• 생명과학회지
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• 제25권2호
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• pp.136-150
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• 2015

Pseudomonas syringae pv. tabaci 11528은 담배를 숙주로 하여 wildfire disease를 일으키는 식물 병원성 세균이다. P. syringae pv. tabaci psyR deletion mutant를 이용하여 swarming motility, tabtoxin 생산능, siderophore 생산능, AHL 생산능 등의 phenotypic test를 수행하였다. psyR deletion mutant는 wild-type 균주보다 swarming motility가 증가하였고, tabtoxin 생산 또한 증가하였다. 하지만 siderophore와 AHL 생산능은 감소하였고 virulence 또한 지연되었다. 이러한 결과로 PsyR이 QS regulator로 작용한다는 사실과 더불어 병원성 유전자의 조절에도 관여한다는 것을 확인하였다. PsyR이 각각의 병원성 유전자의 발현을 조절하는 regulator들에게 미치는 영향을 전사단계에서 확인하기 위해 fur, gacA, psyI, prhI, prhA, hrpR, hrpA 유전자들을 정량적 real-time PCR (qRT-PCR) 방법으로 확인하였다. 또한 PsyR에 의한 병원성 유전자 조절이 DNA상에 직접적으로 결합하여 일어나는 것인지 아니면 다른 경로를 통해 간접적으로 일어나는 것인지를 확인할 필요가 있어 정제한 PsyR 단백질과 병원성 관련 유전자들의 upstream region 서열을 이용하여 electrophoretic mobility shift assay (EMSA)를 수행한 결과 본 연구에서 선정한 병원성 관련 유전자들이 PsyR에 의해 직접적으로 조절되지는 않는다는 사실을 밝혔다.

• 미생물학회지
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• 제41권3호
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• pp.168-176
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• 2005

갯벌 퇴적물에 존재하는 병원성 해양미생물인 Vibrio vulnificus를 신속하고 정확하게 검출하기 위해 PCR, Southern hybridization 방법과 real-time PCR을 수행하여 검출 민감도를 비교하였다. 갯벌 퇴적물로부터 bead beater를 이용한 물리적 방법으로 DNA 조추출액을 얻고 상용화된 키트 (Geneclean turbo Kit)를 이용하여 부식물질(humic substances)을 제거하였다. 병원성에 관련된 3 종의 유전자(hemolysin, vvhA; phosphomannomutase, pmm; metalloprotease, vvpE)를 대상으로 설계한 프라이머 셋을 동시에 사용하는 multiplex PCR 방법과 Southern hybridization과 병행한 방법(PCR/Southern hybridization)을 수행하였다. Real-time PCR은 hemolysin 유전자(vvhA)에 특이한 프라이머와 TaqMan 탐침을 사용하였다. 전처리하지 않은 갯벌 퇴적물의 경우, PCR/Sourthern hybridization과 real-time PCR 방법의 검출 민감도는 퇴적물 1 g 당 약 $10^2$ 개의 세포 수준이었다. 농후처리액(APW; alkaline peptone water)으로 $35^{\circ}C$에서 $2{\~}3$시간, 8시간 중균 배양할 경우 갯벌 퇴적물 1 g 당 $2{\~}10$개 세포가 존재할 때 PCR/Southern hybridization 방법과 real-time PCR 방법으로 각각 검출할 수 있었다. 전처리 과정을 포함하여 real-time PCR은 $6{\~}7$시간, PCR/Sourthern hybridization은 약 36시간이 소요되었다.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.862-868
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• 2014

Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MP-PCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: $5{\times}10^1CFU/ml$ for EHEC, $5{\times}10^3CFU/ml$ for ETEC expressing lt and sth, $5{\times}10^4CFU/ml$ for ETEC expressing stp, $5{\times}10^2CFU/ml$ for EPEC, $5{\times}10^4CFU/ml$ for EAEC, and $5{\times}10^2CFU/ml$ for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.284-289
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• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• 한국축산식품학회지
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• 제38권1호
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• pp.203-208
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• 2018

The objective of this study was to investigate the pathogenicity and antimicrobial resistance of foodborne pathogens isolated from farmstead cheeses. Twenty-seven isolates, including 18 Bacillus cereus, two Escherichia coli, and seven Staphylococcus aureus, were subjected to polymerase chain reaction (PCR) to detect virulence genes and toxin genes, and the antibiotic resistances of the isolates were determined. All E. coli isolates were determined by PCR to be non-pathogenic. Among the 18 B. cereus isolates, 17 isolates (94.4%) were diarrheal type, as indicated by the presence of nheA, entFM, hbIC, cytK and bceT genes, and one isolate (5.6%) was emetic type, based on the presence of the CER gene. Among the seven S. aureus isolates, three (42.9%) had the mecA gene, which is related to methicillin-resistance. Most B. cereus isolates (94.7%) showed antibiotic resistance to oxacillin and penicillin G, and some strains also showed resistance to ampicillin (26.3%), erythromycin (5.3%), tetracycline (10.5%), and vancomycin (5.3%). These results indicate that microbial food safety measures for farmstead cheese must be implemented in Korea because antibiotic resistant foodborne pathogens, with resistance even to vancomycin, harboring virulence genes were found to be present in the final products of farmstead cheese.

• Fisheries and Aquatic Sciences
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• 제14권4호
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• pp.347-354
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• 2011

Aeromonas salmonicida is an important fish pathogen commonly associated with furunculosis in salmonids. Typical A. salmonicida strains have the surface virulence A-layer protein, a major virulence determinant encoded by the vapA gene. In this study, 880 chum salmon Oncorhynchus keta were collected from the east coast of Korea during 2006-2011, including 560 wild adults and 320 artificially hatched fry pools, and the presence of typical A. salmonicida was examined by PCR using the typical A. salmonicida-specific vapA gene primers. The results demonstrated that 34.5% of the samples (304/880 samples) were PCR positive, implying that a typical A. salmonicida infection is highly prevalent among chum salmon in Korea. Twenty typical A. salmonicida isolates were recovered based on their brown pigmentation on Trypticase Soy Agar (TSA) plates, which indicates the existence of the A-layer protein. Further biochemical analyses with the four randomly selected typical A. salmonicida isolates revealed some variations in their amino acid decarboxylation and carbohydrate fermentation activity. A phylogenetic analysis based on the entire vapA gene sequence suggested that the A. salmonicida isolates from chum salmon were clustered with those isolated from Atlantic salmon in Europe. Further study is needed to resolve such an interesting relationship in detail.

• 대한수의학회지
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• 제46권4호
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• pp.371-379
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• 2006

Newcastle disease virus (NDV) is a single-stranded negative sense RNA virus, which has been classified as a member of the Avulavirus genus of the Paramyxoviridae family. It is also one of the most important pathogens in the poultry industry. The glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN), determine the virulence of NDV, and the relevant molecular structures have already been determined. NDV isolates differ in terms of virulence, and at least 2 of 9 genotypes (I-IX) have been shown to co-circulate. Therefore, it is clearly important to differentiate between vaccine strains and field isolates. In vivo pathogenicity tests have been the standard protocol for some time, but molecular methods appear preferable in terms of the rapidity of diagnosis, as well as animal welfare concerns. In this study, we have designed primer sets from HN gene for phylogenetic analysis and restriction enzyme analysis, and from F gene for pathotype-specific RT-PCR. Via the combination of 2 methods, 106 Korean NDV isolates obtained from 1980 to 2005 were differentiated into vaccine strains, and virulent genotypes VI and VII. The genotype VI viruses were only rarely isolated after 1999, and genotype VII, after it was initially isolated from poultry in 1995, recurred in 2000, and then became the main NDV constituting a threat to the Korean poultry industry.

• 한국동물위생학회지
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• 제39권2호
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• pp.87-92
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• 2016

Porcine edema disease (ED) is a communicable disease of pigs caused by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) which expresses F18 fimbriae and/or Stx type 2e (Stx2e). While STEC causes a severe illness including hemorrhagic colitis and hemolytic-uremic syndrome in humans, it induces damage to the vascular endothelium, which results in edema, hemorrhage, and microthrombosis, leading in high mortality in pigs. In the present study, we cultured Stx2e-producing E. coli field isolates from conventional pig farms that experienced sudden deaths previously with symptoms similar to porcine edema disease, which were further investigated with Shiga toxin profiles. A total of 43 strains were identified from the collected samples by F18 or Stx2e specific PCR. Based on the PCR, 42 isolates out of 43 isolates were proved to carry one of F18 or Stx2e genes and 14 isolates to carry both F18 and Stx2e genes. All of the 30 isolates that harbored Stx2e gene induced the cytopathic effect (CPE) in vero cells and especially, the isolate 150229 produced the highest level of Shiga toxin. Therefore, we identified the virulence genes (F18 and Stx2e) and demonstrated Shiga toxin-producing abilities from porcine edema disease causing E. coli filed isolates. These results suggested that one of the isolates could be a vaccine antigen candidate against STEC through further investigating to elicit an immune response.

• Journal of Periodontal and Implant Science
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• 제46권1호
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• pp.35-45
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• 2016

Purpose: Porphyromonas gingivalis fimA is a virulence factor associated with periodontal diseases, but its role in the pathogenesis of peri-implantitis remains unclear. We aimed to evaluate the relationship between the condition of peri-implant tissue and the distribution of P. gingivalis fimA genotypes in Koreans using a new primer. Methods: A total of 248 plaque samples were taken from the peri-implant sulci of 184 subjects. The control group consisted of sound implants with a peri-implant probing depth (PD) of 5 mm or less with no bleeding on probing (BOP). Test group I consisted of implants with a peri-implant PD of 5 mm or less and BOP, and test group II consisted of implants with a peri-implant PD of more than 5 mm and BOP. DNA was extracted from each sample and analyzed a using a polymerase chain reaction (PCR) with P. gingivalis -specific primers, followed by an additional PCR assay to differentiate the fimA genotypes in P. gingivalis-positive subjects. Results: The Prevalence of P. gingivalis in each group did not significantly differ (P>0.05). The most predominant fimA genotype in all groups was type II. The prevalence of type Ib fimA was significantly greater in test group II than in the control group (P<0.05). Conclusions: The fimA type Ib genotype of P. gingivalis was found to play a critical role in the destruction of peri-implant tissue, suggesting that it may be a distinct risk factor for periimplantitis.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.440-448
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• 2021

Helicobacter pylori (H. pylori) establishes long-term infections associated with severe gastric diseases such as peptic ulceration and gastric cancer. Exposure to an antibacterial agent can help regulate the expression levels of its pathogenic genes. In this study, we analyzed the transcriptional changes in H. pylori genes induced by β-caryophyllene. We used next-generation sequencing (NGS) to analyze RNA expression changes, and reverse transcription-polymerase chain reaction (RT-PCR) was performed as required to verify the results. The NGS results showed that 30 out of 1,632 genes were expressed differentially by β-caryophyllene treatment. Eleven genes associated with DNA replication, virulence factors, and T4SS components were significantly downregulated. RT-PCR confirmed that treatment reduced the expression levels of 11 genes. RT-PCR showed the reduced expression of 11 genes (dnaE, dnaN, holB, gyrA, cagA, vacA, secA, flgE, virB2, virB4, and virB8) following β-caryophyllene treatment. These results suggest that β-caryophyllene can modulate various H. pylori pathogenic determinants and be a potential therapeutic agent for H. pylori infection.