식중독 추적조사를 위한 살모넬라속의 분자생물학적 특성 연구

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2023. 2. 강동현.우리나라에서 비장티푸스성 살모넬라균은 병원성대장균, 노로바이러스 등과 함께 다발성 식중독 요인 중 하나로 보고되고 있다. 살모넬라에 의한 식중독은 세계적으로도 보건학적 부담을 초래하고 있으며, 세계 설사 원인의 4대 주요 원인균 중 하나로서 이로 인해 세계인구의 연 10%가 장관성 설사질환을 경험하고 특히 5세이하의 설사를 경험하는 수가 2억2천여명으로 세계보건기구에서는 보고하고 있다. 특히 가벼운 설사 증상도 있지만 생명을 위협하는 경우도 있어 매우 주의가 필요한 대표적 식중독균이다. 살모넬라속은 생물학적으로도 혈청형이 2,500여 종 이상이 보고되고 있어 이에 대한 검사와 관리가 까다로운 특성이 있다. 그 이유는 식중독 원인조사 과정 중 조사 시료에서 분리되는 수많은 살모넬라균들이 동일한지 밝혀내는 과정이 매우 어렵고, 그 결과에 따라 행정적 책임 소재를 판단해야 하므로 신중하게 접근해야 하기 때문이다. 식중독의 원인을 밝히는 매우 중요한 근거가 되는 단계는 식중독 환자 시료에 존재하는 균과 환자가 섭취한 식품과 관련 환경 시료에 존재하는 균이 일치하는지 판단하는 과정이라고 할 수 있다. 본 연구에서는 살모넬라 식중독 원인 규명을 위하여 분리된 살모넬라 균주 간의 일치 여부를 확인하는 실험실적 방법을 살모넬라의 특성 분석 및 분자생물학적 연구를 통해 제시하였다. 이를 위하여 우리나라 소매상에서 유통되는 계육에서 살모넬라의 분포도를 확인하고, 분리된 살모넬라 균의 특성 연구를 통해 유사도를 판단하였다. 유사도를 판단하는 실험실적 과정은 살모넬라의 동정, 혈청형 확인, 병원성 유전자 확인, 펄스장 겔 전기영동(Pulsed-Field Gel Electrophoresis, PFGE), 다좌위 서열 구분법(Multi-locus Sequence Typing, MLST)을 수행하였다. 총 113건의 계육을 대상으로 살모넬라를 분리 동정한 결과 24건의 시료에서부터 24 종의 살모넬라를 분리하여 21.2%의 분포율을 확인하였고, 혈청형은 S. Enteritidis 11건(45.8%), S. Virchow 6건(25%), S. Montevideo, S. Bsilla 각 2건(8.3%), S. Bareilly, S. Dessau, S. Albany 각 1건(4.2%)으로 나타났다. 병원성 유전자는 유전자 6개 (his, invA, stn, sefA, spvC, hin)를 중심으로 PCR을 통해 확인한 결과 모든 분리된 살모넬라 균주는 his, invA, stn에 양성을 보여 식중독을 일으킬 수 있는 병원성을 보유한 것으로 판단되었다. PFGE 결과 24종의 살모넬라는 18개의 그룹으로 나뉘었으며, 64.4~100% 유사도를 보였다. 11종의 S. Enteritidis는 9개 그룹의 유전형으로 구분되고 74.4%의 유사도를 보인 반면, S. Virchow는 5개 그룹의 유전형, 85.9%의 유사도를 보였다. MLST 결과 국내 충청지역 유통 계육에서의 주요 서열유형(Sequence Type)은 ST-11, ST-16이었으며, 수입산 계육과 차이를 보임을 확인할 수 있었다. 따라서 MLST를 통한 서열 유형도 식중독 원인 식품을 규명하는 하나의 표지로 활용될 가능성을 확인하였다. 본연구에서는 2018년 9월에 발생한 전국 규모의 학교 집단식중독의 원인 조사를 사례연구로 원인조사 시료에서 분리된 살모넬라 균종 간의 특성과 유사도를 분석하였다. 발생 시설에서 보존되어 있는 초코케익 제품을 포함, 유통 중인 초코케익 완제품, 제조사의 제조기구(whipper), 케익 제조용 크림의 원료인 난백액 제품 시료에서 56개의 살모넬라 균종을 대상으로 하였다. 이들 살모넬라의 특성은 앞서 분포도 연구와 동일한 방법으로 정확한 동정을 거쳐 혈청형 확인, 병원성 유전자 확인, PFGE를 수행하였으며, 다좌위 서열 구분법은 전장유전체 다좌위 서열 구분법(whole genome Multi-locus Sequence Typing, wgMLST)을 수행하였다. 모든 연구 대상의 살모넬라 균주는 혈청형 확인 결과 7:k:1,5 항원 반응 조합의 S. Thompson이었으며, 병원성 유전자는 invA, his, hin, stn 양성, sefA, spvC 음성을 나타내었다. PFGE 수행 결과 유전형은 1개 그룹의 100% 유사도를 보여 균주 간의 유전자적 차이가 없음을 알 수 있었다. Whole genome MLST는 56개 균종 중 시료 종류와 지역의 중복 균주를 제외한 19개 균종을 대상으로 수행하였다. 그 결과 100% 유사도를 보였으며, 추가적으로 allele 차이를 기준으로 7개의 유전형 그룹으로 구분됨을 확인할 수 있었다. 이러한 실험실적 특성 분석 결과는 본 연구에 포함되지 않았지만 환자 시료와 동일하게 일치함을 확인하여 이를 근거로 2018년 발생한 학교 식중독의 원인 식품은 살모넬라 톰슨이 오염된 난백액으로 제조한 크림이 도포된 초코케익이었으며, 단일 섭취식품에 의한 식중독임을 입증할 수 있었다. 국내에서 S. Thompson 과 관련된 식중독은 본 식중독 발생 이전 두 건이 보고되었으며, 2014년, 2015년 각 1건의 조사과정에서 분리된 균주와 2018년 균주를 대상으로 특성을 분석하였다. 그 결과 세 균주 모두 동일한 특성과 높은 유전자적 유사도(<99.6%)를 보여 본 연구에서 수행한 실험실적 유사도 분석 방법은 향후 오염원 규명에 매우 유용할 것으로 판단된다.In Korea, non-typhoidal Salmonella is reported as one of the key organisms that frequently causing food poisoning along with pathogenic Escherichia coli and norovirus. The major outbreaks linked to Salmonella spp. occurred in 2022 were two reported cases associated with naengmyeon (cold noodle) and gimbap (seaweed roll), both of the two meals used eggs as ingredients. The average number of cases of recent five years from 2018 to 2022 increased 221% compared to previous five years from 2013 to 2017 by non-typhoidal Salmonella outbreaks. Salmonellosis is increasing a health burden worldwide, 10% of the world's four major causes of diarrhea, especially diarrhea under the age of 5, at 220 million yearly by the World Health Organization reports. In particular, it is a representative food poisoning bacteria that needs to be very careful because there are mild diarrhea symptoms but also life-threatening cases. More than 2,500 serotypes have been reported biologically in Salmonella spp., making it difficult to test or manage them. The reason is that it is very difficult to determine whether the numerous Salmonella bacteria isolated from the investigation samples are the same during the investigation of the cause of food poisoning, and it is necessary to carefully approach the results as they determine the administrative responsibility. An important step in determining the cause of food poisoning is the process of determining whether the bacteria present in the food poisoning patient sample match the food consumed by the patient and the bacteria present in the related environmental sample. In this study, a laboratory method that confirming the correlation between isolated Salmonella strains to determine the cause of foodborne outbreak was presented through characteristic analyses and molecular typing studies. In this study, we sought to investigate the various characteristics of Salmonella spp. isolated from raw chicken meats available in Korean markets. The data collected, such as food source of isolation, sampling information, serotype, virulence, and genetic profile including sequence type, were registered in the database for further comparative analysis of the strains isolated from the traceback investigation samples. To characterize serotype, virulence, and gene sequences, we examined 113 domestically distributed chicken meat samples for contamination with Salmonella spp. Phylogenetic analysis was conducted on 24 strains (21.2%) of Salmonella isolated from 113 commercially available chicken meats and by-products, using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Serotyping of the isolated Salmonella spp. revealed S. Enteritidis in 11 strains (45.8%), S. Virchow in 6 strains (25%), S. Montevideo in 2 strains (8.3%), S. Bsilla in 2 strains (8.3%), S. Bareilly in 1 strain (4.2%), S. Dessau in 1 strain (4.2%), and S. Albany in 1 strain (4.2%). The genetic correlation indicated that 24 isolated strains were classified into 18 clusters with a genetic similarity of 64.4-100% between them. Eleven isolated S. Enteritidis strains were classified into 9 genotypes with a sequence identity of 74.4%, whereas the most distantly related S. Virchow was divided into five genotypes with 85.9% identity. Here, the MLST analysis indicated that the major Sequence Type (ST) of the Salmonella spp. isolated from domestic chicken sold in Chungcheong Province belongs to the ST 11 and 16, which differs from the genotype of Salmonella isolated from imported chicken. The differential sequence characteristics can be a genetic marker for identifying causative bacteria for epidemiological investigations of food poisoning. 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 manufacturers 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.ABSTRACT 1 CONTENTS 6 LIST OF FIGURES 9 LIST OF TABLES 10 CHAPTER Ⅰ. GENERAL INTRODUCTION 11 Ⅰ-1. HISTORY OF SALMONELLA THOMPSON OUTBREAK, 2018 12 Ⅰ-2. LIMITATION OF CURRENT OUTBREAK INVESTIGATION 16 Ⅰ-3. OBJECTIVES OF THIS STUDY 19 CHAPTER Ⅱ. PREVALENCE AND CHRACTERISTICS OF SALMONELLA SPP. ISOLATED FROM RAW CHICKEN MEAT IN KOREA 22 Ⅱ-1. INTRODUCTION 23 Ⅱ-2. MATERIALS AND METHODS 26 Ⅱ-2-1. Sampling 26 Ⅱ-2-2. Isolation and identification of Salmonella spp. 27 Ⅱ-2-3. Pathogenic gene analysis using PCR 27 Ⅱ-2-4. Serology testing 30 Ⅱ-2-5. Pulsed-field gel electrophoresis (PFGE) 30 Ⅱ-2-6. Multi-locus sequence typing (MLST) 32 Ⅱ-3. RESULTS 33 Ⅱ-3-1. Prevalence of Salmonella spp. from raw chicken meat 33 Ⅱ-3-2. Distribution of Salmonella serotypes 34 Ⅱ-3-3. PCR targeted to pathogenic genes 36 Ⅱ-3-4. Comparison of isolates of Salmonella spp. using PFGE 38 Ⅱ-3-5. Multi-locus sequence typing analysis 40 Ⅱ-4. DISCUSSION 42 CHAPTER Ⅲ. COMPARATIVE GENOMIC ANALYSIS OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR THOMPSON ISOLATES ASSOCIATED WITH FOODBORNE DISEASE OUTBREAKS IN KOREA 49 Ⅲ-1. INTRODUCTION 50 Ⅲ-2. MATERIALS AND METHODS 52 Ⅲ-2-1. S. Thompson isolation 52 Ⅲ-2-2. Serology testing 54 Ⅲ-2-3. Pathogenic gene analysis using PCR 54 Ⅲ-2-4. Pulsed-field gel electrophoresis 57 Ⅲ-2-5. Whole genome multi-locus sequence typing analysis 58 Ⅲ-2-6. Correlation analysis among S. Thompson isolates related to foodborne outbreaks 60 Ⅲ-3. RESULTS 60 Ⅲ-3-1. Isolation and identification of Salmonella spp. 60 Ⅲ-3-2. Confirmation of serotypes 63 Ⅲ-3-3. PCR analysis of pathogenic genes 64 Ⅲ-3-4. PFGE patterns of S. Thompson 65 Ⅲ-3-5. Whole genome multi-locus sequence typing analysis 67 Ⅲ-3-6. Correlation among S. Thompson strains isolated in Korea 70 Ⅲ-4. DISCUSSION 75 CHAPTER Ⅳ. REFERENCES ８２ ABSTRACT IN KOREAN ９８박

NMR study on the active site structure of carboxypeptidase A

Maste

Comparison of the soft tissue profile of Korean seniors and youths with normal occlusion

치의학과/석사[한글] 안모 심미성에 대한 관심 증가로 늦은 나이에 교정 치료를 받는 성인 환자가 증가하고 있다. 특히 40대 이후 교정 치료 시, 노화에 따른 연조직 변화에 관한 이해가 필요하며, 이를 참고하여 치료 계획이 세워져야 할 것이다. 이제까지 노인 안면 연조직에 관련된 자료가 미비한 상태이므로 이 연구를 시행하였다.이 연구는 정상 교합의 한국 노인과 청년 남녀의 안면 연조직 차이를 비교하기 위하여 노인과 청년 남녀별로 각각 30명씩 120명을 선정하여 측모 두부 방사선 규격사진을 촬영하였다. 4개 각 군의 평균 연령은 청년 남자군이 19.77세, 청년 여자군이 19세, 노인 남자군이 64.17세, 노인 여자군이 62.53세였다.다음과 같은 결과를 얻었다.1. 연조직 두께에선 턱 부위 (Pog-Pog'''', Gn-Gn'''', Me-Me'''')를 제외하고는, 전반적으로 노인군이 청년군보다 얇았으며, 남녀 모두 Gn-Gn'''' 두께는 노인군이 청년군보다 통계적으로 유의하게 크게 나타났다 (p<0.05). 상순 두께(UIS-LS)는 남녀 모두 노인군이 청년군보다 작게 나타났다 (p<0.05).2. 연조직 수직 고경에선 남녀 모두 청년군보다 노인군의 상순길이가 길었으며, 통계적으로 유의한 차이가 있었다 (p<0.05). 상순 길이의 차이로 상악 전치 노출량은 남녀 모두 노인이 작았다 (p<0.05). 하안면 고경 (Sn-Gn'''')은 노인 여자군이 청년 여자군보다 통계적으로 유의하게 길었다 (p<0.05).3. 연조직 각도 계측 항목의 superior sulcus angle (Sn-A''''-LS)에선 남녀 모두 노인군이 청년군에 비해 컸다 (p<0.05). Inferior sulcus angle (LI-B''''-Pog'''')은 노인 여자군이 청년 여자군보다 통계적으로 유의하게 컸다 (p<0.05). [영문]As people have a lot of interest in facial aesthetics thesedays, the number of adult patients who have orthodontic treatment at a late age is on the increase. When orthodontists treat adult patients, especially those over forty, it is necessary to understand seniors' facial soft tissue change. With consideration of this change, orthodontists have to make a treatment plan. Until now, as data on facial soft tissue in seniors is insufficient, this study was done.The purpose of this study was to compare the facial soft tissue difference between Korean seniors and youths with normal occlusion in both sexes. To achieve this purpose, each 30 samples for male youths, female youths, male seniors, female seniors, 120 samples in all were selected and 120 lateral roentgenograms were taken.The average age of 4 each groups were: 19.77 years old for the male youth group, 19 for the female youth group, 64.17 for the male seniors group, and 62.53 for the female seniors group.The results were as follows:1. In the soft tissue thickness, generally seniors were thinner than youths except for the soft tissue in the chin (Pog-Pog'''', Gn-Gn'''', Me-Me'''') in both sexes. In Gn-Gn'''', senior groups were significantly thicker than the youth groups in both sexes (p<0.05). In the upper lip thickness (UIS-LS), senior groups were significantly thinner than youth groups in both sexes (p<0.05).2. In the vertical measurements of soft tissue, the upper lip length of senior groups was significantly larger than youth groups in both sexes (p<0.05). Due to the difference of upper lip length between seniors and youths, the exposure of upper incisors from the upper lip was significantly smaller in seniors than in youths in both sexes (p<0.05). The lower facial height (Sn-Gn'''') was larger in seniors than in youths in both sexes, there was a significant difference between female youths and female seniors (p<0.05).3. In the superior sulcus angle (Sn-A''''-LS), seniors were significantly larger than youths in both sexes (p<0.05). In the inferior sulcus angle (LI-B''''-Pog''''), the female senior group was significantly larger than the female youth group (p<0.05).ope

Solid State NMR Studies on Catalytic Mechanism of Carboxypeptidase A andEletromic Structure of High Tc Superconductors

Docto

X선 난반사 세기를 이용한 Mo-Si다층박막의 연구

Maste

Mechanism of intracellular invasion and pro-inflammatory response of Orientia tsutsugamushi through the modulation of calcium signaling

학위논문(석사) --서울대학교 대학원 :의학과 (미생물학전공),2009.8.Maste

Ocean carbon dynamics associated with natural and anthropogenic climate change

DoctorThe ocean dissolves a large amount of the anthropogenic CO2 that is emitted by combustion of fossil fuel, and may thereby moderate future climate change. However, acidification caused by dissolved CO2 affects the carbonate system and significant effects marine biogeochemical cycles and ecosystems. Several modeling studies have assessed possible change in ocean chemistry under various CO2 concentration and emission scenarios. To be accurate, projections of oceanic carbonate chemistry should be made with understanding of natural variability in the oceanic carbonate system. Limited observations indicate that the carbonate chemistry of the surface ocean is significantly influenced by global and regional climate changes, but the effect has not been well constrained. Because of complex interplay among physical and biogeochemical processes, the ocean carbonate system varies substantially among environments and on various time scales. Understanding of how the ocean carbonate system will react to continued increase in anthropogenic CO2 requires research on the influence of climate change on each process. The goal of this thesis is to improve our understanding of climate variability and how it affects the oceanic carbonate system. I conducted research that disclosed a link between carbonate chemistry and large-scale climatic predictors including the El Niño Southern Oscillation (ENSO) and Southern Annular Mode in two different locations: the Chuuk coral reef in the western Pacific (Chapter 2) and the Southern Ocean (Chapter 3). In coastal waters, human inputs of nutrients can lead to excessive production of algae (eutrophication). During photosynthesis, phytoplankton release organic acids that react with protons during seawater titration, and thereby contribute to total alkalinity. The third study of the thesis work concentrates on evaluating a non-conservative variation of total alkalinity caused by organic acid accumulation (Chapter 4). As the amount of anthropogenic CO2 in the ocean increase, adverse effects of ocean acidification on coral reefs will increase in future oceanic environments. The carbonate system in coral reef environments is controlled by complex interactions of internal processes, including net community calcification (calcification minus dissolution) and net community production (organic matter production minus respiration), and external processes, such as the intrusion of surrounding seawater and air-sea CO2 exchange. The 6-year weekly records of carbonate chemistry conditions in Chuuk lagoon, located in the coral-rich western Pacific Ocean, showed that during weak intrusion of ambient seawater from the surrounding open ocean two internal biological processes (calcification and respiration) reinforced each other and collectively lowered the pH of the reef water for extended periods, of a few to several months. Our study indicates that periods of low wind speed weaken the intrusion of ambient water, thereby increasing the residence time of reef water; as a result, respiration and calcification increase its acidification. This finding is not an isolated phenomenon, but may be widespread in the coral-rich western Pacific Ocean, which hosts 50% of global coral reefs and in which the degree of ambient water intrusion is closely associated with the wind speed change associated with the ENSO. To estimate the effect of climate variability on the Southern Ocean’s capacity to uptake anthropogenic CO2, I investigated hydrographic and carbon parameters in the eastern South Pacific Ocean along the 110°W meridian from 67°S to 21°N. I used seawater δ13C datasets collected in 1994 and 2008 to determine the change in the anthropogenic CO2 inventory during this period. The δ13C change in each of the latitudinal bands was primarily caused by inputs of anthropogenic CO2 by transport and air-sea exchange. More than 50% of the total anthropogenic CO2 was added to the subpolar band by the northward movement of Antarctic Intermediate Water (AAIW) from the south; and air-sea exchange. I also calculated the ratio of the temporal change in δ13C to the change in dissolved inorganic carbon, which is a measure of the efficiency of oceanic uptake of anthropogenic CO2. The change in preformed δ13C and DIC suggests that the ratio for AAIW in 1994 (−0.017‰ (μmol kg−1) −1) was greater than that in 2008 (−0.010‰ (μmol kg−1) −1); this result means that the efficiency of CO2 uptake by the Southern Ocean was lower in 2008 relative than in 1994. AAIW remained at the surface for a shorter period in 2008 than in 1994, and thus would have taken up less atmospheric CO2 prior to subduction. The projected reduction in this ratio indicates a relative weakening of CO2 uptake by the Southern Ocean in the period. The assessment of the carbonate system builds on precise measurement of four measureable properties of the carbon dioxide system; total alkalinity, total inorganic carbon, CO2 fugacity (fCO2), and pH. In seawater dissolved organic acids produced by phytoplankton dissociate into equal amounts of hydrogen ions and conjugated bases, which maintain the seawater alkalinity (AT) unchanged. However, the resulting conjugate bases react with protons during seawater titration and thereby contribute to the alkalinity (AT−ORG) whereas the contributions of other species (e.g. HCO3−, CO32−, B(OH)4−) to AT are proportionally lowered. Production of such dissolved organic acids was confirmed in each of six phytoplankton cultures and in a coastal environment. In phytoplankton monocultures with initial concentrations of ~70 µM nitrate and ~5 µM phosphate, the contribution of organic acids to seawater alkalinity (AT−ORG) was found to be 15−40 μmol kg−1 AT−ORG upon the complete consumption of added nutrients; the magnitude of AT−ORG depended on the phytoplankton species involved. In the coastal environment as high as 15 μmol kg−1 of AT−ORG was found. If the effect of organic acids on seawater AT is not accounted for, the concentrations of inorganic carbon components calculated from pairs of carbon parameters, including AT, is inaccurate in culture studies and in studies of productive coastal environments

Surface Treatment Method of a Device using Nano Mask

나노 마스크를 이용한 소자의 표면 처리 방법에 대해 개시된다. 개시된 표면 처리 방법은 구조체 및 나노 입자들 사이에 정전기적 인력이 작용하도록 하여 구조체 표면에 나노 입자를 용이하게 결합하도록 하여 건식 식각 공정에 의해 구조체 표면에 굴곡 형상을 형성할 수 있다