Updated on 2021/12/16

写真a

 
MIYOSHI Shinichi
 
Organization
Medicine, Dentistry and Pharmaceutical Sciences Professor
Position
Professor
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Degree

  • 薬学修士 ( 岡山大学 )

  • Doctor of Philosophy ( Osaka University )

Research Interests

  • Protein toxin

  • Environmental microorganism

  • Protease

  • Bioremediation

  • プロテアーゼ

  • バイオレメディエーション

  • Vibrio

  • 蛋白質毒素

  • 環境微生物

  • ビブリオ

Research Areas

  • Life Science / Pharmaceutical hygiene and biochemistry

  • Life Science / Bacteriology

  • Life Science / Pharmaceutical hygiene and biochemistry

  • Life Science / Pharmacology

  • Life Science / Hygiene and public health (laboratory)

  • Life Science / Hygiene and public health (non-laboratory)

  • Life Science / Medical management and medical sociology

  • Life Science / Medical management and medical sociology

  • Life Science / Hygiene and public health (laboratory)

  • Life Science / Hygiene and public health (non-laboratory)

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Education

  • Okayama University    

    - 1985

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  • Okayama University   薬学研究科   製薬化学

    - 1985

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    Country: Japan

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  • Okayama University    

    - 1983

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  • Okayama University   薬学部   製薬化学

    - 1983

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    Country: Japan

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Research History

  • - 岡山大学医歯薬学総合研究科 教授

    2005

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  • - Professor,Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,Okayama University

    2005

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  • 国際協力事業団インド下痢症対策プロジェクト 短期専門家 未設定

    2002

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  • 米国コロラド州立大学 生化学・分子生物学科 博士研究員 未設定

    1998 - 1999

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  • 米国コロラド州立大学 生化学・分子生物学科 博士研究員 未設定

    1994 - 1995

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Professional Memberships

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Committee Memberships

  • 日本防菌防黴学会   防菌防黴誌編集委員  

    2011   

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    Committee type:Academic society

    日本防菌防黴学会

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  • 日本防菌防黴学会   理事  

    2011   

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    Committee type:Academic society

    日本防菌防黴学会

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  • 日本薬学会   衛生薬学教科担当教員会議委員  

    2010   

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    Committee type:Academic society

    日本薬学会

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  • 日本薬学会   日本薬学会学会賞第1次選考委員  

    2010   

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    Committee type:Academic society

    日本薬学会

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  • 日米医学協力研究会 コレラ・細菌性腸管感染症専門部会   研究員  

    2009   

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    Committee type:Academic society

    日米医学協力研究会 コレラ・細菌性腸管感染症専門部会

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  • 日本薬学会   日本薬学会第130年会組織委員  

    2009   

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    Committee type:Academic society

    日本薬学会

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  • 日本細菌学会   評議員  

    2008   

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    Committee type:Academic society

    日本細菌学会

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  • 日本細菌学会   広報委員会委員  

    2008   

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    Committee type:Academic society

    日本細菌学会

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  • 日本食品微生物学会   評議員  

    2008   

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    Committee type:Academic society

    日本食品微生物学会

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  • 日本薬学会   中国四国支部幹事  

    2007 - 2008   

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    Committee type:Academic society

    日本薬学会

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  • 日本薬学会   環境・衛生部会試験法委員会委員  

    2007   

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    Committee type:Academic society

    日本薬学会

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  • 日本薬学会   フォーラム2007 衛生薬学・環境トキシコロジー実行委員会委員  

    2007   

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    Committee type:Academic society

    日本薬学会

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  • 日本薬学会   代議員  

    2007   

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    Committee type:Academic society

    日本薬学会

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  • 日本薬学会   環境・衛生部会試験法委員会微生物試験法専門委員会委員長  

    2007   

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    Committee type:Academic society

    日本薬学会

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  • 日本薬学会   放射薬学教科担当教員会議委員  

    2006   

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    Committee type:Academic society

    日本薬学会

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  • 毒素シンポジウム   運営委員  

    2005 - 2007   

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    Committee type:Academic society

    毒素シンポジウム

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  • 日本防菌防黴学会   評議員  

    2005   

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    Committee type:Academic society

    日本防菌防黴学会

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  • 日本防菌防黴学会   Biocontrol Science編集委員  

    2005   

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    Committee type:Academic society

    日本防菌防黴学会

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  • 日本薬学会   環境・衛生部会試験法委員会微生物試験法専門委員会専門委員  

    2003   

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    Committee type:Academic society

    日本薬学会

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  • 日本薬学会   中国四国支部幹事  

    2001 - 2002   

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    Committee type:Academic society

    日本薬学会

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  • 日本細菌学会   中国四国支部評議員  

    1999   

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    Committee type:Academic society

    日本細菌学会

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Papers

  • Low Viability of Cholera Toxin-Producing Vibrio cholerae O1 in the Artificial Low Ionic Strength Aquatic Solution.

    Subha Sankar Paul, Eizo Takahashi, Goutam Chowdhury, Shin-Ichi Miyoshi, Tamaki Mizuno, Asish K Mukhopadhyay, Shanta Dutta, Keinosuke Okamoto

    Biological & pharmaceutical bulletin   43 ( 8 )   1288 - 1291   2020.8

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    It has been well known that Vibrio cholerae inhabit in environmental water. As many patients infected with cholera toxin-producing V. cholerae O1 (toxigenic V. cholerae O1) emerge in Kolkata, India, it has been thought that toxigenic V. cholerae O1 is easily detected in environmental water in Kolkata. However, we could not isolate toxigenic V. cholerae O1 from environmental water in Kolkata, though NAG Vibrio (generic name of V. cholerae non-O1/non-O139) is constantly detected. To clear the reason for the non-isolation of toxigenic V. cholerae O1, we examined the viability of V. cholera O1 and NAG Vibrios in the artificial low ionic strength aquatic solution. We found that the viability of toxigenic V. cholerae O1 in the solution is low, but that of NAG Vibrios is high. Subsequently, we examined the viability of NAG Vibrios possessing cholera toxin gene (ctx) in the same condition and found that the viability of these NAG Vibrios is low. These results indicate that the existence of ctx in V. cholerae affects the viability of V. cholerae in the aquatic solution used in this experiment. We thought that there was closely relation between the low viability of toxigenic V. cholerae O1 in the artificial low ionic strength aquatic solution and the low frequency of isolation of the strain from environmental water.

    DOI: 10.1248/bpb.b20-00350

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  • Genomic characterization of antibiotic resistance-encoding genes in clinical isolates of Vibrio cholerae non-O1/non-O139 strains from Kolkata, India: generation of novel types of genomic islands containing plural antibiotic resistance genes. International journal

    Daichi Morita, Eizo Takahashi, Masatomo Morita, Makoto Ohnishi, Tamaki Mizuno, Shin-Ichi Miyoshi, Devarati Dutta, Thandavarayan Ramamurthy, Goutam Chowdhury, Asish K Mukhopadhyay, Keinosuke Okamoto

    Microbiology and immunology   64 ( 6 )   435 - 444   2020.6

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    Non-O1/non-O139 nontoxigenic Vibrio cholerae associated with cholera-like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non-O1/non-O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole-genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance-determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non-O1/non-O139 strains through the action of newly generated genomic islands.

    DOI: 10.1111/1348-0421.12790

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  • Genomic and biological features of Plasmodium falciparum resistance against antimalarial endoperoxide N-89 Reviewed

    Masayuki Morita, Kosuke Hayashi, Akira Sato, Akiko Hiramoto, Osamu Kaneko, Rena Isogawa, Yuji Kurosaki, Shin-ichi Miyoshi*, Kyung-Soo Chang, Yusuke Wataya, Hye-Sook Kim

    Gene   716 ( x )   144016 - x   2019.10

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    DOI: 10.1016/j.gene.2019.144016

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  • Pharmacokinetic analysis of new synthetic antimalarial N-251 Reviewed

    Kazuaki Okada, Akira Sato, Akiko Hiramoto, Rena Isogawa, Yuji Kurosaki, Kazutaka Higaki, Shin-ichi Miyoshi*, Kyung-Soo Chang, Hye-Sook Kim

    Tropical Medicine and Health   47 ( x )   40 - x   2019.7

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    DOI: 10.1186/s41182-019-0167-4

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  • Comparative proteomic analysis to characterize temperature-induced viable but non-culturable and resuscitation states in Vibrio cholerae Reviewed

    Anusuya Debnath, Tamaki Mizuno, Shin-Ichi Miyoshi*

    Microbiology   165 ( 7 )   737 - 746   2019.5

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    DOI: 10.1099/mic.0.000798

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  • Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system Reviewed

    Tomoka Kawase, Fumi Miura, Anusuya Denbath, Kinuyo Imakura, Shin-Ichi Miyoshi*

    Protein expression and purification   149 ( x )   13 - 16   2018.9

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    DOI: 10.1016/j.pep.2018.04.004

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  • Antibacterial Activities of Surfactants in the Laundry Detergents and Isolation of the Surfactant Resistant Aquatic Bacteria

    Yoko Maehara, Shin-Ichi Miyoshi

    BIOCONTROL SCIENCE   22 ( 4 )   229 - 232   2017.12

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC ANTIBACTERIAL & ANTIFUNGAL AGENTS, JAPAN  

    Linear alkylbenzene sulfonate (LAS) and polyoxyethylene lauryl ether (POLE) are major surfactants contained in the laundry detergents. In the present study, the antibacterial activities of the surfactants to aquatic microorganisms were compared. When freshwater samples from a small river in Okayama city were treated with each of the surfactants, only LAS showed the significant antibacterial activity. Several strains, which survived after the treatment with 2.0% LAS, were isolated and identified by sequencing of 16S rDNA. All strains were classified into the family Enterobacteriaceae. However, this family was not a major member of the aquatic microflora, suggesting that the bacteria in Enterobacteriaceae have a common property of LAS-resistance in the river water.

    DOI: 10.4265/bio.22.229

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  • Comparative genome analysis of VSP-II and SNPs reveals heterogenic variation in contemporary strains of Vibrio cholerae O1 isolated from cholera patients in Kolkata, India

    Daisuke Imamura, Masatomo Morita, Tsuyoshi Sekizuka, Tamaki Mizuno, Taichiro Takemura, Tetsu Yamashiro, Goutam Chowdhury, Gururaja P. Pazhani, Asish K. Mukhopadhyay, Thandavarayan Ramamurthy, Shin-ichi Miyoshi, Makoto Kuroda, Sumio Shinoda, Makoto Ohnishi

    PLOS NEGLECTED TROPICAL DISEASES   11 ( 2 )   1600 - 1606   2017.2

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    Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epi-center for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years.

    DOI: 10.1371/journal.pntd.0005386

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  • Regulation systems of protease and hemolysin production in Vibrio vulnificus

    Abdelaziz Elgaml, Shin-ichi Miyoshi

    MICROBIOLOGY AND IMMUNOLOGY   61 ( 1 )   1 - 11   2017.1

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    Vibrio vulnificus is a gram-negative halophilic estuarine bacterium. It is an opportunistic human pathogen causing rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. A range of virulence factors have been proposed to play a role in pathogenesis of the bacterium during human and/or eel infection. Among these factors, the metalloprotease (V. vulnificus protease: VVP) and the cytolytic toxin (V. vulnificus hemolysin: VVH) are significantly important. VVP elicits the characteristic edematous and hemorrhagic skin damage. On the other hand, VVH exhibits powerful hemolytic and cytolytic activities and it also contributes to the bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels were recently found to produce a serine protease termed as V. vulnificus serine protease (VvsA) instead of VVP. Similar to VVP, VvsA may also possess various toxic activities such as collagenolytic, cytotoxic and edema-forming activity. In this review, we clarify the regulation of V. vulnificus VVP, VVH and VvsA in terms of expression at mRNA and protein level. The explanation is given on the basis of quorum sensing system, which is dependent on the bacterial cell density. In addition, we also outline the role of environmental factors and global regulators, such as histone-like nucleoid structuring protein, cyclic AMP receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation. Altogether, we compiled the cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus.

    DOI: 10.1111/1348-0421.12465

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  • 食品・医薬品・環境分野等の微生物試験法および微生物汚染の制御に関する最近の話題7 「衛生試験法・注解2015」収載微生物試験法

    三好伸一

    防菌防黴   45 ( 5 )   277 - 279   2017

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  • Regulation of Vibrio mimicus metalloprotease (VMP) production by the quorum-sensing master regulatory protein, LuxR

    El-Shaymaa Abdel-Sattar, Shin-ichi Miyoshi, Abdelaziz Elgaml

    JOURNAL OF BASIC MICROBIOLOGY   56 ( 10 )   1051 - +   2016.10

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    Vibrio mimicus is an estuarine bacterium, while it can cause severe diarrhea, wound infection, and otitis media in humans. This pathogen secretes a relatively important toxin named V. mimicus metalloprotease (VMP). In this study, we clarified regulation of the VMP production according to the quorum-sensing master regulatory protein named LuxR. First, the full length of luxR gene, encoding LuxR, was detected in V. mimicus strain E-37, an environmental isolate. Next, the putative consensus binding sequence of LuxR protein could be detected in the upstream (promoter) region of VMP encoding gene, vmp. Finally, the effect of disruption of luxR gene on the expression of vmp and production of VMP was evaluated. Namely, the expression of vmp was significantly diminished by luxR disruption and the production of VMP was severely altered. Taken together, here we report that VMP production is under the positive regulation of the quorum-sensing master regulatory protein, LuxR.

    DOI: 10.1002/jobm.201600002

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  • Indolo[3,2-b]quinoline Derivatives Suppressed the Hemolytic Activity of Beta-Pore Forming Toxins, Aerolysin-Like Hemolysin Produced by Aeromonas sobria and Alpha-Hemolysin Produced by Staphylococcus aureus

    Eizo Takahashi, Chiaki Fujinami, Teruo Kuroda, Yasuo Takeuchi, Shin-ichi Miyoshi, Sakae Arimoto, Tomoe Negishi, Keinosuke Okamoto

    BIOLOGICAL & PHARMACEUTICAL BULLETIN   39 ( 1 )   114 - 120   2016.1

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    In an attempt to discover inhibitory compounds against pore-forming toxins, some of the major toxins produced by bacteria, we herein examined the effects of four kinds of indolo[3,2-b]quinoline derivatives on hemolysis induced by the aerolysin-like hemolysin (ALH) of Aeromonas sobria and also by the alpha-hemolysin of Staphylococcus aureus. The results showed that hemolysis induced by ALH was significantly reduced by every derivative, while that induced by alpha-hemolysis was significantly reduced by three out of the four derivatives. However, the degrees of reduction induced by these derivatives were not uniform. Each derivative exhibited its own activity to inhibit the respective hemolysin. Compounds 1 and 2, which possessed the amino group bonding the naphthalene moiety at the C-11 position of indolo[3,2-b]quinoline, had strong inhibitory effects on the activity of ALH. Compound 4 which consisted of benzofuran and quinoline had strong inhibitory effects on the activity of alpha-hemolysin. These results indicated that the amino group bonding the naphthalene moiety of compounds 1 and 2 assisted in their ability to inhibit ALH activity, while the oxygen atom at the 10 position of compound 4 strengthened its interaction with alpha-hemolysin. These compounds also suppressed the hemolytic activity of the supernatant of A. sobria or A. hydrophila, suggesting that these compounds were effective at the site of infection of these bacteria.

    DOI: 10.1248/bpb.b15-00708

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  • Role of the Histone-Like Nucleoid Structuring Protein (H-NS) in the Regulation of Virulence Factor Expression and Stress Response in Vibrio vulnificus

    Abdelaziz Elgaml, Shin-Ichi Miyoshi

    BIOCONTROL SCIENCE   20 ( 4 )   263 - 274   2015.12

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC ANTIBACTERIAL & ANTIFUNGAL AGENTS, JAPAN  

    Temperature is one of the important parameters regulating the expression of virulence factors in bacteria. The global regulator, a histone-like nucleoid structuring protein (H-NS), is known to play a crucial role in this regulation. In the present study, we first clarified the role of H-NS in the temperature-dependent regulation of virulence factor production in Vibrio vulnificus, including that of the cytolytic toxin (V vulnificus hemolysin: VVH) and the proteolytic enzyme (V. vulnificus protease: VVP). The expression of hns itself was subjected to temperature regulation, where hns was expressed more at 26 degrees C than at 37 degrees C. VVH production and the expression of its gene vvhA were increased by disruption of the hns gene. H-NS appeared to affect the vvhA expression by the well-documented transcriptional silencing mechanism. On the other hand, hns disruption resulted in the reduction of VVP production and the expression of its gene vvpE. H-NS was suggested to positively regulate vvpE expression through the increase in the level of the rpoS mRNA. Moreover, H-NS was found to contribute to the survival of V vulnificus in stressful environments. When compared to the wild type strain, the hns mutant exhibited reduced survival rates when subjected to acidic pH, hyperosmotic and oxidative stress.

    DOI: 10.4265/bio.20.263

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  • Presence of Nitric Oxide-Sensing Systems in the Human Pathogen Vibrio vulnificus

    Abdelaziz Elgaml, Shin-ichi Miyoshi

    BIOCONTROL SCIENCE   20 ( 3 )   199 - 203   2015.9

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC ANTIBACTERIAL & ANTIFUNGAL AGENTS, JAPAN  

    Vibrio vulnificus is a halophilic estuarine bacterium, but this species causes fatal septicemia in humans. V. vulnificus may encounter many kinds of stresses either in the natural environment or in the human body. One of the striking stresses is the exposure to the reactive oxygen species including nitric oxide (NO). The present study revealed that NO could participate in the regulation of the V. vulnificus community behavior. When the bacterium was cultivated in the presence of sub-lethal doses of an NO donor, the expression of the genes encoding NO-detoxifying enzymes was significantly increased. The NO donor was also found to cause significant increase in production of a metalloprotease, a putative virulence factor, by the bacterium.

    DOI: 10.4265/bio.20.199

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  • Development of a real-time loop-mediated isothermal amplification assay for the sensitive and rapid detection of Listeria monocytogenes

    L. Ye, Y. Li, J. Zhao, Z. Zhang, H. Meng, H. Yan, S. -i. Miyoshi, L. Shi

    LETTERS IN APPLIED MICROBIOLOGY   61 ( 1 )   85 - 90   2015.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    A real-time loop-mediated isothermal amplification (RealAmp) assay for the detection of Listeria was developed. The RealAmp assay, using primers specific for the hemolysin-encoding hlyA gene, was verified using Listeria monocytogenes strains (n=58) from different regions of the world. Both the sensitivity and specificity of the RealAmp assay were high. The RealAmp assay could detect 10(3)CFUml(-1) within 30min. A comparative evaluation of the RealAmp assay, the API Listeria assay, and the real-time PCR assay revealed that the RealAmp assay is simpler, faster, and has a higher specificity than the other two assays.
    Significance and Impact of the StudyConventional culture and molecular detection methods are always time consuming and require a specific laboratory infrastructure, thereby restricting their use for the rapid detection and diagnosis of pathogens. A real-time loop-mediated isothermal amplification (RealAmp) assay performed by ESEtube scanner to rapidly detect Listeria monocytogenes isolated from food was developed. The results showed that the RealAmp assay using the tube scanner was more efficient and precise than the conventional API Listeria assay and the real-time PCR assay.

    DOI: 10.1111/lam.12429

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  • International Collaborative Research on Infectious Diseases by Japanese Universities and Institutes in Asia and Africa, with a Special Emphasis on J-GRID

    Sumio Shinoda, Daisuke Imamura, Tamaki Mizuno, Shin-Ichi Miyoshi, Thandavrayan Ramamurthy

    BIOCONTROL SCIENCE   20 ( 2 )   77 - 89   2015.6

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    In developed countries including Japan, malignant tumor (cancer), heart disease and cerebral apoplexy are major causes of death, but infectious diseases are still responsible for a high number of deaths in developing countries, especially among children aged less than 5 years. World Health Statistics published by WHO reports a high percentage of mortality from infectious diseases in children, and many of these diseases may be subject to transmission across borders and could possibly invade Japan.
    Given this situation, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan initiated Phase I of the Program of Founding Research Centers for Emerging and Reemerging Infectious Disease, which ran from FY 2005 to 2009, and involved 8 Japanese universities and 2 research centers. The program was established for the following purposes: 1) creation of a domestic research structure to promote the accumulation of fundamental knowledge about infectious diseases, 2) establishment of 13 overseas research collaboration centers in 8 countries at high risk of emerging and reemerging infections and at which Japanese researchers are stationed and conduct research in partnership with overseas instructors, 3) development of a network among domestic and overseas research centers, and 4) development of human resources.
    The program was controlled under MEXT and managed by the RIKEN Center of Research Network for Infectious Diseases (Riken CRNID). Phase II of the program was set up as the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), and has been running in FY 2010-2014.
    Phase III will start in April 2015, and will be organized by the newly established Japanese governmental organization "Japan Agency for Medical Research and Development (AMED)'', the so-called Japanese style NIH.
    The Collaborative Research Center of Okayama University for Infectious Diseases in India (CRCOUI) was started up in 2007 at the National Institute of Cholera and Enteric Disease, Kolkata, India. Major projects of CRCOUI are concerned with diarrheal diseases such as, 1) active surveillance of diarrhea! patients, 2) development of dysentery vaccines, 3) viable but nonculturable (VBNC) Vibrio cholerae, and 4) pathogenic mechanisms of various diarrhogenic microorganisms.
    This review article outlines project of J-GRID and CRCOUI which the authors carried out collaboratively with NICED staff members.

    DOI: 10.4265/bio.20.77

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  • Stepwise changes in viable but nonculturable Vibrio cholerae cells

    Daisuke Imamura, Tamaki Mizuno, Shin-ichi Miyoshi, Sumio Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   59 ( 5 )   305 - 310   2015.5

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    Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC-state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT-29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. These characteristic changes in VBNC-state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.

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  • インドにおける感染症研究の連携:岡山大学インド感染症共同研究センターとコレラおよび腸管感染症研究所 (NICED)

    篠田 純男, 今村 大輔, 水野 環, 三好 伸一

    最新医学   70 ( 4 )   738 - 744   2015

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  • Defensive Effects of Human Intestinal Antimicrobial Peptides against Infectious Diseases Caused by Vibrio mimicus and V. vulnificus

    Shin-Ichi Miyoshi, Hiroto Ikehara, Mika Kumagai, Tamaki Mizuno, Tomoka Kawase, Yoko Maehara

    BIOCONTROL SCIENCE   19 ( 4 )   199 - 203   2014.12

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    Of human pathogenic Vibrio species, V mimicus causes gastroenteritis whereas V vulnificus causes fatal septicemia after consumption of contaminated seafood. These two pathogens produce hemolytic toxins termed V mimicus hemolysin (VMH) and V vulnificus hemolysin (VVH), respectively. These toxins elicit the cytolysis of various eukaryotic cells, as well as erythrocytes. The human intestine secretes cationic antimicrobial peptides (AMPs) to prevent infectious diseases. Paneth cells in the small intestine secrete a-defensin 5 (HD-5) and epithelial cells in the large intestine produce LL-37. In the present study, we examined the bactericidal activities of AMPs against V mimicus and V vulnificus. Although HD-5 showed no bactericidal activity, LL-37 revealed significant activity against both Vibrio species, suggesting that neither V mimicus nor V vulnificus can multiply in the large intestine. We also tested whether AMPs had the ability to inactivate the hemolytic toxins. Only HD-5 was found to inactivate VMH, but not VVH, in a dose-dependent manner through the direct binding to VMH. Therefore, it is considered that V mimicus cannot penetrate the small intestinal epithelium because the cytolytic action of VMH is inactivated by HD-5.

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  • A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin

    Tamaki Mizuno, Ayako Nanko, Yoko Maehara, Sumio Shinoda, Shin-Ichi Miyoshi

    MICROBIOLOGY AND IMMUNOLOGY   58 ( 9 )   503 - 512   2014.9

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    Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N-terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin-like substrate specificity was purified from the bacterial culture supernatant. The N-terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg(151)Ser(152) bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.

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  • Evaluation of real-time loop-mediated isothermal amplification (RealAmp) for rapid detection of Mycobacterium tuberculosis from sputum samples

    Yiming Li, Lei Shi, Anqi Pan, Weiwei Cao, Xun Chen, Hecheng Meng, He Yan, Shin-ichi Miyoshi, Lei Ye

    JOURNAL OF MICROBIOLOGICAL METHODS   104   55 - 58   2014.9

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    Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) leads to serious health problems as a chronic respiratory infectious disease. Here we established a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) using a portable ESE Quant tube scanner as a convenient rapid detection method for MTB. The method efficacy from sputum samples was further investigated, and the reaction time was only 20 min with the detection limit low to 10(2) CPU/ml concentration of MTB. We assessed a total of 1067 samples by the RealAmp assay, comparing the results with smear microscopy and conventional culture methods. To examine whether the failure to detect TB by culturing is due to low sensitivity or true absence, we examined the culture negative samples by commercial real time PCR MTB detection kit, and the results were compared with RealAmp. The data showed that RealAmp assay had a higher positive rate than that of sputum smear and culture methods. RealAmp had a sensitivity of 96.70% and a specificity of 91.55% when compared with culture. In addition, its sensitivity and specificity were 95.29% and 86.88% respectively compared with examination of smear samples using light microscopy. The sensitivity of RealAmp in comparison to real time PCR was 98.25% and specificity was 99.11% in validation of culture negative samples. The present study revealed the newly established RealAmp assay as a convenient, efficient, sensitive and specific method that could be an alternative for rapid detection of MTB and a tool to validate culture and smear negative samples. Furthermore, the portability of the ESE Quant tube scanner also contributed to the promising application for grassroots and field detection of MTB. (C) 2014 Elsevier B.V. All rights reserved.

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  • Isolation of viable but nonculturable Vibrio cholerae O1 from environmental water samples in Kolkata, India, in a culturable state

    Mitsutoshi Senoh, Jayeeta Ghosh-Banerjee, Tamaki Mizuno, Sumio Shinoda, Shin-ichi Miyoshi, Takashi Hamabata, G. Balakrish Nair, Yoshifumi Takeda

    MICROBIOLOGYOPEN   3 ( 2 )   239 - 246   2014.4

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    Previously, we reported that viable but nonculturable (VBNC) Vibrio cholerae was converted into a culturable state by coculture with several eukaryotic cell lines including HT-29 cells. In this study, we found that a factor converting VBNC V. cholerae into a culturable state (FCVC) existed in cell extracts of eukaryotic cells. FCVC was nondialyzable, proteinase K-sensitive, and stable to heating at <60 degrees C for 5 min. We prepared thiosulfate citrate bile salts sucrose (TCBS) plates with FCVC (F-TCBS plates). After confirming that VBNC V. cholerae O1 and O139 formed typical yellow colonies on F-TCBS plates, we tried to isolate cholera toxin gene-positive VBNC V. cholerae from environmental water samples collected in urban slum areas of Kolkata, India and succeeded in isolating V. cholerae O1 El Tor variant strains harboring a gene for the cholera toxin. The possible importance of VBNC V. cholerae O1 as a source of cholera outbreaks is discussed.

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  • Effects of temperature, growth phase and luxO-disruption on regulation systems of toxin production in Vibrio vulnificus strain L-180, a human clinical isolate

    Abdelaziz Elgaml, Kazutaka Higaki, Shin-ichi Miyoshi

    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY   30 ( 2 )   681 - 691   2014.2

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    Vibrio vulnificus is a halophilic estuarine bacterium while it causes fatal septicemia or necrotizing wound infections in humans. This pathogen secretes the metalloprotease (V. vulnificus protease: VVP) and the cytolysin (V. vulnificus hemolysin: VVH) as protein toxins; however, their production was coordinated in response to the bacterial cell density. This regulation is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). In the present study, we investigated effects of disruption of luxO encoding a central response regulator of the QS circuit, as well as effects of temperature and growth phase, on the toxin production by V. vulnificus. Disruption of luxO was found to increase VVP production and expression of its gene vvpE. The expression of smcR, crp and rpoS, of which products positively regulate vvpE expression, and luxS encoding the AI-2 synthetase were also significantly increased. On the other hand, the luxO disruption resulted in reduction of VVH production and expression of its gene vvhA. Expression of other two genes affecting the QS circuit, luxT and rpoN, were also significantly decreased. The regulation systems of VVP production were found to exert their action during the stationary phase of the bacterial growth and to be operated strongly at 26 A degrees C. By contrast, those of VVH production apparently started at the log phase and were operated more effectively at 37 A degrees C.

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  • Activity of Collaborative Research Center of Okayama University for Infectious Disease in India

    Shinoda S, Imamura D, Mizuno T, Miyoshi S

    J Disast Res   9 ( 5 )   774 - 783   2014

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  • Regulation system of serine protease production in Vibrio vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel

    Abdelaziz Elgaml, Kazutaka Higaki, Shin-ichi Miyoshi

    AQUACULTURE   416   315 - 321   2013.12

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    Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in cultured eels or shrimps, as well as in immunocompromised humans. An extracellular metalloprotease has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA. In the present study, we first clarified the regulatory characteristics of the VvsA production in V. vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel. V. vulnificus coordinates expression of virulence genes in response to the bacterial cell density, which is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). When cultivated at 26 degrees C, the vvsA expression was closely related with expression of the luxS gene encoding the synthase of the AI-2 precursor LuxS. Both VvsA and AI-2 were sufficiently secreted at early stationary phase of the bacterial growth. In contrast, when cultivated at 37 degrees C, far less amounts of the AI-2 and VvsA were produced even at the stationary phase. Disruption of the luxS gene was found to decrease significantly the vvsA expression and VvsA production. Disruption of luxO encoding the central response regulator of the QS circuit caused an increase in the vvsA expression and VvsA production at the logarithmic growth phase. These findings indicate that VvsA production is positively regulated by the V. vulnificus LuxS-dependent QS system, which operated more effectively at 26 degrees C than at 37 degrees C. (C) 2013 Elsevier B.V. All rights reserved.

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  • Extracellular proteolytic enzymes produced by human pathogenic Vibrio species

    Shin-ichi Miyoshi

    FRONTIERS IN MICROBIOLOGY   4   1 - 8   2013.11

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    Bacteria in the genus Vibrio produce extracellular proteolytic enzymes to obtain nutrients via digestion of various protein substrates. However, the enzymes secreted by human pathogenic species have been documented to modulate the bacterial virulence. Several species including Vibrio cholerae and V vulnificus are known to produce thermolysin-like metalloproteases termed vibriolysin. The vibriolysin from V vulnificus, a causative agent of serious systemic infection, is a major toxic factor eliciting the secondary skin damage characterized by formation of the hemorrhagic brae. The vibriolysin from intestinal pathogens may play indirect roles in pathogenicity because it can activate protein toxins and hemagglutinin by the limited proteolysis and can affect the bacterial attachment to or detachment from the intestinal surface by degradation of the mucus layer. Two species causing wound infections, V alginolyticus and V parahaemolyticus, produce another metalloproteases so-called collagenases. Although the detailed pathological roles have not been studied, the collagenase is potent to accelerate the bacterial dissemination through digestion of the protein components of the extracellular matrix. Some species produce cymotrypsin-like serine proteases, which may also affect the bacterial virulence potential. The intestinal pathogens produce sufficient amounts of the metalloprotease at the small intestinal temperature; however, the metalloprotease production by extra-intestinal pathogens is much higher around the body surface temperature. On the other hand, the serine protease is expressed only in the absence of the metalloprotease.

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  • Ecological Study of Pathogenic Vibrios in Aquatic Environments

    Sumio Shinoda, Yuki Furumai, Sei-Ichi Katayama, Tamaki Mizuno, Shin-Ichi Miyoshi

    BIOCONTROL SCIENCE   18 ( 1 )   53 - 58   2013.3

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    An ecological study of pathogenic vibrios in aquatic environments of Okayama was carried out. The number of Vibrio parahaemolyticus detected in the sea area was comparatively smaler than that found in the survey of about two decades ago. Various reasons for the decrease in the case of food poisoning by V. parahaemolyticus have been suggested but the lower number of the vibrio in aquatic environments may be one explanation. Although the number of V. vulnificus was also not as large, most of the isolates possessed the pathogenic genes, vvp and vvh, suggesting the potential for fatal pathogenicity to patients having underlying diseases. As for V. cholerae, some non-O1/non-O139 serovar isolates were detected in a fresh water area, and many of them had hlyA, the gene for hemolysin which acts as a pathogenic factor in sporadic cases of diarrhea. Thus, the total number of pathogenic vibrios detected was not of concern. However, the marine products of these areas are shipped in wide area and are for general consumption. Therefore, it is necessary to continue to survey pathogenic vibrios in aquatic environments in order to ensure food hygiene.

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  • ビブリオ・バルニフィカス感染

    三好伸一

    化学療法の領域   29 ( 7 )   1454 - 1459   2013

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  • Vibriolysin

    Shin-ichi Miyoshi, Keinosuke Okamoto, Eizo Takahashi

    Handbook of Proteolytic Enzymes (Third Ed)   3   579 - 582   2013

  • Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells

    Mitsutoshi Senoh, Jayeeta Ghosh-Banerjee, Thandavarayan Ramamurthy, Rita R. Colwell, Shin-ichi Miyoshi, G. Balakrish Nair, Yoshifumi Takeda

    MICROBIOLOGY AND IMMUNOLOGY   56 ( 5 )   342 - 345   2012.5

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    Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells.

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  • Possible variation of the human oral bacterial community after wearing removable partial dentures by DGGE

    Xiao Zhu, Shaohai Wang, Yihai Gu, Xiaoyu Li, Hui Yan, He Yan, Shin-ichi Miyoshi, Lei Shi

    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY   28 ( 5 )   2229 - 2236   2012.5

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    Although it is well-known that variations of the microbial community in a specific location of human body may be associated with some diseases, the developing change of the oral microbiota related to oral diseases before and after wearing the removable partial dentures (RPD) is not completely understood. In this study, three kinds of samples (saliva, supra- and subgingival plaque, and oral mucosal surfaces) were collected from the 10-patients group at three different times: before, 1-month and 6-months after the treatment. Ten healthy adults were also selected as the control group. Denaturing gradient gel electrophoresis was applied to identify the bacterial profiles and to analyze the dynamics of the oral microbial population in the pre- and post-therapy. The ANOVA of Repeated Measurement Data indicated that, in the saliva and mucosal surfaces, wearing RPDs caused significant change of numbers of amplicons. As many as 607 amplicons were chosen to cut out and re-amplify by PCR. After cloning and sequencing, a total of 16 bacterial genera were identified. The health-associated genera such as Streptococcus, Neisseria, Rothia, Corynebacterium, Leptotrichia, Gemella, Veillonella, Selenomona and Actinomyces tended to decrease, whereas the disease-associated species including Streptococcus mutans tended to increase. In general, wearing RPDs influenced the diversity of the bacterial species in the oral microbial ecosystem. It is noteworthy that the oral environment will be changed from the healthy status towards the disease status after the treatment.

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  • An extracellular serine protease produced by Vibrio vulnificus NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel

    Shin-ichi Miyoshi, Jiyou Wang, Keizo Katoh, Mitsutoshi Senoh, Tamaki Mizuno, Yoko Maehara

    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY   28 ( 4 )   1633 - 1639   2012.4

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    Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in immunocompromised humans, cultured eels or shrimps. An extracellular metalloprotease VVP/VvpE has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA, but not VVP/VvpE. In the present study, we found that these strains had lost the 80 kb genomic region including the gene encoding VVP/VvpE. We also purified VvsA from the culture supernatant through ammonium sulfate fractionation, gel filtration and ion-exchange column chromatography, and the enzyme was demonstrated to be a chymotrypsin-like protease, as well as those from some vibrios. The gene vvsA was shown to constitute an operon with a downstream gene vvsB, and several Vibrio species were found to have orthologues of vvsAB. These findings indicate that the genes vvp/vvpE and vvsAB might be mobile genetic elements.

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  • Development of a sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli

    Takashi Kurakawa, Hiroyuki Kubota, Hirokazu Tsuji, Kazunori Matsuda, Takashi Asahara, Takuya Takahashi, Thandavarayan Ramamurthy, Takashi Hamabata, Eizo Takahashi, Shin-ichi Miyoshi, Keinosuke Okamoto, Asish K. Mukhopadhyay, Yoshifumi Takeda, Koji Nomoto

    MICROBIOLOGY AND IMMUNOLOGY   56 ( 1 )   10 - 20   2012.1

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    A sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was developed for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli by using specific primers. Counts of the enteric pathogens spiked in human stools were quantified at the lower detection limit of 103 cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 105 to 106 cells/g stool. The bacterial counts determined by RT-qPCR were almost equivalent to those determined by the culture method and fluorescence in situ hybridization (FISH) during the course of in vitro culture. Bacterial rRNA in the stools was stable for at least 4 weeks when the stools were kept as the suspensions in RNA-stabilizing agent, RNAlater (R), even at 37oC. These data suggested that the rapid and high sensitive rRNA-targeted RT-qPCR was applicable for the accurate quantification of viable enteric pathogens, such as V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli.

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  • Characterization and horizontal transfer of class 1 integrons in Salmonella strains isolated from food products of animal origin

    Hecheng Meng, Zhigang Zhang, Miaorui Chen, Yongyu Su, Lin Li, Shin-ichi Miyoshi, He Yan, Lei Shi

    INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY   149 ( 3 )   274 - 277   2011.10

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    A total of 81 Salmonella isolates from retail meats and seafood in Hebei province, China, were assayed for the presence of and horizontal transfer of class 1 integrons. By the PCR screening for the integrons, class 1 integron was detected from strains in serotypes of Derby, Indiana, London and Choleraesuis, which were isolated from pork, chicken or seafood: however, two isolates contained the empty integron that lacked the resistance cassette, a potential hotspot for development of the multidrug resistance. In contrast, two other isolates had the antibiotic resistance gene cassettes within the class 1 integron, which were dfrA1-aadA1 and aadB-cmlA, respectively. The conjugation experiments demonstrated the plasmid-mediated transfer of the class 1 integrons. Furthermore, each of the integrons was transmitted to Streptococcus mutans via natural gene transformation. These findings suggest the possible transfer of class 1 integrons from foodborne pathogens to human residential bacteria via horizontal gene transfer. (C) 2011 Elsevier B.V. All rights reserved.

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  • Inactivation of Vibrio vulnificus hemolysin through mutation of the N- or C-terminus of the lectin-like domain

    Shin-ichi Miyoshi, Yuki Abe, Mitsutoshi Senoh, Tamaki Mizuno, Yoko Maehara, Hiroshi Nakao

    TOXICON   57 ( 6 )   904 - 908   2011.5

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    Vibrio vulnificus is an etiological agent causing serious systemic infections in the immu-nocompromised humans or cultured eels. This species commonly produces a hemolytic toxin consisting of the cytolysin domain and the lectin-like domain. For hemolysis, the lectin-like domain specifically binds to cholesterol in the erythrocyte membrane, and to form a hollow oligomer, the toxin is subsequently assembled on the membrane. The cytolysin domain is essential for the process to form the oligomer. Three-dimensional structure model revealed that two domains connected linearly and the C-terminus was located near to the joint of the domains. Insertion of amino acid residues between two domains was found to cause inactivation of the toxin. In the C-terminus, deletion, substitution or addition of an amino acid residue also elicited reduction of the activity. However, the cholesterol-binding ability was not affected by the mutations. These results suggest that mutation of the C- or N-terminus of the lectin-like domain may result in blockage of the toxin assembly. (C) 2011 Elsevier Ltd. All rights reserved.

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  • Proteases Produced by Vibrios

    Sumio Shinoda, Shin-Ichi Miyoshi

    BIOCONTROL SCIENCE   16 ( 1 )   1 - 11   2011.3

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    Bacteria of the genus Vibrio are normal habitants of the aquatic environment but the some species are believed to be human pathogens. Pathogenic vibrios produce various pathogenic factors, and the proteases are also recognized to play pathogenic roles in the infection: the direct: roles by digesting many kinds of host proteins or indirect roles by processing other pathogenic protein factors. Especially VVP from Vibrio vulnificus is thought to be a major pathogenic factor of the vibrio. Although HA/P, the V. cholerae hemagglutinin/protease, is not a direct toxic factor of cholera vibrio, its significance is an undeniable fact. Production of HA/P is regulated together with major pathogenic factors such as CT (cholera toxin) or TCP (toxin co-regulated pilus) by a quorum-sensing system. HA/P is necessary for full expression of pathogenicity of the vibrio by supporting growth and translocation in the digestive tract. Processing of protein toxins such as CT or El Tor hemolysin is also an important pathogenic role.

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  • Defensive Effects of Human Antimicrobial Peptide alpha-Defensins against Enterococcus faecalis

    Shin-ichi Miyoshi, Kenta Koyama, Tamaki Mizuno, Minoru Kashihara, Yoko Maehara, Hiroshi Nakao

    JOURNAL OF HEALTH SCIENCE   56 ( 5 )   618 - 622   2010.10

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    Cationic and amphiphilic antimicrobial peptides (AMPs) such as alpha-defensins and cathelicidins are factors related to innate immunity. In the present study, we examined the protective effects of two AMPs, human neutrophil peptide-3 and alpha-defensin-5, against the opportunistic pathogen Enterococcus faecalis (E. faecalis). The alpha-defensins had dose-dependent bactericidal activity, whereas they showed no synergistic effect on the antimicrobial actions of antibiotics. Although AMPs often neutralize bacterial bioactive products, neither alpha-defensin reduced the proteolytic activity of GelE, a toxic protease from E. faecalis. On the other hand, the alpha-defensins were found to be fairly stable even in the presence of excess amounts of GelE. These results indicate that alpha-defensins may be defensive factors against E. faecalis in humans.

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  • Prevalence and antimicrobial resistance of Salmonella in retail foods in northern China

    He Yan, Lin Li, M. Jahangir Alam, Sumio Shinoda, Shin-ichi Miyoshi, Lei Shi

    INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY   143 ( 3 )   230 - 234   2010.10

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    A total of 387 retail meat, seafood and milk powder samples were collected from nine cities in northern China in 2005 and screened for the presence of Salmonella. Salmonella strains isolated were subjected to serotyping and antimicrobial susceptibility testing. Salmonella was isolated from 81 (20.9%, 81/387) samples and classified into 23 serotypes. The isolates were frequently resistant to sulfamethoxazole (86.4%), sulfamethoxazole/trimethoprim (48.1%), nalidixic acid (30.9%). tetracycline (19.8%), carboxybenzylpenicillin (17.3%), amoxicillin (17.3%) and ampicillin (16.0%). The multiple resistance (resistance to >= 3 antibiotics) was found in 29.6% (n = 24) isolates. Additionally, 4 isolates from chicken displayed the ACSSuTNx profile, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline and nalidixic acid, in particular, strain HBS084 showing the resistance to as many as 20 antibiotics. Salmonella from chicken showed the higher frequency of antimicrobial resistance. Our findings indicate that in northern China food products of animal origin can be a source of exposure for consumers to multiresistant Salmonella strains. (c) 2010 Elsevier B.V. All rights reserved.

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  • Assimilation of Metal Ions Bound to Porphyrins or Porphyrin-Peptides by Vibrio vulnificus, a Human Pathogen Inhabiting Estuarine and Marine Environments

    Shin-Ichi Miyoshi, Tomoko Sasaki, Nahoko Kaku, Takaharu Inoue, Natsuki Uozumi, Yoko Maehara, Hiroshi Nakao

    BIOCONTROL SCIENCE   15 ( 1 )   1 - 6   2010.3

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    Vibrio vulnificus, a ubiquitous microorganism in aquatic environments, causes serious septicemia to the immunocompromised host. In addition to protoheme, this species can utilize Fe-TCPP [ferric tetrakis (4-carboxyphenyl) porphine] as an iron source. In the present study, heme c bound covalently to the protein in cytochrome c, as well as the Fe-TCPP complex formed with a nanopeptide with a high affinity, was found to be useful iron sources for V. vulnificus. This bacterium was also revealed to use Zn-TCPP as a single zinc source. However, other metalloporphyrins such as Mn-TCPP and Pt-TCPP delayed the bacterial growth in the broth containing Fe-TCPP, suggesting interference in the iron assimilation. These results indicate that V. vulnificus may acquire metal ions from both free and peptide-bound metalloporphyrins.

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  • Specific detection of viable Salmonella cells by an ethidium monoazide-loop mediated isothermal amplification (EMA-LAMP) method Reviewed

    Lu Y, Yang W, Shi L, Li I, Alam MJ, Guo S and *Miyoshi S

    Journal of Health Science   54 ( 6 )   686 - 691   2009.12

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  • Specific detection of viable Salmonella cells by an ethidium monoazide-loop mediated isothermal amplification (EMA-LAMP) method

    Yuxia Lu, Weiqing Yang, Lei Shi, N. Li, Muhammad Jahangir Alam, Siyuan Guo, Shin-Ichi Miyoshi

    Journal of Health Science   55 ( 5 )   820 - 824   2009.10

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    The persistence of DNA after the cell death causes a major issue in aspects of medical or biological studies. The signal from viable bacterial cells cannot be distinguished from the dead cells in the conventional DNA-based detection methods. In the present study, the loop-mediated isothermal amplification (LAMP) method combined with the ethidium monoazide (EMA) treatment was applied for specific detection of viable, but not dead, Salmonella cells. For this method (EMA-LAMP), we designed a series of primers, which recognize six distinct sequences of the target invA gene conserved in Salmonella. The invA gene of the viable cells was remarkably amplified within 1 hr when as small amounts as 100 fg of DNA was subjected to EMA-LAMP. Because EMA selectively penetrated into the dead cells and bound covalently to DNA, the gene of the dead cells could not be amplified. This study offers a novel DNA-based method to distinguish the viable bacterial cells from the dead cells. ©2009 The Pharmaceutical Society of Japan.

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  • Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease

    Tamaki Mizuno, Syed Z. Sultan, Yoshimi Kaneko, Tomonaga Yoshimura, Yoko Maehara, Hiroshi Nakao, Tomofusa Tsuchiya, Sumio Shinoda, Shin-ichi Miyoshi

    FEBS JOURNAL   276 ( 3 )   825 - 834   2009.2

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    Vibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is secreted as an 80 kDa precursor, which is later converted to the 66 kDa mature toxin through removal of an N-terminal propeptide via cleavage of the Arg151-Ser152 bond. In this article, we investigate the role of the endogenous metalloprotease (V. mimicus protease) in the maturation of V. mimicus hemolysin. In vitro experiments using purified proteins showed that, although it activated the precursor at the early stage via cleavage of the Asn157-Val158 bond, V. mimicus protease finally converted the activated and physiologically maturated toxin to a 51 kDa protein through removal of the C-terminal polypeptide. This 51 kDa derivative was unable to lyse erythrocytes because of its inability to bind to the erythrocyte membrane. Vibrio mimicus protease-negative strains were found to produce high levels of V. mimicus hemolysin at the logarithmic phase of bacterial growth and maintained high hemolytic activity even at the stationary phase. These findings indicate that, although it is not directly related to toxin maturation in vivo, V. mimicus protease can modulate the activity of V. mimicus hemolysin and/or its precursor through limited proteolysis.

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  • Role of the Enterotoxic Hemolysin in Pathogenicity of Vibrio mimicus

    Tho Li, Akiko Kobayashi, Noriko Takata, Tomonaga Yoshimura, Yoko Maehara, Tomofusa Tsuchiya, Shin-ichi Miyoshi

    JOURNAL OF HEALTH SCIENCE   54 ( 6 )   686 - 691   2008.12

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    Vibrio mimicus (V mimicus) is a causative agent of human gastroenteritis and food poisoning. Although several toxic or virulence factors have been isolated from the bacterium, an enterotoxic hemolysin is a sole toxin produced by all clinical isolates. In the present study, we found that the antibody against the hemolysin significantly inhibited the fluid-accumulating action of the living cells inoculated into a rabbit ileal loop, and that the hemolysin gene (vmhA) was probably expressed by the bacterium in the ileal loop. Additionally, in spit of the comparable motility and similar proteome profiles, a vmhA mutant revealed the reduced fluid-accumulating activity. Theses findings suggest that the hemolysin contributes to full virulence of V. mimicus.

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  • Differential gene expression and extracellular secretion of the collagenolytic enzymes by the pathogen Vibrio parahaemolyticus

    Shin-ichi Miyoshi, Yuko Nitanda, Kaori Fujii, Kiyomi Kawahara, Tao Li, Yoko Maehara, Thandavarayan Ramamurthy, Yoshifumi Takeda, Sumio Shinoda

    FEMS MICROBIOLOGY LETTERS   283 ( 2 )   176 - 181   2008.6

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    Vibrio parahaemolyticus, a causative agent of wound infections as well as food poisoning, harbors two collagenase genes: vppC and prtV. When cultivated at 26 degrees C in gelatin broth supplemented with 3.0% NaCl, significant collagenolytic activity was detected in the culture supernatant at the early stationary phase. Native polyacrylamide gel electrophoresis analysis revealed a 90-kDa protein, and N-terminal amino acid sequencing showed that this protein was VppC, generated through truncation of 72 N-terminal amino acid residues. Additionally, significant expression of only vppC was observed by reverse transcriptase PCR. By contrast, a vppC-negative mutant constructed through single crossover homologous recombination secreted a 50-kDa-collagenolytic enzyme; however, this enzyme was a serine protease that was reported previously. These results suggest that VppC is a primary extracellular collagenase produced by V. parahaemolyticus.

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  • Variation of extracellular proteases produced by Vibrio vulnificus clinical isolates: Genetic diversity of the metalloprotease gene (vvp), and serine protease secretion by vvp-negative strains

    Jiyou Wang, Tomoko Sasaki, Yoko Maehara, Hiroshi Nakao, Tomofusa Tsuchiya, Shin-ichi Miyoshi

    MICROBIAL PATHOGENESIS   44 ( 6 )   494 - 500   2008.6

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    Vibrio vulnificus is a causative agent of septicemia or wound infection in human and eel; however. the genetic variation between human and eel isolates has been reported. In the present study, the difference in the vvp gene encoding a tissue-damaging metalloprotease Was (type B vvp) was 95.2% identical with that of strain L-180 from human blood investigated. The acne of strain E86 from a diseased eel (type A vvp). PCR using oligonucleotide primers designed to differentiate two types of the acne showed that eel avirulent strains (9 isolates) commonly carry type A ut;p. whereas eel virulent strains (18 isolates) revealed significant genetic variation. The vvp genes From strains including strain E86 were placed on type B while those from 3 strains, were on type A. other strains were found to he negative but analysis Showed that they secreted a serine protease (VVA0302) instead of the negative, but PAGE and amino acid sequencing metalloprotease. This protease is all orthologue of a toxic protease from Vibrio parahaemolyticus a human pathogen causing wound infection as well Lis gastroenteritis. These findings suggest that, in addition to metalloprotease. the extracellular serine protease may contribute to pathogenicity of V. vulnificus. (C) 2008 Elsevier Ltd. All rights reserved.

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  • Molecular epidemiological studies of Vibrio cholerae in Bengal region

    Sumio Shinoda, Tomoko Nakagawa, Nobuyuki Hirakawa, Shin-Ichi Miyoshi, Eiji Arakawa, Thandavarayan Ramamurthy, B. Dutta, Shah M. Faruque, Gopinath Balakrish Nair

    BIOCONTROL SCIENCE   13 ( 1 )   1 - 8   2008.3

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    Vibrio cholerae isolates from environmental and clinical origins in the Bengal region in which epidemics of cholera break out periodically were analyzed with particular emphasis on the molecular epidemiological features. The presence of the virulence genes (ctxA, tcpA and toxR) in the isolates was analyzed by the PCR (polymerase chain reaction) method. PFGE (pulsed-field gel electrophoresis) was performed to determine the clonal relationships between the clinical and environmental strains. Antiblograms and O serovars of the isolates were also examined. O1 and O139 strains from both clinical and environmental sources were all positive for the three virulence genes while non-O1/non-O139 strains from both sources were all negative for ctxA and tcpA but positive for toxR. PFGE patterns of recent isolates of O1 and O139 were similar in each serovar regardless of origin, suggesting a clonal relationship between the clinical and environmental strains, although comparison with past isolates or isolates from different geographical area showed some differences.

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  • The crucial amino acid residue related to inactivation of Vibrio vulnificus hemolysin

    Mitsutoshi Senoh, Yuka Okita, Sumio Shinoda, Shin-ichi Miyoshi

    MICROBIAL PATHOGENESIS   44 ( 1 )   78 - 83   2008.1

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    Vibrio vulnificus. all opportunistic human pathogen causing fetal septicemia, produces a 50-kDa pore-forming toxin as a virulence factor. This toxin consists of 451 amino acid residues: however. there are two types of this toxin on the basis of the difference of some,amino acid residues, type 1 (Leu(281), Ser(415), Asn(435)/Asp(435), Asn(438)) and type 2 (Ile(281), Asn(415), Asn(435), Thr(438)). In the present study, two characteristic properties of type 2 toxin that was elaborated by V. vulnificus cells or synthesized by the in vitro system were compared to those of type 1 toxin. Type 2 toxin was found to be more resistant to spontaneous inactivation at 37 degrees C and to specific inactivation by cholesterol. On the other hand a variant of type 2 toxin (Asp(435), Asn(438)) showed the same properties as type 1 toxin. The replacement of the 438th Asn to Thr (N438T), but not the 435th Asp to Asn (D435N). resulted in reversion of the variant type 2 toxin to typical type 2 toxin. These findings indicate that a single amino acid residue. Thr(438), may be critical for higher stability of type 2 toxin. (C) 2007 Elsevier Ltd. All rights reserved.

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  • ビブリオ・バルニフィカスの病原性

    三好伸一

    化学療法の領域   24 ( 6 )   879 - 884   2008

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  • A plasmidic class 1 integron from five Pseudomonas aeruginosa clinical strains harbored aacA4 and nonsense-mutated cmIA1 gene cassettes

    He Yan, Lei Shi, Shinji Yamasaki, Xinhui Li, Yicheng Cao, Lin Li, Liansheng Yanga, Shin-ichi Miyoshi

    JOURNAL OF HEALTH SCIENCE   53 ( 6 )   750 - 755   2007.12

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    Five strains of multidrug-resistant Pseudomonas aeruginosa (P aeruginosa) were isolated from inpatients at a local hospital in China. The most frequent resistance was to cefoperazone, ciprofloxacin, ceftriaxone, cefotaxime, gentamicin, piperacillin, trimethoprim sulfamethoxazole, and tobramycin. These strains were found to contain the class 1 integron, in which the 2360-bp gene cassettes were flanked by 5'- and 3'-conserved segments. Sequence analysis revealed that the gene cassettes contained aacA4 and cmlA1 genes; however, the latter gene had a nonsense mutation resulting in the production of a truncated protein. To the best of our knowledge, this is the first report of a nonsense mutation in the cmlA1 gene. Moreover, the R aeruginosa strains showed identical profiles in pulsed-field gel electrophoresis, suggesting that they were derived from the same clone. These results emphasize the importance of controlling the spread of multidrug-resistant pathogens in hospitals.

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  • Growth phase dependant activation of the precursor of Vibrio mimicus hemolysin (Pro-VMH)

    Zafar Sultan, Thmaki Mizuno, Aki Sakurai, Noriko Takata, Keinosuke Okamoto, Shin-ichi Miyoshi

    JOURNAL OF HEALTH SCIENCE   53 ( 4 )   430 - 434   2007.8

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    Vibrio mimicus (V mimicus), a causative agent of gastroenteritis and food poisoning, secretes a 63-kDa enterotoxic hemolysin as the most potent virulence factor. The vmhA gene encoding an 83-kDa precursor of the hemolysin was expressed from the early to late log phase of the bacterial growth, and the 79-kDa inactive protoxin was detected from the culture supernatant in the same growth phase. The N-terminal amino acid sequence of the protoxin was determined to be NH2-Asn-Ile-Ser-Asp-Pro-Val indicating cleavage of the Ala(25)-Asn(26) bond by a signal peptidase. In contrast, the hemolytic activity and the mature hemolysin in the culture supernatant were detected only at the late log phase. The maturation of the hemolysin, therefore, is suggested to be achieved by two-step processing, cleavage of the signal peptide followed by the growth phase-dependent removal of the 16-kDa propeptide.

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  • Analysis of genetic determinants involved in multiresistance in clinical strains isolated from renal transplantation recipients in Guangzhou, China

    Lei Shi, Yali Kou, Lin Li, Shin-ichi Miyoshi

    JOURNAL OF HEALTH SCIENCE   53 ( 2 )   185 - 189   2007.4

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    In the present study, we examined the antibiotic sensitivity of 19 bacterial strains [5 coagulase-negative Staphylococcus, 2 methicillin-resistant Staphylococcus aureus (S. aureus), 2 Enterococcus faecium (E. faecium), 5 Escherichia coli (E. coli), 3 Cedecea sp., 1 Klebsiella pneumoniae (K. pneumoniae), and 1 Burkholderia cepacia (B. cepacia)], which were isolated from renal transplantation patients using the Kirby-Bauer method. We also investigated the production of beta-lactamase and extended-spectrum beta-lactamase (ESBL), and the presence of the integrase gene (inI1) and resistance gene cassette. Among the 19 strains tested, all displayed severe multiresistance, and 12 strains produced beta-lactamase, in which 6 strains were ESBL positive. Eleven strains were revealed to possess the class 1 integron; however, neither class 2 nor 3 was detected. Additionally, 3 drug resistance genes, aadA2, dfrA17, and aadA5, were found in some strains. The results indicate that the horizontal transfer of the beta-lactamase gene and/or the class 1 integron may contribute significantly to the spread of multiresistant bacteria among renal transplantation patients.

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  • Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells

    Akira Takahashi, Shin-ichi Miyoshi, Noriko Takata, Masayuki Nakano, Akiko Hamamoto, Kazuaki Mawatari, Nagakatsu Harada, Sumio Shinoda, Yutaka Nakaya

    CELLULAR MICROBIOLOGY   9 ( 3 )   583 - 595   2007.3

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    Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and I-125 efflux of the cells. The VMH-induced increases in Isc and I-125 efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and I-125 efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+-dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.

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  • Vibrio vulnificus infection and metalloprotease

    Shin-ichi Miyoshi

    JOURNAL OF DERMATOLOGY   33 ( 9 )   589 - 595   2006.9

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    Vibrio vulnificus is ubiquitous in aquatic environments; however, it occasionally causes serious and often fatal infections in humans. These include invasive septicemia contracted through consumption of raw seafood, as well as wound infections acquired through contact with brackish or marine waters. In most cases of septicemia, the patients have underlying disease(s), such as liver dysfunction or alcoholic cirrhosis, and the secondary skin lesions including cellulitis, edema and hemorrhagic bulla appear on the limbs. Although V. Vul produces various virulent factors including polysaccharide capsule, type IV pili, hemolysin and proteolytic enzymes, the 45-kDa metalloprotease may be a causative factor of the skin lesions, because the purified protease enhances vascular permeability through generation of chemical mediators and also induces serious hemorrhagic damage through digestion of the vascular basement membrane. As well as other bacteria, V. Vul can regulate the protease production through the quorum-sensing system depending on bacterial cell density. However, this system operates efficiently at 25 degrees C, but not at 37 degrees C. Therefore, V. vulnificus may produce sufficient amounts of the protease only in the interstitial tissue of the limbs, in which temperature is lower than the internal temperature of the human body.

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  • Vibrio vulnificus溶血毒素の成熟過程

    沖田祐佳, 妹尾充敏, 三好伸一, 篠田純男

    臨床と微生物   33 ( 3 )   309 - 309   2006.5

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  • Biodegradation of dichloromethane by the polyvinyl alcohol-immobilized methylotrophic bacterium Ralstonia metallidurans PD11

    C Miyake-Nakayama, H Ikatsu, M Kashihara, M Tanaka, M Arita, S Miyoshi, S Shinoda

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   70 ( 5 )   625 - 630   2006.5

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    A dichloromethane (DCM)-degrading bacterium, Ralstonia metallidurans PD11 NBRC 101272, was immobilized in a polyvinyl alcohol (PVA) gel to use in a bioreactor for DCM treatment. After 4-month incubation of PVA gel beads with R. metallidurans PD11 and DCM in a mineral salt medium, the cells were tightly packed in the mesh of the gel. Forty beads of the gel in 10 ml of a batch system model showed effective activity degrading 500 and 1,000 mg l(-1) DCM within 2 and 3 h, respectively. Although reduction of pH due to accumulation of chloride ion liberated from DCM decreased the activity, it was recovered by adjustment to neutral pH. The activity of the immobilized cells was not affected by addition of nutrients which were preferentially utilized by R. metallidurans PD11, unlike the activity of the free-living cells. A continuous flow system with a column was more effective for rapid degradation of DCM. Thus, the PVA gel-immobilized cell of R. metallidurans PD11 is thought to be a prospective candidate to develop the bioreactor.

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  • Growth phase-dependent production of a toxic metalloprotease by Vibrio vulnificus

    SI Miyoshi, S Sultan, Y Yasuno, S Shinoda

    TOXIN REVIEWS   25 ( 1 )   19 - 30   2006.4

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    Vibrio vulnificus, a ubiquitous estuarine bacterium, is divided into two groups based on the virulence potential. Group 1 is a causative agent of human fatal septicemia, which is characterized by edematous and hemorrhagic secondary skin lesions on the limbs. This human pathogen secretes a toxic metalloprotease as an important virulence determinant. The protease can evoke the skin damage through enhancement of the vascular permeability and destruction of the capillaries. Group 2 is an etiological agent of epizooticus in the cultured eels. Significant levels of metalloprotease as well as autoinducer, a well-known signal molecule of cell-density dependent regulatory system for production of virulence determinants, were found to be secreted by both the groups at early stationary phase but not middle logarithmic phase when the bacteria were cultivated at 25 degrees C. Expression of the protease gene also was found to be increased several times at the stationary phase. Therefore, the autoinducer molecules that had accumulated might have accelerated the expression of the protease gene. In contrast, when cultivated at 37 degrees C, far less amounts of the protease and autoinducer were produced even at the stationary phase. Most patients suffering from V. vulnificus septicemia have underlying diseases causing elevation of the serum iron level. When incubated in human serum supplemented with ferric chloride at 37 degrees C, only Group 1 V. vulnificus showed steady bacterial multiplication and protease production but secretion of significant level of autoinducer could not be detected. Hence, the protease production by Group 1 V. vulnificus in human serum containing ferric ion is also dependent on the bacterial growth; however, protease production in this condition does not require the accumulation of the autoinducer.

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  • 原因子としてのビブリオ属菌プロテアーゼ

    篠田純男, 三好伸一

    日本細菌学雑誌   61(2):261-271   2006

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  • Presence of LuxS/Al-2 based quorum-sensing system in Vibrio mimicus: LuxO controls protease activity

    Z Sultan, S Miyosh, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   50 ( 5 )   407 - 417   2006

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    Presence of the quorum-sensing regulation system in Vibrio mimicus was investigated. The culture supernatants of V mimicus strains were found to possess AI-2 autoinducer like activity, and the strains were found to harbor the genes which are homologous to luxS, luxO, and luxR of V harveyi. These genes of V harveyi have been shown to be important components of V harveyi-like quorum-sensing system. The luxO gene homologue known to encode LuxO, the central component of the regulation system, was disrupted, and effects on protease and hemolysin activity were studied. Disruption of luxO gene resulted in the increased protease activity, but the hemolysin activity did not vary considerably.

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  • Proteolytic activation of Vibrio mimicus (Vm) major outer membrane protein haemagglutinin (HA) with Vm-HA/protease: Implication for understanding bacterial adherence

    Munirul Alam, Shin-ichi Miyoshi, Kabir Uddin Ahmed, Nur A. Hasan, Ken-ichi Tomochika, Surnio Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   50 ( 11 )   845 - 850   2006

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    Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm-HA/protease) and a major outer membrane protein-HA (Vm-OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm-HA/protease and Vm-OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm-OMPHA was confirmed by SDS-PAGE. The proteolytic activation of the native cell-associated Vm-ONIPHA by limited proteolysis was also demonstrated in several V mimicus strains. Proteolytic activation of OMPRA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V mimicus HAs in the adherence of the bacterium.

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  • Molecular characterization of a multidrug-resistant strain of enteroinvasive Escherichia coli O164 isolated in Japan

    AM Ahmed, S Miyoshi, S Shinoda, T Shimamoto

    JOURNAL OF MEDICAL MICROBIOLOGY   54 ( 3 )   273 - 278   2005.3

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    Enteroinvasive Escherichia coli (EIEC) O164 strain RIMD05091045 was isolated from a travelling patient suffering from diarrhoea at the Osaka airport quarantine facility in Japan. The strain showed multidrug resistance against streptomycin, spectinomycin, co-trimoxazole (trimethoprim/sulfamethoxazole) and ampicillin, and reduced susceptibility to ciprofloxacin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing three genes, a dihydrofolate reductase type XII gene, dfrXII, which confers resistance to trimethoprim, an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin, and an ORF of unknown function. Southern blot hybridization and conjugation experiments showed that the class 1 integron was located on a transferable plasm id that was less than 90 kb in size. The resistance of EIEC 0164 to ampicillin was found to be due to the presence of TEM-1 beta-lactamase. On the other hand, a single mutation that has not previously been described, P1 58-to-S, was detected downstream of the quinolone-resistance-determining region of parC of topoisomerase IV and may be responsible for the reduced susceptibility to ciprofloxacin in this strain.

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  • Evaluation of the biofilm-forming ability and genetic typing for clinical isolates of Pseudomonas aeruginosa by enterobacterial repetitive Intergenic consensus-based PCR

    WQ Yang, L Shi, WX Jia, XL Yin, JY Su, YL Kou, Yi, X, S Shinoda, S Miyoshi

    MICROBIOLOGY AND IMMUNOLOGY   49 ( 12 )   1057 - 1061   2005

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    Biofilm formation is an important phenotype associated with chronic Pseudomonas aeruginosa infections. In the present study, a total of 48 P. aeruginosa strains isolated from clinical specimens were examined for their biofilm-forming ability using a microtiter plate method. The different biofilm-forming abilities were demonstrated among the strains; however, most strains formed a larger biofilm than strain PAO1, a reference strain. The genetic typing was also carried out by enterobacterial repetitive intergenic consensus-based polymerase chain reaction. Although they were divided into five groups (A to E), most of the strains showing the higher biofilm-forming ability were found to be in groups D and E, suggesting a significant relationship between the biofilm-forming ability and the genetic group.

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  • The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains: Comparison of nucleotide sequences and application to the genetic grouping

    M Senoh, S Miyoshi, K Okamoto, B Fouz, C Amaro, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   49 ( 6 )   513 - 519   2005

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    Vibrio vulnificus can be divided into two groups on the basis of pathogenesis. Group 1 is pathogenic only to humans, whereas group 2 is pathogenic to eels and occasionally to humans. Although both groups produce a 50-kDa cytotoxin-hemolysin (V vulnificus hemolysin; VVH), the toxins are different. In the present study, the nucleotide sequence of the toxin gene (vvhA) of strain CDC B3547 (a group 2 strain) was determined, and the deduced amino acid sequence was compared to that of strain L-180 (a group I strain). The nucleotide sequence of vvhA of strain CDC B3547 was about 96 % identical with that of strain L-180, which results in a difference of 3 amino acid residues in the C-terminal lectin domain of VVH. Nevertheless, two primer sets for polymerase chain reaction could be designed to differentiate the toxin gene of each strain. When 27 V vulnificus clinical isolates were tested, group 1 strains (9 strains) were shown to react only to the primers designed for vvhA of strain L-180; whereas, the gene of group 2 strains (18 strains) could be amplified with the primers for vvhA of strain CDC B3547. These findings may lead to development of a novel genetic grouping system related to the virulence potential or to the host range.

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  • A hemolysin of Vibrio mimicus (VMH) stimulates cells to produce ATP and cyclic AMP which appear to be secretory mediators

    YS Li, K Okamoto, E Takahashi, S Miyoshi, S Shinoda, T Tsuji, Y Fujii

    MICROBIOLOGY AND IMMUNOLOGY   49 ( 1 )   73 - 78   2005

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    The hemolysin of Vibrio mimicus (VMH) is a pore-forming toxin with both enterotoxiic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently. we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP-dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.

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  • Isolation and characterization of a 1,3-dichloro-2-propanol-degrading bacterium

    R Yonetani, H Ikatsu, C Miyake-Nakayama, E Fujiwara, Y Maehara, S Miyoshi, H Matsuoka, S Shinoda

    JOURNAL OF HEALTH SCIENCE   50 ( 6 )   605 - 612   2004.12

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    Arthrobacter sp. strain PY1, a bacterium having the ability to degrade 1.3-dichlotopropanol (1.3-DCP), was isolated from a soil sample of a chemical plant. Strain PY1 degraded 1000 mg/l (7.75 mM) of 1.3-DCP completely within 7 days, and the ability was elevated by acclimatization up to 4000 mg/l/week. Addition of nutrients such as peptone, glucose or glycerol showed no or slight effect on the degrading activity. These results suggest that strain PY1 is a useful organism in a biological control system for 1,3-DCP pollution. The ability to degrade 1.3-DCP was induced by addition of 1.3-DCP to the culture of strain PY1. A 1.3-DCP-degrading enzyme (Deh-PY1) was purified from the cytoplasmic fraction of strain PY1 by fractionation with ammonium sulfate, hydrophobic chromatography and anion exchange chromatography. Purified Deh-PY1 is a tetramer of a homogeneous subunit having a molecular weight of 20 kDa. Analysis of the N-terminal amino acid sequence of Deh-PY1 showed that the 31 residues were quite similar to those of known 1.3-DCP-dehalogenases of other organisms. Arthrobarter sp. strain AD2 and Corynebacterium sp. strain N-1074, although some differences in composition or enzymatic characteristics were observed. The Km value and Vmax of Deh-PY1 were 2.67 mM and 7.81 mumol/min/mg, respectively. and the optimum reaction temperature and pH were 40-50degreesC and 9.5-10.5.

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  • Generation of active fragments from human zymogens in the brady kinin-generating cascade by extracellular proteases from Vibrio vulnificus and V-parahaemolyticus

    S Miyoshi, H Watanabe, T Kawase, H Yamada, S Shinoda

    TOXICON   44 ( 8 )   887 - 893   2004.12

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    Vibrio vulnificus is an opportunistic human pathogen causing septicemia, and the infection is characterized by formation of the edematous. skin lesions on limbs. This pathogenic species secretes a thermolysin-like metalloprotease as a virulence determinant. The metalloprotease was confirmed to activate human factor XII-plasma kallikrein-kinin cascade that results in liberation of bradykinin, a chemical mediator enhancing the vascular permeability, from high-molecular weight kininogen. Namely, the metalloprotease showed to generate active fragments by cleavage of Arg-Ile, Arg-Val or Gly-Leu peptide bond in human zymogens (plasma prekallikrein and factor XII). In spite of induction of the sufficient vascular permeability-enhancing and edema-forming reaction in the guinea pig model, a serine protease from V. parahaemolyticus, a human pathogen causing primarily watery diarrhea, showed far less ability to activate and to cleave the human zymogens. These results in part may explain why only V. vulnificus often causes serious edematous skin damages in humans. (C) 2004 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.toxicon.2004.08.013

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  • Regulation system for protease production in Vibrio vulnificus

    T Kawase, S Miyoshi, Z Sultan, S Shinoda

    FEMS MICROBIOLOGY LETTERS   240 ( 1 )   55 - 59   2004.11

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    Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to consumption of raw seafoods. This human pathogen secretes a metalloprotease (VVP) that evokes enhancement of the vascular permeability and disruption of the capillaries. Production of microbial proteases is generally induced at early stationary phase of its growth. This cell density dependent regulation of VVP production in V vulnificus known to be the quorum-sensing. When V. vulnificus was cultivated in Luria-Bertani (LB) medium, accumulation of the autoinducer, the signal molecule operating the quorum-sensing system, was detected. Moreover, expression of the vvp gene encoding VVP was found to be closely related with expression of the luxS gene that encode the synthase of the autoinducer precursor (luxS). These findings may indicate VVP production is controlled by the quorum-sensing system in LB medium. Futhermore, this system functioned more effectively at 26 degreesC than at 37 degreesC. When incubated at 37 degreesC in human serum supplemented with ferric chloride, production of VVP and expression of vvp increased in proportion to the concentration of ferric ion; whereas, expression of luxS was not increased. This suggests that VVP production in human serum containing ferric ion may be regulated mainly by the system other than the quorum-sensing system. (C) 2604 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.femsle.2004.09.023

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  • High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans

    H Watanabe, S Miyoshi, T Kawase, K Tomochika, S Shinoda

    MICROBIAL PATHOGENESIS   36 ( 3 )   117 - 123   2004.3

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    Vibrio vulnificus biotype 1, a causative agent of fatal septicemia or wound infection in humans, is known to produce a toxic metalloprotease as an important virulence determinant. V. vulnificus biotype 2 (serovar E), a primary eel pathogen, was found to elaborate an extracellular metalloprotease that was indistinguishable from that of biotype 1. The potential of V. vulnificus biotype 1 for production of the metalloprotease was compared with biotype 2 and other human non-pathogenic Vibrio species (Vibrio anguillarum and Vibrio proteolyticus). When cultivated at 25degreesC in tryptone-yeast extract broth supplemented with 0.9% NaCl, all bacteria multiplied sufficiently and secreted significant amounts of the metalloprotease. However, at 37degreesC with 0.9% NaCl, V. anguillarum neither grew nor produced the metalloprotease. In human serum, only V. vulnificus biotype 1 revealed a steady multiplication accompanied with production of the extracellular metalloprotease. This prominent ability of biotype 1 in growth and protease production may contribute to cause serious systemic diseases in humans. (C) 2003 Elsevier Ltd. All rights reserved.

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  • An exocellular cytolysin produced by Vibrio vulnificus CDC B3547, a clinical isolate in biotype 2 (Serovar E)

    SI Miyoshi, A Morita, T Teranishi, KI Tomochika, S Yamamoto, S Shinoda

    JOURNAL OF TOXICOLOGY-TOXIN REVIEWS   23 ( 1 )   111 - 121   2004

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    Vibrio vulnificus biotype 2, a primary eel pathogen, is also an opportunistic pathogen for humans. The strains in this biotype secrete a cytolysin into the culture medium. The cytolysin from the strain CDC B3547 (ATCC 33817), which was originally isolated from a human leg wound, can disrupt various kinds of eukaryotic cells including erythrocytes and mast cells, and artificial vesicles, liposomes. The cytolysin is a 50 kDa single-chain protein and is categorized into the pore-forming toxins. After binding tightly to the cell-membrane cholesterol in a temperature-independent manner, the toxin molecules assemble each other in a temperature-dependent manner, forming a small transmembrane pore. When incubated with a metalloprotease from the same species, the cytolysin is converted to the nicked toxin composed of some peptide chains, joined with disulfide bond(s). This nicked toxin is more hydrophilic while maintaining comparable cytolytic activity.

    DOI: 10.1081/TXR-120030650

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  • Distribution of virulence-associated genes in Vibrio mimicus isolates from clinical and environmental origins

    S Shinoda, T Nakagawa, L Shi, K Bi, Y Kanoh, K Tomochika, S Miyoshi, T Shimada

    MICROBIOLOGY AND IMMUNOLOGY   48 ( 7 )   547 - 551   2004

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    Distribution of virulence-associated genes in Vibrio mimicus was studied including the toxin genes ctxA, tdh, st and vmh and the genes necessary for regulation of toxin production, toxR, toxS, toxT, tcpA and tcpP. Approximately half of clinical V mimicus isolates possessed one or more genes encoding V cholerae enterotoxic factors such as ctxA, tdh and st. All of the clinical and environmental isolates possessed vmh encoding V mimicus hemolysin (VMH). The ctxA encoding cholera toxin was detected in only 2 strains, 5% of the clinical isolates. Furthermore, there were very few strains possessing tcpP and toxT needed for the expression of ctxA. These results may suggest that VMH is a more important pathogenic factor than well recognized toxins such as cholera toxin (CT) in V mimicus infection.

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  • Identification and characterization of class 1 integron resistance gene cassettes among Salmonella strains isolated from healthy humans in China

    HM Zhang, L Shi, L Li, SY Guo, XM Zhang, S Yamasaki, S Miyoshi, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   48 ( 9 )   639 - 645   2004

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    Twenty-three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in-F and in-B primers showed the existence of amplicons; of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1-positive isolates, respectively. Sequence analysis revealed that the 1,009-bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664-bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all-round surveillance of the antibiotic resistance of pathogens.

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  • Isolation and characterization of a new dichloromethane degrading bacterium, Ralstonia metallidurans, PD11

    Chizuko Miyake-Nakayama, Sachiyo Masujima, Hisayoshi Ikatsu, Shin-Ichi Miyoshi, Sumio Shinoda

    Biocontrol Science   9 ( 4 )   89 - 93   2004

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    The widely used organic solvent dichloromethane (DCM) has various toxic effects including carcinogenesis. We isolated a DCM-degrading bacterium Ralstonia metallidurans PD11 from drainage water which grew with DCM as a sole carbon source. PD11 was a methylotrophic bacterium with the ability to grow with C1 compounds such as methanol or methylamine. Although the existence of methylotrophic bacteria having DCM-degrading ability has been reported, there has been no report on Ralstonia sp. to date. The DCM-degrading activity of PD11 was increased by acclimatization, finally reaching a level to degrade 2,500 mg DCM/l within a week. The cell-free extract of PD11 showed DCM-degrading activity by liberating chloride which was stimulated by addition of glutathione, suggesting that the DCM dehalogenationg enzyme could be classified into the glutathione S-transferase super family.

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  • Evidence that temporally alternative expression of the Vibrio vulnificus elastase prevents proteolytic inactivation of hemolysin

    RJ Eun, JH Lee, HS Jeong, UY Park, DH Lee, GJ Woo, S Miyoshi, SH Choi

    JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY   13 ( 6 )   1021 - 1026   2003.12

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    Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. Among the putative virulence factors are an elastase, elastolytic protease, and a cytolytic hemolysin. Effects of the elastase on the hemolysin were assessed by evaluating changes of hemolytic activities either in the presence or absence of the protease. Although hemolytic activity in the culture supernatant was lowered by the purified elastase added in vitro, the cellular level of hemolytic activity was unaffected by the mutation of vvpE encoding the elastase. Growth kinetic studies revealed that hemolysin reached its maximum level in the exponential phase of growth, and the elastase appeared at the onset of the stationary phase. These results have provided insight into the regulation of virulence factors: temporally coordinate regulation of virulence factors is essential for the overall success of the pathogen during pathogenesis.

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  • Identification and characterization of genes required for biosynthesis and transport of the siderophore vibrioferrin in Vibrio parahaemolyticus

    T Tanabe, T Funahashi, H Nakao, SI Miyoshi, S Shinoda, S Yamamoto

    JOURNAL OF BACTERIOLOGY   185 ( 23 )   6938 - 6949   2003.12

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    In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, L-alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon,pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA. Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

    DOI: 10.1128/JB.185.23.6938-6949.2003

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  • Studies on pathogenic Vibrio parahaemolyticus during a warm weather season in the Seto Inland Sea, Japan

    MJ Alam, SI Miyoshi, S Shinoda

    ENVIRONMENTAL MICROBIOLOGY   5 ( 8 )   706 - 710   2003.8

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    Vibrio parahaemolyticus is a potentially pathogenic bacterium, occurring naturally in estuarine and marine environments throughout the world. The incidence of this organism in an aquatic environment depends upon many ecofactors. Sea water and organic material were collected during the warm weather season from a coast of the Seto Inland Sea, Japan, and analysed to determine V. parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 99% of samples were positive for V. parahaemolyticus with densities of 3 to> 1400 cells per 100 ml of water or 10 g of organic samples by the most-probable-number (MPN)-PCR technique, but only 76.6% were positive by the conventional MPN culture technique, with densities ranging from 3 to> 1400 cells per 100 ml of water or 10 g of organics. Furthermore, the tdh and trh genes were positive in 41.5% and 8.5% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN culture procedure. The difference in detection between the MPN culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.

    DOI: 10.1046/j.1462-2920.2003.00458.x

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  • Histamine-releasing reaction induced by the N-terminal domain of Vibrio vulnificus metalloprotease

    S Miyoshi, K Kawata, M Hosokawa, K Tomochika, S Shinoda

    LIFE SCIENCES   72 ( 20 )   2235 - 2242   2003.4

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    A zinc metalloprotease secreted by Vibrio vulnificus, an opportunistic human pathogen causing septicemia and wound infection, stimulates exocytotic histamine release from rat mast cells. This protease consists of two functional domains: the N-terminal domain that catalyzes proteolytic reaction and the C-terminal domain that promotes the association with a protein substrate or cell membrane. Like the intact protease, the N-terminal domain alone also induced histamine release from rat peritoneal mast cells in a dose- and time-dependent manner. However, the reaction induced was apparently weak and went on more slowly. The nickel-substituted protease or its N-terminal domain, each of which has the reduced proteolytic activity due to decreased affinity to a substrate, showed much less histamine-releasing activity. When injected into the rat dorsal skin, the N-terminal domain also evoked enhancement of the hypodermic vascular permeability, while the activity was comparable to that of the protease. Taken together, the protease may stimulate histamine release through the action of the catalytic center of the N-terminal domain on the target substance(s) on the mast cell membrane. The C-terminal domain may support the in vitro action of the N-terminal domain by coordination of the association of the protease with the membrane, but it may not modulate the in vivo action. (C) 2003 Elsevier Science Inc. All rights reserved.

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  • Vibrio vulnificus induces macrophage apoptosis in vitro and in vivo

    T Kashimoto, S Ueno, M Hanajima, H Hayashi, Y Akeda, S Miyoshi, T Hongo, T Honda, N Susa

    INFECTION AND IMMUNITY   71 ( 1 )   533 - 535   2003.1

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    In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of V. vulnificus.

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  • An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization, and gene cloning

    S Miyoshi, Y Sonoda, H Wakiyama, MM Rahman, K Tomochika, S Shinoda, S Yamamoto, K Tobe

    MICROBIAL PATHOGENESIS   33 ( 3 )   127 - 134   2002.9

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    An exocellular metalloprotease produced by Vibrio fluvialis, an enteropathogenic vibrio, was purified and characterized. The metalloprotease (V. fluvialis protease [VFP]) was found to have very similar characteristics to V. vulnificus protease, including a molecular mass of 45 kDa, sensitivity to chelating agents or competitive inhibitors for thermolysin-like metalloproteases, and the substrate specificity. The structural gene for VFP was also cloned, and its nucleotide sequence was determined. The deduced amino acid sequence confirmed that VFP was a member of the thermolysin family. VFP, like V. vulnificus protease, showed the haemagglutinating, permeability-enhancing and haemorrhagic activities in addition to the proteolytic activity toward oligopeptide, casein or elastin. (C) 2002 Elsevier Science Ltd. All rights reserved.

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  • Induction of an outer membrane protein of 78 kDa in Vibrio vulnificus cultured in the presence of desferrioxamine B under iron-limiting conditions

    H Aso, S Miyoshi, H Nakao, K Okamoto, S Yamamoto

    FEMS MICROBIOLOGY LETTERS   212 ( 1 )   65 - 70   2002.6

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    Although Vibrio vulnificus is known to be able to utilize ferrioxamine B as an iron source, its outer membrane receptor remains to be determined. In this study, we found that V. vulnificus expressed a new outer membrane protein of 78 kDa when grown in the presence of desferrioxamine B under iron-limiting conditions. The desferrioxamine B-dependent iron uptake was only observed in bacterial cells expressing this protein. Furthermore, non-denaturing polyacrylamide gel electrophoresis followed by autoradiography of the outer membrane preparation containing the 78-kDa protein preincubated with [Fe-55]ferrioxamine B provided a single radioactive band in which the 78-kDa outer membrane protein was present as the major component. These lines of evidence suggest that the inducible 78-kDa protein may serve as the cell-surface receptor for ferrioxamine B. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

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  • Identification and characterization of pvuA, a gene encoding the ferric vibrioferrin receptor protein in Vibrio parahaemolyticus

    T Funahashi, K Moriya, S Uemura, S Miyoshi, S Shinoda, S Narimatsu, S Yamamoto

    JOURNAL OF BACTERIOLOGY   184 ( 4 )   936 - 946   2002.2

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    We previously reported that Vibrio parahaemolyticus expresses two outer membrane proteins of 78 and 83 kDa concomitant with production of siderophore vibrioferrin in response to iron starvation stress and that these proteins are the ferric vibrioferrin receptor and heme receptor, respectively (S. Yamamoto, T. Akiyama, N. Okujo, S. Matsuura, and S. Shinoda, Microbiol. Immunol. 39:759-766, 1995; S. Yamamoto, V. Hara, K. Tomochika, and S. Shinoda, FEMS Microbiol. Lett. 128:195-200, 1995). In this study, the Fur titration assay (FURTA) system was applied to isolate DNA fragments containing a potential Fur box from a genomic DNA library of V. parahaemolyticus WP1. Sequencing a 3.2-kb DNA insert in one FURTA-positive clone revealed that an amino acid sequence deduced from a partial gene, which was preceded by a full-length gene (psuA) encoding a receptor for a siderophore of unknown origin, was consistent with the N-terminal amino acid sequence of the 78-kDa ferric vibrioferrin receptor. Then, the full-length gene (pvuA) encoding the ferric vibrioferrin receptor was cloned and characterized. The deduced protein encoded by pvuA displayed the highest similarity (31% identity; 48% similarity) to RumA, a ferric rhizoferrin receptor of Morganella morganii. Primer extension and Northern blot analyses indicated that psuA and pvuA constitute an operon which is transcribed from a Fur-repressed promoter upstream of psuA. The product of the pvuA gene and its function were confirmed by generating a pvuA-disrupted mutant, coupled with genetic complementation studies. A mutant with disruption in the upstream psuA gene also displayed a phenotype impaired in the utilization of ferric vibrioferrin.

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  • Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds

    S Miyoshi, T Kamei, Y Ota, C Masunaga, Y Izuhara, K Tomochika, S Shinoda, S Yamamoto

    FEMS MICROBIOLOGY LETTERS   208 ( 1 )   77 - 81   2002.2

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    Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron-tetra(4-sulfonatophenyl)porphyrin (Fe-TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe-TCPP, but not Fe-TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe-TPPS. When inoculated into human serum supplemented with Fe-TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S0378-1097(02)00448-2

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  • Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan

    MJ Alam, KI Tomochika, SI Miyoshi, S Shinoda

    FEMS MICROBIOLOGY LETTERS   208 ( 1 )   83 - 87   2002.2

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    Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to > 1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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  • ビブリオのVNC菌とその衛生学的問題

    友近健一, 三好伸一, 篠田純男

    Bokin Bobai   30(2):85-90   2002

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  • Purification of a serine protease of Vibrio parahaemolyticus and its characterization

    M Ishihara, A Kawanishi, H Watanabe, K Tomochika, S Miyoshi, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   46 ( 4 )   299 - 303   2002

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    A 50 kDa protease designated as VPPI was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and Fractogel EMD TMAE 650 ion-exchange chromatography. VPPI was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium-dependent serine protease. N-terminal amino acid sequence of VPPI was quite similar to that of V. metschnikovii protease and antibody against VPPI inhibited the activity of V metschnikovii protease, suggesting the similarity of the two proteases. It was demonstrated that VPPI or its related protease widely distribute in not only V parahaemolyticus but also V alginolyticus.

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  • The C-terminal domain promotes the hemorrhagic damage caused by Vibrio vulnificus metalloprotease

    S Miyoshi, K Kawata, K Tomochika, S Shinoda, S Yamamoto

    TOXICON   39 ( 12 )   1883 - 1886   2001.12

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    Vibrio vulnificus, an opportunistic human pathogen, produces a 45-kDa zinc metalloprotease (V. vulnificus protease; VVP) as an important virulence determinant. VVP injected intradermally into the dorsal skin causes the hemorrhagic damage through specific degradation of type IV collage in the vascular basement membrane. The N-terminal 35-kDa polypeptide (VVP-N), the catalytic domain, also evoked the hemorrhagic skin reaction within minutes. However, the hemorrhagic activity of VVP-N was one-third of that of VVP. Besides, the proteolytic activity of VVP-N toward the reconstituted basement membrane or type IV collagen was found to be about 50 % of VVP. VVP-N, like VVP, was quickly inactivated by an equimolar amount Of alpha (2)-macroglobulin, a broad-spectrum plasma protease inhibitor, These findings indicate that the C-terminal 10-kDa polypeptide, the substrate-binding domain mediating the effective binding to protein substrates, functions to augment the hemorrhagic reaction of VVP. (C) 2001 Elsevier Science Ltd. All rights reserved.

    DOI: 10.1016/S0041-0101(01)00171-4

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  • 環境中のチトクロームP450産生細菌による内分泌撹乱化学物質の分解

    篠田純男, 加藤安成, 友近健一, 広部滋末, 三好伸一, 井勝久喜

    岡山大学環境計測共同利用施設年報 しぶかわ   ( 22 )   18 - 24   2001

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  • Detection of virulence associated genes in clinical strains of Vibrio mimicus

    KW Bi, S Miyoshi, K Tomochika, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   45 ( 8 )   613 - 616   2001

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    A total of 42 clinical strains of Vibrio mimicus were examined for the presence of virulence associated genes toxR, toxS, toxT, tcpP, ctx and tcpA by PCR assay. Almost all strains were shown to have the toxR gene, while the toxS gene was found in 27 strains. On the other hand, five strains possessed both toxT and tcpP genes, but others had neither. Only two strains were positive for amplification of the ctx gene, whereas no PCR product with tcpA primers was detected. The results indicate the incomplete copies of virulence cascade in V mimicus strains. The pathogenesis and epidemic potential of this species is also discussed.

    DOI: 10.1111/j.1348-0421.2001.tb01292.x

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  • Detection of viable Vibrio mimicus by reverse transcription-polymerase chain reaction

    K. Bi, S. I. Miyoshi, L. Shi, K. I. Tomochika, S. Shinoda

    Biocontrol Science   6 ( 2 )   81 - 86   2001

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    Differentiating viable cells from nonviable cells is of considerable importance in the monitoring of food-borne pathogens. A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect mRNA from the phospholipase gene (phl) of Vibrio mimicus. Viable V. mimicus cells were killed by heat or ethanol treatment and kept for various periods at room temperature. Total RNA from V. mimicus was extracted, treated with DNase and subjected to RT-PCR with primers for the phl gene. The phl mRNA was detected in the viable cells, but it gradually disappeared when the killed cells were left at room temperature and became undetectable after 8 h of storage. Furthermore, RT-PCR generated a 493 bp fragment from the total RNA extracted from as few as about 103 organisms, confirming the sensitivity of the assay. The amplification of the phl mRNA was specific for V. mimicus, as no amplification was found when fifteen other Vibrio species and seven related organisms were tested. The results indicated a good relationship between the detection of the phl mRNA and viability of V. mimicus cells because the phl transcript is rapidly degraded upon cell death. This work shows the usefulness of RT-PCR as a sensitive method for the specific detection of viable V. mimicus.

    DOI: 10.4265/bio.6.81

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  • Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other Vibrios

    MJ Alam, K Tomochika, S Miyoshi, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   45 ( 5 )   393 - 397   2001

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    We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios, We observed similar seasonality of V. parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and K parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CPU on nutrient agar (with 2% NaCl) did not vary so much during the study period.

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  • Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus

    R Kimoto, T Funahashi, N Yamamoto, S Miyoshi, S Narimatsu, S Yamamoto

    MICROBIOLOGY AND IMMUNOLOGY   45 ( 2 )   135 - 142   2001

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    Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli, The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes. Furthermore, when grown under iron-limiting conditions, E. coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions, These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.

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  • Isolation of dichloromethane-degrading bacteria from drainage water

    H Kawata, C Nakayama, M Sakamoto, H Ikatsu, S Miyoshi, K Tomochika, S Shinoda

    JOURNAL OF HEALTH SCIENCE   46 ( 3 )   187 - 191   2000.6

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    The biodegrading ability of drainage water from research laboratories to dichloromethane (DCM) and chloroform (CF) was surveyed. When DCM was used as a sole carbon source in a synthetic mineral salt medium, some water samples showed ability to degrade DCM, and DCM-degrading bacteria were isolated from them, whereas no samples showed CF degradation activity. Two isolates, strain P3310, a Flavimonas sp., and strain G31, a Chryseobacterium sp., were used for further investigations. Both strains were able to use DCM as a carbon source for growth and also grow in complex media containing other carbon sources, suggesting they were facultative methylotroph. Both strains needed 6 days at 30 degrees C to completely degrade 200 mg/l of DCM with the first isolated cells, but this was shortened to 2 days with the first subculture, suggesting they were acclimatized. Although the DCM-degrading activity of strain G31 was inhibited by addition of other carbon sources such as peptone or glucose, that of strain P3310 was not affected. Thus, strain P3310 may be a useful candidate for bioremediation to eliminate DCM from drainage.

    DOI: 10.1248/jhs.46.187

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  • Cloning and characterization of the ddc homolog encoding L-2,4-diaminobutyrate decarbosylase in Enterobacter aerogenes

    S Yamamoto, N Mutoh, D Tsuzuki, H Ikai, H Nakao, S Shinoda, S Narimatsu, S Miyoshi

    BIOLOGICAL & PHARMACEUTICAL BULLETIN   23 ( 5 )   649 - 653   2000.5

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    L-2,4-Diaminobutyrate decarboxylase (DABA DC) catalyzes the formation of 1,3-diaminopropane (DAP) from DABA. In the present study, the ddc gene encoding DABA DC from Enterobacter aerogenes ATCC 13038 was cloned and characterized, Determination of the nucleotide sequence revealed an open reading frame of 1470 bp encoding a 53659-Da protein of 490 amino acids, whose deduced NH2-terminal sequence was identical to that of purified DABA DC from E. aerogenes. The deduced amino acid sequence was highly similar to those of Acinetobacter baumannii and Haemophilus influenzae DABA DCs encoded by the ddc genes. The lysine-307 of the E. aerogenes DABA DC was identified as the pyridoxal 5'-phosphate binding residue by site-directed mutagenesis, Furthermore, PCR analysis revealed the distribution of E. aerogenes ddc homologs in some other species of Enterobacteiacene. Such a relatively wide occurrence of the ddc homologs implies biological significance of DABA DC and its product DAP.

    DOI: 10.1248/bpb.23.649

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  • A snake venom inhibitor to muscarinic acetylcholine receptor (mAChR): Isolation and interaction with cloned human mAChR

    S Miyoshi, AT Tu

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   377 ( 2 )   290 - 295   2000.5

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    An inhibitor to the muscarinic acetylcholine receptor (mAChR) was purified from the venom of Crotalus atrox (western diamondback rattlesnake). The inhibitor was found to be a 30-kDa homodimer protein with phospholipase A, activity. In order to determine the subtype selectivity of the purified inhibitor, the inhibitory effect on the binding of two orthosteric antagonists, [H-3]quinuclidinyl benzilate ([H-3]QNB) and [[H-3]N-methylscopolamine methyl chloride ([H-3]NMS), to five subtypes of cloned human mAChR was tested. The purified inhibitor reduced the binding of [H-3]QNB and/or [H-3]NMS to all subtypes of the mAChR while showing the highest inhibitory effect on the M-5 subtype. The K-d values of the receptors for the antagonists were increased in the presence of the inhibitor; however, the B-max values were not changed. The effects of the purified inhibitor on the dissociation of [H-3]NMS from the receptors were also investigated. Dissociation of the antagonist was remarkably slowed down by addition of the inhibitor. These findings may suggest an allosteric action of the purified inhibitor. In addition, the present study indicates that the presence of mAChR inhibitors is quite common in snake venoms. (C) 2000 Academic Press.

    DOI: 10.1006/abbi.2000.1784

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  • Microbial metalloproteases and pathogenesis

    S Miyoshi, S Shinoda

    MICROBES AND INFECTION   2 ( 1 )   91 - 98   2000.1

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    Zinc metalloproteases produced by human pathogenic microorganisms show a wide variety of pathological actions. In local infections, the proteases cause necrotic or hemorrhagic tissue damage through digestion of structural components of the ground substance, and also form edematous lesions through generation of inflammatory mediators, while in systemic infections, the proteases act as a synergist-ic virulence factor through disordered proteolysis of many plasma proteins. Clostridial neurotoxins, Bacteroides fragilis enterotoxin and Bacillus anthracis lethal factor are also zinc metalloproteases. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

    DOI: 10.1016/S1286-4579(00)00280-X

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  • Presence of hemolysin genes (vmh, tdh and hlx) in isolates of Vibrio mimicus determined by polymerase chain reaction

    L Shi, S Miyoshi, KW Bi, M Nakamura, M Hiura, K Tomochika, S Shinoda

    JOURNAL OF HEALTH SCIENCE   46 ( 1 )   63 - 65   2000.1

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    A total of 120 strains of Vibrio mimicus, 51 clinical and 69 environmental were examined for the presence of three types of hemolysin genes (vmh, tdh and hlx) by PCR. Ninety-six percent (115) of the strains contained at least one of these hemolysin genes. Only 5 strains from the environment were missing all three hemolysin genes, The tdh was only found in 20 of the 51 clinical isolates of V, mimicus, This mag indicate that the tdh gene is a virulence determinant of V. mimicus. More than 90% of the strains isolated from both the environment and patients possessed the vmh gene. Two clinical isolates possessed the hlx gene alone and had no other enterotoxic factors.

    DOI: 10.1248/jhs.46.63

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  • Enteropathogenic factors produced by vibrios other than cholera toxin

    S. Shinoda, S. I. Miyoshi

    Journal of Natural Toxins   9 ( 3 )   231 - 249   2000

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  • Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction

    K Bi, L Shi, Y Maehara, S Miyoshi, K Tomochika, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   44 ( 2 )   149 - 153   2000

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    A total of 51 Vibrio minicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR), The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups, AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. minicus.

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  • Dichloromethane-degrading properties of bacteria isolated from environmental water

    H. Ikatsu, H. Kawata, C. Nakayama, S. I. Miyoshi, K. I. Tomochika, T. Katsu, S. Shinoda

    Biocontrol Science   5 ( 2 )   117 - 120   2000

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    Degradation of dichloromethane (DCM) by two environmental isolates, Flevimones sp. strain P3310 and Chryseobacterum sp. strain G31, were studied. The ability of the strains was raised to degrade 3,000 mg/l of DCM by acclimatization, although the original isolates could degrade less than 500 mg/l. The first step in the degradation process was dechlorination, and the liberated chloride ions caused the reduction of pH and the bacterial growth
    the addition of phosphate salts, however, restored the growth and the degrading ability of the culture by increasing the buffer capacity. The DCM-degrading activity was also detected in the cell-free extract and the culture-supernatant. These results suggest that the isolates or their products are possible candidates for bioremediation to eliminate DCM pollution.

    DOI: 10.4265/bio.5.117

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  • Characterization of Vibrio parahaemolyticus manganese-resistant mutants in reference to the function of the ferric uptake regulatory protein

    T Funahashi, C Fujiwara, M Okada, S Miyoshi, S Shinoda, S Narimatsu, S Yamamoto

    MICROBIOLOGY AND IMMUNOLOGY   44 ( 12 )   963 - 970   2000

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    In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs), Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V; parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.

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  • Isolation and characterization of cytochrome P450-producing bacteria from various environments

    H. Ikatsu, Y. Kino, N. Kawahara, M. Adachi, S. I. Miyoshi, K. I. Tomochika, S. Shinoda

    Biocontrol Science   5 ( 2 )   111 - 116   2000

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    Eight strains of cytochrome P450 (P450)-producing bacteria were isolated from M9 medium containing a P450-inducer as the sole carbon source from the environment. Strains EP1 to EP6 utilizing 2-ethoxyphenol as the sole carbon source were isolated from the soil of a weed-filled field, the water of a paddy field, laboratory effluent, domestic effluent and river water. Strain MP1 utilizing 2-methoxyphenol and strain CP1 utilizing camphor were isolated from oil-polluted soil and the soil of a weed-filled field, respectively. This suggests the distribution of P450-producing bacteria in the environment. Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone) inhibited the growth of P450-producing bacteria on the media containing a P450-inducer as the sole carbon source, strongly suggesting that the P450 is involved in the catabolism of the inducer.

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  • Effects of Vibrio vulnificus metalloprotease on the capillaries: Pathological actions and inactivation by α-macroglobulin

    S. I. Miyoshi

    Yakugaku Zasshi   120 ( 11 )   1149 - 1157   2000

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    Vibrio vulnificus is an opportunistic human pathogen causing wound infection and septicemia, characterized by hemorrhagic and edematous damage to the skin of limbs. When injected into the dorsal skin, an extracellular metalloprotease from this vibrio (V. vulnificus protease: VVP) enhanced the vascular permeability through activation of the Hageman factor-plasma kallikrein-kinin cascade and/or stimulation of exocytotic histamine release. Additionally, VVP caused the hemorrhagic skin lesion through disorganization of the vascular basement membrane layer due to specific degradation of type IV collagen, which is known to form the backbone structure of the basement membrane. However, injected VVP was quickly inactivated by a plasma glycoprotein, α-macroglobulin, at a molar ratio of 1 : 1. This glycoprotein was leaked from the capillaries by the actions of VVP, which resulted in in situ inactivation by physical entrapment. When VVP (45000 Da) was incubated at 37°C, a 35000 Da fragment was generated by the autocatalytic removal of a 10000Da C-terminal polypeptide. This N-terminal fragment showed significant proteolytic activity, however, because of a markedly decreased affinity to the protein substrates, its permeability-enhancing and hemorrhagic activity was reduced to less than 50%. These findings indicate that the C-terminal polypeptide is not essential for but promotes skin reactions caused by VVP.

    DOI: 10.1248/yakushi1947.120.11_1149

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  • Purification and characterization of 2-ethoxyphenol-induced cytochrome P450 from Corynebacterium sp strain EP1

    N Kawahara, H Ikatsu, H Kawata, S Miyoshi, K Tomochika, S Sinoda

    CANADIAN JOURNAL OF MICROBIOLOGY   45 ( 10 )   833 - 839   1999.10

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    A soluble cytochrome P450 (P450(EP1A)) induced by 2-ethoxyphenol was purified to apparent homogeneity from Corynebacterium sp. strain EP I. The P450(EP1A) showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 45 kDa. The GO-reduced difference spectra of P450(EP1A) had a Soret maximum at 447.6 nm. The substrate difference spectra with 2-ethoxyphenol showed an absorption maximum at 394.0 nm. The purified P450(EP1A) degraded 2-ethoxyphenol in an assay system composed of spinach ferredoxin-NADP(+) oxidoreductase and NADPH. The reaction activity decreased to 1.4% of its original activity by addition of CO. The existence of catechol in the reaction mixture was confirmed after the metabolic reaction, indicating that P450EP1A catalyzes O-dealkylation of 2-ethoxyphenol. In addition to 2-ethoxyphenol, the P450(EP1A) metabolized 2-methoxyphenol, 1,1,1-trichloroethane, carbon tetrachloride. benzene, and toluene.

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  • Muscarinic acetylcholine receptor (mAChR) inhibitor from snake venom: Interaction with subtypes of human mAChR

    S Miyoshi, AT Tu

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   369 ( 1 )   114 - 118   1999.9

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    Snake venoms can contain a variety of well-studied neurotoxins, especially nicotinic acetylcholine receptor inhibitor, normally called postsynaptic neurotoxin. Karlsson first reported muscarinic acetylcholine receptor (mAChR) inhibitor from snake venom. In a previous study in our laboratory, we found a mAChR inhibitor from Naja naja sputatrix venom that bound to rat brain synaptosomes. Brain synaptosomes contain all subtypes of mAChRs, and thus the exact selectivity of the inhibitor could not be determined. mAChR inhibitor from N. naja sputatrix venom was purified and the binding to all human mAChR subtypes (M1, M2, M3, M4, and M5) was investigated and is reported in this communication. The inhibitor bound to all subtypes of the human mAChR, but showed considerably high selectivity for the R15 subtype. It was also found that the reduction of disulfide bonds in the inhibitor eliminated the binding to the mAChR. This suggests that a specific tertiary conformation maintained by disulfide bonds is essential for binding to the mAChR. An oligo peptide, QIHDNCYNE, comparable to a part of the inhibitor molecule, was synthesized and studied for its binding to the mAChR. The synthetic peptide did not show any binding activity, suggesting this portion of the inhibitor molecule is not involved in mAChR binding. The selective binding of the M5 mAChR subtype to antagonists has not yet been reported. Therefore, the purified inhibitor reported in this communication may be a useful tool to clarify the mechanism of muscarinic cholinergic transmission. (C) 1999 Academic Press.

    DOI: 10.1006/abbi.1999.1321

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  • The ability of Vibrio vulnificus to use a synthetic hydrophilic heme compound, Fe-TPPS, as a single iron source

    S Miyoshi, T Kamei, Y Inami, Y Ota, S Yamamoto, K Tomochika, S Shinoda

    FEMS MICROBIOLOGY LETTERS   172 ( 1 )   73 - 77   1999.3

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    Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we tested whether this pathogen also uses a synthetic heme compound, Fe-alpha,beta,gamma,delta-tetraphenylporphine tetrasulfonic acid (Fe-TPPS), as an iron source. When inoculated into a medium containing Fe-TPPS, V. vulnificus L-180 multiplication was seen to be dependent on the concentration of the synthetic heme compound; a mutant lacking the ability to utilize protoheme did not multiply. Cells of the strain grown under the iron-restricted condition showed time-dependent uptake of Fe-TPPS. The ability to use either protoheme or Fe-TPPS was significantly reduced by the addition of an excess amount of free TPPS or Cu-TPPS. The data suggest that, V. vulnificus may assimilate Fe-TPPS, at least partially, through the same system as that for protoheme. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S0378-1097(99)00016-6

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  • Vibrio vulnificusの亜鉛金属プロテアーゼに関する研究

    三好伸一

    日本細菌学雑誌   54(4):763-772   1999

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  • Siderophore production of Vibrio parahaemolyticus strains from different sources (vol 43, pg 909, 1999)

    S Yamamoto, N Okujo, S Miyoshi, S Shinoda, S Narimatsu

    MICROBIOLOGY AND IMMUNOLOGY   43 ( 10 )   993 - 993   1999

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    Vibrio parahaemolyticus strains isolated from different sources were assayed for their ability to produce a siderophore, vibrioferrin, under iron-limited growth conditions. The mean value +/- standard error of mean (mu M vibrioferrin in spent culture supernatant/optical density at 660 nm) was 832.3 +/- 66.9 for clinical isolates (n=44), which was significantly higher (P<0.01) than those for food isolates (461.0 +/- 66.5; n=37) and coastal isolates (378.8 +/- 37.2; n=26). This suggests that greater productivity of vibrioferrin by clinical isolates may be associated with a selective advantage for survival and proliferation under conditions of iron-limitation such as in the intestine.

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  • The hemagglutinating action of Vibrio vulnificus metalloprotease

    S Miyoshi, K Kawata, K Tomochika, S Shinoda

    MICROBIOLOGY AND IMMUNOLOGY   43 ( 1 )   79 - 82   1999

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    Vibrio vulnificus protease (VVP), a 45-kDa zinc metalloprotease, consists of two functional domains: an N-terminal 35-kDa polypeptide having endoproteinase activity, and a C-terminal 10-kDa polypeptide that mediates the binding of VVP to the erythrocyte membrane. Therefore, VVP, but not its N-terminal endoproteinase domain alone, has agglutinating activity to rabbit erythrocytes. When a single zinc atom in the catalytic center was substituted by treatment with CuCl2 or NiCl2, proteolytic and hemagglutinating activities were reduced by Ni substitution but not by Cu substitution, Cu-treated 35-kDa polypeptide showed sufficient affinity of the catalytic center and weak binding ability to the erythrocyte membrane, but the Ni-treated polypeptide did not. These results suggest that the binding of endoproteinase domain to membrane is also necessary for hemagglutination.

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  • Role of protease on the adherence and enterotoxicity of Vibrio mimicus(共著)

    ALAM M, MIYOSHI S, SONODA Y, CHOWDHURY M A R, TOMOCHIKA K, SHINODA S

    Worl J. Microbiol. Biotechnol.   13 ( 1 )   37 - 41   1997

  • Some properties nicked Vibrio vulnificus hemolysin.

    MIYOSHI S, FUJII S, TOMOCHIKA K, SHINODA S

    Microbial Pathog.   23 ( 4 )   235 - 239   1997

  • 腸管出血性大腸菌O157:H7感染症(共著)

    篠田純男, 山本重雄, 友近健一, 三好伸一

    衛生化学   43 ( 1 )   1 - 14   1997

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Books

  • 薬学領域の環境衛生学

    廣川書店  2017  ( ISBN:9784567476706

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  • 有害微生物の制御と管理−現場対応への実践的な取り組み−

    テクノシステム  2016  ( ISBN:9784924728776

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  • 菌・カビを知る・防ぐ・60の知恵

    化学同人  2015  ( ISBN:9784759815993

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  • 標準微生物学 改訂第12版

    医学書院  2015 

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  • 衛生試験法・注解2015

    金原出版  2015 

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  • 微生物の簡易迅速検査法

    テクノシステム  2013 

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  • Handbook of Proteolytic Enzymes (Third Edition)

    Elsevier Academic Press  2013 

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  • 腸炎ビブリオ 第Ⅳ集

    近代出版  2013 

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  • シンプル微生物学 改訂第5版

    南江堂  2011 

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  • 病原菌の今日的意味 改訂4版

    医薬ジャーナル  2011 

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  • 食品微生物学辞典

    中央法規出版  2010 

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  • 環境衛生の科学(第2版)

    三共出版  2010 

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  • 衛生試験法・注解2010

    金原出版  2010 

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  • 薬毒物分析学辞典

    廣川書店  2009 

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  • Comprehensive sourcebook of bacterial protein toxins, Third Edition

    Elsevier Academic Press  2006 

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  • 衛生試験法・注解2005

    金原出版  2005 

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  • 毒物・中毒用語辞典

    化学同人  2005 

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  • Handbook of Proteolytic Enzymes (Second Edition)

    Elsevier Academic Press  2004 

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  • 細菌毒素ハンドブック

    サイエンスフォーラム  2002 

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  • 事件からみた毒-トリカブトからサリンまで

    化学同人  2001 

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  • 環境衛生の科学

    三共出版  2001 

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MISC

  • インドコルカタ地域環境水中のVBNC Vibrio choleraeの生態

    水野環, 今村大輔, 妹尾充敏, 竹田美文, 三好伸一, 篠田純男

    日本防菌防黴学会年次大会要旨集   40th   234   2013.9

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    Language:Japanese  

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  • インドコルカタ市における環境水からのVBNC Vibrio choleraeの単離(Isolation of VBNC Vibrio cholerae from Environmental Water samples in Kolkata, India)

    水野 環, 今村 大輔, 妹尾 充敏, 三好 伸一, 竹田 美文, 篠田 純男

    日本細菌学雑誌   68 ( 1 )   134 - 134   2013.2

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    Language:English   Publisher:日本細菌学会  

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  • インドコルカタ市におけるVBNCコレラ菌の水環境汚染に関する調査研究

    三好伸一, 水野環, 妹尾充敏, 竹田美文, 篠田純男

    日本国際保健医療学会学術大会プログラム・抄録集   27th   132   2012.11

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    Language:Japanese  

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  • VBNCコレラ菌のヒト結腸上皮細胞HT‐29由来のconversion factor

    水野環, 妹尾充敏, 三好伸一, 竹田美文, 篠田純男

    微生物シンポジウム講演要旨集   24th   54 - 55   2012

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    Language:Japanese  

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  • Vibrio vulnificus溶血毒素の性状を左右するアミノ酸残基

    妹尾充敏, 三好伸一, 篠田純男

    日本薬学会年会要旨集   126th ( 3 )   95 - 95   2006.3

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    Language:Japanese   Publisher:(公社)日本薬学会  

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  • 溶血毒素遺伝子によるVibrio vulnificusの型別

    妹尾充敏, 三好伸一, 岡本敬の介, 篠田純男

    日本細菌学雑誌   59 ( 1 )   281 - 281   2004.2

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    Language:Japanese   Publisher:日本細菌学会  

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  • 溶血毒素遺伝子によるVibrio vulnificusのTyping

    妹尾充敏, 三好伸一, 岡本敬の介, 篠田純男

    日本食品微生物学会学術総会講演要旨集   24th   66   2003.10

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Presentations

  • メタゲノム解析によるコレラ患者下痢便中のコレラ菌の定量的解析

    ?橋栄造, 三好伸一*, 元岡大祐, 中村昇太, 飯田哲也, 岡本敬の介

    第72回日本細菌学会中国・四国支部総会  日本細菌学会

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    Event date: 2019.11.23 - 2019.11.24

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:米子  

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  • コレラ流行株における大規模なゲノム領域の増加

    今村大輔, 越智 郁, 水野 環, 三好伸一*, 佐藤 勉

    第60回日本熱帯医学会大会  日本熱帯医学会

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    Event date: 2019.11.8 - 2019.11.10

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:宜野湾  

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  • インド コルカタ市の環境水中の病原性Vibrio choleraeの調査および単離株の病原性

    ?橋栄造, 森田昌知, 大西 真, 三好伸一*, 岡本敬の介

    第60回日本熱帯医学会大会  日本熱帯医学会

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    Event date: 2019.11.8 - 2019.11.10

    Language:Japanese   Presentation type:Poster presentation  

    Venue:宜野湾  

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  • インド・コルカタ地域の環境水中でのコレラ菌の生息に関する研究

    岡本敬の介, ?橋栄造, 三好伸一*, 元岡大祐, 中村昇太, 飯田哲也

    第53回ビブリオシンポジウム  ビブリオシンポジウム

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    Event date: 2019.10.25 - 2019.10.26

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:名古屋  

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  • Metagenomic sequencing analysis of stool sample of diarrhea patients indicates the presence of carrier of Vibrio cholerae O1 in Kolkata, India International conference

    Okamoto K, Takahashi E, Miyoshi S*, Mukhopadhyay AK, Dutta S, Motooka D, Nakamura S, Iida T

    Asian-African Research Forum on Emerging and Reemerging Infections 2019  国立研究開発法人日本医療研究開発機構

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    Event date: 2019.9.5 - 2019.9.6

    Language:English   Presentation type:Oral presentation (general)  

    Venue:Sapporo, Japan  

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  • Examination of virulent Vibrio cholerae inhabiting environmental water in Kolkata and their survival in water International conference

    Takahashi E, Chowdhury G, Mukhopadhyay AK, Mizuno T, Miyoshi S*, Okamoto K

    Asian-African Research Forum on Emerging and Reemerging Infections 2019  国立研究開発法人日本医療研究開発機構

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    Event date: 2019.9.5 - 2019.9.6

    Language:English   Presentation type:Oral presentation (general)  

    Venue:Sapporo, Japan  

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  • Collaborative Research Center of Okayama University for Infectious Diseases International conference

    Miyoshi S*, Okamoto K, Takahashi E, Ohnishi M

    Asian-African Research Forum on Emerging and Reemerging Infections 2019  国立研究開発法人日本医療研究開発機構

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    Event date: 2019.9.5 - 2019.9.6

    Language:English   Presentation type:Poster presentation  

    Venue:Sapporo, Japan  

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  • Comparative proteomic analysis to characterize temperature induced VBNC and resuscitation state in Vibrio cholerae International conference

    Debnath A, Mizuno T, Miyoshi S*

    Asian-African Research Forum on Emerging and Reemerging Infections 2019  国立研究開発法人日本医療研究開発機構

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    Event date: 2019.9.5 - 2019.9.6

    Language:English   Presentation type:Oral presentation (general)  

    Venue:Sapporo, Japan  

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  • Inhibition of vibrio motility by human LL-37 and its derivatives International conference

    Miyoshi S*, Mizuno T

    Asian-African Research Forum on Emerging and Reemerging Infections 2019  国立研究開発法人日本医療研究開発機構

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    Event date: 2019.9.5 - 2019.9.6

    Language:English   Presentation type:Oral presentation (general)  

    Venue:Sapporo, Japan  

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  • インド コルカタ環境水中に生息する病原性遺伝子を保有するV. choleraeの調査

    ?橋栄造, 水野 環, 三好伸一*, 岡本敬の介

    日本薬学会第139年会  日本薬学会

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    Event date: 2019.3.20 - 2019.3.23

    Language:Japanese   Presentation type:Poster presentation  

    Venue:千葉  

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  • Analysis of Vibrio cholerae inhabiting environmental water in Kolkata, India International conference

    Takahashi E, Chowdhury G, Mukhopadhyay AK, Mizuno T, Miyoshi S*, Okamoto K

    United States-Japan Cooperative Medical Sciences Program: 53rd Year Joint Panel Conference Cholera and Other Bacterial Enteric Infections  US-Japan Cooperative Medical Sciences

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    Event date: 2019.2.28 - 2019.3.1

    Language:English   Presentation type:Poster presentation  

    Venue:Hanoi, Vietnam  

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  • Genetic characteristics and changing antimicrobial resistance of V. fluvialis isolated from hospitalized diarrhoeal patients in Kolkata, India International conference

    Chowdhury G, Ramamurthy T, Ghosh A, Takahashi E, Miyoshi S*, Dutta ., Mukhopadhyay AK, Okamoto K

    United States-Japan Cooperative Medical Sciences Program: 53rd Year Joint Panel Conference Cholera and Other Bacterial Enteric Infections  US-Japan Cooperative Medical Sciences

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    Event date: 2019.2.28 - 2019.3.1

    Language:English   Presentation type:Poster presentation  

    Venue:Hanoi, Vietnam  

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  • Characterization and significance of viable but non-culturable (VBNC) Vibrio cholerae from environmental water samples of Kolkata, India International conference

    Sarkar A, Takahashi E, Miyoshi S*, Okamoto K

    United States-Japan Cooperative Medical Sciences Program: 53rd Year Joint Panel Conference Cholera and Other Bacterial Enteric Infections  US-Japan Cooperative Medical Sciences

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    Event date: 2019.2.28 - 2019.3.1

    Language:English   Presentation type:Poster presentation  

    Venue:Hanoi, Vietnam  

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  • コレラ下痢症患者便を中心としたインド・コルカタ地域での下痢便のメタゲノム解析

    ?橋 栄造, 森田 大地, 三好 伸一*, Dutta S, Mukhopadhyay AK, Chowdhury G, 元岡 大祐, 中村 昇太, 飯田 哲也, 岡本 敬の介

    第52回ビブリオシンポジウム  ビブリオシンポジウム

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    Event date: 2018.10.25

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:猪苗代  

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  • インド コルカタの環境水に生息する病原性Vibrio choleraeの性状解析

    ?橋 栄造,水野 環,三好 伸一*,岡本 敬の介

    第71回日本細菌学会中国・四国支部総会  日本細菌学会中国・四国支部

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    Event date: 2018.10.6 - 2018.10.7

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:松山  

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  • Studies on genotypic and phenotypic variant of Vibrio cholerae strains from environmental water in Kolkata, India International conference

    The 17th Awaji Internatina Forum on Infectin and Immunity 

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    Event date: 2018.9.4 - 2018.9.7

    Language:English   Presentation type:Poster presentation  

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  • Properties of ctx-positive Vibrio cholerae NAG strains isolated from environmental water in Kolkata

    akahashi E, Morita D, Chowdhury G, Mukhopadhyay AK, Mizuno T, Miyoshi S*, Okamoto K

    第91回日本細菌学会総会  日本細菌学会

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    Event date: 2018.3.27 - 2018.3.29

    Language:Japanese   Presentation type:Poster presentation  

    Venue:福岡  

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  • 2007〜2014年間にコルカタ市の患者より分離されたVibrio cholerae O1株の保有するSXT elementと流行株の変化の解析

    森田 大地, 水野 環, 今村 大輔, Mukhopadhyay AK, 三好 伸一*, 篠田 純男, ?橋 栄造, 岡本 敬の介

    第91回日本細菌学会総会  日本細菌学会

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    Event date: 2018.3.27 - 2018.3.29

    Language:Japanese   Presentation type:Poster presentation  

    Venue:福岡  

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  • Analysis of extracellular proteases of bacteria which inhabit aquatic environments International conference

    Takahashi E, Morita D, Chowdhury G, Mukhopadhyyay AK, Miyoshi S*, Okamoto K

    The 14th Asian Coference on Diarrhoeal Disease and Nutrition  2017 

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    Event date: 2017.10.30 - 2017.11.1

    Language:English   Presentation type:Poster presentation  

    Venue:Kochi, India  

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  • Vibrio choleraeの保有する可動性伝達因子SXT elementの解析

    森田 大地, 大西 真, 森田 昌知, 水野 環, 今村 大輔, 三好 伸一*, 篠田 純男, Mukhopadhyay AK, 高橋 栄造, 岡本 敬の介

    第51回ビブリオシンポジウム  2017  ビブリオシンポジウム

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    Event date: 2017.10.20

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:石垣島  

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  • コルカタ市で分離されたVibrio cholerae O1の保有する薬剤耐性遺伝子の解析

    森田 大地, 水野環, 今村 大輔, Asish K. Mukhopadhyay, 三好 伸一*, 篠田 純男, ?橋 栄造, 岡本 敬の介

    第70回日本細菌学会中国・四国支部総会  2017  日本細菌学会中国・四国支部

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    Event date: 2017.10.14 - 2017.10.15

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:東広島  

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  • 災害時における食中毒とその対策について

    三好 伸一*

    第44回日本防菌防黴学会年次大会  2017  日本防菌防黴学会

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    Event date: 2017.9.26 - 2017.9.27

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:大阪  

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  • 岡山市内環境水のコレラ菌を含むビブリオ・コレレ汚染に関する研究

    梁 勇, 吉川 真矢, 水野 環, 三好 伸一*

    第44回日本防菌防黴学会年次大会  2017  日本防菌防黴学会

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    Event date: 2017.9.26 - 2017.9.27

    Language:Japanese   Presentation type:Poster presentation  

    Venue:大阪  

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  • Comparison of proteome profiles between culture, viable but non-culturable (VBNC) and recovery state in Vibrio cholerae International conference

    Anusuya D, Mizuno T, Miyoshi S*

    第16回あわじしま感染症・免疫フォーラム  2017  大阪大学微生物研究所等

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    Event date: 2017.9.5 - 2017.9.8

    Language:English   Presentation type:Poster presentation  

    Venue:淡路島  

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  • Serratia marcescensにおけるクロルヘキシジンに対する馴化・耐性機構の解析

    星川 果南, 山本 幸子, 峠 雄太, 芳賀 仁美, 篠原 佳那子, 近藤 有馬, 熊谷 孝則, 的場 康幸, 三好 伸一*, 小川 和加野, 黒田 照夫

    第29回微生物シンポジウム  2017  日本薬学会

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    Event date: 2017.8.29 - 2017.8.30

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:呉  

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  • Characterization of the viable but non-culturable (VBNC) and recovery state in Vibrio cholerae

    Mizuno T, Debnath A, Miyoshi S*

    日米コレラ部会日本側総会  2017  日米コレラ部会

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    Event date: 2017.8.8

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:京都  

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  • Production of extracellular proteases of bacteria which inhabit aquatic environments International conference

    akahashi E, Morita D, Miyoshi S*, Okamoto K

    IUMS 15th International Congress of Bacteriology and Applied Microbiology  2017  IUMS

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    Event date: 2017.7.17 - 2017.7.21

    Language:English   Presentation type:Oral presentation (general)  

    Venue:Sigapore  

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  • Analysis of antiboitic resistant gene variation in Vibrio cholerae isolated from clinical patients and environmental water in Kolkata from 2007-2014 International conference

    Morita D, Mizuno T, Imamura D, Takahashi E, Mukhopadhyay AK, Miyoshi S*, Shinoda S, Okamoto K

    IUMS 15th International Congress of Bacteriology and Applied Microbiology  2017  IUMS

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    Event date: 2017.7.17 - 2017.7.21

    Language:English   Presentation type:Oral presentation (general)  

    Venue:Sigapore  

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  • コルカタ市において臨床及び環境水より分離されたVibrio choleraeの保有する抗薬剤耐性遺伝子の解析

    森田 大地, 水野 環, 今村 大輔, Mukhopadhyay AK, 三好 伸一*, 篠田 純男, 岡本 敬の介

    第90回日本細菌学会総会  2017  日本細菌学会

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    Event date: 2017.3.19 - 2017.3.21

    Language:Japanese   Presentation type:Poster presentation  

    Venue:仙台  

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  • コルカタ市で単離されたVibrio choleraeの環境分離株及び臨床分離株の病原因子の比較研究

    水野 環, 森田 大地, Mukhopadhyay AK, 今村 大輔, 篠田 純男, 三好 伸一*

    第90回日本細菌学会総会  2017  日本細菌学会

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    Event date: 2017.3.19 - 2017.3.21

    Language:Japanese   Presentation type:Poster presentation  

    Venue:仙台  

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  • Effects of disruption of genes expressed during VBNC phase of Vibrio cholerae on survival under starvation International conference

    Imamura D, Mizuno T, Miyoshi S*, Shinoda S

    第90回日本細菌学会総会  2017  US-Japan Cooperative Medical Sciences

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    Event date: 2017.2.7 - 2017.2.10

    Language:English   Presentation type:Poster presentation  

    Venue:Seoul, Korea  

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  • Effects of disruption of genes expressed during VBNC phase of Vibrio cholerae on survival under starvation

    The 19th International Conference on Emerging Infectious Diseases (EID) in the Pacific Rim. US-Japan Joint Panel Conference on Cholera and Other Bacterial Enteric Infections  2017 

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  • Properties of exotoxins produced by Aeromonas species

    Gut Microbiome 2016: an international perspective  2016 

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  • コレラ菌を含むビブリオ・コレレの水環境汚染に関する日印両国での比較研究

    日本防菌防黴学会第43回年次大会  2016 

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  • Modulation of the bacterial virulence by proteolytic enzymes

    The 7th Vibrio conference 2016  2016 

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  • Effects of disruption of genes expressed during VBNC phase of Vibrio cholerae on survival under starvation

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2016 

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  • インド・コルカタにおけるコレラ流行株の特徴と変化

    第50回腸炎ビブリオシンポジウム  2016 

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  • 腸炎ビブリオのNa+耐性機構に関わる遺伝子の同定

    第50回腸炎ビブリオシンポジウム  2016 

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  • 環境水由来のVibrio cholerae環境分離株のPathogenicity islandの多様性

    日本防菌防黴学会第43回年次大会  2016 

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  • 腸炎ビブリオの新規抗菌物質排出ポンプの解析

    第69回日本細菌学会中国・四国支部総会  2016 

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  • Functional role of N- and C-terminal amino acids in the structural subunits of colonization factor CS6 expressed by enterotoxigenic Escherichia coli

    The 15th Awaji International Forum on Infection and Immunity  2016 

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  • Comparative genomic analysis reveals heterogeneity in VSP-II genomic island of El Tor variant Vibrio cholerae in Kolkata, India

    50th US-Japan Cooperative Medical Science Program Conference on Cholera and Other Bacterial Enteric Infections  2016 

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  • Whole genome analysis reveals heterogeneity of VSP-II and genetic shifts of Vibrio cholerae O1 clinical isolates in Kolkata India

    The 7th Vibrio conference 2016  2016 

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  • コルカタにおける2007-2014年コレラ流行株の全ゲノム解析によって明らかになったVSP-IIの変化

    第89回日本細菌学会総会  2016 

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  • コルカタ市環境水から単離されたVibiro choleraeのVPIとCTXΦの多様性

    第89回日本細菌学会総会  2016 

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  • コルカタ市環境水から単離されたVibiro choleraeのVPIの多様性

    第49回腸炎ビブリオシンポジウム  2015 

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  • VBNC stage specific proteins in Vibrio cholerae

    49th US-Japan Cooperative Medical Science Program Conference on Cholera and Other Bacterial Enteric Infections  2015 

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  • Inductive effect of skim milk on the production of serine protease by Aeromonas spp

    49th US-Japan Cooperative Medical Science Program Conference on Cholera and Other Bacterial Enteric Infections  2015 

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  • Inductive effect of casein phophopeptide on the production of serine protease by Aeromonas spp

    第88回日本細菌学会総会  2015 

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  • Whole genome analysis reveals VSP-II genotype shift of Vibrio cholerae O1 clinical isolates between 2007 and 2014 in Kolkata, India

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2015 

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  • インドコルカタ市の環境水からのVibrio choleraeの単離と保有遺伝子解析

    第88回日本細菌学会総会  2015 

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  • VBNC状態に特徴的なコレラ菌タンパク質の探索

    第88回日本細菌学会総会  2015 

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  • すぐそばにいる微生物

    日本防菌防黴学会第42回年次大会  2015 

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  • インドコルカタ市の環境水から単離されたVibrio choleraeのtcpA配列の多様性

    日本防菌防黴学会第42回年次大会  2015 

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  • コルカタにおける2007-2014年コレラ流行株の全ゲノム解析によって明らかになったVSP-IIの変化

    第68回日本細菌学会中国・四国支部総会  2015 

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  • ランドリー用洗剤に抵抗性を示す土壌細菌の単離・同定と性状解析

    日本防菌防黴学会第42回年次大会  2015 

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  • Whole genome analysis reveals VSP-II genotype shift of Vibrio cholerae O1 clinical isolates between 2007 and 2014 in Kolkata, India

    Microcon-2015  2015 

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  • 岡山市内の環境水のビブリオ・コレレおよびビブリオ属細菌汚染に関する調査研究

    第68回日本細菌学会中国・四国支部総会  2015 

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  • インドコルカタ市の環境水中から単離されたVibrio choleraeの保有遺伝子解析

    第48回腸炎ビブリオシンポジウム  2014 

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  • Isolation of VBNC Vibrio cholerae from environmental water sample in Kolkata, India

    Asian-African Research Forum on Emerging and Reemerging Infections 2013  2014 

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  • Analysis of outer membrane proteins and extracellular proteins of Vibrio mimicus incubated sub-lytic doses of antimicrobial peptides

    Asian-African Research Forum on Emerging and Reemerging Infections 2013  2014 

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  • Time course changes and conversion of VBNC Vibrio cholerae, a suggested state in environmental water

    Asian-African Research Forum on Emerging and Reemerging Infections 2013  2014 

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  • Vibrio choleraeの環境中での細胞状態と、VBNC細胞の変化に関する研究

    第87回日本細菌学会総会  2014 

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  • Time course changes and conversion of VBNC Vibrio cholerae, a suggested state in environmental water

    48th US-Japan Cooperative Medical Science Program Conference on Cholera and Other Bacterial Enteric Infections  2014 

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  • Analysis of outer membrane proteins produced by human pathogenic vibrios incubated with sub-lytic doses of antimicrobial peptides

    48th US-Japan Cooperative Medical Science Program Conference on Cholera and Other Bacterial Enteric Infections  2014 

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  • 微生物試験法 食中毒菌の個別試験法 腸管出血性大腸菌

    日本薬学会第134年会  2014 

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  • Isolation of VBNC Vibrio cholerae from environmental water sample, Kolkata, India, 2013

    International Union of Microbiological Societies Congresses 2014  2014 

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  • クドア・セプテンプンクタータ試験法

    日本薬学会第134年会  2014 

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  • 微生物試験法 汚染指標細菌試験法 腸内細菌科菌群

    日本薬学会第134年会  2014 

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  • Inductive effect of skim milk on the production of serine protease by Aeromonas spp

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2014 

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  • Aeromonas sobriaセリンプロテアーゼのスキムミルクによる産生亢進

    第61回トキシンシンポジウム  2014 

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  • Characterization of the viable but nonculturable state in Vibrio cholerae

    International Union of Microbiological Societies Congresses 2014  2014 

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  • VBNC stage specific proteins in Vibrio cholerae

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2014 

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  • インドコルカタ市の環境水から単離されたVBNC Vibrio cholerae

    日本防菌防黴学会第41回年次大会  2014 

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  • ジクロロメタン分解菌が保有する特異的遺伝子の解析

    日本防菌防黴学会第41回年次大会  2014 

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  • Aeromonas sobria溶血毒に対するインドロキノリン化合物活性抑制作用

    第67回日本細菌学会中国・四国支部総会  2014 

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  • コレラ菌のVBNC状態に特徴的なタンパク質の探索

    第67回日本細菌学会中国・四国支部総会  2014 

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  • インドコルカタ市環境水からのHT-29細胞破砕抽出物を用いたVBNCコレラ菌の単離

    第47回腸炎ビブリオシンポジウム  2013 

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  • Detection of VBNC Vibrio cholerae from the environmental water in Kolkata, India

    Asian-African Research Forum on Emerging and Reemerging Infections 2013  2013 

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  • ランプ法を用いた食品中での各種食中毒微生物病原因子遺伝子の検出

    日本薬学会第133年会  2013 

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  • 微生物試験法 環境病原性細菌試験法 リステリア・モノサイトゲネス

    日本薬学会第133年会  2013 

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  • Modulation of Vibrio mimicus infection by human antimicrobial peptides

    Asian-African Research Forum on Emerging and Reemerging Infections 2013  2013 

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  • インドコルカタ市における環境水からのVBNC Vibrio cholerae の単離

    第86回日本細菌学会総会  2013 

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  • 環境水中のVibrio choleraeが形成していると考えられるVBNC細胞の経時的変化

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2013 

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  • 亜致死量抗菌ペプチドに曝された病原ビブリオの外膜タンパク質の解析

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2013 

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  • 食中毒にかからないために

    日本防菌防黴学会第41回通常総会及び付設講演会  2013 

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  • Isolation of VBNC Vibrio cholerae from environmental water sample

    5th Congress of European Microbiologists (FEMS)  2013 

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  • クリーニング用洗剤および溶剤に含まれる化学物質に抵抗性を示す環境微生物の解析

    日本防菌防黴学会第40回年次大会  2013 

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  • 環境水中のVibrio choleraeが形成していると考えられるVBNC細胞の経時的変化

    第66回日本細菌学会中国・四国支部総会  2013 

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  • インドコルカタ地域環境水中のVBNC Vibrio choleraeの生態

    日本防菌防黴学会第40回年次大会  2013 

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  • Quorum sensing regulation of virulence factors expression in Vibrio vulnificus

    Bioactive Okayama: Food and Health, Okayama, Japan  2012 

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  • Significance of Vibrio mimicus trypsin-like protease (VmtA) for maturation of Vibrio mimicus hemolysin

    Asian-African Research Forum on Emerging and Reemerging Infections 2012  2012 

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  • VBNCコレラ菌のヒト結腸上皮細胞HT-29由来のconversion factor

    第24回微生物シンポジウム  2012 

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  • ヒト腸管抗菌ペプチドのビブリオ感染症に対する抑制効果

    日本防菌防黴学会第39回年次大会  2012 

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  • 腸炎ビブリオの鉄獲得系と生体内蔵職能の検討,及び抗ビブリオフェリン受容体モノクローナル抗体の作成

    第85回日本細菌学会総会  2012 

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  • Defensive effects of human antimicrobial peptide on infectious diseases by vibrios

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2012 

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  • Defensive effects of human antimicrobial peptide on infectious diseases by vibrios

    47th US-Japan Cooperative Medical Science Program Conference on Cholera and Other Bacterial Enteric Infections  2012 

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  • ビブリオ属細菌に対するヒト抗菌ペプチドの感染症抑制効果

    第50回日本薬学会・日本薬剤師会・日本病院薬剤師会中国四国支部学術大会  2011 

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  • A hemolytic factor other than Vibrio mimicus hemolysin (VMH) produced by a clinical strain

    日米医学協力研究会コレラ・細菌性腸管感染症専門部会日本側総会  2011 

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  • 家庭用洗剤に含まれる界面活性剤の環境微生物への影響

    日本防菌防黴学会第38回年次大会  2011 

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  • 微生物試験法 汚染指標細菌試験法 腸内細菌科

    日本薬学会第131年会  2011 

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  • 微生物試験法 核酸増幅法 細菌および真菌のDNA塩基配列解析による同定法

    日本薬学会第131年会  2011 

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  • A trypsin-like serine protease of Vibrio mimicus involved in maturation of a heat-labile hemolysin

    International Union of Microbiological Societies 2011 Congress  2011 

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  • Assimilation of ferric ions bound to porphyrins by Vibrio vulnificus, the human and eel pathogen inhabitting estuarine and marine environments

    International Union of Microbiological Societies 2011 Congress  2011 

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  • ビブリオ属細菌に対するヒト抗菌ペプチドの感染症抑制効果

    第32回日本食品微生物学会学術総会  2011 

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  • A hemolytic factor other than Vibrio mimicus hemolysin (VMH) produced by a clinical strain

    46th US-Japan Cholera and Other Bacterial Enteric Infections Joint Panel Meeting 2011  2011 

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  • ビブリオ・ミミカスにおけるトリプシン様プロテアーゼ遺伝子(vmtA)の保有状況と溶血毒素の活性化に対する影響

    第64回日本細菌学会中国・四国支部総会  2011 

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  • 腸炎ビブリオのvibrioferrinを介する鉄獲得系と病原性

    第63回日本細菌学会中国・四国支部総会  2010 

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  • Purification and characterization of Vibrio vulnificus serine protease (VvsA): a potential pathogenic factor

    The 3rd International Symposium for Future Technology Creating Better Human Health and Society  2010 

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  • Vibrio mimicusヘモリジンの成熟化に関与する菌体外プロテアーゼ

    第57回トキシンシンポジウム  2010 

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  • ビブリオ・バルニフィカスの産生する溶血毒素の変異導入によるトキソイド化

    日本防菌防黴学会第37回年次大会  2010 

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  • Role of Vibrio mimicus trypsin-like protease in maturation of an enterotoxic hemolysin

    2010年度日米医学協力研究会 コレラ・細菌性腸管感染症専門部会 日本側総会  2010 

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  • ビブリオ・ミミカス溶血毒素の成熟化過程

    第22回微生物シンポジウム  2010 

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  • A novel endogenous protease involved in maturation of Vibrio mimicus hemolysin

    Asia-African Research Forum on Emerging and Reemerging Infections 2010  2010 

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  • Role of Vibrio mimicus trypsin-like protease in maturation of an enterotoxic hemolysin

    45th Joint Conference on Cholera and Other Bacterial Enteric Infections Panel  2010 

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  • ヒト抗菌ペプチドのビブリオ属細菌に対する影響

    第63回日本細菌学会中国・四国支部総会  2010 

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  • ドメイン連結部分の変異によるVibrio vulnificus溶血毒素のトキソイド化

    第43回腸炎ビブリオシンポジウム  2009 

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  • Vibrio minicusの産生する毒素活性化作用を有する新奇プロテアーゼ

    第82回日本細菌学会総会  2009 

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  • 抗菌ペプチドの腸球菌Enterococcus faecalisの生存に及ぼす影響

    日本防菌防黴学会第36回年次大会  2009 

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  • Vibrio mimicusヘモリジンの成熟化に関与する新奇プロテアーゼ

    第62回日本細菌学会中国・四国支部総会  2009 

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  • 微生物試験法 細菌一般試験法 免疫学的検査法

    日本薬学会第129年会  2009 

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  • A novel extracellular protease mediating maturation of Vibrio mimicus hemolysin

    日米医学協力研究会 コレラ・細菌性腸管感染症専門部会 日本側総会  2009 

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  • 腸炎ビブリオのシデロフォアvibrioferrinを介する鉄獲得系と病原性

    第48回日本薬学会・日本薬剤師会・日本病院薬剤師会 中国四国支部学術大会  2009 

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  • ビブリオ・バルニフィカスの金属プロテアーゼ遺伝子に関する研究

    第48回日本薬学会・日本薬剤師会・日本病院薬剤師会 中国四国支部学術大会  2009 

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  • ビブリオ・バルニフィカスの増殖と金属ポルフィリン

    日本食品微生物学会30周年記念学術総会  2009 

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  • ビブリオ・バルニフィカス金属プロテアーゼの無細胞系での合成

    第48回日本薬学会・日本薬剤師会・日本病院薬剤師会 中国四国支部学術大会  2009 

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  • ヒト抗菌ペプチドdefensinsのEnterococcus faecalisの生存に対する影響

    第29回日本食品微生物学会学術総会  2008 

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  • ビブリオ・バルニフィカスの溶血毒素(hemolysin)の活性に必要なアミノ酸残基の解析

    日本薬学会第128年会  2008 

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  • 新規抗菌薬の開発を指向したヘム鉄獲得系の解析

    日本防菌防黴学会第35回年次大会  2008 

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  • Vibrio vulnificus溶血毒素の活性発現におけるC末端アミノ酸残基の効果

    第61回日本細菌学会中国・四国支部総会  2008 

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  • Vibrio vulnificus NCIMB 2137株の分泌するセリンプロテアーゼの精製と性状の解析

    第47回日本薬学会・日本薬剤師会・日本病院薬剤師会 中国四国支部学術大会  2008 

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  • Modulation of the activity of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease

    43rd Joint Conference on Cholera and Other Bacterial Enteric Infections Panel  2008 

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  • Vibrio mimicusのトランポゾン変異株の単離と解析

    第47回日本薬学会・日本薬剤師会・日本病院薬剤師会 中国四国支部学術大会  2008 

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  • Vibrio mimicus溶血毒素の成熟化に関与するプロテアーゼ

    フォーラム2007 衛生薬学・環境トキシコロジー  2007 

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  • プロテアーゼ遺伝子に基づいたVibrio vulnificusのグループ分け

    第80回日本細菌学会総会  2007 

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  • Vibrio mimicusの産生する溶血毒は2種類の経路により腸管上皮細胞のClイオン分泌を促進する

    第80回日本細菌学会総会  2007 

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  • Vibrio mimicusヘモリジンのプロテアーゼによる限定分解と溶血活性の変化

    第80回日本細菌学会総会  2007 

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  • 食中毒菌のPCR法による迅速同定

    日本薬学会第127年会  2007 

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  • Pore formation of Vibrio mimicus hemolysin in lipid bilayers

    107th Annual Meeting of American Society for Microbiology  2007 

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  • 菌数測定:最確数(MPN)法

    日本薬学会第127年会  2007 

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  • ノロウイルス検査法

    日本薬学会第127年会  2007 

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  • Vibrio vulnificus臨床分離株のプロテアーゼの多様性

    日本薬学会・日本薬剤師会・日本病院薬剤師会 中国四国支部学術大会  2007 

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  • Vibrio mimicusヘモリジンの金属プロテアーゼによる溶血活性の変化

    第54回毒素シンポジウム  2007 

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  • The crucial amino acid residues for stability and activity of Vibrio vulnificus hemolysin

    International Symposium of Vibrio vulnificus and Its Infectious Diseases  2006 

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  • Proteolytic inactivation of an enterotoxic hemolysin produced by Vibrio mimicus

    FOOD MICRO 2006  2006 

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  • Vibrio cholerae の選択的単離法の開発

    第18回微生物シンポジウム  2006 

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  • Virulence factor(s) produced by Vibrio mimicus

    FOOD MICRO 2006  2006 

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  • Amino acid residues relevant for hemolytic activity of Vibrio vulnificus

    FOOD MICRO 2006  2006 

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  • Control of protease activity by LuxO in Vibrio mimicus: evidence of the presence of active V. harveyi-like quorum sensing network

    41st Joint Conference on Cholera and Other Bacterial Enteric Infections Pannel  2006 

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  • Vibrio mimicusヘモリジンの限定分解と活性の変化

    第40回腸炎ビブリオシンポジウム  2006 

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  • Vibrio vulnificusのヘム利用系の特異性

    第18回微生物シンポジウム  2006 

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  • 新規抗菌薬の開発を指向したヘム利用系の解析

    日本防菌防黴学会第33回年次大会  2006 

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  • Vibrio mimicusの病原因子の探索

    第53回毒素シンポジウム  2006 

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  • Vibrio vulnificus hemolysinの活性発現に重要なアミノ酸残基

    第53回毒素シンポジウム  2006 

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  • Vibrio vulnificus 溶血毒素の性状を左右するアミノ酸残基

    日本薬学会第126年会  2006 

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  • Vibrio vulnificus E86株のvvp遺伝子: 塩基配列の決定及びグループ分けへの応用

    第44回日本薬学会中国四国支部学術大会  2005 

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  • ベンガル地域で分離されたVibrio cholerae分子疫学的,病原学的研究

    第78回日本細菌学会総会  2005 

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  • ビブリオ・バル二フィカスにおける毒素産生のクォーラム・センシング調節について

    第78回日本細菌学会総会  2005 

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  • Aeromonas sobriaの産生する溶血毒素の下痢発現におけるATPの関与

    第78回日本細菌学会総会  2005 

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  • ジクロロメタン分解菌Ralstonia metallidurans PD11株の固定化とバイオリアクターへの応用

    日本防菌防黴学会第32回年次大会  2005 

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  • ベンガル地域で分離されたVibrio choleraeの分子疫学的,病原学的研究

    日本防菌防黴学会第32回年次大会  2005 

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  • コレラ流行地域で分離されたVibrio choleraeにおける毒素遺伝子の分布

    第52回毒素シンポジウム  2005 

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  • Quorum-sensing in Vibrio mimicus

    The 11th International Congress of Bacteriology and Applied Microbiology: International Union of Microbiological Societies  2005 

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  • The new genetic typing of Vibrio vulnificus strains: comparison of nucleotide sequence of the hemolysin gene vvhA

    The 11th International Congress of Bacteriology and Applied Microbiology; International Union of Microbiological Societies  2005 

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  • 溶血毒素遺伝子(vvhA)によるVibrio vulnificusの遺伝学的型別

    第52回毒素シンポジウム  2005 

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  • Immobilization of a dichloromethane degrading bacterium Ralstonia metallidurans PD11 for waste treatment

    he 11th International Congress of Bacteriology and Applied Microbiology; International Union of Microbiological Societies  2005 

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  • 限定分解を受けたVibrio mimicus溶血毒素の活性

    第39回腸炎ビブリオシンポジウム  2005 

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  • Vibrio vulnificus溶血毒素の成熟過程

    第39回腸炎ビブリオシンポジウム  2005 

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  • Immobilization of a dichloromethane degrading bacterium Ralstonia metallidurans PD11 for waste treatment

    7th Symposium on Asian Academic Network for Environmental Safety and Waste Management  2005 

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  • Vibrio vulnificusのdesRAに基づくグルーピング

    第39回腸炎ビブリオシンポジウム  2005 

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  • Vibrio parahaemolyticusの二種類のコラゲナーゼの比較研究

    第39回腸炎ビブリオシンポジウム  2005 

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  • インド・ベンガル地域で単離された Vibrio cholerae臨床株・環境株の 分子生物学的比較研究

    第39回腸炎ビブリオシンポジウム  2005 

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  • Vibrio vulnificus株間におけるdesferal利用遺伝子desRAの比較

    第44回日本薬学会中国四国支部学術大会  2005 

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  • Vibrio vulnificus infection and metalloprotease

    The 14th Japan-Korea Joint Meeting of Dermatology  2005 

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  • 発酵食品テンペから分離されたEnterococcus faecalisの産生するプロテアーゼ

    第26回日本食品微生物学会学術総会  2005 

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  • ビブリオ・バル二フィカスにおけるクォーラム・センシング調節

    第44回日本薬学会中国四国支部学術大会  2005 

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  • Molecular epidemiological study of Vibrio cholerae in Bengal region

    Fortieth anniversary United States-Japan cooperative medical science program  2004 

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  • ジクロロメタン分解菌 Ralstonia metallidurans PD11 株の分解特性及びバイオリアクターへの応用

    日本薬学会第124年会  2004 

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  • 1,3-dichrolo-2-propanol 分解菌 Arthrobavter sp. PY1 株の分解特性

    日本薬学会第124年会  2004 

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  • Aeromonas sobria の産生する溶血毒素の下痢発症機序の解析

    第77回日本細菌学会総会  2004 

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  • Vibrio vulnificus のプロテアーゼ産生における AI-2 依存調節系の重要性

    第77回日本細菌学会総会  2004 

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  • ベンガル地域におけるコレラ関連細菌の分子疫学的研究

    日本薬学会第124年会  2004 

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  • 衛生試験法における赤痢菌とコレラ菌の検出法に関する提案

    日本薬学会第124年会  2004 

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  • ベンガル地域で分離されたナグビブリオの分子疫学的および病原学的研究

    日本防菌防黴学会第31回年次大会  2004 

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  • ジクロロメタン分解菌 Ralstonia metallidurans PD11 株の分解特性及びバイオリアクターへの応用

    日本防菌防黴学会第31回年次大会  2004 

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  • 溶血毒素遺伝子による Vibrio vulnificus の型別

    第77回日本細菌学会総会  2004 

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  • Vibrio vulnificus 溶血毒素(VVH)の分子生物学的研究

    第57回日本細菌学会中国・四国支部総会  2004 

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  • The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains: comparison of nucleotide sequences and application to the genetic typing

    The 7th Kora-Japan international symposium on microbiology  2004 

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  • 1,3-dichloro-2-propanol 分解菌 Arthrobacter sp. PY1 株の分解特性

    日本防菌防黴学会第31回年次大会  2004 

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  • Growth-phase dependence of protease production by Vibrio vulnificus, an etiological agent of fatal food-borne disease

    The 19th international ICFMH symposium  2004 

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  • ビブリオ・バルニフィカス溶血毒素オペロン(vvhBA)の機能解析

    第43回日本薬学会中国四国支部学術大会  2004 

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  • Molecular characterization of a multidrug-resistant strain of enteroinvasive Escherichia coli O164 isolated in Japan

    The 7th Korea-Japan international symposium on microbiology  2004 

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  • Arthrobacter sp. PY1 株固定化担体を用いた 1,3-dichloro-2-propanol の分解

    第43回日本薬学会中国四国支部学術総会  2004 

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  • Vibrio vulnificus の金属プロテアーゼ産生における AI-2 依存性調節系の関与

    第38回腸炎ビブリオシンポジウム  2004 

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  • Presence of Vibrio harveyi signaling sysyem-2 like quorum sensing system in V. mimicus

    Fortieth anniversary United States-Japan cooperative medical science program  2004 

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  • ビブリオ・バルニフィカスにおける毒素産生のクォーラム・センシング調節について

    第43回日本薬学会中国四国支部学術大会  2004 

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  • Vibrio mimicus が保有するクォーラムセンシング調節遺伝子とシグナル分子

    第38回腸炎ビブリオシンポジウム  2004 

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  • Vibrio parahaemolyticusの産生するセリンプロテアーゼ -その病原因子としての可能性-

    第76回日本細菌学会総会  2003 

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  • ジクロメタン分解菌Ralstonia metallidurans PD11株:その性質とジクロ分解処理への応用について

    日本薬学会第123年会  2003 

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  • ビブリオ・バルニフィカス金属プロテアーゼにおけるクォーラム・センシング調節

    日本薬学会第123年会  2003 

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  • ビブリオ・バルニフィカス金属プロテアーゼの産生におけるクォーラム・センシング調節

    日本薬学会第123年会  2003 

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  • 脂肪族ハロアルコール分解菌の探索とその分解活性

    日本薬学会第123年会  2003 

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  • Identification and characterization of class 1 integron-mediated resistance among Salmonella strains

    第24回日本食品微生物学会学術総会  2003 

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  • Aeromonas sobriaの分泌する溶血毒とその類似毒素の下痢誘発機構の比較研究

    第76回日本細菌学会総会  2003 

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  • ジクロロメタン分解菌Ralostonia metallidurans PD11株 -その性状とジクロロメタン分解処理への応用-

    日本薬学会第123年会  2003 

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  • 脂肪属ハロアルコール分解菌の探索とその分解活性

    日本薬学会第123年会  2003 

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  • Aeromonas sobriaの産生する溶血毒素の下痢発現機構の解析

    第76回日本細菌学会総会  2003 

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  • Arthrobacter sp. strain PY1による1.3-dichloro-2-propanolの分解

    2003年度日本防菌防黴学会若手の会  2003 

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  • ベンガル地域におけるVibrio cholerae及び関連細菌の分子疫学的研究

    第76回日本細菌学会総会  2003 

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  • 脂肪族ハロアルコール分解菌(PY1株)の分解活性

    日本防菌防黴学会第30回年次大会  2003 

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  • ジクロロメタン分解菌Ralostonia sp. PD11株の応用に向けた基礎的研究及び遺伝学的研究

    日本防菌防黴学会第30回年次大会  2003 

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  • 非定型Vibrio choleraeおよびVibrio mimicusのsucrose利用能に関する分子生物学的解析

    第76回日本細菌学会総会  2003 

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  • ベンガル地域におけるVibrio choleraeの分離及び遺伝学的解析

    日本防菌防黴学会第30回年次大会  2003 

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  • 溶血毒素遺伝子によるVibrio vulnificusのtyping

    第24回日本食品微生物学会学術総会  2003 

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  • ジクロロメタン分解菌Ralostonia metallidurans PD11株 -バイオリアクター応用に関する検討および遺伝学的検討について-

    フォーラム2003:衛生薬学・環境トキシコロジー  2003 

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  • 腸炎ビブリオセリンプロテアーゼの血管透過性亢進活性

    第50回毒素シンポジウム  2003 

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  • 東南アジア伝統的発酵食品テンペから分離した乳酸菌Enterococcus faecalis TH10酸性物質の抗菌性について

    第24回日本食品微生物学会学術総会  2003 

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  • ベンガル地域におけるコレラ菌およびナグビブリオの分子疫学的研究

    第37回腸炎ビブリオシンポジウム  2003 

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  • 人喰い菌ビブリオ・バルニフィカスの毒素産生調節システム:細胞間コミュニケーション

    第42回日本薬学会中国四国支部学術大会  2003 

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  • ジクロロメタン分解菌Ralstonia metallidurans PD11株の分解能力の検討

    第42回日本薬学会中国四国支部学術総会  2003 

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  • ビブリオ・バルニフィカスの溶血毒素遺伝子の塩基配列の決定と型別への応用

    第42回日本薬学会中国四国支部学術総会  2003 

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  • Vibrio vulnificusにおける金属プロテアーゼの産生調節機構

    第36回腸炎ビブリオシンポジウム  2002 

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  • ビブリオ金属プロテアーゼの病原性:病原菌と非病原菌の金属プロテアーゼの比較

    第75回日本細菌学会総会  2002 

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  • Vibrio vulnificus臨床分離株はマクロファージにアポトーシスを誘導する

    第75回日本細菌学会総会  2002 

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  • 非定型Vibrio choleraeにおけるsucrose利用能欠損原因の解析

    第75回日本細菌学会総会  2002 

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  • 腸炎ビブリオにおけるvibrioferrinを介する鉄獲得系遺伝子群の解析

    第75回日本細菌学会総会  2002 

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  • Vibrio vulnificusにおける外因性シデロフォアによる外膜レセプターの誘導と遺伝子解析

    第75回日本細菌学会総会  2002 

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  • ジクロメタン分解菌の分離とその分解能力の検討

    日本防菌防黴学会第29回年次大会  2002 

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  • Aeromonas sobriaの産生する溶血毒素の下痢発現機構

    第55回日本細菌学会中国四国支部総会  2002 

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  • Acinetobacter baumanniiにおけるacinetobactinを介する鉄獲得系遺伝子群の解析

    第75回日本細菌学会総会  2002 

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  • Vibrio mimicus分離株における病原遺伝子の分布

    日本防菌防黴学会第29回年次大会  2002 

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  • Vibrio vulnificusの亜鉛金属プロテアーゼ産生におけるクォーラムセンシングの関与

    第55回日本細菌学会中国四国支部総会  2002 

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  • Vibrio parahaemolyticusの産生するセリンプロテアーゼ:その病原因子としての可能性

    第41回日本薬学会中国四国支部学術大会  2002 

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  • Vibrio vulnificusにおけるaerobactinによる当該外膜レセプターの発現誘導機構

    第55回日本細菌学会中国四国支部総会  2002 

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  • Vibrio mimicusにおけるaerobactinオペロンの発現調節と遺伝子破壊による機能解析

    第55回日本細菌学会中国四国支部総会  2002 

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  • Acinetobacter baumanniiにおけるacnetobactin(鉄輸送キレーター)生合成遺伝子群の転写制御および生合成経路について

    第41回日本薬学会中国四国支部学術大会  2002 

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  • 腸炎ビブリオにおけるferric aerobactinに対する外膜受容体遺伝子のクローニングと解析

    第41回日本薬学会中国四国支部学術大会  2002 

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  • 非定型Vibrio choleraeおよびVibrio mimicusのsucrose利用能欠損原因の解析

    第41回日本薬学会中国四国支部学術大会  2002 

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  • An enterotoxic hemolysin produced by Vibrio mimicus

    International symposium on toxins and natural products  2002 

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  • Functional domains of a zinc metalloprotease from Vibrio vulnificus

    International symposium on toxins and natural products  2002 

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  • 脂肪族ハロアルコールの微生物分解

    第41回日本薬学会中国四国支部学術大会  2002 

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  • ジクロメタン分解菌の分離とその分解能力の検討

    第41回日本薬学会中国四国支部学術大会  2002 

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  • Vibrio属菌におけるaerobactin利用系遺伝子群の多様性について

    第36回腸炎ビブリオシンポジウム  2002 

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  • 腸炎ビブリオの産生するプロテアーゼに関する研究

    第36回腸炎ビブリオシンポジウム  2002 

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  • Acinetobacter Baumanniiにおける鉄獲得系遺伝子の解析

    第40回日本薬学会中国・四国支部学術大会  2001 

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  • Vibrio vulnificusプロテアーゼの赤血球凝集作用

    第40回日本薬学会中国・四国支部学術大会  2001 

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  • 芳香族ハロアルコール分解菌の探索とその分解活性

    第40回日本薬学会中国・四国支部学術大会  2001 

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  • 非定型コレラ菌および類縁菌におけるスクロース利用能欠損の解析

    第40回日本薬学会中国・四国支部学術大会  2001 

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  • Vibrio mimicusにおける病原遺伝子の分布

    第40回日本薬学会中国・四国支部学術大会  2001 

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  • 環境分離菌が産生するチトクロムP450による内分泌撹乱化学物質の分解

    第40回日本薬学会中国・四国支部学術大会  2001 

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  • Vibrio属菌フェリチン遺伝子(ftn)のクローニングと解析

    第40回日本薬学会中国・四国支部学術大会  2001 

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  • 腸炎ビブリオの鉄獲得系遺伝子の解析

    第35回腸炎ビブリオシンポジウム  2001 

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  • 病原ビブリオの鉄獲得機構

    第74回日本細菌学会総会  2001 

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  • 腸炎ビブリオVBNC菌の再生過程の解析

    第74回日本細菌学会総会  2001 

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  • Vibrio vulnificusプロテアーゼの赤血球凝集活性

    第74回日本細菌学会総会  2001 

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  • 腸炎ビブリオは3種類のferric siderophoreに対する外膜レセプターを発現する

    第74回日本細菌学会総会  2001 

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  • Vibrio mimicusにおけるaerobactinオペロンの遺伝子解析

    第74回日本細菌学会総会  2001 

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  • Vibrio mimicus臨床分離株の病原因子遺伝子

    第74回日本細菌学会総会  2001 

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  • Pathogenic factors of Vibrio vulnificus

    11th World Congress of Food Science and Technology  2001 

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  • 腸炎ビブリオのirgAB遺伝子:クローニング,発現調節とIrgAの菌体外分泌について

    第74回日本細菌学会総会  2001 

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  • Acinetobacter baumanniiの鉄製御遺伝子の単離と解析

    第74回日本細菌学会総会  2001 

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  • 病原細菌Vibrio vulnificusのヘム獲得機構の特異性

    第11回金属の関与する生体関連反応シンポジウム  2001 

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  • 腸炎ビブリオにおける鉄レギュロン関連遺伝子の解析

    第11回金属の関与する生体関連反応シンポジウム  2001 

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  • 海水中からの腸炎ビブリオの検出方法に関する研究

    第28回日本防菌防黴学会  2001 

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  • 環境分離菌が産生するチトクロムP450による内分泌撹乱化学物質の分解

    第28回日本防菌防黴学会  2001 

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  • 限定分解を受けたVibrio mimicus溶血毒素の生物活性

    第48回毒素シンポジウム  2001 

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  • Vibrio choleraeおよびVibrio mimicusのsucrose利用能に関する遺伝学的解析

    第54回日本細菌学会中国・四国支部総会  2001 

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  • Biological activities of the proteolyzed derivative from Vibrio mimicus hemolysin

    10th European Workshop Conference on Bacterial Pritein Toxins  2001 

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  • Vibrio parahaemolyticusの鉄獲得系:1. vibrioferin生合成遺伝子の解析

    第54回日本細菌学会中国・四国支部総会  2001 

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  • Vibrio parahaemolyticusの鉄獲得系:2. ferric vibrioferin輸送遺伝子の解析

    第54回日本細菌学会中国・四国支部総会  2001 

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  • ヒト非病原性ビブリオ属細菌の分泌する金属プロテアーゼに関する研究

    第54回日本細菌学会中国・四国支部総会  2001 

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  • Vibrio vulnificusにおけるferric aerobactin receptor遺伝子の解析

    第54回日本細菌学会中国・四国支部総会  2001 

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  • 腸炎ビブリオが産生するプロテアーゼに関する研究

    第35回腸炎ビブリオシンポジウム  2001 

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  • ヒト血清中に認められた細菌DNAの解析

    第54回日本細菌学会中国・四国支部総会  2001 

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  • Pathological actions of Vibrio vulnificus metalloprotease on the capillaries

    2nd Workshop on Natural Toxins  2000 

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  • Vibrio fluvialisの産生する金属プロテアーゼ

    第73回日本細菌学会総会  2000 

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  • Hemorrhagic damage caused by Vibrio vulnificus metalloprotease

    The Millennium for Microbiology Meeting  2000 

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  • Identification and characterization of a gene encoding the outer membrane receptor for heme in Vibrio mimicus

    The Millennium for Microbiology Meeting  2000 

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  • 腸炎ビブリオferric vibrioferrinレセプターのクローニングと解析

    第73回日本細菌学会総会  2000 

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  • Vibrio mimicus腸管付着因子としての主要外膜タンパク質の菌株間における多様性の解析

    第73回日本細菌学会総会  2000 

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  • Vibrio mimicusヘモリジン(VMH)の作用に及ぼすメンブレンポテンシャルの役割

    第47回毒素シンポジウム  2000 

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  • 岡山県児島湾において高頻度に分離されたtdh遺伝子保有腸炎ビブリオにおける病原因子の解析

    第34回腸炎ビブリオシンポジウム  2000 

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  • Isolation and characterization of a novel cytochrome P450 from environmental bacteria

    The Millennium for Microbiology Meeting  2000 

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  • ガラガラヘビ(Crotalus atrox)の毒液に含まれるムスカリン性アセチルコリン受容体阻害因子

    第47回毒素シンポジウム  2000 

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  • 腸炎ビブリオの鉄製御外膜蛋白をコードする遺伝子(pfuA,pvuA,pauA)のクローニングと解析

    第34回腸炎ビブリオシンポジウム  2000 

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  • 腸炎ビブリオpvuA(ferric vibrioferrin receptor)遺伝子のクローニングと解析

    第53回日本細菌学会中国・四国支部総会  2000 

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  • 海水より分離した腸炎ビブリオにおいて高頻度に検出されたtdhおよびtrh遺伝子の解析

    第53回日本細菌学会中国・四国支部総会  2000 

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  • Acinetobacter baumanniiにおける鉄制御遺伝子群の単離と解析

    第39回日本薬学会中国・四国支部学術大会  2000 

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  • Vibrio mimicusヘムレセプター遺伝子(mhuA)のクローニングと転写調節について

    第39回日本薬学会中国・四国支部学術大会  2000 

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  • Vibrio vulnificusヘモリジン(VVH)の構造と活性

    第53回日本細菌学会中国・四国支部総会  2000 

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  • 環境分離株の産生するチトクロムP450の精製と性質解析

    第39回日本薬学会中国・四国支部学術大会  2000 

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  • Vibrio vulnificusプロテアーゼの赤血球凝集作用

    第39回日本薬学会中国・四国支部学術大会  2000 

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Awards

  • 日本薬学会 中国四国支部奨励賞

    1999  

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    Country:Japan

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  • 日本細菌学会 黒屋奨学賞

    1999  

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Class subject in charge

  • Chemistry of Public Health 1 (2021academic year) 1st semester  - 火1,火2

  • Chemistry of Public Health 2 (2021academic year) Second semester  - 火1,火2

  • Microbiology (2021academic year) 3rd and 4th semester  - 金3,金4

  • Microbiology (2021academic year) 3rd and 4th semester  - 金3,金4

  • Environmental Micbiology (2021academic year) special  - その他

  • Life Science 2 (2021academic year) Late  - その他

  • Research Projects and Practicals: Environmental Health and Microbiology I (2021academic year) special  - その他

  • Lecture and Research Projects: Environmental Health and Microbiology I (2021academic year) special  - その他

  • Research Projects and Practicals: Environmental Health and Microbiology II (2021academic year) special  - その他

  • Lecture and Research Projects: Environmental Health and Microbiology II (2021academic year) special  - その他

  • Practice in Hygienic Pharmaceutical Sciences (2021academic year) Third semester  - その他6~9

  • Practice in Hygienic Pharmaceutical Sciences (2021academic year) Third semester  - その他6~9

  • Health Chemistry 3 (2021academic year) 1st semester  - 金1,金2

  • Health Chemistry 3 (2021academic year) 1st semester  - 金1,金2

  • Health Chemistry 4 (2021academic year) Second semester  - 金1,金2

  • Health Chemistry 4 (2021academic year) Second semester  - 金1,金2

  • Health Chemistry 6 (2021academic year) Fourth semester  - 水3,水4,水5

  • Health Chemistry 6 (2021academic year) Fourth semester  - 水3,水4,水5

  • Microbiology (2020academic year) 3rd and 4th semester  - 金3,金4

  • Microbiology (2020academic year) 3rd and 4th semester  - 金3,金4

  • Microbiology (2020academic year) 3rd and 4th semester  - 金3,金4

  • Environmental Micbiology (2020academic year) special  - その他

  • Life Science 2 (2020academic year) special  - その他

  • Research Projects and Practicals: Environmental Health and Microbiology I (2020academic year) special  - その他

  • Lecture and Research Projects: Environmental Health and Microbiology I (2020academic year) special  - その他

  • Research Projects and Practicals: Environmental Health and Microbiology II (2020academic year) special  - その他

  • Lecture and Research Projects: Environmental Health and Microbiology II (2020academic year) special  - その他

  • Practice in Hygienic Pharmaceutical Sciences (2020academic year) special  - その他

  • Practice in Hygienic Pharmaceutical Sciences (2020academic year) special  - その他

  • Health Chemistry 3 (2020academic year) 1st semester  - 金1,金2

  • Health Chemistry 3 (2020academic year) 1st semester  - 金1,金2

  • Health Chemistry 4 (2020academic year) Second semester  - 金1,金2

  • Health Chemistry 4 (2020academic year) Second semester  - 金1,金2

  • Health Chemistry 6 (2020academic year) Fourth semester  - 水3,水4

  • Health Chemistry 6 (2020academic year) Fourth semester  - 水3,水4

  • Health Chemistry II (2020academic year) 1st-4th semester  - [第1学期]金1,金2, [第2学期]水3,水4, [第3学期]水3,水4, [第4学期]水3,水4

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