• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.672-677
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• 2005

The standard reference collection of strains for E. coli, the ECOR collection, was analyzed by a genome-based typing method. Seventy-one ECOR strains were subjected to repetitive extragenic palindromic PCR genome fingerprinting with BOX primers (BOX-PCR). Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 28 genotypes in the ECOR collection. Shannon's entropy-based diversity index was 3.07, and the incident-based coverage estimator indicated potentially 420 genotypes among E. coli populations. Chi-square test of goodness-of-fit showed statistically significant association between the genotypes defined by BOX-PCR fingerprinting and the groups previously defined by multi-locus enzyme electrophoresis. This study suggests that the diversification of E. coli strains in natural populations is actively ongoing, and rep-PCR fingerprinting is a convenient and reliable method to type E. coli strains for the purposes ranging from ecology to quarantine.ine.

• Journal of Microbiology and Biotechnology
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• 제11권5호
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• pp.788-797
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• 2001

The usefulness of three PCR methods were evaluated for the epidemiological typing of Staphylococcus aureus: an enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic element PCR (REP-PCR), and 16S-23S intergenic spacer PCR (ITS-PCR). The analysis was performed using a collection of S. aureus strains comprised of 6 reference and 79 isolates from patients with various diseases. Among the 85 S. aureus strains tested, 6 references and 6 isolates were found to be susceptible to methicillin, whereas the remaining 73 isolates were resistant to it. PCR methods are of special concern, as conventional phenotypic methods are unable to clearly distinguish among methicillin-resistant S. aureus (MRSA) strains. The ability of the techniques to detect different unrelated types was found to be as follows: ERIC-PCR, 19 types; REP-PCR, 36 types; and ITS-PCR, 14 types. On the basis of combining the ERIC, REP, and ITS fingerprints, the 85 S. aureus strains were grouped into 56 genetic types (designated G1 to G56). The diversities for the 85 S. aureus strains, calculated according to Simpson\`s index, were 0.88 for an ERIC-PCR, 0.93 for a REP-PCR, and 0.48 for an ITS-PCR, and the diversity increased up to 0.97 when an ERIC-PCR and REP-PCR were combined. The above discrimination indices imply that the genetic heterogeneity of S. aureus strains is high. Accordingly, this study demonstrates that DNA sequences from highly conserved repeats of a genome, particularly a combination of ERIC sequences and REP elements, are a convenient and accurate tool for the subspecies-specific discrimination and epidemiologic tracking of S. aureus.

• 한국식품과학회지
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• 제37권4호
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• pp.611-617
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• 2005

본 연구에서는 E. coli. Salmonella, Shigella, Vibrio 등 4속의 주요 식중독유발 그람 음성 세균들을 대상으로 반복성 염기서열인 REP DNA sequence를 응용한 REP-PCR을 실시하였다. 이전의 보고에서 이들 4속의 식중독 유발세균 중 각각 혹은 일부를 대상으로 반복성 염기서열을 이용한 PCR을 적용한 사례는 있지만 그때 적용한 primer, PCR 반응조건 및 전기영동조건 등이 다양하였다. 그러므로 본 연구에서는 이와같은 4속의 세균들에 대하여 최적화된 동일한 primer와 PCR 반응조건 및 전기영동조건을 표준조건으로서 적용하였다. 그 결과로서 모든 4속의 식중독 세균 균주마다 REP-PCR 후 생성되는 fingerprinting pattern에서 속마다 1-3개의 공통적이며 독특한 band가 생성되는 것이 확인되어 이러한 pattern을 이용한 속 수준의 분리 동정과 그와 같은 주요 band들 이외의 부수적인 band들을 고려하여 종 수준까지의 분리도 가능함을 확인하였다. 따라서 본 연구를 통하여 반복적 DNA 염기서열을 이용한 REP-PCR이 주요 식중독 세균의 분리 동정 방법으로 사용될 수 있음을 확인하였다. 또한 본 연구를 통하여 얻은 결과는 더 많은 속(genus)의 식중독세균을 대상으로 한 새로운 분리 동정 방법을 확립하기 위하여 사용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.169-178
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• 2010

The microbial community structure in Thailand soils contaminated with low and high levels of arsenic was determined by denaturing gradient gel electrophoresis. Band pattern analysis indicated that the bacterial community was not significantly different in the two soils. Phylogenetic analysis obtained by excising and sequencing six bands indicated that the soils were dominated by Arthrobacter koreensis and $\beta$-Proteobacteria. Two hundred and sixty-two bacterial isolates were obtained from arsenic-contaminated soils. The majority of the As-resistant isolates were Gramnegative bacteria. MIC studies indicated that all of the tested bacteria had greater resistance to arsenate than arsenite. Some strains were capable of growing in medium containing up to 1,500 mg/l arsenite and arsenate. Correlations analysis of resistance patterns of arsenite resistance indicated that the isolated bacteria could be categorized into 13 groups, with a maximum similarity value of 100%. All strains were also evaluated for resistance to eight antibiotics. The antibiotic resistance patterns divided the strains into 100 unique groups, indicating that the strains were very diverse. Isolates from each antibiotic resistance group were characterized in more detail by using the repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique with ERIC primers. The PCR products were analyzed by agarose gel electrophoresis. The genetic relatedness of 100 bacterial fingerprints, determined by using the Pearson product-moment similarity coefficient, showed that the isolates could be divided into four clusters, with similarity values ranging from 5-99%. Although many isolates were genetically diverse, others were clonal in nature. Additionally, the arsenic-resistant isolates were examined for the presence of arsenic resistance (ars) genes by using PCR, and 30% of the isolates were found to carry an arsenate reductase encoded by the arsC gene.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.599-606
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• 2004

Gram-positive antagonistic bacilli were isolated from agricultural soils for possible use in biocontrol of plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and/or Pythium ultimum. Among the 65 antagonistic Gram-positive soil isolates, 22 strains were identified as Bacillus species by 16S rDNA sequence analyses. Four strains, including DF14, especially exhibited multiple antagonistic properties against the three damping-off fungi. Genotypic properties of the Bacillus isolates were characterized by rapid molecular fingerprinting methods using repetitive extragenic palindromic-PCR (REP-PCR), ribosomal intergenic spacer-length polymorphisms (RIS-LP), 16S rDNA PCR-restriction fragment length polymorphisms (PCR-RFLP), and strain-specific PCR assays. The results indicated that the REP-PCR method was more valuable than the RIS-LP and 16S rDNA PCR-RFLP analyses as a rapid and reliable approach for bacilli typing and identification. The use of strain-specific primers designed based on 16S rDNA sequence comparisons enabled it to be possible to selectively detect a strain, DF14, which is being used as a biocontrol agent against damping-off fungi.

• 대한임상검사과학회지
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• 제48권4호
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• pp.327-334
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• 2016

최근 사람과 가축에 항균제의 과도한 사용으로 감염병을 일으키는 병원성 세균들의 항균제 내성이 증가하고 있다. 본 연구에서는 PCR과 염기서열분석법을 이용하여 충청지역 일개의 대학병원에 의뢰된 임상검체와 같은 지역에서 사육된 닭으로부터 분리된 P. mirabilis 균주를 대상으로 16S ribosomal RNA methyltransferase(RMTase) 유전자와 integron을 조사하였다. 또한 Repetitive extragenic palindromic sequence-based PCR (REP-PCR)을 이용하여 P. mirabilis 균주들의 역학적 연관성 조사하였다. 총 38균주의 P. mirabilis 중에서 임상검체로부터 분리된 7균주 (18.4%)만이 RMTases 유전자를 가지고 있었는데 이들은 모두 amikacin, tobramycin, 및 gentamicin에 내성을 나타냈다. 또한 대상균주 중 23균주(60.5%)가 class 1 integron을 가지고 있는 것으로 나타났으며 class 2 및 class 3 integron은 검출되지 않았다. 본 연구에서 확인된 integrons에는 aminoglycoside 내성유전자(aadA2, aadA5, aadA7, 및 aacCA5), ${\beta}$-lactmam 내성유전자($bla_{PSE}$), erythromycin 내성유전자(ereA), lincosamides 내성유전자(linF), 및 trimethoprim 내성유전자(dfrA12, dfrA17 및 dfrA32)등이 유전자 카세트로 포함되어 있었다. 본 연구결과 RMTase 유전자는 임상검체로부터 분리된 P. mirabilis 균주에만 확산되어 있었던 반면 class 1 integrons는 임상검체와 닭으로부터 분리된 P. mirabilis 균주에 광범위하게 확산되어 있음을 확인할 수 있었다. 게다가 닭으로부터 분리된 균주 중에는 동일한 REP-PCR 밴드패턴을 보인 균주들이 있었는데 이는 닭들 사이에서 P. mirabilis 균주가 수평확산 되었음을 의미한다. P. mirabilis 균주에서 항균제 내성유전자의 확산을 막기 위해서는 내성유전자 지속적인 모니터링과 감시가 필요할 것으로 사료된다.

• Food Science and Biotechnology
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• 제17권6호
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• pp.1171-1177
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• 2008

This study was undertaken to classify Enterobacter sakazakii isolates from 13 powdered infant formula products, 25 powdered weaning diet products, and 33 weaning diet ingredients on polymerse chain reaction (PCR) methods. The numbers of the isolates from 1 powdered infant formula product, 7 powdered weaning diet products, and 6 weaning diet ingredients were 1, 14, and 8, respectively. The contaminated ingredients were 1 rice powder, 2 millet powders, 2 vegetable powders, and 1 fruit and vegetable premix. PCR with the primer of repetitive extragenic palindromic element (REP-PCR) and random amplification of polymorphic DNA(RAPD) were effective in discriminating among the isolates, but tRNA-PCR and PCR with the primer of l6S-23S internal transcribed spacer (ITS-PCR) were not. Some of E. sakazakii isolates from vegetable powders, fruit and vegetable premix, and millets powders were classified into the clonal groups based on the DNA patterns in the REP-PCR and RAPD analysis. A close genetic relationship among the isolates from some of the powdered weaning diet products and the rice powder was also detected in the cluster analysis based on the DNA patterns in RAPD.

• 미생물학회지
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• 제41권1호
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• pp.60-66
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• 2005

국내산 한우, 흑염소, 돼지, 개, 닭 및 마우스 둥을 포함한 각종 동물과 사람환자에서 분리된 MRSA 균주를 포함하여 총 116주의 S. aureus 균주를 확보하여 이들 균주들의 유전학적 다양성을 분석하고자 시도하였다. 이를 위하여 쉽고, 편리하며 많이 활용되고 있는 PCR을 이용하여, 개별 분석기법에 따른 유전학적 특성을 파악하는 것은 물론 활용한 기법들 중에서 유전자수준에서 가장 효율적이며 뛰어난 감별능력을 지닌 기법을 선발하고자 하는데 궁극적인 목적을 두었다. 이를 위해서 통계학적 인 수치에 따라 객관적인 분석방법으로서 Simpson's index of diversity (SID)를 산출하여 성적상호간을 비교 및 평가하였다. 공시한 총 99 주에 대한 4M primer를 이용한 RAPD 성적에 근거하여 산출한 SID 값은 0.915의 양호한 간이 산출되었다. 같은 방법으로 충 98 주에 대한 RA primer를 이용한 RAPD성적을 토대로 산출한 SID 값은 0.874로 확인되었다. 한편 총 107주에 대한 ERIC-PCR을 수행한 종합분석 성적에서 공시균주들은 모두 10종의 유전형(genotype)으로 구분되었고, EM-type 는 14주가 포함되어 가장 대표적 인 유전형으로 분류되었으며, 이 그룹에는 사람유래의 6주가 포함되어 가장 지배적인 유전형으로 확인되었다. 또한 DNA profile에 근거한 덴드로그람을 작성하고 SID간을 산출하였던 바, SDI 0.929의 신뢰도 높은 성적을 산출하였다. 반면에 공시한 총 108주의 S. aureus균주에 대한 종합적인 REP-PCR 성적에서 모두20종의 유전형으로 세분되었고 RB-type은 17주로 가장 많은 균주가 포함되었다. 작성된 덴드로그람 성적에서 산출한 SID 값은 우리의 연구에서 수행한 PCR에 기초한 유전자 분석기법들 중에서는 가장 높은 0.930으로 확인되었다. 결론적으로 RAPD 기법 중에서는 4M primer를 활용한RAPD가 보다 효율적임을 확인할 수 있었고, REP-PCR과 ERIC-PCR의 양자의 성적은 거의 비슷하여 적용했던 PCR분석기법 중에서는 이들 분석기법이 가장 우수한 감별능력을 확보한 분석법으로 최종 선발할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.13-24
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• 2012

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.

• 대한임상검사과학회지
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• 제45권2호
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• pp.43-47
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• 2013

This study was designed to evaluate the prevalence of ESBL genes and monitor antimicrobial resistance pattern in Escherichia coli, isolated from a hospital and a community. We tested 200 E. coli strains isolated in the hospitals and community in Chungcheong area from January to March 2012. Antimicrobial susceptibilities were tested by using the disk diffusion method. A search for ESBL genes was conducted by PCR amplification, and the genotypes were determined by direct nucleotide sequence analysis of the amplified products. An Epidemiologic study was performed by repetitive extragenic palindromic sequence-based PCR (REP-PCR). The percentage of ESBL-producing isolates was 17% for hospital associated E. coli and 11% for community associated E. coli. The ESBL gene sequencing results showed that the most common ESBL in E. coli was CTX-M-14 (19/28), followed by CTX-M-15 (9/28). The REP-PCR study also showed the genetic diversity, but there was no difference between the hospital and community associated E. coli. In this study, the most common types of class A ESBLs identified were CTX-M in the hospital and the community in Chungcheong area. ESBL-producing E. coli isolates showed diverse clonality.