSVTA around Cys238 were also identical in all $H_2S$- producing E. coli strains. Diverse genetic relatedness among the isolates was observed by pulsed-field gel electrophoresis analysis. These results suggested that $H_2S$-producing E. coli strains were not derived from a specific clone and $H_2S$ production in E. coli is not associated with virulence genes.

• Journal of Microbiology
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• 제44권1호
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• pp.106-112
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• 2006

In this study, the genetic diversities of multi-resistant Salmonella typhimurium (ST) isolates were analyzed via the application of both pulsed field gel electrophoresis (PFGE) and Polymerase chain reaction (PCR) analysis methods, using 6 kinds of primers (REP, ERIC, SERE, BOX, P-1254 and OPB-17). And their discriminative abilities (DA) were also compared in order to determine the most effective and reliable analysis method. 118 S. typhimurium isolates, cultured from diverse animals and human patients in Korea beginning in 1993, were analyzed and subjected to a comparison of Simpson's index of diversity (SID), using both PFGE and PCR methods. PFGE by XbaI enzyme digestion allowed for discrimination into 9 pulsotypes, with high SID values (0.991) on the genomic DNA level. This shows that PFGE is a very discriminative genotypic tool, and also that multiple clones of S. typhimurium isolates had existed in domestic animals and humans in Korea since 1993. However, we could ultimately not to trace the definitive sources or animal reservoirs of specific S. typhimurium isolates examined in this study. Depending on the SID values, the combined method (7 kinds of method) was found to be the most discriminative method, followed by (in order) SERE-PCR, REP-PCR, ERIC-PCR, PFGE & OPB-17 (RAPD), P-1254 (RAPD), and BOX-PCR at the $80\%$ clone cut-off value. This finding suggests that the REP-PCR method (which utilizes 4 primer types) may be an alternative tool to PFGE for the genotyping of S. typhimurium isolates, with comparable cost, time, and labor requirement. The establishment of a highly reliable and discriminatory method for epidemiologic analysis is considered necessary in order for researchers to trace the sources of specific pathogens and, consequently, to control and prevent the spread of epidemic S. typhimurium isolates to humans.

• Journal of Microbiology and Biotechnology
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• 제26권9호
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• pp.1643-1649
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• 2016

The aims of this study were to characterize the molecular epidemiological profiles of CTX-M-producing uropathogenic Escherichia coli isolates from a tertiary hospital in Daejeon, Korea, and to investigate the genetic diversity and compare the prevalence of sequence types (STs) in different areas. Extended spectrum β-lactamase-producing E. coli strains isolated from urine were analyzed for CTX-M, integrons, and insertion sequence common regions (ISCRs) by PCR and sequencing. Multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), phylogenetic analysis, and rep-PCR were also used for molecular typing of the isolates. Of 80 CTX-M producers, 31 and 46 expressed CTX-M-15 and CTX-M-14, respectively. MLST analysis indicated that the most prevalent ST was ST131 (n = 34, 42.5%), followed by ST38 (n = 22, 27.5%), ST405 (n = 8, 10.0%), and ST69 (n = 6, 7.5%). Most CTX-M producers harbored class 1 integrons. ST131 strains belonged to phylogenetic group B2 and showed identical rep-PCR patterns, whereas ST69, ST38, and ST405 strains belonged to phylogenetic group D; the ST38 and ST405 strains displayed the same rep-PCR pattern, respectively. ST131 and ST38 isolates showed 21 and 19 distinct types, respectively, by PFGE. In Daejeon, D-ST38 CTX-M-14 producers were relatively more prevalent than in other countries and Korean cities. Our results indicate that CTX-M-producing E. coli isolates belonged mostly to ST131 or ST38 and were more related to hospital-onset than to community-onset infections and that the bla_CTX-M gene may vary according to the ST.

• Journal of Microbiology and Biotechnology
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• 제20권6호
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• pp.1042-1052
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• 2010

Forty-seven Salmonella Typhimurium (33 zoonotic, 14 clinical) strains were tested for antimicrobial resistance using the standard disk diffusion method. The presence of relevant resistance genes and class 1 integrons were investigated by using PCR. Pulsed-field gel electrophoresis (PFGE) and plasmid profiling were carried out to determine the genomic diversity of Salmonella Typhimurium. Approximately 57.4% of the S. Typhimurium strains were multidrug resistant (MDR) and showed high resistance rates to tetracycline (70.2%), sulfonamides (57.4%), streptomycin (53.1%), ampicillin (29.7%), nalidixic acid (27.6%), kanamycin (23.4%), chloramphenicol (21.2%), and trimethoprim (19.1%). Resistance towards cephalosporins was noted for cephalothin (27.6%), cephradine (21.2%), amoxicillin clavulanic acid (17.0%), and cephalexin (17.0%). Resistance genes, $bla_{TEM}$, strA, aadA, sul1, sul2, tetA, tetB, and tetC, were detected among the drug-resistant strains. Thirtythree strains (70.2%) carried class 1 integrons, which were grouped in 9 different profiles. DNA sequencing identified sat, aadA, pse-1, and dfrA genes in variable regions on class 1 integrons. Thirty-five strains (74.4%) were subtyped to 22 different plasmid profiles, each with 1-6 plasmids (2.0 to 95 kb). PFGE subtyped the 47 strains into 39 profiles. In conclusion, high rates of multidrug resistance were found among the Malaysian Salmonella Typhimurium strains. The emergence of multidrug-resistant Salmonella Typhimurium to cephalosporin antibiotics was also observed. The strains were very diverse and no persistent clone was observed. The emergence of MDR Salmonella Typhimurium is a worldwide problem, and this report provides information for the better understanding of the prevalence and epidemiology of MDR S. Typhimurium in Malaysia.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1276-1284
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• 2006

Listeria monocytogenes is a well-known high-risk foodborne pathogen that grows at refrigeration temperature and is responsible for outbreaks of listeriosis. We report here the incidence of L. monocytogenes in fresh chicken carcasses and present genetic diversity of L. monocytogenes isolates. In this study, 25 g of chicken carcasses from markets in Korea were examined according to the FDA method, and presumptive isolates were confirmed by multiplex PCR assay. L. monocytogenes isolates were analyzed by Pulsed-Field Gel Electrophoresis using restriction enzymes, ApaI and AscI, to obtain strain-specific DNA fragments profiles. Antimicrobial resistance of L. monocytogenes strains against generally used antibiotics (Penicillin G, Kanamycin, Tetracycline, Vancomycin, Cephalothin, Rifampicin, Erythromycin, Ampicillin, Gentamicin, Streptomycin, and Chloramphenicol) were analyzed by NCCLS protocols to examine the presence of antimicrobial resistance in natural L. monocytogenes. Of a total 274 chicken samples, 81 samples (29.6%) were positive for L. monocytogenes. Listeria innocua (50.1%), Listeria welshimeri (6.9%), and Listeria grayi (11.3%) were also detected. PFGE analysis, using restriction enzymes ApaI and AscI, showed 27 pulsotypes of L. monocytogenes. Antimicrobial resistance analysis confirmed the existence of antimicrobial resistance for penicillin G and tetracycline in isolated L. monocytogenes strains.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.338-346
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• 2018

Two molecular epidemiologic methods, IS6110 restriction fragment length polymorphism (IS6110-RFLP) and 24-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR), are used worldwide in studies of Mycobacterium tuberculosis (MTB). Conversely, because of its poor resolution, pulsed-field gel electrophoresis (PFGE) is not widely used for MTB. In this study, we improved the 24-locus MIRU-VNTR and PFGE protocols and compared the effectiveness of these approaches for the molecular typing of MTB using 75 clinical isolates obtained from a cohort investigation of high-risk populations infected with MTB. The 24-locus MIRU-VNTR method demonstrated superior discriminatory ability, followed by PFGE and IS6110-RFLP. Next, we analyzed six isolates with clear epidemiologic connections; that is, isolates from patients who attended the same school. IS6110-RFLP and PFGE identified these samples as the same type. By contrast, according to MIRU-VNTR, two isolates differed from four other isolates at one locus each; one isolate was identified as Mtub29 and the other as QUB-26. In summary, the 24-locus MIRU-VNTR assay was the most useful molecular typing method among the three methods investigated due to its discriminatory power, short time required, and availability as an epidemiologic investigation tool. PFGE was the second-best method. Compared with the other loci assessed in the 24-locus MIRU-VNTR assay, the Mtub29 and QUB-26 loci appeared to exhibit greater variability during transmission.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.162-170
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• 2018

Non-typhoidal Salmonella is one of the main pathogens causing food-borne illness in humans, with up to 20% of cases resulting from consumption of pork products. Over the gastroenteritis signs, multidrug resistant Salmonella has arisen. In this study, pan-susceptible phenotypic strains of Salmonella enterica serotype Weltevreden recovered from pig production chain in Chiang Mai, Thailand during 2012-2014 were chosen for analysis. The aim of this study was to use whole genome sequencing (WGS) data with an emphasis on antimicrobial resistance gene investigation to assess their pathogenic potential and genetic diversity determination based on whole genome Multilocus Sequence Typing (wgMLST) to expand epidemiological knowledge and to provide additional guidance for disease control. Analyis using ResFinder 3.0 for WGS database tracing found that one of pan-susceptible phenotypic strain carried five classes of resistance genes: aminoglycoside, beta-lactam, phenicol, sulfonamide, and tetracycline associated genes. Twenty four and 36 loci differences were detected by core genome Multilocus Sequence Typing (cgMLST) and pan genome Multilocus Sequence Typing (pgMLST), respectively, in two matching strains (44/13 vs A543057 and A543056 vs 204/13) initially assigned by conventional MLST and Pulsed-field Gel Electrophoresis (PFGE). One hundread percent discriminant ability can be achieved using the wgMLST technique. WGS is currently the ultimate molecular technique for various in-depth studies. As the findings stated above, a new of "gold standard typing method era" for routine works in genome study is being set.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.106-113
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• 2002

One-hundred and twenty-four trimethoprim-resistant Shigella sonnei isolates extracted in Korea during the last two decades were investigated for their epidemiological relationship and mechanisms of resistance to trimethoprim. The S. sonnei isolates were distributed into two groups by three different epidemiological tools: biotyping, antibiogram, and pulsed-field gel electrophoresis. One group contained the isolates from the 1980s and the other group included the isolates from the 1990s. The geometric mean MICs of trimethoprim in S. sonnei isolates from the 1980s and 1990s were found to be $672.9{\mu}g/ml\;and\;>2,048{\mu}g/ml$, respectively. Trimethoprim resistance was associated with dfrA5, dfrA12, and dfrA13 genes in the isolates from the 1980s, dfrA1, dfrA5, and dfrA12 in the isolates from 1991, and dfrA1 and dfrA12 in the isolates from 1992 to 1999. The dfrA1 gene was located downstream of the intI2 gene in Tn7, which was located on chromosome. Some dfrA12 genes were found as gene cassettes in the class 1 integron. The dfrA5 and dfrA13 genes were located on conjugative plasmids. These results suggested that a clonal change occurred in S. sonnei isolates in Korea during the last two decades and that dfr genes located on different transposable genetic elements had gradually changed.

• Genomics & Informatics
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• 제10권4호
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• pp.249-255
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• 2012

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular- weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.