항산균 배양 검사시 액체배지와 고체배지 병행에 의한 효율성 분석

권승직,김영미,이정섭,김성한,박미선,김동혁 대한결핵 및 호흡기학회 2015 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.120 No.0

항산균 배양에 있어 액체배지는 고체배지에 비하여 양성률이 높고 배양시간이 짧지만, 오염률이 높고 분리배양이 어려운 단점을 지녀 고체배양과 병행하는 것이 적절하다고 알려져 있다. 이에 항산균 검출률을 높이기 위해 결핵의심 검체 630개를 대상으로 Ogawa배지와 7H10배지의 양성률을 비교, 분석하였다. 본 연구에서 MGIT배지의 양성률은 14.28%(90/630)로 나타났으며, 오염률은 4.76%(30/630)로 산출되었고, 액체배지에서만 증식한 검체는 9건 이었다. Ogawa배지의 양성률은 10.00%(63/630), 오염률은 3.17%(20/630)로 나타났으며, Ogawa배지에서만 검출된 균주는 10건이었다. 7H10배지의 경우 양성률 8.41%(53/630), 오염률 2.38%(15/360)로 나타났으며 단독배양검체는 4건으로 나타났다. MGIT배지와 Ogawa배지를 병행하였을 경우, 116건의 검체에서 균주가 배양되었으며, 7H10배지와 병행하였을 때 110건의 양성검체를 확인할 수 있었다. 본 결과를 통해 MGIT배지를 기본으로 Ogawa배지를 병행하는 것이 7H10을 병행하는 것에 비해 항산균 검출에 도움이 되는 것을 확인할 수 있었다.

PCR-DGGE를 이용한 누룩에서의 미생물 다양성 분석

Seung Jik Kwon(권승직),Jae Hak Sohn(손재학) 한국생명과학회 2012 생명과학회지 Vol.22 No.1

누룩은 탁주와 약주의 제조를 위한 당화효소와 알코올발효를 위한 미생물의 공급원으로서 제품의 맛과 품질을 결정하는 중요한 역할을 한다. 본 연구에서는 산성누룩의 세균과 진균의 다양성을 조사하기 위해 순수분리 종과 16S 및 28S rRNA gene를 대상으로 한 PCR-DGGE를 이용한 분석을 수행하였다. 누룩 내 세균의 수는 2.7×10? CFU/g이었으며 순수분리와 PCR-DGGE 분석에서 우점종은 Kocuria spp., Pantoea spp., Lactobacillus spp., Pediococcous spp., Weissella spp., Staphylococcus spp. 그 외 endophytic bacterium, uncultured gamma-proteobacteria, uncultured Cyanobacteria와 Actinobacteria였다. PCR-DGGE profile에서 주된 우점종은 Pediococcous pentosaceus와 uncultured Cyanobacteria 이었다. 누룩 내 진균의 수는 3.5×10? CFU/g이었으며 순수분리와 PCR-DGGE 분석에서 우점종은 Trichomonascus spp., Pichia spp., Torulaspora spp., Wickerhamomyces spp., Sacharomycopsis spp., Lichtheimia spp., Mucor spp., Rhizopus spp., Aspergillus spp., Cladosporium spp.였다. PCR-DGGE profile에서 주된 우점종은 Pichia kudriavzevii와 Aspergillus oryzae이었다. PCR-DGGE 기술은 본 연구에서 누룩의 미생물군집을 평가하기 위해 처음으로 사용되었으며 미생물 다양성을 설명하는 데 효과적임을 입증하였다. Nuruk plays a significant role in the flavor and quality of Takju and Yakju, which are produced through saccharification and alcohol fermentation by various microorganisms. In this study, we identified microbial strains isolated from a plate count and PCR-denaturing gradient gel electrophoresis (DGGE) analysis targeting the 16S and 28S rRNA genes, in order to characterize bacterial and fungal diversity in Sansung Nuruk. The numbers of bacteria and fungi in Nuruk were 1.5×10? CFU/g and 2.2×10? CFU/g, respectively. The 16S rRNA gene sequence indicated that the predominant bacteria in the isolates and PCR-DGGE profile of Nuruk were Kocuria spp., Pantoea spp., Lactobacillus spp., Pediococcus spp., Weissella spp., Staphylococcus spp., endophytic bacterium, uncultured Gamma-proteobacteria, uncultured Cyanobacteria, and Actinobacteria. Dominant bacteria from the PCR-DGGE profile were Pediococcous pentosaceus and uncultured Cyanobacteria. The 28S rRNA gene sequence indicated the predominant fungi in the isolates and PCR-DGGE profile to be Trichomonascus spp. Pichia spp., Torulaspora spp., Wickerhamomyces spp., Sacharomycopsis spp., Lichtheimia spp., Mucor spp., Rhizopus spp. Aspergillus spp., and Cladosporium spp. Dominant fungi from the PCR-DGGE profile were Pichia kudriavzevii and Aspergillus oryzae. The PCR-DGGE technique was used for the first time in this study to assess a microbial community in Nuruk and proved to be an effective protocol for profiling microbial diversity.

PCR-DGGE를 이용한 막걸리발효에서 미생물 다양성 분석

Seung Jik Kwon(권승직),Tae-Young Ahn(안태영),Jae Hak Sohn(손재학) 한국생명과학회 2012 생명과학회지 Vol.22 No.2

금정산성 막걸리<SUP>®</SUP>는 전통적인 수제누룩과 쌀로부터 발효된 한국의 전통적인 술이다. 본 연구에서는 막걸리 발효기간 동안 세균과 진균의 다양성을 특성화하기 위해 16S와 28S rRNA 유전자를 목적으로 하는 PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) 분석을 수행하였다. 막걸리 발효기간 동안 PCR-DGGE profile에서 검출된 세균은 16S rRNA 유전자 서열에 기초한 동정결과 Lactobacillus spp. (L. curvatus, L. kisonensis, L. plantarum, L. sakei 및 L. gasseri), Pediococcus spp. (P. acidilactici, P. parvulus, P. agglomerans및 P. pentosaceus), Pantoea spp. (P. agglomerans 및 P. ananatis) 그리고 Citrobacter freundii로 총 12종이었으며, 배양2일 이후 L. curvatus가 주된 우점 종을 형성하였다. 반면 PCR-DGGE profile에서 검출된 진균은 28S rRNA 유전자 서열에 기초한 동정결과 Pichia kudriavzevii, Saccharomyces cerevisiae, Asidia idahoensis, Kluyveromyces marxianus, Saccharomycopsis fibuligera 및 Torulaspora delbrueckii로 6종이었으며 주된 우점 진균은 배양0일에서 2일에 P. kudriavzevii에서 배양 3일에서 6일에 S. cerevisiae로 전환되었다. 결과적으로 PCR-DGGE분석은 막걸리발효기간 동안 미생물의 구조와 다양성을 이해하는 데 유용한 도구임을 보여주었다. Kumjungsansung-Makgeolli<SUP>®</SUP> is a traditional Korean rice wine that is fermented from traditional nuruk and rice. In this study, we performed the PCR-denaturing gradient gel electrophoresis (DGGE) analysis targeting the 16S and 28S rRNA genes to characterize bacterial and fungal diversity during Makgeolli fermentation. The predominant bacteria in the PCR-DGGE profile during Makgeolli fermentation were Lactobacillus spp. (Lactobacillus curvatus, L. kisonensis, L. plantarum, L. sakei, and L. gasseri), Pediococcus spp. (P. acidilactici, P. parvulus, P. agglomerans, and P. pentosaceus), Pantoea spp. (P. agglomerans and P. ananatis), and Citrobacter freundii; these were identified on the base of analysis of 16S rRNA gene sequences. The dominant bacterium during Makgeolli fermentation was L. curvatus. The predominant fungi in PCR-DGGE profile during Makgeolli fermentation were Pichia kudriavzevii, Saccharomyces cerevisiae, Asidia idahoensis, Kluyveromyces marxianus, Saccharomycopsis fibuligera, and Torulaspora delbrueckii, and these were identified on the basis of analysis of 28S rRNA gene sequences. The dominant fungal species during Makgeolli fermentation changed from P. kudriavzevii at 0-2 days incubation to S. cerevisiae at 3-6 days incubation. This study suggests that PCR-DGGE analysis could be a suitable tool for the understanding of microbial diversity and structure during Makgeolli fermentation.

Determination the Criteria of Application Line Probe Assay (LPA) in Sputum Samples Comparing Ziehl-Neelsen and Fluorescent Auramine-Rhodamine stain

김영미,권승직,이정섭,김성한,박미선,김동혁 대한결핵 및 호흡기학회 2015 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.120 No.-

Rapid detection of drug resistant TB in sputum specimens is important to diagnosis and treatment. The Line Probe Assay (LPA) endorsed by WHO to apply on direct sputum but limited in smear-positive specimens. Light-emitting diode microscopy showed higher sensitivity and lower specificity than optical light microscopy. Therefore, we analyse the results of ZN and AR stain to establish the applicable standards for LPA. A total of 115 MTB gene detected sputum specimens was stained with ZN and AR smear microscopy and conducted with GenoType MTBDRplus. The results of LPA assay was compared to sputum smear assays. The interpretable results from LPA were 56 (48.7%). Seventy-six (66.08%) were AR smear-positive and 48 (41.74%) were ZN smear-positive. In ZN stain, 11/67 (16.42%) were ZN negative, 13/16 (81.25%) were scanty positive, 14/14 (100%) were 1+, 14/14 (100%) were 2+ and 4/4 (100%) were 3+ can be interpreted in LPA results. None of smear-negative, 7/22 (31.82%) of scanty positive, 20/25 (80.00%) of 1+, 18/18 (100%) of 2+ and 11/11 (100%) of 3+ is readable in AR stain with LPA results. Our study suggested that the criteria of LPA application should different in ZN, from scanty and AR, from 1+.

MGIT배지와 LJ배지의 조합을 통한 항산균 배양 검출률 향상

이정섭,김영미,권승직,김성한,박미선,김동혁 대한결핵 및 호흡기학회 2015 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.120 No.-

세계보건기구에서는 항산균 배양시 배양시간과 검출률이 우수한 액체배양을 권고하고 있지만 고체배지에서만 배양되는 경우도 존재하므로 양배지 모두 병용하는 것이 항산균 검출에 도움이 된다고 알려져 있다. 현 공공진단 부문에서는 오염률 감소와 검출률 증가를 위해 2개의 고체배지를 사용하지만 액체-고체배양 병용시 고체배지 개수에 따른 효율성을 알아보고자 한다. 총 636건의 호흡기 검체를 MGIT배지 1개와 LJ배지 2개에 접종하여 양성률 및 오염률을 비교분석하였다. MGIT에서는 배양 양성 35(5.5%)건,오염 27(4.2%)건이며 MGIT에서만 배양된것은 한 건도 없었다. LJ 한 개에서 배양했을 경우 양성 30(4.7%)건, 오염 29(4.6%)건이 배양 되었고 LJ 두 개에서 배양했을 경우 양성33(5.2%)건, 오염 18(2.8%)건이 배양 되었으며 LJ에서만 검출된 것은 6(18.2%)건으로 확인되었다. MGIT과 LJ1개 배양 시 양성검출은 총35(5.5%)건, MGIT과 LJ2개 배양시 양성 검출은 41(6.4%)건으로 확인되었다. 알려진 바와 같이 액체배양과 고체배양을 병용하는 것이 항산균 검출에 더 효율적으로 확인되었다. 또한 고체배지를 1개만 배양하는 것 보다는 2개를 배양했을 경우 검출률 증가와 오염률을 감소하는데 있어 더 효과적이라고 판단된다.

Molecular Typing of Mycobacterium intracellulare Using Pulsed-Field Gel Electrophoresis, Variable- Number Tandem-Repeat Analysis, Mycobacteria Interspersed Repetitive-Unit-Variable-Number Tandem Repeat Typing, and Multilocus Sequence Typing: Molecular Ch

전세미,임나라,권승직,심태선,박미선,김범준,김성한 질병관리본부 2014 Osong Public Health and Research Persptectives Vol.5 No.3

Objectives: Mycobacterium intracellulare is the major causative agent of nontuberculous mycobacteria-related pulmonary infections. The strain typing of M. intracellulare is important for the treatment and control of its infections. We compared the discrimination capacity and effective value of four different molecular typing methods. Methods: Antibiotic susceptibility testing, hsp65 and rpoB sequencing, pulsedfield gel electrophoresis (PFGE), multilocus sequence typing (MLST), mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR), and VNTR assay targeting 44 M. intracellulare isolates obtained from patients with pulmonary infections were performed. Results: All the antibiotic susceptibility patterns had no association with the molecular and sequence types tested in this study; however, the molecular and sequence types were related with each other. PFGE gave best results for discriminatory capacity, followed by VNTR, MLST, and MIRU-VNTR. Conclusion: The high discriminatory power of PFGE, VNTR, and MLST is enough for differentiating between reinfection and relapse, as well as for other molecular epidemiological usages. The MLST could be regarded as a representative classification method, because it showed the clearest relation with the sequence types.

P-24 Drug resistance profile of isolates from recurrent pulmonary tuberculosis patients to rifampin and isoniazid

이길수,송승은,권승직,박미선,김성한,김동혁 대한결핵 및 호흡기학회 2016 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.121 No.-

The pattern of anti-tubercular drug resistance varied from region and country and at different periods of time. The aim of the present study was to determine the antibiotic resistance profile of Mycobacterium tuberculosis isolated from all relapsed pulmonary tuberculosis patients who were admitted to public health center during 2014-2015. Mutations in specific regions of the katG, inhA, ahpC, and rpoB genes were analyzed by sequencing, and compared to correlation to minimal inhibitory concentration (MIC) and absolute concentration test. In the rifampin-resistance strains, the mutations were identified in the codons 441, 485, 516, 520, 526, 531, and 533 of rpoB gene. The most prevalent point mutations were Ser-Leu at the rpoB531 codon (37.5%). Resistance to isoniazid was associated with mutations found in the katG (67.4%), inhA (26.1%), and ahpC genes (6.5%). The most prevalent mutations were Ser-Thr at the katG315 codon (44.2%). It was noted that five mutations within the region of rpoB, and 9 mutations in katG (5), inhA (3), ahpC (1) region of phenotypically susceptible strains were detected. And most single mutations in the inhA promoter region were associated with low-level resistance to isoniazid (0.12 ≤ MIC ≤ 1.0 ㎍/㎖, 81.8%). These discordant results between molecular and phenotypic tests suggest that further study is needed to identify the influence of these gene mutations to prognosis of TB patients.

A case of Mycobacterium africanum infection in Korea : monitoring result from National TB typing service

송승은,이정섭,김영미,하지민,권승직,유재일,김지은,이소담,박미선,김동혁 대한결핵 및 호흡기학회 2018 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.126 No.-

The increase rate of tuberculosis (TB) among foreigners in Korea possesses important significance in public health. In this study, we report a case of Mycobacterium africanum which was first isolated in Korea from the results of National TB typing service. From the results of spoligotyping and 24-loci MIRU-VNTR, phylogenetic tree analysis suggested that this isolate was closed to M. africanum. And corresponding to TB-insight database, the isolate was identified as M. africanum lineage 2. The isolate was further characterized by performing standard biochemical identification tests, rpoB and hsp65 gene sequencing, and drug susceptibility testing test for first and second line by using the MGIT 960 system. The results of these tests provide the isolate is M. africanum and this bacteria is pan-susceptible to anti-tubercle drugs. From the epidemiological data, the patient was 25-year-old man from Liberia who had entered Korea in February 2016. After 1.5 years later, he had been registered at the public health center as TB patient in October 2017 and completely recovered after standard 6 month treatment. It seems to be the patient would be exposed the bacteria in his own country. Monitoring the genotype of TB is important to distinguish imported TB from domestic TB to identify the route of infection.

Molecular epidemiologic analysis to track TB transmission in high schools in Korea, 2015-2017

이정섭,하지민,김영미,송승은,권승직,유재일,이소담,박미선,김동혁 대한결핵 및 호흡기학회 2018 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.126 No.-

Tuberculosis (TB) in high schools is a major public health concern because of increasing the opportunity for transmission to other students. To estimate levels of transmission and genetic diversity of M. tuberculosis in high school level, we analyzed a total of 176 culture-positive TB isolates from 141 high schools with molecular and epidemiological data in Korea, from 2015-2017. Of these, 58 TB patients were reported from 22 schools with multiple episodes (2-9 episodes in a case). The results of molecular strain typing with 24-loci MIRU-VNTR and spoligotyping revealed that 105 strains (59.7%) had unique genotypic profiles and remains were categorized into 23 clusters. Systemic investigation with genetic and contact tracing results of all 23 genotypic clusters, 9 cases of high schools with multiple TB episodes were related with recent transmission. Of all recent transmission cases, except 1 cases, were school mate transmission and remain case was spread between teachers. Statistical analysis of TB transmission rate revealed that high school setting is not significant higher than other congregate facilities (OR=1.8 (0.7-4.6)). However, age-dependent analysis suggested that under 19 years group are the highest risk group for attributable to recent transmission (OR=3.8 (2.0-7.0)). Our data showed the current status of TB transmission in high schools in Korea and careful interpretation of molecular epidemiology data would help in contact investigations to proper response.