論文 - 井口 純
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Iguchi A., Ueno Y., Hoshinoo K., Okuno M., Uemura R., You G., Ogura Y., Takamatsu D.
Scientific Reports 15 ( 1 ) 2025年4月
担当区分：筆頭著者, 責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Scientific Reports
The bovine respiratory disease complex (BRDC) is a global issue affecting dairy and beef farms and is of major concern due to the high morbidity and mortality rates in calves, as well as decreased production it causes, resulting in significant economic losses. Mannheimia haemolytica is one of the secondary pathogens associated with BRDC. M. haemolytica is classified into 12 serotypes based on capsular antigens. In addition to the prevalent serotypes A1, A2, and A6, strains belonging to other serotypes also cause respiratory diseases in cattle and other ruminants, necessitating a method for their rapid and easy identification. In this study, we organized the capsule biosynthesis genes based on genome information from all serotype strains and designed 11 PCR primer pairs targeting serotype-specific genes, which could individually identify serotypes A14/A16, which possess homologous genes, as well as all other serotypes. Additionally, we developed two multiplex PCR kits that include these serotype-specific and M. haemolytica species-specific primers. Specificity testing using reference strains confirmed that these kits can simultaneously and clearly identify both the species and their serotypes. The PCR-based system described here could be a valuable tool for subtyping M. haemolytica strains in epidemiological studies and surveillance efforts in cattle and other reservoir animals. This study also carefully compared and discussed the differences between the capsule synthesis genes of A8 and A14 from previously published and those obtained in this study.
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Infectious endophthalmitis associated with umbilical infection in Japanese black calf: a case report 査読あり 国際共著
Sato R., Iguchi A., Uemura R., Tsujita H., Steiner A.
Frontiers in Veterinary Science 12 2025年4月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Frontiers in Veterinary Science
A 3-day-old Japanese black calf presented with a swollen and tender umbilical cord and diffusely cloudy and keratoconus eyes. Abdominal ultrasonography confirmed mild enlargement of both umbilical arteries and the urachus with a hyperechoic lumen. Additionally, a hyperechogenic structure suggestive of pus was noted near the abdominal wall. Fluorescein staining revealed corneal epithelial injury, whereas slit lamp examination identified corneal edema, increased corneal thickness, and keratitis with vascularization of the corneal stroma. Based on these findings, diagnoses of omphaloarteritis, omphalourachitis, and bullous keratitis were made. Both umbilical arteries and the urachus were surgically removed; both ocular globes were covered with a third eyelid flap, which was released 30 days postoperatively. On the follow-up, ocular ultrasonography indicated bleeding or fibrin deposits in the vitreous body of the right ocular globe. Because intraocular inflammation was suspected, anterior aqueous humor was collected from the right ocular globe, and bacterial examination was performed with the umbilical artery abscess, urachal abscess, and intraabdominal pus collected intraoperatively. Escherichia coli was isolated from the umbilical artery abscess, urachal abscess, intraabdominal pus, and aqueous humor, and all isolates exhibited identical genotypes. These findings suggest that endophthalmitis occurred as a result of the hematogenous spread of bacteria originating from septic umbilical cord remnants and that ocular ultrasonography is useful for assessing intraocular pathologies.
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Elbastawesy A.M., Awasthi S.P., Hatanaka N., Hinenoya A., Iguchi A., Ombarak R.A., Deeb A.M.M., Yamasaki S.
International Dairy Journal 162 2025年3月
掲載種別：研究論文（学術雑誌） 出版者・発行元：International Dairy Journal
Milk and dairy products are popular in Egyptian diets, but their contamination with Escherichia coli, poses health risks. This study investigated the prevalence of potentially pathogenic and antimicrobial-resistant E. coli in raw milk and dairy products from Kafrelsheikh and Algarbia Governorates, Egypt. Two hundred ten samples including raw buffalo milk, goat milk, Domiati cheese, Domiati cheese with pepper, rayeb, and yogurt were analyzed. The prevalence of E. coli was 26.2%, with the highest occurrence in buffalo milk (68.0%) and the lowest in rayeb (7.5%). Based on ERIC-PCR, eighty-four non-clonal E. coli strains were selected and further characterized. Among tested virulence genes, adhesion genes such as lpfAO113 and ehaA, were the most prevalent. Toxin-encoding genes such as astA, cdt, cnf, and hlyA were also detected. The cytotoxic and hemolytic activity of cdt, cnf, and hylA carrying E. coli were confirmed on CHO cells and sheep blood agar, respectively. Twenty-three (27.4%) strains showed resistance to one or more antimicrobials, and 10 (11.9%) strains exhibited multidrug resistance (MDR). Among 12 antimicrobials tested resistance against ampicillin, streptomycin and tetracycline was the highest. Phylogenetic analysis and O-genotyping indicated clinically significant strains such as Og103, Og157 and OgGp9. Notably, two OgGp9 strains were OgGp9:Hg18 and phylogenetic group D, like those associated with a large diarrheal outbreak caused by milk consumption in Japan, in 2021. Interestingly, these two strains harbored a complete type 3 secretion system 2 locus (ETT2) and one of these strains was MDR. These findings indicate that these dairy products were contaminated with potentially pathogenic and multidrug-resistant E. coli. This is the first report to analyze E. coli contamination in Domiati cheese with pepper and detect OgGp9:Hg18 outbreak-associated strains with ETT2 and MDR in Egypt.
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下痢原性大腸菌食中毒事例におけるO血清群およびH型の遺伝子型判定PCR検査法の活用 査読あり
小田切 正昭, 加藤 直樹, 曽根 美紀, 土屋 彰彦, 近藤 貴英, 井口 純
日本食品微生物学会雑誌 41 ( 3 ) 119 - 123 2024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：日本食品微生物学会
Diarrheagenic <i>Escherichia coli</i> strains are mainly classified into the following five pathotypes; enteropathogenic <i>E. coli</i>, enterotoxigenic <i>E. coli</i>, enteroinvasive <i>E. coli</i>, enterohemorrhagic <i>E. coli</i> and enteroaggregative <i>E. coli</i>. <i>E. coli</i> strains of various pathotypes can be further classified by their serotypes combined by O-serogroup and H-type. Generally in Japan, the commercial O-antisera and H-antisera are used to serotype strains. These serotypes provide useful information in investigations of outbreaks and epidemiological studies. In May 2021, a large-scale food poisoning outbreak thought to be caused by <i>E. coli</i> occurred in Saitama City. Serotyping of 21 <i>E. coli</i> strains carrying virulence genes isolated from patients and an asymptomatic cooking worker was carried out using a commercially available kit, but most of the strains (57%) could not be serotyped. Therefore, in this study, we tried the multiplex PCR methods including <i>E. coli</i> O-genotyping PCR and <i>E. coli</i> H-genotyping PCR. As a result, only 5% of strains could not be determined their Og-types, and the main genotype in this case was found to be Og104:Hg4 (12/21 strains) carrying <i>astA</i>+<i>aggR</i> or <i>estA2</i>+<i>elt</i>+<i>astA</i>+<i>aggR</i>. In conclusion, we believe that Og-typing PCR and Hg-typing PCR are effective methods in investigating cases caused by pathogenic <i>E. coli</i> belonging to rare pathotypes.
DOI： 10.5803/jsfm.41.119
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Lee K., Iguchi A., Terano C., Hataya H., Isobe J., Seto K., Ishijima N., Akeda Y., Ohnishi M., Iyoda S.
Microbiology spectrum 12 ( 1 ) 2024年1月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Microbiology spectrum
IMPORTANCE: Hemolytic uremic syndrome (HUS) is a life-threatening disease caused by Shiga toxin-producing Escherichia coli (STEC) infection. The treatment approaches for STEC-mediated typical HUS and atypical HUS differ, underscoring the importance of rapid and accurate diagnosis. However, specific detection methods for STECs other than major serogroups, such as O157, O26, and O111, are limited. This study focuses on the utility of PCR-based O-serotyping, serum agglutination tests utilizing antibodies against the identified Og type, and isolation techniques employing antibody-conjugated immunomagnetic beads for STEC isolation. By employing these methods, we successfully isolated a STEC strain of a minor serotype, O76:H7, from a HUS patient.
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Iguchi A., Takemura T., Ogura Y., Nguyen T.T.H., Kikuchi T., Okuno M., Tokizawa A., Iwashita H., Pham H.Q.A., Doan T.H., Tran N.L., Tran T.L., Nguyen T.H., Tran T.H., Pham T.N.L., Dao T.D., Vu T.M.H., Nguyen T.N., Vu H., Nguyen V.T., Vu T.T.H., Le T.H., Lai T.A., Ngo T.C., Hasebe F., Nguyen D.T., Yamashiro T.
PLoS Neglected Tropical Diseases 17 ( 4 ) 2023年4月
掲載種別：研究論文（学術雑誌） 出版者・発行元：PLoS Neglected Tropical Diseases
Background Diarrheagenic Escherichia coli (DEC) is a group of bacterial pathogens that causes life-threatening diarrhea in children in developing countries. However, there is limited information on the characteristics of DEC isolated from patients in these countries. A detailed genomic analysis of 61 DEC-like isolates from infants with diarrhea was performed to clarify and share the characteristics of DEC prevalent in Vietnam. Principal findings DEC was classified into 57 strains, including 33 enteroaggregative E. coli (EAEC) (54.1%), 20 enteropathogenic E. coli (EPEC) (32.8%), two enteroinvasive E. coli (EIEC) (3.3%), one enterotoxigenic E. coli (ETEC), and one ETEC/EIEC hybrid (1.6% each), and surprisingly into four Escherichia albertii strains (6.6%). Furthermore, several epidemic DEC clones showed an uncommon combination of pathotypes and serotypes, such as EAEC Og130: Hg27, EAEC OgGp9:Hg18, EAEC OgX13:H27, EPEC OgGp7:Hg16, and E. albertii EAOg1:HgUT. Genomic analysis also revealed the presence of various genes and mutations associated with antibiotic resistance in many isolates. Strains that demonstrate potential resistance to ciprofloxacin and ceftriaxone, drugs recommended for treating childhood diarrhea, accounted for 65.6% and 41%, respectively. Significance Our finding indicate that the routine use of these antibiotics has selected resistant DECs, resulting in a situation where these drugs do not provide in therapeutic effects for some patients. Bridging this gap requires continuous investigations and information sharing regarding the type and distribution of endemic DEC and E. albertii and their antibiotic resistance in different countries.
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Suzuki Y., Shimizu H., Tamai S., Hoshiko Y., Maeda T., Nukazawa K., Iguchi A., Masago Y., Ishii S.
Environmental Monitoring and Assessment 195 ( 2 ) 2023年2月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Environmental Monitoring and Assessment
Waterborne diseases due to pathogen contamination in water are a serious problem all over the world. Accurate and simultaneous detection of pathogens in water is important to protect public health. In this study, we developed a method to simultaneously detect various pathogenic Escherichia coli by sequencing the amplicons of multiplex PCR. Our newly designed multiplex PCR amplified five genes for pathogenic E. coli (uidA, stx1, stx2, STh gene, and LT gene). Additional two PCR assays (for aggR and eae) were also designed and included in the amplicon sequencing analysis. The same assays were also used for digital PCR (dPCR). Strong positive correlations were observed between the sequence read count and the dPCR results for most of the genes targeted, suggesting that our multiplex PCR-amplicon sequencing approach could provide quantitative information. The method was also successfully applied to monitor the level of pathogenic E. coli in river water and wastewater samples. The approach shown here could be expanded by targeting genes for other pathogens.
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Habets A., Touzain F., Lucas P., Huong N.T.T., Iguchi A., Crombé F., Korsak N., Piérard D., Saulmont M., Cox E., Engelen F., Mainil J., Thiry D.
Veterinary Sciences 9 ( 9 ) 2022年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Veterinary Sciences
Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (AE) lesions and cause non-bloody diarrhea in mammals. A minority of bovine EPEC belong to one of the ten classical serotypes of human and bovine AE-STEC. The purpose of this study was to identify five non-classical O serotypes (O123/186, O156, O177, O182, and O183) among bovine EPEC and to characterize their virulence repertoires by whole genome sequencing. Around 40% of the 307 EPEC from 307 diarrheic calves, 368 EPEC from 47 healthy cattle, and 131 EPEC from 36 healthy calves in dairy farms were analyzed. Serotype O177 was the most frequent among EPEC from diarrheic and healthy calves, while the O156 was the most frequent in healthy cattle. The genomic analysis identified different H serotypes, MLSTypes, and/or eae gene subtypes among the O156 and O177 EPEC, while the O182 was homogeneous. The virulence gene profiles of bovine EPEC were closely related to each other and to the profiles of ten bovine and human AE-STEC. These results emphasize the need for additional studies to identify more O:H serotypes of bovine EPEC and to elucidate their origin and evolution of EPEC with regard to AE-STEC belonging to the same O:H serotypes.
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Murase K., Arakawa E., Izumiya H., Iguchi A., Takemura T., Kikuchi T., Nakagawa I., Thomson N.R., Ohnishi M., Morita M.
Microbial Genomics 8 ( 8 ) 2022年8月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Microbial Genomics
Approximately 200 O-serogroups of Vibrio cholerae have already been identified; however, only 2 serogroups, O1 and O139, are strongly related to pandemic cholera. The study of non-O1 and non-O139 strains has hitherto been limited. Nevertheless, there are other clinically and epidemiologically important serogroups causing outbreaks with cholera-like disease. Here, we report a comprehensive genome analysis of the whole set of V. cholerae O-serogroup reference strains to provide an overview of this important bacterial pathogen. It revealed structural diversity of the O-antigen biosynthesis gene clusters located at specific loci on chromosome 1 and 16 pairs of strains with almost identical O-antigen biosynthetic gene clusters but differing in serologi-cal patterns. This might be due to the presence of O-antigen biosynthesis-related genes at secondary loci on chromosome 2.
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Ono T., Taniguchi I., Nakamura K., Nagano D.S., Nishida R., Gotoh Y., Ogura Y., Sato M.P., Iguchi A., Murase K., Yoshimura D., Itoh T., Shima A., Dubois D., Oswald E., Shiose A., Gotoh N., Hayashi T.
Microbial genomics 8 ( 3 ) 2022年3月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Microbial genomics
Serratia marcescens is an important nosocomial pathogen causing various opportunistic infections, such as urinary tract infections, bacteremia and sometimes even hospital outbreaks. The recent emergence and spread of multidrug-resistant (MDR) strains further pose serious threats to global public health. This bacterium is also ubiquitously found in natural environments, but the genomic differences between clinical and environmental isolates are not clear, including those between S. marcescens and its close relatives. In this study, we performed a large-scale genome analysis of S. marcescens and closely related species (referred to as the 'S. marcescens complex'), including more than 200 clinical and environmental strains newly sequenced here. Our analysis revealed their phylogenetic relationships and complex global population structure, comprising 14 clades, which were defined based on whole-genome average nucleotide identity. Clades 10, 11, 12 and 13 corresponded to S. nematodiphila, S. marcescens sensu stricto, S. ureilytica and S. surfactantfaciens, respectively. Several clades exhibited distinct genome sizes and GC contents and a negative correlation of these genomic parameters was observed in each clade, which was associated with the acquisition of mobile genetic elements (MGEs), but different types of MGEs, plasmids or prophages (and other integrative elements), were found to contribute to the generation of these genomic variations. Importantly, clades 1 and 2 mostly comprised clinical or hospital environment isolates and accumulated a wide range of antimicrobial resistance genes, including various extended-spectrum β-lactamase and carbapenemase genes, and fluoroquinolone target site mutations, leading to a high proportion of MDR strains. This finding suggests that clades 1 and 2 represent hospital-adapted lineages in the S. marcescens complex although their potential virulence is currently unknown. These data provide an important genomic basis for reconsidering the classification of this group of bacteria and reveal novel insights into their evolution, biology and differential importance in clinical settings.
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Diversity of the Tellurite Resistance Gene Operon in Escherichia coli 査読あり
Nguyen T.T.H., Kikuchi T., Tokunaga T., Iyoda S., Iguchi A.
Frontiers in Microbiology 12 2021年5月
担当区分：責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Frontiers in Microbiology
Tellurite is highly toxic to most bacteria owing to its strong oxidative ability. However, some bacteria demonstrate tellurite resistance. In particular, some Escherichia coli strains, including Shiga toxin-producing E. coli O157:H7, are known to be resistant to tellurite. This resistance is involved in ter operon, which is usually located on a prophage-like element of the chromosome. The characteristics of the ter operon have been investigated mainly by genome analysis of pathogenic E. coli; however, the distribution and structural characteristics of the ter operon in other E. coli are almost unknown. To clarify these points, we examined 106 E. coli strains carrying the ter operon from various animals. The draft genomes of 34 representative strains revealed that ter operons were clearly classified into four subtypes, ter-type 1–4, at the nucleotide sequence level. Complete genomic sequences revealed that operons belonging to three ter-types (1, 3, and 4) were located on the prophage-like elements on the chromosome, whereas the ter-type 2 operon was located on the IncHI2 plasmid. The positions of the tRNASer, tRNAMet, and tRNAPhe indicated the insertion sites of elements carrying the ter operons. Using the PCR method developed in this study, 106 strains were classified as type 1 (n = 66), 2 (n = 13), 3 (n = 8), and 4 (n = 17), and two strains carried both types 1 and 2. Furthermore, significant differences in the minimum inhibitory concentration (MIC) of tellurite were observed between strains carrying ter-type 4 and the others (p < 0.05). The ter-type was also closely related to the isolation source, with types 2 and 4 associated with chickens and deer, respectively. This study provided new insights related not only to genetic characteristics of the ter operons, but also to phenotypic and ecological characteristics that may be related to the diversity of the operon.
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Le P.Q., Awasthi S.P., Hatanaka N., Hinenoya A., Hassan J., Ombarak R.A., Iguchi A., Tran N.T.T., Dao K.V.T., Vien M.Q., Le H.X., Do H.T., Yamamoto Y., Yamasaki S.
International Journal of Food Microbiology 346 2021年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：International Journal of Food Microbiology
The aim of the study was to assess the presence of genes in ESBL-producing E. coli (ESBL-Ec) isolated from retail raw food in Nha Trang, Vietnam. A total of 452 food samples comprising chicken (n = 116), pork (n = 112), fish (n = 112) and shrimp (n = 112) collected between 2015 and 2017 were examined for the prevalence of ESBL-Ec. ESBL-Ec were detected in 46.0% (208/452) of retail food samples, particularly in 66.4% (77/116), 55.4% (62/112), 42.0% (47/112) 19.6% (22/112) of chicken, pork, fish and shrimp, respectively. Sixty-five out of the 208 (31.3%) ESBL-Ec isolates were positive for mcr genes including mcr-1, mcr-3 and both mcr-1 and mcr-3 genes in 56/208 (26.9%), 1/208 (0.5%) and 8/208 (3.9%) isolates, respectively. Particularly, there was higher prevalence of mcr-1 in ESBL-Ec isolates from chicken (53.2%, 41/77) in comparison to shrimp (22.7%, 5/22), pork (11.3%, 7/62) and fish (6.4%, 3/47). mcr-3 gene was detected in co-existence with mcr-1 in ESBL-Ec isolates from shrimp (9.1%, 2/22), pork (8.1%, 5/62) and fish (2.1%, 1/47) but not chicken. The 65 mcr-positive ESBL-Ec (mcr-ESBL-Ec) were colistin-resistant with the MICs of 4–8 μg/mL. All mcr-3 gene-positive isolates belonged to group A, whereas phylogenetic group distribution of isolates harboring only mcr-1 was B1 (44.6%), A (28.6%) and D (26.8%). PFGE analysis showed diverse genotypes, although some isolates demonstrated nearly clonal relationships. S1-PFGE and Southern hybridization illustrated that the mcr-1 and mcr-3 genes were located either on chromosomes or on plasmids. However, the types of mcr genes were harbored on different plasmids with varied sizes of 30–390 kb. Besides, the ESBL genes of CTX-M-1 or CTX-M-9 were also detected to be located on plasmids. Noteworthy, co-location of CTX-M-1 with mcr-1 or mcr-3 genes on the same plasmid was identified. The conjugation experiment indicated that the mcr-1 or mcr-3 was horizontally transferable. All mcr-ESBL-Ec isolates were multidrug resistance (resistance to ≥3 antimicrobial classes). Moreover, β-Lactamase-encoding genes of the CTX-M-1 (78.5%), CTX-M-9 (21.5%), TEM (61.5%) groups were found in mcr-ESBL-Ec. The astA gene was detected in 27 (41.5%) mcr-ESBL-Ec isolates demonstrating their potential virulence. In conclusion, mcr-1 and mcr-3 genes existed individually or concurrently in ESBL-Ec isolates recovered from retail raw food in Nha Trang city, which might further complicate the antimicrobial-resistant situation in Vietnam, and is a possible health risk for human.
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Lee K., Iguchi A., Uda K., Matsumura S., Miyairi I., Ishikura K., Ohnishi M., Seto J., Ishikawa K., Konishi N., Obata H., Furukawa I., Nagaoka H., Morinushi H., Hama N., Nomoto R., Nakajima H., Kariya H., Hamasaki M., Iyoda S.
Emerging Infectious Diseases 27 ( 5 ) 1509 - 1512 2021年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Emerging Infectious Diseases
We report a fatal case of hemolytic uremic syndrome with urinary tract infection in Japan caused by Shiga toxin- producing Escherichia coli. We genotypically identifi ed the isolate as OX18:H2. Whole-genome sequencing revealed 3 potentially pathogenic lineages (OX18:H2, H19, and H34) that have been continuously isolated in Japan.
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OKUNO Kentaro, AWASTHI Sharda Prasad, KOPPRIO Germán A., IGUCHI Atsushi, HATANAKA Noritoshi, HINENOYA Atsushi, LARA Rubén José, YAMASAKI Shinji
Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｖｅｔｅｒｉｎａｒｙ Ｍｅｄｉｃａｌ Ｓｃｉｅｎｃｅ 2021年4月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：公益社団法人 日本獣医学会
The aims of this study were to investigate prevalence, O-genotype, and virulence gene profile including Shiga toxin 2 gene-subtype of Stx-producing <i>Escherichia coli</i> (STEC) in beef cattle from the Bahía Blanca in Argentina. Rectal swabs were collected from 283 beef cattle in 2012. <i>stx</i> genes were detected in 90 (32%) out of the 283 rectal swabs by <i>stx</i> gene-specific PCR assay. The positive cases were 13 with <i>stx1</i>, 58 with <i>stx2,</i> and 19 with both <i>stx1</i> and <i>stx2</i>. Among 90 <i>stx</i> gene-positive samples, 45 STEC strains were isolated, which included 3 <i>stx1</i>, 34 <i>stx2</i>, and eight <i>stx1</i> and <i>stx2 </i>genes positive isolates. O-genotyping grouped 45 STEC strains into 19 different O-genotypes such as Og8, Og145, Og171, Og185 (4 from each), Og22, Og153, Og157 (3 from each) and others. Various <i>stx2</i> gene-subtypes were identified in 42 STEC strains: 13 positive cases for <i>stx2a</i>, 11 for <i>stx2c</i>, 3 for <i>stx2g</i>, 10 for <i>stx2a</i> and <i>stx2d</i>, 4 for <i>stx2a</i> and <i>stx2c</i>, and 1 for <i>stx2b</i>, <i>stx2c</i> and <i>stx2g</i>. <i>efaI</i> gene, generally prevalent in clinical strains, was detected in relatively high in the STEC strains. These data suggest that <i>stx2a</i> and <i>stx2c</i> were distributed not only in O145 and O157 but also in minor O-genotypes of STEC in Argentina.
DOI： 10.1292/jvms.21-0002
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Virulence profile of diarrhoeagenic <i>Escherichia coli</i> from the Western region of Ghana 査読あり
Prah Isaac, Iwanaga Shiroh, Ablordey Anthony, Saito Ryoichi, Ayibieke Alafate, Huong Nguyen Thi Thu, Iguchi Atsushi, Mahazu Samiratu, Sato Wakana, Hayashi Takaya, Yamaoka Shoji, Suzuki Toshihiko
Japanese Journal of Infectious Diseases 74 ( 2 ) 115 - 121 2021年3月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：国立感染症研究所 Japanese Journal of Infectious Diseases 編集委員会
Diarrhoeagenic <i>Escherichia coli</i> (DEC), an important agent of infectious diarrhoea, is constantly evolving, making it necessary for its periodic monitoring. Unfortunately, DEC genotypes in Ghana remain uncharacterised. We focused on characterising the molecular serotypes, virulence factors, multilocus sequence types, and the phylogenetic relatedness among different DEC pathotypes recovered from stool samples of paediatric patients with diarrhoea symptoms from the Western region of Ghana. We detected all five common DEC pathotypes, with majority being enterotoxigenic <i>E. coli</i> (ETEC) isolates harbouring the heat-labile enterotoxin gene. The DEC strains exhibited a diverse serotypic identity with novel and other outbreak strains. Sequence type (ST)38, ST316, and ST1722 were the most prevalent STs, and clonal complex (CC)10 was the most common CC. A close evolutionary distance was observed among most of the isolates. Coli surface antigen 6 was the most prevalent (44%, n = 11) ETEC-specific colonisation factor. Nearly all of the isolates harboured <i>lpfA</i>, and the frequencies of other virulence genes, such as <i>pap</i> and <i>cnf1,</i> were 7.9% and 18.4%, respectively. This study provides insights into the important and novel genotypes circulating in the Western region of Ghana that should be monitored for public health.
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Nguyen T.T.H., Iguchi A., Ohata R., Kawai H., Ooka T., Nakajima H., Iyoda S.
Journal of Clinical Microbiology 59 ( 3 ) 2021年3月
記述言語：英語 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 an important foodborne pathogen. Although most cases of STEC infection in humans are due to O157 and non-O157 serogroups, there are also reports of infection with STEC strains that cannot be serologically classified into any O serogroup (O-serogroup untypeable [OUT]). Recently, it has become clear that even OUT strains can be subclassified based on the diversity of O-antigen biosynthesis gene cluster (O-AGC) sequences. Cattle are thought to be a major reservoir of STEC strains belonging to various serotypes; however, the internal composition of OUT STEC strains in cattle remains unknown. In this study, we screened 366 STEC strains isolated from healthy cattle by using multiplex PCR kits including primers that targeted novel O-AGC types (Og types) found in OUT E. coli and Shigella strains in previous studies. Interestingly, 94 (25.7%) of these strains could be classified into 13 novel Og types. Genomic analysis revealed that the results of the in silico serotyping of novel Og-type strains were perfectly consistent with those of the PCR experiment. In addition, it was revealed that a dual Og81OgSB17-type strain carried two types of O-AGCs from E. coli O8 and Shigella boydii type 17 tandemly inserted at the locus, with both antigens expressed on the cell surface. The results of this comprehensive analysis of cattle-derived STEC strains may help improve our understanding of the strains circulating in the environment. Additionally, the DNA-based serotyping systems used in this study could be used in future epidemiological studies and risk assessments of other STEC strains.
DOI： 10.1128/JCM.02624-20
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Iguchi A., Nishii H., Seto K., Mitobe J., Lee K., Konishi N., Obata H., Kikuchi T., Iyoda S.
Journal of Clinical Microbiology 58 ( 11 ) 2020年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Clinical Microbiology
The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.
DOI： 10.1128/JCM.01493-20
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Jikumaru A., Ishii S., Fukudome T., Kawahara Y., Iguchi A., Masago Y., Nukazawa K., Suzuki Y.
Journal of Bioscience and Bioengineering 130 ( 1 ) 76 - 81 2020年7月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Bioscience and Bioengineering
The quantification of pathogens is important for assessing water safety and preventing disease outbreaks. Culture-independent approaches, such as quantitative PCR (qPCR) and digital PCR (dPCR), are useful techniques for quantifying pathogens in water samples. However, since pathogens are usually present at low concentrations in water, it is necessary to concentrate microbial cells before extracting their DNA. Many existing microbial concentration methods are inefficient or take a long time to perform. In this study, we applied a coagulation and foam separation method to concentrate environmental water samples of between 1000 and 5000 mL to 100 μL of DNA (i.e., a 1–5 × 10 -fold concentration). The concentration process took <1 h. The DNA samples were then used to quantify various target pathogens using dPCR. One gene, the Shiga toxin gene (stx ) of Shiga toxin-producing Escherichia coli, was detected at 32 copies/100 mL in a river water sample. The coagulation and foam concentration method followed by dPCR reported herein is a fast, sensitive, and reliable method to quantify pathogen genes in environmental water samples. 4 2
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飼育犬から分離された<i>Escherichia albertii</i> の性状
Duangtathip Karn, Nguyen Thi Thu Huong, 井口 純, 三澤 尚明, 谷口 喬子
日本獣医師会雑誌 73 ( 4 ) 191 - 194 2020年4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：公益社団法人 日本獣医師会
<i>Escherichia albertii</i> は新しく認定された腸管病原性細菌で，わが国でも集団食中毒事例が報告されている．本菌は特徴的な生化学性状に乏しく，腸管病原性大腸菌（EPEC）や腸管出血性大腸菌（EHEC）との鑑別が難しい．われわれは，健康な飼育犬の糞便から，<i>E. albertii</i> を分離・同定した．本分離株は，病原性関連遺伝子（<i>eae</i> ）及び細胞膨張化致死毒素（<i>cdt</i> ）を保有していたが，志賀毒素遺伝子（<i>stx2f</i> ）は保有していなかった．また，既報と同様，ペニシリン，アンピシリン及びエリスロマイシンに耐性を示した．飼育犬から<i>E. albertii</i> が分離されたことより，犬が人への感染源となり得る可能性が示唆された．
DOI： 10.12935/jvma.73.191
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Ooka T, Seto K, Ogura Y, Nakamura K, Iguchi A, Gotoh Y, Honda M, Etoh Y, Ikeda T, Sugitani W, Konno T, Kawano K, Imuta N, Yoshiie K, Hara-Kudo Y, Murakami K, Hayashi T, Nishi J.
Microbial genomics 5 ( 11 ) e000314 - - 2019年11月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Arimizu Y, Kirino Y, Sato MP, Uno K, Sato T, Gotoh Y, Auvray F, Brugere H, Oswald E, Mainil JG, Anklam KS, Döpfer D, Yoshino S, Ooka T, Tanizawa Y, Nakamura Y, Iguchi A, Morita-Ishihara T, Ohnishi M, Akashi K, Hayashi T, Ogura Y.
Genome Research 29 ( 9 ) 1495 - 1505 2019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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Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing 査読あり
Banjo M, Iguchi A, Seto K, Kikuchi T, Harada T, Scheutz F, Iyoda S
Journal of Clinical Microbiology 56 ( 6 ) 2018年5月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Thiry D., De Rauw K., Takaki S., Duprez J., Iguchi A., Piérard D., Korsak N., Mainil J.
Journal of Applied Microbiology 124 ( 3 ) 867 - 873 2018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：Journal of Applied Microbiology
© 2017 The Society for Applied Microbiology Aims: The purpose of this survey was to estimate the respective prevalence of the ‘gang of seven’ and ‘non-gang of seven’ serotypes of Shigatoxigenic and enteropathogenic Escherichia coli and to identify the O80:H2 serotype in 245 intestinal contents collected at two slaughterhouses in Belgium in 2014. Methods and Results: After overnight enrichment growth, the 69 intestinal contents testing positive with PCR targeting the eae, stx1 and stx2 genes were inoculated onto four agar media. Of the 2542 colonies picked up, 677 from 59 samples were PCR confirmed. The most frequent virulotypes were eae+ in 47 (80%) samples, stx2+ in 20 (34%) samples and eae+ stx1+ in 16 (27%) samples. PCR-positive colonies belonged to different virulotypes in 36 samples. No colony was O80-positive, whereas two eae+ colonies from two samples were O26:H11, 50 eae+ stx1+ and eae+ from eight samples were O103:H2 and two eae+ stx1+ stx2+ colonies from one sample were O157:H7. Conclusions: The ‘non-gang of seven’ serotypes are more frequent than the ‘gang of seven’ serotypes and the O80:H2 serotype was not detected among Shigatoxigenic and enteropathogenic Escherichia coli in the intestines of cattle at these two slaughterhouses. Significance and Impact of the Study: Although the identification protocols of Shigatoxigenic Escherichia coli focus on the ‘gang of seven’ serotypes, several other serotypes can be present with possible importance in public health. Innovative selective identification procedures should be designed.
DOI： 10.1111/jam.13677
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Bacterial flora analysis of coliforms in sewage, river water, and ground water using MALDI-TOF mass spectrometry 査読あり
Suzuki Y, Niina K, Matsuwaki T, Nukazawa K, Iguchi A
Journal of Environmental Science and Health. Part A, Toxic/hazardous Substances & Environmental Engineering 53 ( 2 ) 160 - 173 2018年1月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Comparison of Three Molecular Subtyping Methods among O157 and Non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Japanese Cattle 査読あり
Nakamura H, Iguchi A, Maehara T, Fujiwara K, Fujiwara A, Ogasawara J
Japanese Journal of Infectious Diseases 7 ( 1 ) 45 - 50 2018年1月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Enteropathogenic Escherichia coli O80:H2 in Young Calves with Diarrhea, Belgium 査読あり
Thiry D, Saulmont M, Takaki S, De Rauw K, Duprez JN, Iguchi A, Piérard D, Mainil JG
Emerging Infectious Diseases 23 ( 12 ) 2093 - 2095 2017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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An untypeable enterotoxigenic Escherichia coli represents one of the dominant types causing human disease 査読あり
Iguchi A, von Mentzer A, Kikuchi T, Thomson NR
Microbial Genomics 3 ( 9 ) 2017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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Shiga Toxin Subtypes and Virulence Genes in Escherichia coli Isolated from Cattle 査読あり
Akiyama Yumi, Futai Hiroko, Saito Etsuko, Ogita Kenichi, Sakae Hiroshi, Fukunaga Masaharu, Tsuji Hidetaka, Chikahira Masatsugu, Iguchi Atsushi
Japanese Journal of Infectious Diseases 70 ( 2 ) 181 - 185 2017年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：国立感染症研究所 Japanese Journal of Infectious Diseases 編集委員会
Subtypes of <i>stx1</i> and <i>stx2</i> in 45 Shiga toxin-producing <i>Escherichia coli</i> (STEC) strains isolated from cattle were investigated by PCR. Only subtype <i>stx1a</i> was detected among all the <i>stx1</i>-positive strains. The major <i>stx2</i> subtype was <i>stx2a</i> followed by <i>stx2d</i>, <i>stx2c</i>, <i>stx2b</i>, and <i>stx2g</i> in decreasing order of frequency. <i>stx2c</i> was found in strains of serotypes O157 and O174. <i>stx2d</i> was found in 11 strains. These strains were confirmed by DNA sequencing to carry both the activatable tail and the END motif; all were <i>eae</i>-negative, and 3 contained <i>stx2d</i> as the only <i>stx</i>. <i>stx2g</i> was found in 2 strains in association with <i>stx2a</i>, <i>estA1</i>, and <i>astA</i>. In addition, 7 hybrid strains of shigatoxigenic and enterotoxigenic <i>E. coli</i> (STEC/ETEC) were found to harbor one or both of <i>stx1a</i> and <i>stx2a</i> (<i>stx1a</i>/<i>stx2a</i>) and <i>estA1</i>. Among 27 serotypes of STEC strains isolated from cattle, O157:H7 and O109:H– strains were <i>eae</i>-positive. Other putative adhesin genes, such as <i>saa</i>, <i>iha</i>, <i>espP</i>, and <i>lpfA</i><sub>O113</sub> were detected in more than 12 serotypes.
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Characterization of Shiga toxin-producing Escherichia coli from feces of sika deer (Cervus nippon) in Japan using PCR binary typing analysis to evaluate their potential human pathogenicity. 査読あり
Kabeya H, Sato S, Oda S, Kawamura M, Nagasaka M, Kuranaga M, Yokoyama E, Hirai S, Iguchi A, Ishihara T, Kuroki T, Morita-Ishihara T, Iyoda, Terajima J, Ohnishi M, Maruyama S
Journal of Veterinary Medical Science 79 ( 5 ) 834 - 841 2017年5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 Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC. 査読あり
Fakih I, Thiry D, Duprez JN, Saulmont M, Iguchi A, Piérard D, Jouant L, Daube G, Ogura Y, Hayashi T, Taminiau B, Mainil JG
Veterinary Microbiology 2017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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Identification of a New Virulent Clade in Enterohemorrhagic Escherichia coli O26:H11/H- Sequence Type 29 査読あり
Nozomi Ishijima, Ken-ichi Lee, Tomomi Kuwahara、Haruyuki Nakayama-Imaohji、Saori Yoneda、Atsushi Iguchi、Yoshitoshi Ogura、Tetsuya Hayashi、Makoto Ohnishi、Sunao Iyoda
Scientific Reports 2017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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大腸菌のO抗原合成遺伝子領域の解析と検査法の確立 招待あり 査読あり
井口 純
日本細菌学雑誌 71 ( 4 ) 209 - 215 2016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：日本細菌学会
大腸菌はO血清群を指標として細分類され, 特に病原性大腸菌の調査や研究においては, 分離株の優先すべき特徴としてO血清群の判定が行われる。O血清群の多様性はO抗原合成遺伝子群の多様性と関連しており, それぞれのO抗原合成遺伝子群に特徴的な塩基配列はO血清群の判定に利用できる。我々はこれまでに定められているO1からO187までのすべてのO血清群におけるO抗原合成遺伝子群の配列を決定し, 保存性と特異性の両面における特徴を明らかにした。さらにその解析結果を基に, O血清群を網羅的に判定できる2つの遺伝学的な手法を開発した。一つはPCRを基礎とするマルチプレックスPCR法（<i>E. coli</i> O-genotyping PCR）で, もう一つは<i>wzx/wzy</i>と<i>wzm/wzt</i>の配列セットを用いたBLAST検索による方法（SerotypeFinder）である。いずれの方法で得られる結果も表現型に準じており, 今後, 大腸菌における調査や研究において幅広い利用が期待される。
DOI： 10.3412/jsb.71.209
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Secondary Shiga toxin–producing Escherichia coli infection, Japan, 2010–2012 査読あり
Morita-Ishihara T., Iyoda S., Iguchi A., Ohnishi M.
Emerging Infectious Diseases 22 ( 12 ) 2181 - 2184 2016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：Emerging Infectious Diseases
© 2016, Centers for Disease Control and Prevention (CDC). All rights reserved. To evaluate the potential public health risk caused by secondary Shiga toxin–producing Escherichia coli (STEC) infections in Japan, we investigated the prevalence and characteristics of STEC isolated from healthy adults during 2010–2012. Although prevalence among healthy adults was high, most STEC organisms displayed characteristics rarely found in isolates from symptomatic patients.
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Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131. 査読あり
Naoko Imuta, Tadasuke Ooka, Kazuko Seto, Ryuji Kawahara, Toyoyasu Koriyama, Tsuyoshi Kojyo, Atsushi Iguchi, Koichi Tokuda, Hideki Kawamura, Kiyotaka Yoshiie, Yoshitoshi Ogura, Tetsuya Hayashi, Junichiro Nishia
Journal of Clinical Microbiology 2016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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Six novel O genotypes from Shiga toxin-producing Escherichia coli 査読あり
Iguchi A., Iyoda S., Seto K., Nishii H., Ohnishi M., Mekata H., Ogura Y., Hayashi T.
Frontiers in Microbiology 7 ( MAY ) 2016年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Frontiers in Microbiology
© 2016 Iguchi, Iyoda, Seto, Nishii, Ohnishi, Mekata, Ogura and Hayashi. Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny.
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Prevalence and pathogenic potential of Escherichia coli isolates from raw milk and raw milk cheese in Egypt 査読あり
Ombarak RA, Hinenoya A, Awasthi SP, Iguchi A, Shima A, Elbagory AR, Yamasaki S
International Journal of Food Microbiology 2016年3月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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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 査読あり
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 2015年12月
記述言語：日本語 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 査読あり
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 2015年12月
記述言語：日本語 掲載種別：研究論文（学術雑誌）
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Multiplex Real-Time PCR Assays for Screening of Shiga Toxin 1 and 2 Genes, Including All Known Subtypes, and Escherichia coli O26-, O111-, and O157-Specific Genes in Beef and Sprout Enrichment Cultures 査読あり
Harada T, Iguchi A, Iyoda S, Seto K, Taguchi M, Kumeda Y
Journal of Food Protection 2015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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Joensen K., Tetzschner A., Iguchi A., Aarestrup F., Scheutz F.
Journal of Clinical Microbiology 53 ( 8 ) 2410 - 2426 2015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 Clinical Microbiology
Copyright © 2015, American Society for Microbiology. All Rights Reserved. Accurate and rapid typing of pathogens is essential for effective surveillance and outbreak detection. Conventional serotyping of Escherichia coli is a delicate, laborious, time-consuming, and expensive procedure. With whole-genome sequencing (WGS) becoming cheaper, it has vast potential in routine typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-based in silico serotyping of E. coli applicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). All E. coli isolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific SerotypeFinder CGE tool. SerotypeFinder was evaluated on 682 E. coli genomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen genes wzx, wzy, wzm, and wzt and the flagellin genes fliC, flkA, fllA, flmA, and flnA were detected in 569 and 508 genome sequences, respectively. SerotypeFinder for WGS-based O and H typing predicted 560 of 569 O types and 504 of 508 H types, consistent with conventional serotyping. In combination with other available WGS typing tools, E. coli serotyping can be performed solely from WGS data, providing faster and cheaper typing than current routine procedures and making WGS typing a superior alternative to conventional typing strategies.
DOI： 10.1128/JCM.00008-15
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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.
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Iguchi A., Iyoda S., Seto K., Ohnishi M.
Journal of Clinical Microbiology 49 ( 10 ) 3678 - 3680 2011年10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
This is the first report of the isolation of Shiga toxin-producing Escherichia coli (STEC) strains whose O antigens were genetically and serologically identical to those of Shigella boydii type 10, from human feces. The novel STEC O serogroup may be widespread in Japan and associated with diarrhea and hemorrhagic colitis. Copyright © 2011, American Society for Microbiology. All Rights Reserved.
DOI： 10.1128/JCM.01197-11
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Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli
Iguchi A., Shirai H., Seto K., Ooka T., Ogura Y., Hayashi T., Osawa K., Osawa R.
PLoS ONE 6 ( 8 ) 2011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：PLoS ONE
Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-a ntigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci. © 2011 Iguchi et al.
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Iguchi A., Umekawa N., Maegawa T., Tsuruta H., Odamaki T., Xiao J., Osawa R.
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology 99 ( 3 ) 457 - 471 2011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：Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology
The polymorphism of ORFs encoding putative cell-surface adhesins was investigated in Bifidobacterium longum subsp. longum. Firstly, we performed a PCR assay targeting 15 ORFs encoding putative adhesion proteins, which included 8 ORFs with a sortase targeting LPXTG motif, in 42 strains of different pulsotypes isolated from fecal samples from 12 human individuals. We found a variability in the presence of an ORF, BL0675, which encodes a putative fimbrial subunit protein. We sequenced ORFs corresponding to BL0675 in the 42 strains and adjacent ORFs corresponding to BL0674 and BL0676. The results indicated that ORFs corresponding to BL0675 were highly polymorphic with five variant types (i.e. A-, B-, C-, D-, and E-types). Meanwhile, BL0674 and BL0676, which encode an additional putative fimbrial subunit protein and a fimbrial-associated sortase-like protein, were highly conserved. Subsequent quantitative polymerase chain reaction (qPCR) assays targeting the variant types in 89 human fecal samples revealed that A-type was the most commonly distributed (74.2%), followed by B-type (59.6%), D-type (31.5%), E-type (32.6%) and C-type (5.6% prevalence). Since BL0675 is considered to be a fimbrial protein with glycoprotein-binding ability, the proteins encoded by the five variant types of BL0675 may have specific binding properties to various carbohydrate structures expressed on the human intestinal wall, thereby allowing B. longum to colonize the intestine in a host-specific manner. © 2010 Springer Science+Business Media B.V.
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Nlec, a type III secretion protease, compromises NF-kB activation by targeting p65/rela
Yen H., Ooka T., Iguchi A., Hayashi T., Sugimoto N., Tobe T.
PLoS Pathogens 6 ( 12 ) 2010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：PLoS Pathogens
The NF-kB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-kB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-kB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-kB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other antiinflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-kB activation by the direct cleavage of NF-kB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-kB pathway and accounts for most of the immune suppression caused by EHEC/EPEC. © 2010 Yen et al.
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Ogura Y., Ooka T., Iguchi A., Toh H., Asadulghani M., Oshima K., Kodama T., Abe H., Nakayama K., Kurokawa K., Tobe T., Hattori M., Hayashi T.
Proceedings of the National Academy of Sciences of the United States of America 106 ( 42 ) 17939 - 17944 2009年10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：Proceedings of the National Academy of Sciences of the United States of America
Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5-5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.
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Ooka T., Terajima J., Kusumoto M., Iguchi A., Kurokawa K., Ogura Y., Asadulghani M., Nakayama K., Murase K., Ohnishi M., Iyoda S., Watanabe H., Hayashi T.
Journal of Clinical Microbiology 47 ( 9 ) 2888 - 2894 2009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
Enterohemorrhagic Escherichia coli O157 (EHEC O157) is a food-borne pathogen that has raised worldwide public health concern. The development of simple and rapid strain-typing methods is crucial for the rapid detection and surveillance of EHEC O157 outbreaks. In the present study, we developed a multiplex PCR-based strain-typing method for EHEC O157, which is based on the variability in genomic location of IS629 among EHEC O157 strains. This method is very simple, in that the procedures are completed within 2 h, the analysis can be performed without the need for special equipment or techniques (requiring only conventional PCR and agarose gel electrophoresis systems), the results can easily be transformed into digital data, and the genes for the major virulence markers of EHEC O157 (the stx1, stx2, and eae genes) can be detected simultaneously. Using this method, 201 EHEC O157 strains showing different XbaI digestion patterns in pulsed-field gel electrophoresis (PFGE) analysis were classified into 127 types, and outbreak-related strains showed identical or highly similar banding patterns. Although this method is less discriminatory than PFGE, it may be useful as a primary screening tool for EHEC O157 outbreaks. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
DOI： 10.1128/JCM.00792-09
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Leopold S., Magrini V., Holt N., Shaikh N., Mardis E., Cagno J., Ogura Y., Iguchi A., Hayashi T., Mellmanng A., Karch H., Besser T., Sawyer S., Whittam T., Tarr P.
Proceedings of the National Academy of Sciences of the United States of America 106 ( 21 ) 8713 - 8718 2009年5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：Proceedings of the National Academy of Sciences of the United States of America
Single nucleotide polymorphisms (SNPs) in stable genome regions provide durable measurements of species evolution. We systematically identified each SNP in concatenations of all backbone ORFs in 7 newly or previously sequenced evolutionarily instructive pathogenic Escherichia coli O157:H7, O157:H -, and O55:H7. The 1,113 synonymous SNPs demonstrate emergence of the largest cluster of this pathogen only in the last millennium. Unexpectedly, shared SNPs within circumscribed clusters of organisms suggest severely restricted survival and limited effective population sizes of pathogenic O157:H7, tenuous survival of these organisms in nature, source-sink evolutionary dynamics, or, possibly, a limited number of mutations that confer selective advantage. A single large segment spanning the rfb-gnd gene cluster is the only backbone region convincingly acquired by recombination as O157 emerged from O55. This concatenomic analysis also supports using SNPs to differentiate closely related pathogens for infection control and forensic purposes. However, constrained radiations raise the possibility of making false associations between isolates.
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Asadulghani M., Ogura Y., Ooka T., Itoh T., Sawaguchi A., Iguchi A., Nakayama K., Hayashi T.
PLoS Pathogens 5 ( 5 ) 2009年5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：PLoS Pathogens
Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1-Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1-Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities. © 2009 Asadulghani et al.
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Iguchi A., Thomson N., Ogura Y., Saunders D., Ooka T., Henderson I., Harris D., Asadulghani M., Kurokawa K., Dean P., Kenny B., Quail M., Thurston S., Dougan G., Hayashi T., Parkhill J., Frankel G.
Journal of Bacteriology 91 ( 1 ) 347 - 354 2009年1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 Bacteriology
Enteropathogenic Escherichia coli (EPEC) was the first pathovar of E. coli to be implicated in human disease; however, no EPEC strain has been fully sequenced until now. Strain E2348/69 (serotype O127:H6 belonging to E. coli phylogroup B2) has been used worldwide as a prototype strain to study EPEC biology, genetics, and virulence. Studies of E2348/69 led to the discovery of the locus of enterocyte effacement-encoded type III secretion system (T3SS) and its cognate effectors, which play a vital role in attaching and effacing lesion formation on gut epithelial cells. In this study, we determined the complete genomic sequence of E2348/69 and performed genomic comparisons with other important E. coli strains. We identified 424 E2348/69-specific genes, most of which are carried on mobile genetic elements, and a number of genetic traits specifically conserved in phylogroup B2 strains irrespective of their pathotypes, including the absence of the ETT2-related T3SS, which is present in E. coli strains belonging to all other phylogroups. The genome analysis revealed the entire gene repertoire related to E2348/69 virulence. Interestingly, E2348/69 contains only 21 intact T3SS effector genes, all of which are carried on prophages and integrative elements, compared to over 50 effector genes in enterohemorrhagic E. coli O157. As E2348/69 is the most-studied pathogenic E. coli strain, this study provides a genomic context for the vast amount of existing experimental data. The unexpected simplicity of the E2348/69 T3SS provides the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
DOI： 10.1128/JB.01238-08
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Ogura Y., Abe H., Katsura K., Kurokawa K., Asadulghani M., Iguchi A., Ooka T., Nakayama K., Yamashita A., Hattori M., Tobe T., Hayashi T.
Journal of Bacteriology 190 ( 21 ) 6948 - 6960 2008年11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 Bacteriology
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are diarrheagenic pathogens that colonize the intestinal tract through the formation of attaching and effacing lesions, induced by effectors translocated via a type III secretion system (T3SS) encoded on the locus of enterocyte effacement (LEE). In EHEC O157, numerous virulence factors, including around 40 T3SS effectors, have been identified. Most of them are encoded on genomic islands (GEIs) such as prophages and integrative elements. For EPEC, however, no systematic search of GEIs and virulence-related genes carried therein has been done, and only a limited number of virulence factors have been identified so far. In this study, we performed a systemic and genome-wide survey of the GEIs in strain B171-8, one of the prototype strains of EPEC, by the combined use of whole-genome PCR scanning and fosmid mapping and identified 22 large GEIs, including nine lambda-like prophages, three P2-like prophages, the LEE, and three additional integrative elements. On these prophages and integrative elements, we found genes for a set of T3SS proteins, a total of 33 T3SS effectors or effector homologues, and 12 other virulence factors which include five nonfimbrial adhesins. Most of the T3SS effector families identified are also present in EHEC O157, but B171-8 possesses a significantly smaller number of effectors. Not only the presence or absence of Shiga toxin genes but also the difference in the T3SS effector repertoire should be considered in analyzing the pathogenicity of EPEC and EHEC strains. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
DOI： 10.1128/JB.00625-08
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Iguchi A., Ooka T., Ogura Y., Asadulghani, Nakayama K., Frankel G., Hayashi T.
Microbiology 154 ( 2 ) 559 - 570 2008年2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
Typical enteropathogenic Escherichia coli (EPEC) O55:H7 is regarded as the closest relative of enterohaemorrhagic E. coli (EHEC) O157:H7. Both serotypes usually express the γ1 intimin subclass and trigger actin polymerization by the Tir-TccP pathway. However, atypical O55:H7 strains capable of triggering actin polymerization via the Tir-Nck pathway have recently been identified. In this study, we investigated the genotypic differences and phylogenetic relationships between typical and atypical O55:H7 strains. We show that the atypical O55:H7 strains, which express the θ intimin subclass and lack both tccP and tccP2, belong to an E. coli lineage distinct from the typical O55:H7 and from the EPEC O55:H6, which also uses the Tir-Nck actin polymerization pathway. We conducted genomic comparisons of the chromosomal regions covering the O-antigen gene cluster and its flanking regions between the three O55 lineages by RFLP analysis of PCR products and DNA sequencing analysis of about 65 kb chromosomal regions. This unexpectedly revealed that horizontal transfer of large fragments (≥40 kb) encoding the O55-antigen gene cluster and part of the neighbouring colanic acid gene cluster was involved in the emergence of the three O55 E. coli lineages. The data provide new insights into the mechanisms involved in the generation of a wide variety of O-serotypes in Gram-negative bacteria. © 2008 SGM.
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Iguchi A., Iyoda S., Watanabe H., Osawa R.
Current Microbiology 54 ( 1 ) 14 - 19 2007年1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：Current Microbiology
We investigated the relationship between expression of the O side chain of outer membrane lipopolysaccharide (LPS) and infection by a Shiga toxin 2 (Stx2)-converting phage in normal and benign strains of Escherichia coli. Of 19 wild-type E. coli strains isolated from the feces of healthy subjects, those with low-molecular-weight LPS showed markedly higher susceptibility to lytic and lysogenic infection by Stx2 phages than those with high-molecular-weight LPS. All lysogens produced infectious phage particles and Stx2. The Stx-negative E. coli O157:H7 strain ATCC43888 with an intact O side chain was found to be resistant to lysis by an Stx2 phage and lysogenic infection by a recombinant Stx2 phage, whereas a rfbE mutant deficient in the expression of the O side chain was readily infected by the phage and yielded stable lysogens. The evidence suggests that an O side chain deficiency leads to the creation of new pathotypes of Shiga toxin-producing E. coli (STEC) within the intestinal microflora. © 2006 Springer Science+Business Media, Inc.
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Iguchi A., Iyoda S., Terajima J., Watanabe H., Osawa R.
Gene 372 ( 1-2 ) 199 - 207 2006年5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：Gene
It is known that XbaI-digested chromosomal DNAs of strains of Escherichia coli O157:H7 exhibit a wide variety in pulsed-field gel electrophoresis (PFGE) fragment patterns, which is used for epidemiological surveillance of this important pathogen. The variety in the restriction enzyme-digestion patterns suggests a wide genomic diversity, however, only a few studies have been conducted to investigate involvement of large-scale chromosomal rearrangements in development of the diversity. In this study, through rounds of subculturing E. coli O157:H7 strain EDL933, naturally occurring genome variation in the isolated derivatives was investigated. By comparing the PFGE patterns among clonally related derivatives, we found five types of large-scale inversions taking place within the chromosome. The five inversions found were across the replication axis and ranged from 250-kb to 1.4-Mb long, and all the corresponding recombination sites were associated with prophages or phage-like regions. Four inversions out of the five were resulted from recombination between pairs of lambda-like prophages disturbing the symmetry of the origin and terminus of the replication axis. These observations indicate that those prophage regions represent some of the hot spots for intrachromosomal recombination within the E. coli O157:H7 chromosome, where recombination between the prophage regions results not only in the large chromosomal inversions but might also in generation of chimeric phages. © 2006 Elsevier B.V. All rights reserved.
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Okura M., Osawa R., Iguchi A., Takagi M., Arakawa E., Terajima J., Watanabe H.
Microbiology and Immunology 48 ( 10 ) 787 - 790 2004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：Microbiology and Immunology
A PCR-based assay to identify pandemic group Vibrio parahaemolyticus has been developed. The assay employs an oligonucleotide primer pair derived from the group-specific sequence of an arbitrarily primed-PCR fragment, which is located in the genome encoding a "hypothetical protein," approximately 80% homologous to the Mn2+ and Fe2+ transporter of the NRAMP family of V. vulnificus. The assay distinguished the pandemic group from other V. parahaemolyticus strains by yielding a 235-bp specific amplicon, and can be a useful diagnostic tool for identification of pandemic group strains.
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Okura M., Osawa R., Iguchi A., Arakawa E., Terajima J., Watanabe H.
Journal of Clinical Microbiology 41 ( 10 ) 4676 - 4682 2003年10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
A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the "pandemic group") were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS/new or orf8, that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (tdh) gene also belonged to this cluster and possessed the toxRS/new sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence. The evidence suggests that neither the toxRS/new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other V. parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS/new sequence (651 bp).
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Sata S., Fujisawa T., Osawa R., Iguchi A., Yamai S., Shimada T.
Applied and Environmental Microbiology 69 ( 3 ) 1858 - 1860 2003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：Applied and Environmental Microbiology
An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.
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Iguchi A., Osawa R., Kawano J., Shimizu A., Terajima J., Watanabe H.
Current Microbiology 46 ( 3 ) 224 - 227 2003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：Current Microbiology
Escherichia coli K-12 lysogens of three different Shiga toxin 2 (Stx2)-encoding bacteriophages were examined for variability in their pulsed-field gel electrophoresis (PFGE) fragment patterns. The PFGE fragment patterns could be classified into three types (i.e., PFGE types B, C, and D). For the PFGE type D, a 255-kbp fragment present in the original K-12 strain was apparently shifted by the size of Stx 2-encoding phage genomic DNA (ca. 65 kbp) to the position at 320 kbp. In contrast, the types B and C showed the above fragment shift plus further 6- and 10-fragment differences, respectively, from the original K-12 strain. The evidence suggests that even a single genetic event like lysogeny can cause marked genotypic modification of the host strain.
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Iguchi A., Osawa R., Kawano J., Shimizu A., Terajima J., Watanabe H.
Journal of Clinical Microbiology 40 ( 8 ) 3079 - 3081 2002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 Clinical Microbiology
Three clinical strains of enterohemorrhagic Escherichia coli O157:H7 which were subcultured repeatedly or stored at room temperature over a 25-week period showed appreciable variations in their pulsed-field gel electrophoresis fragment patterns. The variations could be explained by a couple of spontaneous genetic events at most and thus did not invalidate the genetic lineage of the strains.