본 연구에서는 대표적인 신선 잎채소류인 상추의 세척 단계에서 초음파 (37 kHz)와 염소 (100~300 ppm)의 병용 처리 후 냉장 ~ 실온저장 (10~25C)에 따른 이 식품 중의 Salmonella Typhimurium 의 성장예측모델을 개발하였다. 1차 모델 개발을 위해 Gompertz 방정식을 활용하여 각기 다른 실험 조건에서의 S. Typhimurium의 생육도 (SGR과 LT)를 조사했다 . 본 방정식에 의한 1차 모델 개발시 R2가 0.92 이상으로 우수하게 나타났으며 저장온도가 낮을수록 초음파에 사용된 염소의 농도가 높을수록 SGR 값은 감소하였고 LT 값은 증가하였다 . 이를 바탕으로 2 차 polynomial 모델을 개발하여 다양한 통계적 지표 (R2, MSE, Af 및 Bf)를 통해 분석한 결과 개발된 모델의 적합성을 확인할 수 있었다 . 따라서 개발된 모델이 초음파와 염소의 병용 세척에 따른 저장 중 상추에 대한 S. Typhimurium의 성장예측모델로 사용 가능하다고 판단되어지며 , 신선 잎채소류에서의 식중독을 예방하고 미생물학적 위생관리기준을 설정하는데 기초자료로 활용될 수 있을 것으로 사료된다 .

The novel serogroup of Bacillus thuringiensis serovar mogi (H3a3b3d) was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. Plasmids from B. thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. In this study, the genome sequence of the strain was determined. The 6.0-Mb genome of B. thuringiensis mogi contains three replicons: a circular chromosome (5.40-Mb) encoding 5,652 predicted open reading frames (ORFs), and two megaplasmids, pMOGI364 (364 564 bp) and pMOGI222 (222 348 bp). The G+C contents of these replicons ranged from 31.3% to 34.2% for pMOGI364 and pMOGI222, respectively. There are six putative cry genes, cry19Bb1, cry73Aa, cry20Bb1, cry27Ab1, cry4Aa and cry56Ba1, distributed on these two megaplasmids. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative PCR (qPCR) from the wild type strain. Also, these cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization.

Plasmids are crucial for determining the pathogenicity and host range of organisms of the Bacillus thuringiensis strains. In this research, a novel serogroup of B. thuringiensis serovar mogi (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained two megaplasmids (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that there are 7 putative cry genes, cry19Bb1, cry73Aa, cry40orf2, cry20Bb1, cry27Ab1, cry56Ba1 and cry39orf2, distributed on the two different megaplasmids, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and p1KSD, which is a recombinant expression vector containing cyt1Aa promoter combined with the STAB-SD sequence, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative PCR (qPCR) from the wild type strain. These results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

Bacillus thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative real-time PCR (qrtPCR) from the wild type strain as well as transformant strains. The results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. B. thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.

Bacillus thuringiensis (Bt) strain K4 was isolated from fallen leaves which had been collected at a forest stand in Mungyeong city, Republic of Korea. The flagellated vegetative cells of Bt K4 were agglutinated with the H3 reference antiserum among 55 reference H-antisera. In a further test to identify subfactors, 3b and 3d monospecific antisera were reactive to the cells, followed up with introducing a novel serogroup of 3a3b3d, designated as serovar mogi. The strain K4 had mosquitocidal activity against Dipteran larvae, Anopheles sinensis and Culex pipiens pallens, with no Lepidopteran toxicity observed. The SDS-PAGE profile of K4 crystal protein, ovoidal-shaped, included several bands ranging from 30-75 kDa. Four putative peptides, Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected from the bands by a nano-LC-ESI-IT MS analysis. Through a thermal asymmetric interlaced PCR, cry19Ba, cry40ORF2 and cry27Aa genes were partially cloned from K4 strain. Three cry genes were further found in the strain by a 454 pyrosequencing, ending up to showing 58%, 39% and 84% homology in amino acids with Cry56Aa, Cry8Ba and Cry39ORF2 toxins, respectively. This novel 3a3b3d type strain, B. thuringiensis subsp. mogi, can be used as a good resource for studying unknown mosquitocidal cry genes.

The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.

Bacillus thuringiensis 1-3 (Bt 1-3) which was isolated from a Korean soil sample showed high insecticidal activity against Aedes aegypti as well as Plutella xylostella. The isolate was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with cry gene specific primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A gene, differing from spp. aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D. We modified the plasmid capture system (PCS) to clone plasmid from Bt 1-3 through in vitro transposition. Fifty-three clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified according to similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its 20 putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family.

In order to investigate phenotype and genotype of Salmonella enterica subsp. enterica serovar Enteritidis, Forty-eight S. Enteritidis isolates from diarrhea patients were analysed using antimicrobial resistance typing, Phage typing, and Pulsed field gel electrophoresis in Seoul from 2004 to 2005. All of S. Enteritidis were resistant to streptomycin(SM, 37.5%), ampicillin(AM, 43.8%), t icarcillin(TIC, 43.8%), chloramphenicol(CM, 29.2%), t etracycline(TE, 10 .4%) and nalidixic acid(NA, 18.8%) among 16 antimicrobial drugs. Of 48 S. Enteritidis, 8 isolates(16.7%) were resistant to 1 drug, 3 isolates( 6.3%) to 2 drugs, 1 isolate (2.1%) to 3 drugs and 17 isolates(35.7%) to 4 drugs. The basic pattern of 4 drugs resistance was SM, TIC, TE, and CM but 1 drug resistant isolates represent all nalidixic acid resistance. Among 30 antibiotic r esistant S . Enteritidis, 2 1 isolates(70 %) were phage type 2 1, 8 i solates(26.7%) were phage t ype 2 3 and 1 isolate( 3.3%) was RDNC, respectively. Of the phage types observed, all of phage type 23(8 isolates) were nalidixic acid resistant and phage type 21 were AM-TIC-SM-CM multi-resistance(13 isolates; 43.3%), AM-TIC-SM-TE(4 isolates; 13.3%), AM-TIC-SM(1 isolate; 3.3%), AM-TIC-CM(1 isolate; 3.3%), and AM-TIC(2 isolates; 6.7%) resistance and 1 isolate of RDNC was NA-TE resistance. PFGE divided the isolates into two major clusters, A(n=14) and B(n=14). There were four different resistance profiles with resistance to AM, TIC, SM, TE, NA within PFGE A. Also resistance to AM, TIC, SM, CM was common within PFGE B. The PFGE A strains typed as PT21(n=5), PT23(n=8), and RDNC(n=1), While all the PFGE B strains typed as PT21(n=14). In consequence, there was the highly significant concurrence between resistance typing, phage typing and PFGE.

In order to investigate the resistance patterns and molecular characteristics of resistance gene of 9 NA-resistant Salmonella enterica subsp. enterica serovar Enteritidis, Total of 101 isolates were isolated from stool sampes from 2005 to 2006. Among them, 48 isolates(47.5%) were identified S. Enteritidis, 24 isolates(23.8%) S. Typhi, 8 isolates(42.5%) S. Typhimurium, 7 isolates(6.9%) S. Paratyphi A and 14 isolates (13.9%) others Salmonellas. All of S. Enteritidis were resistant to ampicillin(43.8%), ticarcillin(43.8%), streptomycin(37.5%), chloramphenicol(29.2%), tetracycline(10.4%) and nalidixic acid(18.8%), respectively. All nalidixic acid-resistance isolates represented one point mutation in the quinolone resistance determining region(QRDR) of gyrA gene. Among them, 8(89%) isolates were substituted Tyr for Ser at position amino acid 83th or 1(11%) isolate was substituted Asn for Asp at position amino acid 87th. In consequence, Continued surveillance of NA-resistance among non-Typhi S. entetica isolates is needed to mitigate the increasing prevalence of nalidixic acid resistance.

To investigate antimicrobial susceptibility and relationships of the multidrug resistance and phage types of 49 Salmonella enterica serovar Typhimurium isolated from diarrhea patients in Seoul between 1999 and 2002, we analyed stranis by serotyping, antimicrobial susceptibility test and phage typing. Out of 2,705 samples examined for the causative agent from diarrhea patients, total of 512 isolates were confirmed to be genus Salmonella. Out of 512 isolates, 49 isolates(9.6%) were identified as S. Typhimurium. All of the S. Typhimurium strains represented 100% susceptibility to ceftriaxone, cefoxitin, amikacin and ciprofloxacin and were over 90% susceptibility to gentamicin, cephalothin and kanamycin, but were resistant to tetracycline(75.5%), streptomycin(59.2%), sulfonamides(55.1%), ticarcillin(36.7%), ampicillin( 28.6%), and chloramphenicol(20.4%). Out of 49 S. Typhimurium, only 3 isolates(20.4%) were resistant to one drug, 6 isolates(12.2%) to two drugs and 12 isolates(24.5%) to 3 drugs, 1 isolate(2.0%) to 4 drugs, 2 isolates (4.1%) to 5 drugs, and 6 isolates(12.2%) to 6 drugs and 5 isolates (10.2%) to 7 drugs. Out of 49 S. Typhimurium, 10 isolates(20.4%) were DT195, 5 isolates(10.2%) were DT193 and DT206, 4 isolates(8.2%) were DT104L, DT146, DT203 and RDNC, respectively. Phage types observed the resistant patterns of more than 6 drugs were DT104L, DT193, DT194, DT195 and DT67, but those of 3 drugs representing S-S3-TE type was DT195.

The prevalence and serotype of food-borne pathogens was investigated from 888 samples of chilled meat, 222 samples of packed frozen meat and 117 samples of imported frozen meat during the period from March 1996 to October 1998. Isolation rates of pathogens associated with food poisoning were revealed in order of Staphyloccus aureus, Campylobacter jejuni/coli, Listeria monocytogenes and Salmonella spp, but Escherichia coli O157:H7 was not isolated in all of the meat samples. Amusingly, Cczmpylobacter jejuni/coli were isolated highly in refrigerated meat, but was not isolated in packed frozen meat. L. monocytogenes was encounted higher isolation frequency in packed frozen chicken meat than in refrigerated chicken meat. In the distribution of serotypes of isolates, most isolates of Sta. aureus classified as enterotoxin type C and D. All of the Salmonella spp. isolated from pork were diagnosed group A and most of isolates from chicken meat were grouped B and D. Most of L. monocytogenes isolated from chicken meat were grouped type 1 and a few number of isolates classified as type 4.

지금까지 많은 연구가 되어 있지 않던 Bacillus thuringiensis serover. darmstadiensis의 내독소를 Rengorafin-76 단계적 기울기 원심분리로 분리하여 전자 현미경으로 관찰하여 이중피라미드 구조를 가진 독소 단백질을 확인하였으며 B. thuringiensis serover. kurstaki HD1의 독소 생성유전자와 B. thuringiensis serovar. darmsladiensis의 유전자가 유사성이 있다는 보고를 근거로 하여 B. thuringiensis serovar. HD1의 독소생성유전자를 가진 프로브(pUYBT 9044)로 이용하여 colony hybridization 및 southern hybridization한 결과 2.6Kb EcoRI 단편 및 Southern hybridizationg한 결과 2.6Kb EcoRI 단편 및 3.6Kb HindⅢ 단편을 선발할 수 있었다. 이들 단편들은 B. thuringiensis serovar.kurstaki HD1 독소 유전자와 hybridization시 유사성이 있었다. 특히 3.5Kb HindⅢ 단편은 2.6Kb EcoRI 단편에 클로닝되어 있는 1.8Kb의 HD1 독소유전자와 유사성이 있는 부분을 공유하고 있었으며 1.0Kb정도의 EcoRI-HindⅢ 부분이 더 삽입한 것을 알 수 있었다.

Bacillus thuringiensis var. thuringiensis H1균을 4종류의 배지에서 배지 조성 및 pH 조건에 따라서 성장 양상과 아포와 내독소 결정체 생산을 조사했다. 1. 4개의 M-배지중 pH 9인 M-3배지에서 BTT의 증식량과 내독소 결정체 및 아포 생산수가 가장 높았다. 150ml배양에서 BTT의 생산량은 3.218g이었고, 생존 아포수는 ml당 개였고, 내독소 결정체의 회수된 무게비는 20.05%였다. 2. M-1배지에서 BTT의 세대시간은 평균 47.6분이었고, M-2에서는 평균 49.6분, M-3에서는 평균 132.9분, M-4에서는 평균 110.2분이었다. 3. M-배지의 초기 pH는 72시간 배양 후에 pH 로 변화하였으며, 최적 배양 pH는 이었다.