論文 - 井口 純
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Escherichia coli O-Genotyping PCR: a Comprehensive and Practical Platform for Molecular O Serogrouping 査読あり
Iguchi A, Iyoda S, Seto K, Morita-Ishihara T, Scheutz F, Ohnishi M Pathogenic E. coli Working Group in Japan
Journal of Clinical Microbiology 53 ( 8 ) 2427 - 2432 2015年8月
記述言語：英語 掲載種別：研究論文（学術雑誌）
DOI： 10.1128/JCM.00321-15
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Rapid and easy in silico serotyping of Escherichia coli using Whole genome sequencing (WGS) data 査読あり
Joensen KG, Tetzschner AM, Iguchi A, Aarestrup FM, Scheutz F
Journal of Clinical Microbiology 53 ( 8 ) 2410 - 2426 2015年8月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster 査読あり
Iguchi A, Iyoda S, Kikuchi T, Ogura Y, Katsura K, Ohnishi M, Hayashi T, Thomson NR
DNA Research 2015年2月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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腸管出血性大腸菌の主要なO血清群と病原性遺伝子を判定するOne-shotマルチプレックスPCR法の開発と評価
井口 純, 秋吉 充子, 伊豫田 淳, 大西 真
日本食品微生物学会雑誌 32 ( 4 ) 215 - 218 2015年
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：日本食品微生物学会
Enterohemorrhagic <i>E. coli</i> (EHEC) or Shiga toxin-producing <i>E. coli</i> is known as an important food-borne pathogen, and O157 is the most frequently reported O serogroups of EHEC strains associated with bloody diarrhea and hemolytic-uremic syndrome worldwide. Subsequently O26, O111, O103, O145, O121 and O165 serogroups of EHEC were also frequently isolated from patients with severe diseases in Japan. The O serogrouping of EHEC is essential in outbreak investigations and surveillance. In a previous study, we developed a comprehensive and practical platform for molecular O-serogrouping of <i>E. coli</i> strains, named as <i>E. coli</i> O-genotyping PCR (Iguchi, A., <i>et al</i>.: J. Clin. Microbiol., in press), targeting unique sequences on each O-antigen biosynthesis gene cluster. Based on the PCR system, we now developed an EHEC-specific multiplex PCR system, named as "MP-1 plus," targeting seven major EHEC O serogroups and three virulence genes, <i>stx1</i>, <i>stx2</i> and <i>eae</i>. This primer set contains 10 primer pairs that amplify products with different sizes, and validation studies using reference and wild strains showed that the results were reliable enough. The one-shot PCR method reported here might be a promising tool for the identification and subtyping of EHEC strains for outbreak investigations as well as for the surveillance.
DOI： 10.5803/jsfm.32.215
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さまざまなO血清群に属する志賀毒素産生性大腸菌の市販選択培地上での生育特性
秋吉 充子, 中村 寛海, 伊豫田 淳, 石原 朋子, 加藤 結子, 井口 純
日本食品微生物学会雑誌 32 ( 4 ) 192 - 198 2015年
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：日本食品微生物学会
STECの主要なO血清群（O157, O111, O26, O103, O121, O145）の選択分離には，CT-SMACなどの亜テルル酸塩が添加された寒天平板培地が広く用いられている．しかし，主要なO血清群を除くさまざまなO血清群STECに対しては，STEC選択培地の有用性はあまり評価されていない．本研究ではCT-SMACを含む4種類の市販STEC選択培地（クロモアガーSTEC, XM-EHEC, “KBM” EHEC発色基質培地）を用いて，さまざまなO血清群に属するSTEC 152株の生育特性を調べた．その結果，16種類のO血清群に属する36株は4種類すべての選択培地で生育し，それらすべてが亜テルル酸塩耐性に関与する<i>terA</i>を保有していることがPCRで確認された．そのうち27株は<i>eae</i>陽性STECであり，残る9株は<i>eae</i>陰性STECであった．本研究で明らかとなったO血清群（またはO genotype）に基づく亜テルル酸塩添加選択培地での生育特性と<i>ter</i>オペロンの保有分布は，分離標的とされるSTECのO血清群が明らかな場合に，培地を選ぶうえで有用な情報になると考えられた．
DOI： 10.5803/jsfm.32.192
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Honda A., Hirose M., Sankai T., Yasmin L., Yuzawa K., Honsho K., Izu H., Iguchi A., Ikawa M., Ogura A.
Experimental Animals 64 ( 1 ) 31 - 37 2015年
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Experimental Animals
© 2015 Japanese Association for Laboratory Animal Science. Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.
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Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution
Von Mentzer A., Connor T., Wieler L., Semmler T., Iguchi A., Thomson N., Rasko D., Joffre E., Corander J., Pickard D., Wiklund G., Svennerholm A., Sjöling Å., Dougan G.
Nature Genetics 46 ( 12 ) 1321 - 1326 2014年12月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Nature Genetics
© 2014 Nature America, Inc. All rights reserved. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea, produce heat-stable and/or heat-labile enterotoxins and at least 25 different colonization factors that target the intestinal mucosa. The genes encoding the enterotoxins and most of the colonization factors are located on plasmids found across diverse E. coli serogroups. Whole-genome sequencing of a representative collection of ETEC isolated between 1980 and 2011 identified globally distributed lineages characterized by distinct colonization factor and enterotoxin profiles. Contrary to current notions, these relatively recently emerged lineages might harbor chromosome and plasmid combinations that optimize fitness and transmissibility. These data have implications for understanding, tracking and possibly preventing ETEC disease.
DOI： 10.1038/ng.3145
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Identification of Enterococcus faecium and Enterococcus faecalis as vanC-type Vancomycin-Resistant Enterococci (VRE) from sewage and river water in the provincial city of Miyazaki, Japan 査読あり
Nishiyama M, Iguchi A, Suzuki Y
Journal of Environmental Science and Health, Part A Toxic/Hazardous Substances and Environmental Engineering 2014年12月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Identification of enterotoxigenic Escherichia coli (ETEC) clades with significant long-term global distribution. 査読あり
von Mentzer A, Connor T, Wieler LH, Semmler T, Iguchi A, Thomson NR, Rasko DA, Joffre E, Corander J, Pickard D, Wiklund G, Svennerholm A, Sj_ling A, Dougan G.
Nature Genetics 2014年12月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Single-step generation of rabbits carrying a targeted allele of Tyrosinase gene using CRISPR/Cas9 査読あり
Honda A, Hirose M, Sankai T, Yasmin L, Yuzawa K, Honsho K, Izu H, Iguchi A, Ikawa M, Ogura A
Experimental Animals 2014年9月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Identification of O Serotypes, Genotypes and Virulotypes of Shiga Toxin-Producing Escherichia coli Isolates Including Non-O157 from Beef Cattle in Japan 査読あり
Mekata H, Iguchi A, Kawano K, Kirino Y, Kobayashi I, Misawa N
Journal of Food Protection 2014年8月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Phylogenetic clades 6 and 8 of enterohemorrhagic Escherichia coli O157:H7 with particular stx subtypes are more frequently found in isolates from hemolytic uremic syndrome patients than from asymptomatic carriers 査読あり
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Open Forum Infectious Diseases 2014年8月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Genome Evolution and Plasticity of Serratia marcescens, an Important Multidrug-Resistant Nosocomial Pathogen 査読あり
Iguchi A, Nagaya Y, Pradel E, Ooka T, Ogura Y, Katsura K, Kurokawa K, Oshima K, Hattori M, Parkhill J, Sebaihia M, Coulthurst SJ, Gotoh N, Thomson NR, Ewbank JJ, Hayashi T.
Genome Biology and Evolution 2014年8月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Mekata H., Iguchi A., Kawano K., Kirino Y., Kobayashi I., Misawa N.
Journal of Food Protection 77 ( 8 ) 1269 - 1274 2014年8月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Food Protection
Bovines are recognized as an important reservoir of Shiga toxin-producing Escherichia coli (STEC). Although STEC strains are significant foodborne pathogens, not all of the STEC held by cattle are pathogenic, and which type of STEC that will become epidemic in humans is unpredictable. Information about the prevalence of serotype and virulence gene distribution in beef cattle is insufficient to develop monitoring and controlling activities for a food safety and security program. Thus, this study investigated the prevalence of O157 and non-O157 STEC in Japanese beef cattle and characterized the isolates by the type of O antigen and several virulence markers to help predict the pathogenicity. In this study, 64.2% (176 of 274) of enrichment cultures of fecal samples collected from an abattoir and farms were stx<sup>1</sup> and/or stx<sup>2</sup> positive by PCR. STEC strains were isolated from 22.1% (39 of 176) of the positive fecal samples, and these isolates represented 17 types of O antigen (O1, O2 or O50, O5, O8, O55, O84, O91, O109, O113, O136, O150, O156, O157, O163, O168, O174, and O177). Two selective media targeting major STEC groups, cefixime-tellurite sorbitol MacConkey agar and CHROMagar O26/O157, allowed isolation of a variety of STEC strains. The most frequently isolated STEC was O113 (8 of 39), which has previously been reported as a cause of foodborne infections. Although most of the O113 STEC isolated from infected patients possessed the enterohemolysin (hlyA) gene, none of the O113 STEC cattle isolates possessed the hlyA gene. The second most common isolate was O157 (6 of 39), and all these isolates contained common virulence factors, including eae, tir, lpf<inf>1</inf>, lpf<inf>2</inf>, and hlyA. This study shows the prevalence of O157 and non-O157 STEC in Japanese beef cattle and the relationship of O antigen and virulotypes of the isolates. This information may improve identification of the source of infection, developing surveillance programs or the current understanding of virulence factors of STEC infections. Copyright ©, International Association for Food Protection.
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Electricity generation from cattle manure slurry by cassette-electrode microbial fuel cells 査読あり
Inoue K*, Ito T, Kawano Y, Iguchi A, Miyahara M, Suzuki Y, Watanabe K
Journal of bioscience and bioengineering 116 ( 5 ) 610 - 615 2013年10月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Osawa K, Shigemura K, Iguchi A, Shirai H, Imayama T, Seto K, Raharjo D, Fujisawa M, Osawa R, Shirakawa T
Microbiology and immunology 57 ( 9 ) 616 - 623 2013年9月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Osawa K., Shigemura K., Iguchi A., Shirai H., Imayama T., Seto K., Raharjo D., Fujisawa M., Osawa R., Shirakawa T.
Microbiology and Immunology 57 ( 9 ) 616 - 623 2013年9月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Microbiology and Immunology
Escherichia coli O157 strains belonging to a distinct lineage and expressing different O-antigen (Oag) lengths were isolated. Although the function of wzz in E. coli has not been adequately investigated, this gene is known to be associated with regulation of Oag length. Using E. coli O157:H7 ATCC43888 (wild-type), several wzz mutants of E. coli O157, including a wzz deletion mutant, were generated and the relationship between the length of Oag modulated by the wzz gene and sensitivities to serum complement investigated. SDS-PAGE, immunoblot analyses and sensitivity tests to human serum complement were performed on these strains. The lengths of the O157-antigen could be modulated by the wzz gene mutations and were classified into long, intermediate and short groups. The short chain mutant was more serum sensitive than the wild-type strain and the other wzz mutants (P<0.001). In conclusion, Oag chain length modulated by the wzz gene in E. coli O157 influences its sensitivities to serum complement. The present findings suggest that E. coli O157 strains with intermediate or long length Oag chains might show greater resistance to serum complement than those with short chains. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.
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Susceptibility of Pseudomonas aeruginosa isolates collected from river water in Japan to antipseudomonal agents 査読あり
Suzuki Y, Kajii S, Nishiyama M, Iguchi A
Science of The Total Environment 15 148 - 154 2013年4月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Iguchi A., Iyoda S., Ohnishi M.
Journal of Clinical Microbiology 50 ( 9 ) 2894 - 2900 2012年9月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Clinical Microbiology
Shiga toxin-producing Escherichia coli (STEC) is one of the most important groups of food-borne pathogens, and STEC strains belonging to the serotype O103:H2 can cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. STEC O103:non-H2 strains are also sometimes isolated from human patients, but their genetic characteristics and role in significant human enteric disease are not yet understood. Here, we investigated 17 STEC O103:non-H2 strains, including O103:H11, O103:H25, O103:HUT (UT [untypeable]), and O103:H- (nonmotile) isolated in Japan, and their characteristics were compared to those of STEC O103:H2 and other serotype STEC strains. Sequence analyses of fliC and eae genes revealed that strains possessed any of the following combinations: fliC-H2/eae-epsilon, fliC-H11/eae-beta1, and fliC-H25/eae-theta, where fliC-H2, -H11, and -H25 indicate fliC genes encoding H2, H11, and H25 flagella antigens, respectively, and eae-epsilon, -beta1, and -theta indicate eae genes encoding epsilon, beta1, and theta subclass intimins, respectively. Phylogenetic analysis based on the sequences of seven housekeeping genes demonstrated that the O103:H11/[fliC-H11] and O103:H25/[fliC-H25] strains formed two distinct groups, different from that of the O103:H2/[fliC-H2] strains. Interestingly, a group consisting of O103:H11 strains was closely related to STEC O26:H11, which is recognized as a most important non-O157 serotype, suggesting that the STEC O103:H11 and STEC O26:H11 clones evolved from a common ancestor. The multiplex PCR system for the rapid typing of STEC O103 strains described in the present study may aid clinical and epidemiological studies of the STEC O103:H2, O103:H11, and O103:H25 groups. In addition, our data provide further insights into the high variability of STEC stains with emerging new serotypes. Copyright © 2012, American Society for Microbiology. All Rights Reserved.
DOI： 10.1128/JCM.00789-12
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Murase K., Ooka T., Iguchi A., Ogura Y., Nakayama K., Asadulghani M., Islam M., Hiyoshi H., Kodama T., Beutin L., Hayashi T.
Microbiology 158 ( 3 ) 746 - 758 2012年3月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Microbiology
Among three haemolysins identified thus far in Escherichia coli, alpha-haemolysin (HlyA) is encoded on the pathogenicity islands of extraintestinal pathogenic strains, while enterohaemolysin (EhxA) is encoded on the virulence plasmids of enterohaemorrhagic E. coli (EHEC) strains. In contrast, the gene for haemolysin E (HlyE) is located on the E. coli chromosome backbone and is therefore widely distributed among E. coli strains. However, because hlyE gene expression is repressed by the H-NS protein and because the gene has been disrupted in many strains, its haemolytic activity cannot be detected in wild-type strains by routine screening on blood agar plates. In this study, we found that the HlyE-derived haemolytic activity of enteropathogenic E. coli (EPEC) O55:H7 can be detected after anaerobic cultivation on a washed blood agar plate (EHX plate) that is used to detect the production of EhxA.We also found that the haemolytic activity of EHECO157:H7 observed on EHX plates under aerobic and anaerobic growth conditions is derived from EhxA and HlyE, respectively; this differential expression of the two haemolysins occurs at the transcriptional level. Our analysis of 60 E. coli strains of various pathotypes and phylogenies for their repertoires of haemolysin genes, haemolytic phenotypes and hlyE gene sequences revealed that HlyE activity can generally be detected on EHX plates under anaerobic growth conditions if the gene is intact. Furthermore, our results indicate that hlyE gene inactivation occurred in three of the five E. coli lineages (phylogroups A, B1 and B2), which demonstrates phylogroup-specific gene disruption patterns. © 2012 SGM.