Papers - SHIBAYAMA Keigo

Characterization of three novel Helicobacter species infecting stomachs of dogs and cats: Helicobacter gastrocanis sp. nov., Helicobacter gastrofelis sp. nov., and Helicobacter felistomachi sp. nov. Reviewed

Emiko Rimbara, Sae Aoki, Masato Suzuki, Hirotaka Kobayashi, Taisuke Nakagawa, Yuko Goto-Koshino, Sachiyo Nomura, Wan-Ying Du, Hidenori Matsui, Shigetarou Mori, Keigo Shibayama, Tsuyoshi Kenri, Koichi Ohno

Frontiers in Microbiology     2025.1

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2024.1459401

A nationwide survey of antimicrobial resistance of Escherichia coli isolated from broiler chickens in Malawi. Reviewed International coauthorship

Chisembe P, Suzuki M, Dao DT, Njunga G, Nkhoma J, Mthilakuwili L, Kinoshita-Daitoku R, Kuroda E, Kimura K, Shibayama K

JAC Antimicrobial Resistance   Vol. 6 ( 6 )   2024.12

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/jacamr/dlae200

Antimicrobial-resistant Helicobacter pylori in Japan: Report of nationwide surveillance for 2018-2020.

Okimoto T, Ando T, Sasaki M, Ono S, Kobayashi I, Shibayama K, Chinda D, Tokunaga K, Nakajima S, Osaki T, Sugiyama T, Kato M, Murakami K

Helicobacter   Vol. 29 ( 1 ) page： e13028   2024.1

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DOI： 10.1111/hel.13028

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Characterization of HcaA, a novel autotransporter protein in Helicobacter cinaedi, and its role in host cell adhesion.

Aoki S, Mori S, Matsui H, Shibayama K, Kenri T, Rimbara E

mSphere   Vol. 8 ( 6 ) page： e0040323   2023.12

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Helicobacter cinaedi infects the human gut and causes invasive infections such as bacteremia and cellulitis through bacterial translocation. However, the mechanism by which H. cinaedi attaches to host cells and establishes infection remains unclear. This study aimed to investigate the relationship between a novel putative autotransporter protein, H. cinaedi autotransporter protein A (HcaA), and its role in pathogenicity. The cytotoxicity of H. cinaedi infection in colon epithelial cell lines (Caco-2 and HT29) was assessed using a lactate dehydrogenase assay, and it was found that cytotoxicity significantly decreased upon HcaA knockout. Adhesion assays further revealed that the HcaA-knockout strain showed significantly reduced attachment to the human epithelial colorectal adenocarcinoma (Caco-2) cell line compared to that of the wild-type strain. Moreover, the recombinant HcaA protein demonstrated strong adhesion properties to the human monocytic cell line (U937). The adhesive activity was diminished when the Arg-Gly-Asp (RGD) motif in HcaA was replaced with RAD, indicating that the RGD motif in HcaA is crucial for host cell adhesion. To determine the role of HcaA in H. cinaedi infection in vivo, C57BL/6 mice were orally infected with wild-type and HcaA-knockout H. cinaedi strains. Bacterial colonization was assessed 7, 14, and 28 days post-infection. At 7 days post-infection, colonization was significantly lower in mice infected with the HcaA-knockout strain compared to those infected with the wild-type strain. In conclusion, our findings suggest that HcaA, a novel putative autotransporter protein in H. cinaedi, plays a significant role as an adhesin in establishing colonization.

DOI： 10.1128/msphere.00403-23

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Methicillin-resistant Staphylococcus aureus-induced staphylococcal scalded skin syndrome in a preterm infant, and subsequent toxigenic analysis.

Fukaura R, Terashima-Murase C, Ota M, Noda H, Oka K, Ishihara Y, Shibayama K, Akiyama M

The Journal of dermatology     2023.8

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Language：English   Publisher：Journal of Dermatology  

DOI： 10.1111/1346-8138.16929

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In Vitro Susceptibility of <i>Bacillus cereus</i> Spores of Blood Isolates and Healthy Skin Isolates to 10% Povidone-iodine

ISHIHARA Yuka, SHIBAYAMA Keigo, SHAMOTO Ikue, USAMI Hisae, OHTA Michio

Japanese Journal of Infection Prevention and Control   Vol. 38 ( 2 ) page： 57 - 60   2023.3

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Language：Japanese   Publisher：Japanese Society for Infection Prevention and Control  

<p>The susceptibility of <i>Bacillus cereus</i> spores to 10% povidone-iodine was measured at 0°C, 20°C, and 37°C, respectively. The spores of 15 <i>B. cereus</i> blood isolates and 15 isolates from healthy adult skin, respectively, were used for the measurement. The spore solution was prepared 9 days after the culture of <i>B. cereus</i> on NGKG plates, and spore formation was confirmed microscopically. No <i>B. cereus</i> strain in the spore form was highly resistant to any conditions of 10% povidone-iodine treatment. Approximately 99% of the spores of most strains were killed after a 5-min treatment with 10% povidone-iodine irrespective of treatment temperature, whereas 1% of spores survived a 30-min treatment. The spores of 5 blood isolates and 3 healthy skin isolates were more sensitive to 10% povidone-iodine, and only 0.1% of the spores survived the 5-min treatment. No apparent differences in susceptibility to povidone-iodine between the blood and skin isolates were observed. From these results, 5-min treatment using 10% povidone-iodine for skin disinfection is applicable when contamination of <i>B. cereus</i> spores on the skin is expected to be mild or low.</p>

DOI： 10.4058/jsei.38.57

CiNii Research

Rate of Colonization and Serotypes of <i>Streptococcus pneumoniae</i> Isolated from Healthy Adult Community Residents after the Regulatory Approval of PPSV23

ISHIHARA Yuka, YAGI Tetsuya, SHIBAYAMA Keigo, OHTA Michio

Kansenshogaku Zasshi   Vol. 96 ( 3 ) page： 82 - 90   2022.5

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Language：Japanese   Publisher：The Japanese Association for Infectious Diseases  

<p>The purpose of this study was to determine the pharyngeal colonization rate and serotypes of <i>Streptococcus pneumoniae</i> in healthy adults in our community by our modified PCR detection method. We set up a new surveillance system for surveying the pharyngeal pneumococcal colonization rate in healthy adults. The pharyngeal colonization rate and the serotypes of <i>S. pneumoniae</i> were investigated in 449 randomly selected healthy adults living in the community between 2014 and 2020, after the launch of the 23-valent pneumococcal polysaccharide vaccine (PPSV23). The 449 examinees included 361 adults aged 65 years or over, who were eligible to receive the PPSV23 vaccine. We performed PCR-based<i> lytA</i> detection using our modified procedure and found pharyngeal <i>S. pneumoniae</i> colonization in 207 examinees (46.1%), a significantly high rate as compared with that reported previously. We then determined the serotypes of the colonizing <i>S. pneumoniae</i> in the examinees by PCR using serotype-specific primers. The PPSV23 serotypes of <i>S. pneumoniae</i> were detected in the pharynx of 188 examinees. Serotype 18C was the most frequently detected, and serotypes 14 and 4 were also detected at a high frequency. The colonization rate of <i>S. pneumoniae</i> belonging to the PCV7 serotypes was 67.6%, while the rate for the PCV13 serotypes was 73.4%. Since the colonization rate of <i>S. pneumoniae</i> belonging to the PPSV23 serotypes was 88.9%, PPSV23 as well as PCV13 were found to be applicable to elderly people in the district.</p><p>Of the elderly examinees age 65 or over, 53.5% had received the PPSV23 vaccine before this study. The pharyngeal colonization rate of <i>S. pneumoniae</i> in the group that had already received the PPSV23 vaccine was 38.9%, whereas that in the group that had not received the PPSV23 vaccine was 49.4%. The colonization rate was not influenced by prior influenza vaccination or the contact frequency of the elderly examinees with their grandchildren. Conclusion; We found a much higher rate, as compared with previous reports, of pharyngeal <i>S. pneumoniae</i> colonization in local community-dwelling adults using our modified PCR method. We also found no apparent serotype changes in the <i>S. pneumoniae</i> colonizing the pharynx in local community-dwelling adults after the introduction of the PCV13 or PPSV23 vaccine.</p>

DOI： 10.11150/kansenshogakuzasshi.96.82

CiNii Research

Surveillance of multi-drug resistance phenotypes in Staphylococcus aureus in Japan and correlation with whole-genome sequence findings. Open Access

Hosaka Y, Yahara K, Clark A, Kitagawa H, Hisatsune J, Sugai M, Shibayama K, Stelling J

The Journal of hospital infection   Vol. 123   page： 34 - 42   2022.5

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Language：English   Publisher：Journal of Hospital Infection  

Background: Antimicrobial resistance in Staphylococcus aureus imposes a high disease burden. Both phenotypic and genotypic monitoring are key to understanding and containing emerging resistant strains. Aim: Phenotypic monitoring of emerging resistance in S. aureus and correlation of priority strain phenotypes with whole-genome sequencing (WGS) findings. Methods: Antimicrobial susceptibility test results of >40,000 isolates from 213 participating hospitals from 2011 to 2019 were exported from the national Japan Nosocomial Infections Surveillance (JANIS) database. Longitudinal and geographic distribution and prevalence of distinct multi-drug resistance phenotypes (‘resistance profiles’) of S. aureus were examined among hospitals and prefectures. We further conducted a genome sequence analysis of strains with specific resistance profiles of concern. Findings: The overall prevalence of meticillin-resistant S. aureus (MRSA) decreased from 40.3% to 35.1% from 2011 to 2019. However, among dozens of S. aureus resistance profiles, only one profile of a type of MRSA, exhibited a statistically significant increase in inpatient frequency, exceeding 10% during the nine years. This MRSA profile showed resistance to oxacillin, erythromycin and levofloxacin. Analysis of WGS results of S. aureus isolates with this phenotype revealed that most belonged to clonal complex 8, and all carried SCCmec IV, typical of community-acquired MRSA. Conclusion: Tracking distinct resistance profiles deepened our understanding of the overall decrease in MRSA and led to recognition of the emergence of a new resistance phenotype. This study provides a model for future epidemiological research on antimicrobial resistance correlating multi-drug resistance phenotypes with selective genome sequencing, which can be applied to other bacterial species.

DOI： 10.1016/j.jhin.2022.02.011

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Emergence of mobile tigecycline resistance gene tet(X4)-harbouring Shewanella xiamenensis in a water environment

Trung Duc Dao, Kasuga Ikuro, Hirabayashi Aki, Dong Tu Nguyen, Hien Thi Tran, Vu Hieu, Linh Tuyet Ngoc Pham, Thi My Hanh Vu, Hasebe Futoshi, Ha Thanh Nguyen, Trang Le Thi, Tran Hoang Huy, Shibayama Keigo, Takemura Taichiro, Suzuki Masato

JOURNAL OF GLOBAL ANTIMICROBIAL RESISTANCE   Vol. 28   page： 140 - 142   2022.3

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DOI： 10.1016/j.jgar.2021.12.029

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Helicobacter suis SNTW101株の培養成功と完全ゲノム配列の決定

林原 絵美子, 松井 英則, 鈴木 仁人, 中村 正彦, 柴山 恵吾, 徳永 健吾

日本ヘリコバクター学会学術集会プログラム・抄録集   Vol. 26回   page： 58 - 58   2020.12

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Language：Japanese   Publisher：(一社)日本ヘリコバクター学会  

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Helicobacter suis胃粘膜感染と病態発症

鈴木 仁人, 林原 絵美子, 松井 英則, 徳永 健吾, 柴山 恵吾

日本ヘリコバクター学会学術集会プログラム・抄録集   Vol. 26回   page： 73 - 73   2020.12

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胃MALTリンパ腫におけるHelicobacter suis感染の関与

徳永 健吾, 林原 絵美子, 松井 英則, 鈴木 仁人, 大崎 敬子, 井田 陽介, 三好 佐和子, 長濱 清隆, 大野 亜希子, 三好 潤, 柴山 恵吾, 久松 理一, 岡本 晋

Gastroenterological Endoscopy   Vol. 62 ( Suppl.2 ) page： 2101 - 2101   2020.10

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Language：Japanese   Publisher：(一社)日本消化器内視鏡学会  

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FOCUS 輸入感染症-耐性菌を中心に

柴山 恵吾

検査と技術   Vol. 48 ( 7 ) page： 684 - 687   2020.7

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Language：English   Publisher：株式会社医学書院  

DOI： 10.11477/mf.1543208043

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特集 薬剤耐性(AMR)対策 厚生労働省院内感染対策サーベイランス(JANIS)からみたAMR対策の課題と展望

柴山 恵吾

公衆衛生   Vol. 81 ( 10 ) page： 798 - 803   2017.10

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Language：English   Publisher：株式会社医学書院  

DOI： 10.11477/mf.1401208754

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Expanding distributions of red back spiders and bites in Japan from 2011 to 2013

Yamagishi Takuya, Arai Satoru, Hifumi Toru, Sawabe Kyoko, Yamamoto Akihiko, Shibayama Keigo, Ato Manabu, Taki Hisashi, Goka Koichi, Oishi Kazunori

Medical Entomology and Zoology   Vol. 67 ( 4 ) page： 219 - 221   2016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japan Society of Medical Entomology and Zoology  

<p>In Japan, fourteen bites by the red back spider, <i>Latrodectus hasseltii</i>, were reported in four hospitals between 2011 and 2013 in a survey of sentinel hospitals. The distribution of the spider and the areas in which patients were bitten by the spider both expanded geographically each year. Although fatalities or severely ill patients are yet to be reported in Japan, stockpiles of the antivenom and communication with the public and medical professionals should be considered.</p>

DOI： 10.7601/mez.67.219

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Understanding Procedural Knowledge for Solving Arithmetic Task by Externalization

Miwa, K; Terai, H; Shibayama, K

INTELLIGENT TUTORING SYSTEMS, ITS 2016   Vol. 9684   page： 3 - 12   2016

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Language：English   Publisher：Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics)  

Students build cognitive models for solving a cryptarithmetic task in a learning environment that enables them to formally describe various types of procedural knowledge in a group learning setting in which each student is allowed to refer to the procedural rules described by the other group members. Experimental evaluation showed that: (1) three-quarters of participants successfully constructed valid models with the system, and (2) participants learned to describe procedural knowledge more precisely not only for the training task (cryptarithmetic task) but also for a transfer task (bug identification for a multi-column subtraction problem).

DOI： 10.1007/978-3-319-39583-8_1

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アンチバイオグラム

柴山 恵吾

ファルマシア   Vol. 51 ( 4 ) page： 342_2 - 342_2   2015

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：公益社団法人 日本薬学会  

国，地域，病院，または病棟ごとに分離された細菌の薬剤感受性試験のデータを集計し，それぞれの菌種で各種抗菌薬について耐性，中間，感性の菌がどれくらいの割合であるのかを図や表にしたもの．薬剤耐性菌対策を検討していくための重要な基本情報となる．また，臨床現場における治療薬のガイドラインを作成する際には，重要な参考情報となる．さらに，病院や病棟ごとでデータの経時的な変化を見たり，そのデータを地域や国全体のデータと比較することで，感染制御がうまくできているかどうかを評価することにも用いられる．

DOI： 10.14894/faruawpsj.51.4_342_2

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日本における薬剤耐性菌の状況

柴山 恵吾

ファルマシア   Vol. 51 ( 4 ) page： 324 - 328   2015

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：公益社団法人 日本薬学会  

20世紀に入ってからペニシリンをはじめとする様々な抗菌薬が開発され，人類にとって大きな脅威である感染症から多くの人が救われてきた．しかし一方で，新たな抗菌薬が開発されると間もなく，その薬剤に耐性を獲得した細菌が出現し，薬剤の普及とともに拡散してきた．これまでに耐性が出現しなかった抗菌薬はなく，これは今後も続くと考えられる．近年，薬剤耐性菌は国境を越えて拡散し，世界的な問題になっている．菌種や薬剤によっては，耐性菌の割合が数年で大きく増加しているものもある．WHOは2013年に薬剤耐性菌に関する諮問グループを組織し，2014年に薬剤耐性菌に関する報告書を出した．WHOは，この中で今後取り組むべき課題の1つとして，薬剤耐性菌のグローバルサーベイランスの強化を挙げている．<br>日本においては，薬剤耐性菌のサーベイランスとして厚生労働省院内感染対策サーベイランス（Japan Nosocomial Infection Surveillance：JANIS）事業がある．本稿では，JANISの2013年の公開情報2）のデータを用いて，主要な各種耐性菌の国内での状況を紹介する．さらに，近年特に問題になっているカルバペネム耐性の腸内細菌科細菌について，日本でよく検出される耐性遺伝子や外国の状況との比較なども説明する．また，JANISが各参加医療機関個別に感染対策に活用していただくことを目的に返している報告書についても触れる．

DOI： 10.14894/faruawpsj.51.4_324

CiNii Research

28-P2PM-017 本邦におけるAcinetobacter spp.の分離状況と耐性獲得状況(感染制御(その他)1,一般演題(ポスター),新時代を拓く医療薬学フロンティア)

木村 修徳, 松井 真理, 鈴木 里和, 鈴木 仁人, 柴山 恵吾, 三浦 布紗子, 永野 真久, 安森 奈緒子, 井上 大奨, 平木 洋一, 真鍋 健一, 河野 文夫

日本医療薬学会年会講演要旨集   Vol. 24 ( 0 ) page： 421   2014.8

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：一般社団法人 日本医療薬学会  

DOI： 10.20825/amjsphcs.24.0_421_6

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Genome sequence of a strain of the human pathogenic bacterium Pseudomonas alcaligenes that caused bloodstream infection Reviewed

Suzuki M., Suzuki S., Matsui M., Hiraki Y., Kawano F., Shibayama K.

Genome Announcements   Vol. 1 ( 5 )   2013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Genome Announcements  

Pseudomonas alcaligenes, a Gram-negative aerobic bacterium, is a rare opportunistic human pathogen. Here, we report the whole-genome sequence of P. alcaligenes strain MRY13-0052, which was isolated from a bloodstream infection in a medical institution in Japan and is resistant to antimicrobial agents, including broad-spectrum cephalosporins and monobactams.

DOI： 10.1128/genomeA.e00919-13

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Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase Reviewed

SHIBAYAMA Keigo, TAKEUCHI Hiroaki, WACHINO Jun-ichi, MORI Shigetarou, ARAKAWA Yoshichika

Microbiology and immunology   Vol. 55 ( 6 ) page： 408 - 417   2011.6

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Virulence Genes, Quinolone and Fluoroquinolone Resistance, and Phylogenetic Background of Uropathogenic <i>Escherichia coli</i> Strains Isolated in Japan Reviewed

Kawamura-Sato Kumiko, Yoshida Risa, Shibayama Keigo, Ohta Michio

Japanese Journal of Infectious Diseases   Vol. 63 ( 2 ) page： 113 - 115   2010.3

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Language：English   Publisher：National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee  

<p>A total of 312 uropathogenic <i>Escherichia coli</i> strains were isolated from clinical specimens in 7 hospitals in Aichi Prefecture, Japan. Among them, 113 strains were resistant to quinolone, and 49 strains were resistant to fluoroquinolone. Phylogenetic group B2 was most prevalent in both susceptible strains (148 of 199 strains, 74.4%) and resistant strains (quinolone-resistant strains, 73 of 113 strains, 64.6%; fluoroquinolone-resistant strains, 40 of 49 strains, 81.6%). The resistant strains showed a significantly lower prevalence of virulence genes <i>papA</i>, <i>hlyA</i>, and <i>cnf1</i> than did the susceptible strains, and this observation was further obvious when compared within B2 group strains. Among the 40 fluoroquinolone-resistant strains belonging to group B2, 37 (92.5%) strains carried PAI<i>usp</i> subtype IIa, 36 strains of which carried E84V mutation in <i>par</i>C, whereas none of the 9 strains belonging to group D carried PAI<i>usp</i> subtype IIa, and only one strain carried the mutation. These observations indicate that the differences of phenotypes including resistance of quinolone and carriage of virulence genes are associated with the complex context of genetic background, including the phylogenetic group and PAI<i>usp</i> subtype.<tt> </tt></p>

DOI： 10.7883/yoken.63.113

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Protective effect against 17β-estradiol on neuronal apoptosis in hippocampus tissue following transient ischemia/recirculation in Mongolian gerbils via down-regulation of tissue transglutaminase activity

Fujita K., Kato T., Shibayama K., Imada H., Yamauchi M., Yoshimoto N., Miyachi E., Nagata Y.

Neurochemical Research   Vol. 31 ( 8 ) page： 1059 - 1068   2006.8

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Language：English   Publisher：Neurochemical Research  

We analyzed the protective effect of 17β-estradiol (17β-ED) injection against delayed neuronal death in the hippocampus tissue of the brain in Mongolian gerbils after transient ischemia/recirculation treatment, especially in relation with bcl-2 gene expression and enzymatic activity changes of caspase-3 and tissue transglutaminase (tTGase). Daily intraperitoneal injection of 17β-ED to the animal after the ischemia stimulated the expression of an apoptosis suppressor gene, bcl-2, in the hippocampal tissue for a week. The gradually increasing apoptotic enzyme activity of caspase-3 and increased number of TUNEL positive fragmented neuronal nuclei caused by ischemic attack in the gerbil brain were clearly suppressed by 17β-ED administration. The reduced activity and enzyme protein of tTGase, a neurodegenerative marker of apoptosis in the hippocampus after ischemia, were also restored to nearly normal levels by 17β-ED injection. These results suggest that daily 17β-ED administration to the gerbil after transient ischemic insult with progressing neuronal deteriorative changes in hippocampus tissue can effectively prevent apoptotic changes through a molecular cascade involving gene expression regulation. © Springer Science+Business Media, Inc. 2006.

DOI： 10.1007/s11064-006-9114-y

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[H. pylori infection and epithelial cell apoptosis]. Invited

Keigo Shibayama, Yoshichika Arakawa

Nihon rinsho. Japanese journal of clinical medicine   Vol. 63 Suppl 11   page： 156 - 60   2005.11

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H. pylori infection and epithelial cell apoptosis Invited

Shibayama K., Arakawa Y.

Nippon rinsho. Japanese journal of clinical medicine   Vol. 63 Suppl 11   page： 156 - 160   2005.11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nippon rinsho. Japanese journal of clinical medicine  

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H.pylori感染胃上皮細胞におけるアポトーシス

柴山 恵吾

日本臨床 (in press)     2005

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Helicobacter pyloriにおけるアモキシシリン耐性について

柴山 恵吾

日本ヘリコバクター学会雑誌 (in press)     2005

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Growth Competition of Macrolide-Resistant and -Susceptible Helicobacter pylori Strains Reviewed

KANAI Kyoko, SHIBAYAMA Keigo, SUZUKI Satowa, WACHINO Jun-ichi, ARAKAWA Yoshichika

Microbiology and Immunology   Vol. 48 ( 12 ) page： 977 - 980   2004.12

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Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Reviewed International journal

Keiko Yokoyama, Yohei Doi, Kunikazu Yamane, Hiroshi Kurokawa, Naohiro Shibata, Keigo Shibayama, Tetsuya Yagi, Haru Kato, Yoshichika Arakawa

Lancet (London, England)   Vol. 362 ( 9399 ) page： 1888 - 93   2003.12

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BACKGROUND: Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. METHODS: We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. FINDINGS: An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. INTERPRETATION: Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.

DOI： 10.1016/S0140-6736(03)14959-8

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PCR Typing of Genetic Determinants for Metallo-β-Lactamases and Integrases Carried by Gram-Negative Bacteria Isolated in Japan, with Focus on the Class 3 Integron Reviewed

Naohiro Shibata, Yohei Doi, Kunikazu Yamane, Tetsuya Yagi, Hiroshi Kurokawa, Keigo Shibayama, Haru Kato, Kumiko Kai, Yoshichika Arakawa

Journal of Clinical Microbiology   Vol. 41 ( 12 ) page： 5407 - 13   2003.12

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From January 2001 to December 2002, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-β-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (blaIMP-1), while only 7 and 67 strains carried the IMP-2 gene (blaIMP-2) and the VIM-2 gene (blaVIM-2), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the intI1 gene, and 4 strains carrying both the intI1 and intI3 genes were reidentified as Pseudomonas putida harboring blaIMP-1. Of these four P. putida strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357 bla IMP-1 carriers, 116, 53, 51, 47, and 30 strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, P. putida/fluorescens, Serratia marcescens, and Acinetobacter baumannii, respectively. Four strains carrying blaIMP-2 were reidentified as P. putida. Sixty-three P. aeruginosa strains and four P. putida strains carried blaVIM-2. Of 427 intI1-positive strains, 180, 53, 51, 47, and 35 were identified as P. aeruginosa, A. xylosoxidans, P. putida/fluorescens, S. marcescens, and A. baumannii, respectively. In the present study, it was confirmed that strains carrying blaIMP-1 with a class 1 integron are the most prevalent type in Japan, although several intI3 carriers have also been identified sporadically in this country.

DOI： 10.1128/JCM.41.12.5407-5413.2003

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A new TEM-derived extended-spectrum β-lactamase (TEM-91) with an R164C substitution at the ω-loop confers ceftazidime resistance Reviewed

Hiroshi Kurokawa, Naohiro Shibata, Yohei Doi, Keigo Shibayama, Kazunari Kamachi, Tetsuya Yagi, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 47 ( 9 ) page： 2981 - 3   2003.9

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A new plasmid-mediated TEM-derived extended-spectrum β-lactamase, TEM-91, was identified in a ceftazidime-resistant (MIC, >128 μg per ml) Escherichia coli strain isolated in 1996 in Japan. TEM-91 has three amino acid substitutions, R164C, M184T, and E240K, compared with TEM-1 penicillinase. The isoelectric point (pI), Km, and kcat of TEM-91 for ceftazidime were 5.7, 179 μM, and 29.0 s-1, respectively. The Ki of clavulanic acid for ceftazidime hydrolysis was 30.3 nM.

DOI： 10.1128/AAC.47.9.2981-2983.2003

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A novel apoptosis-inducing protein from Helicobacter pylori Reviewed

Keigo Shibayama, Kazunari Kamachi, Noriyo Nagata, Tetsuya Yagi, Toshi Nada, Yohei Doi, Naohiro Shibata, Keiko Yokoyama, Kunikazu Yamane, Haru Kato, Yoshitsugu Iinuma, Yoshichika Arakawa

Molecular Microbiology   Vol. 47 ( 2 ) page： 443 - 51   2003.1

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Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori-mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited γ-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-L-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial γ-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant γ-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that γ-glutamyl transpeptidase plays a significant role in H. pylori-mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial γ-glutamyl transpeptidase function.

DOI： 10.1046/j.1365-2958.2003.03305.x

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Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate Reviewed

Yohei Doi, Naohiro Shibata, Keigo Shibayama, Kazunari Kamachi, Hiroshi Kurokawa, Keiko Yokoyama, Tetsuya Yagi, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 46 ( 8 ) page： 2427 - 34   2002.8

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An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The β-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of β-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sull-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of blaCMY-9 and ended with a truncated 3′ conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. blaCMY-9 was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of blaCMY-9 from some environmental microorganisms such as aeromonads.

DOI： 10.1128/AAC.46.8.2427-2434.2002

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Trends in antimicrobial-drug resistance in Japan Reviewed

Yoshichika Arakawa, Yasuyoshi Ike, Mitsuaki Nagasawa, Naohiro Shibata, Yohei Doi, Keigo Shibayama, Tetsuya Yagi, Takeshi Kurata

Emerging Infectious Diseases   Vol. 6 ( 6 ) page： 572 - 575   2000

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Multidrug resistance in gram-positive bacteria has become common worldwide. In Japan until recently, gram-negative bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia marcescens were controlled by carbapenems, fluoroquinolones, and aminoglycosides. However, several of these microorganisms have recently developed resistance against many antimicrobial drugs.

DOI： 10.3201/eid0606.000604

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β-Lactamase inhibitors have antibacterial activities against Helicobacter pylori Reviewed

HORII Toshinobu, KIMURA Taku, SATO-KAWAMURA Kumiko, NADA Tochi, SHIBAYAMA Keigo, OHTA Michio

J Infect Chemother.   Vol. 5 ( 4 ) page： 206 - 207   1999.12

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β-Lactamase inhibitors have antibacterial activities against Helicobacter pylori Reviewed

Horii T., Kimura T., Sato-Kawamura K., Nada T., Shibayama K., Ohta M.

Journal of Infection and Chemotherapy   Vol. 5 ( 4 ) page： 206 - 207   1999.12

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Recently, it was reported that amoxicillin-clavulanate has slightly higher activity than amoxicillin against Helicobacter pylori. In this study, we evaluated the in-vitro antibacterial activity of β-lactamase inhibitors against H. pylori. We investigated the susceptibility of 30 H. pylori strains to β-lactamase inhibitors, including clavulanate, sulbactam, and tazobactam. In short-term bactericidal studies, a clinical isolate of H. pylori NU27 was exposed to 1 x minimum inhibitory concentration (MIC) of the β-lactamase inhibitors, amoxicillin, clarithromycin, and amoxicillin-clavulanate for 3 and 6h. The MICs90 for these β-lactamase inhibitors were 2, 4, and 2 mg/l, respectively. The short-term bactericidal studies showed that these β- lactamase inhibitors decreased viable counts of H. pylori during 6-h exposure at 1 x MIC. Our results suggest that β-lactamase inhibitors have in-vitro antibacterial activity against H. pylori. Amoxicillin and clavulanate used in combination resulted in increased antibacterial activity.

DOI： 10.1007/s101560050036

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Role of multiple efflux pumps in Escherichia coli in indole expulsion Reviewed

Kumiko Kawamura-Sato, Keigo Shibayama, Toshinobu Horii, Yoshitsugu Iimuma, Yoshichika Arakawa, Michio Ohta

FEMS Microbiology Letters   Vol. 179 ( 2 ) page： 345 - 352   1999.10

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Escherichia coli chromosome encodes several multidrug transporters. Despite their protective function against antibacterial agents, the specific physiological actions of these transporters are not fully understood. E. coli produces indole, a metabolite of tryptophan, under physiological conditions. Defined inactivation of the acrEF gene, the product of which is known as an energy-dependent multiple drug efflux pump, decreased indole excretion while reintroduction of the acrEF gene restored it. A ΔacrEF mutant accumulated more intracellular indole than the parent. This mutant was more susceptible to the growth-inhibitory effect of indole than the parent. These results indicate that the AcrEF system plays a significant role in indole efflux. Copyright (C) 1999 Federation of European Microbiological Societies.

DOI： 10.1016/S0378-1097(99)00433-4

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Are Autoantibodies against Lewis Antigens Involved in the Pathogenesis of Helicobacter pylori-Induced Peptic Ulcers? Reviewed

KAMIYA Kenji, ARISAWA Tomiyasu, GOTO Hidemi, SHIBAYAMA Keigo, HORII Toshinobu, HAYAKAWA Tetsuo, OHTA Michio

MICROBIOLOGY and IMMUNOLOGY   Vol. 43 ( 5 ) page： 403 - 408   1999.5

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We examined whether anti-Lewis x (Le^x) and y (Le^y) autoantibodies affect the pathogenesis of <i>Helicobacter pylori</i>-induced peptic ulcers. Of 11 patients with peptic ulcers, 10 patients had both anti-Le^x and -Le^y immunoglobulin G (IgG) antibodies, and 1 patient had only anti-Le^x antibody. After successful eradication, we measured the serum titer of anti-Le^x and -Le^y antibodies. Six patients had a reduction of the titers of anti-Le^x and/or -Le^y antibodies, whereas no notable changes were detected in 5 patients in the follow-up. This result suggests that anti-Le^x and -Le^y autoantibodies had no critical role in the development of <i>H. pylori</i>-induced peptic ulcer.

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Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR Reviewed

Shibayama K., Ohsuka S., Sato K., Yokoyama K., Horii T., Ohta M.

FEMS Microbiology Letters   Vol. 174 ( 1 ) page： 105 - 109   1999.5

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FEMS Microbiology Letters  

Escherichia coli K-12 WaaR is a non-processive α-1,2 glucosyltransferase, involved in the synthesis of the R-core of lipopolysaccharide. WaaR possesses the four conserved structural regions I, II, III and IV, each presumably involved in the mechanistic function in catalysis. Regions I and III contain the pair of strictly conserved Asp residues. Asp-129, 131 (region I) and 215, 217 (region III) of WaaR were individually converted to Asn by the site-directed mutagenesis of the waaR gene. All mutated enzymes were inactive, supporting the model for an α-glycosyl transfer reaction where the pair of strictly conserved aspartic acid residues in regions I and III play a critical role in the catalytic function. Copyright (C) 1999 Federation of European Microbiological Societies.

DOI： 10.1016/S0378-1097(99)00127-5

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Antibacterial Activities of New Synthetic Divalent Cation Chelators Reviewed

ASHOORI Mandana, OHTA Michio, OHSUKA Shinji, SHIBAYAMA Keigo, HORII Toshinobu, UEDA Minoru, KUROSAKI Hiromasa

MICROBIOLOGY and IMMUNOLOGY   Vol. 43 ( 4 ) page： 311 - 316   1999.4

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A series of new synthetic ligand compounds which chelate divalent cations was examined for the antibacterial activities of the compounds. Only 2 of 14 synthetic chelators, 9-<i>trans</i>-anthryl-1, 4, 8, 11-tetraazatetradecane (No. 6) and bis(2-pyridyl)methylamine (No. 13) showed antibacterial activities, whereas none of the diamines, hydrophilic triamines nor tetramines showed antibacterial activities. Chelators No. 6 and No. 13 inhibited the growth of both Gram-negative and -positive bacteria at doses of 25-200μg/ml, comparable to those of common antibiotics such as polymixin B, fosfomycin and macrolides. Ethylenediaminetetraacetate (EDTA) potentiated these antibacterial activities, whereas an inhibitory effect of Mg²⁺ on the MICs of these chelators was observed. Moreover, these chelators enhanced the leakage of periplasmic β-lactamase. It is therefore suggested that chelators No. 6 and No. 13 disrupt both the membranes and cytoplasmic functions of bacteria, resulting in cell death.

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Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli O26 Reviewed

Horii T., Kimura T., Sato K., Shibayama K., Ohta M.

Antimicrobial Agents and Chemotherapy   Vol. 43 ( 4 ) page： 789 - 793   1999.4

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We evaluated the susceptibilities of 129 Shiga-like toxin-producing Escherichia coli (STEC) isolates to various antibiotics. The numbers of isolates for which MICs were high (≥128 μg/ml) were as follows: 5 for fosfomycin, 14 for ampicillin, 1 for cefaclor, 6 for kanamycin, 22 for tetracycline, and 2 for doxycycline. For two isolates of STEC O26 MICs of fosfomycin were high (1,024 and 512 μg/ml, respectively). Conjugation experiments and glutathione S-transferase assays suggested that the fosfomycin resistance in these isolates was determined not by a plasmid but chromosomally. The amount of active intracellular fosfomycin in these STEC isolates was 100- to 200-fold less than that in E. coli C600 harboring pREFTT47B408 in the presence of either L-α-glycerophosphate or glucose-6- phosphate. Cloning, sequencing, and Northern blot analysis demonstrated that the transcriptional level of the murA gene encoding UDP-N-acetylglucosamine enolpyruvoyl transferase in these isolates was greater than that in E. coli C600. Our results suggest that the fosfomycin resistance in these STEC isolates is due to concurrent effects of alteration of the glpT and/or uhp transport systems and of the enhanced transcription of the murA gene.

DOI： 10.1128/aac.43.4.789

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Growth Conditions of and Emetic Toxin Production by Bacillus cereus in a Defined Medium with Amino Acids Reviewed

AGATA Norio, OHTA Michio, MORI Masashi, SHIBAYAMA Keigo

MICROBIOLOGY and IMMUNOLOGY   Vol. 43 ( 1 ) page： 15 - 18   1999.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Center For Academic Publications Japan  

The growth and emetic toxin (cereulide) production of <i>Bacillus cereus</i> strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by <i>B. cereus</i>. The addition of high levels (50mM) of leucine, isoleucine and glutamlc acid decreased the toxin production. Other amino acids had no effect at this concentration.

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Antibacterial Activities of New Synthetic Divalent Cation Chelators Reviewed

Ashoori Mandana, Ohta Michio, Ohsuka Shinji, Shibayama Keigo, Horii Toshinobu, Ueda Minoru, Kurosaki Hiromasa

Japanese Journal of Microbiology   Vol. 43 ( 4 ) page： 311 - 316   1999

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Center For Academic Publications Japan  

A series of new synthetic ligand compounds which chelate divalent cations was examined for the antibacterial activities of the compounds. Only 2 of 14 synthetic chelators, 9-<i>trans</i>-anthryl-1, 4, 8, 11-tetraazatetradecane (No. 6) and bis(2-pyridyl)methylamine (No. 13) showed antibacterial activities, whereas none of the diamines, hydrophilic triamines nor tetramines showed antibacterial activities. Chelators No. 6 and No. 13 inhibited the growth of both Gram-negative and -positive bacteria at doses of 25-200μg/ml, comparable to those of common antibiotics such as polymixin B, fosfomycin and macrolides. Ethylenediaminetetraacetate (EDTA) potentiated these antibacterial activities, whereas an inhibitory effect of Mg²⁺ on the MICs of these chelators was observed. Moreover, these chelators enhanced the leakage of periplasmic β-lactamase. It is therefore suggested that chelators No. 6 and No. 13 disrupt both the membranes and cytoplasmic functions of bacteria, resulting in cell death.

CiNii Research

Antibacterial activities of new synthetic divalent cation chelators Reviewed

Ashoori M., Ohta M., Ohsuka S., Shibayama K., Horii T., Ueda M., Kurosaki H.

Microbiology and Immunology   Vol. 43 ( 4 ) page： 311 - 316   1999

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Microbiology and Immunology  

A series of new synthetic ligand compounds which chelate divalent cations was examined for the antibacterial activities of the compounds. Only 2 of 14 synthetic chelators, 9-trans-anthryl-1, 4, 8, 11-tetraazatetradecane (No. 6) and bis(2-pyridyl)methylamine (No. 13) showed antibacterial activities, whereas none of the diamines, hydrophilic triamines nor tetramines showed antibacterial activities. Chelators No. 6 and No. 13 inhibited the growth of both Gram-negative and -positive bacteria at doses of 25-200 μg/ml, comparable to those of common antibiotics such as polymixin B, fosfomycin and macrolides. Ethylenediaminetetraacetate (EDTA) potentiated these antibacterial activities, whereas an inhibitory effect of Mg2+ on the MICs of these chelators was observed. Moreover, these chelators enhanced the leakage of periplasmic β-lactamase. It is therefore suggested that chelators No. 6 and No. 13 disrupt both the membranes and cytoplasmic functions of bacteria, resulting in cell death.

DOI： 10.1111/j.1348-0421.1999.tb02410.x

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Are autoantibodies against Lewis antigens involved in the pathogenesis of Helicobacter pylori-induced peptic ulcers? Reviewed

Kamiya K., Arisawa T., Goto H., Shibayama K., Horii T., Hayakawa T., Ohta M.

Microbiology and Immunology   Vol. 43 ( 5 ) page： 403 - 408   1999

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Microbiology and Immunology  

We examined whether anti-Lewis x (Le(x)) and y (Le(y)) autoantibodies affect the pathogenesis of Helicobacter pylori-induced peptic ulcers. Of 11 patients with peptic ulcers, 10 patients had both anti-Le(x) and -Le(y) immunoglobulin G (IgG) antibodies, and 1 patient had only anti-Le(x) antibody. After successful eradication, we measured the serum titer of anti- Le(x) and -Le(y) antibodies. Six patients had a reduction of the titers of anti-Le(x) and/or -Le(y) antibodies, whereas no notable changes were detected in 5 patients in the follow-up. This result suggests that anti-Le(x) and - Le(y) autoantibodies had no critical role in the development of H. pylori- induced peptic ulcer.

DOI： 10.1111/j.1348-0421.1999.tb02423.x

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Are Autoantibodies against Lewis Antigens Involved in the Pathogenesis of <i>Helicobacter pylori</i>-Induced Peptic Ulcers? Reviewed

Kamiya Kenji, Arisawa Tomiyasu, Goto Hidemi, Shibayama Keigo, Horii Toshinobu, Hayakawa Tetsuo, Ohta Michio

Japanese Journal of Microbiology   Vol. 43 ( 5 ) page： 403 - 408   1999

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Center For Academic Publications Japan  

We examined whether anti-Lewis x (Le^x) and y (Le^y) autoantibodies affect the pathogenesis of <i>Helicobacter pylori</i>-induced peptic ulcers. Of 11 patients with peptic ulcers, 10 patients had both anti-Le^x and -Le^y immunoglobulin G (IgG) antibodies, and 1 patient had only anti-Le^x antibody. After successful eradication, we measured the serum titer of anti-Le^x and -Le^y antibodies. Six patients had a reduction of the titers of anti-Le^x and/or -Le^y antibodies, whereas no notable changes were detected in 5 patients in the follow-up. This result suggests that anti-Le^x and -Le^y autoantibodies had no critical role in the development of <i>H. pylori</i>-induced peptic ulcer.

CiNii Research

Growth Conditions of and Emetic Toxin Production by <i>Bacillus cereus</i> in a Defined Medium with Amino Acids Reviewed

Agata Norio, Ohta Michio, Mori Masashi, Shibayama Keigo

Japanese Journal of Microbiology   Vol. 43 ( 1 ) page： 15 - 18   1999

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Center For Academic Publications Japan  

The growth and emetic toxin (cereulide) production of <i>Bacillus cereus</i> strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by <i>B. cereus</i>. The addition of high levels (50mM) of leucine, isoleucine and glutamlc acid decreased the toxin production. Other amino acids had no effect at this concentration.

CiNii Research

Growth conditions of and emetic toxin production by Bacillus cereus in a defined medium with amino acids Reviewed

Agata N., Ohta M., Mori M., Shibayama K.

Microbiology and Immunology   Vol. 43 ( 1 ) page： 15 - 18   1999

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Microbiology and Immunology  

The growth and emetic toxin (cereulide) production of Bacillus cereus strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by B. cereus. The addition of high levels (50 mM) of leucine, isoleucine and glutamic acid decreased the toxin production. Other amino acids had no effect at this concentration.

DOI： 10.1111/j.1348-0421.1999.tb02367.x

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Streptococcal pyrogenic exotoxin F (SpeF) causes permeabilization of lung blood vessels Reviewed

Matsumoto M., Ishikawa N., Saito M., Shibayama K., Horii T., Sato K., Ohta M.

Infection and Immunity   Vol. 67 ( 9 ) page： 4307 - 4311   1999

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Infection and Immunity  

Acute respiration distress syndrome (ARDS) is a typical complication in toxic shock-like syndrome (TSLS) caused by Streptococcus pyogenes. An isolated perfused rat lung model was used to identify the causative agent of ARDS in TSLS in this study. Some crude preparations separated from the culture supernatants of S. pyogenes isolates caused rapid increases in the weight of perfused lungs, indicating vascular permeabilization. Six samples from M type 1 and 3 isolates from TSLS and pharyngitis patients showed strong permeabilization activity, whereas preparations from isolates of other M types (although the number of isolates examined was limited) were negative. The active substance was purified to a single band by sodium dodecyl sulfate- polyacrylamide gel electrophoresis using various columns, and the N-terminal amino acid sequence was determined. The resultant sequence of eight amino acids was identical to SpeF (mitogenic factor). Moreover, the vascular permeabilization activity of the purified band was abolished with anti-SpeF antiserum prepared by immunizing with the purified SpeF. This activity was also neutralized by the antiserum prepared by immunizing with a synthetic peptide derived from the published SpeF sequence. These results suggested that streptococcal SpeF is a major cause of permeabilization of lung blood vessels and sufficient for the pathogenesis of ARDS under the conditions of TSLS caused by S. pyogenes.

DOI： 10.1128/iai.67.9.4307-4311.1999

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Heterogeneity of Phenotypic and Genotypic Traits Including Organic-Acid Resistance in Escherichia coli O157 Isolates Reviewed

HORII Toshinobu, BARUA Soumitra, KIMURA Taku, KASUGAI Shin, SATO Kumiko, SHIBAYAMA Keigo, ICHIYAMA Satoshi, OHTA Michio

MICROBIOLOGY and IMMUNOLOGY   Vol. 42 ( 12 ) page： 871 - 874   1998.12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Center For Academic Publications Japan  

We undertook an epidemiologic study for the sensitivity of both Shiga-like toxin (Slt)-producing <i>Escherichia coli</i> (STEC) O157 and non-STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The <i>E. coli</i> O157 isolates examined showed a wide variety of organic-acid susceptibility patterns. <i>E. coli</i> O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.

CiNii Research

Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI) Reviewed

Shibayama K., Ohsuka S., Tanaka T., Arakawa Y., Ohta M.

Journal of Bacteriology   Vol. 180 ( 20 ) page： 5313 - 5318   1998.10

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Journal of Bacteriology  

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α- glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.

DOI： 10.1128/jb.180.20.5313-5318.1998

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Heterogeneity of Phenotypic and Genotypic Traits Including Organic-Acid Resistance in <i>Escherichia coli</i> O157 Isolates Reviewed

Horii Toshinobu, Barua Soumitra, Kimura Taku, Kasugai Shin, Sato Kumiko, Shibayama Keigo, Ichiyama Satoshi, Ohta Michio

Japanese Journal of Microbiology   Vol. 42 ( 12 ) page： 871 - 874   1998

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Center For Academic Publications Japan  

We undertook an epidemiologic study for the sensitivity of both Shiga-like toxin (Slt)-producing <i>Escherichia coli</i> (STEC) O157 and non-STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The <i>E. coli</i> O157 isolates examined showed a wide variety of organic-acid susceptibility patterns. <i>E. coli</i> O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.

CiNii Research

Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like β-lactamase genes Reviewed

Yagi T., Kurokawa H., Senda K., Ichiyama S., Ito H., Ohsuka S., Shibayama K., Shimokata K., Kato N., Ohta M., Arakawa Y.

Antimicrobial Agents and Chemotherapy   Vol. 41 ( 12 ) page： 2606 - 2611   1997.12

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Escherichia coli HKY56, which demonstrated resistance to various β- lactams except carbapenems, was isolated from the throat swab of an inpatient in 1994. Conjugal transfer of cephem resistance from HKY56 to E. coli CSH2 was not successful. Three cefotaxime-resistant E. coli clones harboring plasmid pMRE001, pMRE002, or pMRE003, each of which carried a 3.4-, 5.8-, or 6.2-kb EcoRI fragment insert, respectively, were obtained from HKY56. Although restriction analysis suggested their different origins, these clones showed similar profiles of resistance to various β-lactams. The sequence of 10 amino acid residues at the N terminus of β-lactamase purified from E. coli HB101(pMRE001) was identical to that of Toho-1. This Toho-1-like β- lactamase-1 (TLB-1) was able to hydrolyze cefoperazone and cefotaxime efficiently, but it failed to hydrolyze cephamycins. A Toho-1-specific DNA probe was hybridized with three distinct EcoRI fragments derived from the chromosomal DNA of strain HKY56, and these fragments corresponded to DNA inserts carried by pMRE001, pMRE002, and pMRE003, respectively. PCR and Southern hybridization analysis suggested that all six cephem-resistant E. coli strains, strains HKY273, HKY285, HKY288, HKY305, HKY316, and HKY335, which were isolated in 1996 at the same hospital where strain HKY56 had been isolated, also possessed multiple Toho-1-like β-lactamase (TLB) genes, and the hybridization patterns obtained with the Toho-1-specific probe were quite similar among these six isolates. The DNA fingerprinting patterns observed by pulsed-field gel electrophoresis revealed that among the E. coli isolates tested, all isolates except HKY56 possessed a similar genetic background. These findings suggested that E. coli strains that carry chromosomally multiplied TLB genes may have been proliferating and transmitted among patients in the same hospital.

DOI： 10.1128/aac.41.12.2606

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RobA-Induced Multiple Antibiotic Resistance Largely Depends on the Activation of the AcrAB Efflux Reviewed

TANAKA Toshihiko, HORII Toshinobu, SHIBAYAMA Keigo, SATO Kumiko, OHSUKA Shinji, ARAKAWA Yoshichika, YAMAKI Ken-ichi, TAKAGI Kenzo, OHTA Michio

MICROBIOLOGY and IMMUNOLOGY   Vol. 41 ( 9 ) page： 697 - 702   1997.9

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RobA is a member of the XylS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in <i>Escherichia coli</i>. In this study, we introduced a multicopy <i>robA</i> plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB-deficient mutant. We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression. Interestingly, a <i>ΔompF</i> mutant (TK007) became more sensitive when pMEP1 was introduced. Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an <i>acrA1</i>^- mutant was observed. Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of <i>micF</i>.

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RobA-induced multiple antibiotic resistance largely depends on the activation of the AcrAB efflux Reviewed

Tanaka T., Horii T., Shibayama K., Sato K., Ohsuka S., Arakawa Y., Yamaki K.I., Takagi K., Ohta M.

Microbiology and Immunology   Vol. 41 ( 9 ) page： 697 - 702   1997

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RobA is a member of the XyIS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in Escherichia coli. In this study, we introduced a multicopy robA plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB-deficient mutant. We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression. Interestingly, a ΔompF mutant (TK007) became more sensitive when pMEP1 was introduced. Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an acrA1- mutant was observed. Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of micF.

DOI： 10.1111/j.1348-0421.1997.tb01913.x

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Effects of coronavirus disease 2019 on the spread of respiratory-transmitted human-to-human bacteria Reviewed

Kajihara T, Yahara K, Kamigaki T, Hirabayashi A, Hosaka Y, Kitamura N, Shimbashi R, Suzuki M, Sugai M, Shibayama K

Journal of Infection   Vol. 89 ( 2 )   2024.8

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DOI： 10.1016/j.jinf.2024.106201

ect Hirabayashi A, Yahara K, Oka K, Kajihara T, Ohkura T, Hosaka Y, Shibayama K, Sugai M, Yagi T. Comparison of disease and economic burden between MRSA infection and MRSA colonization in a university hospital: a retrospective data integration study. Reviewed

Hirabayashi A, Yahara K, Oka K, Kajihara T, Ohkura T, Hosaka Y, Shibayama K, Sugai M, Yagi T.

Antimicrob Resist Infect Control   Vol. 13 ( 1 )   2024.2

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DOI： 10.1186/s13756-024-01383-8

Protocol for detecting Helicobacter suis infection in gastric biopsies and serum by PCR and ELISA International journal Open Access

Hidenori Matsui, Masato Suzuki, Sae Aoki, Keigo Shibayama, Kengo Tokunaga, Hidekazu Suzuki, Katsuhiro Mabe, Tsuyoshi Kenri, Emiko Rimbara

STAR Protocols   Vol. 4 ( 4 ) page： 102556 - 102556   2023.12

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Infection with Helicobacter suis, which causes many cases of gastric disease, is not reliably diagnosed. Here, we present a protocol for detecting H. suis infection. We describe steps for collecting gastric biopsies and sera from patients, preparing DNA for PCR, and targeting the H. suis-specific gene. We then define procedures for inoculating biopsies onto primary agar plates and transferring colonies to secondary agar plates. Finally, we detail whole-genome sequencing of bacteria and assess H. suis infection in sera with ELISA. For complete details on the use and execution of these protocols, please refer to Matsui et al.1.

DOI： 10.1016/j.xpro.2023.102556

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【上部消化管疾患の残された課題】NHPHによる胃疾患の疫学と病態

徳永 健吾, 井田 陽介, 三好 佐和子, 大野 亜希子, 久松 理一, 林原 絵美子, 松井 英則, 鈴木 仁人, 柴山 恵吾, 大崎 敬子

Progress in Medicine   Vol. 43 ( 9 ) page： 813 - 815   2023.9

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Temporal shifts in the predominant carbapenemase gene types among carbapenemase-producing Klebsiella pneumoniae isolated in Bangkok, Thailand, during 2013–2016

Noriko Sakamoto, Warawut Laolerd, Yukihiro Akeda, Yo Sugawara, Daisuke Motooka, Norihisa Yamamoto, Dan Takeuchi, Rathina Kumar Shanmugakani, Isao Nishi, Masato Suzuki, Keigo Shibayama, Tetsuya Iida, Pitak Santanirand, Kazunori Tomono, Shigeyuki Hamada

Journal of Medical Microbiology   Vol. 72 ( 6 )   2023.6

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Introduction. Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global threat to public health and clinical practice.

Hypothesis/Gap Statement. In Thailand, reports describing CPEs carrying bla_NDM and bla_OXA-48-like genes have been increasing recently; however, data on detailed plasmid analysis and temporal shift of sequence type and carbapenemase type are limited.

Aim. In this study, we analysed whole-genome sequencing (WGS) data of clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) to reveal the molecular epidemiology of CPKP in a tertiary-care hospital in Bangkok, Thailand.

Methodology. Seventy-seven non-duplicated CPKP isolates collected during 2013–2016 were examined for their drug-resistance genes, sequence types and phylogenetic relationships.

Results. All the tested isolates possessed carbapenemase gene(s), and the major type of carbapenemase gene in 2014–2015 was bla_NDM-1, whereas isolates in 2016 harboured more bla_OXA-232 than bla_NDM-1. Other carbapenemase gene variants, such as bla_NDM-4, bla_NDM-5, bla_OXA-48, bla_OXA-181 and bla_IMP-14 were detected in some CPKP isolates. Furthermore, this study revealed that CPKP co-harbouring two genes, bla_NDM-1 and bla_OXA-232 or bla_OXA-181, emerged during this period. Notably, such isolates co-carrying the two carbapenemase genes emerged in three different sequence types, even in a single hospital, and then spread clonally. The WGS of CPKP revealed a temporal shift of the predominant carbapenemase genes from bla_NDM-1 to bla_OXA-232 along with a variation in other carbapenemase gene types within a span of 4 years.

Conclusion. Our findings suggest that a substantial change in CPE types occurred in Thailand and potentially in Southeast Asian countries.

DOI： 10.1099/jmm.0.001711

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NHPHの基礎と臨床の新知見 日本におけるNon-Helicobacter pylori Helicobacter species感染の実態調査 中間報告

徳永 健吾, 塚平 俊久, 間部 克裕, 鈴木 秀和, 下山 克, 杉本 光繁, 沖本 忠義, 松井 英則, 柴山 恵吾, 太田 浩良, 村上 和成, 加藤 元嗣, 林原 絵美子

日本ヘリコバクター学会学術集会プログラム・抄録集   Vol. 29回   page： 100 - 100   2023.6

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Helicobacter ailurogastricus in Patient with Multiple Refractory Gastric Ulcers, Japan Reviewed International journal

Masaya Sano, Emiko Rimbara, Masato Suzuki, Hidenori Matsui, Miwa Hirai, Sae Aoki, Tsuyoshi Kenri, Keigo Shibayama, Hidekazu Suzuki

Emerging Infectious Diseases   Vol. 29 ( 4 ) page： 833 - 835   2023.4

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We report the isolation of Helicobacter ailurogastricus, a Helicobacter species that infects cats and dogs, from a person with multiple refractory gastric ulcers. In addition to H. suis, which infects pigs, Helicobacter species that infect cats and dogs should be considered as potential gastric pathogens in humans.

DOI： 10.3201/eid2904.221807

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Detection of Bartonella quintana (Hyphomicrobiales: Bartonellaceae) Among Day Laborers in Osaka, Japan, 2009-2010. International journal

Toshinori Sasaki, Mayumi Matsuoka, Kyoko Sawabe, Haruhiko Isawa, Keigo Shibayama, Mutsuo Kobayashi

Journal of medical entomology   Vol. 60 ( 2 ) page： 408 - 411   2023.3

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Bartonella quintana is a gram-negative bacterium causing trench fever, an illness historically acquired by soldiers during World War I. More recently, outbreaks of trench fever have been reported in those experiencing homelessness in the United States, France, Russia, and Tokyo, as well as in children in Nepal and persons in Ethiopia. Reports of B. quintana infection outside of Tokyo are rare in Japan. The aim of this study was to examine body lice and blood obtained from people staying in shelters in Osaka (2009-2010) for B. quintana via polymerase chain reaction and enzyme-linked immunosorbent assays. Day laborers were defined as homeless individuals and shelter residents in this study. We detected genes of B. quintana in body lice by PCR and antibodies against B. quintana. The positive rate of B. quintana genes was 6/10 (60%) in body lice and the seroprevalence (IgG) of B. quintana was 4/10 (40%). This demonstrates that trench fever was endemic in people staying in shelters in Osaka in 2009-2010.

DOI： 10.1093/jme/tjad001

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敗血症患者血液由来および健常者皮膚分離Bacillus cereus株芽胞のポビドンヨード感受性

石原 由華, 柴山 恵吾, 社本 生衣, 宇佐美 久枝, 太田 美智男

日本環境感染学会誌   Vol. 38 ( 2 ) page： 57 - 60   2023.3

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Bacillus cereus芽胞のポビドンヨードに対する感受性を血液由来15株と健常者皮膚分離15株の芽胞を用いて作用時間と温度を変えて検討した.いずれの処理条件でも高度耐性株はなく,多くの菌株で10%ポビドンヨード処理5分で約99%の芽胞が殺菌されたが,残りの約1%の芽胞は30分処理後も生残した.皮膚由来株5株,血液由来株3株は5分処理後に生菌数が約0.1%になった.作用温度の違いによるポビドンヨードの効果には大きな差が無く,また血液由来株と皮膚由来株の間に差がほとんど見られなかった.以上によりB.cereus芽胞による皮膚汚染が軽度なら10%ポビドンヨードによる消毒が可能と考えられた.(著者抄録)

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Association between the proportion of laparoscopic approaches for digestive surgeries and the incidence of consequent surgical site infections, 2009–2019: A retrospective observational study based on national surveillance data in Japan International journal Open Access

Toshiki Kajihara, Koji Yahara, Aki Hirabayashi, Yumiko Hosaka, Norikazu Kitamura, Motoyuki Sugai, Keigo Shibayama

PLOS ONE   Vol. 18 ( 2 ) page： e0281838 - e0281838   2023.2

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Background

Surgical site infections (SSIs) are among the most common healthcare-associated infections. Laparoscopy is increasingly being used in various surgical procedures. However, no study has examined the association between the proportion of laparoscopic procedures and the incidence of SSIs in digestive surgery using nationwide surveillance data.

Methods

We retrospectively investigated national SSI surveillance data from the Japan Nosocomial Infections Surveillance between 2009 and 2019. The annual trend of the SSI rate and the proportion of laparoscopic procedures were assessed, focusing on five major digestive surgeries. This was based on data from 109,544 (appendix surgery), 206,459 (gallbladder surgery), 60,225 (small bowel surgery), 363,677 (colon surgery), and 134,695 (rectal surgery) procedures. The effect of a 10% increase in the proportion of laparoscopic procedures on the reduction of the SSI rate was estimated using mixed-effect logistic regression.

Findings

The average SSI rate of the five digestive surgeries decreased from 11.8% in 2009 to 8.1% in 2019. The proportion of laparoscopic procedures in each of the five digestive surgeries increased continuously (p&lt;0.001). The SSI rate for laparoscopic procedures was always lower than that for open procedures. The results were consistent between all and core hospitals participating in the surveillance. The odds ratios of the 10% increase in the proportion of laparoscopic procedures for five digestive surgeries were always &lt;0.950 (p&lt;0.001).

Conclusion

An increase in the proportion of laparoscopic procedures was associated with a reduction in the SSI rate in digestive surgeries.

DOI： 10.1371/journal.pone.0281838

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黄色ブドウ球菌における多剤耐性表現型のサーベイランスとWGS所見との相関性(Surveillance of multidrug resistance phenotypes in S. aureus and correlation with WGS findings)

矢原 耕史, 保阪 由美子, Clarck Adam, 北川 浩樹, 久恒 順三, 菅井 基行, 柴山 恵吾, Stelling John

日本細菌学雑誌   Vol. 78 ( 1 ) page： 105 - 105   2023.2

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Helicobacter cinaediの新規オートトランスポーター蛋白質HcaAの特徴(Characterization of novel autotransporter protein HcaA in Helicobacter cinaedi)

青木 沙恵, 森 茂太郎, 松井 英則, 柴山 恵吾, 見理 剛, 林原 絵美子

日本細菌学雑誌   Vol. 78 ( 1 ) page： 87 - 87   2023.2

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ヘリコバクター・スイスとヘリコバクター・ピロリ感染の世界初の同時診断(The world's first simultaneous diagnostic approach of H. suis and H. pylori infection)

松井 英則, 林原 絵美子, 青木 沙恵, 柴山 恵吾, 鈴木 仁人

日本細菌学雑誌   Vol. 78 ( 1 ) page： 68 - 68   2023.2

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今月の主題 Non-H. pylori Helicobacter胃炎と周辺疾患 主題症例 除菌にて完全寛解したHelicobacter suis感染胃MALTリンパ腫の1例

徳永 健吾, 大野 亜希子, 井田 陽介, 三好 佐和子, 神保 陽子, 林原 絵美子, 松井 英則, 柴山 恵吾, 鈴木 仁人, 大崎 敬子, 太田 浩良, 二階 雪野, 長濱 清隆, 柴原 純二, 岡本 晋, 久松 理一

胃と腸   Vol. 58 ( 1 ) page： 87 - 94   2023.1

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Language：English   Publisher：株式会社医学書院  

DOI： 10.11477/mf.1403203095

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胃MALTリンパ腫とNon-Helicobacter pylori Helicobacterの関連

徳永 健吾, 林原 絵美子, 柴山 恵吾, 鈴木 仁人, 松井 英則, 太田 浩良, 井田 陽介, 三好 佐和子, 岡本 晋, 楠原 光謹, 大野 亜希子, 久松 理一, 大崎 敬子

日本ヘリコバクター学会誌   Vol. 24 ( 2 ) page： 154 - 159   2023.1

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近年、H.pylori(H.p.)感染者の多くが除菌を受け、また衛生環境が良くなったことでH.p.感染者は減少したが、一方でH.p.以外のHelicobacter(NHPH)による胃疾患が相対的に増加している。胃型NHPHには、H.suis、H.felis、H.heilmannii、H.salomonisなどが存在する。1)H.p.感染の胃MALTリンパ腫、2)H.p.未感染の胃MALTリンパ腫、3)胃MALTリンパ腫とNHPH感染、4)NHPH感染胃MALTリンパ腫の内視鏡像、5)H.p.未感染胃MALTリンパ腫に対する除菌効果、6)NHPH感染胃MALTリンパ腫に対する除菌効果、について概説した。

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2023&ichushi_jid=J05115&link_issn=&doc_id=20230216410011&doc_link_id=%2Fdy6jahel%2F2023%2F002402%2F019%2F0154-0159%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fdy6jahel%2F2023%2F002402%2F019%2F0154-0159%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

非HP非NSAID・難治性胃・十二指腸潰瘍の診断と治療 非H.pylori非NSAIDs特発性胃・十二指腸潰瘍におけるNHPH含む成因の検討

徳永 健吾, 大野 亜希子, 楠原 光謹, 林原 絵美子, 松井 英則, 鈴木 仁人, 柴山 恵吾, 大崎 敬子, 三好 佐和子, 井田 陽介, 岡本 晋, 久松 理一

日本消化管学会雑誌   Vol. 7 ( Suppl. ) page： 164 - 164   2023.1

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Genotyping of Mycoplasma pneumoniae strains isolated in Japan during 2019 and 2020: spread of p1 gene type 2c and 2j variant strains. International journal Open Access

Tsuyoshi Kenri, Tsutomu Yamazaki, Hitomi Ohya, Michio Jinnai, Yoichiro Oda, Sadasaburo Asai, Rikako Sato, Nobuhisa Ishiguro, Tomohiro Oishi, Atsuko Horino, Hiroyuki Fujii, Toru Hashimoto, Hiroshi Nakajima, Keigo Shibayama

Frontiers in microbiology   Vol. 14   page： 1202357 - 1202357   2023

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We characterized 118 Mycoplasma pneumoniae strains isolated from three areas of Japan (Saitama, Kanagawa, and Osaka) during the period of 2019 and 2020. Genotyping of the p1 gene in these strains revealed that 29 of them were type 1 lineage (29/118, 24.6%), while 89 were type 2 lineage (89/118, 75.4%), thereby indicating that type 2 lineage was dominant in this period. The most prevalent variant of type 2 lineage was type 2c (57/89, 64%), while the second-most was type 2j, a novel variant identified in this study (30/89, 33.7%). Type 2j p1 is similar to type 2 g p1, but cannot be distinguished from reference type 2 (classical type 2) using the standard polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) with HaeIII digestion. Thus, we used MboI digestion in the PCR-RFLP analysis and re-examined the data from previous genotyping studies as well. This revealed that most strains reported as classical type 2 after 2010 in our studies were actually type 2j. The revised genotyping data showed that the type 2c and 2j strains have been spreading in recent years and were the most prevalent variants in Japan during the time-period of 2019 and 2020. We also analyzed the macrolide-resistance (MR) mutations in the 118 strains. MR mutations in the 23S rRNA gene were detected in 29 of these strains (29/118, 24.6%). The MR rate of type 1 lineage (14/29, 48.3%) was still higher than that of type 2 lineage (15/89, 16.9%); however, the MR rate of type 1 lineage was lower than that found in previous reports published in the 2010s, while that of type 2 lineage strains was slightly higher. Thus, there is a need for continuous surveillance of the p1 genotype and MR rate of M. pneumoniae clinical strains, to better understand the epidemiology and variant evolution of this pathogen, although M. pneumoniae pneumonia cases have decreased significantly since the COVID-19 pandemic.

DOI： 10.3389/fmicb.2023.1202357

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Genomic Epidemiological Analysis of Antimicrobial-Resistant Bacteria with Nanopore Sequencing. International journal

Masato Suzuki, Yusuke Hashimoto, Aki Hirabayashi, Koji Yahara, Mitsunori Yoshida, Hanako Fukano, Yoshihiko Hoshino, Keigo Shibayama, Haruyoshi Tomita

Methods in molecular biology (Clifton, N.J.)   Vol. 2632   page： 227 - 246   2023

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Antimicrobial-resistant (AMR) bacterial infections caused by clinically important bacteria, including ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and mycobacteria (Mycobacterium tuberculosis and nontuberculous mycobacteria), have become a global public health threat. Their epidemic and pandemic clones often accumulate useful accessory genes in their genomes, such as AMR genes (ARGs) and virulence factor genes (VFGs). This process is facilitated by horizontal gene transfer among microbial communities via mobile genetic elements (MGEs), such as plasmids and phages. Nanopore long-read sequencing allows easy and inexpensive analysis of complex bacterial genome structures, although some aspects of sequencing data calculation and genome analysis methods are not systematically understood. Here we describe the latest and most recommended experimental and bioinformatics methods available for the construction of complete bacterial genomes from nanopore sequencing data and the detection and classification of genotypes of bacterial chromosomes, ARGs, VFGs, plasmids, and other MGEs based on their genomic sequences for genomic epidemiological analysis of AMR bacteria.

DOI： 10.1007/978-1-0716-2996-3_16

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A Nationwide Plasmidome Surveillance in Thailand Reveals a Limited Variety of New Delhi Metallo-β-Lactamase-Producing Carbapenem-Resistant <i>Enterobacteriaceae</i> Clones and Spreading Plasmids International journal

Ryuichiro Abe, Yukihiro Akeda, Noriko Sakamoto, Dan Takeuchi, Yo Sugawara, Norihisa Yamamoto, Anusak Kerdsin, Yuki Matsumoto, Daisuke Motooka, Warawut Laolerd, Pitak Santanirand, Masato Suzuki, Keigo Shibayama, Kazunori Tomono, Tetsuya Iida, Shigeyuki Hamada

Journal of Clinical Microbiology   Vol. 60 ( 12 ) page： e0108022   2022.11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Despite frequent identification of plasmids carrying carbapenemase genes, the transfer of plasmids carrying carbapenemase genes is not well recognized in clinical settings because of technical limitations. To investigate the detailed mechanisms of the spread of carbapenem-resistant Enterobacteriaceae (CRE), we performed multifaceted genomic surveillance of CRE isolates in Thailand and analyzed their plasmidome.

DOI： 10.1128/jcm.01080-22

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Characterization of HcaA, a novel autotransporter protein in Helicobacter cinaedi, and its role in host cell adhesion

Sae Aoki, Shigetarou Mori, Hidenori Matsui, Keigo Shibayama, Tsuyoshi Kenri, Emiko Rimbara

bioRxiv     2022.11

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DOI： 10.1101/2022.11.18.517170

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Development of a serological assay to simultaneously identify <i>Helicobacter suis</i> and <i>Helicobacter pylori</i> infection International journal Open Access

Hidenori Matsui, Emiko Rimbara, Masato Suzuki, Kengo Tokunaga, Hidekazu Suzuki, Masaya Sano, Takashi Ueda, Hitoshi Tsugawa, Sohachi Nanjo, Akira Takeda, Makoto Sasaki, Shuichi Terao, Tsuyoshi Suda, Sae Aoki, Keigo Shibayama, Hiroyoshi Ota, Katsuhiro Mabe

iScience   Vol. 26 ( 4 ) page： 106522 - 106522   2022.10

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BackgroundHelicobacter suis hosted by hogs is the most-prevalent gastric non-Helicobacter pylori Helicobacter species (NHPH) found in humans with gastric diseases. However, there is no reliable method for diagnosing H. suis infection in clinical practice without gastric biopsy specimens.MethodsHelicobacter infection was diagnosed by using routine methods for detecting H. pylori infection and three other methods (PCR, culture accompanied by genomic analyses, and microscopy) for detecting NHPH and H. pylori infection using gastric biopsy specimens. Then, an ELISA was developed to detect serum anti-H. suis and anti-H. pylori antibody titers. Findings The serum specimens (n=101) were divided into specimens with (n=20) and without H. suis infection (n=81), or into specimens with (n=17) or without H. pylori infection (n=64) after excluding H. pylori infection-eradicated specimens (n=20). The cut-off values of the optical density in the ELISA were used to judge Helicobacter infections. The sensitivity and specificity were 100% and 92.5%, respectively, for identifying H. suis infection, or 88.2% and 87.5%, respectively, for identifying H. pylori infection, and these levels were considered acceptable for clinical application. We detected no other NHPH infections by PCR or culture in this study.Interpretation We developed the world′s first ELISA to simultaneously diagnose H. suis and H. pylori infection, which is available for large-scale medical examination. The eradication of H. suis was confirmed by the time-dependent decline of anti-H. suis antibody titers. Funding This work was supported by MEXT/JSPS KAKENHI (JP19H03474, JP20K08365) and AMED (JP20fk0108148, JP20fk0108133).

DOI： 10.1101/2022.10.10.22280809

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Strand-biased circularizing integrative elements spread tmexCD-toprJ gene clusters encoding RND-type multidrug efflux pumps by repeated transpositions

Trung Duc Dao, Hirokazu Yano, Taichiro Takemura, Aki Hirabayashi, Le Thi Trang, Hoang Huy Tran, Keigo Shibayama, Futoshi Hasebe, Ikuro Kasuga, Masato Suzuki

bioRxiv     2022.9

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Antimicrobial resistance genes (ARGs) are associated with mobile genetic elements (MGEs) that conscript useful genes into the human-microbe and microbe-microbe battlefields. Thus, under intense selective pressure, ARGs have been constantly adapting and evolving, spreading among microbes. tmexCD-toprJ gene clusters, which encode resistance-nodulation-cell division (RND)-type efflux pumps, confer multidrug-resistance to clinically important antimicrobials, including tigecycline. Noteworthily, these gene clusters have emerged in gram-negative bacteria in humans, animals, and the environment worldwide by MGE-mediated transfer. Here we show a hidden MGE, strand-biased circularizing integrative element (SE), that is recently recognized to mediate transpositions of ARGs, associated with the spread of tmexCD-toprJ gene clusters. We identified multidrug-resistant isolates of Aeromonas species in a water environment in Vietnam that harbored multiple copies of tmexCD-toprJ in their chromosomes that were associated with SEs. In particular, Aeromonas hydrophila NUITM-VA1 was found to harbor two copies of a novel variant of tmexC3.3D3.3-topJ1 within cognate SEs, whereas Aeromonas caviae NUITM-VA2 harbored four copies of a novel variant of tmexC2D2.3-topJ2 within cognate SEs. Based on the nature of SE to incorporate a neighboring sequence into the circular form and reinsert it into target sites during transposition, we identified the order of intragenomic movements of tmexCD-toprJ gene clusters. Altogether, our findings suggest that most known subgroups of tmexCD-toprJ and their subvariants underwent transpositions among bacterial chromosomes and plasmids via SEs. Hence, a tmexCD-toprJ gene cluster ancestor may have been initially mobilized via SE, subsequently spreading among bacteria and evolving in new hosts.

DOI： 10.1101/2022.09.22.508988

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Emergence of plasmid-mediated RND-type efflux pump gene cluster tmexCD-toprJ in Shewanella xiamenensis in a water environment

Trung Duc Dao, Taichiro Takemura, Ikuro Kasuga, Aki Hirabayashi, Nguyen Thi Nga, Pham Hong Quynh Anh, Nguyen Dong Tu, Le Thi Trang, Hoang Huy Tran, Keigo Shibayama, Futoshi Hasebe, Masato Suzuki

bioRxiv     2022.9

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The emergence of the mobile resistance-nodulation-division (RND)-type efflux pump tmexCD-toprJ gene cluster that confers multidrug resistance (MDR), including tigecycline resistance, in gram-negative bacteria poses a global public health threat. However, the spread of such clinically important antimicrobial resistance genes (ARGs) in the natural environment has not yet been well investigated. In this study, we investigated MDR aquatic bacteria in Vietnam. A carbapenem- and tigecycline-resistant Shewanella xiamenensis isolate NUITM-VS2 was obtained from urban drainage in Hanoi, Vietnam, in October 2021. S. xiamenensis NUITM-VS2 showed resistance to most antimicrobials tested, including tigecycline, tetracyclines, carbapenems, cephalosporins, fluoroquinolone, and aminoglycosides. Whole-genome analysis was performed by long- and short-read sequencing, resulting in the complete genome sequence consisting of one chromosome and five plasmid sequences. ARGs and plasmid replicons in the genome were detected using ResFinder with the custom ARG database, including all known tigecycline resistance genes, and PlasmidFinder, respectively. A 152.2-kb IncC plasmid, pNUITM-VS2_2, co-carried two mobile tigecycline resistance genes, tet(X4) and tmexC3.1D3.1-toprJ1. In addition, a 24.8-kb untypeable plasmid, pNUITM-VS2_4, carried the carbapenemase gene blaNDM-1. pNUITM-VS2_2 was transferred to Escherichia coli by conjugation, which simultaneously conferred high-level resistance against many antimicrobials, including tigecycline. To the best of our knowledge, this is the first report of the detection of the mobile RND-type efflux pump gene cluster tmexCD-toprJ in Shewanella species. Our results provide genetic evidence of the complexity of the dynamics of clinically important ARGs among aquatic bacteria, which could be important reservoirs for ARGs in the natural environment.

DOI： 10.1101/2022.09.15.508154

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Clonal dissemination of carbapenem-resistant <i>Klebsiella pneumoniae</i> ST16 co-producing NDM-1 and OXA-232 in Thailand Reviewed International journal Open Access

Ryuichiro Abe, Yukihiro Akeda, Dan Takeuchi, Noriko Sakamoto, Yo Sugawara, Norihisa Yamamoto, Anusak Kerdsin, Yuki Matsumoto, Daisuke Motooka, Warawut Leolerd, Pitak Santanirand, Masato Suzuki, Keigo Shibayama, Kazunori Tomono, Tetsuya Iida, Shigeyuki Hamada

JAC-Antimicrobial Resistance   Vol. 4 ( 4 ) page： dlac084   2022.8

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Abstract

Background

Klebsiella pneumoniae ST258 and ST11 carrying blaKPC are among the most widespread carbapenem-resistant K. pneumoniae strains worldwide. Our carbapenem-resistant Enterobacteriaceae surveillance in Thailand revealed a nationwide dissemination of K. pneumoniae ST16 isolates carrying blaNDM-1 and blaOXA-232.

Objectives

To analyse the genomic details of this nationwide dissemination by focusing on plasmids and virulence factors.

Methods

Using WGS data of 119 K. pneumoniae ST16 isolates carrying blaNDM-1 obtained in our previous surveillance study, clonality of chromosomes and plasmids of the isolates with carriage of virulence factors was evaluated.

Results

Of the 119 isolates, 111 carried plasmid pKP151_NDM1, and all 104 isolates harbouring blaOXA-232 carried plasmid pKP151_OXA232. These 104 K. pneumoniae ST16 isolates showing chromosomal clonality possessed both pKP151_NDM1 and pKP151_OXA232, demonstrating clonal dissemination of K. pneumoniae ST16 with these plasmids. The isolates had essentially similar virulence factors as those of K. pneumoniae ST16 clones carrying blaKPC, which were recently reported as highly invasive clones in Brazil.

Conclusions

The potential global dissemination of these invasive clones with resistance to several antibiotics highlights the importance of appropriate monitoring and strict standard precautions.

DOI： 10.1093/jacamr/dlac084

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Helicobacter suis胃粘膜感染と病態発症

鈴木 仁人, 林原 絵美子, 松井 英則, 徳永 健吾, 柴山 恵吾

日本ヘリコバクター学会誌   Vol. 24 ( 1 ) page： 2 - 9   2022.5

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Helicobacter suisはブタが自然宿主と考えられている細菌で、ヒト胃での持続感染は胃粘膜関連リンパ組織(MALT)リンパ腫などの胃関連疾患の発症に関連していることが指摘されている。H.suisは過去にブタから単離培養されていたが、ヒトからの分離はなされていなかったため、ヒトにおいてH.suis感染と胃関連疾患発症の因果関係の証明は不完全なままとなっていた。本研究では、われわれはヒト胃から直接的にH.suisを分離し、in vitroで培養することに成功した。H.suisはHelicobacter pyloriと異なり、中性pHで著しく生存率が低下するため、胃生検の輸送には低pH培地を用いた。最終的に胃MALTリンパ腫を含む胃関連疾患患者3名からH.suisを分離することができた。患者全員において抗菌治療によりH.suisの除菌に成功し、内視鏡所見、病理組織学的所見が有意に改善された。H.suis臨床分離株をマウスに経口感染させると、胃粘膜で持続感染し、胃局所および全身性の炎症反応が惹起され、感染4ヵ月後には胃粘膜上皮化生が進行することが確認された。また、感染マウスの胃からH.suisが分離されたことから、本菌はヒトの胃関連疾患の起因菌としてコッホの原則を満たす形で初めて証明された。臨床分離株を用いたH.suisの比較ゲノム解析により、各分離株の染色体にはIV型分泌機構関連遺伝子などの株特異的な病原因子をコードする可塑性の高い領域が存在していること、ヒトとブタの分離株は遺伝的に非常に似ており、ブタ-ヒト感染の可能性があることが示唆された。(著者抄録)

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Helicobacter suis胃粘膜感染と病態発症

鈴木 仁人, 林原 絵美子, 松井 英則, 徳永 健吾, 柴山 恵吾

日本ヘリコバクター学会誌   Vol. 24 ( 1 ) page： 2 - 9   2022.5

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Language：Japanese   Publisher：(一社)日本ヘリコバクター学会  

Helicobacter suisはブタが自然宿主と考えられている細菌で、ヒト胃での持続感染は胃粘膜関連リンパ組織(MALT)リンパ腫などの胃関連疾患の発症に関連していることが指摘されている。H.suisは過去にブタから単離培養されていたが、ヒトからの分離はなされていなかったため、ヒトにおいてH.suis感染と胃関連疾患発症の因果関係の証明は不完全なままとなっていた。本研究では、われわれはヒト胃から直接的にH.suisを分離し、in vitroで培養することに成功した。H.suisはHelicobacter pyloriと異なり、中性pHで著しく生存率が低下するため、胃生検の輸送には低pH培地を用いた。最終的に胃MALTリンパ腫を含む胃関連疾患患者3名からH.suisを分離することができた。患者全員において抗菌治療によりH.suisの除菌に成功し、内視鏡所見、病理組織学的所見が有意に改善された。H.suis臨床分離株をマウスに経口感染させると、胃粘膜で持続感染し、胃局所および全身性の炎症反応が惹起され、感染4ヵ月後には胃粘膜上皮化生が進行することが確認された。また、感染マウスの胃からH.suisが分離されたことから、本菌はヒトの胃関連疾患の起因菌としてコッホの原則を満たす形で初めて証明された。臨床分離株を用いたH.suisの比較ゲノム解析により、各分離株の染色体にはIV型分泌機構関連遺伝子などの株特異的な病原因子をコードする可塑性の高い領域が存在していること、ヒトとブタの分離株は遺伝的に非常に似ており、ブタ-ヒト感染の可能性があることが示唆された。(著者抄録)

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Establishment of a reference panel of Helicobacter pylori strains for antimicrobial susceptibility testing. International journal Open Access

Kenji Yokota, Takako Osaki, Shunji Hayashi, Shin-Ichi Yokota, Hiroaki Takeuchi, Emiko Rimbara, Hinako Ojima, Toyotaka Sato, Hideo Yonezawa, Keigo Shibayama, Kengo Tokunaga, Shigeru Kamiya, Kazunari Murakami, Mototsugu Kato, Toshiro Sugiyama

Helicobacter   Vol. 27 ( 3 ) page： e12874   2022.3

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BACKGROUND: Eradication treatment for Helicobacter pylori gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese H. pylori strains for antimicrobial susceptibility testing. METHOD: A total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined. RESULTS: Three H. pylori strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for Helicobacter Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella-based horse-serum-containing agar medium were as follows: JSHR3 (CLR 16 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), JSHR6 (CLR 0.016 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), and JSHR31 (CLR 16 μg/ml, AMX 1 μg/ml and MNZ 64 μg/ml). CONCLUSIONS: A reference panel of H. pylori JSHR strains was established. The panel consisted of JSHR6, which was antibiotic-susceptible, JSHR3, which was CLR-resistant, and JSHR31, which was multi-resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.

DOI： 10.1111/hel.12874

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The First Reported Case of Bordetella pertussis Bacteremia in a Patient With Human Immunodeficiency Virus Infection. International journal Open Access

Yuji Wakimoto, Nao Otsuka, Yasuaki Yanagawa, Kentaro Koide, Kazunari Kamachi, Keigo Shibayama, Katsuji Teruya, Yoshimi Kikuchi, Shinichi Oka

Open forum infectious diseases   Vol. 9 ( 3 ) page： ofac020   2022.3

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We describe a case of bacteremia in a human immunodeficiency virus-infected patient caused by a Bordetella pertussis strain lacking 2 major virulence factors, filamentous hemagglutinin and fimbriae. Although B pertussis bacteremia is uncommon, physicians should be aware that even attenuated B pertussis strains can cause invasive infection in immunocompromised patients. Bordetella pertussis is a gram-negative coccobacillus that causes a severe paroxysmal coughing disease known as whooping cough or pertussis. Bordetella pertussis colonizes the epithelial cells of the human respiratory tract, and the organisms are typically isolated from nasopharynx. We describe a case of B pertussis bacteremia in a patient with human immunodeficiency virus (HIV) infection. Interestingly, the isolate recovered from blood culture did not produce the major virulence factors, filamentous hemagglutinin (FHA) and fimbriae (FIM). Previously, 3 cases of B pertussis bacteremia were reported in the literature. We discuss the features of B pertussis bacteremia.

DOI： 10.1093/ofid/ofac020

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Emergence of mobile tigecycline resistance gene tet(X4)–harboring Shewanella xiamenensis in a water environment Reviewed International journal

Trung Duc Dao, Ikuro Kasuga, Aki Hirabayashi, Dong Tu Nguyen, Hien Thi Tran, Hieu Vu, Linh Tuyet Ngoc Pham, Thi My Hanh Vu, Futoshi Hasebe, Ha Thanh Nguyen, Trang Le Thi, Hoang Huy Tran, Keigo Shibayama, Taichiro Takemura, Masato Suzuki

Journal of Global Antimicrobial Resistance   Vol. 28   page： 140 - 142   2022.3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

OBJECTIVES: Tigecycline resistance mediated by the mobile tigecycline-inactivating enzyme gene tet(X) in Gram-negative bacteria is an emerging concern for global public health. However, limited information is available on the distribution of tet(X) in the natural environment. In this study, we investigated the presence of tet(X) in environmental Gram-negative bacteria. METHODS: A carbapenem- and tigecycline-resistant Shewanella xiamenensis isolate (NUITM-VS1) was obtained from an urban drainage in Hanoi, Vietnam, in March 2021. Whole-genome sequencing analysis was performed by long- and short-read sequencing, resulting in a complete genome sequence. Antimicrobial resistance genes (ARGs) in the genome were detected based on the custom ARG database, including all known tigecycline resistance genes. RESULTS: Shewanella xiamenensis isolate NUITM-VS1 harboured the tet(X4) gene and the blaOXA-48 carbapenemase gene on the chromosome. tet(X4) was flanked by IS91 family transposase genes, suggesting that the acquisition of tet(X4) was mediated by this mobile gene element (MGE), whereas no MGE was found surrounding blaOXA-48, consistent with previous findings that blaOXA-48-like β-lactamase genes are species-specific intrinsic ARGs in Shewanella spp. CONCLUSION: To the best of our knowledge, this is the first report of a tet(X4)-harbouring Shewanella sp. isolate. Our results provide genetic evidence of the complexity of the dynamics of clinically important ARGs among bacteria in the water environment.

DOI： 10.1016/j.jgar.2021.12.022

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H.pyloriと胃がん以外の疾患 ここまで解明された Helicobacter suisを中心に

徳永 健吾, 林原 絵美子, 鈴木 仁人, 柴山 恵吾, 松井 英則

日本胃癌学会総会記事   Vol. 94回   page： 528 - 528   2022.3

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H.pyloriと胃がん以外の疾患 ここまで解明された Helicobacter suisを中心に

徳永 健吾, 林原 絵美子, 鈴木 仁人, 柴山 恵吾, 松井 英則

日本胃癌学会総会記事   Vol. 94回   page： 528 - 528   2022.3

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Genomic characterization of triple-carbapenemase-producing Acinetobacter baumannii Reviewed International journal Open Access

Ken-Ichi Oinuma, Masato Suzuki, Arata Sakiyama, Taishi Tsubouchi, Kozo Saeki, Kanako Sato, Mamiko Niki, Koichi Yamada, Keigo Shibayama, Hiroshi Kakeya, Yukihiro Kaneko

JAC-Antimicrobial Resistance   Vol. 3 ( 4 ) page： dlab191   2021.12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

<title>Abstract</title>
<sec>
<title>Objectives</title>
To characterize Acinetobacter baumannii OCU_Ac16a, a clinical isolate co-harbouring three acquired carbapenemase genes, blaNDM-1, blaTMB-1, and blaOXA-58, and assess the clinical significance of so-called multiple-carbapenemase producers.


</sec>
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<title>Methods</title>
OCU_Ac16a and its close relative, OCU_Ac16b, which lacks the blaNDM-1, were isolated from sputum cultures of a patient at Osaka City University Hospital. We subjected these strains to whole-genome analysis, particularly focusing on the genetic context of each carbapenemase gene. The transmissibility and functionality of each carbapenemase gene were analysed by conjugation and transformation experiments and antimicrobial susceptibility tests.


</sec>
<sec>
<title>Results</title>
bla TMB-1 was located in a class 1 integron on the chromosome, whereas blaNDM-1 and blaOXA-58 were found on plasmids named pOCU_Ac16a_2 and pOCU_Ac16a_3, respectively. pOCU_Ac16a_2 (which exhibited highly efficient self-transmissibility) and pOCU_Ac16a_3 (which did not show transmissibility but could be introduced into another A. baumannii strain via electroporation) could both confer carbapenem resistance (MICs ≥512 and ≥32 mg/L, respectively) on the recipient strain. The functionality of blaTMB-1 was evident from the high resistance of OCU_Ac16b to ceftazidime and cefepime (MICs ≥256 and 48 mg/L, respectively), and the high resistance of OCU_Ac16a to cefiderocol (MIC 32 mg/L) could be explained by the additive effect of blaNDM-1 and blaTMB-1.


</sec>
<sec>
<title>Conclusions</title>
Our data revealed the genomic organization of OCU_Ac16a and demonstrated that all the carbapenemase genes are functional, each contributing to the extremely high broad-spectrum resistance of OCU_Ac16a to β-lactams. As multiple-carbapenemase producers can be serious health threats as drug-resistant pathogens and disseminators of carbapenemase genes, close attention should be paid to their emergence.


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DOI： 10.1093/jacamr/dlab191

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Potential spread of mcr-9-carrying IncHI2 plasmids in Enterobacter hormaechei in Vietnam. Reviewed International journal

Van Thi Thu Ha, Linh Dieu Tran, Nguyen Thi Tuyet Mai, Aki Hirabayashi, Son Thai Nguyen, Hoang Huy Tran, Keigo Shibayama, Masato Suzuki

Journal of global antimicrobial resistance   Vol. 27   page： 332 - 334   2021.11

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OBJECTIVES: Mobile colistin resistance (mcr) genes are widely distributed around the world. To date, ten major variants of mcr genes are known (mcr-1 to mcr-10). However, only a few instances of Enterobacterales isolates harbouring mcr genes other than mcr-1 have been reported in Vietnam. The aim of this study was to investigate mcr-harbouring antimicrobial-resistant Enterobacterales isolates in Vietnam. METHODS: Two mcr-9-harbouring Enterobacter hormaechei clinical isolates (NIHE14-1904 and MH17-539M) were obtained from medical institutions in Hanoi, Vietnam, in 2014 and 2017, respectively. Their genomes and plasmid sequences were analysed by short-read and long-read sequencing. Subsequently, comparative sequence analysis of their mcr-9-carrying plasmids was performed. RESULTS: Strains NIHE14-1904 and MH17-539M belonged to sequence types ST916 and ST66, respectively, according to the Enterobacter cloacae multilocus sequence typing (MLST) scheme. NIHE14-1904 and MH17-539M harboured the mcr-9 gene on similar IncHI2 plasmids, namely pNIHE14-1904-mcr9 (373.1 kb) and pMH17-539M-mcr9 (289.3 kb), respectively. These plasmids were also highly identical to widespread IncHI2 plasmids that are often associated with mcr genes. CONCLUSION: For the first time, mcr-9-harbouring Enterobacterales isolates were detected in Vietnam, which carried mcr-9 on IncHI2 plasmids. The prevalence of such plasmids needs to be monitored in the future owing to their high dissemination.

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Expansion of KPC–producing Enterobacterales in four large hospitals in Hanoi, Vietnam Reviewed International journal

Tran Dieu Linh, Nguyen Hoai Thu, Keigo Shibayama, Masato Suzuki, LayMint Yoshida, Pham Duy Thai, Dang Duc Anh, Tran Nhu Duong, Hong Son Trinh, Vu Phuong Thom, Luu Thi Vu Nga, Nguyen Thi Kim Phuong, Bui Thanh Thuyet, Timothy R Walsh, Le Viet Thanh, Anne-Laure Bañuls, H Rogier van Doorn, Tran Van Anh, Tran Huy Hoang

Journal of Global Antimicrobial Resistance   Vol. 27   page： 200 - 211   2021.10

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OBJECTIVES: The incidence of carbapenem resistance among nosocomial Gram-negative bacteria in Vietnam is high and increasing, including among Enterobacterales. In this study, we assessed the presence of one of the main carbapenemase genes, blaKPC, among carbapenem-resistant Enterobacterales (CRE) from four large hospitals in Hanoi, Vietnam, between 2010 and 2015, and described their key molecular characteristics. METHODS: KPC-producing Enterobacterales were detected using conventional PCR and were further analysed using S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and whole-genome sequencing (WGS) for sequence typing and genetic characterisation. RESULTS: blaKPC genes were detected in 122 (20.4%) of 599 CRE isolates. blaKPC-carrying plasmids were diverse in size. Klebsiella pneumoniae harbouring blaKPC genes belonged to ST15 and ST11, whereas KPC-producing Escherichia coli showed more diverse sequence types including ST3580, ST448, ST709 and ST405. Genotypic relationships supported the hypothesis of circulation of a population of 'resident' resistant bacteria in one hospital through the years and of transmission among these hospitals via patient transfer. WGS results revealed co-carriage of several other antimicrobial resistance genes and three different genetic contexts of blaKPC-2. Among these, the combination of ISEcp1-blaCTX-M and ISKpn27-blaKPC-ΔISKpn6 on the same plasmid is reported for the first time. CONCLUSION: We describe the dissemination of blaKPC-expressing Enterobacterales in four large hospitals in Hanoi, Vietnam, since 2010, which may have started earlier, along with their resistance patterns, sequence types, genotypic relationship, plasmid sizes and genetic context, thereby contributing to the overall picture of the antimicrobial resistance situation in Enterobacterales in Vietnam.

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Impact of the COVID-19 pandemic on the surveillance of antimicrobial resistance. Reviewed International journal

Aki Hirabayashi, Toshiki Kajihara, Koji Yahara, Keigo Shibayama, Motoyuki Sugai

The Journal of hospital infection   Vol. 117   page： 147 - 156   2021.9

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BACKGROUND: The impact of the coronavirus disease (COVID-19) pandemic on antimicrobial resistance (AMR) is a major concern. AIM: To compare the number of patients and isolation rate of antimicrobial-resistant bacteria before and after the beginning of the COVID-19 pandemic using the comprehensive national surveillance data. METHODS: We utilized comprehensive surveillance data, collected in the Japan Nosocomial Infections Surveillance program, which included a total of 16.7 million samples of 5.9 million tested patients from >1,300 hospitals. We compared the number of patients and isolation rate of five bacteria between 2019 and 2020, including antimicrobial-susceptible and -resistant bacteria of Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. FINDINGS: The number of patients and isolation rate of S. aureus and methicillin-resistant S. aureus decreased slightly; those of S. pneumoniae and penicillin-resistant S. pneumoniae decreased by 60%; and those of third-generation cephalosporin-resistant K. pneumoniae increased. The isolation rate of the remaining bacteria apparently increased, although the number of patients decreased. This was due to a substantial decrease in the total number of tested patients (the denominator of the isolation rate), which was larger than that of the number of patients (the numerator of the isolation rate). Consistent results were obtained when the same data were re-aggregated using the procedure of the World Health Organization Global Antimicrobial Resistance Surveillance System, demonstrating the general importance of this problem. CONCLUSION: Surveillance data during the COVID-19 pandemic must be carefully interpreted based on examination of the numerator, denominator and background factors that affect the denominator.

DOI： 10.1016/j.jhin.2021.09.011

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Resistance mechanisms and genetic relatedness among carbapenem-resistant Pseudomonas aeruginosa isolates from three major hospitals in Hanoi, Vietnam (2011-15). International journal Open Access

Hai Anh Tran, Thi Ngoc Bich Vu, Son Tung Trinh, Dieu Linh Tran, Ha My Pham, Thi Hong Hanh Ngo, Minh Thao Nguyen, Nhu Duong Tran, Duy Thai Pham, Duc Anh Dang, Keigo Shibayama, Masato Suzuki, Lay-Myint Yoshida, Hong Son Trinh, Viet Thanh Le, Phuong Thom Vu, Thi Vu Nga Luu, Anne-Laure Bañuls, Khanh Linh Trinh, Van Anh Tran, Huy Hoang Tran, H Rogier van Doorn

JAC-antimicrobial resistance   Vol. 3 ( 3 ) page： dlab103   2021.9

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Background: MDR bacteria including carbapenem-resistant Pseudomonas aeruginosa are recognized as an important cause of hospital-acquired infections worldwide. This investigation seeks to determine the molecular characterization and antibiotic resistance genes associated with carbapenem-resistant P. aeruginosa. Methods: We conducted WGS and phylogenetic analysis of 72 carbapenem-resistant P. aeruginosa isolated from hospital-acquired infection patients from August 2011 to March 2015 in three major hospitals in Hanoi, Vietnam. Results: We identified three variants of IMP gene, among which bla IMP-15 was the most frequent (n = 34) in comparison to bla IMP-26 (n = 2) and bla IMP-51 (n = 12). We observed two isolates with imipenem MIC >128 mg/L that co-harboured bla IMP-15 and bla DIM-1 genes and seven isolates (imipenem MIC > 128 mg/L) with a bla KPC-1 gene from the same hospital. MLST data shows that these 72 isolates belong to 18 STs and phylogenetic tree analysis has divided these isolates into nine groups. Conclusions: Our results provide evidence that not only bla IMP-26 but other IMP variants such as bla IMP-15 and bla IMP-51 genes and several STs (ST235, ST244, ST277, ST310, ST773 and ST3151) have been disseminating in healthcare settings in Vietnam. In addition, we report the emergence of two isolates belonging to ST1240 and ST3340 that harboured two important carbapenemase genes (bla IMP-15 and bla DIM-1) and seven isolates belonging to ST3151 of P. aeruginosa that carried the bla KPC-1 gene in Vietnam, which could potentially cause serious restricted availability of treatment options in healthcare settings.

DOI： 10.1093/jacamr/dlab103

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Helicobacter suis胃粘膜感染と病態発症

鈴木 仁人, 林原 絵美子, 松井 英則, 徳永 健吾, 柴山 恵吾

日本ヘリコバクター学会学術集会プログラム・抄録集   Vol. 27回   page： 147 - 147   2021.9

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イヌおよびネコの胃生検組織から分離された2つの新種Helicobacter属菌

青木 沙恵, 鈴木 仁人, 森 茂太郎, 柴山 恵吾, 大野 耕一, 見理 剛, 林原 絵美子

日本ヘリコバクター学会学術集会プログラム・抄録集   Vol. 27回   page： 196 - 196   2021.9

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アシネトバクター属菌に拡散しているcGAS遺伝子陽性プラスミド

鈴木 仁人, 浅井 さとみ, 平林 亜希, 氣駕 恒太朗, 相羽 由詞, 渡邊 真弥, 梅澤 和夫, 宮地 勇人, 崔 龍洙, 柴山 恵吾

緑膿菌感染症研究会講演記録   Vol. 55回   page： 20 - 20   2021.9

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Helicobacter基礎研究の最前線 Helicobacter suisなどのNon-Helicobacter pylori Helicobacterの細菌学的特徴およびゲノム比較

林原 絵美子, 鈴木 仁人, 徳永 健吾, 間部 克裕, 南條 宗八, 松井 英則, 青木 沙恵, 森 茂太郎, 大野 耕一, 柴山 恵吾, 見理 剛

日本ヘリコバクター学会学術集会プログラム・抄録集   Vol. 27回   page： 173 - 173   2021.9

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A Transferable IncC-IncX3 Hybrid Plasmid Cocarrying blaNDM-4, tet(X), and tmexCD3-toprJ3 Confers Resistance to Carbapenem and Tigecycline. International journal Open Access

Aki Hirabayashi, Trung Duc Dao, Taichiro Takemura, Futoshi Hasebe, Le Thi Trang, Nguyen Ha Thanh, Hoang Huy Tran, Keigo Shibayama, Ikuro Kasuga, Masato Suzuki

mSphere   Vol. 6 ( 4 ) page： e0059221 - 6   2021.8

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Tigecycline is a last-resort antimicrobial against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, have emerged in China and have spread possibly worldwide. Tet(X) family proteins function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, to the best of our knowledge we report a CPE isolate harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes strain, NUITM-VK5, was isolated from an urban drainage in Vietnam in 2021, and a plasmid, pNUITM-VK5_mdr, cocarrying tet(X) and tmexCD3-toprJ3 along with the carbapenemase gene blaNDM-4 was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor reduced TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different backbones. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and the environment. Thus, the future global spread of multidrug resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis. IMPORTANCE Tigecycline is important as a last-resort antimicrobial and effective against antimicrobial-resistant bacteria, such as carbapenem-producing Enterobacterales (CPE), whose infections are difficult to treat with antimicrobials. Since 2019, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, and their variants have been reported mainly from China, and it has become important to understand their epidemiological situation and detailed genetic mechanisms. In this study, we identified a bacterial isolate coharboring tet(X) and tmexCD-toprJ on the same plasmid. A Klebsiella aerogenes isolate in Vietnam carried both these tigecycline resistance genes on a transferable plasmid leading to high-level resistance to multiple clinically important antimicrobials, including carbapenem and tigecycline, and could actually transfer the plasmid to other bacteria. The spread of such a multidrug resistance plasmid among bacterial pathogens should be of great concern because there are few antimicrobials to combat bacteria that have acquired the plasmid.

DOI： 10.1128/mSphere.00592-21

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Disease burden of bloodstream infections caused by antimicrobial-resistant bacteria: A population-level study, Japan, 2015-2018. Reviewed International journal

Shinya Tsuzuki, Nobuaki Matsunaga, Koji Yahara, Keigo Shibayama, Motoyuki Sugai, Norio Ohmagari

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases   Vol. 108   page： 119 - 124   2021.7

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BACKGROUND: Antimicrobial resistance (AMR) is a global health problem. However, quantitative evaluation of its disease burden is challenging. This study aimed to estimate the disease burden of bloodstream infections (BSIs) caused by major antimicrobial-resistant bacteria in Japan between 2015 and 2018 in terms of disability-adjusted life-years (DALYs). METHODS: DALYs of BSIs caused by nine major antimicrobial-resistant bacteria in Japan were estimated using comprehensive national surveillance data of all routine bacteriological test results from more than 1400 hospitals between 2015 and 2018. The methodology of Cassini et al. was modified to enable comparison of the present results with those in other countries. RESULTS: It was estimated that 137.9 [95% uncertainty interval (UI) 130.7-145.2] DALYs per 100,000 population were attributable to BSIs caused by nine antimicrobial-resistant bacteria in 2018. Methicillin-resistant Staphylococcus aureus (MRSA), fluoroquinolone-resistant Escherichia coli (FQREC) and third-generation cephalosporin-resistant E. coli (3GREC) accounted for 87.2% overall. The burden did not decrease during the study period and was highest in people aged ≥65 years. CONCLUSION: The results revealed, for the first time, the disease burden of BSIs caused by nine major antimicrobial-resistant bacteria in Japan. The estimated disease burden associated with AMR in Japan is substantial and has not begun to decrease. Notably, the burden from FQREC and 3GREC has increased steadily, and that from MRSA is larger in Japan than in the European Union/European Economic Area, whereas the burden from other bacteria is comparatively small. These results are expected to provide useful information for healthcare policy makers for prioritizing interventions for AMR.

DOI： 10.1016/j.ijid.2021.05.018

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Molecular ruler of the attachment organelle in Mycoplasma pneumoniae. Reviewed International journal

Daisuke Nakane, Kohki Murata, Tsuyoshi Kenri, Keigo Shibayama, Takayuki Nishizaka

PLoS pathogens   Vol. 17 ( 6 ) page： e1009621   2021.6

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Length control is a fundamental requirement for molecular architecture. Even small wall-less bacteria have specially developed macro-molecular structures to support their survival. Mycoplasma pneumoniae, a human pathogen, forms a polar extension called an attachment organelle, which mediates cell division, cytadherence, and cell movement at host cell surface. This characteristic ultrastructure has a constant size of 250-300 nm, but its design principle remains unclear. In this study, we constructed several mutants by genetic manipulation to increase or decrease coiled-coil regions of HMW2, a major component protein of 200 kDa aligned in parallel along the cell axis. HMW2-engineered mutants produced both long and short attachment organelles, which we quantified by transmission electron microscopy and fluorescent microscopy with nano-meter precision. This simple design of HMW2 acting as a molecular ruler for the attachment organelle should provide an insight into bacterial cellular organization and its function for their parasitic lifestyles.

DOI： 10.1371/journal.ppat.1009621

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Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae. Reviewed International journal

Takashi Hamabata, Mitsutoshi Senoh, Masaaki Iwaki, Ayae Nishiyama, Akihiko Yamamoto, Keigo Shibayama

Microorganisms   Vol. 9 ( 5 )   2021.4

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Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

DOI： 10.3390/microorganisms9050927

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Association between the frequency of surgeries for video-assisted thoracic surgery and the incidence of consequent surgical site infections: a retrospective observational study based on national surveillance data. Reviewed International journal

Toshiki Kajihara, Koji Yahara, Aki Hirabayashi, Hitomi Kurosu, Motoyuki Sugai, Keigo Shibayama

BMC infectious diseases   Vol. 21 ( 1 ) page： 363 - 363   2021.4

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BACKGROUND: The association between the frequency of surgeries and the incidence of surgical site infections (SSIs) has been reported for various surgeries. However, no previous study has explored this association among video-assisted thoracic surgeries (VATS). Hence, we aimed to investigate the association between the frequency of surgeries and SSI in video-assisted thoracic surgeries. METHODS: We analyzed the data of 26,878 thoracic surgeries, including 21,154 VATS, which were collected during a national surveillance in Japan between 2014 and 2018. The frequency of surgeries per hospital department was categorized into low (< 50/year), moderate (50-100/ year), and high (> 100/year). Chi-squared test or Fisher's exact test was used for discrete explanatory variables, whereas Wilcoxon's rank-sum test or Kruskal-Wallis test was used for continuous explanatory variables. Univariate analysis of the department groups was conducted to explore confounding factors associated with both SSIs and the department groups. We used a multiple logistic regression model focusing on VATS and stratified by the National Nosocomial Infections Surveillance System (NNIS) risk index. RESULTS: The rates of SSIs in the hospital groups with low, moderate, and high frequency of surgeries were 1.39, 1.05, and 1.28%, respectively. In the NNIS risk index 1 stratum, the incidence of SSIs was significantly lower in the moderate-frequency of surgeries group than that in the other groups (odds ratio [OR]: vs. low-frequency of surgeries: 2.48 [95% confidence interval [CI]: 1.20-5.13], P = 0.0143; vs. high-frequency of surgeries: 2.43 [95% CI: 1.44-4.11], P = 0.0009). In the stratum of NNIS risk indices 2 and 3, the incidence of SSI was significantly higher in the low-frequency of surgeries group (OR: 4.83, 95% CI: 1.47-15.93; P = 0.0095). CONCLUSION: The result suggests that for departments with low-frequency of surgeries, an increase in the frequency of surgeries to > 50 per department annually potentially leads to a decrease in the incidence of SSIs. This occurs through an increase in the experience of the departmental surgeons and contributes to the improvement of VATS outcomes in thoracic surgeries.

DOI： 10.1186/s12879-021-06050-6

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Subtype screening of blaIMP genes using bipartite primers for DNA sequencing.

Ryuji Kawahara, Masanori Watahiki, Yuko Matsumoto, Kaoru Uchida, Makiko Noda, Kanako Masuda, Chiemi Fukuda, Yuki Abe, Yukiko Asano, Kazunori Oishi, Keigo Shibayama, Hiroto Shinomiya

Japanese journal of infectious diseases   Vol. 74 ( 6 ) page： 592 - 599   2021.3

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Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

DOI： 10.7883/yoken.JJID.2020.926

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Isolation and characterization of Helicobacter suis from human stomach. International journal

Emiko Rimbara, Masato Suzuki, Hidenori Matsui, Masahiko Nakamura, Misako Morimoto, Chihiro Sasakawa, Hiroki Masuda, Sachiyo Nomura, Takako Osaki, Noriyo Nagata, Keigo Shibayama, Kengo Tokunaga

Proceedings of the National Academy of Sciences of the United States of America   Vol. 118 ( 13 ) page： e2026337118   2021.3

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Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured H. suis directly from human stomachs. Unlike Helicobacter pylori, the viability of H. suis decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated H. suis from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with H. suis clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because H. suis could be isolated from the stomachs of infected mice, our findings satisfied Koch's postulates. Although further prospective clinical studies are needed, H. suis, like H. pylori, is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of H. suis using complete genomes of clinical isolates revealed that the genome of each H. suis isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system-associated genes, and that H. suis isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.

DOI： 10.1073/pnas.2026337118

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Japan Nosocomial Infections Surveillance (JANIS): Current Status, International Collaboration, and Future Directions for a Comprehensive Antimicrobial Resistance Surveillance System. Reviewed

Toshiki Kajihara, Koji Yahara, Aki Hirabayashi, Keigo Shibayama, Motoyuki Sugai

Japanese journal of infectious diseases   Vol. 74 ( 2 ) page： 87 - 96   2021.3

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Japan Nosocomial Infections Surveillance (JANIS) is one of the largest national antimicrobial resistance (AMR) surveillance systems in the world. The JANIS Clinical Laboratory division collects comprehensive specimen-based data from diagnostic microbiology laboratories of participating hospitals to monitor the isolation rate of 11 major bacteria and specific AMR bacteria, and creates antibiograms of approximately 20 bacterial species. Data on the JANIS web database system are also annually tabulated and shared with the WHO Global Antimicrobial Resistance Surveillance System. To create a network of international AMR surveillance systems among Asian countries, Japan is developing an international web database system named ASIan Antimicrobial Resistance Surveillance Network (ASIARS-Net) based on the JANIS system; ASIARS-Net is an open-source database and confidentially available at almost no cost. JANIS continues to evolve in multiple directions; some are discussed at the end of this review.

DOI： 10.7883/yoken.JJID.2020.499

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Rapid and simple SNP genotyping for Bordetella pertussis epidemic strain MT27 based on a multiplexed single-base extension assay. Reviewed International journal

Kazunari Kamachi, Shu-Man Yao, Chuen-Sheue Chiang, Kentaro Koide, Nao Otsuka, Keigo Shibayama

Scientific reports   Vol. 11 ( 1 ) page： 4823 - 4823   2021.3

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Multilocus variable-number tandem repeat analysis (MLVA) is widely used for genotyping of Bordetella pertussis, the causative bacteria for pertussis. However, MLVA genotyping is losing its discriminate power because prevalence of the epidemic MT27 strain (MLVA-27) is increasing worldwide. To address this, we developed a single nucleotide polymorphism (SNP) genotyping method for MT27 based on multiplexed single-base extension (SBE) assay. A total of 237 MT27 isolates collected in Japan during 1999-2018 were genotyped and classified into ten SNP genotypes (SG1 to SG10) with a Simpson's diversity index (DI) of 0.79 (95% CI 0.76-0.82). Temporal trends showed a marked increase in the genotypic diversity in the 2010s: Simpson's DI was zero in 1999-2004, 0.16 in 2005-2009, 0.83 in 2010-2014, and 0.76 in 2015-2018. This indicates that the SNP genotyping is applicable to the recently circulating MT27 strain. Additionally, almost all outbreak-associated MT27 isolates were classified into the same SNP genotypes for each outbreak. Multiplexed SBE assay allows for rapid and simple genotyping, indicating that the SNP genotyping can potentially be a useful tool for subtyping the B. pertussis MT27 strain in routine surveillance and outbreak investigations.

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Emergence of a plasmid-borne tigecycline resistance in Klebsiella pneumoniae in Vietnam. International journal

Aki Hirabayashi, Van Thi Thu Ha, An Van Nguyen, Son Thai Nguyen, Keigo Shibayama, Masato Suzuki

Journal of medical microbiology   Vol. 70 ( 3 )   2021.3

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Tigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, tmexCD1-toprJ1, encoding the resistance-nodulation-cell division (RND) efflux pump was identified on plasmids in Klebsiella pneumoniae isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified K. pneumoniae isolates harbouring the tmexCD1-toprJ1 genes outside of China for the first time. Two tigecycline-resistant K. pneumoniae isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb tmexCD1-toprJ1-carrying plasmid with IncFIB and IncHI1B replicons. The tmexCD1-toprJ1 gene cluster was surrounded by several mobile gene elements, including IS26, and the plasmids had high sequence identity with that of K. pneumoniae isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by tmexCD1-toprJ1-carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.

DOI： 10.1099/jmm.0.001320

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Nationwide multicenter questionnaire surveys on countermeasures against antimicrobial resistance and infections in hospitals. Reviewed International journal

Jung-Ho Shin, Seiko Mizuno, Takuya Okuno, Hisashi Itoshima, Noriko Sasaki, Susumu Kunisawa, Mitsuo Kaku, Makiko Yoshida, Yoshiaki Gu, Daiichi Morii, Keigo Shibayama, Norio Ohmagari, Yuichi Imanaka

BMC infectious diseases   Vol. 21 ( 1 ) page： 234 - 234   2021.2

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BACKGROUND: The goals of the National Action Plan on Antimicrobial Resistance (AMR) of Japan include "implementing appropriate infection prevention and control" and "appropriate use of antimicrobials," which are relevant to healthcare facilities. Specifically, linking efforts between existing infection control teams and antimicrobial stewardship programs was suggested to be important. Previous studies reported that human resources, such as full-time equivalents of infection control practitioners, were related to improvements in antimicrobial stewardship. METHODS: We posted questionnaires to all teaching hospitals (n = 1017) regarding hospital countermeasures against AMR and infections. To evaluate changes over time, surveys were conducted twice (1st survey: Nov 2016, 2nd survey: Feb 2018). A latent transition analysis (LTA) was performed to identify latent statuses, which refer to underlying subgroups of hospitals, and effects of the number of members in infection control teams per bed on being in the better statuses. RESULTS: The number of valid responses was 678 (response rate, 66.7%) for the 1st survey and 559 (55.0%) for the 2nd survey. More than 99% of participating hospitals had infection control teams, with differences in activity among hospitals. Roughly 70% had their own intervention criteria for antibiotics therapies, whereas only about 60 and 50% had criteria established for the use of anti-methicillin-resistant Staphylococcus aureus antibiotics and broad-spectrum antibiotics, respectively. Only 50 and 40% of hospitals conducted surveillance of catheter-associated urinary tract infections and ventilator-associated pneumonia, respectively. Less than 50% of hospitals used maximal barrier precautions for central line catheter insertion. The LTA identified five latent statuses. The membership probability of the most favorable status in the 2nd study period was slightly increased from the 1st study period (23.6 to 25.3%). However, the increase in the least favorable status was higher (26.3 to 31.8%). Results of the LTA did not support a relationship between increasing the number of infection control practitioners per bed, which is reportedly related to improvements in antimicrobial stewardship, and being in more favorable latent statuses. CONCLUSIONS: Our results suggest the need for more comprehensive antimicrobial stewardship programs and increased surveillance activities for healthcare-associated infections to improve antimicrobial stewardship and infection control in hospitals.

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Invasive Haemophilus influenzae type a infection and polyarthritis in a 72-year-old Japanese man: A case report. Reviewed International journal

Tsuneaki Kenzaka, Ken Goda, Mayumi Kubota, Kosuke Takayanagi, Tsuyoshi Kenri, Keigo Shibayama, Hozuka Akita

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy   Vol. 27 ( 7 ) page： 1084 - 1088   2021.2

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Haemophilus influenzae is a small, nonmotile, non-spore-forming bacterium classified into 6 serotypes (a to f) and non-typeable strains that lack a capsule. Although H. influenzae serotype a (Hia) is prevalent in Canada, the United States, Brazil, Australia, across the African continent, and several other locations, it has not been reported in Japan thus far. Our case was of a 72-year-old Japanese man who sought medical consultation after presenting with chills, fever, and polyarthritis. Cultures of blood and synovial fluid from the left knee revealed H. influenzae infection. Diagnostic imaging showed poor contrast regions in both kidneys, fluid retention around both knee joints, the left shoulder joint, and both elbow joints. Subsequently, the patient was diagnosed with invasive H. influenzae infection accompanied by polyarthritis and renal infarction. 16S ribosomal RNA gene sequencing revealed that the bacterial strain was Hia. The patient was treated with antimicrobial agents and arthroscopic curettage. We present a case of invasive Hia infection accompanied by polyarthritis and renal infarction. To the best of our knowledge, this is the first case of Hia infection in Japan. The case is very rare considering that the disease occurred in an elderly patient who developed polyarthritis.

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Detection of Bartonella quintana infection among the Homeless Population in Tokyo, Japan, from 2013-2015.

Toshinori Sasaki, Tomohide Adachi, Kazuto Itoh, Mayumi Kubota, Takuya Yamagishi, Maki Hirao, Haruhiko Isawa, Kazunori Oishi, Keigo Shibayama, Kyoko Sawabe

Japanese journal of infectious diseases   Vol. 74 ( 5 ) page： 411 - 415   2021.1

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Several outbreaks of trench fever caused by Bartonella quintana, occurred in soldiers during World Wars I and II. Although the number of trench fever cases has been decreasing worldwide, the disease has been reported among the homeless population in both developing and developed countries. The current prevalence of B. quintana infection in Japan is unclear. We collected blood and body louse (Pediculus humanus humanus) samples from homeless inpatients who had body lice at the time of emergency hospitalization in Tokyo from January 2013 to March 2015. Patients were tested for B. quintana infections using culture method, polymerase chain reaction, and indirect immunofluorescence assay (IFA). Among the 29 patients tested, the presence of Bartonella spp. was confirmed by genomic sequencing of DNA extracted from the samples from 2 patients (blood culture performed for 13 out of 15 patients), and from body louse samples of 20 patients (69%). Immunoglobulin G against B. quintana was detected in 10 patients (34.5%) at a cut-off titer of 1:256 in IFA. B. quintana infection was detected in samples obtained between 2013 and 2015 in Tokyo and needs to be on the list of differential diagnoses performed for febrile homeless individuals.

DOI： 10.7883/yoken.JJID.2020.505

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Emergence of DIM-1 and KPC-1 Genes Associated Carbapenem-Resistant Pseudomonas Aeruginosa Isolates in Three Major Hospitals in Hanoi, Vietnam (2010-2015) Reviewed

Tran Hai Anh, Tran Huy Hoang, Vu Thi Ngoc Bich, Trinh Son Tung, Tran Dieu Linh, Pham Ha My, Ngo Thi Hong Hanh, Nguyen Minh Thao, Tran Nhu Duong, Pham Duy Thai, Dang Duc Anh, Keigo Shibayama, Masato Suzuki, Lay-Myint Yoshida, Hong Son Trinh, Le Viet Thanh, Vu Phuong Thom, Luu Thi Vu Nga, Anne-Laure Bañuls, Trinh Khanh Linh, Tran Van Anh, H Rogier van Doorn

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<title>Abstract</title>
<bold>Background:</bold> Multidrug-resistant bacteria including carbapenem resistant <italic>Pseudomonas aeruginosa </italic>are recognised as an important cause of hospital-acquired infections worldwide. To determine the molecular characterisation and antibiotic resistant genes associated with carbapenem-resistant <italic>P. aeruginosa</italic>. <bold>Methods:</bold> we conducted whole-genome sequencing and phylogenetic analysis of 72 carbapenem-resistant <italic>P. aeruginosa </italic>isolated from hospital-acquired infection patients from 2010 to 2015 in three major hospitals in Hanoi, Vietnam. <bold>Results:</bold> We identified three variants of IMP genes, among which IMP-15 gene was the most frequent (n= 34) in comparison to IMP-26 (n= 2) and IMP-51 (n=12). We observed two isolates with imipenem MIC &gt;128mg/L that co-harboured IMP-15 and DIM-1 genes and seven isolates (imipenem MIC&gt; 128mg/L) with KPC-1 gene from the same hospital. MLST data showed that sequence types (ST) of 72 isolates were classified into 18 STs and phylogenetic tree analysis divided these isolates into nine groups. <bold>Conclusion:</bold> Our results provide evidence that not only IMP-26, but other variants of IMPs like IMP-15 and IMP-51 genes and several STs (ST235, ST244, ST277, ST310, ST773 and ST3151) have been disseminated in health care settings in Vietnam. Also, we report the first finding in Vietnam that two isolates belonging to ST1240 and ST3340 harboured two important carbapenemase genes (IMP-15 and, DIM-1) and seven isolates belonging to ST3151 of <italic>P. aeruginosa </italic>carried the KPC-1 gene, which could be a potential cause of seriously restricted available treatment options in healthcare settings.

DOI： 10.21203/rs.3.rs-143498/v1

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Plasmid analysis of NDM metallo-β-lactamase-producing Enterobacterales isolated in Vietnam. Reviewed International journal Open Access

Aki Hirabayashi, Koji Yahara, Satomi Mitsuhashi, So Nakagawa, Tadashi Imanishi, Van Thi Thu Ha, An Van Nguyen, Son Thai Nguyen, Keigo Shibayama, Masato Suzuki

PloS one   Vol. 16 ( 7 ) page： e0231119   2021

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Carbapenem-resistant Enterobacterales (CRE) represent a serious threat to public health due to the lack of treatment and high mortality. The rate of antimicrobial resistance of Enterobacterales isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene blaNDM, spread via plasmids among Enterobacterales in Vietnam. In this study, we characterized blaNDM-carrying plasmids in Enterobacterales isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-nonsusceptible isolates from a medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 13 blaNDM-positive isolates, including isolates of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Morganella morganii, and Proteus mirabilis, were further sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Almost identical 73 kb IncFII(pSE11)::IncN hybrid plasmids carrying blaNDM-1 were found in a P. mirabilis isolate and an M. morganii isolate. A 112 kb IncFII(pRSB107)::IncN hybrid plasmid carrying blaNDM-1 in an E. coli isolate had partially identical sequences with a 39 kb IncR plasmid carrying blaNDM-1 and an 88 kb IncFII(pHN7A8)::IncN hybrid plasmid in a C. freundii isolate. 148-149 kb IncFIA(Hl1)::IncA/C2 plasmids and 75-76 kb IncFII(Yp) plasmids, both carrying blaNDM-1 were shared among three sequence type 11 (ST11) isolates and three ST395 isolates of K. pneumoniae, respectively. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to blaNDM-1. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.

DOI： 10.1371/journal.pone.0231119

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On-Site Genomic Epidemiological Analysis of Antimicrobial-Resistant Bacteria in Cambodia With Portable Laboratory Equipment. Reviewed International journal Open Access

Aki Hirabayashi, Hideji Yanagisawa, Hiromizu Takahashi, Koji Yahara, Philipp Boeing, Bethan Wolfenden, Vandarith Nov, Vichet Lorn, Mom Veng, Vuth Ann, Chau Darapheak, Keigo Shibayama, Masato Suzuki

Frontiers in microbiology   Vol. 12   page： 675463 - 675463   2021

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The rapid emergence of carbapenemase-producing gram-negative bacteria (CPGNB) is a global threat due to the high mortality of infection and limited treatment options. Although there have been many reports of CPGNB isolated from Southeast Asian countries, to date there has been no genetic analysis of CPGNB isolated from Cambodia. Sequence-based molecular epidemiological analysis enables a better understanding of the genotypic characteristics and epidemiological significance of antimicrobial-resistant (AMR) bacteria in each country, and allows countries to enact measures related to AMR issues. In this study, we performed on-site genomic epidemiological analysis of CPGNB isolated in Cambodia using a portable laboratory equipment called Bento Lab, which combines a PCR thermal cycler, microcentrifuge, gel electrophoresis apparatus, and LED transilluminator, along with the MinION nanopore sequencer. PCR targeting of major carbapenemase genes using Bento Lab revealed that two Escherichia coli isolates and one Acinetobacter baumannii isolate harbored carbapenemase genes: bla NDM, bla OXA-48, and bla OXA-23, respectively. The results of phenotypic diagnostic tests for CPGNB, such as the carbapenem inactivation method and double-disk diffusion test using a specific inhibitor of metallo-β-lactamases, were consistent with their AMR genotypes. Whole-genome sequencing analysis using MinION revealed that bla NDM-5 gene was carried on a 93.9-kb plasmid with IncFIA/IncFIB/IncFII/IncQ1 replicons, and bla OXA-181 gene was carried on a 51.5-kb plasmid with the IncX3 replicon in E. coli isolates. bla OXA-23 was encoded in two locations on the chromosome of A. baumannii. Plasmids carrying bla NDM-5 or bla OXA-181 in E. coli were highly structurally identical to plasmids prevalent in Enterobacterales in China and other countries, suggesting that they disseminated from a common evolutionary origin. Our findings demonstrate the potential impact of portable laboratory equipment on AMR bacteria research in hospitals and research centers with limited research facilities, and provide the first glimpse into the genomic epidemiology of CPGNB in Cambodia.

DOI： 10.3389/fmicb.2021.675463

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Complete Genome Sequence of a Japanese Clinical Isolate of Haemophilus influenzae Type a Strain TAMBA230. Reviewed International journal

Mayumi Kubota, Tsuyoshi Kenri, Yuko Sasaki, Keigo Shibayama, Kosuke Takayanagi, Tsuneaki Kenzaka

Microbiology resource announcements   Vol. 9 ( 48 )   2020.11

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Haemophilus influenzae causes severe infections such as pneumonia and meningitis. Here, we report the complete genome of H. influenzae type a strain TAMBA230, which was isolated in 2019 from a patient exhibiting bacteremia. This represents the first case in Japan of an H. influenzae type a strain associated with invasive infection.

DOI： 10.1128/MRA.01069-20

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Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae. Reviewed International journal

David Vizarraga, Akihiro Kawamoto, U Matsumoto, Ramiro Illanes, Rosa Pérez-Luque, Jesús Martín, Rocco Mazzolini, Paula Bierge, Oscar Q Pich, Mateu Espasa, Isabel Sanfeliu, Juliana Esperalba, Miguel Fernández-Huerta, Margot P Scheffer, Jaume Pinyol, Achilleas S Frangakis, Maria Lluch-Senar, Shigetarou Mori, Keigo Shibayama, Tsuyoshi Kenri, Takayuki Kato, Keiichi Namba, Ignacio Fita, Makoto Miyata, David Aparicio

Nature communications   Vol. 11 ( 1 ) page： 5188 - 5188   2020.10

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Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

DOI： 10.1038/s41467-020-18777-y

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Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans. Reviewed International journal

Tsuyoshi Sekizuka, Chihiro Katsukawa, Makoto Kuroda, Keigo Shibayama, Ken Otsuji, Mitsumasa Saito, Akihiko Yamamoto, Masaaki Iwaki

Emerging infectious diseases   Vol. 26 ( 10 ) page： 2457 - 2459   2020.10

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We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

DOI： 10.3201/eid2610.200086

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Macrolide-Resistant Bordetella pertussis, Vietnam, 2016-2017. Reviewed International journal

Kazunari Kamachi, Hong T Duong, Anh D Dang, T Hai, Do Do, Kentaro Koide, Nao Otsuka, Keigo Shibayama, Ha Thi Thu Hoang

Emerging infectious diseases   Vol. 26 ( 10 ) page： 2511 - 2513   2020.10

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Macrolide-resistant Bordetella pertussis emerged in Vietnam during 2016-2017. Direct analyses of swab samples from 10 patients with pertussis revealed a macrolide-resistant mutation, A2047G, in the 23S rRNA. We identified the MT104 genotype of macrolide-resistant B. pertussis (which is prevalent in mainland China) and its variants in these patients.

DOI： 10.3201/eid2610.201035

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National trend of blood-stream infection attributable deaths caused by Staphylococcus aureus and Escherichia coli in Japan. Reviewed International journal

Shinya Tsuzuki, Nobuaki Matsunaga, Koji Yahara, Yoshiaki Gu, Kayoko Hayakawa, Aki Hirabayashi, Toshiki Kajihara, Motoyuki Sugai, Keigo Shibayama, Norio Ohmagari

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy   Vol. 26 ( 4 ) page： 367 - 371   2020.4

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There has been scarce evidence about deaths due to blood stream infection (BSI) in Japan so far. The main objective of this study is to understand the epidemiological trend of deaths caused by BSIs due to Staphylococcus aureus and Escherichia coli including Methicillin-resistant S. aureus (MRSA) and fluoroquinolone-resistant E. coli (FQREC) at national level. We annually estimated the number of BSI caused by S. aureus and E. coli between 2011 and 2017 across Japan using comprehensive data of bacterial culturing and drug susceptibilities collected in Japan Nosocomial Infection Surveillance (JANIS). The number of death was estimated by using BSI mortality obtained from previous studies in Japan. The number of BSI death attributable to S. aureus was estimated to 17,412 in 2011 and 17,157 in 2017, respectively, out of the whole population (126.8 million) in Japan. Among them, cases attributed to MRSA accounted for 5924 (34.0%) in 2011, and decreased to 4224 (24.6%) cases in 2017. On the other hand, the number of BSI death attributable to E. coli was estimated to 9044 in 2011 and increased to 14,016 in 2017. Among them, cases attributed to FQREC accounted for 2045 (22.6%) in 2011 and increased to 3915 (27.9%) cases in 2017. The number of BSI death attributable to MRSA has been decreasing and that attributable to FQREC has been increasing. This study provides the first annual estimate of disease burden of BSI caused by antimicrobial resistant (AMR) bacteria in Japan, and basis for formulating health policy to deal with AMR.

DOI： 10.1016/j.jiac.2019.10.017

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Single-Tube Multiplex Polymerase Chain Reaction for the Detection of Genes Encoding Enterobacteriaceae Carbapenemase. Reviewed

Masanori Watahiki, Ryuji Kawahara, Masahiro Suzuki, Miyako Aoki, Kaoru Uchida, Yuko Matsumoto, Yuko Kumagai, Makiko Noda, Kanako Masuda, Chiemi Fukuda, Seiya Harada, Keiko Senba, Masato Suzuki, Mari Matsui, Satowa Suzuki, Keigo Shibayama, Hiroto Shinomiya

Japanese journal of infectious diseases   Vol. 73 ( 2 ) page： 166 - 172   2020.3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee  

A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.

DOI： 10.7883/yoken.JJID.2019.041

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A prolonged multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales due to horizontal transmission of the IncN plasmid. Reviewed International journal

Takuya Yamagishi, Mari Matsui, Tsuyoshi Sekizuka, Hiroaki Ito, Munehisa Fukusumi, Tomoko Uehira, Miyuki Tsubokura, Yoshihiko Ogawa, Atsushi Miyamoto, Shoji Nakamori, Akio Tawa, Takahisa Yoshimura, Hideki Yoshida, Hidetetsu Hirokawa, Satowa Suzuki, Tamano Matsui, Keigo Shibayama, Makoto Kuroda, Kazunori Oishi

Scientific reports   Vol. 10 ( 1 ) page： 4139 - 4139   2020.3

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A multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales (IMP-6-CPE) occurred at an acute care hospital in Japan. This study was conducted to understand the mechanisms of IMP-6-CPE transmission by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and whole-genome sequencing (WGS), and identify risk factors for IMP-6-CPE acquisition in patients who underwent abdominal surgery. Between July 2013 and March 2014, 22 hospitalized patients infected or colonized with IMP-6-CPE (Escherichia coli [n = 8], Klebsiella oxytoca [n = 5], Enterobacter cloacae [n = 5], Klebsiella pneumoniae [n = 3] and Klebsiella aerogenes [n = 1]) were identified. There were diverse PFGE profiles and sequence types (STs) in most of the species except for K. oxytoca. All isolates of K. oxytoca belonged to ST29 with similar PFGE profiles, suggesting their clonal transmission. Plasmid analysis by WGS revealed that all 22 isolates but one shared a ca. 50-kb IncN plasmid backbone with blaIMP-6 suggesting interspecies gene transmission, and typing of plasmids explained epidemiological links among cases. A case-control study showed pancreatoduodenectomy, changing drains in fluoroscopy room, continuous peritoneal lavage and enteric fistula were associated with IMP-6-CPE acquisition among the patients. Plasmid analysis of isolates in an outbreak of IMP-6-CPE suggested interspecies gene transmission and helped to clarify hidden epidemiological links between cases.

DOI： 10.1038/s41598-020-60659-2

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Complete Genome Sequence of Helicobacter suis Strain SNTW101c, Originally Isolated from a Patient with Nodular Gastritis. Reviewed International journal

Emiko Rimbara, Masato Suzuki, Hidenori Matsui, Masahiko Nakamura, Hirotaka Kobayashi, Shigetarou Mori, Keigo Shibayama

Microbiology resource announcements   Vol. 9 ( 1 )   2020.1

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Helicobacter suis strain SNTW101c, which was originally obtained from a patient with nodular gastritis, has been maintained in mouse stomach because of difficulty culturing it in vitro Recently, we succeeded in culturing this strain in vitro Here, we report the complete genome sequence of H. suis strain SNTW101c.

DOI： 10.1128/MRA.01340-19

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Geographical distribution of Enterobacterales with a carbapenemase IMP-6 phenotype and its association with antimicrobial use: An analysis using comprehensive national surveillance data on antimicrobial resistance. Reviewed International journal

Aki Hirabayashi, Koji Yahara, Toshiki Kajihara, Motoyuki Sugai, Keigo Shibayama

PloS one   Vol. 15 ( 12 ) page： e0243630   2020

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Enterobacterales resistant to carbapenems, a class of last-resort antimicrobials, are ranked as an "urgent" and "critical" public health hazard by CDC and WHO. IMP-type carbapenemase-containing Enterobacterales are endemic in Japan, and blaIMP-6 is one of the notable carbapenemase genes responsible for the resistance. The gene is plasmid-encoded and confers resistance to meropenem, but not to imipenem. Therefore, IMP-6-producing Enterobacterales isolates are occasionally overlooked in clinical laboratories and are referred to as 'stealth-type'. Since previous reports in Japan were confined only to some geographical regions, their distribution across prefectures and the factors affecting the distribution remain unclear. Here, we revealed the dynamics of the geographical distribution of Enterobacterales with IMP-6 phenotype associated with antimicrobial use in Japan. We utilized comprehensive national surveillance data of all routine bacteriological test results from more than 1,400 hospitals in 2015 and 2016 to enumerate Escherichia coli and Klebsiella pneumoniae isolates with the antimicrobial susceptibility pattern (phenotype) characteristic of IMP-6 (imipenem susceptible, meropenem resistant), and to tabulate the frequency of isolates with the phenotype for each prefecture. Isolates were detected in approximately half of all prefectures, and combined analysis with the national data of antimicrobial usage revealed a statistically significant association between the frequency and usage of not carbapenems but third-generation cephalosporins (p = 0.006, logistic mixed-effect regression) and a weaker association between the frequency and usage of fluoroquinolones (p = 0.043). The usage of third-generation cephalosporins and fluoroquinolones may select the strains with the IMP-6 phenotype, and contribute to their occasional spread. We expect the findings will promote antimicrobial stewardship to reduce the spread of the notable carbapenemase gene.

DOI： 10.1371/journal.pone.0243630

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Corrigendum: Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori. Reviewed International journal

Hirokazu Yano, Md Zobaidul Alam, Emiko Rimbara, Tomoko F Shibata, Masaki Fukuyo, Yoshikazu Furuta, Tomoaki Nishiyama, Shuji Shigenobu, Mitsuyasu Hasebe, Atsushi Toyoda, Yutaka Suzuki, Sumio Sugano, Keigo Shibayama, Ichizo Kobayashi

Frontiers in microbiology   Vol. 11   page： 596598 - 596598   2020

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[This corrects the article DOI: 10.3389/fmicb.2020.01628.].

DOI： 10.3389/fmicb.2020.596598

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Comparison of de-duplication methods used by WHO Global Antimicrobial Resistance Surveillance System (GLASS) and Japan Nosocomial Infections Surveillance (JANIS) in the surveillance of antimicrobial resistance. Reviewed International journal

Toshiki Kajihara, Koji Yahara, John Stelling, Sergey Romualdovich Eremin, Barbara Tornimbene, Visanu Thamlikitkul, Aki Hirabayashi, Eiko Anzai, Satoyo Wakai, Nobuaki Matsunaga, Kayoko Hayakawa, Norio Ohmagari, Motoyuki Sugai, Keigo Shibayama

PloS one   Vol. 15 ( 6 ) page： e0228234   2020

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A major issue in the surveillance of antimicrobial resistance (AMR) is "de-duplication" or removal of repeated isolates, for which there exist multiple methods. The World Health Organization (WHO) Global Antimicrobial Resistance Surveillance System (GLASS) requires de-duplication by selecting only the first isolate of a given bacterial species per patient per surveillance period per specimen type per age group, gender, and infection origin stratification. However, no study on the comparative application of this method has been reported. The objective of this study was to evaluate differences in data tabulation between the WHO GLASS and the Japan Nosocomial Infections Surveillance (JANIS) system, which counts both patients and isolates after removing repeated isolates of the same bacterial species isolated from a patient within 30 days, regardless of specimen type, but distinguishing isolates with change of antimicrobial resistance phenotype. All bacterial data, consisting of approximately 8 million samples from 1795 Japanese hospitals in 2017 were exported from the JANIS database, and were tabulated using either the de-duplication algorithm of GLASS, or JANIS. We compared the tabulated results of the total number of patients whose blood and urine cultures were taken and of the percentage of resistant isolates of Escherichia coli for each priority antibiotic. The number of patients per specimen type tabulated by the JANIS method was always smaller than that of GLASS. There was a small (< 3%) difference in the percentage of resistance of E. coli for any antibiotic between the two methods in both out- and inpatient settings and blood and urine isolates. The two tabulation methods did not show considerable differences in terms of the tabulated percentages of resistance for E. coli. We further discuss how the use of GLASS tabulations to create a public software and website that could help to facilitate the understanding of and treatment against AMR.

DOI： 10.1371/journal.pone.0228234

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Improved penicillin susceptibility of Streptococcus pneumoniae and increased penicillin consumption in Japan, 2013-18. Reviewed International journal

Shinya Tsuzuki, Takayuki Akiyama, Nobuaki Matsunaga, Koji Yahara, Keigo Shibayama, Motoyuki Sugai, Norio Ohmagari

PloS one   Vol. 15 ( 10 ) page： e0240655   2020

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OBJECTIVES: To examine the association between penicillin susceptibility of Streptococcus pneumoniae and penicillin consumption in Japan. METHODS: We used Japan Nosocomial Infection Surveillance data on the susceptibility of S. pneumoniae and sales data obtained from IQVIA Services Japan K.K. for penicillin consumption. We analysed both sets of data by decomposing them into seasonality and chronological trend components. The cross-correlation function was checked using Spearman's rank correlation coefficient to examine the correlation between susceptibility and consumption. RESULTS: After adjusting for seasonality, the susceptibility of S. pneumoniae to penicillins gradually improved (55.7% in 2013 and 60.6% in 2018, respectively) and penicillin consumption increased during the same period (0.76 defined daily doses per 1,000 inhabitants per day [DID] in 2013, and 0.89 DID in 2018). The results showed positive cross-correlation (coefficient 0.801, p-value < 0.001). In contrast, cephalosporin consumption decreased (3.91 DID in 2013 and 3.19 DID in 2018) and showed negative cross-correlation with susceptibility of S. pneumoniae to penicillins (coefficient -0.981, p-value < 0.001). CONCLUSIONS: The rates of penicillin-susceptible S. pneumoniae isolates did not negatively correlate with penicillin consumption at the population level. Increased penicillin consumption might not impair the penicillin susceptibility of S. pneumoniae.

DOI： 10.1371/journal.pone.0240655

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Periodic Genotype Shifts in Clinically Prevalent Mycoplasma pneumoniae Strains in Japan. Reviewed International journal

Tsuyoshi Kenri, Masato Suzuki, Tsuyoshi Sekizuka, Hitomi Ohya, Yoichiro Oda, Tsutomu Yamazaki, Hiroyuki Fujii, Toru Hashimoto, Hiroshi Nakajima, Chihiro Katsukawa, Makoto Kuroda, Keigo Shibayama

Frontiers in cellular and infection microbiology   Vol. 10   page： 385 - 385   2020

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Nationwide increases in Mycoplasma pneumoniae pneumonia cases in Japan were reported in 2011, 2012, 2015, and 2016. In this study, we isolated 554 M. pneumoniae strains in 4 areas in Japan (Kanagawa, Okayama, Osaka, and Saitama) between 2006 and 2019, and performed genotyping analysis. More than 80% of the strains isolated in 2011 and 2012 harbored type 1 p1 adhesin gene; however, strains harboring type 2 or its variant p1 gene increased in 2015 and 2016 and dominated after 2017. These findings suggested that a shift in the prevalent genotype of M. pneumoniae clinical strains occurred recently in Japan. More than 90% of the type 1 strains isolated after 2010 harbored macrolide-resistance mutations in their 23S rRNA gene, whereas most type 2 lineage strains had no such mutations. Consequently, the increase in type 2 lineage strains in Japan has reduced the macrolide resistance rate of clinical M. pneumoniae strains. During this analysis, we also identified M. pneumoniae strains carrying a novel variant type 1 p1 gene, and we classified it as type 1b. We then sequenced the genomes of 81 selected M. pneumoniae strains that we collected between 1976 and 2017 in Japan, and compared them with 156 M. pneumoniae genomes deposited in public databases to provide insights into the interpretation of M. pneumoniae genotyping methods, including p1 typing, multiple-locus variable-number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST), and typing by 8 single-nucleotide polymorphism markers (SNP-8). As expected, p1 typing, MLST, and SNP-8 results exhibited good correlation with whole-genome SNP analysis results in terms of phylogenetic relationships; however, MLVA typing results were less comparable to those of the other methods. MLVA may be useful for the discrimination of strains derived from a single outbreak within a limited area; however, is not reliable for classification of strains collected from distantly separated areas at different time points. This study showed the usefulness of genome-based comparison of M. pneumoniae for molecular epidemiology. Genome sequencing of more strains will improve our understanding of global propagation routes of this pathogen and evolutionary aspects of M. pneumoniae strains.

DOI： 10.3389/fcimb.2020.00385

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Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori. Reviewed International journal

Hirokazu Yano, Md Zobaidul Alam, Emiko Rimbara, Tomoko F Shibata, Masaki Fukuyo, Yoshikazu Furuta, Tomoaki Nishiyama, Shuji Shigenobu, Mitsuyasu Hasebe, Atsushi Toyoda, Yutaka Suzuki, Sumio Sugano, Keigo Shibayama, Ichizo Kobayashi

Frontiers in microbiology   Vol. 11   page： 1628 - 1628   2020

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Epigenetic DNA base methylation plays important roles in gene expression regulation. We here describe a gene expression regulation network consisting of many DNA methyltransferases each frequently changing its target sequence-specificity. Our object Helicobacter pylori, a bacterium responsible for most incidence of stomach cancer, carries a large and variable repertoire of sequence-specific DNA methyltransferases. By creating a dozen of single-gene knockout strains for the methyltransferases, we revealed that they form a network controlling methylome, transcriptome and adaptive phenotype sets. The methyltransferases interact with each other in a hierarchical way, sometimes regulated positively by one methyltransferase but negatively with another. Motility, oxidative stress tolerance and DNA damage repair are likewise regulated by multiple methyltransferases. Their regulation sometimes involves translation start and stop codons suggesting coupling of methylation, transcription and translation. The methyltransferases frequently change their sequence-specificity through gene conversion of their target recognition domain and switch their target sets to remodel the network. The emerging picture of a metamorphosing gene regulation network, or firework, consisting of epigenetic systems ever-changing their specificity in search for adaptation, provides a new paradigm in understanding global gene regulation and adaptive evolution.

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Genomic Diversity, Virulence, and Antimicrobial Resistance of <i>Helicobacter suis</i> Isolated from Human Stomachs Reviewed

Emiko Rimbara, Masato Suzuki, Hidenori Matsui, Masahiko Nakamura, Misako Morimoto, Chihiro Sasakawa, Takako Osaki, Noriyo Nagata, Keigo Shibayama, Kengo Tokunaga

SSRN Electronic Journal     2020

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DOI： 10.2139/ssrn.3663972

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A novel multilocus variable-number tandem repeat analysis for Bordetella parapertussis. Reviewed International journal

Kazunari Kamachi, Nao Otsuka, Rei Fumimoto, Kensuke Ozawa, Shu-Man Yao, Chuen-Sheue Chiang, Laurence Don Wai Luu, Ruiting Lan, Keigo Shibayama, Mineo Watanabe

Journal of medical microbiology   Vol. 68 ( 11 ) page： 1671 - 1676   2019.11

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Purpose. Human-adapted Bordetella parapertussis is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted B. parapertussis.Methodology. Four highly polymorphic variable number tandem repeat (VNTR) loci in the B. parapertussis genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of B. parapertussis.Results. Multiplex PCR-based MLVA characterized 53 B. parapertussis reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86-0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type.Conclusion. Our novel MLVA method has high discriminatory power for genotyping human B. parapertussis. Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks.

DOI： 10.1099/jmm.0.001095

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Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone. Reviewed International journal

Daisuke Asai, Tadashi Fukuda, Kazunori Morokuma, Daiki Funamoto, Yuko Yamaguchi, Takeshi Mori, Yoshiki Katayama, Keigo Shibayama, Hideki Nakashima

Macromolecular bioscience   Vol. 19 ( 10 ) page： e1900167   2019.10

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Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

DOI： 10.1002/mabi.201900167

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DNA gyrase could be a crucial regulatory factor for growth and survival of Mycobacterium leprae. Reviewed International journal

Hyun Kim, Yasuo Fukutomi, Chie Nakajima, Youn Uck Kim, Shigetarou Mori, Keigo Shibayama, Noboru Nakata, Yasuhiko Suzuki

Scientific reports   Vol. 9 ( 1 ) page： 10815 - 10815   2019.7

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Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25-30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.

DOI： 10.1038/s41598-019-47364-5

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Genome-Based Epidemiological Analysis of 13 Acinetobacter Strains Isolated from Blood Cultures of Hospitalized Patients from a University Hospital in Japan. Reviewed

Ken-Ichi Oinuma, Masato Suzuki, Kiyotaka Nakaie, Kanako Sato, Kozo Saeki, Arata Sakiyama, Etsuko Takizawa, Makoto Niki, Mamiko Niki, Koichi Yamada, Keigo Shibayama, Hiroshi Kakeya, Yukihiro Kaneko

Japanese journal of infectious diseases   Vol. 72 ( 4 ) page： 274 - 280   2019.7

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The genus Acinetobacter comprises many species that can cause infectious diseases. Despite their importance as nosocomial pathogens, the clinical distributions of individual species or clones are not well understood. In this study, we aimed to characterize 13 Acinetobacter strains isolated from blood cultures from Osaka City University Hospital. We conducted whole-genome sequencing to reveal their genetic background. We also performed PCR-based open reading frame typing (POT) and compared the results with those of multilocus sequence typing (MLST) to confirm its reliability as a genotyping method. Although biochemical analysis suggested that most isolates were A. baumannii, genomic analysis revealed that the collection of Acinetobacter isolates comprised six different species, with non-baumannii Acinetobacter species representing the majority. All strains possessed an inherent ADC-type β-lactamase gene, whereas the distribution of OXA-type enzymes was limited to A. baumannii, A. pittii, and A. colistiniresistens. While MLST properly discriminated four A. baumannii strains as different clones, POT failed to distinguish three of the four A. baumannii strains from each other, highlighting a potential pitfall that may be encountered when applying POT to non-epidemiological A. baumannii strains.

DOI： 10.7883/yoken.JJID.2018.403

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Novel Sequence Type in Bacillus cereus Strains Associated with Nosocomial Infections and Bacteremia, Japan. Reviewed International journal

Reiko Akamatsu, Masato Suzuki, Keiji Okinaka, Teppei Sasahara, Kunikazu Yamane, Satowa Suzuki, Daisuke Fujikura, Yoshikazu Furuta, Naomi Ohnishi, Minoru Esaki, Keigo Shibayama, Hideaki Higashi

Emerging infectious diseases   Vol. 25 ( 5 ) page： 883 - 890   2019.5

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Bacillus cereus is associated with foodborne illnesses characterized by vomiting and diarrhea. Although some B. cereus strains that cause severe extraintestinal infections and nosocomial infections are recognized as serious public health threats in healthcare settings, the genetic backgrounds of B. cereus strains causing such infections remain unknown. By conducting pulsed-field gel electrophoresis and multilocus sequence typing, we found that a novel sequence type (ST), newly registered as ST1420, was the dominant ST isolated from the cases of nosocomial infections that occurred in 3 locations in Japan in 2006, 2013, and 2016. Phylogenetic analysis showed that ST1420 strains belonged to the Cereus III lineage, which is much closer to the Anthracis lineage than to other Cereus lineages. Our results suggest that ST1420 is a prevalent ST in B. cereus strains that have caused recent nosocomial infections in Japan.

DOI： 10.3201/eid2505.171890

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Detection of mcr-1-mediated colistin resistance in E. coli isolate from imported chicken meat from Brazil. Reviewed International journal

Naoko Chiba, Koichi Tanimoto, Junzo Hisatsune, Motoyuki Sugai, Keigo Shibayama, Haruo Watanabe, Haruyoshi Tomita

Journal of global antimicrobial resistance   Vol. 16   page： 249 - 250   2019.3

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DOI： 10.1016/j.jgar.2019.02.004

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IncFII plasmid carrying antimicrobial resistance genes in Shigella flexneri: Vehicle for dissemination. Reviewed International journal

Dhiviya Prabaa Muthuirulandi Sethuvel, Shalini Anandan, Naveen Kumar Devanga Ragupathi, Revathi Gajendiran, Makoto Kuroda, Keigo Shibayama, Balaji Veeraraghavan

Journal of global antimicrobial resistance   Vol. 16   page： 215 - 219   2019.3

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OBJECTIVES: Plasmids harbouring antimicrobial resistance determinants in clinical strains are a significant public-health concern worldwide. The present study investigated such plasmids in clinical isolates of Shigella flexneri. METHODS: A total of 162 Shigella isolates were obtained from stool specimens in the year 2015. Among the 70 multidrug-resistant (MDR) Shigella spp., 27 S. flexneri isolates were randomly selected for further characterisation. Antimicrobial resistance genes (ARGs) and plasmid incompatibility (Inc) types were analysed. RESULTS: IncFII plasmids were found in 63% (17/27) of the studied S. flexneri isolates. ARGs such as dhfr1a (81%), sulII (74%), blaOXA (74%), blaTEM (33%), blaAmpC (30%), qnrS (15%) and qnrB (4%) were identified by PCR, whereas blaCTX-M was not detected. Next-generation sequencing of a representative S. flexneri IncFII-type plasmid (pSF470) revealed the presence of blaTEM1-B, blaDHA-1, qnrB10, mphA, sulI, sulII, strA, strB and tetR ARGs along with the intI1 integrase gene. In addition, pMLST analysis showed that the replicon belonged to F2:A-:B- type. CONCLUSIONS: This study helps to know the prevalent plasmid types in MDR Shigella isolates and will improve our understanding of resistance dissemination among enteric bacteria. ARGs in plasmids further highlight the importance of such studies in enteric bacteria.

DOI： 10.1016/j.jgar.2018.10.014

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The recent trend of MRSA surveillance in Japanese health care facilities

Tsuzuki, S, Matsunaga, N, Hayakawa, K, Suzuki, Y, Noda, A, Yamagishi, K, Yahara, K, Tsutsui, A, Shibayama, K, Noda, H, Omagari, N, Nishiura, H

INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES   Vol. 79 ( 1 ) page： 49 - 49   2019.2

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DOI： 10.1016/j.ijid.2018.11.131

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Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae. Reviewed International journal

Tsuyoshi Kenri, Yoshito Kawakita, Hisashi Kudo, U Matsumoto, Shigetarou Mori, Yukio Furukawa, Yuhei O Tahara, Keigo Shibayama, Yuuki Hayashi, Munehito Arai, Makoto Miyata

Biochemical and biophysical research communications   Vol. 508 ( 4 ) page： 1050 - 1055   2019.1

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Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

DOI： 10.1016/j.bbrc.2018.11.132

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Genetic characterization of Mycoplasma pneumoniae isolated in Osaka between 2011 and 2017: Decreased detection rate of macrolide-resistance and increase of p1 gene type 2 lineage strains. Reviewed International journal

Chihiro Katsukawa, Tsuyoshi Kenri, Keigo Shibayama, Kazuo Takahashi

PloS one   Vol. 14 ( 1 ) page： e0209938   2019

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We characterized 419 Mycoplasma pneumoniae isolates collected between 2011 and 2017 in Osaka prefecture of Japan. This analysis revealed high prevalence of macrolide-resistant M. pneumoniae (MRMP) in Osaka during 2011 and 2014 with annual detection rates of MRMP strains between 71.4% and 81.8%. However, in 2015 and after, the detection rate of MRMP decreased significantly and did not exceed 50%. Genotyping of the p1 gene of these isolates showed that most of MRMP strains harbored type 1 p1 gene. In contrast, strains expressing p1 gene type 2 or its variant were largely macrolide-susceptible M. pneumoniae (MSMP) strains. There was a strong correlation between p1 gene genotype and the presence of mutations conferring macrolide resistance in M. pneumoniae isolated in Osaka. These results indicate that lower incidence of MRMP strains in Osaka from 2015 was associated with the relative increase of p1 gene type 2 lineage strains. During these experiments, we also isolated three M. pneumoniae strains that showed irregular typing pattern in the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the p1 gene. Two of these strains harbored new variants of type 2 p1 gene and were designated as type 2f and 2g. The remaining strain with an irregular typing pattern had a large deletion in the p1 operon.

DOI： 10.1371/journal.pone.0209938

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Molecular epidemiological analysis and risk factors for acquisition of carbapenemase-producing Enterobacter cloacae complex in a Japanese university hospital. Reviewed International journal

Nobuyuki Tetsuka, Aki Hirabayashi, Akane Matsumoto, Keisuke Oka, Yuki Hara, Hiroshi Morioka, Mitsutaka Iguchi, Yuka Tomita, Masato Suzuki, Keigo Shibayama, Tetsuya Yagi

Antimicrobial resistance and infection control   Vol. 8   page： 126 - 126   2019

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Background: To clarify the molecular epidemiology of carbapenem-resistant Enterobacter cloacae complex (CREC) and the risk factors for acquisition of carbapenemase-producing E. cloacae complex (CPEC). Methods: Using clinical CREC isolates detected in a Japanese university hospital over 4 years, carbapenemase production was screened with phenotypic methods. Carbapenemase genes were analysed by PCR and sequencing. Molecular epidemiological analyses were conducted with repetitive extragenic palindromic (REP)-PCR and multilocus sequence typing (MLST). CRECs were identified to the subspecies level by hsp60 sequencing. Whole-genome sequencing of plasmids was conducted. A case-control study was performed to identify risk factors for acquisition of CPEC among patients with CREC. Results: Thirty-nine CRECs including 20 CPECs carrying blaIMP-1 were identified. Patients with CPEC had longer hospital stay before detection (26.5 days vs. 12 days, p = 0.008), a urinary catheter (odds ratio [OR], 5.36; 95% confidence interval [CI], 1.14-30.9; p = 0.023), or intubation (OR, 7.53; 95% CI, 1.47-53.8; p = 0.008) compared to patients without CPEC. Four genetically closely related CPEC clusters were observed, which showed that three of four CPEC clusters corresponded to E. asburiae (ST 53), E. hormaechei subsp. steigerwaltii (ST 113 and ST 1047) and E. cloacae subsp. cloacae (ST 513) by MLST and hsp60 sequencing. Seven representative plasmids shared structures with class I integron containing blaIMP-1 and IncHI2A replicon type. Conclusions: A longer hospital stay, presence of a urinary catheter, and intubation are risk factors for CPEC acquisition. In addition to horizontal transmission of genetically indistinguishable CPECs, IncHI2A plasmid carrying blaIMP-1 appeared to be transferred among genetically different ECs.

DOI： 10.1186/s13756-019-0578-3

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Seroprevalence of IgA and IgM antibodies to Bordetella pertussis in healthy Japanese donors: Assessment for the serological diagnosis of pertussis. Reviewed International journal

Rei Fumimoto, Nao Otsuka, Hajime Kamiya, Tomimasa Sunagawa, Keiko Tanaka-Taya, Kazunari Kamachi, Keigo Shibayama

PloS one   Vol. 14 ( 7 ) page： e0219255   2019

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Pertussis is a human respiratory infection caused by the gram-negative bacterium, Bordetella pertussis. To evaluate the pertussis burden and vaccine efficacy, diagnosis and epidemiological surveillance should be based on accurate and valid diagnostic methods. Recently, the serodiagnostic tests Novagnost Bordetella pertussis IgA and IgM were approved in Japan for pertussis diagnostics. Although the anti-pertussis toxin (PT) IgG assay has been used for pertussis diagnosis worldwide, little is known about the anti-B. pertussis IgA and IgM assays. In this study, serum samples from 460 healthy donors were examined to determine the seroprevalence of anti-B. pertussis IgA and IgM in a Japanese population, and its correlation with donor age. Our data demonstrated that anti-B. pertussis IgA and IgM are positively and negatively correlated with age (r = 0.27, r = -0.37; P < 0.001, respectively). Age-specific analysis revealed high titers of anti-B. pertussis IgA in adults (46-50 years), while anti-B. pertussis IgM titers were high in schoolchildren (6-10, 11-15 years). When applying the arbitrary cut-off values for these ages, 17.6% and 39.5% of healthy donors were interpreted as pertussis-positive or indeterminate with anti-B. pertussis IgA (46-50 years) and IgM (11-15 years) titers, respectively. Overall, our findings indicated that the Novagnost Bordetella pertussis IgA and IgM testing could be greatly affected by subject age, limiting its value for pertussis diagnosis.

DOI： 10.1371/journal.pone.0219255

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Development of vaccine for Clostridium difficile infection using membrane fraction of nontoxigenic Clostridium difficile. Reviewed International journal

Mitsutoshi Senoh, Masaaki Iwaki, Akihiko Yamamoto, Haru Kato, Tadashi Fukuda, Keigo Shibayama

Microbial pathogenesis   Vol. 123   page： 42 - 46   2018.10

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Although standard antibiotic therapy is performed for diarrhea and pseudomembranous colitis caused by Clostridium difficile, a high recurrence rate of C. difficile infection (CDI) remains a major problem. We previously showed that a membrane fraction of nontoxigenic C. difficile (ntCDMF) was effective as a vaccine antigen by in vitro experiments. In this study, we examined whether ntCDMF had an in vivo effect in animal challenge experiments. By intrarectal immunization with ntCDMF, the number of C. difficile cells in feces of mice was decreased approximately 99% compared to the control mice. In addition, survival rate of C. difficile-challenged hamsters was increased almost 30% by immunization with ntCDMF. These results showed that ntCDMF could be a practical vaccine candidate.

DOI： 10.1016/j.micpath.2018.06.039

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Effect of methicillin-resistant Staphylococcus aureus in Japan. Reviewed International journal

Hironori Uematsu, Kazuto Yamashita, Seiko Mizuno, Susumu Kunisawa, Keigo Shibayama, Yuichi Imanaka

American journal of infection control   Vol. 46 ( 10 ) page： 1142 - 1147   2018.10

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BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is the most common antimicrobial-resistant organism identified in Japanese health care facilities. This study analyzed the clinical and economic burdens attributable to methicillin resistance in S aureus in Japanese hospitals. METHODS: We retrospectively investigated data from 14,905 inpatients of 57 hospitals combined with data from nosocomial infection surveillance and administrative claim databases. The participants were inpatients with admission from April 1, 2014, to discharge on March 31, 2016. The outcomes were evaluated according to length of stay, hospital charges, and in-hospital mortality. We compared the disease burden of MRSA infections with methicillin-susceptible S aureus (MSSA) infections based on patients' characteristics and onset periods. RESULTS: We categorized 7,188 and 7,717 patients into MRSA and MSSA groups, respectively. The adjusted effects of the MRSA group were 1.03-fold (95% confidence interval [CI] 1.01-1.05) and 1.04-fold (95% CI, 1.01-1.06), respectively, with an odds ratio of 1.14 (95% CI, 1.02-1.27). CONCLUSIONS: The results of this study found that patient severity and onset delays were positively associated with both MRSA and burden and that the effect of methicillin resistance remained significant after adjustment.

DOI： 10.1016/j.ajic.2018.04.214

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A nationwide population-based surveillance of invasive Haemophilus influenzae diseases in children after the introduction of the Haemophilus influenzae type b vaccine in Japan. Reviewed International journal

Shigeru Suga, Naruhiko Ishiwada, Yuko Sasaki, Hideki Akeda, Junichiro Nishi, Kenji Okada, Mikiya Fujieda, Megumi Oda, Kazutoyo Asada, Takashi Nakano, Akihiko Saitoh, Mitsuaki Hosoya, Takehiro Togashi, Mayumi Matsuoka, Kouji Kimura, Keigo Shibayama

Vaccine   Vol. 36 ( 38 ) page： 5678 - 5684   2018.9

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BACKGROUND: Haemophilus influenzae type b (Hib) vaccine was introduced as a voluntary vaccine in December 2008 and was included in the national routine immunization program in April 2013 in Japan. Currently, no nationwide data are available to evaluate the effectiveness of Hib vaccine in Japan. METHODS: To evaluate the effectiveness of Hib vaccine in Japan, nationwide active population-based surveillance of culture-proven invasive infections caused by H. influenzae in children was performed in 2008-2017 in 10 prefectures in Japan (covering approximately 23% of the total Japanese population). Clinical data were recorded on a standardized case report form. Capsular type and antimicrobial susceptibility of the H. influenzae isolates were examined. The incidence rate ratio (IRR) and its confidence interval (CI) were calculated to compare data from 5 years before and that from after the introduction of the national routine Hib vaccine immunization program. RESULTS: During the 10-year study period, 566 invasive H. influenzae disease cases including 336 meningitis cases were identified. The average number of invasive H. influenzae disease cases among children <5 years of age during 2013-2017 decreased by 93% (IRR: 0.07, 95%CI 0.05-0.10, p < 0.001) compared with those occurring during 2008-2012. Hib strains have not been isolated from invasive H. influenzae disease cases since 2014; however, non-typeable H. influenzae and H. influenzae type f isolates have been noted as causes of invasive H. influenzae diseases among children <5 years in the post-Hib vaccine era. CONCLUSIONS: After the governmental subsidization of the Hib vaccine, invasive Hib disease cases decreased dramatically in the study population, as per our surveillance. Continuous surveillance is necessary to monitor the effectiveness of Hib vaccine and for detecting any emerging invasive capsular types.

DOI： 10.1016/j.vaccine.2018.08.029

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Clinical and Bacteriologic Analysis of Nontypeable Haemophilus influenzae Strains Isolated from Children with Invasive Diseases in Japan from 2008 to 2015. Reviewed International journal

Sachiko Naito, Noriko Takeuchi, Misako Ohkusu, Azusa Takahashi-Nakaguchi, Hiroki Takahashi, Naoko Imuta, Junichiro Nishi, Keigo Shibayama, Mayumi Matsuoka, Yuko Sasaki, Naruhiko Ishiwada

Journal of clinical microbiology   Vol. 56 ( 7 ) page： e00141   2018.7

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Haemophilus influenzae type b (Hib) conjugate vaccines have led to dramatic reductions in Hib disease among young children worldwide. Nontypeable H. influenzae (NTHi) is now the major cause of invasive H. influenzae infections. We investigated the clinical characteristics of invasive NTHi diseases among children in Japan, to clarify the pathogenicity of isolated NTHi strains. The mortality rate was 10.7%, with deaths occurring mainly among children with underlying comorbidities. Biotypes II and III were the most common, and most strains (64.3%) had multiple amino acid substitutions at the Asp-350, Ser-357, Ser-385, and/or Met-377 sites of penicillin-binding protein 3. Two strains were β-lactamase positive and ampicillin-clavulanate resistant. Biofilm indices varied widely, and IS1016 was detected in 10.7% of the strains tested. Moreover, there was wide variation in the characteristics of invasive NTHi strains. NTHi strains, showing great genetic diversity, are responsible for most invasive H. influenzae infections in children in the postvaccine era. Continuous monitoring of NTHi strains responsible for invasive diseases in children is important to detect changes in the epidemiology of invasive H. influenzae infections in the postvaccine era.

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Trends and patterns of national antimicrobial consumption in Japan from 2004 to 2016 Reviewed

Atsuko Tsutsui, Koji Yahara, Keigo Shibayama

Journal of Infection and Chemotherapy   Vol. 24 ( 6 ) page： 414 - 421   2018.6

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Frequent use of broad-spectrum antimicrobial classes has been reported in Japan
however, little is known about the long-term trend of national antimicrobial consumption, and that of individual agents. This study analyzed the national sales data of systemic antimicrobials from 2004 to 2016, derived from the IMS Japan Pharmaceutical Market database, to assess the consumption patterns of antimicrobial classes and agents in Japan. The number of defined daily doses per 1000 inhabitants per day (DID) was calculated for each antimicrobial agent. During the last 13 years, total antimicrobial consumption fluctuated by only 5% around the average of 14.41 DID. In 2016, the most used class was macrolides (32%), followed by cephalosporins (28%) and fluoroquinolones (19%). Oral agents comprised a large proportion (93%) of antimicrobial consumption. The most used agent, clarithromycin, accounted for 25% of all oral compounds used in 2016. The consumption of oral agents with high bioavailability, such as fluoroquinolones, amoxicillin, and sulfamethoxazole/trimethoprim increased, whereas that of cephalosporins decreased. In 2016, ceftriaxone was the most consumed parenteral agent, followed by cefazolin. The consumption of parenteral agents increased after 2009 when high-dose regimens of piperacillin/tazobactam, meropenem, and ampicillin/sulbactam were approved by the health insurance system. National antimicrobial consumption has been stable over the last 13 years. Moreover, shifts in the use of agents with high bioavailability and those approved for high-dose regimens were observed. However, the increased use of broad-spectrum agents is worrisome. A multifaceted approach is required to reduce overall antimicrobial consumption.

DOI： 10.1016/j.jiac.2018.01.003

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Complete Genome Sequence of Mycobacterium marinum ATCC 927T, Obtained Using Nanopore and Illumina Sequencing Technologies. Reviewed International journal

Mitsunori Yoshida, Hanako Fukano, Yuji Miyamoto, Keigo Shibayama, Masato Suzuki, Yoshihiko Hoshino

Genome announcements   Vol. 6 ( 20 )   2018.5

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Mycobacterium marinum is a slowly growing, broad-host-range mycobacterial species. Here, we report the complete genome sequence of a Mycobacterium marinum type strain that was isolated from tubercles of diseased fish. This sequence will provide essential information for future taxonomic and comparative genome studies of its relatives.

DOI： 10.1128/genomeA.00397-18

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Genetic shifts in methicillin-resistant Staphylococcus aureus epidemic clones and toxin gene profiles in Japan: Comparative analysis among pre-epidemic, epidemic and post-epidemic phases Reviewed

Shunsuke Osaka, Katsuko Okuzumi, Shota Koide, Kiyoko Tamai, Tomoaki Sato, Koichi Tanimoto, Haruyoshi Tomita, Masahiro Suzuki, Yukiko Nagano, Keigo Shibayama, Yoshichika Arakawa, Noriyuki Nagano

Journal of Medical Microbiology   Vol. 67 ( 3 ) page： 392 - 399   2018.3

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Purpose. The decline in methicillin-resistant Staphylococcus aureus (MRSA) isolation rates has become a general observation worldwide, including Japan. We hypothesized that some genetic shift in MRSA might cause this phenomenon, and therefore we investigated the genetic profiles among MRSA clinical isolates obtained from three different epidemic phases in Japan. Methodology. A total of 353 MRSA isolates were selected from 202 medical facilities in 1990 (pre-epidemic phase), 2004 (epidemic phase) and 2016 (post-epidemic phase). Molecular typing was performed by PCR detection of 22 genes using the polymerase chain reaction (PCR)-based ORF typing (POT) system, including an additional eight genes including small genomic islets and seven toxin genes. Results. Isolates with a POT1 of score 93, identified as presumed clonal complex (pCC)5-staphylococcal cassette chromosome mec (SCCmec) type II including ST5-SCCmec type II New York/Japan clone, represented the major epidemic MRSA lineage in 1990 and 2004. In 2016, however, a marked decrease in isolates with a POT1 score of 93, along with changes in the epidemiology of toxin genes carried, was noted, where the carriers of tst genes including the tst-sec combination were markedly reduced, and those possessing the seb gene alone were markedly increased. Rather, isolates with a POT1 score of 106, including pCC1 or pCC8 among the isolates with SCCmec type IV, which often links to communityassociated MRSA, were predominant. Interestingly, the pCC1 and pCC8 lineages were related to sea and tst-sec carriage, respectively. Conclusions. Over time, a transition in MRSA genetic profiles from a POT1 score of 93 in 1990 and 2004 to 106 in 2014 was found in Japan.

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Rapid and easy detection of low-level resistance to vancomycin in methicillin-resistant staphylococcus aureus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry Reviewed

Kota Asakura, Takuya Azechi, Hiroshi Sasano, Hidehito Matsui, Hideaki Hanaki, Motoyasu Miyazaki, Tohru Takata, Miwa Sekine, Tomoiku Takaku, Tomonori Ochiai, Norio Komatsu, Keigo Shibayama, Yuki Katayama, Koji Yahara

PLoS ONE   Vol. 13 ( 3 ) page： e0194212   2018.3

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Vancomycin-intermediately resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are associated with treatment failure. hVISA contains only a subpopulation of cells with increased minimal inhibitory concentrations, and its detection is problematic because it is classified as vancomycin-susceptible by standard susceptibility testing and the gold-standard method for its detection is impractical in clinical microbiology laboratories. Recently, a research group developed a machine-learning classifier to distinguish VISA and hVISA from vancomycin-susceptible S. aureus (VSSA) according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data. Nonetheless, the sensitivity of hVISA classification was found to be 76%, and the program was not completely automated with a graphical user interface. Here, we developed a more accurate machine-learning classifier for discrimination of hVISA from VSSA and VISA among MRSA isolates in Japanese hospitals by means of MALDI-TOF MS data. The classifier showed 99% sensitivity of hVISA classification. Furthermore, we clarified the procedures for preparing samples and obtaining MALDI-TOF MS data and developed all-in-one software, hVISA Classifier, with a graphical user interface that automates the classification and is easy for medical workers to use
it is publicly available at https://github.com/bioprojects/hVISAclassifier. This system is useful and practical for screening MRSA isolates for the hVISA phenotype in clinical microbiology laboratories and thus should improve treatment of MRSA infections.

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Complete Genome Sequence of a Type Strain of Mycobacterium abscessus subsp. bolletii, a Member of the Mycobacterium abscessus Complex. Reviewed International journal

Mitsunori Yoshida, Hanako Fukano, Yuji Miyamoto, Keigo Shibayama, Masato Suzuki, Yoshihiko Hoshino

Genome announcements   Vol. 6 ( 5 )   2018.2

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Mycobacterium abscessus subsp. bolletii is a rapidly growing mycobacterial organism for which the taxonomy is unclear. Here, we report the complete genome sequence of a Mycobacterium abscessus subsp. bolletii type strain. This sequence will provide essential information for future taxonomic and comparative genome studies of these mycobacteria.

DOI： 10.1128/genomeA.01530-17

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Distribution and Molecular Characterization of Acinetobacter baumannii International Clone II Lineage in Japan. Reviewed International journal

Mari Matsui, Masato Suzuki, Masahiro Suzuki, Jun Yatsuyanagi, Masanori Watahiki, Yoichi Hiraki, Fumio Kawano, Atsuko Tsutsui, Keigo Shibayama, Satowa Suzuki

Antimicrobial agents and chemotherapy   Vol. 62 ( 2 )   2018.2

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Multidrug-resistant (MDR) Acinetobacter spp. have been globally disseminated in association with the successful clonal lineage Acinetobacter baumannii international clone II (IC II). Because the prevalence of MDR Acinetobacter spp. in Japan remains very low, we characterized all Acinetobacter spp. (n = 866) from 76 hospitals between October 2012 and March 2013 to describe the entire molecular epidemiology of Acinetobacter spp. The most prevalent species was A. baumannii (n = 645; 74.5%), with A. baumannii IC II (n = 245) accounting for 28.3% of the total. Meropenem-resistant isolates accounted for 2.0% (n = 17) and carried ISAba1-blaOXA-23-like (n = 10), blaIMP (n = 4), or ISAba1-blaOXA-51-like (n = 3). Multilocus sequence typing of 110 representative A. baumannii isolates revealed the considerable prevalence of domestic sequence types (STs). A. baumannii IC II isolates were divided into the domestic sequence type 469 (ST469) (n = 18) and the globally disseminated STs ST208 (n = 14) and ST219 (n = 4). ST469 isolates were susceptible to more antimicrobial agents, while ST208 and ST219 overproduced the intrinsic AmpC β-lactamase. A. baumannii IC II and some A. baumannii non-IC II STs (e.g., ST149 and ST246) were associated with fluoroquinolone resistance. This study revealed that carbapenem-susceptible A. baumannii IC II was moderately disseminated in Japan. The low prevalence of acquired carbapenemase genes and presence of domestic STs could contribute to the low prevalence of MDR A. baumannii A similar epidemiology might have appeared before the global dissemination of MDR epidemic lineages. In addition, fluoroquinolone resistance associated with A. baumannii IC II may provide insight into the significance of A. baumannii epidemic clones.

DOI： 10.1128/AAC.02190-17

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Mutations in Genes Encoding Penicillin-Binding Proteins and Efflux Pumps Play a Role in β-Lactam Resistance in Helicobacter cinaedi. Reviewed International journal

Emiko Rimbara, Shigetarou Mori, Hyun Kim, Masato Suzuki, Keigo Shibayama

Antimicrobial agents and chemotherapy   Vol. 62 ( 2 )   2018.2

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β-Lactams are often used to treat Helicobacter cinaedi infections; however, the mechanism underlying β-lactam resistance is unknown. In this study, we investigated β-lactam resistance in an H. cinaedi strain, MRY12-0051 (MICs of amoxicillin [AMX] and ceftriaxone [CRO], 32 and 128 μg/ml; obtained from human feces). Based on a comparative whole-genome analysis of MRY12-0051 and the CRO-susceptible H. cinaedi strain MRY08-1234 (MICs of AMX and CRO, 1 and 4 μg/ml; obtained from human blood), we identified five mutations in genes encoding penicillin-binding proteins (PBPs), including two in pbpA, one in pbp2, and two in ftsI Transformation and penicillin binding assays indicated that CRO resistance was mainly associated with mutations in pbpA; mutations in ftsI also led to increased resistance to AMX. Knocking out cmeB and cmeD, which encode resistance-nodulation-division-type efflux pump components, in H. cinaedi type strain CCUG18818 (AMX MIC, 4 to 8 μg/ml) resulted in 8- and 64-fold decreases, respectively, in the AMX MIC. Hence, MICs of AMX in H. cinaedi become similar to those of Helicobacter pylori isolates in the absence of cmeD In conclusion, the difference in susceptibility to β-lactams between H. pylori and H. cinaedi is explained by differences in efflux pump components. Mutations in pbpA are the primary determinant of high resistance to β-lactams in H. cinaedi.

DOI： 10.1128/AAC.02036-17

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The plasmid-encoded transcription factor ArdK contributes to the repression of the IMP-6 metallo-β-lactamase gene blaIMP-6, leading to a carbapenem-susceptible phenotype in the blaIMP-6-positive Escherichia coli strain A56-1S. Reviewed International journal

Takaya Segawa, Tsuyoshi Sekizuka, Satowa Suzuki, Keigo Shibayama, Mari Matsui, Makoto Kuroda

PloS one   Vol. 13 ( 12 ) page： e0208976   2018

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Carbapenemase-producing Enterobacteriaceae (CPE) are a global concern because these bacteria are resistant to almost all β-lactams. Horizontal interspecies gene transfer via plasmid conjugation has increased the global dissemination of CPE. Recently, an Enterobacteriaceae strain positive for carbapenemase gene but showing a carbapenem-susceptible phenotype was identified, suggesting that these susceptible strains may be challenging to detect solely via antimicrobial susceptibility tests without molecular analysis. Here, we isolated a blaIMP-6 carbapenemase-gene positive but imipenem- and meropenem-susceptible Escherichia coli (ISMS-E) strain A56-1S (imipenem and meropenem minimum inhibitory concentration, ≤ 0.125 mg/L), from a human urine specimen in Japan. A56-1S was carbapenemase negative by the Carba NP test, suggesting that IMP-6 production was low or undetectable. Thus, to characterize the mechanism of this phenotype, a meropenem-resistant E. coli A56-1R strain was obtained using meropenem-selection. A56-1R was positive for carbapenemase production by the Carba NP test, and blaIMP-6 transcription in A56-1R was 53-fold higher than in A56-1S, indicating that blaIMP-6 in A56-1S is negatively regulated at the transcriptional level. Comparative genomic analysis between the two strains revealed that the alleviation of restriction of DNA (ardK) gene encoding a putative transcription factor is disrupted by the IS26 insertion in A56-1R. A cotransformation assay of ardK and the regulatory element upstream of blaIMP-6 showed repression of blaIMP-6 transcription, indicating that ArdK negatively modulates blaIMP-6 transcription. ArdK binding and affinity assays demonstrated that ArdK directly binds to the regulatory element upstream of blaIMP-6 with dissociation constant values comparable to those of general transcription factors. The IMP-6 carbapenemase showed low hydrolytic activity against imipenem, resulting in an imipenem-susceptible and meropenem-resistant (ISMR) phenotype (previously reported as a stealth phenotype). However, the low expression of IMP-6 in the A56-1S strain could be a typical characteristic of ISMS-E due to gene repression, indicating that conventional antimicrobial susceptibility tests might be unable to detect such strains even when using both imipenem and meropenem. Bacteria that exhibit the ISMS phenotype could play a potential role as undetectable reservoirs and might facilitate gene transfer to other organisms while avoiding detection.

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Draft genome sequence of Mycobacterium sp. strain shizuoka-1, a novel mycobacterium isolated from groundwater of a bathing facility in Shizuoka, Japan Reviewed

Mitsunori Yoshida, Shinji Izumiyama, Hanako Fukano, Kanji Sugiyama, Masato Suzuki, Keigo Shibayama, Yoshihiko Hoshino

Genome Announcements   Vol. 5 ( 47 )   2017.11

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Mycobacterium sp. strain shizuoka-1 is a rapidly growing scotochromogenic mycobacterium and was isolated from well water for a bathing facility in Shizuoka Prefecture in Japan. Here, we report the draft sequence of its genome, comprising a 6.5-Mb chromosome. This mycobacterium has 83.1% identity with Mycobacterium rhodesiae, a human pathogen.

DOI： 10.1128/genomeA.01309-17

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Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part II. Experimental Production of Equine Antivenom against the Redback Spider Reviewed

Shigemi Mori, Akira Horita, Akihiro Ginnaga, Yoshinobu Miyatsu, Kyoko Sawabe, Takayuki Matsumura, Manabu Ato, Akihiko Yamamoto, Keigo Shibayama, Satoru Arai, Takuya Yamagishi, Motohide Takahashi, Hisashi Taki, Toru Hifumi

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 70 ( 6 ) page： 635 - 641   2017.11

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This is the first report on large-scale experimental production of an equine antivenom against the redback spider (Latrodectus hasseltii) lived in Japan. We captured 10,000 redback spiders in Japan and prepared the toxoids of crude venom extract, mixed the toxoids with a mineral oil adjuvant, and immunized healthy horses repeatedly over a period of several weeks. Thereafter, we separated the horse plasma, purified the gamma-globulin fraction, and stocked it as a purified antivenom concentrate. Consequently, we manufactured approximately 6,500 vials of a single-dose freeze-dried test lot from a portion of the purified gamma-globulin fraction, equivalent to the extract derived from 520 spiders. This test lot had an antitoxin titer comparable to that of a similar drug commercially available overseas (a liquid preparation), and the other quality met all quality reference specifications based on the Minimum Requirements for Biological Products and other guidelines relevant to existing antivenom drug products in Japan.

DOI： 10.7883/yoken.JJID.2017.125

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Diversity and microevolution of CRISPR loci in Helicobacter cinaedi Reviewed

Junko Tomida, Yuji Morita, Keigo Shibayama, Ken Kikuchi, Tomohiro Sawa, Takaaki Akaike, Yoshiaki Kawamura

PLOS ONE   Vol. 12 ( 10 ) page： e0186241   2017.10

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Helicobacter cinaedi is associated with nosocomial infections. The CRISPR-Cas system provides adaptive immunity against foreign genetic elements. We investigated the CRISPR-Cas system in H. cinaedi to assess the potential of the CRISPR-based microevolution of H. cinaedi strains. A genotyping method based on CRISPR spacer organization was carried out using 42 H. cinaedi strains. The results of sequence analysis showed that the H. cinaedi strains used in this study had two CRISPR loci (CRISPR1 and CRISPR2). The lengths of the consensus direct repeat sequences in CRISPR1 and CRISPR2 were both 36 bp-long, and 224 spacers were found in the 42 H. cinaedi strains. Analysis of the organization and sequence similarity of the spacers of the H. cinaedi strains showed that CRISPR arrays could be divided into 7 different genotypes. Each genotype had a different ancestral spacer, and spacer acquisition/deletion events occurred while isolates were spreading. Spacer polymorphisms of conserved arrays across the strains were instrumental for differentiating closely-related strains collected from the same hospital. MLST had little variability, while the CRISPR sequences showed remarkable diversity. Our data revealed the structural features of H. cinaedi CRISPR loci for the first time. CRISPR sequences constitute a valuable basis for genotyping, provide insights into the divergence and relatedness between closelyrelated strains, and reflect the microevolutionary process of H. cinaedi.

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Integration of DPC and clinical microbiological data in Japan reveals importance of confirming a negative follow-up blood culture in patients with MRSA bacteremia Reviewed

Naoki Miyamoto, Koji Yahara, Rie Horita, Tomomi Yano, Naotaka Tashiro, Daiichi Morii, Atsuko Tsutsui, Kenichiro Yaita, Keigo Shibayama, Hiroshi Watanabe

JOURNAL OF INFECTION AND CHEMOTHERAPY   Vol. 23 ( 10 ) page： 687 - 691   2017.10

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Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is one of the commonest and most life-threatening of all infectious diseases. The morbidity and mortality rates associated with MRSA bacteremia are higher than those associated with bacteremia caused by other pathogens. A common guideline in MRSA bacteremia treatment is to confirm bacteremia clearance through additional blood cultures 2-4 days after initial positive cultures and as needed thereafter. However, no study has presented statistical evidence of how and to what extent confirming a negative follow-up blood culture impacts clinical outcome. We present this evidence for the first time, by combining clinical microbiological data of blood cultures and the DPC administrative claims database; both had been systematically accumulated through routine medical care in hospitals. We used electronic medical records to investigate the clinical background and infection source in detail. By analyzing data from a university hospital, we revealed how survival curves change when a negative follow-up blood culture is confirmed. We also demonstrated confirmation of a negative culture is significantly associated with clinical outcomes: there was a more than three-fold increase in mortality risk (after adjusting for clinical background) if a negative blood culture was not confirmed within 14 days of the initial positive blood culture. Although we used data from only one university hospital, our novel approach and results will be a basis for future studies in several hospitals in Japan to provide statistical evidence of the clinical importance of confirming a negative follow-up blood culture in bacteremia patients, including those with MRSA infections. (C) 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jiac.2017.07.009

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Molecular epidemiology of Bordetella pertussis in Cambodia determined by direct genotyping of clinical specimens Reviewed

Takumi Moriuchi, Ork Vichit, Yong Vutthikol, Md. Shafiqul Hossain, Chham Samnang, Kohei Toda, Varja Grabovac, Yukihiro Hiramatsu, Nao Otsuka, Keigo Shibayama, Kazunari Kamachi

INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES   Vol. 62   page： 56 - 58   2017.9

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Objectives: This study sought to determine the genotypes of circulating Bordetella pertussis, the causative agent of pertussis, in Cambodia by direct molecular typing of clinical specimens.
Methods: DNA extracts from nasopharyngeal swabs obtained from 82 pertussis patients in 20082016 were analyzed by multilocus variable-number tandem repeat analysis (MLVA). B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter ptxP were also investigated by DNA sequence-based typing.
Results: Forty-four DNA extracts (54%) yielded a complete MLVA profile, and these were sorted into 8 MLVA types (MT18, MT26, MT27, MT29, MT43, MT72, MT95, and MT200). MT27 and MT29, which are common in developed countries, were the predominant strain types (total 73%). The predominant profile of virulence-associated allelic genes was the combination of ptxP3/ptxA1/prn2/fim3A (48%). MT27 strains were detected during the entire study period, whereas MT29 strains were only found in 2014-2016.
Conclusions: The B. pertussis population in Cambodia, where a whole-cell pertussis vaccine (WCV) has been continuously used, resembled those observed previously in developed countries where acellular pertussis vaccines are used. Circulating B. pertussis strains in Cambodia were distinct from those in other countries using WCVs. (C) 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

DOI： 10.1016/j.ijid.2017.07.015

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Analysis of multidrug resistant group B streptococci with reduced penicillin susceptibility forming small, less hemolytic colonies Reviewed

Hirotsugu Banno, Kouji Kimura, Yosuke Tanaka, Tsuyoshi Sekizuka, Makoto Kuroda, Wanchun Jin, Jun-ichi Wachino, Keiko Yamada, Keigo Shibayama, Yoshichika Arakawa

PLOS ONE   Vol. 12 ( 8 ) page： e0183453   2017.8

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Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for elderly adults. Until now, nearly all GBS with reduced penicillin susceptibility (PRGBS) have shown beta-hemolytic activity and grow on sheep blood agar. However, we have previously reported three PRGBS clinical isolates harboring a CylK deletion that form small less hemolytic colonies. In this study, we examined the causes of small, less hemolytic colony formation in these clinical isolates. Isogenic strains were sequenced to identify the mutation related to a small colony size. We identified a 276_277insG nucleic acid insertion in the thiamin pyrophosphokinase (tpk) gene, resulting in premature termination at amino acid 103 in TPK, as a candidate mutation responsible for small colony formation. The recombinant strain Delta tpk, which harbored the 276_277insG insertion in the tpk gene, showed small colony formation. The recombinant strain.cylK, which harbored the G379T substitution in cylK, showed a reduction in hemolytic activity. The phenotypes of both recombinant strains were complemented by the expression of intact TPK or CylK, respectively. Moreover, the use of Rapid ID 32 API and VITEK MS to identify strains as GBS was evaluated clinical isolates and recombinant strains. VITEK MS, but not Rapid ID 32 API, was able to accurately identify the strains as GBS. In conclusion, we determined that mutations in tpk and cylK caused small colonies and reduced hemolytic activity, respectively, and characterized the clinical isolates in detail.

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A high seroprevalence of antibodies to pertussis toxin among Japanese adults: Qualitative and quantitative analyses Reviewed

Takumi Moriuchi, Nao Otsuka, Yukihiro Hiramatsu, Keigo Shibayama, Kazunari Kamachi

PLOS ONE   Vol. 12 ( 7 ) page： e0181181   2017.7

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In 2013, national serosurveillance detected a high seroprevalence of antibodies to pertussis toxin ( PT) from Bordetella pertussis among Japanese adults. Thus, we aimed to determine the cause(s) of this high seroprevalence, and analyzed the titers of antibodies to PT and filamentous hemagglutinin (FHA) among adults (35-44 years old), young children (4-7 years old), and older children (10-14 years old). Our quantitative analyses revealed that adults had higher seroprevalences of anti-PT IgG and PT-neutralizing antibodies, and similar titers of anti-FHA IgG, compared to the young and older children. Positive correlations were observed between the titers of PT-neutralizing antibodies and anti-PT IgG in all age groups (rs values of 0.326-0.522), although the correlation tended to decrease with age. The ratio of PT-neutralizing antibodies to anti-PT IgG was significantly different when we compared the serum and purified IgG fractions among adults (p = 0.016), although this result was not observed among young and older children. Thus, it appears that some adults had non-IgG immunoglobulins to PT. Our analyses also revealed that adults had high-avidity anti-PT IgG ( avidity index: 63.5%, similar results were observed among the children); however, the adults had lower-avidity anti-FHA IgG (37.9%, p &lt; 0.05). It is possible that low-avidity antiFHA IgG is related to infection with other respiratory pathogens (e.g., Bordetella parapertussis, Haemophilus influenzae, or Mycoplasma pneumoniae), which produces antibodies to FHA-like proteins. Our observations suggest that these adults had been infected with B. pertussis and other pathogen(s) during their adulthood.

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Antibiotic Susceptibilities and Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Stools of Pediatric Diarrhea Patients in Surabaya, Indonesia Reviewed

Eddy Bagus Wasitou, Katsumi Shigemura, Kayo Osawa, Alpha Fardah, Akiho Kanaida, Dadik Raharjo, K. Kuntaman, Usman Hadi, Sugeng Harijono, Subijanto Marto Sudarmo, Tatsuya Nakamura, Keigo Shibayama, Masato Fujisawa, Toshiro Shirakawa

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 70 ( 4 ) page： 378 - 382   2017.7

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The purpose of this study was to investigate extended-spectrum beta-lactamase (ES-BL)-producing Escherichia coli isolates from pediatric (aged 0 to 3 years) diarrhea patients in Surabaya, Indonesia, where this kind of survey is rare; our study included assessment of their antibiotic susceptibilides, as well as ESBL typing, multilocus sequence typing (MLST), and diarrheagenic E. coli (DEC)-typing. ESBL-producing E. coli were detected in 18.8% of all the samples. Many ESBL-producing E. coli had significantly lower susceptibility to gentamicin (p &lt; 0.0001) and the quinolones nalidixic acid (p = 0.004) and ciprofloxacin (p &lt; 0.0001) than non-producers. In ESBL-producing E. coli, 84.0% of strains expressed CTX-M-15 alone or in combination with other ESBL types. MLST revealed that 24.0% of ESBL-producers had sequence type 617, all of which expressed the CTX-M-15 gene; we also detected expression of 3 DEC-related genes: 2 enteroaggregative E. coli genes and 1 enteropathogenic E. coli gene. In conclusion, CTX-M-15-type ESBL-producing E. coli ST617 appear to have spread to Indonesia.

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Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis Reviewed

Stefano Donini, Silvia Garavaglia, Davide M. Ferraris, Riccardo Miggiano, Shigetarou Mori, Keigo Shibayama, Menico Rizzi

PLOS ONE   Vol. 12 ( 4 ) page： e0175815   2017.4

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Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.

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Significant Decrease in Pertactin-Deficient Bordetella pertussis Isolates, Japan Reviewed

Yukihiro Hiramatsu, Yusuke Miyaji, Nao Otsuka, Yoshichika Arakawa, Keigo Shibayama, Kazunari Kamachi

EMERGING INFECTIOUS DISEASES   Vol. 23 ( 4 ) page： 699 - 701   2017.4

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Prevalence of pertactin-lacking Bordetella pertussis isolates has been observed worldwide. In Japan, however, we found that the frequency of pertactin-deficient isolates in 2014-2016 (8%) was significantly lower than the frequency in 2005-2007 (41%), 2008-2010 (35%), and 2011-2013 (25%). This reduction was closely associated with changes in genotypes.

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Reduction in chlorhexidine efficacy against multi-drugresistant Acinetobacter baumannii international clone II Reviewed

M. Hayashi, K. Kawamura, M. Matsui, M. Suzuki, S. Suzuki, K. Shibayama, Y. Arakawa

JOURNAL OF HOSPITAL INFECTION   Vol. 95 ( 3 ) page： 318 - 323   2017.3

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Background: Nosocomial infections caused by Acinetobacter baumannii international clone II (IC II) can cause severe clinical outcomes. Aim: Differential evaluation of bactericidal efficacy of chlorhexidine gluconate (CHX) and benzethonium chloride (BZT) disinfectants against IC II and non-IC II isolates.
Methods: Minimum inhibitory concentrations (MICs) of CHX and BZT were determined for 137 A. baumannii IC II, 99 non-IC II and 69 non-baumannii isolates, further classified according to MIC values into disinfectant-reduced susceptible (DRS) and disinfectantsusceptible (DS) groups. Time-kill curves and minimum bactericidal concentrations (MBCs) were evaluated for representative isolates in each group.
Results: CHX and BZT MIC(90)s for IC II isolates were 100 and 175 mg/L, respectively, but those for non-IC II and non-baumannii isolates were &lt; 100 mg/L. Nevertheless, time-kill curves indicated that CHX and BZT reduced live bacterial cell number by 5 log(10) for IC II and non-IC II isolates within 30 s when used at 1000 mg/L, comparable to practical use concentrations. CHX MBC at 30 s was 1000 mg/L for IC II and non-IC II isolates, and was not influenced by addition of 3% bovine serum albumin (BSA); BZT MBC at 30 s was 100 mg/L without BSA and increased up to 500 mg/L upon addition of BSA. No significant differences in BSA were found between DRS and DS isolates.
Conclusion: CHX and BZT were effective against Acinetobacter spp. including IC II at a concentration of 1000 mg/L and exposure for at least 30 s, but their concentrations should be considered carefully to ensure sufficient effects in both clinical and healthcare settings.(C) 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

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Emergence of New Delhi metallo-beta-lactamase 1 and other carbapenemase-producing Acinetobacter calcoaceticus-baumannii complex among patients in hospitals in Ha Noi, Viet Nam Reviewed

D. N. Tran, H. H. Tran, M. Matsui, M. Suzuki, S. Suzuki, K. Shibayama, T. D. Pham, T. T. Van Phuong, D. A. Dang, H. S. Trinh, C. T. Loan, L. T. V. Nga, H. R. Van Doorn, H. F. L. Wertheim

EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY & INFECTIOUS DISEASES   Vol. 36 ( 2 ) page： 219 - 225   2017.2

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Acinetobacter baumannii is an important cause of multidrug-resistant hospital acquired infections in the world. Here, we investigate the presence of NDM-1 and other carbapenemases among carbapenem-resistant A. baumannii isolated between August 2010 and December 2014 from three large hospitals in Hanoi, Vietnam. We identified 23/582 isolates (4 %) (11 from hospital A, five from hospital B, and seven from hospital C) that were NDM-1 positive, and among them 18 carried additional carbapenemase genes, including seven isolates carrying NDM-1, IMP-1, and OXA-58 with high MICs for carbapenems. Genotyping indicated that NDM-1 carrying A. baumannii have expanded clonally in these hospitals. Five new STs (ST1135, ST1136, ST1137, ST1138, and ST1139) were identified. One isolate carried NDM-1 on a plasmid belonging to the N-repA replicon type; no NDM-1-positive plasmids were identified in the other isolates. We have shown the extent of the carbapenem resistance and the local clonal spread of A. baumannii carrying NDM-1 in these hospitals; coexistence of NDM-1 and IMP-1 is reported for the first time from Vietnam here, and this will further seriously limit future therapeutic options.

DOI： 10.1007/s10096-016-2784-8

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Evaluation of a commercial loop-mediated isothermal amplification assay for diagnosis of Bordetella pertussis infection Reviewed

Kazunari Kamachi, Takumi Moriuchi, Yukihiro Hiramatsu, Nao Otsuka, Keigo Shibayama

JOURNAL OF MICROBIOLOGICAL METHODS   Vol. 133   page： 20 - 22   2017.2

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We evaluated a commercial loop-mediated isothermal amplification (LAMP) assay kit for Bordetella pertussis detection. The LAMP primers were designed to target the pbcP1 allele of the pertussis toxin promoter, but the assay could detect B. pertussis ptxP3 and ptxP8 strains in addition to ptxPl strains, with high analytical sensitivity. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mimet.2016.12.009

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Inter- and intra-strain variability of tandem repeats in Mycoplasma pneumoniae based on next-generation sequencing data Reviewed

Jing Zhang, Xiaohong Song, Marella J. Ma, Li Xiao, Tsuyoshi Kenri, Hongmei Sun, Travis Ptacek, Shaoli Li, Ken B. Waites, T. Prescott Atkinson, Keigo Shibayama, Kevin Dybvig, Yanmei Feng

FUTURE MICROBIOLOGY   Vol. 12 ( 2 ) page： 119 - 129   2017.2

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Aim: To characterize inter-and intra-strain variability of variable-number tandem repeats (VNTRs) in Mycoplasma pneumoniae to determine the optimal multilocus VNTR analysis scheme for improved strain typing. Methods: Whole genome assemblies and next-generation sequencing data from diverse M. pneumoniae isolates were used to characterize VNTRs and their variability, and to compare the strain discriminability of new VNTR and existing markers. Results: We identified 13 VNTRs including five reported previously. These VNTRs displayed different levels of inter-and intra-strain copy number variations. All new markers showed similar or higher discriminability compared with existing VNTR markers and the P1 typing system. Conclusion: Our study provides novel insights into VNTR variations and potential new multilocus VNTR analysis schemes for improved genotyping of M. pneumoniae.

DOI： 10.2217/fmb-2016-0111

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Utilizing the Carba NP test as an indicator of expression level of carbapenemase genes in Enterobacteriaceae Reviewed

Takaya Segawa, Mari Matsui, Masato Suzuki, Atsuko Tsutsui, Makoto Kuroda, Keigo Shibayama, Satowa Suzuki

JOURNAL OF MICROBIOLOGICAL METHODS   Vol. 133   page： 35 - 39   2017.2

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The Carba NP test was developed to detect carbapenemase-producing Enterobacteriaceae, and uses imipenem as the reaction substrate. In Japan, IMP-6 metallo-beta-lactamase (MBL) producers, which are usually resistant to meropenem but susceptible to imipenem, and IMP-1 MBL producers, which are usually resistant to both carbapenems are prevalent. We performed the Carba NP test with IMP-6 and IMP-1 MBL producers, and both types were detected by the Carba NP test with high sensitivity. All IMP-1 MBL producers were detected by the Carba NP test, but the minimum inhibitory concentrations (MICs) of imipenem varied from 0.25 to &gt;32 mu g/mL, and the time to positivity varied from 0 to 30 min. Time to positivity was significantly correlated with expression levels of bla(IMP-1), but not with MICs of imipenem. These results suggested that the Carba NP test can be used as a screening assay for carbapenemase gene expression levels among producers of the same type of carbapenemase. Using this approach, it is possible to determine whether the carbapenem resistance of each carbapenemase-producing Enterobacteriaceae isolate is primarily due to carbapenemase production, or to another mechanism such as outer membrane impermeability. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mimet.2016.12.015

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The proline residue at position 319 of BvgS is essential for BvgAS activation in Bordetella pertussis Reviewed

Yukihiro Hiramatsu, Shuji Yoshino, Yoshiko Yamamura, Nao Otsuka, Keigo Shibayama, Mineo Watanabe, Kazunari Kamachi

PATHOGENS AND DISEASE   Vol. 75 ( 1 )   2017.1

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Bordetella pertussis is the etiological agent of pertussis and produces various virulence factors, including pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN), most of which are positively regulated by the BvgAS two-component sensory transduction system. Here, we describe a B. pertussis isolate not expressing PT, FHA and PRN recovered from a pertussis patient. Sequencing revealed that the bvgS gene of this isolate contains a spontaneous mutation (C&gt; A at position 955) causing the proline residue at position 319 of the BvgS protein to be substituted by threonine. Moreover, loss of PT, FHA and PRN expression was completely restored by complementation with a wild-type bvgAS locus, indicating that this non-synonymous substitution in bvgS leads to impaired BvgS function. Our findings indicate that the proline residue at position 319 in this protein plays an essential role in activation of the BvgAS system and, therefore, subsequent expression of Bvg-regulated virulence factors in B. pertussis.

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Complete genome sequences of the p1 gene type 2b and 2c strains Mycoplasma pneumoniae KCH-402 and KCH-405 Reviewed

Tsuyoshi Kenri, Masato Suzuki, Atsuko Horino, Tsuyoshi Sekizuka, Makoto Kuroda, Hiroyuki Fujii, Toru Hashimoto, Hiroshi Nakajima, Hitomi Ohya, Keigo Shibayama

Genome Announcements   Vol. 5 ( 24 )   2017

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Here, we present the complete genome sequences of Mycoplasma pneumoniae KCH-402 and KCH-405, which are p1 gene type 2b and 2c strains, respectively. These strains harbor variations in the orf6 gene, which encodes the cytadherence-related proteins P40 and P90.

DOI： 10.1128/genomeA.00513-17

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Genome sequence of Clostridium botulinum strain Adk2012 associated with a foodborne botulinum case in Tottori, Japan, in 2012 Reviewed

Hiroshi Asakura, Shiori Yamamoto, Yoshika Momose, Haru Kato, Masaaki Iwaki, Keigo Shibayama

Genome Announcements   Vol. 5 ( 34 )   2017

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We report here a draft genome sequence of Clostridium botulinum Adk2012 responsible for a foodborne botulism case that occurred in Tottori, Japan, in 2012. Its genome size was 2,904,173 bp, with 46 rRNAs and 54 tRNAs, at a coverage of 14.5X.

DOI： 10.1128/genomeA.00872-17

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Mycobacterium avium infection induces the resistance of the interferon-γ response in mouse spleen cells at late stages of infection Reviewed International journal Open Access

Atsuko Masumi, Keiko Mochida, Kazuya Takizawa, Takuo Mizukami, Madoka Kuramitsu, Momoka Tsuruhara, Shigetarou Mori, Keigo Shibayama, Kazunari Yamaguchi, Isao Hamaguchi

Inflammation and Regeneration   Vol. 36 ( 1 ) page： 21 - 21   2016.12

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Background: Bacterial infections cause an increase in the population of hematopoietic stem cells (HSCs). To investigate the downstream factors associated with hematopoietic stem cells, mice are infected with Mycobacterium avium (M. avium). Results: Mycobacterium avium (M. avium) infection induces the enlargement of the spleen and changes in histopathology, including changes to the lineage populations. A dramatic expansion of Lin-c-kit+Sca-1+ (KSL) cells in mouse bone marrow cells and spleen cells was detected 4 weeks after infection with M. avium; however, there was no difference in the engraft activity between infected and un-infected mouse bone marrow cells. We tested the cytokine and cytokine-related gene expression after M. avium infection and found that IFN-γ expression increased and peaked at 4 weeks in both bone marrow and spleen cells. The expression of Sca-1 gene peaked at 4 weeks in the bone marrow but peaked at 2 weeks in spleen cells, although the Sca-1 surface marker peaked at 4 weeks after infection in both bone marrow and spleen cells. Interferon regulatory factor-2 (IRF-2) expression did not change in the bone marrow cells, whereas it decreased in spleen cells at 4 weeks and IRF-1 expression was up-regulated in both bone marrow and spleen cells after infection. However, the up-regulation of IRF-1 was not correlated with IFN-γ expression in the M. avium-infected mouse spleen cells. Conclusions: This finding suggests that the IFN-γ production mediated by M. avium infection alters the population of KSL cells during host defense, and the down-regulation of the IFN-γ response in spleen cells occurs at the late stage after M. avium infection.

DOI： 10.1186/s41232-016-0024-3

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A genome-wide association study identifies a horizontally transferred bacterial surface adhesin gene associated with antimicrobial resistant strains Reviewed

Masato Suzuki, Keigo Shibayama, Koji Yahara

SCIENTIFIC REPORTS   Vol. 6   page： 37811   2016.11

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Carbapenems are a class of last-resort antibiotics; thus, the increase in bacterial carbapenem-resistance is a serious public health threat. Acinetobacter baumannii is one of the microorganisms that can acquire carbapenem-resistance; it causes severe nosocomial infection, and is notoriously difficult to control in hospitals. Recently, a machine-learning approach was first used to analyze the genome sequences of hundreds of susceptible and resistant A. baumannii strains, including those carrying commonly acquired resistant mechanisms, to build a classifier that can predict strain resistance. A complementary approach is to explore novel genetic elements that could be associated with the antimicrobial resistance of strains, independent of known mechanisms. Therefore, we carefully selected A. baumannii strains, spanning various genotypes, from public genome databases, and conducted the first genome-wide association study (GWAS) of carbapenem resistance. We employed a recently developed method, capable of identifying any kind of genetic variation and accounting for bacterial population structure, and evaluated its effectiveness. Our study identified a surface adhesin gene that had been horizontally transferred to an ancestral branch of A. baumannii, as well as a specific region of that gene that appeared to accumulate multiple individual variations across the different branches of carbapenem-resistant A. baumannii strains.

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gamma-Glutamyltranspeptidase is an endogenous activator of Toll-like receptor 4-mediated osteoclastogenesis Reviewed

Sawako Moriwaki, Takeshi Into, Keiko Suzuki, Mutsumi Miyauchi, Takashi Takata, Keigo Shibayama, Shumpei Niida

SCIENTIFIC REPORTS   Vol. 6   page： 35930   2016.10

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Chronic inflammation-associated bone destruction, which is observed in rheumatoid arthritis (RA) and periodontitis, is mediated by excessive osteoclastogenesis. We showed previously that gamma-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, acts as an endogenous activator of such pathological osteoclastogenesis, independent of its enzymatic activity. GGT accumulation is clinically observed in the joints of RA patients, and, in animals, the administration of recombinant GGT to the gingival sulcus as an in vivo periodontitis model induces an increase in the number of osteoclasts. However, the underlying mechanisms of this process remain unclear. Here, we report that Toll-like receptor 4 (TLR4) recognizes GGT to activate inflammation-associated osteoclastogenesis. Unlike lipopolysaccharide, GGT is sensitive to proteinase K treatment and insensitive to polymyxin B treatment. TLR4 deficiency abrogates GGT-induced osteoclastogenesis and activation of NF-kappa B and MAPK signaling in precursor cells. Additionally, GGT does not induce osteoclastogenesis in cells lacking the signaling adaptor MyD88. The administration of GGT to the gingival sulcus induces increased osteoclastogenesis in wild-type mice, but does not induce it in TLR4-deficient mice. Our findings elucidate a novel mechanism of inflammation-associated osteoclastogenesis, which involves TLR4 recognition of GGT and subsequent activation of MyD88-dependent signaling.

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Draft Genome Sequence of Helicobacter suis Strain SNTW101, Isolated from a Japanese Patient with Nodular Gastritis. Reviewed International journal

Hidenori Matsui, Tetsufumi Takahashi, Somay Y Murayama, Ikuo Uchiyama, Katsushi Yamaguchi, Shuji Shigenobu, Masato Suzuki, Emiko Rimbara, Keigo Shibayama, Anders Øverby, Masahiko Nakamura

Genome announcements   Vol. 4 ( 5 ) page： e00934   2016.9

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We present here the draft whole-genome shotgun sequence of an uncultivated strain SNTW101 of Helicobacter suis, which has been maintained in the stomachs of mice. This strain was originally isolated from gastric biopsy specimens of a urea breath test-negative Japanese patient suffering from nodular gastritis.

DOI： 10.1128/genomeA.00934-16

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Pelvic abscess due to <i>Mycoplasma hominis</i> following caesarean section. Reviewed International journal

Mori N, Takigawa A, Kagawa N, Kenri T, Yoshida S, Shibayama K, Aoki Y

JMM case reports   Vol. 3 ( 4 ) page： e005059   2016.8

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INTRODUCTION: Mycoplasma hominis is associated with genito-urinary tract infection and adverse pregnancy outcomes. However, whether the species is a true pathogen or part of the genito-urinary tracts natural flora remains unclear. CASE PRESENTATION: A 41-year-old pregnant woman was admitted to our hospital at 38 weeks and 5 days of gestation owing to premature rupture of the membranes. The patient delivered by caesarean section. Subsequently, the patient complained of lower abdominal pain and had persistent fever. Enhanced computed tomography revealed pelvic abscesses. Gram staining of pus from the abscess and vaginal secretions indicated presence of polymorphonuclear leucocytes but no pathogens. Cultures on blood agar showed growth of pinpoint-sized colonies in an anaerobic environment within 48 h. Although administration of carbapenem and metronidazole was ineffective and we could not fully drain the abscess, administration of clindamycin led to clinical improvement. The isolates 16S rRNA gene and yidC gene sequences exhibited identity with those of M. hominis. CONCLUSION: Physicians should consider M. hominis in cases of pelvic abscesses where Gram staining yields negative results, small colonies are isolated from the abscess and treatment with β-lactam antibiotics is ineffective.

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Correction for Rimbara et al., Draft Genome Sequence of Helicobacter fennelliae Strain MRY12-0050, Isolated from a Bacteremia Patient. Reviewed International journal

Emiko Rimbara, Mari Matsui, Shigetarou Mori, Satowa Suzuki, Masato Suzuki, Hyun Kim, Tsuyoshi Sekizuka, Makoto Kuroda, Keigo Shibayama

Genome announcements   Vol. 4 ( 4 )   2016.7

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DOI： 10.1128/genomeA.00634-16

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BipA Is Associated with Preventing Autoagglutination and Promoting Biofilm Formation in Bordetella holmesii Reviewed

Yukihiro Hiramatsu, Momoko Saito, Nao Otsuka, Eri Suzuki, Mineo Watanabe, Keigo Shibayama, Kazunari Kamachi

PLOS ONE   Vol. 11 ( 7 ) page： e0159999   2016.7

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Bordetella holmesii causes both invasive and respiratory diseases in humans. Although the number of cases of pertussis-like respiratory illnesses due to B. holmesii infection has increased in the last decade worldwide, little is known about the virulence factors of the organism. Here, we analyzed a B. holmesii isolate that forms large aggregates and precipitates in suspension, and subsequently demonstrated that the autoagglutinating isolate is deficient in Bordetella intermediate protein A (BipA) and that this deletion is caused by a frame-shift mutation in the bipA gene. A BipA-deficient mutant generated by homologous recombination also exhibited the autoagglutination phenotype. Moreover, the BipA mutant adhered poorly to an abiotic surface and failed to form biofilms, as did two other B. holmesii autoagglutinating strains, ATCC 51541 and ATCC 700053, which exhibit transcriptional down-regulation of bipA gene expression, indicating that autoagglutination indirectly inhibits biofilm formation. In a mouse intranasal infection model, the BipA mutant showed significantly lower levels of initial lung colonization than did the parental strain (P&lt;0.01), suggesting that BipA might be a critical virulence factor in B. holmesii respiratory infection. Together, our findings suggest that BipA production plays an essential role in preventing autoagglutination and indirectly promoting biofilm formation by B. holmesii.

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A Novel IgM-capture enzyme-linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection Reviewed

Nao Otsuka, Kensei Gotoh, Naoko Nishimura, Takao Ozaki, Yukitsugu Nakamura, Kiyohito Haga, Makoto Yamazaki, Fumio Gondaira, Kenji Okada, Yusuke Miyaji, Hiromi Toyoizumi-Ajisaka, Keigo Shibayama, Yoshichika Arakawa, Kazunari Kamachi

MICROBIOLOGY AND IMMUNOLOGY   Vol. 60 ( 5 ) page： 326 - 333   2016.5

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An ELISA that measures anti-PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti-Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P &lt; 0.001). ROC analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.

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A Epidemiological Investigation of a Nosocomial Outbreak of Multidrug-Resistant Acinetobacter baumannii in a Critical Care Center in Japan, 2011-2012 Reviewed

Hiroto Ushizawa, Yuichiro Yahata, Takeo Endo, Tomoko Iwashima, Michiyo Misawa, Makoto Sonobe, Takuya Yamagishi, Hajime Kamiya, Kazutoshi Nakashima, Tamano Matsui, Mari Matsui, Satowa Suzuki, Keigo Shibayama, Mikio Doi, Fujiko Irie, Shinichi Yamato, Yasuhiro Otomo, Kazunori Oishi

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 69 ( 2 ) page： 143 - 148   2016.3

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In 2011, a multidrug-resistant Acinetobacter baumannii (MDRAB) outbreak occurred at a Japanese critical care center (CCC) in a tertiary care hospital. Multidrug-resistance in Japan is defined as resistance to the antimicrobials amikacin, carbapenem, and fluoroquinolone. We conducted a retrospective epidemiological investigation of this outbreak to identify the risk factors for MDRAB respiratory tract acquisition in this hospital. Cases were defined as hospitalized patients with MDRAB-positive cultures at least 3 days post admission to the CCC between June 1, 2011 and April 20, 2012. Fifteen MDRAB cases were identified, including 3 with infection and 12 with colonization. This case-control study demonstrated that hypoalbuminemia along with carbapenem administration were associated with MDRAB respiratory tract acquisition. Pulsed-field gel electrophoresis analysis and multilocus sequence typing using MDRAB isolates suggested a clonal dissemination of MDRAB strains with sequence type 74 occurred primarily among patients admitted to the CCC. From April 16, 2012, a decreased isolation rate of MDRAB in the hospital occurred after the implementation of the following infection control measures: closing the emergency room, discontinuing admission to the CCC, isolating patients with MDRAB colonization or infection to single room status, and conducting environmental cleaning. No MDRAB cases were detected between March 23 and April 20, 2012.

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A novel experimental platform for toxigenic and non-toxigenic Corynebacterium ulcerans infection in mice Reviewed

Yu Mochizuki, Honami Saeki, Masaaki Iwaki, Hitrotaka Takagi, Keigo Shibayama, Hiromi Amao, Akihiko Yamamoto

PATHOGENS AND DISEASE   Vol. 74 ( 2 )   2016.3

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Corynebacterium ulcerans is a zoonotic pathogen that can produce diphtheria toxin and causes an illness categorized as diphtheria in the European Union because its clinical appearance is similar to that of diphtheria caused by Corynebacterium diphtheriae. Despite the importance of the pathogen in public health, the organism's mechanism of infection has not been extensively studied, especially in experimental animal models. Therefore in the present study we constructed an intranasal infection system for mice. Mice are insensitive to diphtheria toxin and this has the advantage of excluding the cytotoxic effect of the toxin that might interfere with the analysis of the early stage of infection. Both the toxigenic and non-toxigenic C. ulcerans strains were capable of killing mice within 3 days after inoculation at 107 colony-forming units per mouse. In experimentally infected animals, C. ulcerans was detected in the respiratory tract but not in the intestinal tract. The bacterium was also detected in peripheral blood and it disseminated into the lung, kidney and spleen to produce a systemic infection. This experimental infection system provides a platform for analyzing the virulence of C. ulcerans in future studies.

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Linezolid-resistant Staphylococcus epidermidis associated with long-term, repeated linezolid use in a pediatric patient Reviewed

Naruhiko Ishiwada, Akiko Takaya, Asahi Kimura, Masaharu Watanabe, Moeko Hino, Hidemasa Ochiai, Mari Matsui, Keigo Shibayama, Tomoko Yamamoto

JOURNAL OF INFECTION AND CHEMOTHERAPY   Vol. 22 ( 3 ) page： 187 - 190   2016.3

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We report an 8-year-old patient with catheter-related bacteremia caused by linezolid-resistant Staphylococcus epidermidis that was isolated after the long-term, repeated use of linezolid. Three S. epidermidis strains isolated from this patient were bacteriologically analyzed. While the strain isolated prior to linezolid initiation was susceptible to linezolid, two strains after linezolid therapy displayed low-level linezolid susceptibility (MIC, 4 mg/L) and linezolid resistance (MIC, 16 mg/L). T2500A mutation in two copies and G2575T mutations in three copies of 23S rRNA were detected in the low-susceptible strain and the resistant strain, respectively. Linezolid-resistant S. epidermidis infection is rare, but may occur with the long-term administration of linezolid. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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Analyses of short-term antagonistic evolution of Pseudomonas Aeruginosa strain PAO1 and phage KPP22 (Myoviridae family, PB1-like virus genus) Reviewed

Jumpei Uchiyama, Masato Suzuki, Koji Nishifuji, Shin ichiro Kato, Reina Miyata, Tadahiro Nasukawa, Kotoe Yamaguchi, Iyo Takemura-Uchiyama, Takako Ujihara, Hidekatsu Shimakura, Hironobu Murakami, Noriaki Okamoto, Yoshihiko Sakaguchi, Keigo Shibayama, Masahiro Sakaguchi, Shigenobu Matsuzaki

Applied and Environmental Microbiology   Vol. 82 ( 15 ) page： 4482 - 4491   2016

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© 2016, American Society for Microbiology. Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086. The information obtained in this study should be useful for further development of safe and efficient phage therapy.

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A Epidemiological Investigation of a Nosocomial Outbreak of Multidrug-Resistant <i>Acinetobacter baumannii</i> in a Critical Care Center in Japan, 2011&ndash;2012 Reviewed

Ushizawa Hiroto, Matsui Tamano, Matsui Mari, Suzuki Satowa, Shibayama Keigo, Doi Mikio, Irie Fujiko, Yamato Shinichi, Otomo Yasuhiro, Oishi Kazunori, Yahata Yuichiro, Endo Takeo, Iwashima Tomoko, Misawa Michiyo, Sonobe Makoto, Yamagishi Takuya, Kamiya Hajime, Nakashima Kazutoshi

Japanese Journal of Infectious Diseases   Vol. 69 ( 4 ) page： 356 - 356   2016

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Volume 69, no. 2, p. 143&ndash;148, 2016. Page 143, Title should appear as shown below.<br><br>An Epidemiological Investigation of a Nosocomial Outbreak of Multidrug-Resistant <i>Acinetobacter baumannii</i> in a Critical Care Center in Japan, 2011&ndash;2012

DOI： 10.7883/yoken.JJID.2016.E002

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Genome sequence of an Acinetobacter baumannii strain carrying three acquired carbapenemase genes Reviewed

Ken-Ichi Oinuma, Masato Suzuki, Kanako Sato, Kiyotaka Nakaie, Makoto Niki, Etsuko Takizawa, Mamiko Niki, Keigo Shibayama, Koichi Yamada, Hiroshi Kakeya, Yukihiro Kaneko

Genome Announcements   Vol. 4 ( 6 ) page： e01290-16   2016

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The emergence of multiple-carbapenemase-producing Acinetobacter strains has been a serious concern during the past decade. Here, we report the draft genome sequence of an Acinetobacter baumannii strain isolated from a Japanese patient with three acquired carbapenemase genes: blaNDM-1, blaTMB-1, and blaOXA-58.

DOI： 10.1128/genomeA.01290-16

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Genome sequence of the psychrophilic bacterium Tenacibaculum ovolyticum strain da5A-8 isolated from deep seawater Reviewed

Maki Teramoto, Zhenyu Zhai, Ayumi Komatsu, Keigo Shibayama, Masato Suzuki

Genome Announcements   Vol. 4 ( 3 )   2016

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Some bacterial species of the genus Tenacibaculum, including Tenacibaculum ovolyticum, have been known as fish pathogens in the sea. So far, the only published genome sequence for this genus is for Tenacibaculum dicentrarchi, which could also be a fish pathogen. Strain da5A-8, showing 100% identity to the 16S rRNA gene sequence of T. ovolyticum DSM 18103T, was isolated from seawater at a depth of 344min Kochi, Japan, and grew optimally at 10 to 20�C. The genome sequence of strain da5A-8 revealed the possible virulence genes commonly observed in the genus Tenacibaculum.

DOI： 10.1128/genomeA.00644-16

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Simple and rapid detection method for qepA1 by loop-mediated isothermal amplification Reviewed

YAMANE Kunikazu, HORINO Atsuko, SUZUKI Satowa, SHIBAYAMA Keigo, FUSHIMI Shigeko, KATSUYAMA Hironobu

Kawasaki medical journal   Vol. 42 ( 1 ) page： 25 - 29   2016

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Although fluoroquinolone (FQ) has been used for the treatment of various bacterial infectious diseases, its continued use has been problematic given the appearance of FQ-resistant bacteria. However, the recent discovery of four plasmid-mediated quinolone resistance (PMQR) genes comprising qnr, aac(6')Ib-cr, qepA and OqxAB since 1998 has provided insights in the area of FQ-resistance. For practical detection of qepA in microbiology laboratory, a specific, simple, rapid and cost-effective isothermal amplification method designated as LAMP is the good candidate to use. In this study, the development of a new detection method using LAMP to identify qepA1, one variant of the qepA gene, was tried. As the results, the LAMP method using a qepA1-specific LAMP primer set comprising five primerscould detect all four qepA1-positive strains in addition to 17 qepA1-negative strains. The LAMP method is clearly much more advantageous for use in clinical laboratories. Furthermore, the time and accuracy benefits allow for the selection of antibiotics in a clinical setting.

DOI： 10.11482/KMJ-E42(1)25

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Laboratory-based surveillance of pertussis using multitarget real-time PCR in Japan: Evidence for Bordetella pertussis infection in preteens and teens Reviewed

K. Kamachi, S. Yoshino, C. Katsukawa, N. Otsuka, Y. Hiramatsu, K. Shibayama

New Microbes and New Infections   Vol. 8   page： 70 - 74   2015.11

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Between January 2013 and December 2014, we conducted laboratory-based surveillance of pertussis using multitarget real-time PCR, which discriminates among Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii and Mycoplasma pneumoniae. Of 355 patients clinically diagnosed with pertussis in Japan, B. pertussis, B. parapertussis and M. pneumoniae were detected in 26% (n = 94), 1.1% (n = 4) and 0.6% (n = 2), respectively, whereas B. holmesii was not detected. It was confirmed that B. parapertussis and M. pneumoniae are also responsible for causing pertussis-like illness. The positive rates for B. pertussis ranged from 16% to 49%, depending on age. Infants aged ≤ 3 months had the highest rate (49%), and children aged 1 to 4 years had the lowest rate (16%, p &lt
0.01 vs. infants aged ≤ 3 months). Persons aged 10 to 14 and 15 to 19 years also showed high positive rates (29% each)
the positive rates were not statistically significant compared with that of infants aged ≤ 3 months (p ≥ 0.06). Our observations indicate that similar to infants, preteens and teens are at high risk of B. pertussis infection.

DOI： 10.1016/j.nmni.2015.10.001

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Predominance of PCR-ribotypes, 018 (smz) and 369 (trf) of Clostridium difficile in Japan: a potential relationship with other global circulating strains? Reviewed

Mitsutoshi Senoh, Haru Kato, Tadashi Fukuda, Akiko Niikawa, Yoshiko Hori, Hideharu Hagiya, Yoichiro Ito, Hiroshi Miki, Yoshifumi Abe, Kiyoshi Furuta, Hideki Takeuchi, Hirokazu Tajima, Harumi Tominaga, Hideyuki Satomura, Hideaki Kato, Sayuri Morita, Ai Tanada, Toshinori Hara, Miki Kawada, Yuka Sato, Masahiko Takahashi, Akiko Higuchi, Tomoko Nakajima, Yukiko Wakamatsu, Masahiro Toyokawa, Akiko Ueda, Paul Roberts, Fabio Miyajima, Keigo Shibayama

JOURNAL OF MEDICAL MICROBIOLOGY   Vol. 64 ( 10 ) page： 1226 - 1236   2015.10

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Global spread and evolutionary links of an epidemic Clostridium difficile strain (PCR-ribotype 027) have been noted in recent decades. However, in Japan, no outbreaks caused by type 027 have been reported to date. A total of 120 C. difficile isolates from patients at 15 hospitals during non-outbreak seasons between 2011 and 2013 as well as 18 and 21 isolates collected from two hospitals in 2010 and 2009, respectively, in outbreak periods in Japan, were examined. Among these 120 isolates, Japan-ribotypes smz and ysmz (subtype variant of smz) were the most predominant (39.2 %) followed by Japan-ribotype trf (15.8 %). Types smz/ysmz and trf were also concurrently predominant at two hospitals in the outbreak settings. Out of the five binary toxin-positive isolates observed, only one was PCR-ribotype 027 and another PCR-ribotype 078. Type smz was later found to correspond to PCR-ribotype 018. High rates of resistance against gatifloxacin, moxifloxacin, erythromycin and clindamycin were observed in the PCR-ribotype 018 isolates. Interestingly, all trf isolates were toxin A-negative, toxin B-positive, but they did not correspond to PCR-ribotype 017, thus being assigned a new ribotype (PCR-ribotype 369). In conclusion, PCR-ribotypes 018 (smz) and 369 (trf) were identified as major circulating strains in both outbreak and non-outbreak settings in Japan. Given their epidemiological relevance, molecular investigations are warranted to clarify potential evolutionary links with related strains found elsewhere, such as PCR-ribotypes 018 and 017 from Europe and North America.

DOI： 10.1099/jmm.0.000149

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Development of a novel chromogenic method, Penta-well test, for rapid prediction of beta(-)lactamase classes produced in clinical Enterobacteriaceae isolates Reviewed

Tatsuki Mura, Kumiko Kawamura, Jun-ichi Wachino, Keigo Shibayama, Yoshichika Arakawa

DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE   Vol. 83 ( 1 ) page： 25 - 29   2015.9

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We developed a novel chromogenic method, Penta-well test, which enables the rapid detection and classification of beta-lactamases in clinical Enterobacteriaceae isolates. This test is based on a combination of nitrocefin and 3 beta-lactamase inhibitors specific to classes A. B, and/or C, with nitrocefin hydrolysis by beta-lactamases being assessed by optical density measurements at 490 nm. When the cutoff value for each beta-lactamase class was determined (0.09, 0.4, and 0.55 for class A, class B, and class C beta-lactamase producers, respectively), the sensitivity and specificity of classification were 93.5% and 68.8% for class A, 93.8% and 100% for class B, and 86.7% and 100% for class C. respectively. Moreover, this method allowed accurate beta-lactamase classification in 20 of 23 (87.0%) isolates producing plural class of beta-lactamases. Thus, the Penta-well test can provide information that would be useful in the accurate detection and classification of beta-lactamases produced by causative bacteria. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.diagmicrobio.2015.06.002

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The determinant of periodicity in Mycoplasma pneumoniae incidence: an insight from mathematical modelling Reviewed

Ryosuke Omori, Yukihiko Nakata, Heidi L. Tessmer, Satowa Suzuki, Keigo Shibayama

SCIENTIFIC REPORTS   Vol. 5   page： 14473   2015.9

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Until the early 1990s, incidences of Mycoplasma pneumoniae (MP) infection showed three to five year epidemic cycles in multiple countries, however, the mechanism for the MP epidemic cycle has not been understood. Here, we investigate the determinant of periodicity in MP incidence by employing a mathematical model describing MP transmission dynamics. Three candidates for the determinant of periodicity were evaluated: school-term forcing, minor variance in the duration of immunity, and epidemiological interference between MP serotypes. We find that minor variation in the duration of immunity at the population level must be considered essential for the MP epidemic cycle because the MP cyclic incidence pattern did not replicate without it. Minor variation, in this case, is a less dispersed distribution for the duration of immunity than an exponential distribution. Various lengths of epidemic cycles, including cycles typically found in nature, e.g. three to five year cycles, were also observed when there was minor variance in the duration of immunity. The cyclic incidence pattern is robust even if there is epidemiological interference due to cross-immune protection, which is observed in the epidemiological data as negative correlation between epidemics per MP serotype.

DOI： 10.1038/srep14473

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The Other Helicobacters. Reviewed International journal

Bram Flahou, Emiko Rimbara, Shigetarou Mori, Freddy Haesebrouck, Keigo Shibayama

Helicobacter   Vol. 20 Suppl 1   page： 62 - 7   2015.9

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In the past year, a substantial number of (putative) novel Helicobacter species have been described, including Helicobacter himalayensis colonizing the Himalayan marmot and Helicobacter apodemus, colonizing the Korean striped field mouse. In addition, a putative novel gastric Helicobacter species was identified in wild gorillas and chimpanzees, for which the name "Candidatus H. homininae" was proposed. A high incidence of gastric non-H. pylori Helicobacter infection was described in China and multiple case reports have described the involvement of enterohepatic Helicobacter species, especially Helicobacter cinaedi, in a wide range of diseases. Several studies in rodent models further elucidated the mechanisms underlying the development of gastric mucosa-associated lymphoid tissue lymphoma during infection with gastric non-H. pylori Helicobacters. The effects of infection with gastric Helicobacters on the development of neuroinflammation were investigated and several enterohepatic Helicobacter species were shown to affect the composition of the gut microbiota, to influence vaccine efficiency as well as the progression of cancer in distant sites of the body.

DOI： 10.1111/hel.12259

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Purification and functional characterization of diadenosine 5',5‴-P(1),P(4)-tetraphosphate phosphorylases from Mycobacterium smegmatis and Mycobacterium avium. Reviewed International journal

Naoko Honda, Hyun Kim, Emiko Rimbara, Atsushi Kato, Keigo Shibayama, Shigetarou Mori

Protein expression and purification   Vol. 112   page： 37 - 42   2015.8

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We recently demonstrated that the Rv2613c protein from Mycobacterium tuberculosis H37Rv is a novel diadenosine 5',5‴-P(1),P(4)-tetraphosphate (Ap4A) phosphorylase (MtAPA) that forms a tetramer. Mycobacterium avium and Mycobacterium smegmatis express proteins named MAV_3489 and MSMEG_2932, respectively, that are homologous to MtAPA. Here we showed that the MAV_3489 and MSMEG_2932 proteins possess Ap4A phosphorylase activity and enzymatic properties similar to those of MtAPA. Furthermore, gel-filtration column chromatography revealed that MAV_3489 and MSMEG_2932 assembled into homotetramers in solution, indicating that they may also form unique Ap4A-binding sites composed of tetramers.

DOI： 10.1016/j.pep.2015.04.010

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Comparative Analysis of Penicillin-Susceptible and Non-Susceptible Isolates of Group B Streptococci by Multilocus Sequence Typing Reviewed

Ryoko Yamada, Kouji Kimura, Noriyuki Nagano, Yukiko Nagano, Satowa Suzuki, Wanchun Jin, Jun-ichi Wachino, Keiko Yamada, Keigo Shibayama, Yoshichika Arakawa

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 68 ( 4 ) page： 326 - 329   2015.7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL INST INFECTIOUS DISEASES  

Since Group B Streptococcus (GBS, Streptococcus agalactiae) clinical isolates are believed to be uniformly susceptible to beta-lactams, penicillin G has been used as the first-line agent for the prevention and treatment of GBS infections. However, the existence and characteristics of GBS isolates with reduced penicillin susceptibility (PRGBS) have recently been reported in Japan. Moreover, the sequence type (ST) 458 is predominant among the PRGBS in Japan. Although the majority of the PRGBS isolates in Japan have been recovered from respiratory specimens of adults, no information on the genotype of these isolates is available. Therefore, whether ST458 predominates among GBS isolates obtained from such specimens is not known. In this study, we characterized the STs of 38 penicillin-susceptible GBS isolates (PSGBS) recovered from respiratory specimens and compared them to the reported PRGBS STs. ST458, the predominant ST among the PRGBS isolates studied (10/19, 53%), was not found in the PSGBS isolates. Thirty-six PSGBS isolates belonged to the ST1/19/10 group (includes 6 different STs), and the remaining 2 isolates belonged to that of ST23. Further, the PRGBS isolates were divided into the ST1 (3 STs), and ST23 (2 STs) groups. ST458 was not predominant among the PSGBS isolates recovered from respiratory specimens in Japan and may therefore be specific to the PRGBS. Thus, the ST distribution of the PRGBS isolates does not reflect that of the PSGBS.

DOI： 10.7883/yoken.JJID.2014.387

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[A Study on the Clinical Course and Antimicrobial Susceptibility of Mycoplasma pneumoniae in a Community Hospital]. Reviewed

Sakai T, Ishida T, Arita M, Tachibana H, Yoshioka H, Noyama M, Tokioka F, Ito A, Furuta K, Nishiyama A, Hashimoto T, Fujii H, Nakajima H, Kenri T, Shibayama K

Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases   Vol. 89 ( 4 ) page： 458 - 464   2015.7

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Recently, reports of macrolide-resistant strains of <i>Mycoplasma pneumoniae </i>have been increasing. We examined the antimicrobial susceptibility and clinical significance in patients with <i>M. pneumoniae</i>. Seventy patients in whom <i>M. pneumoniae </i>was detected from 2008 to 2012 were included in the study, and compared with patients between 2003 and 2006. There were no macrolide-resistant strains detected in the 38 strains from 2003 to 2006, but from 2008 to 2012, out of the 70 strains 46 (65.7％) were positive for the macrolide resistant 23SrRNA gene mutant (A2063G), which is consistent with recent trends. Comparison between cases of macrolide resistant strains and those with macrolide sensitive strains did not reveal a significant difference in the hospitalization period. The approximate duration of fever was 7 days ; for both cases : those who received effective antimicrobials as the initial therapy, and for those with macrolide sensitive strains. It seems that the duration of fever depends on susceptibility to the initial antimicrobials regardless of macrolide resistance. There were some patients that improved without use of quinolone or minocycline, though macrolide resistant strains were detected. These patients did not reveal extension of the hospital stay nor aggravation of pneumonia. This suggests that a macrolide drug might be the first choice drug for <i>M. pneumoniae </i>even now, and a change of drug should be considered when fever duration is long.

DOI： 10.11150/kansenshogakuzasshi.89.458

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Common isolation of New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae in a large surgical hospital in Vietnam Reviewed

H. H. Tran, S. Ehsani, K. Shibayama, M. Matsui, S. Suzuki, M. B. Nguyen, D. N. Tran, V. P. Tran, D. L. Tran, H. T. Nguyen, D. A. Dang, H. S. Trinh, T. H. Nguyen, H. F. L. Wertheim

EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY & INFECTIOUS DISEASES   Vol. 34 ( 6 ) page： 1247 - 1254   2015.6

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This study sought to monitor the presence of carbapenem-resistant Enterobacteriaceae (CRE) and the proportion New Delhi metallo-beta-lactamase 1 (NDM-1)-producing bacteria between August 2010 and December 2012 in a surgical hospital in Vietnam. We identified 47 CRE strains from a total of 4,096 Enterobacteriaceae isolates (1.1 %) that were NDM-1-positive from 45 patients admitted to 11 different departments, with the majority being from the urology department. The NDM-1 gene was found in seven different species. Genotyping revealed limited clonality of NDM-1-positive isolates. Most of the isolates carried the NDM-1 gene on a plasmid and 17.8 % (8/45) of those were readily transferable. We found five patients at admission and one patient at discharge with NDM-1-positive bacteria in their stool. From 200 screening environmental hospital samples, five were confirmed to be NDM-1-positive and included Acinetobacter species (n = 3) and Enterobacter aerogenes (n = 2). The results reveal that NDM-1-producing Enterobacteriaceae are commonly isolated in patients admitted to a Vietnamese surgical hospital and are also detected in the hospital environment.

DOI： 10.1007/s10096-015-2345-6

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Molecular epidemiology of Bordetella pertussis in the Philippines in 2012-2014 Reviewed

Salvacion Rosario L. Galit, Nao Otsuka, Yuki Furuse, Daryl Joy V. Almonia, Lydia T. Sombrero, Rosario Z. Capeding, Socorro P. Lupisan, Mariko Saito, Hitoshi Oshitani, Yukihiro Hiramatsu, Keigo Shibayama, Kazunari Kamachi

INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES   Vol. 35   page： 24 - 26   2015.6

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Objectives: The present study was designed to determine the genotypes of circulating Bordetella pertussis in the Philippines by direct molecular typing of clinical specimens.
Methods: Nasopharyngeal swabs (NPSs) were collected from 50 children hospitalized with pertussis in three hospitals during 2012-2014. Multilocus variable-number tandem repeat analysis (MLVA) was performed on the DNA extracts from NPSs. B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter, ptxP, were also investigated by DNA sequence-based typing.
Results: Twenty-six DNA extracts yielded a complete MLVA profile, which were sorted into 10 MLVA types. MLVA type 34 (MT34), which is rare in Australia, Europe, Japan, and the USA, was the predominant strain (50%). Seven MTs (MT29, MT32, MT33, and MT283-286, total 42%) were single-locus variants of MT34, while two (MT141 and MT287, total 8%) were double-locus variants of MT34. All MTs had the combination of virulence-associated allelic genes, ptxP1-ptxA1-prn1-fim3A.
Conclusions: The B. pertussis population in the Philippines comprises genetically related strains. These strains are markedly different from those found in patients from other countries where acellular pertussis vaccines are used. The differences in vaccine types between these other countries and the Philippines, where the whole-cell vaccine is still used, may select for distinct populations of B. pertussis. (C) 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

DOI： 10.1016/j.ijid.2015.04.001

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Genome Sequence of a Carbapenem-Resistant Strain of Ralstonia mannitolilytica. Reviewed International journal

Masato Suzuki, Hisaaki Nishio, Kohsuke Asagoe, Kaneyuki Kida, Satowa Suzuki, Mari Matsui, Keigo Shibayama

Genome announcements   Vol. 3 ( 3 )   2015.5

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Ralstonia mannitolilytica, a Gram-negative aerobic bacterium, is an opportunistic human pathogen that is becoming more common in cases of nosocomial infections. We report for the first time the whole-genome sequence analysis of R. mannitolilytica strain MRY14-0246, which carries the intrinsic OXA-443/OXA-22-like and OXA-444/OXA-60-like β-lactamase genes and is resistant to meropenem.

DOI： 10.1128/genomeA.00405-15

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Inhibition of adhesion of Clostridium difficile to human intestinal cells after treatment with serum and intestinal fluid isolated from mice immunized with nontoxigenic C-difficile membrane fraction Reviewed

Mitsutoshi Senoh, Masaaki Iwaki, Akihiko Yamamoto, Haru Kato, Tadashi Fukuda, Keigo Shibayama

MICROBIAL PATHOGENESIS   Vol. 81   page： 1 - 5   2015.4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Diarrhea and pseudomembrane colitis caused by Clostridium difficile infection is a global health concern because of the high recurrence rate after standard antibiotic therapy. Vaccination presents a powerful countermeasure against disease recurrence. In this study, mice vaccinated with the nontoxigenic Clostridium difficile membrane fraction generated a marked immune response to the antigen, as demonstrated by the serum IgG and intestinal fluid IgA levels. Significantly, pretreatment with harvested IgG- and IgA-containing fluids was sufficient to prevent in vitro adhesion of Clostridium difficile to human Caco-2 intestinal cells. These results highlight the potential of nontoxigenic Clostridium difficile membrane fraction as a vaccine candidate for Clostridium difficile infection. (C) 2015 Elsevier Ltd. All rights reserved.

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Molecular characterization of linezolid-resistant CoNS isolates in Japan Reviewed

Akiko Takaya, Asahi Kimura, Yoshiharu Sato, Naruhiko Ishiwada, Masaharu Watanabe, Mari Matsui, Keigo Shibayama, Tomoko Yamamoto

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 70 ( 3 ) page： 658 - 663   2015.3

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Objectives: Linezolid has been reported to remain active against 98% of staphylococci with resistance identified in 0.05% of Staphylococcus aureus and 1.4% of CoNS. The objective of this study was to characterize the linezolid-resistance mechanisms in the linezolid-resistant CoNS strains isolated in Japan.
Methods: Staphylococcus capitis strains exhibiting linezolid MICs &gt;8 mg/L isolated from inpatients between 2012 and 2014 were screened for cfr and mutations in 23S rRNA, L3 and L4 by PCR/sequencing. Isolates were also examined for mutations in the rlmN gene.
Results: S. capitis had six 23S rRNA alleles. Five S. capitis isolates displayed linezolid MICs of 8, 16 and 32 mg/L. G2576U mutations were detected in three, four or five copies of 23S rRNA in all isolates. In two isolates exhibiting the highest linezolid MIC (32 mg/L) there was a large deletion in a single copy of 23S rRNA. Repeated 10 bp sequences were found in both 16S and 23S rRNAs, suggesting deletion by recombination between the repeats. One isolate had the mutation Ala-142 -&gt; Thr in the ribosomal protein L3. All linezolid-resistant isolates also demonstrated mutations in the gene encoding RlmN methyltransferase, leading to Thr-62 -&gt; Met and Gly-148 -&gt; Ser.
Conclusions: Multiple mechanisms appeared to be responsible for the elevated linezolid resistance in S. capitis isolates: a G2576U mutation in different numbers of copies of 23S rRNA, loss of a single copy of 23S rRNA and a mutation in the ribosomal protein L3, suggesting the accumulation of independent mutational events.

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Crystal Structure of IMP-2 Metallo-beta-lactamase from Acinetobacter spp.: Comparison of Active-Site Loop Structures between IMP-1 and IMP-2 Reviewed

Yamaguchi Yoshihiro, Matsueda Satoshi, Matsunaga Kazuyo, Takashio Nobutoshi, Toma-Fukai Sachiko, Yamagata Yuriko, Shibata Naohiro, Wachino Jun-ichi, Shibayama Keigo, Arakawa Yoshichika, Kurosaki Hiromasa

BIOLOGICAL & PHARMACEUTICAL BULLETIN   Vol. 38 ( 1 ) page： 96 - 101   2015.1

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Crystal Structure of IMP-2 Metallo-beta-lactamase from Acinetobacter spp.: Comparison of Active-Site Loop Structures between IMP-1 and IMP-2 Reviewed

Yoshihiro Yamaguchi, Satoshi Matsueda, Kazuyo Matsunaga, Nobutoshi Takashio, Sachiko Toma-Fukai, Yuriko Yamagata, Naohiro Shibata, Jun-ichi Wachino, Keigo Shibayama, Yoshichika Arakawa, Hiromasa Kurosaki

BIOLOGICAL & PHARMACEUTICAL BULLETIN   Vol. 38 ( 1 ) page： 96 - 101   2015.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

IMP-2, a subclass B1 metallo-beta-lactamase (MBL), is a Zn(II)-containing hydrolase. This hydrolase, involved in antibiotic resistance, catalyzes the hydrolysis of the C-N bond of the beta-lactam ring in beta-lactam antibiotics such as benzylpenicillin and imipenem. The crystal structure of IMP-2 MBL from Acinetobacter spp. was determined at 2.3 angstrom resolution. This structure is analogous to that of subclass B1 MBLs such as IMP-1 and VIM-2. Comparison of the structures of IMP-1 and IMP-2, which have an 85% amino acid identity, suggests that the amino acid substitution at position 68 on a beta-strand (beta 3) (Pro in IMP-1 versus Ser in IMP-2) may be a staple factor affecting the flexibility of loop 1 (comprising residues at positions 60-66; EVNGWGV). In the IMP-1 structure, loop 1 adopts an open, disordered conformation. On the other hand, loop 1 of IMP-2 forms a closed conformation in which the side chain of Trp64, involved in substrate binding, is oriented so as to cover the active site, even though there is an acetate ion in the active site of both IMP-1 and IMP-2. Loop 1 of IMP-2 has a more flexible structure in comparison to IMP-1 due to having a Ser residue instead of the Pro residue at position 68, indicating that this difference in sequence may be a trigger to induce a more flexible conformation in loop 1.

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Helicobacter cinaedi-associated Vertebral Osteomyelitis in an Immunocompetent Patient. Reviewed

Satoshi Murata, Hiromichi Suzuki, Setsu Sakamoto, Takamitsu Miki, Emiko Rimbara, Keigo Shibayama, Shinobu Koyama, Kiyoko Tamai, Yuji Yaguchi, Masaki Tada

Internal medicine (Tokyo, Japan)   Vol. 54 ( 24 ) page： 3221 - 4   2015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Internal Medicine  

A 56-year-old previously healthy man was hospitalized due to a 10-day history of neck pain and an elevated C-reactive protein level. Gram-negative spiral bacilli were isolated from his blood, and Helicobacter cinaedi was confirmed using 16S rRNA sequencing. The infectious focus was not identified by initial cervical magnetic resonance imaging (MRI); however, repeated MRI demonstrated prominent high signal intensity in the entire region of the C6-C7 vertebrae and C6/C7 disc space. Furthermore, fluorodeoxyglucose-positron emission tomography/computed tomography showed no significant uptake, other than in the C6-C7 region. The patient was successfully treated with ceftriaxone for six weeks without sequelae.

DOI： 10.2169/internalmedicine.54.4574

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Pararenal Lymphatic Cyst Infection Caused by Helicobacter cinaedi. Reviewed

Yusaku Akashi, Jun Igarashi, Hiromichi Suzuki, Emiko Rimbara, Keigo Shibayama, Sayaka Nin, Kiyoko Tamai, Yuji Yaguchi, Masanari Shiigai, Takehiro Oikawa, Masatsune Suzuki

Internal medicine (Tokyo, Japan)   Vol. 54 ( 11 ) page： 1437 - 40   2015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Internal Medicine  

A 43-year-old man was referred to our hospital for an acute-onset fever and left flank pain. He had been previously diagnosed with lymphangioma, and abdominal computed tomography showed pararenal cysts with fat stranding around the left kidney, of which infection was subsequently confirmed on magnetic resonance imaging. Gram-negative spiral bacilli were isolated from two sets of blood cultures, and Helicobacter cinaedi was identified using 16S rRNA sequencing. The patient was successfully treated with ceftriaxone therapy without recurrence. A multilocus sequence typing analysis indicated the current H. cinaedi strain differed from previous strains isolated in Japan.

DOI： 10.2169/internalmedicine.54.3991

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Roles of Ala-149 in the catalytic activity of diadenosine tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv. Reviewed International journal

Shigetarou Mori, Hyun Kim, Emiko Rimbara, Yoshichika Arakawa, Keigo Shibayama

Bioscience, biotechnology, and biochemistry   Vol. 79 ( 2 ) page： 236 - 8   2015

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Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap4A) phosphorylase from Mycobacterium tuberculosis H37Rv (MtAPA) belongs to the histidine triad motif (HIT) superfamily, but is the only member with an alanine residue at position 149 (Ala-149). Enzymatic analysis revealed that the Ala-149 deletion mutant displayed substrate specificity for diadenosine 5',5'''-P(1),P(5)-pentaphosphate and was inactive on Ap4A and other substrates that are utilized by the wild-type enzyme.

DOI： 10.1080/09168451.2014.973364

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Venomous snake bites: clinical diagnosis and treatment. Reviewed International journal

Toru Hifumi, Atsushi Sakai, Yutaka Kondo, Akihiko Yamamoto, Nobuya Morine, Manabu Ato, Keigo Shibayama, Kazuo Umezawa, Nobuaki Kiriu, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Kenya Kawakita, Yasuhiro Kuroda

Journal of intensive care   Vol. 3 ( 1 ) page： 16 - 16   2015

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Snake bites are life-threatening injuries that can require intensive care. The diagnosis and treatment of venomous snake bites is sometimes difficult for clinicians because sufficient information has not been provided in clinical practice. Here we review the literature to present the proper management of bites by mamushi, habu, and yamakagashi snakes, which widely inhabit Japan and other Asian countries. No definite diagnostic markers or kits are available for clinical practice; therefore, definitive diagnosis of snake-venom poisoning requires positive identification of the snake and observation of the clinical manifestations of envenomation. Mamushi (Gloydius blomhoffii) bites cause swelling and pain that spreads gradually from the bite site. The platelet count gradually decreases due to the platelet aggregation activity of the venom and can decrease to <100,000/mm(3). If the venom gets directly injected into the blood vessel, the platelet count rapidly decreases to <10,000/mm(3) within 1 h after the bite. Habu (Protobothrops flavoviridis) bites result in swelling within 30 min. Severe cases manifest not only local signs but also general symptoms such as vomiting, cyanosis, loss of consciousness, and hypotension. Yamakagashi (Rhabdophis tigrinus) bites induce life-threatening hemorrhagic symptoms and severe disseminated intravascular coagulation with a fibrinolytic phenotype, resulting in hypofibrinogenemia and increased levels of fibrinogen degradation products. Previously recommended first-aid measures such as tourniquets, incision, and suction are strongly discouraged. Once airway, breathing, and circulation have been established, a rapid, detailed history should be obtained. If a snake bite is suspected, hospital admission should be considered for further follow-up. All venomous snake bites can be effectively treated with antivenom. Side effects of antivenom should be prevented by sufficient preparation. Approved antivenoms for mamushi and habu are available. Yamakagashi antivenom is used as an off-label drug in Japan, requiring clinicians to join a clinical research group for its use in clinical practice.

DOI： 10.1186/s40560-015-0081-8

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First Report of Metallo-beta-Lactamase NDM-5-Producing Escherichia coli in Japan Reviewed

Ryuichi Nakano, Akiyo Nakano, Kenji Hikosaka, Sayoko Kawakami, Naohisa Matsunaga, Miwa Asahara, Shinobu Ishigaki, Taiji Furukawa, Masato Suzuki, Keigo Shibayama, Yasuo Ono

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY   Vol. 58 ( 12 ) page： 7611 - 7612   2014.12

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DOI： 10.1128/AAC.04265-14

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An outbreak of bla(OXA-51-like)(-) and bla(OXA-66)(-) positive Acinetobacter baumannii ST208 in the emergency intensive care unit Reviewed

Satomi Asai, Kazuo Umezawa, Hideo Iwashita, Toshio Ohshima, Maya Ohashi, Mika Sasaki, Hideki Hayashi, Mari Matsui, Keigo Shibayama, Sadaki Inokuchi, Hayato Miyachi

JOURNAL OF MEDICAL MICROBIOLOGY   Vol. 63 ( Pt 11 ) page： 1517 - 1523   2014.11

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A series of clinical isolates of drug-resistant (DR) Acinetobacter baumannii with diverse drug susceptibility was detected from eight patients in the emergency intensive care unit of Tokai University Hospital. The initial isolate was obtained in March 2010 (A. baumannii Tokai strain 1); subsequently, seven isolates were obtained from patients (A. baumannii Tokai strains 2-8) and one isolate was obtained from an air-fluidized bed used by five of the patients during the 3 months from August to November 2011. The isolates were classified into three types of antimicrobial drug resistance patterns (RRR, SRR and SSR) according to their susceptibility (S) or resistance (R) to imipenem, amikacin and ciprofloxacin, respectively. Genotyping of these isolates by multilocus sequence typing revealed one sequence type, ST208, whilst that by a DiversiLab analysis revealed two subtypes. All the isolates were positive for bla(OXA-51-like) and bla(OXA-66), as assessed by PCR and DNA sequencing. A. baumannii Tokai strains 1-8 and 10 (RRR, SRR and SSR) had quinolone resistance-associated mutations in gyrA/parC, as revealed by DNA sequencing. The ISAba / upstream of bla(OXA-51-like) and aminoglycoside resistance-associated gene, armA, were detected in A. baumannii Tokai strains 1-7 and 10 (RRR and SRR) as assessed by PCR. Among the genes encoding resistance nodulation division family pumps (adeB, adeG and adeJ) and outer-membrane porins (oprD and carO) overexpression of adeB and adeJ and suppression of oprD and car were seen in isolates of A. baumannii Tokai strain 2 (RRR), as assessed by realtime PCR. Thus, the molecular characterization of a series of isolates of DR A. baumannii revealed the outbreak of ST208 and diverse antimicrobial drug susceptibilities, which almost correlated with differential gene alterations responsible for each type of drug resistance.

DOI： 10.1099/jmm.0.077503-0

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Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011 Reviewed

Tsuyoshi Kenri, Tsuyoshi Sekizuka, Akihiko Yamamoto, Masaaki Iwaki, Takako Komiya, Takashi Hatakeyama, Hiroshi Nakajima, Motohide Takahashi, Makoto Kuroda, Keigo Shibayama

APPLIED AND ENVIRONMENTAL MICROBIOLOGY   Vol. 80 ( 22 ) page： 6954 - 6964   2014.11

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Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/ A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains.

DOI： 10.1128/AEM.02134-14

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Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile Reviewed

Mitsutoshi Senoh, Haru Kato, Tomoko Murase, Hideharu Hagiya, Yasuaki Tagashira, Tadashi Fukuda, Masaaki Iwaki, Akihiko Yamamoto, Keigo Shibayama

MICROBIOLOGY AND IMMUNOLOGY   Vol. 58 ( 11 ) page： 615 - 620   2014.11

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The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5x10(2) colony-forming units per 100mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI.

DOI： 10.1111/1348-0421.12189

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Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria Reviewed

Mitsuaki Nagasawa, Mitsuo Kaku, Kazunari Kamachi, Keigo Shibayama, Yoshichika Arakawa, Keizo Yamaguchi, Yoshikazu Ishii

JOURNAL OF INFECTION AND CHEMOTHERAPY   Vol. 20 ( 9-10 ) page： 635 - 638   2014.9

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Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 mm. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings. (C) 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jiac.2014.08.013

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Molecular characterization of nicotinate phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv, and inhibition of its activity by pyrazinoic acid Reviewed

Mori S., Kim H., Rimbara E., Arakawa Y., Shibayama K.

FEBS JOURNAL   Vol. 281   page： 584 - 585   2014.9

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Penicillin-Susceptible Group B Streptococcal Clinical Isolates with Reduced Cephalosporin Susceptibility Reviewed

Noriyuki Nagano, Yukiko Nagano, Masami Toyama, Kouji Kimura, Keigo Shibayama, Yoshichika Arakawa

JOURNAL OF CLINICAL MICROBIOLOGY   Vol. 52 ( 9 ) page： 3406 - 3410   2014.9

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We characterized penicillin-susceptible group B streptococcal (PSGBS) clinical isolates exhibiting no growth inhibition zone around a ceftibuten disk (CTBr PSGBS). The CTBr PSGBS isolates, for which augmented MICs of cefaclor and ceftizoxime were found, shared a T394A substitution in penicillin-binding protein 2X (PBP 2X) and a T567I substitution in PBP 2B, together with an additional G429S substitution in PBP 2X or a T145A substitution in PBP 1A, although the T145A substitution in the transglycosidase domain of PBP 1A would have no effect on the level of resistance to ceftibuten.

DOI： 10.1128/JCM.01291-14

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The incidence of pediatric invasive Haemophilus influenzae and pneumococcal disease in Chiba prefecture, Japan before and after the introduction of conjugate vaccines Reviewed

Naruhiko Ishiwada, Haruka Hishiki, Koo Nagasawa, Sachiko Naito, Yasunori Sato, Bin Chang, Yuko Sasaki, Kouji Kimura, Makoto Ohnishi, Keigo Shibayama

VACCINE   Vol. 32 ( 42 ) page： 5425 - 5431   2014.9

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The Haemophilus influenzae type b (Hib) vaccine and the heptavalent pneumococcal conjugate vaccine (PCV7) were introduced in Japan in 2008 and 2010, respectively. In 2011, immunization with these two vaccines was encouraged throughout Japan through a governmental program. Children treated in Chiba prefecture for culture-proven invasive H. influenzae disease (IHiD) and invasive Streptococcus pneumoniae disease (IPD) were identified in a prefectural surveillance study from 2008 to 2013. The incidence rate ratio (IRR) and its confidence interval (CI) were calculated to compare the 3 years before and after governmental financial support for vaccination. The average number of IHiD and IPD cases among children &lt;5 years of age in 2011-2013 decreased 84% (IRR: 0.16,95% CI: 0.09-0.26, p &lt; 0.0001) and 51% (IRR: 0.49,95% CI: 0.37-0.63, p &lt; 0.0001) compared with those occurring in 2008-2010. The most common non-PCV7 serotype encountered in 2011 and 2013 was 19A. After governmental subsidization of Hib and PCV7 vaccination, IHiD and IPD decreased in Chiba prefecture, Japan. Continuous surveillance is necessary to determine the effectiveness of these two vaccines and for detection of emerging invasive serotypes. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.vaccine.2014.07.100

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Practical Agar-Based Disk Potentiation Test for Detection of Fosfomycin-Nonsusceptible Escherichia coli Clinical Isolates Producing Glutathione S-Transferases Reviewed

Genki Nakamura, Jun-ichi Wachino, Natsumi Sato, Kouji Kimura, Keiko Yamada, Wanchun Jin, Keigo Shibayama, Tetsuya Yagi, Kumiko Kawamura, Yoshichika Arakawa

JOURNAL OF CLINICAL MICROBIOLOGY   Vol. 52 ( 9 ) page： 3175 - 3179   2014.9

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The number of reports concerning Escherichia coli clinical isolates that produce glutathione S-transferases responsible for fosfomycin resistance (FR-GSTs) has been increasing. We have developed a disk-based potentiation test in which FR-GST producers expand the growth inhibition zone around a Kirby-Bauer disk containing fosfomycin in combination with sodium phosphonoformate (PPF). PPF, an analog of fosfomycin, is a transition-state inhibitor of FosA(PA), a type of FR-GST from Pseudomonas aeruginosa. Considering its mechanism of action, PPF was expected to inhibit a variety of FR-GSTs. In the presence of PPF, zone enlargement around the disk containing fosfomycin was observed for FosA3-, FosA4-, and FosC2-producing E. coli clinical isolates. Moreover, the growth inhibition zone was remarkably enlarged when the Mueller-Hinton (MH) agar plate contained 25 mu g/ml glucose-6-phosphate (G6P). When we retrospectively tested 12 fosfomycin-resistant (MIC, &gt;= 256 mu g/ml) E. coli clinical isolates from our hospital with the potentiation test, 6 FR-GST producers were positive phenotypically by potentiation disk and were positive for FR-GST genes: 5 harbored fosA3 and 1 harbored fosA4. To identify the production of FR-GSTs, we set the provisional cutoff value, 5-mm enlargement, by adding PPF to a fosfomycin disk on the MH agar plates containing G6P. Our disk-based potentiation test reliably identifies FR-GST producers and can be performed easily; therefore, it will be advantageous in epidemiological surveys and infection control of fosfomycin-resistant bacteria in clinical settings.

DOI： 10.1128/JCM.01094-14

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Molecular characterization of nicotinate phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv, and inhibition of its activity by pyrazinoic acid

S. Mori, H. Kim, E. Rimbara, Y. Arakawa, K. Shibayama

FEBS JOURNAL   Vol. 281   page： 584 - 585   2014.9

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Emergence of third-generation cephalosporin-resistant Enterobacteriacae in a Japanese critical care setting. Reviewed International journal

Hagiya H, Murase T, Suzuki M, Otsuka F, Shibayama K

Acute Medicine & Surgery   Vol. 1 ( 4 ) page： 256 - 258   2014.7

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Aim: Extended-spectrum beta-lactamases and AmpC beta-lactamases give resistance to Enterobacteriaceae against cephalosporins, which are important drugs in various clinical settings. Within 5 days, a total of eight strains of Citrobacter freundii and two strains of Escherichia coli, all of which were resistant to third-generation cephalosporins, were isolated from six different patients in a critical care center. We carried out epidemiological and genetic studies to confirm the situation to be an outbreak by a single strain or a beta-lactamase-producing gene. Methods: A review of patients' clinical data, pulsed-field gel electrophoresis epidemiological study against the strains, and polymerase chain reaction analysis for the detection of extended-spectrum beta-lactamase genes (TEM, SHV, CTX-M-1, CTX-M-2, and CTX-M-9) and multiplex polymerase chain reaction analysis for plasmid-mediated AmpC-beta-lactamase genes were carried out. Results: Pulsed-field gel electrophoresis showed none of the subjects were genetically identical organisms. However, all strains possessed either extended-spectrum beta-lactamases (TEM, CTX-M-2, and CTX-M-9) or AmpC-beta-lactamases. One patient carried both extended-spectrum beta-lactamase- and AmpC- beta-lactamase-producing organisms. Conclusion: This study revealed that various types of easily transmittable drug resistance genes exist in the critical care setting concurrently. This case was not an outbreak of such strains, but the present situation might indicate a more serious condition than a mere outbreak of a single resistant strain. Enterobacteriaceae that possess extended-spectrum beta-lactamases or AmpC-beta-lactamases are increasingly common in many medical institutions in Japan, and constitutive monitoring of drug-resistant organisms and proper contact precautions are essential to prevent possible outbreaks.

DOI： 10.1002/ams2.64

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Food-borne botulism in Japan in March 2012 Reviewed

Yoshika Momose, Hiroshi Asakura, Masaru Kitamura, Yumiko Okada, Yutaka Ueda, Yutaro Hanabara, Tomohiro Sakamoto, Tsuyoshi Matsumura, Masaaki Iwaki, Haru Kato, Keigo Shibayama, Shizunobu Igimi

INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES   Vol. 24   page： 20 - 22   2014.7

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In March 2012, two patients were transported urgently to the hospital in Tottori Prefecture, Japan, because of symptoms suggestive of botulism. Botulinum neurotoxin type A was detected in the clinical specimens and the food consumed by the two patients (vacuum packed adzuki-batto, a sweet adzuki bean soup containing noodles). We were able to make a prompt diagnosis of food botulism associated with the consumption of adzuki-batto, from which the causative pathogen Clostridium botulinum Ab was cultured. (C) 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

DOI： 10.1016/j.ijid.2014.01.014

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Characterization of Multidrug-Resistant Group B Streptococci with Reduced Penicillin Susceptibility Forming Small Non-Beta-Hemolytic Colonies on Sheep Blood Agar Plates Reviewed

Hirotsugu Banno, Kouji Kimura, Yosuke Tanaka, Hiromitsu Kitanaka, Wanchun Jin, Jun-ichi Wachino, Keiko Yamada, Keigo Shibayama, Yoshichika Arakawa

JOURNAL OF CLINICAL MICROBIOLOGY   Vol. 52 ( 6 ) page： 2169 - 2171   2014.6

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We isolated and characterized three multidrug-resistant clinical isolates of group B streptococci with reduced penicillin susceptibility (PRGBS) that formed small non-beta-hemolytic colonies on sheep blood agar plates but grew well on chocolate agar plates. They can be overlooked in the bacterial identification step, leading to clinical misdiagnosis and treatment failure.

DOI： 10.1128/JCM.00226-14

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Distribution of carbapenem resistance determinants among epidemic and non-epidemic types of Acinetobacter species in Japan Reviewed

Mari Matsui, Satowa Suzuki, Kunikazu Yamane, Masato Suzuki, Toshifumi Konda, Yoshichika Arakawa, Keigo Shibayama

JOURNAL OF MEDICAL MICROBIOLOGY   Vol. 63 ( Pt 6 ) page： 870 - 877   2014.6

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We performed a comparative molecular analysis on three types of clinically isolated Acinetobacter spp.: epidemic sequence types (STs) of Acinetobacter baumannii (epidemic ST-AB), non-epidemic sequence types of A. baumannii (non-epidemic ST-AB) and non-baumannii Acinetobacter spp. A total of 87 isolates - 46 A. baumannii, 25 A. pittii and 16 A. nosocomialis - from 43 hospitals were analysed. Of these, 31 A. baumannii isolates were ST1 or ST2 according to the Pasteur Institute multilocus sequence typing scheme and were defined as epidemic ST-AB. The other 15 A. baumannii isolates were defined as non-epidemic ST-AB. The epidemic ST-AB isolates harboured the bla(OXA-23-like) gene or had an ISAba1 element upstream of bla(OXA-51-like), or both, whereas non-epidemic ST-AB and non-baumannii Acinetobacter spp. isolates harboured bla(OXA-58-like) or metallo-beta-lactamase genes, or both. The proportion of multidrug-resistant isolates was significantly higher in the epidemic ST-AB isolates (48%) than that in the other types of Acinetobacter isolates (5%) (P&lt;0.05). In addition, epidemic ST-AB isolates exhibited a relatively higher proportion of fluoroquinolone resistance. We demonstrated that, in terms of genotypes and phenotypes of antimicrobial resistance, non-epidemic ST-AB isolates shared more similarity with non-baumannii Acinetobacter spp. isolates than with epidemic ST-AB isolates, regardless of bacterial species. In addition, this study revealed that, even in Japan, where IMP-type metallo-beta-lactamase producers are endemic, epidemic ST-AB harbouring bla(IMP) have not yet emerged.

DOI： 10.1099/jmm.0.069138-0

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A subclass B3 metallo--lactamase found in Pseudomonas alcaligenes Reviewed

Masato Suzuki, Satowa Suzuki, Mari Matsui, Yoichi Hiraki, Fumio Kawano, Keigo Shibayama

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 69 ( 5 ) page： 1430 - 1432   2014.5

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DOI： 10.1093/jac/dkt498

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Chromobacterium violaceum nosocomial pneumonia in two Japanese patients at an intensive care unit Reviewed

Hideharu Hagiya, Tomoko Murase, Masato Suzuki, Keigo Shibayama, Yumi Kokumai, Naoto Watanabe, Miyako Maki, Fumio Otsuka

JOURNAL OF INFECTION AND CHEMOTHERAPY   Vol. 20 ( 1-2 ) page： 139 - 142   2014.1

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Chromobacterium violaceum is sensitive to temperature and the infection is usually confined to tropical or subtropical regions. Since Japan has a warm climate, C. violaceum has been scarcely isolated from clinical specimens. With global warming, however, the geographical distribution of C violaceum infection is likely to change. We report two cases of C violaceum nosocomial pneumonia that occurred at an intensive care center in Japan. C violaceum was first detected from a patient in the same center as a pathogenic organism of pneumonia. Later, the organism was isolated from sputum and a ventilator circuit tube of another patient in the center. The two patients were admitted to the center in nearby beds for several days. All of the pathogens were confirmed to be C violaceum by the nucleic acid sequence of the 16S rRNA gene and were proven to be genetically identical organisms by pulsed field gel electrophoresis. Both patients were managed with well-humidified and heated oxygen using a venturi mask and ventilator to promote excretion of sputum. It was thought that the medical respiratory care devices that provide a humid and warm environment, an optimal condition for proliferation of C violaceum, can contribute to C violaceum infection in a hospital environment. (C) 2013, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jiac.2013.10.001

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Evaluation of a Double-Disk Synergy Test with a Common Metallo-beta-Lactamase Inhibitor, Mercaptoacetate, for Detecting NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Reviewed

Wachino Jun-ichi, Matsui Mari, Hoang Huy Tran, Suzuki Masato, Suzuki Satowa, Shibayama Keigo

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 67 ( 1 ) page： 66 - 68   2014.1

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Evaluation of a Double-Disk Synergy Test with a Common Metallo-beta-Lactamase Inhibitor, Mercaptoacetate, for Detecting NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Reviewed

Jun-ichi Wachino, Mari Matsui, Hoang Huy Tran, Masato Suzuki, Satowa Suzuki, Keigo Shibayama

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 67 ( 1 ) page： 66 - 68   2014.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL INST INFECTIOUS DISEASES  

DOI： 10.7883/yoken.67.66

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Biochemical characterization of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and inhibition of its activity by pyrazinamide. Reviewed International journal

Hyun Kim, Keigo Shibayama, Emiko Rimbara, Shigetarou Mori

PloS one   Vol. 9 ( 6 ) page： e100062   2014

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Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) is a key enzyme in the de novo pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis and a target for the development of new anti-tuberculosis drugs. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide from quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP) through a phosphoribosyl transfer reaction followed by decarboxylation. The crystal structure of QAPRTase from Mycobacterium tuberculosis H37Rv (MtQAPRTase) has been determined; however, a detailed functional analysis of MtQAPRTase has not been published. Here, we analyzed the enzymatic activities of MtQAPRTase and determined the effect on catalysis of the anti-tuberculosis drug pyrazinamide (PZA). The optimum temperature and pH for MtQAPRTase activity were 60°C and pH 9.2. MtQAPRTase required bivalent metal ions and its activity was highest in the presence of Mg2+. Kinetic analyses revealed that the Km values for QA and PRPP were 0.08 and 0.39 mM, respectively, and the kcat values for QA and PRPP were 0.12 and 0.14 [s-1], respectively. When the amino acid residues of MtQAPRTase, which may interact with QA, were substituted with alanine residues, catalytic activity was undetectable. Further, PZA, which is an anti-tuberculosis drug and a structural analog of QA, markedly inhibited the catalytic activity of MtQAPRTase. The structure of PZA may provide the basis for the design of new inhibitors of MtQAPRTase. These findings provide new insights into the catalytic properties of MtQAPRTase.

DOI： 10.1371/journal.pone.0100062

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Effect of antivenom therapy of Rhabdophis tigrinus (Yamakagashi snake) bites. Reviewed International journal

Toru Hifumi, Atsushi Sakai, Akihiko Yamamoto, Masahiro Murakawa, Manabu Ato, Keigo Shibayama, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Akihiko Ginnaga, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 44 - 44   2014

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BACKGROUND: Rhabdophis tigrinus (Yamakagashi snake) is a rear-fanged colubrid snake present throughout Russia and Asia. Its venom induces life-threatening hemorrhagic symptoms and severe disseminated intravascular coagulation with a fibrinolytic phenotype. R. tigrinus antivenom manufactured by the immunization of horses to neutralize the venom has the risk of adverse events such as anaphylaxis and serum sickness disease. It should be used when benefit is greater than the risk of adverse effects; however, its efficacy has not been well evaluated. Although our previous survey of nine cases demonstrated that seven of all cases treated with antivenom survived, the clinical characteristics and prognosis without antivenom administration remained unclear. We assumed that R. tigrinus antivenom administration overlaps self-recovery with supportive care. We aimed to determine the association between antivenom administration and outcome with further analyzed cases. METHODS: We retrospectively reviewed the records of the Japan Snake Institute between January 1, 1973 and December 31, 2013. Antivenom and without antivenom groups were compared with regard to baseline demographic features, treatment-related factors, and outcomes. RESULTS: In total, 34 patients were analyzed (97% male, median age 37.5 years). Twenty-five patients were further examined from our previous study. On admission, the median levels of fibrinogen and fibrinogen degradation products were 35 mg/dL and 200 μg/mL, respectively, and platelet counts were 107,000/mm(3). The median disseminated intravascular coagulation score (defined by the Japanese Association of Acute Medicine) was 5. Antivenom was administered to 19 patients, with a median interval of 32 h between bite and antivenom administration. The in-hospital mortality rate was 12%. In univariate analysis, baseline characteristics and laboratory data were not significantly different between the antivenom and without antivenom groups. Hospital mortality in the antivenom group was significantly better than that in the without antivenom group (0% vs. 26.7%, P = 0.03). Moreover, the number of patients developing renal failure requiring hemodialysis was significantly lower in the antivenom group (5.3% vs. 40.0%, P = 0.03). CONCLUSIONS: In our small retrospective study, antivenom administration was likely to be effective in the management of R. tigrinus bites. Apparently, further research is required to confirm its efficacy.

DOI： 10.1186/s40560-014-0044-5

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Clinical characteristics of yamakagashi (Rhabdophis tigrinus) bites: a national survey in Japan, 2000-2013. Reviewed International journal

Toru Hifumi, Atsushi Sakai, Akihiko Yamamoto, Masahiro Murakawa, Manabu Ato, Keigo Shibayama, Akihiko Ginnaga, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 19 - 19   2014

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BACKGROUND: Yamakagashi (Rhabdophis tigrinus) is a species of pit viper present throughout Russia and Eastern Asia. Although R. tigrinus venom is known to induce life-threatening hemorrhagic symptoms, the clinical characteristics and effective treatment of R. tigrinus bites remain unknown. The present study aimed to clarify these issues. METHODS: Records in the Japan Snake Institute between 2000 and 2013 were retrospectively investigated. The following were determined: patient characteristics, coagulation and fibrinolytic system abnormalities, effect of antivenom treatment, and outcomes. RESULTS: Nine patients (all males; median age, 38 years) with R. tigrinus bites were identified. On admission, the median levels of fibrinogen and fibrinogen degradation products, and platelet counts were 50 mg/dL, 295 μg/mL, and 107,000/mm(3), respectively. The median (minimum-maximum) disseminated intravascular coagulation (DIC) score defined by the Japanese Association of Acute Medicine was 8 (1-8). Antivenom was administered to seven patients, with a median interval of 35 h between bite and antivenom administration. All patients treated with antivenom survived, and the in-hospital mortality rate was 11%. CONCLUSIONS: Patients with R. tigrinus bites presented with DIC of a fibrinolytic phenotype, which can result in life-threatening injury unless appropriate antivenom and DIC treatment are provided.

DOI： 10.1186/2052-0492-2-19

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Clinical characteristics of redback spider bites. Reviewed International journal

Toru Hifumi, Satoshi Fujimi, Takuya Yamagishi, Satoru Arai, Kyoko Sawabe, Akihiko Yamamoto, Manabu Ato, Keigo Shibayama, Akihiko Ginnaga, Nobuaki Kiriu, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Masanobu Kishikawa, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 62 - 62   2014

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BACKGROUND: Redback spiders (Latrodectus hasselti) (RBSs) are venomous spiders that have recently spread to Asia from Australia. Since the first case report in 1997 (Osaka), RBS bites have been a clinical and administrative issue in Japan; however, the clinical characteristics and effective treatment of RBS bites, particularly outside Australia remains unclear. This study aimed to elucidate the clinical characteristics of RBS bites and to clarify the effectiveness of the administration of antivenom for treatment. METHODS: We performed a retrospective questionnaire survey from January 2009 to December 2013 to determine the following: patient characteristics, effect of antivenom treatment, and outcomes. To clarify the characteristics of patients who develop systemic symptoms, we compared patients with localized symptoms and those with systemic symptoms. We also examined the efficacy and adverse effects in cases administered antivenom. RESULTS: Over the 5-year study period, 28 patients were identified from 10 hospitals. Of these, 39.3% were male and the median age was 32 years. Bites most commonly occurred on the hand, followed by the forearm. Over 80% of patients developed local pain and erythema, and 35.7% (10 patients) developed systemic symptoms. Baseline characteristics, vital signs, laboratory data, treatment-related factors, and outcome were not significantly different between the localized and systemic symptoms groups. Six patients with systemic symptoms received antivenom, of whom four experienced symptom relief following antivenom administration. Premedication with an antihistamine or epinephrine to prevent the adverse effects of antivenom was administered in four patients, which resulted in no anaphylaxis. One out of two patients who did not receive premedication developed a mild allergic reaction after antivenom administration that subsided without treatment. CONCLUSIONS: Approximately one third of cases developed systemic symptoms, and antivenom was administered effectively and safely in severe cases. Further research is required to identify clinically applicable indications for antivenom use.

DOI： 10.1186/s40560-014-0062-3

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Rhabdophis tigrinus is not a pit viper but its bites result in venom-induced consumptive coagulopathy similar to many viper bites. Reviewed International journal

Anjana Silva, Toru Hifumi, Atsushi Sakai, Akihiko Yamamoto, Masahiro Murakawa, Manabu Ato, Keigo Shibayama, Akihiko Ginnaga, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 43 - 43   2014

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As a response to the recent article by Hifumi et al. published in the Journal of Intensive Care, the present correspondence clarifies the family-level taxonomy of the yamakagashi (Rhabdophis tigrinus). Further, the relevance of the term 'venom-induced consumptive coagulopathy,' instead of disseminated intravascular coagulation, in describing the procoagulant coagulopathy of R. tigrinus is highlighted.

DOI： 10.1186/s40560-014-0043-6

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Isolation of Genetically Indistinguishable Carbapenem-Resistant and -Susceptible Acinetobacter baumannii Isolates from a Single Patient Reviewed

Mari Matsui, Keigo Shibayama, Yasuhiro Tsuji, Hidetoshi Kamimura, Yoshiharu Karube, Mayumi Yoshida, Yoko Masuda, Yoichi Hiraki, Kazutaka Takaki, Fumio Kawano

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY   Vol. 57 ( 11 ) page： 5781 - 5782   2013.11

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DOI： 10.1128/AAC.01631-13

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Rapid and Reliable Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus agalactiae Reviewed

Kouji Kimura, Hideji Yanagisawa, Jun-ichi Wachino, Keigo Shibayama, Yoshichika Arakawa

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 6 ) page： 546 - 548   2013.11

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Streptococcus agalactiae (group B Streptococcus, GBS) is the leading cause of neonatal sepsis and meningitis and an important pathogen in elderly patients and those with underlying diseases. The diagnosis of GBS infections is primarily based on culture of GBS. Some clinical laboratories perform the Christie-Atkins-Munch-Peterson (CAMP) test for discrimination of GBS from other streptococci. Here, we developed a rapid GBS identification method, i.e., the loop-mediated isothermal amplification (LAMP) method for detecting the cfb gene encoding the CAMP factor. This method detected at least 4 copies of the cfb gene in GBS under isothermal conditions with in a short time (65 degrees C, within 90 min). No inappropriate amplification of nucleotide by this method was observed when the chromosomal DNA of 17 streptococci and enterococci species, other than GBS, were used as templates. In this investigation, we successfully developed a LAMP method for rapid and highly sensitive detection of GBS, which provides a beneficial alternative to the conventional CAMP test.

DOI： 10.7883/yoken.66.546

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Rapid and Reliable Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus agalactiae Reviewed

Kimura Kouji, Yanagisawa Hideji, Wachino Jun-ichi, Shibayama Keigo, Arakawa Yoshichika

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 6 ) page： 546 - 548   2013.11

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Genome Sequence of a Strain of the Human Pathogenic Bacterium Pseudomonas alcaligenes That Caused Bloodstream Infection. Reviewed International journal

Masato Suzuki, Satowa Suzuki, Mari Matsui, Yoichi Hiraki, Fumio Kawano, Keigo Shibayama

Genome announcements   Vol. 1 ( 5 )   2013.10

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Pseudomonas alcaligenes, a Gram-negative aerobic bacterium, is a rare opportunistic human pathogen. Here, we report the whole-genome sequence of P. alcaligenes strain MRY13-0052, which was isolated from a bloodstream infection in a medical institution in Japan and is resistant to antimicrobial agents, including broad-spectrum cephalosporins and monobactams.

DOI： 10.1128/genomeA.00919-13

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Genetic Analysis of Bordetella pertussis Isolates from the 2008-2010 Pertussis Epidemic in Japan Reviewed

Yusuke Miyaji, Nao Otsuka, Hiromi Toyoizumi-Ajisaka, Keigo Shibayama, Kazunari Kamachi

PLoS ONE   Vol. 8 ( 10 ) page： e77165   2013.10

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A large pertussis epidemic occurred between 2008 and 2010 in Japan. To investigate epidemic strains, we analyzed 33 Bordetella pertussis isolates from the epidemic period by sequencing virulence-associated genes (fim3, ptxP, ptxA, and prn) and performing multilocus variable-number tandem repeat analysis (MLVA), and compared these results with those of 101 isolates from non-epidemic, earlier and later time periods. DNA sequencing of the fim3 allele revealed that the frequency of fim3B was 4.3%, 12.8%, 30.3%, and 5.1% within isolates in 2002-2004, 2005-2007, 2008-2010, and 2011-2012, respectively. The isolation rate of the fim3B strain therefore temporarily increased during the epidemic period 2008-2010. In contrast, the frequencies of the virulence-associated allelic variants, ptxP3, ptxA1, and prn2, increased with time during overall study period, indicating that these variants were not directly involved in the occurrence of the 2008-2010 epidemic. MLVA genotyping in combination with analysis of allele types showed that the prevalence of an MT27d strain temporarily increased in the epidemic period, and that this strain carried virulence-associated allelic variants (fim3B, ptxP3, ptxA1, and prn2) also identified in recent epidemic strains of Australia, Europe, and the US. Phenotypic analyses revealed that the serotype Fim3 strain was predominant (≥87%) during all the periods studied, and that the frequency of adhesion pertactin (Prn) non-expressing B. pertussis decreased by half in the epidemic period. All MT27d strains expressed Prn and Fim3 proteins, suggesting that B. pertussis MT27d strains expressing Prn and Fim3B have the potential to cause large epidemics worldwide. © 2013 Miyaji et al.

DOI： 10.1371/journal.pone.0077165

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Role of γ-glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection. Reviewed International journal

Emiko Rimbara, Shigetarou Mori, Hyun Kim, Keigo Shibayama

Microbiology and immunology   Vol. 57 ( 10 ) page： 665 - 73   2013.10

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γ-Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ-glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ-Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ-glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild-type and knockout strains and inflammatory responses evaluated by induction of interleukin-8 (IL-8). IL-8 response was significantly decreased by knockout of the γ-glutamyltranspeptidase-encoding gene but not by knockout of the asparaginase-encoding gene. Additionally, IL-8 induction by infection with the H. pylori wild-type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL-8 induction caused by γ-glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ-glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.

DOI： 10.1111/1348-0421.12089

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Draft Genome Sequence of Helicobacter fennelliae Strain MRY12-0050, Isolated from a Bacteremia Patient. Reviewed International journal

Emiko Rimbara, Mari Matsui, Shigetarou Mori, Satowa Suzuki, Masato Suzuki, Hyun Kim, Tsuyoshi Sekizuka, Makoto Kuroda, Keigo Shibayama

Genome announcements   Vol. 1 ( 4 )   2013.8

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Helicobacter fennelliae, a human enterohepatic pathogen, causes bacteremia and colitis. We isolated H. fennelliae strain MRY12-0050 from a female patient; this strain was isolated from 2 other patients from the same hospital during the same period, suggesting human-to-human transmission. This is the first report of an H. fennelliae genome sequence.

DOI： 10.1128/genomeA.00512-13

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Rapid discrimination of Acinetobacter baumannii international clone II lineage by pyrosequencing SNP analyses of bla(OXA-51-like) genes Reviewed

Mari Matsui, Satowa Suzuki, Masato Suzuki, Yoshichika Arakawa, Keigo Shibayama

JOURNAL OF MICROBIOLOGICAL METHODS   Vol. 94 ( 2 ) page： 121 - 124   2013.8

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We found that Acinetobacter baumannii international clone II generally possesses unique GTA sequence at nucleotide positions 106-108 in the bla(OXA-51-like) genes. We exploited this to develop an easy and rapid method for discrimination of international clone II from other A. baumannii by employing pyrosequencing analyses of single nucleotide polymorphisms. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mimet.2013.05.014

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Genome Sequences of Multidrug-Resistant Acinetobacter baumannii Strains from Nosocomial Outbreaks in Japan. Reviewed International journal

Masato Suzuki, Mari Matsui, Satowa Suzuki, Emiko Rimbara, Satomi Asai, Hayato Miyachi, Tohru Takata, Yoichi Hiraki, Fumio Kawano, Keigo Shibayama

Genome announcements   Vol. 1 ( 4 )   2013.7

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Acinetobacter baumannii has emerged worldwide as an important nosocomial pathogen in medical institutions. Here, we present the draft genome sequences of A. baumannii strains MRY09-0642, MRY10-0558, and MRY12-0277 that were isolated from nosocomial outbreaks in Japan between 2008 and 2012 and that are resistant to antimicrobial agents, including carbapenems, fluoroquinolones, and aminoglycosides.

DOI： 10.1128/genomeA.00476-13

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Helicobacter cinaedi and Helicobacter fennelliae transmission in a hospital from 2008 to 2012. Reviewed International journal

Emiko Rimbara, Shigetarou Mori, Hyun Kim, Mari Matsui, Satowa Suzuki, Shunji Takahashi, Satoshi Yamamoto, Masaya Mukai, Keigo Shibayama

Journal of clinical microbiology   Vol. 51 ( 7 ) page： 2439 - 42   2013.7

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Forty-six Helicobacter cinaedi isolates from the same hospital were analyzed by multilocus sequence typing. Most H. cinaedi isolates exhibited clonal complex 9 and were mainly isolated from immunocompromised patients in the same ward. Three Helicobacter fennelliae isolates were obtained from the same ward and exhibited the same pulsed-field gel electrophoresis patterns. All isolates were resistant to clarithromycin and ciprofloxacin. H. cinaedi and H. fennelliae must be carefully monitored to prevent nosocomial infection.

DOI： 10.1128/JCM.01035-13

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High cephalosporin resistance due to amino acid substitutions in PBP1A and PBP2X in a clinical isolate of group B Streptococcus Reviewed

Kouji Kimura, Jun-ichi Wachino, Hiroshi Kurokawa, Mari Matsui, Satowa Suzuki, Kunikazu Yamane, Noriyuki Nagano, Keigo Shibayama, Yoshichika Arakawa

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 68 ( 7 ) page： 1533 - 1536   2013.7

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Group B Streptococcus (GBS; Streptococcus agalactiae) has been regarded as uniformly susceptible to penicillins. However, we recently reported the existence of GBS with reduced penicillin susceptibility (PRGBS), with amino acid substitutions in penicillin-binding protein (PBP) 2X. Although most PRGBS show high MICs of ceftizoxime (464 mg/L) and cefotaxime (0.121 mg/L), those for strain B1 are exceptionally high (ceftizoxime MIC 256 mg/L and cefotaxime MIC 2 mg/L). We previously found an amino acid substitution (G539S) neighbouring the conserved K(540)TG motif in PBP1A in addition to the PRGBS-specific amino acid substitution Q557E in PBP2X of B1. The aim of this study was to reveal the effect of the amino acid substitutions in PBP1A and PBP2X of B1 on the high cephalosporin resistance.
A ceftizoxime competition assay was performed to reveal the PBPs that are the main targets of ceftizoxime. We generated two allelic exchange mutants from -lactam-susceptible GBS BAA-611. BAA-611 (B1PBP2X) contained the PBP2X gene derived from B1 and BAA-611 (B1PBP2X, B1PBP1A) contained both the PBP2X and the PBP1A gene derived from B1. These allelic exchange mutants and strain B1 were subjected to susceptibility testing.
The ceftizoxime competition assay revealed that PBP1A and PBP2X were the main targets of ceftizoxime. Although the MICs of ceftizoxime and cefotaxime for BAA-611 (B1PBP2X) were 64 and 0.5 mg/L, respectively, BAA-611 (B1PBP2X, B1PBP1A) showed high cephalosporin resistance (ceftizoxime MIC 256 mg/L and cefotaxime MIC 2 mg/L) comparable to B1.
The high cephalosporin resistance of GBS was caused by amino acid substitutions in PBP1A and PBP2X.

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Ability of the VITEK 2 system to detect group B streptococci with reduced penicillin susceptibility (PRGBS) Reviewed

Kouji Kimura, Noriyuki Nagano, Yukiko Nagano, Jun-ichi Wachino, Keigo Shibayama, Yoshichika Arakawa

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 68 ( 6 ) page： 1442 - 1444   2013.6

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DOI： 10.1093/jac/dkt008

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Detection of Tripoli metallo--lactamase 2 (TMB-2), a variant of bla(TMB-1), in clinical isolates of Acinetobacter spp. in Japan Reviewed

Satowa Suzuki, Mari Matsui, Masato Suzuki, Akira Sugita, Yoko Kosuge, Nobuhiro Kodama, Yasuko Ichise, Keigo Shibayama

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 68 ( 6 ) page： 1441 - 1442   2013.6

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DOI： 10.1093/jac/dkt031

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Screening for Group B Streptococci with Reduced Penicillin Susceptibility in Clinical Isolates Obtained between 1977 and 2005 Reviewed

Kimura Kouji, Nishiyama Yasunobu, Shimizu Seiichi, Wachino Jun-ichi, Matsui Mari, Suzuki Satowa, Yamane Kunikazu, Shibayama Keigo, Arakawa Yoshichika

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 3 ) page： 222 - 225   2013.5

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Screening for Group B Streptococci with Reduced Penicillin Susceptibility in Clinical Isolates Obtained between 1977 and 2005 Reviewed

Kouji Kimura, Yasunobu Nishiyama, Seiichi Shimizu, Jun-ichi Wachino, Mari Matsui, Satowa Suzuki, Kunikazu Yamane, Keigo Shibayama, Yoshichika Arakawa

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 3 ) page： 222 - 225   2013.5

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Group B streptococcus (GBS; Streptococcus agalactiae) is a leading cause of neonatal invasive infections, and until recently, it was thought to be completely susceptible to penicillin. However, we recently identified several clinical GBS isolates with reduced penicillin susceptibility (PRGBS) whose minimum inhibitory concentrations of penicillin were &gt;0.12 mu g/ml, which is above the susceptibility breakpoint set by the Clinical and Laboratory Standards Institute. These PRGBS were isolated between 1995 and 2005 in Japan; whether these PRGBS existed in Japan before 1995 is unknown. In the study described here, we screened for PRGBS among 349 clinical GBS isolates obtained in Japan between 1977 and 2005 using the previously developed disk diffusion method for the detection of PRGBS. With this method, we selected 6 PRGBS candidates and confirmed that 1 isolate was PRGBS, using agar dilution method, including oxacillin, ceftizoxime, and penicillin-binding protein 2X (PBP2X) gene sequencing analysis. This isolate was obtained from sputum in 2005, and we could not detect PRGBS isolates before 1995 in this investigation.

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Hemin-binding proteins as potent markers for serological diagnosis of infections with bartonella quintana Reviewed

Mayumi Matsuoka, Toshinori Sasaki, Naomi Seki, Mutsuo Kobayashi, Kyoko Sawabe, Yuko Sasaki, Keigo Shibayama, Tsuguo Sasaki, Yoshichika Arakawa

Clinical and Vaccine Immunology   Vol. 20 ( 4 ) page： 620 - 626   2013.4

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It is difficult to distinguish infections with different Bartonella species using commercially available immunofluorescence (indirect immunofluorescent antibody [IFA]) assay kits. To identify appropriate proteins for serodiagnosis of Bartonella quintana infections, we investigated the antigenicity of B. quintana proteins using sera from homeless people with high B. quintana IgG titers in IFA assay. These sera reacted strongly to an outer membrane protein, hemin-binding protein D (HbpD). Further, serum from an endocarditis patient infected with B. quintana reacted to HbpB and HbpD. To locate the antigenic sites within the proteins, we generated deletion mutants of HbpB and HbpD. Amino acid residues 89 to 220 of HbpB and 151 to 200 of HbpD were identified as the minimum regions required for recognition by these sera. Several oligopeptides comprising parts of the minimum regions of HbpB and HbpD were synthesized, and their immunoreactivity with the above-mentioned sera was tested by enzyme-linked immunosorbent assay (ELISA). Serum from the endocarditis patient reacted similarly to synthetic peptides HbpB2 (amino acid residues 144 to 173 of HbpB) and HbpD3 (151 to 200 residues of HbpD), while sera from the other subjects reacted to HbpD3. These results indicate that synthetic peptides HbpB2 and HbpD3 might be suitable for developing serological tools for differential diagnosis of B. quintana infections from other Bartonella infections. © 2013, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/CVI.00717-12

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Active Screening of Group B Streptococci with Reduced Penicillin Susceptibility and Altered Serotype Distribution Isolated from Pregnant Women in Kobe, Japan Reviewed

Kouji Kimura, Kousaku Matsubara, Go Yamamoto, Keigo Shibayama, Yoshichika Arakawa

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 2 ) page： 158 - 160   2013.3

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Group B streptococcus (GBS; Streptococcus agalactiae) is a leading cause of neonatal invasive infections and was believed to be fully susceptible to penicillin. However, we recently identified several clinical GBS isolates with reduced penicillin susceptibility (PRGBS), which were mainly isolated from respiratory specimens of elderly people. An investigation of both the isolation rate of PRGBS and the serotype distribution among pregnant women is crucial to decisions regarding optimal prevention and strategies for GBS treatment in neonates. We collected 141 GBS isolates from vaginal specimens of 122 pregnant women in a hospital in Kobe, Japan, from 2007 to 2008. Of the 141 GBS isolates, 139 were subjected to antimicrobial susceptibility testing based on the results of screening for PRGBS by the disk diffusion method. All 139 isolates were susceptible to penicillin G, ampicillin, cefotaxime, cefepime, and meropenem; no PRGBS isolates were detected. However, the rates of erythromycin and clindamycin resistance in the isolates were 10.1% and 5.0%, respectively, which are much higher than the values previously reported in Japan. Serotypes VI and VIII accounted for 26% of GBS; a markedly decreased percentage from the rates observed around the year 2000. These findings suggested that penicillin remains an effective means of intrapartum antibiotic prophylaxis in Japan.

DOI： 10.7883/yoken.66.158

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Active Screening of Group B Streptococci with Reduced Penicillin Susceptibility and Altered Serotype Distribution Isolated from Pregnant Women in Kobe, Japan Reviewed

Kimura Kouji, Matsubara Kousaku, Yamamoto Go, Shibayama Keigo, Arakawa Yoshichika

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 2 ) page： 158 - 160   2013.3

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High frequency of fluoroquinolone- and macrolide-resistant streptococci among clinically isolated group B streptococci with reduced penicillin susceptibility Reviewed

Kouji Kimura, Noriyuki Nagano, Yukiko Nagano, Satowa Suzuki, Jun-ichi Wachino, Keigo Shibayama, Yoshichika Arakawa

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 68 ( 3 ) page： 539 - 542   2013.3

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Recently several clinical isolates of Streptococcus agalactiae [also known as group B Streptococcus (GBS)] that have acquired reduced penicillin susceptibility (PRGBS) by amino acid substitutions in the penicillin-binding protein 2X have emerged. The frequency of fluoroquinolone (FQ)- and macrolide-resistant streptococci among PRGBS is not yet known.
Fifty-seven GBS [19 PRGBS and 38 penicillin-susceptible GBS (PSGBS)], isolated from different medical institutions in Japan, were studied. For GBS, the MICs of penicillin G, levofloxacin and erythromycin were determined using the agar dilution method. Nineteen PRGBS were previously confirmed as genetically diverse streptococci by PFGE. Further, the mechanisms underlying penicillin, FQ and macrolide non-susceptibility/resistance were analysed.
The frequency of non-susceptibility to FQs among PSGBS was 18.4 (7/38), whereas that among PRGBS was 100 (19/19). The frequency of resistance to erythromycin among PSGBS was 7.9 (3/38), while that among PRGBS was 47.4 (9/19). Statistical significance was determined using Fishers exact test between reduced penicillin susceptibility and FQ non-susceptibility (P0.0001) and macrolide resistance (P0.0012). The resistance/non-susceptibility mechanisms among PRGBS were diverse, suggesting that the PRGBS examined were not clonal.
PRGBS isolates tend to show resistance to FQs and/or macrolides. Because the drug choice for treating these multidrug-resistant GBS is more limited than that for usual GBS, these strains may present future public health challenges.

DOI： 10.1093/jac/dks423

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Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae strains containing new delhi metallo-beta-lactamase isolated from two patients in Vietnam Reviewed

Tran Huy Hoang, Heiman Wertheim, Nguyen Binh Minh, Tran Nhu Duong, Dang Duc Anh, Tran Thi Lan Phuong, Trinh Hong Son, Hidemasa Izumiya, Makoto Ohnishi, Keigo Shibayama, Nguyen Tran Hien

Journal of Clinical Microbiology   Vol. 51 ( 1 ) page： 373 - 374   2013.1

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DOI： 10.1128/JCM.02322-12

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First Report of OXA-48 Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli in Japan from a Patient Returned from Southeast Asia Reviewed

Nagano Noriyuki, Endoh Yasunobu, Nagano Yukiko, Toyama Masami, Matsui Mari, Shibayama Keigo, Arakawa Yoshichika

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 1 ) page： 79 - 81   2013.1

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First Report of OXA-48 Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli in Japan from a Patient Returned from Southeast Asia Reviewed

Noriyuki Nagano, Yasunobu Endoh, Yukiko Nagano, Masami Toyama, Mari Matsui, Keigo Shibayama, Yoshichika Arakawa

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 1 ) page： 79 - 81   2013.1

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DOI： 10.7883/yoken.66.79

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Effects of Simultaneous Immunization of Haemophilus influenzae Type b Conjugate Vaccine and Diphtheria-Tetanus-Acellular Pertussis Vaccine on Anti-Tetanus Potencies in Mice, Guinea Pigs, and Rats Reviewed

Tadashi Fukuda, Masaaki Iwaki, Takako Komiya, Keigo Shibayama, Motohide Takahashi, Hideki Nakashima

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 1 ) page： 41 - 45   2013.1

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Haemophilus influenzae type b vaccine conjugated with tetanus toxoid (HibT) was licensed for use in childhood immunization in Japan in 2007. As adsorbed diphtheria-tetanus-acellular pertussis (DTaP) combined with HibT vaccine has not been introduced in Japan, DTaP and HibT vaccines are injected at separate sites with a similar immunization schedule. There are various interfering or stimulatory effects between components of combined vaccines contained in DTaP and HibT vaccines. In this study, we investigated the effect of HibT containing combination vaccines on anti-tetanus potencies by using animal models (mouse, guinea pig, and rat). HibT vaccine and HibT components of imported DTaP-HibT vaccine alone showed comparable or higher anti-tetanus potency than DTaP vaccine and DTaP-containing components of combination vaccines. Mixing these components before injection resulted in potencies greater than the sum of individual potencies. Injecting individual components at separate sites in animals resulted in potency roughly equivalent to the sum of the individual potencies. These results provide useful information regarding the use of HibT-containing multivalent vaccines in childhood immunization.

DOI： 10.7883/yoken.66.41

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Structural Insights into the Subclass B3 Metallo-beta-Lactamase SMB-1 and the Mode of Inhibition by the Common Metallo-beta-Lactamase Inhibitor Mercaptoacetate Reviewed

Jun-ichi Wachino, Yoshihiro Yamaguchi, Shigetarou Mori, Hiromasa Kurosaki, Yoshichika Arakawa, Keigo Shibayama

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY   Vol. 57 ( 1 ) page： 101 - 109   2013.1

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A novel subclass B3 metallo-beta-lactamase (MBL), SMB-1, recently identified from a Serratia marcescens clinical isolate, showed a higher hydrolytic activity against a wide range of beta-lactams than did the other subclass B3 MBLs, i.e., BJP-1 and FEZ-1, from environmental bacteria. To identify the mechanism underlying the differences in substrate specificity among the subclass B3 MBLs, we determined the structure of SMB-1, using 1.6-angstrom diffraction data. Consequently, we found that SMB-1 reserves a space in the active site to accommodate beta-lactam, even with a bulky R1 side chain, due to a loss of amino acid residues corresponding to F31 and L226 of BJP-1, which protrude into the active site to prevent beta-lactam from binding. The protein also possesses a unique amino acid residue, Q157, which probably plays a role in recognition of beta-lactams via the hydrogen bond interaction, which is missing in BJP-1 and FEZ-1, whose K-m values for beta-lactams are particularly high. In addition, we determined the mercaptoacetate (MCR)-complexed SMB-1 structure and revealed the mode of its inhibition by MCR: the thiolate group bridges to two zinc ions (Zn1 and Zn2). One of the carboxylate oxygen atoms of MCR makes contact with Zn2 and Ser221, and the other makes contact with T223 and a water molecule. Our results demonstrate the possibility that MCR could be a potent inhibitor for subclass B3 MBLs and that the screening technique using MCR as an inhibitor would be effective for detecting subclass B3 MBL producers.

DOI： 10.1128/AAC.01264-12

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Identification of VanN-Type Vancomycin Resistance in an Enterococcus faecium Isolate from Chicken Meat in Japan Reviewed

Takahiro Nomura, Koichi Tanimoto, Keigo Shibayama, Yoshichika Arakawa, Shuhei Fujimoto, Yasuyoshi Ike, Haruyoshi Tomita

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY   Vol. 56 ( 12 ) page： 6389 - 6392   2012.12

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Five VanN-type vancomycin-resistant Enterococcus faecium strains were isolated from a sample of domestic chicken meat in Japan. All isolates showed low-level resistance to vancomycin (MIC, 12 mg/liter) and had the same pulsed-field gel electrophoresis profile. The vancomycin resistance was encoded on a large plasmid (160 kbp) and was expressed constitutively. The VanN-type resistance operon was identical to the first resistance operon to be reported, with the exception of a 1-bp deletion in vanT(N) and a 1-bp substitution in vanS(N).

DOI： 10.1128/AAC.00747-12

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Identification of Mycoplasma pneumoniae type 2b variant strains in Japan Reviewed

Tsuyoshi Kenri, Hitomi Ohya, Atsuko Horino, Keigo Shibayama

JOURNAL OF MEDICAL MICROBIOLOGY   Vol. 61 ( 11 ) page： 1633 - 1635   2012.11

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DOI： 10.1099/jmm.0.046441-0

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Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan. Reviewed International journal

Emiko Rimbara, Shigetarou Mori, Mari Matsui, Satowa Suzuki, Jun-Ichi Wachino, Yoshiaki Kawamura, Zeli Shen, James G Fox, Keigo Shibayama

Journal of clinical microbiology   Vol. 50 ( 8 ) page： 2553 - 60   2012.8

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Helicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized. However, methods for analyzing the molecular epidemiology of H. cinaedi are not yet established. A genotyping method involving multilocus sequence typing (MLST) was developed and used to analyze 50 H. cinaedi isolates from Japanese hospitals in addition to 6 reference strains. Pulsed-field gel electrophoresis (PFGE) results were also compared with the MLST results. Based on the genomic information from strain CCUG18818, 21 housekeeping genes were selected as candidates for MLST and were observed to have high homology (96.5 to 100%) between isolates. Following a comparison of the 21 housekeeping genes from 8 H. cinaedi isolates, 7 genes were chosen for MLST, revealing 14 sequence types (STs). The isolates from 3 hospitals belonged to the same STs, but the isolates from the other 4 hospitals belonged to different STs. Isolates belonging to ST6 were analyzed by PFGE and showed similar, but not identical, patterns between isolates. Isolates belonging to ST9, ST10, and ST11, which belonged to the same clonal complex, had the same pattern. All isolates were found to contain mutations in GyrA and the 23S rRNA gene that confer ciprofloxacin and clarithromycin resistance, respectively, in H. cinaedi. These results raise concerns about the increase in H. cinaedi isolates resistant to clarithromycin and ciprofloxacin in Japan.

DOI： 10.1128/JCM.06810-11

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Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay Reviewed

Nao Otsuka, Shuji Yoshino, Kimiko Kawano, Hiromi Toyoizumi-Ajisaka, Keigo Shibayama, Kazunari Kamachi

MICROBIOLOGY AND IMMUNOLOGY   Vol. 56 ( 7 ) page： 486 - 489   2012.7

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A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.

DOI： 10.1111/j.1348-0421.2012.00465.x

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Transmission of Bordetella holmesii during Pertussis Outbreak, Japan Reviewed

Hajime Kamiya, Nao Otsuka, Yuka Ando, Fumito Odaira, Shuji Yoshino, Kimiko Kawano, Hirokazu Takahashi, Toshihide Nishida, Yoshio Hidaka, Hiromi Toyoizumi-Ajisaka, Keigo Shibayama, Kazunari Kamachi, Tomimasa Sunagawa, Kiyosu Taniguchi, Nobuhiko Okabe

EMERGING INFECTIOUS DISEASES   Vol. 18 ( 7 ) page： 1166 - 1169   2012.7

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We describe the epidemiology of a pertussis outbreak in Japan in 2010-2011 and Bordetella holmesii transmission. Six patients were infected; 4 patients were students and a teacher at the same junior high school. Epidemiologic links were found between 5 patients. B. holmesii may have been transmitted from person to person.

DOI： 10.3201/eid1807.120130

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Reply to Letter to the Editor regarding Shibayama etal.: Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase Reviewed

Shibayama K.

Microbiology and Immunology   Vol. 56 ( 6 ) page： 422 - 422   2012.6

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DOI： 10.1111/j.1348-0421.2012.00459.x

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Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C-diphtheriae NCTC 13129 prophage Reviewed

Tsuyoshi Sekizuka, Akihiko Yamamoto, Takako Komiya, Tsuyoshi Kenri, Fumihiko Takeuchi, Keigo Shibayama, Motohide Takahashi, Makoto Kuroda, Masaaki Iwaki

BMC MICROBIOLOGY   Vol. 12   page： 72   2012.5

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Background: Corynebacterium ulcerans can cause a diphtheria-like illness, especially when the bacterium is lysogenized with a tox gene-carrying bacteriophage that produces diphtheria toxin. Acquisition of toxigenicity upon phage lysogenization is a common feature of C. ulcerans and C. diphtheriae. However, because of a lack of C. ulcerans genome information, a detailed comparison of prophages has not been possible between these two clinically important and closely related bacterial species.
Results: We determined the whole genome sequence of the toxigenic C. ulcerans 0102 isolated in Japan. The genomic sequence showed a striking similarity with that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of C. diphtheriae. The 0102 genome contained three distinct prophages. One of these, (sic)CULC0102-I, was a tox-positive prophage containing genes in the same structural order as for tox-positive C. diphtheriae prophages. However, the primary structures of the individual genes involved in the phage machinery showed little homology between the two counterparts.
Conclusion: Taken together, these results suggest that the tox-positive prophage in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae NCTC 13129.

DOI： 10.1186/1471-2180-12-72

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Nosocomial spread of multidrug-resistant group B streptococci with reduced penicillin susceptibility belonging to clonal complex 1 Reviewed

Noriyuki Nagano, Yukiko Nagano, Masami Toyama, Kouji Kimura, Takashi Tamura, Keigo Shibayama, Yoshichika Arakawa

Journal of Antimicrobial Chemotherapy   Vol. 67 ( 4 ) page： 849 - 856   2012.4

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Background: Multiple group B Streptococcus (GBS) isolates with reduced penicillin susceptibility (PRGBS) were recovered from several patients, hence a probable nosocomial transmission of PRGBS in a hospital setting was suspected. Methods: Ten PRGBS recovered from eight patients in a general hospital were characterized. Sequence analysis of genes for penicillin-binding proteins (PBPs) and quinolone resistance-determining regions (QRDRs) of gyrA, gyrB and parC was performed, and the macrolide resistance genes were detected by PCR. Genetic relatedness among the isolates was examined by PFGE and multilocus sequence typing. Results: All the PRGBS had the key amino acid substitution V405A, together with F395L, R433H, H438Y and G648A in PBP 2X and T567I in PBP 2B. A 23S rRNA methylase gene, erm(B), was also found in all 10 PRGBS strains. PFGE analysis revealed considerable genetic relatedness among the isolates. Isolates of pulsotype I were obtained from four patients in ward A and one patient in ward B, while isolates of pulsotypes II and III were obtained from two patients in ward B and one patient in ward C, respectively. Isolates of pulsotype I were resistant to levofloxacin (MIC >8 mg/L) and had the following amino acid substitutions in the QRDRs: S81L in GyrA, E476K in GyrB and S79Y in ParC. However, pulsotype II strains resistant to levofloxacin (MIC 8 mg/L) had no change in GyrA, but changes in GyrB (E476K) and ParC (S79Y). All 10 PRGBS strains belonged to serotype VI and ST458 (where ST stands for sequence type). Conclusions: This is the first description of the nosocomial spread of multidrug-resistant PRGBS strains belonging to the genetic lineage ST458. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

DOI： 10.1093/jac/dkr546

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Complete genome sequence of Mycoplasma pneumoniae type 2a strain 309, isolated in Japan Reviewed

Tsuyoshi Kenri, Atsuko Horino, Mari Matsui, Yuko Sasaki, Satowa Suzuki, Mitsuo Narita, Hitomi Ohya, Norio Okazaki, Keigo Shibayama

Journal of Bacteriology   Vol. 194 ( 5 ) page： 1253 - 1254   2012.3

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Mycoplasma pneumoniae strain 309, a type 2a (subtype 2 variant) strain of this bacterium, has variations in the P1 protein, which is responsible for attachment of the bacterium to host cells. Here, we report the complete genome sequence of M. pneumoniae strain 309 isolated from a pneumonia patient in Japan. © 2012, American Society for Microbiology.

DOI： 10.1128/JB.06553-11

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Crystallization and preliminary X-ray analysis of the subclass B3 metallo-β-lactamase SMB-1 that confers carbapenem resistance Reviewed

Jun Ichi Wachino, Yoshihiro Yamaguchi, Shigetarou Mori, Yuriko Yamagata, Yoshichika Arakawa, Keigo Shibayama

Acta Crystallographica Section F: Structural Biology and Crystallization Communications   Vol. 68 ( 3 ) page： 343 - 346   2012.3

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The carbapenem-hydrolyzing subclass B3 metallo-β-lactamase SMB-1 was expressed in Escherichia coli and purified. Diffraction data were collected from two types of SMB-1 crystals that were obtained under different conditions. One crystal (SMB-1a) belonged to the trigonal space group P31 with unit-cell parameters a = b = 67.83, c = 48.67 Å, while the other crystal (SMB-1b) also belonged to space group P3 1 but with unit-cell parameters a = b = 67.25, c = 46.83 Å. Both crystals contained one molecule per asymmetric unit. Initial phases were determined by molecular replacement; further refinement and model building are in progress. © 2012 International Union of Crystallography All rights reserved.

DOI： 10.1107/S1744309112004691

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Prevalence and genetic characterization of pertactin-deficient bordetella pertussis in Japan Reviewed

Nao Otsuka, Hyun Ja Han, Hiromi Toyoizumi-Ajisaka, Yukitsugu Nakamura, Yoshichika Arakawa, Keigo Shibayama, Kazunari Kamachi

PLoS ONE   Vol. 7 ( 2 ) page： e31985   2012.2

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The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn -) B. pertussis was observed in Japan. The Prn - isolate was first discovered in 1997, and 33 (27%) Prn - isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn - isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn - isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn - isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn - isolates belong to MLVA-186, and 6 and 3 Prn - isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn - clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn - isolates have a higher growth potential than the Prn + back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn - strains have arisen and that Prn expression is not essential for fitness under these conditions. © 2012 Otsuka et al.

DOI： 10.1371/journal.pone.0031985

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Predominance of sequence type 1 group with serotype VI among group B streptococci with reduced penicillin susceptibility identified in japan Reviewed

Kouji Kimura, Noriyuki Nagano, Yukiko Nagano, Jun ichi Wachino, Satowa Suzuki, Keigo Shibayama, Yoshichika Arakawa

Journal of Antimicrobial Chemotherapy   Vol. 66 ( 11 ) page： 2460 - 2464   2011.11

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Background: Although group B Streptococcus (GBS; i.e. Streptococcus agalactiae) has been considered to be uniformly susceptible to β-lactams, GBS isolates with reduced penicillin susceptibility (PRGBS) have been reported from Japan and North America. In this study, PRGBS from Japan were characterized by multilocus sequence typing (MLST) and the results compared with data on PRGBS reported from the USA. Methods: Twenty-eight clinical isolates of PRGBS recovered in Japan (including 22 isolates previously analysed by PFGE) were analysed by MLST and eBURST (http://eburst.mlst.net/). Results: Twenty-three isolates were found to belong to the sequence type 1 (ST1) group (11 ST458, 7 ST1, 3 ST297, 1 ST358 and 1 ST4), while the remaining 5 isolates formed the ST23 group. Among 11 ST458 and 7 ST1 isolates, 9 and 4 were serotype VI, respectively, indicating a probable correlation between the ST1 group and serotype VI for PRGBS in Japan. Conclusions: PRGBS in Japan could be classified into at least two ST groups, ST1 and ST23, which are genetically different from the ST19 PRGBS isolated in the USA, though five allele variations were seen between ST1 and ST19, implying a slight genetic relatedness. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

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SMB-1, a novel subclass B3 metallo-β-lactamase, associated with ISCR1 and a class 1 integron, from a carbapenem-resistant Serratia marcescens clinical isolate Reviewed

Jun Ichi Wachino, Hiroyuki Yoshida, Kunikazu Yamane, Satowa Suzuki, Mari Matsui, Takuya Yamagishi, Atsuko Tsutsui, Toshifumi Konda, Keigo Shibayama, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 55 ( 11 ) page： 5143 - 5149   2011.11

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A carbapenem-resistant Serratia marcescens strain, 10mdr148, was identified in a Japanese hospital in 2010. The carbapenem resistance of this strain was attributed to the production of a novel metallo-β-lactamase (MBL), named SMB-1 (Serratia metallo-β-lactamase). SMB-1 possessed a zinc binding motif, H(Q)XHXDH (residues 116 to 121), H196, and H263 and was categorized as a member of subclass B3 MBL. SMB-1 has 75% amino acid identity with the most closely related MBL, AMO1, of uncultured bacterium, recently identified through the metagenomic analysis of apple orchard soil. The introduction of bla SMB-1 into Escherichia coli conferred resistance to a variety of β-lactam antibiotics, penicillins, cephalosporins, and carbapenems, but not aztreonam, a resistance pattern consistent with those of other MBLs. SMB-1 demonstrated high k cat values of >500 s -1 for carbapenems, resulting in the highest hydrolyzing efficiency (k cat/K m) among the agents tested. The hydrolyzing activity of SMB-1 was well inhibited by chelating agents. The bla SMB-1 gene was located on the chromosome of S. marcescens strain 10mdr148 and at the 3′ end of the ISCR1 element in complex with a typical class 1 integron carrying aac(6′)-Ib and catB3 gene cassettes. Downstream of bla SMB-1, the second copy of the 3′ conserved segment and ISCR1 were found. To our knowledge, this is the first subclass B3 MBL gene associated with an ISCR1 element identified in an Enterobacteriaceae clinical isolate. A variety of antibiotic resistance genes embedded with ISCR1 have been widely spread among Enterobacteriaceae clinical isolates, thus the further dissemination of bla SMB-1 mediated by ISCR1 transposition activity may become a future concern. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

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Structural insights into the novel diadenosine 5′,5‴-P ¹,P⁴-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv Reviewed International journal

Shigetarou Mori, Keigo Shibayama, Jun Ichi Wachino, Yoshichika Arakawa

Journal of Molecular Biology   Vol. 410 ( 1 ) page： 93 - 104   2011.7

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Rv2613c is a diadenosine 5′,5‴-P1,P 4-tetraphosphate (Ap4A) phosphorylase from Mycobacterium tuberculosis H37Rv. Sequence analysis suggests that Rv2613c belongs to the histidine triad (HIT) motif superfamily, which includes HIT family diadenosine polyphosphate (ApnA) hydrolases and Ap4A phosphorylases. However, the amino acid sequence of Rv2613c is more similar to that of HIT family ApnA hydrolases than to that of typical Ap4A phosphorylases. Here, we report the crystal structure of Rv2613c, which is the first structure of a protein with ApnA phosphorylase activity, and characterized the structural basis of its catalytic activity. Our results showed that the structure of Rv2613c is similar to those of other HIT superfamily proteins. However, Asn139, Gly146, and Ser147 in the active site of Rv2613c replace the corresponding Gln, Gln, and Thr residues that are normally found in HIT family ApnA hydrolases. Furthermore, analyses of Rv2613c mutants revealed that Asn139, Gly146, and Ser147 are important active-site residues and that Asn139 has a critical role in catalysis. The position of Gly146 might influence the phosphorylase activity. In addition, the tetrameric structure of Rv2613c and the presence of Trp160 might be essential for the formation of the Ap4A binding site. These structural insights into Rv2613c may facilitate the development of novel structure-based inhibitors for treating tuberculosis. © 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2011.04.059

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Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase Reviewed International journal

Keigo Shibayama, Hiroaki Takeuchi, Jun ichi Wachino, Shigetarou Mori, Yoshichika Arakawa

Microbiology and Immunology   Vol. 55 ( 6 ) page： 408 - 417   2011.6

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Asparaginase was purified from Helicobacter pylori 26695 and its pathophysiological role explored. The Km value of asparagine was 9.75 ± 1.81 μM at pH 7.0, and the optimum pH range was broad and around a neutral pH. H. pylori asparaginase converted extracellular asparagine to aspartate. H. pylori cells were unable to take up extracellular asparagine directly. Instead, aspartate produced by the action of the asparaginase was transported into H. pylori cells, where it was partially converted to β-alanine. Asparaginase exhibited striking cytotoxic activity against histiocytic lymphoma cell line U937 cells via asparagine deprivation. The cytotoxic activity of live H. pylori cells against U937 cells was significantly diminished by deletion of the asparaginase gene, indicating that asparaginase functions as a cytotoxic agent of the bacterium. The cytotoxic effect was negligible for gastric epithelial cell line AGS cells, suggesting that the effect differs across host cell types. An asparaginase-deficient mutant strain was significantly less capable of colonizing Mongolian gerbils. Since asparagine depletion by exogenous asparaginase has been shown to suppress lymphocyte proliferation in vivo, the present results suggest that H. pylori asparaginase may be involved in inhibition of normal lymphocyte function at the gastric niche, allowing H. pylori to evade the host immune system. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

DOI： 10.1111/j.1348-0421.2011.00333.x

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Report of an international collaborative study to evaluate the suitability of multiplex PCR as an identity assay for different sub-strains of BCG vaccine Reviewed International journal

Kevin Markey, Mei M. Ho, Babna Choudhury, Masaaki Seki, Liu Ju, Luiz R.R. Castello-Branco, Sunil Gairola, Aihua Zhao, Keigo Shibayama, Murielle Andre, Michael J. Corbel

Vaccine   Vol. 28 ( 43 ) page： 6964 - 6969   2010.10

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Current methods for the identification of BCG vaccine in quality control settings involve acid-fast staining with microscopic examination. However, this method is unable to distinguish the many different sub-strains of BCG, or to differentiate BCG strains from virulent members of the Mycobacterium tuberculosis complex. A multiplex PCR (mPCR) which uses six target regions in mycobacteria has been developed to identify specific sub-strains of BCG. This study reports the findings from an international collaborative study to assess the accuracy, robustness and reproducibility of this mPCR method to differentiate BCG sub-strains. The method was found to fulfil these criteria successfully and was able to distinguish BCG sub-strains in vaccine preparations. The majority of the participants in the study generated the expected PCR product profiles indicating the method is also robust. © 2010 Elsevier Ltd.

DOI： 10.1016/j.vaccine.2010.08.045

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RmtC introduces G1405 methylation in 16S rRNA and confers high-level aminoglycoside resistance on Gram-positive microorganisms Reviewed

Jun Ichi Wachino, Keigo Shibayama, Kouji Kimura, Kunikazu Yamane, Satowa Suzuki, Yoshichika Arakawa

FEMS Microbiology Letters   Vol. 311 ( 1 ) page： 56 - 60   2010.10

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Seven plasmid-mediated 16S rRNA methyltransferases (MTases), RmtA, RmtB, RmtC, RmtD, RmtE, ArmA, and NpmA, conferring aminoglycoside resistance have so far been found in Gram-negative pathogenic microorganisms. In the present study, by performing an RNase protection assay, primer extension, and HPLC, we confirmed that RmtC indeed methylates at the N7 position of nucleotide G1405 in 16S rRNA as found in ArmA and RmtB. RmtC has an MTase activity specific for the bacterial 30S ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but not for the naked 16S rRNA, as seen in ArmA, RmtB, and NpmA. All seven 16S rRNA MTases have been found exclusively in Gram-negative bacilli to date, and no plasmid-mediated 16S rRNA MTase has been reported in Gram-positive pathogenic microorganisms. Thus, we checked whether or not the RmtC could function in Gram-positive bacilli, and found that RmtC could indeed confer high-level resistance to gentamicin and kanamycin in Bacillus subtilis and Staphylococcus aureus. 16S rRNA MTases seemed to be functional to some extent in any bacterial species, regardless of the provenance of the 16S rRNA MTase gene responsible for aminoglycoside resistance. © 2010 Federation of European Microbiological Societies.

DOI： 10.1111/j.1574-6968.2010.02068.x

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Profile of Expression of Helicobacter pylori gamma-glutamyltranspeptidase. Reviewed International journal

Jun-Ichi Wachino, Keigo Shibayama, Satowa Suzuki, Kunikazu Yamane, Shigetarou Mori, Yoshichika Arakawa

Helicobacter   Vol. 15 ( 3 ) page： 184 - 92   2010.6

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BACKGROUND: Helicobacter pylori produces gamma-glutamyltranspeptidase (GGT), a potential virulence factor involved in induction of host cell apoptosis. Regulation of the production of this protein is not known. METHODS: The transcription start sites were determined by primer extension analysis. Transcription level of the GGT gene was examined by measuring the mRNA by RT-PCR and expression level of GGT protein was examined by Western blot analysis under different conditions. RESULTS: Two transcription start sites were identified; thymine at 78-bp upstream and adenine at 79-bp upstream from the ATG codon of the GGT gene. There was a possible -10 consensus promoter sequence (ATTAAT), but no apparent -35 consensus sequence was found. The transcription of the mRNA and the expression of the protein were at almost constant level during the course of culture. The mRNA level increased by exposure to low pH; however, the actual protein expression level remained almost constant. Addition of glutamine or glutamate did not affect the mRNA level and the protein expression level to a remarkable degree, nor did co-culture with AGS cells affect the GGT activity level. CONCLUSION: It was suggested that H. pylori GGT is constitutively expressed under various conditions.

DOI： 10.1111/j.1523-5378.2010.00755.x

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Virulence genes, quinolone and fluoroquinolone resistance, and phylogenetic background of uropathogenic Escherichia coli strains isolated in Japan Reviewed

Kawamura-Sato K.

Japanese Journal of Infectious Diseases   Vol. 63 ( 2 ) page： 113 - 115   2010.4

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Virulence Genes, Quinolone and Fluoroquinolone Resistance, and Phylogenetic Background of Uropathogenic Escherichia coli Strains Isolated in Japan Reviewed

Kawamura-Sato Kumiko, Yoshida Risa, Shibayama Keigo, Ohta Michio

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 63 ( 2 ) page： 113 - 115   2010.3

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Virulence genes, quinolone and fluoroquinolone resistance, and phylogenetic background of uropathogenic Escherichia coli strains isolated in Japan. Reviewed

Kawamura-Sato K, Yoshida R, Shibayama K, Ohta M

Japanese journal of infectious diseases   Vol. 63 ( 2 ) page： 113 - 5   2010.3

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Purification and molecular characterization of a novel diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv Reviewed International journal

Shigetarou Mori, Keigo Shibayama, Jun ichi Wachino, Yoshichika Arakawa

Protein Expression and Purification   Vol. 69 ( 1 ) page： 99 - 105   2010.1

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In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5′-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5′-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the Km values for Ap4A and phosphate were 0.10 and 0.94 mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif. © 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2009.09.010

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Crystallization and preliminary X-ray analysis of the diadenosine 5′,5′′′-P ¹,P ⁴-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv Reviewed International journal

Shigetarou Mori, Keigo Shibayama, Jun Ichi Wachino, Yoshichika Arakawa

Acta Crystallographica Section F: Structural Biology and Crystallization Communications   Vol. 66 ( 3 ) page： 279 - 281   2010

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A novel diadenosine 5′,5′′′-P 1,P 4-tetraphosphate (Ap4A) phosphorylase (Rv2613c) from Mycobacterium tuberculosis H37Rv has been crystallized by the sitting-drop vapour-diffusion method. The crystal belonged to space group C2, with unit-cell parameters a = 101.5, b = 63.6, c = 79.1 Å, Β = 110.9°. The diffraction of the crystals extended to 1.9 Å resolution. The asymmetric unit is expected to contain two molecules of Rv2613c, with a corresponding crystal volume per protein weight (VM) of 2.41 Å3 Da-1 and a solvent content of 49.1%. This is the first report of a crystal of Ap4A phosphorylase. © 2010 International Union of Crystallography All rights reserved.

DOI： 10.1107/S174430910905444X

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The nosocomial transmission of Helicobacter cinaedi infections in immunocompromised patients Reviewed

Koichiro Minauchi, Shunji Takahashi, Toshiya Sakai, Makoto Kondo, Keigo Shibayama, Yoshichika Arakawa, Masaya Mukai

Internal Medicine   Vol. 49 ( 16 ) page： 1733 - 1739   2010

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Background We encountered 15 cases of Helicobacter cinaedi (H. cinaedi) infection between March and July 2008. Patient, Method, and Result The underlying diseases were hematological malignancies in a majority of cases, many of which received chemotherapy. All patients had a fever. The fever was followed by cellulitis in three, a skin rash in six, pain in the lower limbs in three, and diarrhea in three cases. We analyzed the bacterial 23S rRNA genes. The fifteen strains were divided according to base sequence into Groups A, B, and C, respectively. All four cases in Group A were women and all ten in Group C were men, indicating that the gender of the patient corresponded precisely to the genotypes of the separated bacilli in these two groups. These findings also suggested the strong possibility of nosocomial spread. Conclusion It is highly likely that H. cinaedi infections have been overlooked due to the difficulties encountered in culturing the bacterium. The possibility of septicemia caused by H. cinaedi should be suspected especially in immunocompromised patients such as those undergoing chemotherapy, with symptoms such as fever, rash, arthritis, cellulitis, leg pain, and other systemic or local symptoms. © 2010 The Japanese Society of Internal Medicine.

DOI： 10.2169/internalmedicine.49.3649

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A novel insertion sequence, IS1642, of Mycobacterium avium, which forms long direct repeats of variable length. Reviewed International journal

Zhenyu Piao, Keigo Shibayama, Shigetarou Mori, Jun-ichi Wachino, Yoshichika Arakawa

FEMS microbiology letters   Vol. 291 ( 2 ) page： 216 - 21   2009.2

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A new insertion sequence (IS), IS1642, was identified in a Mycobacterium avium strain isolated from a human patient. IS1642 had a size of 1642 bp and contained a single ORF encoding a probable transposase of 503 amino acid residues homologous (79% identity) to that of IS1549 found in Mycobacterium smegmatis. The IS1642 included imperfect inverted repeats (5'-cctgacttttatca-3', 5'-tgataaaagtcggg-3') on its ends, and was flanked by direct repeats of variable length ranging from 5 to 161 bp. It was suggested that the IS1642 was widely distributed in many M. avium strains of human patients, and the Southern blot profile of IS1642 was very diverse among the strains examined. The transposition event of IS1642 was observed by in vitro repeated passages, showing that the IS1642 is actually a transposable element. In light of these characteristics, IS1642 could be a new useful marker when genotyping with high discrimination is required.

DOI： 10.1111/j.1574-6968.2008.01459.x

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The sodium-dependent D-glucose transport protein of Helicobacter pylori Reviewed International journal

Georgios Psakis, Massoud Saidijam, Keigo Shibayama, Julia Polaczek, Kim E. Bettaney, Jocelyn M. Baldwin, Stephen A. Baldwin, Ryan Hope, Lars Oliver Essen, Richard C. Essenberg, Peter J.F. Henderson

Molecular Microbiology   Vol. 71 ( 2 ) page： 391 - 403   2009.1

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Helicobacter pylori is a Gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize d-glucose and/or d-galactose in both oxidative and fermentative pathways via a Na +-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na+-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled d-glucose, d-galactose and 2-deoxy-d-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. d-Mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-d-glucose, d-mannose, d-glucose or d-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family. © 2008 The Authors.

DOI： 10.1111/j.1365-2958.2008.06535.x

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[Molecular mechanism of Helicobacter pylori-mediated pathogenesis]. Reviewed

Keigo Shibayama

Nihon saikingaku zasshi. Japanese journal of bacteriology   Vol. 63 ( 2-4 ) page： 387 - 90   2008.12

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DOI： 10.3412/jsb.63.387

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First molecular characterization of group B streptococci with reduced penicillin susceptibility Reviewed International journal

Kouji Kimura, Satowa Suzuki, Jun Ichi Wachino, Hiroshi Kurokawa, Kunikazu Yamane, Naohiro Shibata, Noriyuki Nagano, Haru Kato, Keigo Shibayama, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 52 ( 8 ) page： 2890 - 2897   2008.8

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Group B Streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 μg/ml, 2 to 8 μg/ml, and 4 to 128 μg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of β-lactam-insusceptible GBS from a phenotypic standpoint. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.00185-08

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Novel plasmid-mediated 16S rRNA m¹A1408 methyltransferase, NpmA, found in a clinically isolated Escherichia coli strain resistant to structurally diverse aminoglycosides Reviewed

Jun Ichi Wachino, Keigo Shibayama, Hiroshi Kurokawa, Kouji Kimura, Kunikazu Yamane, Satowa Suzuki, Naohiro Shibata, Yasuyoshi Ike, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 51 ( 12 ) page： 4401 - 4409   2007.12

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We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.00926-07

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New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate Reviewed

Kunikazu Yamane, Jun Ichi Wachino, Satowa Suzuki, Kouji Kimura, Naohiro Shibata, Haru Kato, Keigo Shibayama, Toshifumi Konda, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 51 ( 9 ) page： 3354 - 3360   2007.9

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Plasmid-mediated Qnr and AAC(6′)-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrB-TolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.00339-07

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Profile of expression of Helicobacter pylari gamma-glutamyltranspeptidase

K. Shibayama, J. Wachino, S. Suzuki, Y. Arakawa

HELICOBACTER   Vol. 12 ( 4 ) page： 412 - 412   2007.8

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16S rRNA methylase-producing, gram-negative pathogens, Japan Reviewed

Kunikazu Yamane, Jun Ichi Wachino, Satowa Suzuki, Naohiro Shibata, Haru Kato, Keigo Shibayama, Kouji Kimura, Kumiko Kai, Satoshi Ishikawa, Yoshiyuki Ozawa, Toshifumi Konda, Yoshichika Arakawa

Emerging Infectious Diseases   Vol. 13 ( 4 ) page： 642 - 646   2007.4

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To investigate the exact isolation frequency of 16S rRNA methylase-producing, gram-negative pathogenic bacteria, we tested 87,626 clinical isolates from 169 hospitals. Twenty-six strains from 16 hospitals harbored 16S rRNA methylase genes, which suggests sparse but diffuse spread of pan-aminoglycoside-resistant microbes in Japan.

DOI： 10.3201/eid1304.060501

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Metabolism of glutamine and glutathione via γ-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: Possible significance in the pathophysiology of the organism Reviewed International journal

Keigo Shibayama, Jun Ichi Wachino, Yoshichika Arakawa, Massoud Saidijam, Nicholas G. Rutherford, Peter J.F. Henderson

Molecular Microbiology   Vol. 64 ( 2 ) page： 396 - 406   2007.4

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γ-Glutamyltranspeptidase (GGT) is a periplasmic enzyme of Helicobacter pylori implicated in its pathogenesis towards mammalian cells. We have cloned and expressed the H. pylori strain 26695 recombinant GGT protein in Escherichia coli and purified it to homogeneity. The purified protein exhibited hydrolysis activity with very high affinities for glutamine and glutathione shown by apparent Km values lower than 1 μM. H. pylori cells were unable to take up extracellular glutamine and glutathione directly. Instead, these substances were hydrolysed to glutamate by the action of GGT outside the cells. The glutamate produced was then transported by a Na+-dependent reaction into H. pylori cells, where it was mainly incorporated into the TCA cycle and partially utilized as a substrate for glutamine synthesis. These observations show that one of the principle physiological functions of H. pylori GGT is to enable H. pylori cells to utilize extracellular glutamine and glutathione as a source of glutamate. As glutamine and glutathione are important nutrients for maintenance of healthy gastrointestinal tissue, their depletion by the GGT enzyme is hypothesized to account for the damaging of mammalian cells and the pathophysiology of H. pylori. © 2007 The Authors.

DOI： 10.1111/j.1365-2958.2007.05661.x

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Quantification of two variant strains contained in freeze-dried Japanese BCG vaccine preparation by real-time PCR Reviewed

Keigo Shibayama, Keiko Mochida, Tetsuya Yagi, Shigetaro Mori, Yoshichika Arakawa, Saburo Yamamoto

Biologicals   Vol. 35 ( 2 ) page： 139 - 143   2007.4

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Japanese bacillus Calmette-Guerin (BCG) vaccine preparation contains two types of variant strains, Type I, which has a 22-base-pair deletion in the RD16 region, and Type II, which has an identical sequence to those of other BCG strains. In this study, we established a method to quantify the percentage of variant strain Type II contained in freeze-dried BCG product with real-time PCR. With this method we examined the master seed lot Tokyo 172, two secondary seed lots, Tokyo 172-1 and Tokyo 172-2, which were produced from Tokyo 172, and four commercial lots produced form Tokyo 172-1. Tokyo 172, Tokyo 172-1, and Tokyo 172-2 contained 55.1%, 19.5%, and 3.6% of Type II variant strain, respectively. Commercial lots contained 1.5%, 4.5%, 7.4%, and 4.3% of Type II variant strain, respectively. These results indicated that the two variant strains contained in the master seed lot continued to coexist in subsequently produced lots with a decrease in population of variant strain Type II. This method would be useful for quality control of commercial Japanese BCG vaccine preparations. © 2006 The International Association for Biologicals.

DOI： 10.1016/j.biologicals.2006.07.005

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Usefulness of adult bovine serum for Helicobacter pylori culture media Reviewed

Keigo Shibayama, Mitsuaki Nagasawa, Takafumi Ando, Masaaki Minami, Jun Ichi Wachino, Satowa Suzuki, Yoshichika Arakawa

Journal of Clinical Microbiology   Vol. 44 ( 11 ) page： 4255 - 4257   2006.11

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Fetal bovine serum (FBS) and adult bovine serum (BS) exhibited bactericidal activity against Helicobacter pylori at various levels, which were higher in BS than in FBS. The bactericidal activity was inactivated by heat treatment at 56°C for 30 min. Our results demonstrated that heat-treated BS is a useful serum source of H. pylori culture medium. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/JCM.00477-06

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PCR classification of CTX-M-type β-lactamase genes identified in clinically isolated gram-negative bacilli in Japan Reviewed

Naohiro Shibata, Hiroshi Kurokawa, Yohei Doi, Tetsuya Yagi, Kunikazu Yamane, Jun Ichi Wachino, Satowa Suzuki, Kouji Kimura, Satoshi Ishikawa, Haru Kato, Yoshiyuki Ozawa, Keigo Shibayama, Kumiko Kai, Toshifumi Konda, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 50 ( 2 ) page： 791 - 795   2006.2

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Of 1,456 strains isolated from 2001 to 2003 demonstrating resistance to either oxyimino-cephalosporin, 317 strains, isolated in 57 of 132 clinical facilities, were found to harbor bla/CTX-M genes by PCR. Fifty-seven, 161, and 99 strains harbored bla/CTX-M genes belonging to the blaCTX-M-1, bla/CTX-M-2, and bla/CTX-M-9 clusters, respectively. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.50.2.791-795.2006

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Novel plasmid-mediated 16S rRNA methylase, RmtC, found in a Proteus mirabilis isolate demonstrating extraordinary high-level resistance against various aminoglycosides Reviewed

Jun Ichi Wachino, Kunikazu Yamane, Keigo Shibayama, Hiroshi Kurokawa, Naohiro Shibata, Satowa Suzuki, Yohei Doi, Kouji Kimura, Yasuyoshi Ike, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 50 ( 1 ) page： 178 - 184   2006.1

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Proteus mirabilis ARS68, which demonstrated a very high level of resistance to various aminoglycosides, was isolated in 2003 from an inpatient in Japan. The aminoglycoside resistance of this strain could not be transferred to recipient strains Escherichia coli CSH-2 and E. coli HB101 by a general conjugation experiment, but E. coli DHSα was successfully transformed by electroporation with the plasmid of the parent strain, ARS68, and acquired an unusually high degree of resistance against aminoglycosides. Cloning and sequencing analyses revealed that the presence of a novel 16S rRNA methylase gene, designated rmtC, was responsible for resistance in strain ARS68 and its transformant. The G+C content of rmtC was 41.1%, and the deduced amino acid sequences of the newly identified 16S rRNA methylase, RmtC, shared a relatively low level of identity (£29%) to other plasmid-mediated 16S rRNA methylases, RmtA, RmtB, and ArmA, which have also been identified in pathogenic gram-negative bacilli. Also, RmtC shared a low level of identity (≤28%) with the other 16S rRNA methylases found in aminoglycoside-producing actinomycetes. The purified histidine-tagged RmtC clearly showed niethyltransferase activity against E. coli 16S rRNA in vitro. rmtC was located downstream of an ISEcp1-like element containing trip A. Several plasmid-mediated 16S rRNA methylases have been identified in pathogenic gram-negative bacilli belonging to the family Enterobacteriaceae, and some of them are dispersing worldwide. The acceleration of aminoglycoside resistance among gram-negative bacilli by producing plasmid-mediated 16S rRNA methylases, such as RmtC, RmtB, and RmtA, may indeed become an actual clinical hazard in the near future. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.50.1.178-184.2006

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Pathophysiological role of Helicobacter pylori gamma-glutamyltranspeptidase

Shibayama K, Wachino J, Arakawa Y, Saidijam M, Rutherford N, Henderson P

Helicobacter   Vol. 11 ( 4 ) page： 355-356   2006

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Active membrane transport and receptor proteins from bacteria Reviewed

M. Saidijam, K. E. Bettaney, G. Szakonyi, G. Psakis, K. Shibayama, S. Suzuki, J. L. Clough, V. Blessie, A. Abu-Bakr, S. Baumberg, J. Meuller, C. K. Hoyle, S. L. Palmer, P. Butaye, K. Walravens, S. G. Patching, J. O'Reilly, N. G. Rutherford, R. M. Bill, D. I. Roper, M. K. Phillips-Jones, P. J.F. Henderson

Biochemical Society Transactions   Vol. 33 ( 4 ) page： 867 - 872   2005.8

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A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides. ©2005 Biochemical Society.

DOI： 10.1042/BST0330867

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Practical methods using boronic acid compounds for identification of class C β-lactamase-producing Klebsiella pneumoniae and Escherichia coli Reviewed

Tetsuya Yagi, Jun Ichi Wachino, Hiroshi Kurokawa, Satowa Suzuki, Kunikazu Yamane, Yohei Doi, Naohiro Shibata, Haru Kato, Keigo Shibayama, Yoshichika Arakawa

Journal of Clinical Microbiology   Vol. 43 ( 6 ) page： 2551 - 2558   2005.6

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Detection of the resistance mediated by class C β-lactamases remains a challenging issue, considering that transferable plasmid-mediated class C β-lactamases are of worldwide concern. Methods for the identification of strains that produce extended-spectrum β-lactamases (ESBLs) or metallo-β-lactamases (MBLs) have been developed and applied for routine use in clinical microbiology laboratories, but no practical methods for identification of plasmid-mediated class C producers have been established to date. We therefore developed three simple methods for clinical microbiology laboratories that allow identification of plasmid-mediated class C β-lactamase-producing bacteria using a boronic acid derivative, 3-aminophenylboronic acid (APB), one of the specific inhibitors of class C β-lactamases. Detection by the disk potentiation test was based on the enlargement of the growth-inhibitory zone diameter (by greater than or equal to 5 mm) around a Kirby-Bauer disk containing a ceftazidime (CAZ) or a cefotaxime (CTX) disk in combination with APB. In a double-disk synergy test, the discernible expansion of the growth-inhibitory zone around the CAZ or the CTX disk toward a disk containing APB was indicative of class C β-lactamase production. A greater than or equal to eightfold decrease in the MIC of CAZ or CTX in the presence of APB was the criterion for detection in the microdilution test. By using these methods, Escherichia coli and Klebsiella pneumoniae isolates producing plasmid-mediated class C β-lactamases, ACT-1, CMY-2, CMY-9, FOX-5, LAT-1, and MOX-1, were successfully distinguished from those producing other classes of β-lactamases, such as ESBLs and MBLs. These methods will provide useful information needed for targeted antimicrobial therapy and better infection control. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/JCM.43.6.2551-2558.2005

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Inhibitor-sensitive AmpC β-lactamase variant produced by an Escherichia coli clinical isolate resistant to oxyiminocephalosporins and cephamycins Reviewed

Yohei Doi, Jun Ichi Wachino, Masaji Ishiguro, Hiroshi Kurokawa, Kunikazu Yamane, Naohiro Shibata, Keigo Shibayama, Keiko Yokoyama, Haru Kato, Tetsuya Yagi, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 48 ( 7 ) page： 2652 - 2658   2004.7

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Escherichia coli HKY28, a ceftazidime-resistant strain isolated from a urine specimen in Japan, produced an inhibitor-sensitive AmpC β-lactamase variant. The deduced amino acid sequence of the enzyme contained a number of substitutions and a tripeptide deletion (Gly286-Ser287-Asp288) compared with the sequence of native AmpC of E. coli. When the deletion was reverted by a 9-base insertion at the relevant site of ampC in the clone, the typical inhibitor-resistant phenotype of AmpC was restored, while at the same time the levels of resistance to ceftazidime, cefpirome, and cefepime were reduced eightfold or more. Molecular modeling studies indicated that a structural change took place in the H-10 helix as a result of the deletion, and this change caused an alteration of the substrate binding site, leading to a unique phenotype analogous to that of inhibitor-sensitive class A extended-spectrum β-lactamases. The degree of inhibition was greater with sulbactam and tazobactam than with clavulanic acid. To our knowledge, this is the first report to have characterized an E. coli ampC that encodes chromosomal AmpC β-lactamase sensitive to the available β-lactamase inhibitors.

DOI： 10.1128/AAC.48.7.2652-2658.2004

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Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates Reviewed

Kunikazu Yamane, Yohei Doi, Keiko Yokoyama, Tetsuya Yagi, Hiroshi Kurokawa, Naohiro Shibata, Keigo Shibayama, Haru Kato, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 48 ( 6 ) page： 2069 - 2074   2004.6

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Nine Pseudomonas aeruginosa strains showing very high levels of resistance to various aminoglycosides have been isolated from clinical specimens in seven separate Japanese hospitals in five prefectures since 1997. These strains harbor the newly identified 16S rRNA methylase gene (rmtA). When an rmtA gene probe was hybridized with genomic DNAs of the nine strains digested with EcoRI, two distinct patterns were observed. The 11.1- and 15.8-kb regions containing the rmtA genes of strains AR-2 and AR-11, respectively, were sequenced and compared. In strain AR-2, a transposase gene-like sequence (sequence 1) and a probable tRNA ribosyltransferase gene (orfA) were located upstream of rmtA, and a Na+/H+ antiporter gene-like sequence (sequence 2) was identified downstream of rmtA. This 6.2-kbp insert (the rmtA locus) was flanked by 262-bp κγ elements. Part of the orfQ gene adjacent to an inverted repeat was found outside of the rmtA locus. In strain AR-11, the rmtA gene and sequence 2 were found, but the 5′ end of the orfA gene was truncated and replaced with IS6100. An orfQ-orfI region was present on each side of the rmtA gene in strain AR-11. The G+C content of the rmt4 gene was about 55%, and since the newly identified rmtA gene may well be mediated by some mobile genetic elements such as Tn5041, further dissemination of the rmtA gene could become an actual clinical problem in the near future.

DOI： 10.1128/AAC.48.6.2069-2074.2004

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Spread of novel aminoglycoside resistance gene aac(6′)-Iad among Acinetobacter clinical isolates in Japan Reviewed

Yohei Doi, Jun Ichi Wachino, Kunikazu Yamane, Naohiro Shibata, Tetsuya Yagi, Keigo Shibayama, Haru Kato, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 48 ( 6 ) page： 2075 - 2080   2004.6

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A novel aminoglycoside resistance gene, aac(6′)-Iad, encoding aminoglycoside 6′-N-acetyltransferase, was identified in Acinetobacter genospecies 3 strain A.51. The gene encoded a 144-amino-acid protein, which shared modest identity (up to 36.7%) with some of the aminoglycoside 6′-N-acetyltransferases. The results of high-pressure liquid chromatography assays confirmed that the protein is a functional aminoglycoside 6′-N-acetyltransferase. The enzyme conferred resistance to amikacin, tobramycin, sisomicin, and isepamicin but not to gentamicin. The prevalence of this gene among Acinetobacter clinical isolates in Japan was then investigated. Of 264 Acinetobacter sp. strains isolated from geographically diverse areas in Japan in 2002, 16 were not susceptible to amikacin, and aac(6′)-Iad was detected in 7. Five of the producers of aminoglycoside 6′ -N-acetyltransferase type Iad were identified as Acinetobacter baumannii, and two were identified as Acinetobacter genospecies 3. These results suggest that aac(6′)-Iad plays a substantial role in amikacin resistance among Acinetobacter spp. in Japan.

DOI： 10.1128/AAC.48.6.2075-2080.2004

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[Helicobacter pylori infection and epithelial cell signaling in gastric pathogenesis]. Reviewed

Keigo Shibayama, Yoshichika Arakawa

Nihon saikingaku zasshi. Japanese journal of bacteriology   Vol. 59 ( 2 ) page： 415 - 24   2004.5

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DOI： 10.3412/jsb.59.415

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Plasmid-Mediated 16S rRNA Methylase in Serratia marcescens Conferring High-Level Resistance to Aminoglycosides Reviewed

Yohei Doi, Keiko Yokoyama, Kunikazu Yamane, Jun Ichi Wachino, Naohiro Shibata, Tetsuya Yagi, Keigo Shibayama, Haru Kato, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 48 ( 2 ) page： 491 - 496   2004.2

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Serratia marcescens S-95, which displayed an unusually high degree of resistance to aminoglycosides, including kanamycins and gentamicins, was isolated in 2002 from a patient in Japan. The resistance was mediated by a large plasmid which was nonconjugative but transferable to an Escherichia coli recipient by transformation. The gene responsible for the aminoglycoside resistance was cloned and sequenced. The deduced amino acid sequence of the resistance gene shared 82% identity with RmtA, which was recently identified as 16S rRNA methylase conferring high-level aminoglycoside resistance in Pseudomonas aeruginosa. Histidine-tagged recombinant protein showed methylation activity against E. coli 16S rRNA. The novel aminoglycoside resistance gene was therefore designated rmtB. The genetic environment of rmtB was further investigated. The sequence immediately upstream of rmtB contained the right end of transposon Tn3, including blaTEM, while an open reading frame possibly encoding a transposase was identified downstream of the gene. This is the first report describing 16S rRNA methylase production in S. marcescens. The aminoglycoside resistance mechanism mediated by production of 16S rRNA methylase and subsequent ribosomal protection used to be confined to aminoglycoside-producing actinomycetes. However, it is now identified among pathogenic bacteria, including Enterobacteriaceae and P. aeruginosa in Japan. This is a cause for concern since other treatment options are often limited in patients requiring highly potent aminoglycosides such as amikacin and tobramycin.

DOI： 10.1128/AAC.48.2.491-496.2004

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Growth competition of macrolide-resistant and -susceptible Helicobacter pylori strains Reviewed

Kyoto Kanai, Keigo Shibayama, Satowa Suzuki, Jun Ichi Wachino, Yoshichika Arakawa

Microbiology and Immunology   Vol. 48 ( 12 ) page： 977 - 980   2004

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We examined population dynamics in a mixed culture of clonally related macrolide-resistant and -susceptible Helicobacter pylori strains isolated from a single patient. The resistant strain had a macrolide resistance-conferring A2143G mutation in the 23S rRNA gene. The growth rate of these two strains did not apparently differ when cultured separately. On the other hand, by conducting sequential passage of a mixed culture of the resistant and the susceptible strains, the ratio of the resistant strain to the susceptible strain in the culture typically decreased per passage, indicating that the resistance imposed a significant disadvantage on bacterial fitness in the population.

DOI： 10.1111/j.1348-0421.2004.tb03628.x

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A novel apoptosis-inducing protein from Helicobacter pylori Reviewed

Shibayama K, Kamachi K, Nagata N, Yagi T, Nada T, Doi YH, Shibata N, Yokoyama K, Yamane K, Kato H, Iinuma Y, Arakawa Y

MOLECULAR MICROBIOLOGY   Vol. 47 ( 2 ) page： 443 - 451   2003.1

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A novel apoptosis-inducing protein from Helicobacter pylori.

Shibayama K, Kamachi K, Yagi T, Yamane K, Doi Y, Shibata N, Kato H, Arakawa, Y

Helicobacter   Vol. 8 ( 4 ) page： 354   2003

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Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions Reviewed

Shibayama K, Doi Y, Shibata N, Yagi T, Nada T, Iinuma Y, Arakawa Y

INFECTION AND IMMUNITY   Vol. 69 ( 5 ) page： 3181 - 3189   2001.5

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Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions Reviewed

K. Shibayama, Y. Doi, N. Shibata, T. Yagi, T. Nada, Y. Iinuma, Y. Arakawa

Infection and Immunity   Vol. 69 ( 5 ) page： 3181 - 3189   2001.5

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The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pylori infection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressed H. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.

DOI： 10.1128/IAI.69.5.3181-3189.2001

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A preliminary survey of extended-spectrum β-lactamases (ESBLs) in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Japan Reviewed

Tetsuya Yagi, Hiroshi Kurokawa, Naohiro Shibata, Keigo Shibayama, Yoshichika Arakawa

FEMS Microbiology Letters   Vol. 184 ( 1 ) page： 53 - 56   2000.3

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We conducted a survey of extended-spectrum β-lactamases (ESBLs) among 16 805 Escherichia coli and 9794 Klebsiella pneumoniae clinical isolates recovered from 196 separate medical institutions during the period January 1997 to January 1998. Using the criteria for minimal inhibitory concentrations (MICs) of oxyimino-cephalosporins of ≥8 μg ml-1 and confirmation by double-disk test, we detected 15 E. coli and 34 K. pneumoniae isolates producing ESBLs. Genotypes of ESBLs determined by PCR with type- specific primers included one TEM-derived and 24 SHV-derived ESBLs, in addition to 24 Toho-1-type ESBLs, one of the major types of ESBLs reported in Japan. Nucleotide sequence analysis of SHV-specific PCR products revealed that SHV-12 was the dominant type of SHV-derived ESBL. In addition, we also identified TEM-26 and SHV-2. This is the first report characterizing TEM- and SHV-derived ESBLs in Japan. (C) 2000 Published by Elsevier Science B.V.

DOI： 10.1016/S0378-1097(00)00016-1

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Medium compositions and culture conditions for the assay of fosfomycin susceptibility by Etest

Toshinobu Horii, Taku Kimura, Takuya Odagiri, Keigo Shibayama, Michio Ohta

Journal of Infection and Chemotherapy   Vol. 6 ( 1 ) page： 30 - 34   2000

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The Etest (AB Biodisk, Solna, Sweden), a susceptibility testing method, was used for fosfomycin and was evaluated by comparison with the agar dilution method for 73 clinical isolates of Escherichia coli, including two resistant strains, and an optimal Etest method for fosfomycin was determined. Media and culture conditions greatly affected the minimum inhibitory concentrations (MICs) determined by the Etest for fosfomycin, as shown for the agar dilution methods. Our results showed that the most favorable conditions for the Etest for fosfomycin were with Mueller-Hinton agar (Becton-Dickinson Japan, Tokyo, Japan) under aerobic conditions. However, the MICs for the resistant strains were much higher than those determined by agar dilution methods, using Nutrient agar (Becton-Dickinson Japan) under anaerobic conditions. The addition of glucose-6-phosphate did not significantly affect the Etest results.

DOI： 10.1007/s101560050046

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A New SHV-derived extended-spectrum β-lactamase (SHV-24) that hydrolyzes ceftazidime through a single-amino-acid substitution (D179G) in the Ω-loop Reviewed

Hiroshi Kurokawa, Tetsuya Yagi, Naohiro Shibata, Keigo Shibayama, Kazunari Kamachi, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy   Vol. 44 ( 6 ) page： 1725 - 1727   2000

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A new SHV-derived extended-spectrum β-lactamase (SHV-24) conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin was identified in Japan. This enzyme was encoded by a transferable 150-kb plasmid from an Escherichia coli clinical isolate. The pI and K(m) for CAZ of this enzyme were 7.5 and 30 μM, respectively. SHV-24 was found to have a D179G substitution in the Ω-loop of the enzyme.

DOI： 10.1128/AAC.44.6.1725-1727.2000

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Convenient test for screening metallo-β-lactamase-producing gram- negative bacteria by using thiol compounds Reviewed

Yoshichika Arakawa, Naohiro Shibata, Keigo Shibayama, Hiroshi Kurokawa, Tetsuya Yagi, Hiroshi Fujiwara, Masafumi Goto

Journal of Clinical Microbiology   Vol. 38 ( 1 ) page： 40 - 43   2000

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A simple disk diffusion test was constructed for detection of IMP-1-type metallo-β-lactamase-producing gram-negative bacteria. Two Kirby-Bauer disks containing ceftazidime (CAZ) and a filter disk containing a metallo-β- lactamase inhibitor were used in this test. Several IMP-1 inhibitors such as thiol compounds including 2-mercaptopropionic acid, heavy metal salts, and EDTA were evaluated for this test. Two CAZ disks were placed on a Mueller- Hinton agar plate on which a bacterial suspension was spread according to the method recommended by the National Committee for Clinical Laboratory Standards. The distance between the disks was kept to about 4 to 5 cm, and a filter disk containing a metallo-β-lactamase inhibitor was placed near one of the CAZ disks within a center-to-center distance of 1.0 to 2.5 cm. For IMP-1-producing strains, the growth-inhibitory zone between the two disks expanded, while no evident change in the shape of the growth-inhibitory zone was observed for CAZ-resistant strains producing serine β-lactamases such as AmpC or SHV-12. As a result, 2 to 3 μl of undiluted 2-mercaptopropionic acid or mercaptoacetic acid able to block IMP-1 activity gave the most reproducible and clearest results, and CAZ-resistant strains producing AmpC or extended-spectrum β-lactamases were distinguishable from IMP-1 producers by this test. A similar observation was made with IMP-1-producing clinical isolates such as Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter spp., and Alcaligenes xylosoxidans. The specificity and sensitivity of this test were comparable to those of PCR analysis using bla(IMP)-specific primers. Therefore, this convenient test would be valuable for daily use in clinical laboratories.

DOI： 10.1128/JCM.38.1.40-43.2000

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Role of multiple efflux pumps in Escherichia coli in indole expulsion Reviewed

Kawamura-Sato K, Shibayama K, Horii T, Iimuma Y, Arakawa Y, Ohta M

FEMS MICROBIOLOGY LETTERS   Vol. 179 ( 2 ) page： 345 - 352   1999.10

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Worldwide proliferation of carbapenem-resistant gram-negative bacteria Reviewed

Kurokawa H, Yagi T, Shibata N, Shibayama K, Arakawa Y

LANCET   Vol. 354 ( 9182 ) page： 955 - 955   1999.9

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Worldwide proliferation of carbapenem-resistant gram-negative bacteria [9] Reviewed

H. Kurokawa, T. Yagi, N. Shibata, K. Shibayama, Y. Arakawa

Lancet   Vol. 354 ( 9182 ) page： 955 - 955   1999.9

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DOI： 10.1016/S0140-6736(05)75707-X

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Streptococcal pyrogenic exotoxin F (SpeF) causes permeabilization of lung blood vessels Reviewed

Matsumoto M, Ishikawa N, Saito M, Shibayama K, Horii T, Sato K, Ohta M

INFECTION AND IMMUNITY   Vol. 67 ( 9 ) page： 4307 - 4311   1999.9

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Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR Reviewed

Shibayama K, Ohsuka S, Sato K, Yokoyama K, Horii T, Ohta M

FEMS MICROBIOLOGY LETTERS   Vol. 174 ( 1 ) page： 105 - 109   1999.5

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Pathological effect of synthetic cereulide, an emetic toxin of Bacillus cereus, is reversible in mice Reviewed

Keiko Yokoyama, Masafumi Ito, Norio Agata, Minoru Isobe, Keigo Shibayama, Toshinobu Horii, Michio Ohta

FEMS Immunology and Medical Microbiology   Vol. 24 ( 1 ) page： 115 - 120   1999.5

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Cereulide is the causative toxin of the emetic type of food-borne illness caused by Bacillus cereus. This toxin was previously shown to be associated with fulminant liver failure in a human case. Mice were injected i.p. with synthetic cereulide and the development of histopathological changes was examined. Hepatocytes showed mitochondrial swelling with loss of cristae, and dose-dependent increase of small fatty droplets. These microsteatotic hepatocytes were distributed mainly in the pericentral area. At higher cereulide doses, massive degeneration of hepatocytes occurred. The serum values of hepatic enzymes were highest on days 2-3 after the inoculation of cereulide, and rapidly decreased thereafter. General recovery from the pathological changes and regeneration of hepatocytes was observed after 4 weeks. Copyright (C) 1999 Federation of European Microbiological Societies.

DOI： 10.1016/S0928-8244(99)00017-6

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Emergence of fosfomycin-resistant isolates of shiga-like toxin-producing Escherichia coli O26 Reviewed

Horii T, Kimura T, Sato K, Shibayama K, Ohta M

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY   Vol. 43 ( 4 ) page： 789 - 793   1999.4

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Antibacterial activities of new synthetic divalent cation chelators Reviewed

Ashoori M, Ohta M, Ohsuka S, Shibayama K, Horii T, Ueda M, Kurosaki H

MICROBIOLOGY AND IMMUNOLOGY   Vol. 43 ( 4 ) page： 311 - 316   1999

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Are autoantibodies against Lewis antigens involved in the pathogenesis of Helicobacter pylori-induced peptic ulcers? Reviewed

Kamiya K, Arisawa T, Goto H, Shibayama K, Horii T, Hayakawa T, Ohta M

MICROBIOLOGY AND IMMUNOLOGY   Vol. 43 ( 5 ) page： 403 - 408   1999

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Growth conditions of and emetic toxin production by Bacillus cereus in a defined medium with amino acids Reviewed

Agata N, Ohta M, Mori M, Shibayama K

MICROBIOLOGY AND IMMUNOLOGY   Vol. 43 ( 1 ) page： 15 - 18   1999

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Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI) Reviewed

Shibayama K, Ohsuka S, Tanaka T, Arakawa Y, Ohta M

JOURNAL OF BACTERIOLOGY   Vol. 180 ( 20 ) page： 5313 - 5318   1998.10

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Heterogeneity of phenotypic and genotypic traits including organic-acid resistance in Escherichia coli O157 isolates

T Horii, S Barua, T Kimura, S Kasugai, K Sato, K Shibayama, S Ichiyama, M Ohta

MICROBIOLOGY AND IMMUNOLOGY   Vol. 42 ( 12 ) page： 871 - 874   1998

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

We undertook an epidemiologic study for the sensitivity of both Shiga-like toxin (Slt)-producing Escherichia coli (STEC) O157 and non-STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The E. coli O157 isolates examined showed a wide variety of organic-acid susceptibility patterns. E. coli O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.

DOI： 10.1111/j.1348-0421.1998.tb02363.x

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Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple toho-1-like beta-lactamase genes Reviewed

Yagi T, Kurokawa H, Senda K, Ichiyama S, Ito H, Ohsuka S, Shibayama K, Shimokata K, Kato N, Ohta M, Arakawa Y

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY   Vol. 41 ( 12 ) page： 2606 - 2611   1997.12

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RobA-induced multiple antibiotic resistance largely depends on the activation of the AcrAB efflux Reviewed

Tanaka T, Horii T, Shibayama K, Sato K, Ohsuka S, Arakawa Y, Yamaki K, Takagi K, Ohta M

MICROBIOLOGY AND IMMUNOLOGY   Vol. 41 ( 9 ) page： 697 - 702   1997

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