• Fisheries and Aquatic Sciences
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• 제22권8호
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• pp.17.1-17.9
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• 2019

Bacillus is a diverse genus consisting of more than 200 species with extensive genetic diversity. Their beneficial effects in industrial shrimp farming have been well documented. However, little is known about the biodiversity of the Bacillus spp. in this aquaculture system. Taxonomic analysis by 16S rRNA sequencing does not always allow species-level identification of Bacillus spp. In this study, 26 Bacillus isolates from two industrial Litopenaeus vannamei shrimp ponds in Bac Lieu Province, Vietnam, were analyzed for their genetic diversity by multi-locus sequence typing (MLST). A total of 22 sequence types were identified and segregated into four distinct clusters, corresponding to B. subtilis, B. velezensis, B. siamensis, and B. licheniformis. Bacillus subtilis and B. velezensis accounted for more than 73% of the Bacillus isolates. Notably, the MLST scheme exhibited high discriminatory power and might be further simplified to be a convenient method to identify species of the genus Bacillus.

• 생명과학회지
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• 제30권2호
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• pp.122-128
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• 2020

Candida albicans가 칸디다 혈증의 주요 원인 균으로 알려져 있지만, 최근에는 non-albicans Candida (NAC)에 의한 심각한 감염이 증가하고 있다. NAC 중에서 C. glabrata는 두 번째로 병원성을 나타내는 원인 균이지만 C. glabrata의 구조, 역학 등의 기본적인 연구는 거의 없는 실정이다. 따라서 본 연구에서는 다양한 유형의 임상 표본으로부터 분리된 총 102개의 C. glabrata균주로 multi-locus sequence typing (MLST)을 수행하였다. FKS, LEU2, NMT1, TRP1, UGP1 및 URA3을 포함한 6개의 하우스키핑 유전자를 증폭하여 염기서열을 분석하였다. 총 3,345개의 DNA 염기서열 중 49개의 가변 염기서열 부위가 발견되었으며, 그 결과 102개의 균주에서 총 12개의 상이한 서열 유형을 확인하였고, 알려지지 않은 서열 유형(USTs) 중 UST1이 가장 많이 나타났다. 이 연구의 결과는 국내 C. glabrata 항생제 처방에 도움이 될 것으로 사료되며, 한국에서 유행하는 C. glabrata에 대한 추가 연구를 위한 기본 역학자료로 사용될 것으로 기대한다.

• Journal of Microbiology and Biotechnology
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• 제32권12호
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• pp.1605-1614
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• 2022

The strains associated with foodborne Salmonella enterica Thompson outbreaks in Korea have not been identified. Therefore, we characterized S. Thompson strains isolated from chocolate cakes linked to foodborne outbreaks in Korea. A total of 56 strains were isolated from preserved cake products, products in the supply chain distribution, the manufacturer's apparatus, and egg white liquid products used for cream preparation. Subsequently, serological typing, pathogenic gene-targeted polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome multi-locus sequence typing (wgMLST) were performed to characterize these isolates. The antigen formula of all isolates was 7:k:1,5, namely Salmonella enterica subsp. enterica Serovar Thompson. All 56 isolates harbored invA, his, hin, and stn, and were negative for sefA and spvC based on gene-targeted PCR analyses. Based on PFGE results, these isolates were classified into one group based on the same SP6X01.011 pattern with 100% similarity. We selected 19 strains based on the region and sample type, which were subjected to wgMLST. Although the examined strains showed 100% similarity, they were classified into seven clusters based on allelic differences. According to our findings, the cause of these outbreaks was chocolate cake manufactured with egg white liquid contaminated with the same Salmonella Thompson. Additionally, comparative analysis of wgMLST on domestic isolates of S. Thompson from the three outbreaks showed genetic similarities of over 99.6%. Based on the results, the PFGE and wgMLST combination can provide highly resolved phylogeny and reliable evidence during Salmonella outbreak investigations.

• 대한수의학회지
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• 제55권1호
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• pp.31-39
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• 2015

The present study was conducted to determine the full rpoB and eight house-keeping gene sequences of 78 and 35, respectively, avian pathogenic E. coli (APEC) strains. Phylogenetic comparison with 66 E. coli and Shigella strains from GenBank and EMBL was also conducted. Based on the full rpoB sequence, 50 different rpoB sequence types (RSTs) were identified. RST 1 was assigned to a major RST that included 34.7% (50/144) of the analyzed strains. RST 2 to RST 50 were then assigned to other strains with higher nucleotide sequence similarity to RST 1 in order. RST 1, 11, and 23 were mixed with APEC along with human commensal and pathogenic strains while RST 2, 6, 9, 13-15, 22, 24, 25, 33, 34, 36, and 41 were unique to APEC strains. Only five APEC strains grouped into RST 32 and 47, which contained human pathogenic E. coli (HPEC). Thus, most of the APEC strains had genetic backgrounds different from HPEC strains. However, the minor APEC strains similar to HPEC should be considered potential zoonotic risks. The resolution power of multi-locus sequence typing (MLST) was better than RST testing. Nevertheless, phylogenetic analysis of rpoB was simpler and more economic than MLST.

• 대한의생명과학회지
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• 제24권3호
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• pp.221-229
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• 2018

Trichophyton rubrum is one of the well-known pathogenic fungi and causes dermatophytosis and cutaneous mycosis in human world widely. However, there are not an available sequence type (ST) classification methods and previous studies for T. rubrum until now. Therefore, currently, molecular biological tools using their DNA sequences are used for genotype identification and classification. In the present study, in order to characterize the genetic diversity and the phylogenetic relation of T. rubrum clinical isolates, five different housekeeping genes, such as actin (ACT), calmodulin (CAL), RNA polymerase II (RPB2), superoxide dismutase 2 (SOD2), and ${\beta}$-tubulin (BT2) were analyzed using by multilocus sequence typing (MLST). Also, DNA sequence analysis was performed to examine the differences between the sequences of Trichophyton strains and the identified genetic variations sequence. As a result, most of the sequences were shown to have highly matched rates in their housekeeping genes. However, genetic variations were found on three different positions of ${\beta}$-tubulin gene and were shown to have changed from $C{\rightarrow}G$ (1766), $G{\rightarrow}T$ (1876), and $C{\rightarrow}A$ (1886). To confirm the association with T. rubrum inheritance, a phylogenetic tree analysis was performed. It was classified as four clusters, but there was little significant correlation. Even so, MLST analysis is believed to be helpful for determining the genetic variations of T. rubrum in cases where there is more large-scale data accumulation. In conclusion, the present study demonstrated the first MLST analysis of T. rubrum in Korea and explored the possibility that MLST could be a useful tool for studying the epidemiology and evolution of T. rubrum through further studies.

• 대한의생명과학회지
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• 제23권3호
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• pp.223-229
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• 2017

Fungal infections by human pathogenic fungi are increasing globally in elderly, children and immune suppressed or deficient patients. Aspergillus fumigatus is one of the well-known pathogenic fungi and causes aspergilloses in human world widely. However, current identification and classification methods based on its phenotypic characteristics still have limitations. Therefore, currently, molecular biological tools using their DNA sequences are used for genotype identification and classification. In the present study, in order to analyze genetic variations of A. fumigatus clinical isolates, a total of six housekeeping genes were amplified by PCR using specific primer pairs and multi-locus sequence typing (MLST) assay. Results from phylogenetic tree analysis showed that most A. fumigatus strains (88.9%) from respiratory specimens were classified into cluster A and B, and approximately half of A. fumigatus strains (46%) from non-respiratory specimens were classified into cluster C and D. Although the sample size was limited, genetic characteristics of A. fumigatus clinical isolates according to their origins were very similar and well-correlated with other clinical data.

• 대한임상검사과학회지
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• 제50권3호
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• pp.331-336
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• 2018

본 연구에서는 국내 대전 소재 3차 병원 임상에서 분리된 Candida albicans 40 균주를 대상으로 균주 분리원에 따라 7종류의 housekeeping gene에 대한 염기서열 변이를 확인하고 MLST 분석을 통해 균주간의 계통학적 연관성에 대한 연구를 수행하여 다음과 같은 결과를 얻었다. Phylogenetic tree 분석 결과 sub-cluster 1로 central line blood (2), others (5), sputum (4), urine (7)을 포함해 총 18개가 분류되었으며, sub-cluster 2로는 central line blood (1), others (5), peripheral blood (6), sputum (1), urine (1)을 포함해 총 14개가 분류되어 동일 채취 부위에서 분리된 균주는 유전학적으로 유사성을 가지고 있을 가능성이 있음을 확인하였다. 앞으로 분리지역과 상병 등에 따른 C. albicans 유전자들의 변이 추세에 대한 자료의 축적과 임상 검체에 따른 계통학적 관계를 추정하여 감염병 연구 및 역학적 감시체계 구축에 도움이 될 것으로 생각된다.

• 대한의생명과학회지
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• 제25권4호
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• pp.407-416
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• 2019

Recent years have seen an increase in the incidence of candidiasis caused by non-albicans Candida (NAC) species. In fact, C. glabrata is now second only to C. albicans as the most common cause of invasive candidiasis. Therefore, the rapid genotyping specifically for C. glabrata is required for early diagnosis and treatment of candidiasis. A number of genotyping assays have been developed to differentiate C. glabrata sequence types (STs), but they have several limitations. In the previous study, multi-locus sequence typing (MLST) has performed with a total of 101 C. glabrata clinical isolates to analyze the prevalent C. glabrata STs in Korea. A total of 11 different C. glabrata STs were identified and, among them, ST-138 was the most commonly classified. Thus, a novel probe-based quantitative PCR (qPCR) assay was developed and evaluated for rapid and accurate identification of the predominant C. glabrata ST-138 in Korea. Two primer pairs and hybridization probe sets were designed for the amplification of internal transcribed spacer 1 (ITS1) region and TRP1 gene. Analytical sensitivity of the probe-based qPCR assay was 100 ng to 10 pg and 100 ng to 100 pg (per 1 μL), which target ITS1 region and TRP1 gene, respectively. This assay did not react with any other Candida species and bacteria except C. glabrata. Of the 101 clinical isolates, 99 cases (98%) were concordant with MLST results. This novel probe-based qPCR assay proved to be rapid, sensitive, highly specific, reproducible, and cost-effective than other genotyping assay for C. glabrata ST-138 identification.

• 미생물학회지
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• 제48권2호
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• pp.116-124
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• 2012

2008년 3월, 서울의 상수원으로 이용되는 한강에서 분리된 Enterococcus 76주를 동정하고 이 균주들에 대한 항생제 감수성과 항생제 내성유전자 구조분석, 교차접합, PFGE 및 MLST를 실시하였다. 분리 균주 중 E. casseliflavus는 25주이었으며, E. faecalis 및 E. hirae가 각각 4주 및 1주이었다. 항생제 감수성을 조사한 결과 15주가 vancomycin에 대해 내성을 나타내었으며 E. faecium 11주와 E. casseliflavus 4주가 VRE인 것을 알 수 있었다. VRE를 대상으로 vanA 유전자 검출을 통해 6주의 E. faecium가 vanA를 가지는 VREF임을 밝혔으며, vanA가 포함된 transposon인 Tn1546 구조를 분석한 결과 Km36과 Km37은 Tn1 type, Km20과 Km38은 Tn2 type 그리고 Km 39와 Km40은 Tn3 type으로 나타났다. PFGE 결과 6주의 VREF 중 Km36과 Km37은 동일한 subtype을 나타내었으며, 나머지 4주는 각기 다른 subtype을 나타냈다. VREF 6주를 대상으로 MLST를 실시한 결과 3주는 ST78, 나머지 3주는 각각 ST18, ST192 및 ST230로 나타났다. 이들의 clonal complex는 모두 병원에서 분리되는 CC17로부터 파생된 것으로 파악되었으며 4주는 CC78에, 나머지 2주는 각각 CC18과 CC192에 포함된 것을 확인할 수 있었다.

• 대한임상검사과학회지
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• 제47권2호
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• pp.71-77
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• 2015

최근 항균제의 광범위한 사용으로 인한 항균제 내성세균의 출현과 확산이 축산환경에서 빈번하게 일어나고 있고 이렇게 출현한 다제 내성세균은 사람에게 직접 전파될 수 있어 큰 문제가 되고 있다. 본 연구는 2013년 7월부터 2014년 7월까지 충청지역의 임상검체(n=44) 및 양계농장의 닭(n=34)에서 분리된 78주의 대장균을 대상으로 이루어졌다. PCR과 염기서열 분석을 통하여 MLST 분석과 class 1 integron의 유전형을 분석하였다. 항균제 감수성 검사는 디스크 확산법으로 시행하였다. 사람 유래 대장균의 경우 ST131 (n=20)과 ST38 (n=15)이 가장 많았고 닭 유래 대장균은 ST752 (n=7)이 가장 많았으며 ST117, ST189, ST69는 각각 4주였다. 사람에서 유래한 대장균은 총 44주 중 25주가 class 1 integron을 가지고 있었다. 반면 닭에서 유래한 대장균은 총 34주 중 11주가 class 1 integron을 가지고 있었다. 충청지역에 분포되어있는 사람 유래 대장균과 닭 유래 대장균의 유전형 및 항균제 내성 패턴에 차이를 보였다.