2022年10月, タイ マヒドン大学, 公衆衛生学 名誉博士号　　　　　　　　　　　　　　　
タイの公衆衛生微生物学への学術的貢献
鈴木 定彦

2021年03月, 公益財団法人結核予防会, 秩父宮妃記念結核予防国際協力功労賞　　　　　　　　　　　　　　　

2010年, 日本ハンセン病学会賞　　　　　　　　　　　　　　　
日本国

Vermicomposting reduces the antimicrobial resistance in livestock waste
Masaru Usui, Akira Fukuda, Takashi Azuma, Yoshihiro Kobae, Yuichi Hori, Mitsutaka Kushima, Satoshi Katada, Chie Nakajima, Yasuhiko Suzuki
Journal of Hazardous Materials Advances, 16, 2024年11月
研究論文（学術雑誌）, Vermicomposting, a process in which housefly larvae are used to decompose organic waste, has attracted attention as a method for managing antimicrobial-resistant bacteria (ARB) in livestock manure. Vermicomposting effectively reduces antimicrobial resistance genes (ARGs) and residual antimicrobials. However, the evaluation of live bacteria, including ARB, remains scarce. Additionally, conventional DNA extraction methods include DNA from dead bacteria, impeding the accurate evaluation of ARG-associated risk in compost and the microbiome. This study assesses the effectiveness of vermicomposting pig manure against antimicrobial resistance (AMR) by evaluating the ARB, ARGs (focusing on DNA from live bacteria), and microbiome associated with vermicomposting processes. Vermicomposting significantly reduces the abundance of bacteria, including ARB, and decreases the ARG (tetA, tetB, blaTEM, and blaCTX−M) copy number in live bacteria. Bacterial community analysis revealed an increase in the abundance of Gammaproteobacteria. Moreover, the vermicomposted samples effectively cultivated myriad plants. Overall, vermicomposting effectively reduces the ARB and ARGs in pig manure, with potential benefits for plant growth and sustainable waste management. Hence, it can be widely applied to treat livestock manure and other organic wastes to combat AMR.

Antibiotic use and adherence to the WHO AWaRe guidelines across 16 hospitals in Zambia: a point prevalence survey.
Joseph Yamweka Chizimu, Steward Mudenda, Kaunda Yamba, Chileshe Lukwesa, Raphael Chanda, Ruth Nakazwe, Misheck Shawa, Herman Chambaro, Harvey K Kamboyi, Aubrey Chichonyi Kalungia, Duncan Chanda, Sombo Fwoloshi, Elimas Jere, Tiza Mufune, Derick Munkombwe, Peter Lisulo, Tebuho Mateele, Jeewan Thapa, Kenneth Kapolowe, Nyambe Sinyange, Cephas Sialubanje, Nathan Kapata, Mirfin Mpundu, Freddie Masaninga, Khalid Azam, Chie Nakajima, Makomani Siyanga, Nathan Nsubuga Bakyaita, Evelyn Wesangula, Martin Matu, Yasuhiko Suzuki, Roma Chilengi
JAC-antimicrobial resistance, 6, 5, dlae170, 2024年10月, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The inappropriate use of antibiotics in hospitals contributes to the development and spread of antimicrobial resistance (AMR). This study evaluated the prevalence of antibiotic use and adherence to the World Health Organization (WHO) Access, Watch and Reserve (AWaRe) classification of antibiotics across 16 hospitals in Zambia. METHODS: A descriptive, cross-sectional study employing the WHO Point Prevalence Survey (PPS) methodology and WHO AWaRe classification of antibiotics was conducted among inpatients across 16 hospitals in December 2023, Zambia. Data analysis was performed using STATA version 17.0. RESULTS: Of the 1296 inpatients surveyed in the 16 hospitals, 56% were female, and 54% were aged between 16 and 50 years. The overall prevalence of antibiotic use was 70%. Additionally, 52% of the inpatients received Watch group antibiotics, with ceftriaxone being the most prescribed antibiotic. Slightly below half (48%) of the inpatients received Access group antibiotics. Compliance with the local treatment guidelines was 53%. CONCLUSIONS: This study found a high prevalence of prescribing and use of antibiotics in hospitalized patients across the surveyed hospitals in Zambia. The high use of Watch group antibiotics was above the recommended threshold indicating non-adherence to the WHO AWaRe guidelines for antibiotic use. Hence, there is a need to establish and strengthen antimicrobial stewardship programmes that promote the rational use of antibiotics in hospitals in Zambia.

WQ-3810, a fluoroquinolone with difluoropyridine derivative as the R1 group exerts high potency against quinolone-resistant Campylobacter jejuni.
Kentaro Koide, Hyun Kim, Matthew V X Whelan, Lawrence P Belotindos, Wimonrat Tanomsridachchai, Ruchirada Changkwanyeun, Masaru Usui, Tadhg Ó Cróinín, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Microbiology spectrum, e0432223, 2024年08月20日, ［国際誌］
英語, 研究論文（学術雑誌）, UNLABELLED: Quinolone-resistant Campylobacter jejuni have been increasing worldwide. Quinolones exert their antibacterial activity by inhibiting DNA gyrase, but most of the isolates acquire quinolone resistance via an amino acid substitution in the A subunit of DNA gyrase. WQ-3810 is a quinolone antibiotic that has been reported to have high potency even to DNA gyrase with amino acid substitutions in several bacterial species; however, there was no information on C. jejuni. Hence, this study aimed to evaluate the activity of WQ-3810 to inhibit wild-type/mutant DNA gyrases of C. jejuni and the bacterial growth for accessing the potency for the treatment of quinolone-resistant C. jejuni infection. The inhibitory activity of WQ-3810 was assessed and compared with ciprofloxacin and nalidixic acid by calculating the half maximal inhibitory concentration (IC50) against wild-type/mutant DNA gyrases. Next, the minimum inhibitory concentration (MIC) of WQ-3810 and five other quinolones was determined for C. jejuni including quinolone-resistant strains with amino acid substitutions in GyrA. Furthermore, the interaction between WQ-3810 and wild-type/mutant DNA gyrase was speculated using docking simulations. The IC50 of WQ-3810 against wild-type DNA gyrase was 1.03 µg/mL and not different from that of ciprofloxacin. However, those of WQ-3810 against mutant DNA gyrases were much lower than ciprofloxacin. The MICs of WQ-3810 ranged <0.016-0.031 µg/mL and were the lowest against both quinolone-susceptible and quinolone-resistant strains among the examined quinolones. The results obtained by the docking simulation agreed well with this observation. WQ-3810 seems to be a promising antimicrobial agent for the infections caused by quinolone-resistant C. jejuni. IMPORTANCE: WQ-3810, a relatively new quinolone antibiotic, demonstrates exceptional antibacterial properties against certain pathogens in previous studies. However, its efficacy against quinolone-resistant Campylobacter jejuni was not previously reported. The prevalence of quinolone-resistant C. jejuni as a cause of foodborne illnesses is increasing, prompting this investigation into the effectiveness of WQ-3810 as a countermeasure. This study revealed high inhibitory activity of WQ-3810 against both wild-type and mutant DNA gyrases of C. jejuni. WQ-3810 was equally efficacious as ciprofloxacin against wild-type DNA gyrases but showed superior effectiveness against mutant DNA gyrases. WQ-3810 also demonstrated the lowest minimum inhibitory concentrations, highlighting its enhanced potency against both susceptible and resistant strains of C. jejuni. This observation was well supported by the results of the in silico analysis. Consequently, WQ-3810 exhibits a higher level of bactericidal activity compared to existing quinolones in combating both susceptible and resistant C. jejuni isolates.

Interplay between Amino Acid Substitution in GyrA and QnrB19: Elevating Fluoroquinolone Resistance in Salmonella Typhimurium.
Pondpan Suwanthada, Siriporn Kongsoi, Sasini Jayaweera, Mwangala Lonah Akapelwa, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
ACS infectious diseases, 10, 8, 2785, 2794, 2024年08月09日, ［国際誌］
英語, 研究論文（学術雑誌）, Globally, there have been increasing reports of antimicrobial resistance in nontyphoidal Salmonella (NTS), which can develop into severe and potentially life-threatening diarrhea. This study focuses on the synergistic effects of DNA gyrase mutations and plasmid-mediated quinolone resistance (PMQR) genes, specifically qnrB19, on fluoroquinolone (FQ) resistance in Salmonella Typhimurium. By utilizing recombinant mutants, GyrAS83F and GyrAD87N, and QnrB19's, we discovered a significant increase in fluoroquinolones resistance when QnrB19 is present. Specifically, ciprofloxacin and moxifloxacin's inhibitory concentrations rose 10- and 8-fold, respectively. QnrB19 was found to enhance the resistance capacity of mutant DNA gyrases, leading to high-level FQ resistance. Additionally, we observed that the ratio of QnrB19 to DNA gyrase played a critical role in determining whether QnrB19 could protect DNA gyrase against FQ inhibition. Our findings underscore the critical need to understand these resistance mechanisms, as their coexistence enables bacteria to withstand therapeutic FQ levels, posing a significant challenge to treatment efficacy.

The first insight into Mycobacterium tuberculosis complex isolates in the lower northern region in Thailand.
Janisara Rudeeaneksin, Supranee Bunchoo, Benjawan Phetsuksiri, Sopa Srisungngam, Ratchaneeporn Khummin, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Transactions of the Royal Society of Tropical Medicine and Hygiene, 118, 8, 527, 536, 2024年08月05日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Tuberculosis (TB) remains an important infectious disease and different genotypes have been reported. This study aimed to investigate the genetic diversity and molecular epidemiology of TB in the lower northern region of Thailand, where genotyping data are limited. METHODS: A total of 159 Mycobacterium tuberculosis complex (MTBC) isolates from this region were genotyped by spoligotyping and the major spoligotypes were further subdivided by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method. RESULTS: Spoligotyping identified 34 types and classified them into 14 clusters. East African-Indian (EAI) groups were the most frequent (44.7%), followed by Beijing (36.5%), with a higher prevalence of drug resistance. By 15-loci MIRU-VNTR typing, the major groups of the Beijing and EAI2_NTB were further differentiated into 44 and 21 subtypes forming 9 and 5 subclusters with cluster rates of 0.26 and 0.44, respectively. The Hunter-Gaston Discriminatory Index among the Beijing and EAI2_NTB groups were 0.987 and 0.931, respectively, indicating high diversity. CONCLUSIONS: This is the first look at the MTBC genotypes in the lower northern region of Thailand, which could aid in understanding the distribution and potential spread of MTBC and Mycobacterium bovis in the target region to support TB control in Thailand.

比較ゲノム解析による乳牛由来黄色ブドウ球菌の国際的な拡散状況の解明　　　　　　　　　　　　　　　
大川 佳穂, 福田 昭, 米山 裕, 戸部 隆太, 中島 千絵, 鈴木 定彦, 臼井 優
北海道獣医師会雑誌, 68, 8, 329, 329, (公社)北海道獣医師会, 2024年08月
日本語

Epidemiology and molecular characterization of Mycobacterium tuberculosis including a drug-resistant strain associated with mortality of Asian elephants in Nepal 2019-2022.
Arjun Pandit, Jeewan Thapa, Amir Sadaula, Yasuhiko Suzuki, Chie Nakajima, Susan K Mikota, Naresh Subedi, Bijaya Kumar Shrestha, Michito Shimozuru, Bhawana Shrestha, Bijendra Raya, Sanjay Chaudhary, Sarad Paudel, Toshio Tsubota
Tuberculosis (Edinburgh, Scotland), 148, 102550, 102550, 2024年07月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Tuberculosis (TB) is an emerging threat to the survival of elephants in Nepal. We investigated the lung tissue samples from nine elephants that died from 2019 to 2022 in Nepal using culture, conventional PCR, and loop-mediated isothermal amplification (LAMP) and then performed genotyping of five PCR-positive isolates to understand the possible transmission dynamics of Mycobacterium tuberculosis (Mtb). Results showed that two-thirds (6/9) of elephants were confirmed to be infected from Mtb by LAMP, 5/9 by PCR, and 4/9 by culture. Genotyping of Mtb isolates showed that elephants were infected with the Indo-Oceanic and Beijing lineages including an isoniazid-resistant Beijing lineage. MIRU-VNTR-based phylogeny, gyrA, and katG sequencing showed the possibility of ongoing transmission of Indo-Oceanic lineages and likely transmission of the drug-resistant Beijing lineage from human to elephant. Implementation of comprehensive surveillance and preventive measures are urgently needed to address this zoonotic disease and protect elephants from TB in Nepal.

Functional analysis of the Mycobacterium bovis AF2122/97 PhoPR system.
Jose Maria Urtasun-Elizari, Ruoyao Ma, Hayleah Pickford, Damien Farrell, Gabriel Gonzalez, Viktor Perets, Chie Nakajima, Yasuhiko Suzuki, David E MacHugh, Apoorva Bhatt, Stephen V Gordon
Tuberculosis (Edinburgh, Scotland), 148, 102544, 102544, 2024年07月14日, ［国際誌］
英語, 研究論文（学術雑誌）, The PhoPR system is a master regulator in Mycobacterium tuberculosis. A key difference between M. tuberculosis and Mycobacterium bovis is a G71I substitution in the M. bovis PhoR orthologue. Functional studies of the M. bovis PhoPR system have generated conflicting findings, with some research suggesting that the M. bovis PhoPR is defective while others indicate it is functional. We sought to revisit the functionality of the M. bovis PhoPR system. To address this, we constructed a phoPR mutant in the reference strain M. bovis AF2122/97. We employed a combination of growth assays and transcriptomics analyses to assess the phenotype of the mutant vs wild type and complemented strains. We found that the M. bovis AF2122/97 ΔphoPR mutant showed a growth defect on solid and liquid media compared to the wild type and complemented strains. The transcriptome of the M. bovis AF2122/97 ΔphoPR mutant was also altered as compared to wild type, including differential expression of genes involved in lipid metabolism and secretion. Our work provides further insight into the activity of PhoPR in M. bovis and underlines the importance of the PhoPR system as a master regulator of gene expression in the Mycobacterium tuberculosis complex.

WQ-3810: A Novel Fluoroquinolone Exhibiting Potency Against Fluoroquinolone-Resistant M.avium(タイトル和訳中)　　　　　　　　　　　　　　　
Jayaweera Sasini, Thapa Jeewan, Nakajima Chie, Suzuki Yasuhiko
日本細菌学雑誌, 79, 2, 268, 268, 日本細菌学会, 2024年07月
英語

Biphasic Medium Using Nicotinamide for Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis.
Waraporn Thuansuwan, Charoen Chuchottaworn, Chie Nakajima, Yasuhiko Suzuki, Nuntaree Chaichanawongsaroj
Antibiotics (Basel, Switzerland), 13, 6, 2024年06月16日, ［国際誌］
英語, 研究論文（学術雑誌）, Reliable drug susceptibility testing of pyrazinamide (PZA) is technically difficult, since PZA activity is pH sensitive. The aim of this study was to evaluate a biphasic medium assay (BMA) for the reliable detection of PZA resistance in Mycobacterium tuberculosis (MTB) using nicotinamide (NIC) as a surrogate for PZA and identifying the appropriate cut-off value for the assay. The PZA susceptibility of 122 multidrug-resistant tuberculosis (MDR-TB) isolates and 39 drug-susceptible tuberculosis (DS-TB) isolates was examined using the BMA with NIC at four different concentrations (250, 500, 1000, and 2000 mg/L) and comparing the results with results from the BACTEC MGIT 960 reference method. Out of 122 MDR-TB isolates, 40 were identified as resistant by the BACTEC MGIT 960 system, of which 92.5% contained mutations within their pncA gene plus promoter region. A minimum inhibitory concentration of NIC ≥ 1000 mg/L was used as the cut-off concentration to define resistance in correlation with the MGIT 960 outcomes. NIC-BMA had a sensitivity of 90.91%, a specificity of 100%, and an accuracy of 97.52% compared with the MGIT 960 method. NIC-BMA is a promising assay to screen PZA resistance in microbiological laboratories without automation or advanced molecular instruments.

Wide distribution of plasmid mediated quinolone resistance gene, qnrS, among Salmonella spp. isolated from canal water in Thailand.
Jirachaya Toyting, Neunghatai Supha, Yuwanda Thongpanich, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki, Fuangfa Utrarachkij
Journal of applied microbiology, 135, 6, 2024年06月03日, ［国際誌］
英語, 研究論文（学術雑誌）, AIMS: This research focused on assessing the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and antimicrobial susceptibility in Salmonella strains isolated from Thai canal water. METHODS AND RESULTS: From 2016 to 2020, 333 water samples were collected from six canals across Bangkok, Thailand. Salmonella spp. was isolated, PMQR genes were detected through polymerase chain reactions, and the antimicrobial susceptibility was examined using the disk diffusion method. The results indicated a 92.2% prevalence of Salmonella spp. in canal water, being serogroups B and C the most frequently detected. Overall, 35.3% of isolates harbored PMQR genes, being qnrS the most prevalent gene (97.2%, n = 137/141). Other PMQR genes, including qnrB, qnrD, oqxAB, and aac(6')-Ib-cr, were detected. Notably, six isolates harbored multiple PMQR genes. Furthermore, 9.3% and 3.8% of the overall isolates were resistant to nalidixic acid (NAL) and ciprofloxacin (CIP), respectively. PMQR-positive isolates showed higher rates of non-susceptibility to both NAL (48.2%, n = 68/141) and CIP (92.2%, n = 130/141) compared to PMQR-negative isolates (NAL: 8.9%, n = 23/258; CIP: 11.2%, n = 30/258). CONCLUSIONS: The high prevalence of Salmonella spp., significant PMQR-positive, and reduced susceptibility isolates in canal water is of public health concern in Bangkok.

Mobile linezolid resistance genes in enterococci derived from livestock compost at Japanese farms(タイトル和訳中)　　　　　　　　　　　　　　　
福田 昭, 中島 千絵, 鈴木 定彦, 臼井 優
日本細菌学雑誌, 79, 2, 121, 121, 日本細菌学会, 2024年06月
英語

Genomic Analysis of Salmonella Isolated from Canal Water in Bangkok, Thailand(タイトル和訳中)　　　　　　　　　　　　　　　
Toyting Jirachaya, Nuanmuang Narong, Utrarachkij Fuangfa, Leekitcharoenphon Pimlapas, Aarestrup Frank, 佐藤 豊孝, Thapa Jeewan, 中島 千絵, 鈴木 定彦
日本細菌学雑誌, 79, 2, 128, 128, 日本細菌学会, 2024年06月
英語

Assessment of antimicrobial resistance laboratory-based surveillance capacity of hospitals in Zambia: findings and implications for system strengthening
K. Yamba, J. Y. Chizimu, S. Mudenda, C. Lukwesa, R. Chanda, R. Nakazwe, B. Simunyola, M. Shawa, A. C. Kalungia, D. Chanda, T. Mateele, J. Thapa, K. Kapolowe, M. L. Mazaba, M. Mpundu, F. Masaninga, K. Azam, C. Nakajima, Y. Suzuki, N. N. Bakyaita, E. Wesangula, M. Matu, R. Chilengi
Journal of Hospital Infection, 148, 129, 137, 2024年06月
研究論文（学術雑誌）, Background: A well-established antimicrobial resistance (AMR) laboratory-based surveillance (LBS) is of utmost importance in a country like Zambia which bears a significant proportion of the world's communicable disease burden. This study assessed the capacity of laboratories in selected hospitals to conduct AMR surveillance in Zambia. Methods: This cross-sectional exploratory study was conducted among eight purposively selected hospitals in Zambia between August 2023 and December 2023. Data were collected using the self-scoring Laboratory Assessment of Antibiotic Resistance Testing Capacity (LAARC) tool. Findings: Of the assessed facilities, none had full capacity to conduct AMR surveillance with varying capacities ranging from moderate (63% (5/8)) to low (38% (3/8)). Some of the barriers of AMR-LBS were the lack of an electronic laboratory information system (63% (5/8)) and the lack of locally generated antibiograms (75% (6/8)). Quality control for antimicrobial susceptibility testing (AST), pathogen identification and media preparation had the lowest overall score among all of the facilities with a score of 14%, 20% and 44%, respectively. The highest overall scores were in specimen processing (79%), data management (78%), specimen collection, transport and management (71%), and safety (70%). Most facilities had standard operating procedures in place but lacked specimen-specific standard operating procedures. Conclusion: The absence of laboratories with full capacity to conduct AMR surveillance hinders efforts to combat AMR and further complicates the treatment outcomes of infectious diseases. Establishing and strengthening LBS systems are essential in quantifying the burden of AMR and supporting the development of local antibiograms and treatment guidelines.

Carbapenem and colistin-resistant hypervirulent Klebsiella pneumoniae: An emerging threat transcending the egyptian food chain.
Rana Fahmi Sabala, Akira Fukuda, Chie Nakajima, Yasuhiko Suzuki, Masaru Usui, Mohamed Elhadidy
Journal of infection and public health, 17, 6, 1037, 1046, 2024年06月, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a great public health problem and is associated with many disease outbreaks and high mortality rates. Alarmingly, K. pneumoniae has been isolated from food in several recent studies. This study aimed to investigate the prevalence and characteristics of CRKP in food samples from Egypt. METHODS: A total of 311 food samples (including 116 minced meat, 92 chicken meat, 75 diced meat, and 28 mutton) were collected from local markets in Egypt and were screened for CRKP with the determination of their antimicrobial resistance profiles. The whole genome sequence was done for 23 CRKP isolates to clarify the relationship between CRKP from food and human cases in Egypt using the SNP core genome. The conjugation probability of the blaNDM-5 harboring plasmid was identified using oriTfinder RESULTS: CRKP was isolated from 11% (35/311) of the samples, with 45.71% (16/35) of them showing resistance to colistin, one of the last-resort options for treating CRKP-mediated infections. In addition to the carbapenem and colistin resistance, the CRKP isolates frequently exhibited resistance to multiple antimicrobials including β-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and chloramphenicol. In addition, most of the CRKP were potentially hypervirulent K. pneumoniae (HvKP) identified as phylogroup Kp1 and of high-risk groups as detected in STs reported in many human outbreaks globally, such as ST383 and ST147. The core-genome phylogeny showed similarities between the isolates from this study and those previously isolated from clinical human samples in Egypt. In addition, analysis of the plasmid on which blaNDM is encoded revealed that several antimicrobial resistance genes such as blaOXA-9, blaCTX-M-15, aac(6')-Ib, qnrS1, and several virulence genes are encoded on the same plasmid. CONCLUSIONS: This study is significant for food safety and public health and is important to further identify the change in the epidemiology of CRKP infections, especially the consumption of contaminated food products.

Association of toxin-producing Clostridioides difficile with piglet diarrhea and potential transmission to humans.
Kouki Takeichi, Akira Fukuda, Chika Shono, Noriyasu Ota, Chie Nakajima, Yasuhiko Suzuki, Masaru Usui
The Journal of veterinary medical science, 2024年05月27日, ［国内誌］
英語, 研究論文（学術雑誌）, The pathogenicity of Clostridioides difficile in piglets remains controversial. It is unknown whether C. difficile control helps protect piglet health. To clarify the association between C. difficile presence and piglet diarrhea, isolates were obtained from piglets with and without diarrhea. In addition, to determine the genetic relationship of C. difficile from pigs and humans, we performed whole-genome sequencing (WGS) of C. difficile isolates. Diarrheal and non-diarrheal stool samples were collected from neonatal piglets from five farms in Japan in 2021. To clarify the relationship between C. difficile derived from pigs and those from human clinical cases, WGS of C. difficile isolates was performed. Toxin-positive C. difficile were significantly more prevalent in piglets with diarrhea, although the overall frequency of C. difficile did not differ between piglets with and without diarrhea. This observation indicates an association between toxin-positive C. difficile and diarrhea in piglets. However, further studies are needed to establish a direct causal relationship and to explore other contributing factors to diarrhea in piglets. WGS results showed that C. difficile sequence type (ST)11 including the hypervirulent PCR ribotype 078 isolates derived from Japanese pigs were closely related to ST11 of overseas strains (human clinical and animal-derived) and a Japanese human clinical strain. Toxin-positive C. difficile may cause diarrhea in piglets and hypervirulent C. difficile are spreading among pigs and human populations worldwide.

Antimicrobial Use Survey and Detection of ESBL-Escherichia coli in Commercial and Medium-/Small-Scale Poultry Farms in Selected Districts of Zambia
Taona Sinyawa, Misheck Shawa, Geoffrey M. Muuka, Fusya Goma, Paul Fandamu, Joseph Yamweka Chizimu, Cynthia Sipho Khumalo, Malala Mulavu, Masuzyo Ngoma, Herman Moses Chambaro, Harvey Kakoma Kamboyi, Masahiro Kajihara, Hirofumi Sawa, Yasuhiko Suzuki, Hideaki Higashi, Geoffrey Mainda, Musso Munyeme, John Bwalya Muma, Christian Owusu Nyantakyi, Beverly Egyir, Bernard Mudenda Hang’ombe
Antibiotics, 13, 5, 467, 467, MDPI AG, 2024年05月20日
研究論文（学術雑誌）, Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers’ usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum β-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88–20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00–0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response.

Genomic analysis of Salmonella isolated from canal water in Bangkok, Thailand.
Jirachaya Toyting, Narong Nuanmuang, Fuangfa Utrarachkij, Neunghatai Supha, Yuwanda Thongpanich, Pimlapas Leekitcharoenphon, Frank M Aarestrup, Toyotaka Sato, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Microbiology spectrum, 12, 5, e0421623, 2024年05月02日, ［国際誌］
英語, 研究論文（学術雑誌）, Antimicrobial resistance (AMR) poses an escalating global public health threat. Canals are essential in Thailand, including the capital city, Bangkok, as agricultural and daily water sources. However, the characteristic and antimicrobial-resistance properties of the bacteria in the urban canals have never been elucidated. This study employed whole genome sequencing to characterize 30 genomes of a causal pathogenic bacteria, Salmonella enterica, isolated from Bangkok canal water between 2016 and 2020. The dominant serotype was Salmonella Agona. In total, 35 AMR genes and 30 chromosomal-mediated gene mutations were identified, in which 21 strains carried both acquired genes and mutations associated with fluoroquinolone resistance. Virulence factors associated with invasion, adhesion, and survival during infection were detected in all study strains. 75.9% of the study stains were multidrug-resistant and all the strains harbored the necessary virulence factors associated with salmonellosis. One strain carried 20 resistance genes, including mcr-3.1, mutations in GyrA, ParC, and ParE, and typhoid toxin-associated genes. Fifteen plasmid replicon types were detected, with Col(pHAD28) being the most common type. Comparative analysis of nine S. Agona from Bangkok and 167 from public databases revealed that specific clonal lineages of S. Agona might have been circulating between canal water and food sources in Thailand and globally. These findings provide insight into potential pathogens in the aquatic ecosystem and support the inclusion of environmental samples into comprehensive AMR surveillance initiatives as part of a One Health approach. This approach aids in comprehending the rise and dissemination of AMR and devising sustainable intervention strategies.IMPORTANCEBangkok is the capital city of Thailand and home to a large canal network that serves the city in various ways. The presence of pathogenic and antimicrobial-resistant Salmonella is alarming and poses a significant public health risk. The present study is the first characterization of the genomic of Salmonella strains from Bangkok canal water. Twenty-two of 29 strains (75.9%) were multidrug-resistant Salmonella and all the strains carried essential virulence factors for pathogenesis. Various plasmid types were identified in these strains, potentially facilitating the horizontal transfer of AMR genes. Additional investigations indicated a potential circulation of S. Agona between canal water and food sources in Thailand. The current study underscores the role of environmental water in an urban city as a reservoir of pathogens and these data obtained can serve as a basis for public health risk assessment and help shape intervention strategies to combat AMR challenges in Thailand.

Effect of WQ-3334 on Campylobacter jejuni carrying a DNA gyrase with dominant amino acid substitutions conferring quinolone resistance.
Nami Miura-Ajima, Pondpan Suwanthada, Siriporn Kongsoi, Hyun Kim, Ruttana Pachanon, Kentaro Koide, Shigetarou Mori, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 2024年04月04日, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of quinolones-resistant Campylobacter jejuni poses a significant challenge. The predominant mechanism of quinolones-resistance in this bacterium involves point mutations in the gyrA, resulting in amino acid substitution from threonine to isoleucine at 86th position, representing more than 90% of mutant DNA gyrase, and aspartic acid to asparagine at 90th position. WQ-3334, a novel quinolone, has demonstrated strong inhibitory activity against various bacteria. This study aims to investigate the effectiveness of WQ-3334, and its analogues, WQ-4064 and WQ-4065, with a unique modification in R1 against quinolones-resistant C. jejuni. METHODS: The structure-activity relationship of the examined drugs was investigated by measuring IC50 and their antimicrobial activities were accessed by MIC against C. jejuni strains. Additionally, in silico docking simulations were carried out using the crystal structure of the Escherichia coli DNA gyrase. RESULT: WQ-3334 exhibited the lowest IC50 against WT (0.188 ± 0.039 mg/L), T86I (11.0 ± 0.7 mg/L) and D90 N (1.60 ± 0.28 mg/L). Notably, DNA gyrases with T86I substitutions displayed the highest IC50 values among the examined WQ compounds. Moreover, WQ-3334 demonstrated the lowest MICs against wild-type and mutant strains. The docking simulations further confirmed the interactions between WQ-3334 and DNA gyrases. CONCLUSION: WQ-3334 with 6-amino-3,5-difluoropyridine-2-yl at R1 severed as a remarkable candidate for the treatment of foodborne diseases caused by quinolones-resistant C. jejuni as shown by the high inhibitory activity against both wild-type and the predominant quinolones-resistant strains.

デラフロキサシンのらい菌DNAジャイレースに対する作用(第2報)
Eomseob Jang, Pondpan Suwanthada, 中島 千絵, 阿戸 学, 向井 徹, 鈴木 定彦
日本ハンセン病学会雑誌, 93, 1, 21, 21, 日本ハンセン病学会, 2024年04月
日本語

Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae from Diseased Broiler Chickens in Lusaka District, Zambia.
Chikwanda Chileshe, Misheck Shawa, Nelson Phiri, Joseph Ndebe, Cynthia Sipho Khumalo, Chie Nakajima, Masahiro Kajihara, Hideaki Higashi, Hirofumi Sawa, Yasuhiko Suzuki, Walter Muleya, Bernard Mudenda Hang'ombe
Antibiotics (Basel, Switzerland), 13, 3, 2024年03月15日, ［国際誌］
英語, 研究論文（学術雑誌）, Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.

Heated scallop-shell powder and lime nitrogen effectively decrease the abundance of antimicrobial-resistant bacteria in aerobic compost
Masatoshi Enami, Akira Fukuda, Michi Yamada, Yoshihiro Kobae, Chie Nakajima, Yasuhiko Suzuki, Masaru Usui
Environmental Technology & Innovation, 103590, 103590, Elsevier BV, 2024年03月, ［査読有り］
研究論文（学術雑誌）

Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaque
Cong Thanh Nguyen, Misako Nakayama, Hirohito Ishigaki, Yoshinori Kitagawa, Akemi Kakino, Marumi Ohno, Masashi Shingai, Yasuhiko Suzuki, Tatsuya Sawamura, Hiroshi Kida, Yasushi Itoh
Virology, 110052, 110052, Elsevier BV, 2024年03月
研究論文（学術雑誌）

Transferable linezolid resistance genes (optrA and poxtA) in enterococci derived from livestock compost at Japanese farms.
Akira Fukuda, Chie Nakajima, Yasuhiko Suzuki, Masaru Usui
Journal of global antimicrobial resistance, 2024年02月07日, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: Linezolid is a last-resort antimicrobial in human clinical settings to treat multidrug-resistant gram-positive bacterial infections. Mobile linezolid resistance genes (optrA, poxtA, and cfr) have been detected in various sources worldwide. However, the presence of linezolid-not-susceptible bacteria and mobile linezolid resistance genes in Japan remains uncertain. Therefore, we clarified the existence of linezolid-not-susceptible bacteria and mobile linezolid resistance genes in farm environments in Japan. METHODS: Enterococci isolates from feces compost collected from 10 pig and 11 cattle farms in Japan in 2021 were tested for antimicrobial susceptibility and possession of mobile linezolid resistance genes. Whole-genome sequencing of optrA and/or poxtA genes positive-enterococci was performed. RESULTS: Of 103 enterococci isolates, 12 from pig farm compost were not-susceptible (2 resistant and 10 intermediate) to linezolid. These 12 isolates carried mobile linezolid resistance genes on plasmids or chromosomes (5 optrA-positive Enterococcus faecalis, 6 poxtA-positive E. hirae or E. thailandicus, and 1 optrA- and poxtA-positive E. faecium). The genetic structures of optrA- and poxA-carrying plasmids were almost identical to those reported in other countries. These plasmids were capable of transferring among E. faecium and E. faecalis strains. The optrA- and poxtA-positive E. faecium belonged to ST324 (clade A2), a high-risk multidrug-resistant clone. The E. faecalis carrying optrA gene on its chromosome was identified as ST593. CONCLUSIONS: Although linezolid is not used in livestock, linezolid-not-susceptible enterococci could be indirectly selected by frequently used antimicrobials, such as phenicols. Moreover, various enterococci species derived from livestock compost may serve as reservoirs of linezolid resistance genes carried on globally disseminated plasmids and multidrug-resistant high-risk clones.

Antimicrobial stewardship situation analysis in selected hospitals in Zambia: findings and implications from a national survey.
Joseph Yamweka Chizimu, Steward Mudenda, Kaunda Yamba, Chileshe Lukwesa, Raphael Chanda, Ruth Nakazwe, Bwalya Simunyola, Misheck Shawa, Aubrey Chichonyi Kalungia, Duncan Chanda, Uchizi Chola, Tebuho Mateele, Jeewan Thapa, Kenneth Kapolowe, Mazyanga Lucy Mazaba, Mirfin Mpundu, Freddie Masaninga, Khalid Azam, Chie Nakajima, Yasuhiko Suzuki, Nathan Nsubuga Bakyaita, Evelyn Wesangula, Martin Matu, Roma Chilengi
Frontiers in public health, 12, 1367703, 1367703, 2024年, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Antimicrobial stewardship (AMS) programs are critical in combating antimicrobial resistance (AMR). In Zambia, there is little information regarding the capacity of hospitals to establish and implement AMS programs. The objective of this study was to conduct a baseline assessment of WHO core elements for an AMS program implementation in eight hospitals in Zambia. MATERIALS AND METHODS: We conducted an exploratory cross-sectional study from September 2023 to December 2023 using a self-scoring Periodic National and Healthcare Facility Assessment Tool from the World Health Organization (WHO) policy guidance on integrated AMS activities in human health. Eight public hospitals were surveyed across the five provinces of Zambia. Data was analyzed using the WHO self-scoring tool and thematic analysis. RESULTS: Overall, 62.5% (6/8) of the facilities scored low (below 60%) in implementing AMS programs. Most facilities had challenges with reporting AMS feedback within the hospital (average score = 46%), Drugs and Therapeutics Committee (DTC) functionality (average score = 49%), AMS actions (average score = 50%), education and training (average score = 54%), and leadership commitment to AMS activities (average score = 56%). The overall score for all AMS core elements was average (56%). All the hospitals (100%) did not have an allocated budget for AMS programs. Finally, there were neither antibiograms to guide antimicrobial utilization nor AMS-trained staff in more than 50% of the hospitals surveyed. CONCLUSION: This study found low AMS implementation in these public hospitals, especially where DTCs were non-functional. The identified challenges and gaps require urgent attention for sustainable multidisciplinary AMS programs.

Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand.
Pramualchai Ketkhao, Fuangfa Utrarachkij, Nattaya Parikumsil, Kritchai Poonchareon, Anusak Kerdsin, Peeraya Ekchariyawat, Pawarut Narongpun, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
PloS one, 19, 7, e0307544, 2024年, ［国際誌］
英語, 研究論文（学術雑誌）, Extraintestinal pathogenic Escherichia coli (ExPEC), especially uropathogenic E. coli (UPEC) are responsible for urinary tract infections (UTIs), while diarrheagenic E. coli (DEC) cause foodborne illnesses. These pathogenic E. coli are a serious threat to human health and a public concern worldwide. However, the evidence on pork E. coli (PEC) harboring UPEC virulence-associated genes is currently limited. Therefore, this study aimed to determine the phylogroups, virulence genes, and their association between PEC and UPEC from UTI patients. In this study, 330 E. coli were obtained from archived stock culture isolated from pork (PEC; n = 165) and urine of patients with UTIs (UPEC; n = 165) during 2014-2022. Phylogroups, UPEC- and diarrheagenic E. coli (DEC) associated virulence genes were assessed using PCR assays. The results showed that phylogroups A (50.3%), and B1 (32.1%) were commonly found among PEC whereas phylogroups B2 (41.8%), and C (25.5%) were commonly detected in the UPEC. PEC and UPEC carried similar virulence-associated genes with different percentages. The most frequent UPEC virulence-associated gene among UPEC, and PEC strains was fimH, (93.3%, and 92.1%), followed by iucC (55.2%, and 12.7%), papC (21.8%, and 4.2%), afaC (22.4%, and 0%), hlyCA (17%, and 0.6%), cnf (16.4%, and 0.6%), and sfa/focDE (8.5%, and 4.8%). Additionally, 6 of 27 UPEC virulence-associated gene patterns were found in both PEC and UPEC strains regardless of phylogroups. Furthermore, the DEC virulence-associated genes were found in only 3 strains, one from PEC harboring eae, and two from UPEC carried fimH-bfpA or afaC-CVD432 indicating hybrid strains. Cluster analysis showed a relationship between PEC and UPEC strains and demonstrated that PEC harboring UPEC virulence-associated genes in pork may be associated with UPEC in humans. Food safety and hygiene practices during pork production chain are important procedures for minimizing cross-contamination of these strains that could be transmitted to the consumers.

Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.
Misheck Shawa, Atmika Paudel, Herman Chambaro, Harvey Kamboyi, Ruth Nakazwe, Luke Alutuli, Tuvshinzaya Zorigt, Taona Sinyawa, Mulemba Samutela, Joseph Chizimu, Manyando Simbotwe, Kyoko Hayashida, Naganori Nao, Masahiro Kajihara, Yoshikazu Furuta, Yasuhiko Suzuki, Hirofumi Sawa, Bernard Hang'ombe, Hideaki Higashi
PloS one, 19, 4, e0302053, 2024年, ［国際誌］
英語, 研究論文（学術雑誌）, Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.

Whole-Genome Investigation of Zoonotic Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolated from Pigs and Humans in Thailand.
Pawarut Narongpun, Pattrarat Chanchaithong, Junya Yamagishi, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Antibiotics (Basel, Switzerland), 12, 12, 2023年12月16日, ［国際誌］
英語, 研究論文（学術雑誌）, Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been widespread globally in pigs and humans for decades. Nasal colonization of LA-MRSA is regarded as an occupational hazard to people who are regularly involved in livestock production. Our previous study suggested pig-to-human transmission caused by LA-MRSA clonal complex (CC) 398, using traditional molecular typing methods. Instead, this study aimed to investigate the zoonotic transmission of LA-MRSA CC398 using whole genome sequencing (WGS) technologies. A total of 63 LA-MRSA isolates were identified and characterized in Thailand. Further, the 16 representatives of LA-MRSA CC9 and CC398, including porcine and worker isolates, were subjected to WGS on the Illumina Miseq platform. Core-genome single nucleotide polymorphism (SNP)-based analyses verify the zoonotic transmission caused by LA-MRSA CC398 in two farms. WGS-based characterization suggests the emergence of a novel staphylococcal cassette chromosome (SCC) mec type, consisting of multiple cassette chromosome recombinase (ccr) gene complexes via genetic recombination. Additionally, the WGS analyses revealed putative multi-resistant plasmids and several cross-resistance genes, conferring resistance against drugs of last resort used in humans such as quinupristin/dalfopristin and linezolid. Significantly, LA-MRSA isolates, in this study, harbored multiple virulence genes that may become a serious threat to an immunosuppressive population, particularly for persons who are in close contact with LA-MRSA carriers.

Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.
Thoko Flav Kapalamula, Joseph Yamweka Chizimu, Mwangala Lonah Akapelwa, David Atomanyi Barnes, Jirachaya Toyting, Precious Bwalya, Linda Basikolo, David Squarre, Herman M Chambaro, Stephen V Gordon, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Research in veterinary science, 164, 105030, 105030, 2023年11月, ［国際誌］
英語, 研究論文（学術雑誌）, We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

Exploration of the novel fluoroquinolones with high inhibitory effect against quinolone-resistant DNA gyrase of Salmonella Typhimurium.
Jirachaya Toyting, Nami Miura, Fuangfa Utrarachkij, Wimonrat Tanomsridachchai, Lawrence P Belotindos, Pondpan Suwanthada, Thoko Flav Kapalamula, Siriporn Kongsoi, Kentaro Koide, Hyun Kim, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Microbiology spectrum, e0133023, 2023年10月05日, ［国際誌］
英語, 研究論文（学術雑誌）, Quinolone-resistant nontyphoidal Salmonella, one of the prominent pathogens causing acute gastroenteritis, has become a public health concern globally. The World Health Organization has ranked fluoroquinolone-resistant Salmonella as a high-priority pathogen for researching and developing new antibiotics. WQ-3034 and WQ-3154 are relatively new synthetic fluoroquinolones with distinctive structures. WQ-3034 has 6-amino-3,5-difluoropyridine-2-yl at R1, 3-hydroxyazetidinyl at R7, and the addition of chlorine atom at R8. WQ-3154 has a similar basic pharmacophore to WQ-3034 except for the modification at R8 with a methyl group. In this study, the inhibitory effect and DNA cleavage effect against wild-type (WT) and mutant Salmonella Typhimurium DNA gyrases of WQ-3034 and WQ-3154 were examined along with WQ-3810 and ciprofloxacin by measuring the drug concentration that inhibits half of the enzyme activity (IC50) and the drug concentration that induces 25% of maximum DNA cleavage (CC25). The minimum inhibitory concentration (MIC) of the compounds was assessed against Salmonella Typhimurium and Salmonella Enteritidis. Among four compounds, WQ-3034 demonstrated the highest inhibitory effect against both WT and mutant Salmonella Typhimurium DNA gyrases with amino acid substitution at codon 83 and/or 87, while ciprofloxacin showed the lowest inhibitory effect. Remarkably, WQ-3034 and WQ-3154 exhibited a significantly higher inhibitory effect than ciprofloxacin against Salmonella Typhimurium DNA gyrase with double amino acid substitution, Ser83Phe-Asp87Asn. Similarly, CC25 of WQ-3034 against mutant Salmonella Typhimurium DNA gyrase was lower than ciprofloxacin. Notably, MICs of WQ-3034 and WQ-3154 were higher than ciprofloxacin. In conclusion, this study revealed that WQ-3034 and WQ-3154 could potentially be effective therapeutic agents against quinolone-resistant nontyphoidal Salmonella. IMPORTANCE Quinolone-resistant nontyphoidal Salmonella is a pressing public health concern, demanding the exploration of novel treatments. In this study, we focused on two innovative synthetic fluoroquinolones, WQ-3034 and WQ-3154. Our findings revealed that these new compounds demonstrate potent inhibitory effects, even against mutant strains that cause resistance to existing quinolones. Hence, WQ-3034 and WQ-3154 could potentially be effective therapeutic agents against quinolone-resistant Salmonella Typhimurium. Furthermore, the data obtained in this study will be baseline information for antimicrobial drug development.

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.
Pondpan Suwanthada, Siriporn Kongsoi, Nami Miura, Lawrence P Belotindos, Chayada Piantham, Jirachaya Toyting, Mwangala L Akapelwa, Ruttana Pachanon, Kentaro Koide, Hyun Kim, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Microbial drug resistance (Larchmont, N.Y.), 2023年10月04日, ［国際誌］
英語, 研究論文（学術雑誌）, Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours.
Naoya Maekawa, Satoru Konnai, Kenji Hosoya, Sangho Kim, Ryohei Kinoshita, Tatsuya Deguchi, Ryo Owaki, Yurika Tachibana, Madoka Yokokawa, Hiroto Takeuchi, Yumiko Kagawa, Satoshi Takagi, Hiroshi Ohta, Yukinari Kato, Satoshi Yamamoto, Keiichi Yamamoto, Yasuhiko Suzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi
PloS one, 18, 10, e0291727, 2023年10月, ［国際誌］
英語, 研究論文（学術雑誌）, Immune checkpoint inhibitors (ICIs) have been developed for canine tumour treatment, and pilot clinical studies have demonstrated their antitumour efficacy in dogs with oral malignant melanoma (OMM). Although ICIs have been approved for various human malignancies, their clinical benefits in other tumour types remain to be elucidated in dogs. Here, we conducted a clinical study of c4G12, a canine chimeric anti-PD-L1 antibody, to assess its safety and efficacy in dogs with various advanced malignant tumours (n = 12) at the Veterinary Teaching Hospital of Hokkaido University from 2018 to 2023. Dogs with digit or foot pad malignant melanoma (n = 4), osteosarcoma (n = 2), hemangiosarcoma (n = 1), transitional cell carcinoma (n = 1), nasal adenocarcinoma (n = 1), B-cell lymphoma (n = 1), or undifferentiated sarcoma (n = 2) were treated with 2 or 5 mg/kg c4G12 every 2 weeks. Treatment-related adverse events of any grade were observed in eight dogs (66.7%), including elevated aspartate aminotransferase (grade 3) in one dog (8.3%) and thrombocytopenia (grade 4) in another dog (8.3%). Among dogs with target disease at baseline (n = 8), as defined by the response evaluation criteria for solid tumours in dogs (cRECIST), one dog with nasal adenocarcinoma and another with osteosarcoma experienced a partial response (PR), with an objective response rate of 25.0% (2 PR out of 8 dogs; 95% confidence interval: 3.2-65.1%). These results suggest that c4G12 is safe and tolerable and shows antitumor effects in dogs with malignant tumours other than OMM. Further clinical studies are warranted to identify the tumour types that are most likely to benefit from c4G12 treatment.

Development of a piezo biosensor for pathogen-specific biopolymer detection using a self-assembly barium titanate/polyvinylidene fluoride composite material
Mariko Takeda, Hayato Yoshino, Haruna Yamazaki, Takamichi Hirata, Takashi Kuroiwa, Chie Nakajima, Yasuhiko Suzuki, Fumio Munakata
Sensors and Actuators A: Physical, 360, 114545, 114545, Elsevier BV, 2023年10月
研究論文（学術雑誌）

Development of a high-affinity anti-bovine PD-1 rabbit-bovine chimeric antibody using an efficient selection and large production system.
Tomohiro Okagawa, Satoru Konnai, Shinya Goto, Yamato Sajiki, Otgontuya Ganbaatar, Kei Watari, Hayato Nakamura, Cai-Xia Wang, Taro Tachibana, Yukinari Kato, Yayoi Kameda, Junko Kohara, Nobuhiro Terasaki, Manabu Kubota, Akira Takeda, Hirofumi Takahashi, Yasuhiko Suzuki, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi
Veterinary research, 54, 1, 82, 82, 2023年09月27日, ［国際誌］
英語, 研究論文（学術雑誌）, Immune checkpoint molecules PD-1/PD-L1 cause T-cell exhaustion and contribute to disease progression in chronic infections of cattle. We established monoclonal antibodies (mAbs) that specifically inhibit the binding of bovine PD-1/PD-L1; however, conventional anti-PD-1 mAbs are not suitable as therapeutic agents because of their low binding affinity to antigen. In addition, their sensitivity for the detection of bovine PD-1 is low and their use for immunostaining PD-1 is limited. To address these issues, we established two anti-bovine PD-1 rabbit mAbs (1F10F1 and 4F5F2) and its chimeric form using bovine IgG1 (Boch1D10F1), which exhibit high binding affinity. One of the rabbit mAb 1D10F1 binds more strongly to bovine PD-1 compared with a conventional anti-PD-1 mAb (5D2) and exhibits marked inhibitory activity on the PD-1/PD-L1 interaction. In addition, PD-1 expression in bovine T cells could be detected with higher sensitivity by flow cytometry using 1D10F1. Furthermore, we established higher-producing cells of Boch1D10F1 and succeeded in the mass production of Boch1D10F1. Boch1D10F1 exhibited a similar binding affinity to bovine PD-1 and the inhibitory activity on PD-1/PD-L1 binding compared with 1D10F1. The immune activation by Boch1D10F1 was also confirmed by the enhancement of IFN-γ production. Finally, Boch1D10F1 was administered to bovine leukemia virus-infected cows to determine its antiviral effect. In conclusion, the high-affinity anti-PD-1 antibody developed in this study represents a powerful tool for detecting and inhibiting bovine PD-1 and is a candidate for PD-1-targeted immunotherapy in cattle.

Retrospective identification of pathogenic mycobacterial species in fish: Mycobacterium pseudoshottsii YM-3, isolated from a yellowtail fish in 1986 in Kochi, Japan
Masayuki Imajoh, Shiomi Yoshida, Lisa Nonaka, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Takayuki Wada
Microbiology Resource Announcements, American Society for Microbiology, 2023年09月15日
研究論文（学術雑誌）, ABSTRACT

The complete genome sequence of mycobacterial strain YM-3, isolated from cultured yellowtail in 1986, was determined. The strain was Mycobacterium pseudoshottsii , a closely related subspecies of Mycobacterium marinum , so the strain was isolated earlier than the first report of the subspecies in 2005.

Molecular Characterisation of Mycobacterium bovis Isolates from Cattle Slaughtered in Adamawa and Gombe States, North-Eastern Nigeria
Sadiq Mohammed Damina, David Atomanyi Barnes, Bitrus Inuwa, Gulak Hussaini Ularamu, Mohammed Bello, Olu Solomon Okaiyeto, Ayuba Caleb Kudi, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Current Issues in Molecular Biology, 45, 7, 6055, 6066, MDPI AG, 2023年07月19日
研究論文（学術雑誌）, Bovine tuberculosis is endemic in Nigeria with control measures as provided by the laws of the country being minimally enforced mostly at the abattoirs only. This study focused on bovine tuberculosis in Adamawa and Gombe States. Tuberculosis lesions were observed in 183 of 13,688 slaughtered cattle in the regions between June and December 2020. Analysis of tissue samples resulted in 17 Mycobacterium bovis isolates, predominantly from Gombe State. Spoligotyping identified four spoligotypes, including SB0944, SB1025, SB1104, and one novel pattern. MIRU-VNTR analysis further differentiated these spoligotypes into eight profiles. All isolates belonged to the Af1 clonal complex. The study emphasises the need for broader coverage and more isolates to comprehensively understand the molecular epidemiology of bovine tuberculosis in Nigeria. To enhance research and surveillance, a cost-effective approach is proposed, utilising a discriminatory VNTR panel comprising five or nine loci. The five-locus panel consists of ETR-C, QUB26, QUB11b, MIRU04, and QUB323. Alternatively, the nine-locus panel includes ETR-A, ETR-B, QUB11a, and MIRU26. Implementing this approach would provide valuable insights into the genetic diversity of M. bovis strains in Nigeria. These findings are crucial for developing effective control measures and minimising the impact of bovine tuberculosis on both animal and human health.

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens.
Shota Fukuzawa, Toyotaka Sato, Kotaro Aoki, Soh Yamamoto, Noriko Ogasawara, Chie Nakajima, Yasuhiko Suzuki, Motohiro Horiuchi, Satoshi Takahashi, Shin-Ichi Yokota
Annals of clinical microbiology and antimicrobials, 22, 1, 60, 60, 2023年07月15日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia
Joseph Yamweka Chizimu, Eddie Samuneti Solo, Precious Bwalya, Thoko Flav Kapalamula, Kaemba Kunkuta Mwale, David Squarre, Misheck Shawa, Patrick Lungu, David Atomanyi Barnes, Kaunda Yamba, Tiza Mufune, Herman Chambaro, Harvey Kamboyi, Musso Munyeme, Bernard Mudenda Hang’ombe, Nathan Kapata, Victor Mukonka, Roma Chilengi, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Antibiotics, 12, 7, 1126, 1126, MDPI AG, 2023年06月29日
研究論文（学術雑誌）, The emergence of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) is a threat to TB control programs in developing countries such as Zambia. Studies in Zambia have applied molecular techniques to understand drug-resistance-associated mutations, circulating lineages and transmission patterns of multi-drug-resistant (MDR) Mycobacterium tuberculosis. However, none has reported genotypes and mutations associated with pre-XDR TB. This study characterized 63 drug-resistant M. tuberculosis strains from the University Teaching Hospital between 2018 and 2019 using targeted gene sequencing and conveniently selected 50 strains for whole genome sequencing. Sixty strains had resistance mutations associated to MDR, one polyresistant, and two rifampicin resistant. Among MDR strains, seven percent (4/60) had mutations associated with pre-XDR-TB. While four, one and nine strains had mutations associated with ethionamide, para-amino-salicylic acid and streptomycin resistances, respectively. All 50 strains belonged to lineage 4 with the predominant sub-lineage 4.3.4.2.1 (38%). Three of four pre-XDR strains belonged to sub-lineage 4.3.4.2.1. Sub-lineage 4.3.4.2.1 strains were less clustered when compared to sub-lineages L4.9.1 and L4.3.4.1 based on single nucleotide polymorphism differences. The finding that resistances to second-line drugs have emerged among MDR-TB is a threat to TB control. Hence, the study recommends a strengthened routine drug susceptibility testing for second-line TB drugs to stop the progression of pre-XDR to XDR-TB and improve patient treatment outcomes.

Colistin-resistant bacteria poses few risks under physiological conditions
Soh Yamamoto, Masaru Usui, Noriko Ogasawara, Wataru Hayashi, Masato Suzuki, Noriyuki Nagano, Chie Nakajima, Yasuhiko Suzuki, Motohiro Horiuchi, Satoshi Takahashi, Shin-ichi Yokota, Yutaka Tamura, Toyotaka Sato
Research Square, Research Square Platform LLC, 2023年06月21日
研究論文（学術雑誌）, Abstract

Globally, 5.0 million people die annually from infections associated with antimicrobial-resistant bacteria, most commonly Escherichia coli¹. As colistin is a last-resort antibiotic for multidrug-resistant bacterial infections, the global spread of plasmid-mediated colistin resistance genes (mcr) gene is considered a major public health risk^2-4. However, the actual health risks of colistin resistance in hazardous bacteria have never been evaluated under physiological conditions. Here, we show that the fitness/virulence and colistin resistance of the pandemic multidrug-resistant E. coli clone ST131⁵ very depending on the acquired colistin resistance determinants and differ between physiological and in vitro conditions. The fitness/virulence of ST131 was unaffected by chromosomal-gene (pmrB) mutations or the acquisition of mcr-5-harbouring plasmids in mouse models. However, the acquisition of mcr-1- or mcr-3-harbouring plasmids attenuated fitness/virulence and promoted colistin susceptibility in human serum. We identified two virulence attenuation factors (vafA and vafB) on the pIncI2_mcr-1 plasmid that hijacked the ST131 transcriptome and inhibited nucleotide synthesis, attenuating colistin resistance. Our results demonstrate that colistin resistance poses much less of a threat than believed^6,7. We suggest that “nonresistance genes,” rather than resistance genes, are important antimicrobial resistance determinants for human health because they determine fitness/virulence and ultimately antimicrobial susceptibility under physiological conditions.

Enhanced Systemic Antitumour Immunity by Hypofractionated Radiotherapy and Anti-PD-L1 Therapy in Dogs with Pulmonary Metastatic Oral Malignant Melanoma.
Tatsuya Deguchi, Naoya Maekawa, Satoru Konnai, Ryo Owaki, Kenji Hosoya, Keitaro Morishita, Motoji Nakamura, Tomohiro Okagawa, Hiroto Takeuchi, Sangho Kim, Ryohei Kinoshita, Yurika Tachibana, Madoka Yokokawa, Satoshi Takagi, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Cancers, 15, 11, 2023年06月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Although immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1 (PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patients with cancer. This study retrospectively examined the therapeutic efficacy of combination therapy (hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oral malignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS) in the no RT (n = 20, free from the effect of RT), previous RT (n = 9, received RT ≤8 weeks prior to the first c4G12 dose), and concurrent RT (n = 10, c4G12 therapy within ±1 week of the first RT fraction) groups were 10%/185 days, 55.6%/283.5 days (p < 0.05 vs. no RT group), and 20%/129 days (p > 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be an effective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety profiles. Further prospective clinical studies are required to confirm the findings of this study.

Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in sheep.
Wisa Tiyamanee, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Otgontuya Ganbaatar, Naoya Maekawa, Rie Hasebe, Yumiko Kagawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Veterinary immunology and immunopathology, 261, 110609, 110609, 2023年05月11日, ［国際誌］
英語, 研究論文（学術雑誌）, Sheep have been used as a large animal experimental model for studying infectious diseases. However, due to a lack of staining antibodies and reagents, immunological studies on sheep have not progressed. The immunoinhibitory receptor programmed death-1 (PD-1) is expressed on T lymphocytes. The interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We previously reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections using anti-bovine PD-L1 monoclonal antibodies (mAbs). Furthermore, we found that blocking antibodies against PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy of cattle. However, the immunological role of the PD-1/PD-L1 pathway in chronic diseases of sheep remains unknown. In this study, we identified cDNA sequences of ovine PD-1 and PD-L1 and examined the cross-activity of anti-bovine PD-L1 mAbs against ovine PD-L1 as well as the expression of PD-L1 in ovine listeriosis. The amino acid sequences of ovine PD-1 and PD-L1 share a high degree of identity and similarity with homologs from ruminants and other mammalian species. Anti-bovine PD-L1 mAb recognized ovine PD-L1 on lymphocytes in the flow cytometric assay. Furthermore, an immunohistochemical staining confirmed the PD-L1 expression on macrophages in the brain lesions of ovine listeriosis. These findings indicated that our anti-PD-L1 mAb would be useful for analyzing the ovine PD-1/PD-L1 pathway. Further research is needed to determine the immunological role of PD-1/PD-L1 in chronic diseases such as BLV infection through experimental infection of sheep.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.
Jeewan Thapa, Joseph Yamweka Chizimu, Soyoka Kitamura, Mwangala Lonah Akapelwa, Pondpan Suwanthada, Nami Miura, Jirachaya Toyting, Tomoyasu Nishimura, Naoki Hasegawa, Yukiko Nishiuchi, Stephen V Gordon, Chie Nakajima, Yasuhiko Suzuki
Microbiology spectrum, e0508822, 2023年04月17日, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.
Thoko Flav Kapalamula, Jeewan Thapa, Kyoko Hayashida, Joseph Chizimu, Wimonrat Tanomsridachchai, Mirriam Ethel Nyenje, Rajab Mkakosya, Chie Nakajima, Yasuhiko Suzuki
Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 10406387231164596, 10406387231164596, 2023年04月08日, ［国際誌］
英語, 研究論文（学術雑誌）, The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

デラフロキサシンのらい菌DNAジャイレースに対する作用
鈴木 定彦, 朴 鐘勲, Pondpan Suwanthada, 中島 千絵
日本ハンセン病学会雑誌, 92, 1, 23, 23, 日本ハンセン病学会, 2023年04月
日本語

Determination of the Prevalence and Antimicrobial Resistance of Enterococcus faecalis and Enterococcus faecium Associated with Poultry in Four Districts in Zambia.
Grace Mwikuma, Henson Kainga, Simegnew Adugna Kallu, Chie Nakajima, Yasuhiko Suzuki, Bernard Mudenda Hang'ombe
Antibiotics (Basel, Switzerland), 12, 4, 2023年03月28日, ［国際誌］
英語, 研究論文（学術雑誌）, The presence of antimicrobial-resistant Enterococci in poultry is a growing public health concern worldwide due to its potential for transmission to humans. The aim of this study was to determine the prevalence and patterns of antimicrobial resistance and to detect drug-resistant genes in Enterococcus faecalis and E. faecium in poultry from four districts in Zambia. Identification of Enterococci was conducted using phenotypic methods. Antimicrobial resistance was determined using the disc diffusion method and antimicrobial resistance genes were detected using polymerase chain reaction and gene-specific primers. The overall prevalence of Enterococci was 31.1% (153/492, 95% CI: 27.1-35.4). Enterococcus faecalis had a significantly higher prevalence at 37.9% (58/153, 95% CI: 30.3-46.1) compared with E. faecium, which had a prevalence of 10.5% (16/153, 95% CI: 6.3-16.7). Most of the E. faecalis and E. faecium isolates were resistant to tetracycline (66/74, 89.2%) and ampicillin and erythromycin (51/74, 68.9%). The majority of isolates were susceptible to vancomycin (72/74, 97.3%). The results show that poultry are a potential source of multidrug-resistant E. faecalis and E. faecium strains, which can be transmitted to humans. Resistance genes in the Enterococcus species can also be transmitted to pathogenic bacteria if they colonize the same poultry, thus threatening the safety of poultry production, leading to significant public health concerns.

Corrigendum to 'Clonal/subclonal changes and accumulation of CTX-M-type β-lactamase genes in fluoroquinolone-resistant Escherichia coli ST131 and ST1193 strains isolated during the past 12 years, Japan' [Journal of Global Antimicrobial Resistance 27 (2021) 150-155].
Yukari Fukushima, Toyotaka Sato, Naoyuki Tsukamoto, Chie Nakajima, Yasuhiko Suzuki, Satoshi Takahashi, Shin-Ichi Yokota
Journal of global antimicrobial resistance, 32, 195, 195, 2023年03月, ［国際誌］
英語

Enhancement of Vaccine-Induced T-Cell Responses by PD-L1 Blockade in Calves.
Tomohiro Okagawa, Satoru Konnai, Hayato Nakamura, Otgontuya Ganbaatar, Yamato Sajiki, Kei Watari, Haruka Noda, Mitsuru Honma, Yukinari Kato, Yasuhiko Suzuki, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi
Vaccines, 11, 3, 2023年03月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Interactions between programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) cause functional exhaustion of T cells by inducing inhibitory signals, thereby attenuating effector functions of T cells. We have developed an anti-bovine PD-L1 blocking antibody (Ab) and have demonstrated that blockade of the interaction between PD-1 and PD-L1 reactivates T-cell responses in cattle. In the present study, we examined the potential utility of PD-1/PD-L1-targeted immunotherapy in enhancing T-cell responses to vaccination. Calves were inoculated with a hexavalent live-attenuated viral vaccine against bovine respiratory infections in combination with treatment with an anti-PD-L1 Ab. The expression kinetics of PD-1 in T cells and T-cell responses to viral antigens were measured before and after vaccination to evaluate the adjuvant effect of anti-PD-L1 Ab. PD-1 expression was upregulated in vaccinated calves after the administration of a booster vaccination. The activation status of CD4+, CD8+, and γδTCR+ T cells was enhanced by the combination of vaccination and PD-L1 blockade. In addition, IFN-γ responses to viral antigens were increased following combinatorial vaccination with PD-L1 blockade. In conclusion, the blockade of the PD-1/PD-L1 interaction enhances T-cell responses induced by vaccination in cattle, indicating the potential utility of anti-PD-L1 Ab in improving the efficacy of current vaccination programs.

タイの運河由来Salmonella属におけるプラスミドを介したキノロン系薬耐性遺伝子の有病率(Prevalence of plasmid-mediated quinolone resistance genes in Salmonella spp. from canals of Thailand)　　　　　　　　　　　　　　　
Toyting Jirachaya, Utrarachkij Fuangfa, Supha Neunghatai, Thongpanich Yuwanda, 中島 千絵, 鈴木 定彦
日本細菌学雑誌, 78, 1, 70, 70, 日本細菌学会, 2023年02月
英語

SalmonellaTyphimuriumのフルオロキノロン耐性に及ぼすGyrAおよびQnrB19のアミノ酸置換の影響(Impact of mutations in GyrA and QnrB19 on resistance to fluoroquinolone in Salmonella Typhimurium)　　　　　　　　　　　　　　　
Suwanthada Pondpan, Thapa Jeewan, 中島 千絵, 鈴木 定彦
日本細菌学雑誌, 78, 1, 105, 105, 日本細菌学会, 2023年02月
英語

Mycobacterium avium gyrAにおけるフルオロキノロン耐性関連変異が耐性へ果たす役割(Role of fluoroquinolone resistance-associated mutations in Mycobacterium avium gyrA to resistance)　　　　　　　　　　　　　　　
Thapa Jeewan, Chizimu Joseph Yamweka, 北村 そよか, Akapelwa Mwangala Lonah, Suwanthada Pondpan, 三浦 菜実, Toyting Jirachaya, 中島 千絵, 鈴木 定彦
日本細菌学雑誌, 78, 1, 105, 105, 日本細菌学会, 2023年02月
英語

タイにおける豚からヒトへのLA-MRSAの人獣共通感染の全ゲノム解析(Whole Genome Analysis of Zoonotic Transmission of LA-MRSA from Pigs to Humans in Thailand)　　　　　　　　　　　　　　　
Narongpun Pawarut, チャンチャイトン・パッタラット, 山岸 潤也, 中島 千絵, 鈴木 定彦
日本細菌学雑誌, 78, 1, 109, 109, 日本細菌学会, 2023年02月
英語

日本で分離されたM.avium臨床分離株におけるLevofloxacinのMICに対するmfpAをエンコードするPRPの影響(The effect of mfpA encoding PRP on the MICs of Levofloxacin in M.avium clinical isolates from Japan)　　　　　　　　　　　　　　　
Akapelwa Mwangala, 会津 勇樹[大内], Chizimu Joseph Yamweka, Kapalamula Thoko Flav, Menda Conscilliah Rhombohl, 西内 由紀子, 鈴木 定彦, 中島 千絵
日本細菌学雑誌, 78, 1, 151, 151, 日本細菌学会, 2023年02月
英語

Prevalence and risk factors of bovine tuberculosis in slaughtered cattle, Malawi.
Thoko Flav Kapalamula, Francis Kawonga, Misheck Shawa, Joseph Chizimu, Jeewan Thapa, Mirriam Ethel Nyenje, Rajhab Sawasawa Mkakosya, Kyoko Hayashida, Stephen Gordon, Chie Nakajima, Musso Munyeme, Bernard M Hang'ombe, Yasuhiko Suzuki
Heliyon, 9, 2, e13647, 2023年02月, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine tuberculosis (bTB) is an infectious disease with significant socioeconomic, animal, and public health impacts. However, the prevalence of bTB remains largely unclear in Malawi due to a paucity of information. Additionally, the existence of multiple risk factors is postulated to enhance bTB transmission in animals. A cross-sectional survey to estimate the prevalence of bTB, animal characteristics and identify associated risk factors was conducted from slaughtered cattle at three major regional abattoirs (southern, central and northern regions) in Malawi. Out of a total of 1547 cattle examined, 154 (9.95%) had bTB-like lesions in various visceral organs and lymph nodes; one sample per animal was collected, processed, and cultured in the in the BACTEC Mycobacterial growth indicator tube (MGIT) 960 system. From the 154 cattle that showed tuberculous like lesions, only 112 were positive on MGIT and 87 were confirmed to have M. bovis based on multiplex PCR. Cattle from the southern region (odds ratio (OR) = 1.96, 95% CI: 1.03-3.85) and central region (OR = 2.00, 95% CI: 1.16-3.56) were more likely presented with bTB-like lesions at slaughter than from the northern region. The risk of having bTB-like lesions was higher in females (OR = 1.51, CI: 1.00-2.29), older cattle (OR = 2.17, CI: 1.34-3.37), and crossbreeds (OR = 1.67, 95% CI: 1.12-2.47) than in males, younger animals, and Malawi Zebu breed, respectively. The high prevalence of bTB is of critical concern and necessitates active surveillance and strengthening of the current control strategies under a One Health (OH) approach at the animal-human interface.

Combined Immune Checkpoint Blockade Enhances Antiviral Immunity against Bovine Leukemia Virus.
Hayato Nakamura, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Kei Watari, Kana Kamitani, Maya Saito, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Journal of virology, 97, 1, e0143022, 2023年01月04日, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.

Sensitive detection of Mycobacterium bovis in spiked milk using a polymerase chain reaction assay
Marvin A. Villanueva, Yasuhiko Suzuki, Chie Nakajima, Haruka Suzuki, Yukari Fukushima, Claro N. Mingala
Archives of Veterinary Science, 28, 4, 1, 6, 2023年
研究論文（学術雑誌）, Bovine tuberculosis is a chronic zoonotic disease that affects both animal and human health and imposes serious public health concerns in the world. Intake of non-pasteurized milk is considered the most probable vehicle for the transmission of pathogenic bacteria. In this study, the detection of Mycobacterium bovis BCG in spiked milk using a polymerase chain reaction was performed. The performance of two DNA extraction methods, CTAB/phenol:chloroform:isoamyl alcohol and EXTRAGENMB were also evaluated. In addition, Mycobacterial concentration was tried to determine using the Standard/Viable Plate Count Method and Spectrophotometric (Turbidimetric) Method. PCR successfully detected M. bovis BCG in spiked milk, detecting approximately up to two bacilli per reaction. The two DNA extraction methods were effective in the isolation of amplifiable DNA, having the advantage of EXTRAGENMB in terms of (1) shorter duration of DNA extraction, (2) less sample manipulation, and (3) ease of execution of the procedure. Quantitative determination of the Mycobacterial population, however, failed to quantify the bacterial concentration per dilution, suggesting that CFU concentration should be considered an approximation. It is expected that this method can be used for the detection of M. bovis in raw milk samples.

Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.
Kyoko Hayashida, Alejandro Garcia, Lavel Chinyama Moonga, Tatsuki Sugi, Kodera Takuya, Mitsuo Kawase, Fumihiro Kodama, Atsushi Nagasaka, Nobuhisa Ishiguro, Ayato Takada, Masahiro Kajihara, Naganori Nao, Masashi Shingai, Hiroshi Kida, Yasuhiko Suzuki, William W Hall, Hirofumi Sawa, Junya Yamagishi
PloS one, 18, 5, e0285861, 2023年, ［国際誌］
英語, 研究論文（学術雑誌）, A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

In vitro evaluation of Lactiplantibacillus plantarum HOKKAIDO strain, effective lactic acid bacteria for calf diarrhea.
Mari Ikehata, Satoru Konnai, Tomohiro Okagawa, Kentaro Abe, Mitsuru Honma, Toru Kitamura, Naoya Maekawa, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Frontiers in veterinary science, 10, 1145445, 1145445, 2023年, ［国際誌］
英語, 研究論文（学術雑誌）, Calf diarrhea adversely affects growth and sometimes results in mortality, leading to severe economic losses to the cattle industry. Antibiotics are useful in the treatment against bacterial diarrhea, but not against viral, protozoan, and antibiotic-resistant bacterial diarrhea. Therefore, there are growing requirements for a novel control method for calf diarrhea. Probiotics have been considered promising candidates for preventive and supportive therapy for calf diarrhea for many years. A recent study has revealed that Lactiplantibacillus plantarum HOKKAIDO strain (Lp-HKD) reduces intestinal pathology and the severity of diarrhea in bovine rotavirus (BRV)-infected calves. Lp-HKD is known to enhance the function of human immune cells and expected to be used as probiotics for humans. Therefore, it is hypothesized that Lp-HKD modulates antiviral immune response in cattle and provide the clinical benefits in BRV-infected calves. However, the detailed mechanism of Lp-HKD-induced immunomodulation remains unknown. Thus, this study aimed to elucidate the immunomodulatory and antiviral effects of Lp-HKD in cattle. Cultivation assay of bovine peripheral blood mononuclear cells (PBMCs) showed that live and heat-killed Lp-HKD stimulates the production of interleukin-1β (IL-1β), IL-6, IL-10, and interferon-γ (IFN-γ) from PBMCs. Stimulation by heat-killed Lp-HKD yielded stronger cytokine production than stimulation by the live Lp-HKD. Additionally, CD14+ monocytes were identified as major producers of IL-1β, IL-6, and IL-10 under Lp-HKD stimulation; however, IFN-γ was mainly produced from immune cells other than CD14+ monocytes. Depletion of CD14+ monocytes from the PBMCs cultivation strongly decreased cytokine production induced by heat-killed Lp-HKD. The inhibition of toll-like receptor (TLR) 2/4 signaling decreased IL-1β and IL-6 production induced by live Lp-HKD and IL-1β, IL-6, and IFN-γ production induced by heat-killed Lp-HKD. Furthermore, live or heat-killed Lp-HKD also activated T cells and their production of IFN-γ and tumor necrosis factor-α. Then, culture supernatants of bovine PBMCs treated with heat-killed Lp-HKD demonstrated antiviral effects against BRV in vitro. In conclusion, this study demonstrated that Lp-HKD activates the functions of bovine immune cells via TLR2/4 signaling and exerts an antiviral effect against BRV through the induction of antiviral cytokines. Lp-HKD could be useful for the prevention and treatment of calf diarrhea through its immune activating effect.

Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors.
Naoya Maekawa, Satoru Konnai, Yumie Asano, Takumi Otsuka, Eri Aoki, Hiroto Takeuchi, Yukinari Kato, Mika K Kaneko, Shinji Yamada, Yumiko Kagawa, Maki Nishimura, Satoshi Takagi, Tatsuya Deguchi, Hiroshi Ohta, Takayuki Nakagawa, Yasuhiko Suzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi
PloS one, 18, 1, e0281143, 2023年, ［国際誌］
英語, 研究論文（学術雑誌）, Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies.
Hiroto Takeuchi, Chie Nakajima, Satoru Konnai, Naoya Maekawa, Tomohiro Okagawa, Masaru Usui, Yutaka Tamura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
PloS one, 18, 1, e0281171, 2023年, ［国際誌］
英語, 研究論文（学術雑誌）, Coagulase-positive Staphylococci express protein A, which binds to host antibodies, to evade the immune system. Taking advantage of its specific binding to antibodies, protein A from Staphylococcus aureus, which is called SpA, is commonly used as an affinity chromatography ligand for human therapeutic antibodies. However, among four canine IgG subclasses (A, B, C, and D), only IgG-B binds to SpA strongly and establishing an efficient and robust purification scheme for canine therapeutic antibodies whose IgG subclass is A, C, or D remains difficult and depends on finding a suitable substitute to SpA. S. pseudintermedius, a major coagulase-positive Staphylococci found in dogs, expresses spsQ gene which is orthologous to S. aureus spa. We hypothesized that to serve S. pseudintermedius to better adapt to the dog immune system, SpsQ would bind to canine IgGs stronger than SpA, making it a better affinity chromatography ligand for canine therapeutic antibodies. To characterize SpsQ, we first determined the spsQ nucleotide sequence from S. pseudintermedius isolates. Based on the identified sequence, we prepared recombinant proteins containing the immunoglobulin-binding domains of SpA (r-SpA) and SpsQ (r-SpsQ) and determined their binding capacity for each canine IgG subclass. The binding capacity of r-SpsQ for IgG-B was almost as high as that of r-SpA. Interestingly, while both r-SpsQ and r-SpA showed no binding to IgG-C, the binding capacity of r-SpsQ for IgG-A and IgG-D was significantly higher than that of r-SpA. Finally, we performed affinity chromatography using r-SpsQ- or r-SpA-immobilized resin and revealed that the recovery rates of IgG-A and IgG-D using r-SpsQ were significantly higher than those using r-SpA. Our findings indicate that SpsQ has a strong potential to be used as an affinity chromatography ligand for canine therapeutic antibodies of subclass A, B, and D.

Prostaglandin E2-Induced Immune Suppression via Cytotoxic T-Lymphocyte Antigen 4 in Paratuberculosis.
Yamato Sajiki, Satoru Konnai, Kei Watari, Tomohiro Okagawa, Akina Tanaka, Satoko Kawaji, Reiko Nagata, Naoya Maekawa, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
Infection and immunity, 90, 10, e0021022, 2022年09月14日, ［国際誌］
英語, 研究論文（学術雑誌）, Paratuberculosis is a chronic enteritis of ruminants caused by the facultative intracellular pathogen Mycobacterium avium subsp. paratuberculosis. The Th1 response inhibits the proliferation of M. avium subsp. paratuberculosis during the early subclinical stage. However, we have previously shown that immune inhibitory molecules, such as prostaglandin E2 (PGE2), suppress M. avium subsp. paratuberculosis-specific Th1 responses as the disease progresses. To date, the mechanism underlying immunosuppression during M. avium subsp. paratuberculosis infection has not been elucidated. Therefore, in the present study, we investigated the function of cytotoxic T-lymphocyte antigen 4 (CTLA-4) expressed by peripheral blood mononuclear cells (PBMCs) from cattle with paratuberculosis because CTLA-4 expression is known to be elevated in T cells under an M. avium subsp. paratuberculosis experimental infection. M. avium subsp. paratuberculosis antigen induced CTLA-4 expression in T cells from cattle experimentally infected with M. avium subsp. paratuberculosis. Interestingly, both PGE2 and an E prostanoid 4 agonist also induced CTLA-4 expression in T cells. In addition, a functional assay with a bovine CTLA-4-immunogobulin fusion protein (CTLA-4-Ig) indicated that CTLA-4 inhibited gamma interferon (IFN-γ) production in M. avium subsp. paratuberculosis-stimulated PBMCs, while blockade by anti-bovine CTLA-4 monoclonal antibody increased the secretion of IFN-γ and tumor necrosis factor alpha production in these PBMCs. These preliminary findings show that PGE2 has immunosuppressive effects via CTLA-4 to M. avium subsp. paratuberculosis. Therefore, it is necessary to clarify in the future whether CTLA-4-mediated immunosuppression facilitates disease progression of paratuberculosis in cattle.

In vitro antibacterial activity of OPS-2071 against Gram-positive and Gram-negative enteropathogenic bacteria.
Daisuke Oka, Ruchirada Changkwanyeun, Tomoyuki Yamaguchi, Chie Nakajima, Yasuhiko Suzuki, Makoto Matsumoto
The Journal of antimicrobial chemotherapy, 2022年09月14日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Enteric infections are a major public health issue in developing countries. Antimicrobial resistance is also a problem for enteric infection. OPS-2071 is a novel quinolone antibiotic with low oral absorption and potent antibacterial activity against Clostridioides difficile. OBJECTIVES: This study was conducted to confirm the antimicrobial activity of OPS-2071 against major enteropathogenic bacteria and to evaluate the risk of emergence of drug resistance. METHODS: The antibacterial activity was evaluated by the agar dilution method. The inhibitory activity against DNA gyrase and topoisomerase IV was determined by supercoiling assay and decatenation assay, respectively. The mutant prevention concentration and frequency of spontaneous resistance were determined by inoculation on drug-containing agar. RESULTS: Compared with the reference drugs, the antibacterial activity of OPS-2071 was more potent against Gram-positive bacteria and Campylobacter jejuni, including quinolone-resistant strains. Against other Gram-negative bacteria, OPS-2071 was comparable to existing quinolones. The inhibitory activities against DNA gyrase with quinolone-resistant mutations closely correlated with the antibacterial activity. Spontaneous resistance to OPS-2071 was not observed in Staphylococcus aureus and Escherichia coli and was lower than that of existing quinolones and higher than that of azithromycin in C. jejuni. The mutant prevention concentration of OPS-2071 was lower than that of tested compounds in S. aureus and C. jejuni and slightly higher than that of existing quinolones in E. coli. CONCLUSIONS: The broad and potent in vitro antibacterial activity and lower risk of drug resistance suggested that OPS-2071 may be useful for enteric infections caused by major pathogens including quinolone-resistant Campylobacter.

Detection of Mutations in pncA in Mycobacterium tuberculosis Clinical Isolates from Nepal in Association with Pyrazinamide Resistance.
Dipti Shrestha, Bhagwan Maharjan, Jeewan Thapa, Mwangala Lonah Akapelwa, Precious Bwalya, Joseph Yamweka Chizimu, Chie Nakajima, Yasuhiko Suzuki
Current issues in molecular biology, 44, 9, 4132, 4141, 2022年09月08日, ［国際誌］
英語, 研究論文（学術雑誌）, Without the proper information on pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis (MTB), PZA is inappropriately recommended for the treatment of both susceptible and multidrug-resistant tuberculosis (MDR-TB) in Nepal. This study aimed to collect information regarding PZA susceptibility in MTB isolates from Nepal by analyzing pncA and its upstream regulatory region (URR). A total of 211 MTB isolates were included in this study. Sequence analysis of pncA and its URR was performed to assess PZA resistance. First-line drug susceptibility testing, spoligotyping, and sequence analysis of rpoB, katG, the inhA regulatory region, gyrA, gyrB, and rrs were performed to assess their association with pncA mutation. Sequencing results reveal that 125 (59.2%) isolates harbored alterations in pncA and its URR. A total of 57 different mutation types (46 reported and 11 novel) were scattered throughout the whole length of the pncA gene. Eighty-seven isolates (41.2%) harbored mutations in pncA, causing PZA resistance in MTB. There was a more significant association of pncA alterations in MDR/pre-extensively drug-resistant (Pre-XDR) TB than in mono-resistant/pan-susceptible TB (p < 0.005). This first report on the increasing level of PZA resistance in DR-TB in Nepal highlights the importance of PZA susceptibility testing before DR-TB treatment.

ネコ腫瘍組織におけるprogrammed death ligand 1(PD-L1)の発現解析　　　　　　　　　　　　　　　
青木 絵理, 今内 覚, 前川 直也, 岡川 朋弘, 竹内 寛人, 佐藤 純平, 浅野 裕美恵, 大塚 拓海, 賀川 由美子, 加藤 幸成, 高木 哲, 鈴木 定彦, 村田 史郎, 大橋 和彦
日本獣医学会学術集会講演要旨集, 165回, [DI1A, 04], (公社)日本獣医学会, 2022年09月
日本語

消毒薬が抗菌薬耐性プラスミドの選択圧となる可能性の解明　　　　　　　　　　　　　　　
川端 結, 石川 北斗, 大枝 夏希, 浅井 鉄夫, 中島 千絵, 鈴木 定彦, 福田 昭, 臼井 優
日本獣医学会学術集会講演要旨集, 165回, [F1P, 03], (公社)日本獣医学会, 2022年09月
日本語

ネコ腫瘍組織におけるprogrammed death ligand 1(PD-L1)の発現解析　　　　　　　　　　　　　　　
青木 絵理, 今内 覚, 前川 直也, 岡川 朋弘, 竹内 寛人, 佐藤 純平, 浅野 裕美恵, 大塚 拓海, 賀川 由美子, 加藤 幸成, 高木 哲, 鈴木 定彦, 村田 史郎, 大橋 和彦
日本獣医学会学術集会講演要旨集, 165回, [DI1A, 04], (公社)日本獣医学会, 2022年09月
日本語

Molecular characterization of mutations in isoniazid- and rifampicin- resistant Mycobacterium tuberculosis isolated in Thailand.
Janisara Rudeeaneksin, Benjawan Phetsuksiri, Chie Nakajima, Yukari Fukushima, Worasak Suthachai, Nattakan Tipkrua, Krairerk Suthum, Nasron Jekloh, Supranee Bunchoo, Sopa Srisungngam, Wiphat Klayut, Shigeyuki Hamada, Yasuhiko Suzuki
Japanese journal of infectious diseases, 2022年08月31日, ［国内誌］
英語, 研究論文（学術雑誌）, Drug-resistant tuberculosis (TB) is a great challenge in TB control. The frequency and mutation characteristics can imply the efficiency of molecular tests for the rapid detection of TB drug resistance. This study examined the existence of mutations in katG and inhA for isoniazid (INH) resistance, and rpoB for rifampicin (RIF) resistance. A total of 178 drug-resistant Mycobacterium tuberculosis (MTB) isolates were analyzed. Mutations in katG encoding and inhA regulatory regions were detected in 136/168 (81.0%) and 29/168 (17.3%), respectively, with the most prominent mutation of Ser315Thr substitution in katG in 126/168 (75.0%), and -15 C > T substitution in the regulatory region of the inhA (26/168; 15.5%). Two distinct katG mutations (Tyr337Cys, 1003InsG) were identified. Of 125 RIF-resistant isolates, 118 (94.4%) carried mutations affecting the 81-bp RIF resistance-determining region (RRDR) with the most commonly affected codons 450, 445, and 435 identified in 74 (59.2%), 26 (20.8%) and 12 (9.6%) isolates, respectively. The genetic mutations were highly associated with phenotypic INH and RIF resistance, and the majority shared similarities with those in previous studies in Thailand and other Asian countries. The data is useful for guiding the use and the improvement of molecular tests for TB-drug resistance.

A high-affinity aptamer with base-appended base-modified DNA bound to isolated authentic SARS-CoV-2 strains wild-type and B.1.617.2 (delta variant).
Hirotaka Minagawa, Hirofumi Sawa, Tomoko Fujita, Shintaro Kato, Asumi Inaguma, Miwako Hirose, Yasuko Orba, Michihito Sasaki, Koshiro Tabata, Naoki Nomura, Masashi Shingai, Yasuhiko Suzuki, Katsunori Horii
Biochemical and biophysical research communications, 614, 207, 212, 2022年07月23日, ［国際誌］
英語, 研究論文（学術雑誌）, Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2.

Disseminated tuberculosis with paradoxical reactions caused by a Mycobacterium tuberculosis strain belonging to the Indo-Oceanic lineage: An imported case in Japan.
Kengo Oshima, Chie Nakajima, Kazushige Hirata, Hironori Hayashi, Eiichi N Kodama, Yukari Fukushima, Yasuhiko Suzuki, Hajime Kanamori, Hiroaki Baba, Tetsuji Aoyagi, Koichi Tokuda, Mitsuo Kaku
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 28, 7, 965, 970, 2022年07月, ［国際誌］
英語, 研究論文（学術雑誌）, Tuberculosis remains a major public health concern. Millions of tuberculosis cases and associated deaths have been reported worldwide. The Indo-Oceanic lineage Mycobacterium tuberculosis is common in Southeast Asia and causes extrapulmonary lesions. Only a few case studies on this lineage with genetic analysis using whole-genome sequencing have been reported in the literature. We present a case of disseminated tuberculosis, characterized by a variety of extrapulmonary lesions and paradoxical reactions, caused by the Indo-Oceanic lineage M. tuberculosis in a woman in Myanmar. A 22-year-old Burmese woman had arthritis in the right knee, with unknown aetiology, and was referred to our hospital. Computed tomography of the trunk revealed multiple nodular shadows in both lungs; swollen mediastinal lymph nodes; and small, low-density areas in the spleen. M. tuberculosis was detected in the sputum sample, joint aspirate, subcutaneous tumor, and exudate. She experienced a variety of paradoxical reactions together with aggressive tuberculosis dissemination in all areas of the body. Whole-genome sequencing of the DNA of MTB obtained from sputum and the right cervical subcutaneous abscess confirmed the Indo-Oceanic lineage of M. tuberculosis, the predominant strain in Myanmar. The Indo-Oceanic lineage M. tuberculosis causes disseminated tuberculosis all over the body including the periungual region. When patients show unusual symptoms, physicians should consider the introduction of new strains from foreign countries. Genetic analyses of the strains are recommended to define and confirm the lineages.

Threat from Mycobacterium orygis-associated tuberculosis in south Asia.
Jeewan Thapa, Stephen V Gordon, Chie Nakajima, Yasuhiko Suzuki
The Lancet. Microbe, 3, 9, e641-e642, 2022年06月14日, ［国際誌］
英語, 研究論文（学術雑誌）

Exploration of serum biomarkers in dogs with malignant melanoma receiving anti-PD-L1 therapy and potential of COX-2 inhibition for combination therapy.
Naoya Maekawa, Satoru Konnai, Yumie Asano, Yamato Sajiki, Tatsuya Deguchi, Tomohiro Okagawa, Kei Watari, Hiroto Takeuchi, Satoshi Takagi, Kenji Hosoya, Sangho Kim, Hiroshi Ohta, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Scientific reports, 12, 1, 9265, 9265, 2022年06月03日, ［国際誌］
英語, 研究論文（学術雑誌）, Immune checkpoint inhibitors (ICIs) such as anti-PD-L1 antibodies are widely used to treat human cancers, and growing evidence suggests that ICIs are promising treatments for canine malignancies. However, only some canine oral malignant melanoma (OMM) cases respond to ICIs. To explore biomarkers predictive of survival in dogs with pulmonary metastatic OMM receiving the anti-PD-L1 antibody c4G12 (n = 27), serum concentrations of prostaglandin E2 (PGE2), cytokines, chemokines, and growth factors were measured prior to treatment initiation. Among 12 factors tested, PGE2, interleukin (IL)-12p40, IL-8, monocyte chemotactic protein-1 (MCP-1), and stem cell factor (SCF) were higher in OMM dogs compared to healthy dogs (n = 8). Further, lower baseline serum PGE2, MCP-1, and vascular endothelial growth factor (VEGF)-A concentrations as well as higher IL-2, IL-12, and SCF concentrations predicted prolonged overall survival. These observations suggest that PGE2 confers resistance against anti-PD-L1 therapy through immunosuppression and thus is a candidate target for combination therapy. Indeed, PGE2 suppressed IL-2 and interferon (IFN)-γ production by stimulated canine peripheral blood mononuclear cells (PBMCs), while inhibition of PGE2 biosynthesis using the COX-2 inhibitor meloxicam in combination with c4G12 enhanced Th1 cytokine production by PBMCs. Thus, serum PGE2 may be predictive of c4G12 treatment response, and concomitant use of COX-2 inhibitors may enhance ICI antitumor efficacy.

ANTIBIOGRAMS AND VIRULENCE GENES OF ESCHERICHIA COLI AND SALMONELLA SP FROM FARM PIGS IN CENTRAL THAILAND
Pornthip Chompook, Apiradee Intarapak, Kanjana Changkaew, Ruttana Pachanon, Apichart Suwannachairob, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul, Norikazu Isoda
Southeast Asian Journal of Tropical Medicine and Public Health, 53, 3, 322, 344, 2022年05月19日
研究論文（学術雑誌）, The increasing global trend of antimicrobial resistance (AMR), especially in pig production, can pose a threat to human health. The study investigated the prevalence of AMR in Escherichia coli and Salmonella serovars from farm pigs in central Thailand. E. coli and Salmonella serovars from pig stool specimens were identified by standard methods, susceptibility to antimicrobials by the disk agar diffusion method and pathogenic E. coli virulence genes by multiplex PCR. E. coli isolates harboring astA were the most predominant (27%), followed by eaeA (9%). E. coli isolates with virulence genes were present significantly more in pigs with loose stool (70%) than those with normal stool (40%) (p-value <0.001). The overall prevalence of Salmonella sp isolates in pigs was 44.3%, with S. Agona serovar predominant (27%). E. coli and Salmonella sp isolates demonstrated predominant resistance to penicillin (71 and 79% respectively), followed by tetracycline (67.5 and 73.1%), then streptomycin (52 and 51%), and sulfonamide (45 and 36%), with multidrug resistance (MDR) (≥3 antimicrobial classes) in 66 and 74% E. coli and Salmonella sp isolates, respectively. E. coli harboring a single astA was significantly associated with MDR phenotype compared to those without any virulence gene (odds ratio = 2.16, 95% confidence interval: 1.09-4.29). In conclusion, the study indicates that farm pigs in central Thailand could be potential sources of foodborne MDR E. coli and Salmonella sp.

新たな置換基を1位に保有するキノロン系抗菌薬とらい菌DNAジャイレースの相互作用
朴 鐘勲, 山口 智之, 会津 勇樹[大内], 小出 健太郎, Pachanon Ruttana, Chizimu Joseph Yamweka, 森 茂太郎, 金 玄, 向井 徹, 中島 千絵, 鈴木 定彦
日本ハンセン病学会雑誌, 91, 1, 27, 27, 日本ハンセン病学会, 2022年05月
日本語

Molecular Characterization of Mycobacterium ulcerans DNA Gyrase and Identification of Mutations Reducing Susceptibility to Quinolones In Vitro.
Hyun Kim, Shigetarou Mori, Tsuyoshi Kenri, Yasuhiko Suzuki
Antimicrobial agents and chemotherapy, 66, 4, e0190221, 2022年04月19日, ［国際誌］
英語, 研究論文（学術雑誌）, Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans. Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase. However, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in M. ulcerans remains unknown. This study investigated the detailed molecular mechanism of M. ulcerans DNA gyrase and the contribution of FQ resistance in vitro using recombinant proteins from the M. ulcerans subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC50 of FQs against Ala91Val and Asp95Gly mutants of M. ulcerans shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type (WT) enzyme. Similarly, the quinolone concentrations required to induce 25% of the maximum DNA cleavage (CC25) was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of the WT/mutant M. ulcerans DNA gyrase and FQ side chains were assessed by molecular docking studies. This was the first elaborative study demonstrating the contribution of mutations in M. ulcerans DNA GyrA subunit to FQ resistance in vitro.

Characterisation of plasmids harbouring qnrA1, qnrS1, and qnrB4 in E. coli isolated in the Philippines from food-producing animals and their products.
Lawrence P Belotindos, Risa Tsunoda, Marvin A Villanueva, Chie Nakajima, Claro N Mingala, Yasuhiko Suzuki
Journal of global antimicrobial resistance, 30, 38, 46, 2022年04月18日, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: Determinants showing plasmid-mediated quinolone resistance, which usually leads to antimicrobial ineffectiveness, have become an emerging clinical problem. In our previous study in the Philippines, a high prevalence of Qnr determinants was found in clinical samples and food-producing animals and their food products. However, no qnr-carrying plasmids have been investigated in animals or animal-derived foods. Hence, in the present, we aimed to characterise qnr-carrying plasmids in Escherichia coli isolated from the food supply chain. METHODS: Plasmids from 44 qnr-positive isolates were assigned to incompatibility groups by PCR-based replicon typing, and the presence of β-lactamase-encoding genes were investigated by PCR. Localisation of qnr in plasmids was determined by S1-PFGE and Southern blot hybridisation. The transferability of qnr-carrying plasmids was examined by conjugation analysis. RESULTS: Overall, 77.3% (95%CI = 62.2 - 88.5) of the isolates harbouring qnr determinants were positive for seven plasmid types, and 56.8% concurrently harboured blaTEM-1. Plasmid IncFrepB was prevalent (65.9%, 95%CI = 50.1 - 79.5) among qnr determinants. Localisation of qnr determinants in IncFrepB and transferability of plasmids was further confirmed. CONCLUSIONS: The current study proved that qnr in E. coli isolated from food-producing animals and their food products could spread via plasmid IncFrepB upon selective pressure with quinolones or other antimicrobials. Therefore, to curb the emergence and spread of qnr-harbouring bacteria in the Philippines, prudent use of antimicrobials in animal production and stricter hygiene and food handling are recommended.

Campylobacter Express Resistance Array for detecting the presence of fluoroquinolone- and macrolide-resistant Campylobacter jejuni and Campylobacter coli in broiler farms
Masaru Usui, Junya Tase, Masanobu Onozaki, Yasuhiko Suzuki, Yutaka Tamura, Chie Nakajima
Journal of Applied Microbiology, 132, 4, 3249, 3255, 2022年04月
研究論文（学術雑誌）, Aims: The aim of the study was to develop a microarray-based method for the detection of antibiotic-resistant Campylobacter in broiler farms to decrease the risk of contamination of chicken meat. Methods and Results: A combination of DNA microarray and primer extension for rapid and simultaneous detection of fluoroquinolone- and macrolide-resistant Campylobacter jejuni/Campylobacter coli, termed Campylobacter Express Resistance Array (CAMERA), was used to analyse chicken caecal droppings. CAMERA assays could detect at least 105 colony forming units of C. jejuni/C. coli g−1 of chicken caecal contents spiked with C. jejuni/C. coli. To compare the CAMERA method and direct culturing method for screening antibiotic-resistant C. jejuni/C. coli in poultry farms, chicken caecal droppings obtained from 42 poultry houses were analysed using both methods. In total, 95.2% of the results (40/42 poultry houses) obtained using the CAMERA and culturing method were identical. In the remaining two poultry houses, the CAMERA could detect the prevalent strain of C. jejuni/C. coli based on results of the culturing method. Conclusions: The culturing method required >3 days to isolate and identify antibiotic-resistant C. jejuni/C. coli. In contrast, the CAMERA required only 6 h. Significance and Impact of the Study: This method can facilitate quick screening and control of fluoroquinolone- and macrolide-resistant C. jejuni/C. coli in broiler farms.

Campylobacter Express Resistance Array for detecting the presence of fluoroquinolone- and macrolide-resistant Campylobacter jejuni and Campylobacter coli in broiler farms.
Masaru Usui, Junya Tase, Masanobu Onozaki, Yasuhiko Suzuki, Yutaka Tamura, Chie Nakajima
Journal of applied microbiology, 132, 4, 3249, 3255, 2022年04月, ［国際誌］
英語, 研究論文（学術雑誌）, AIMS: The aim of the study was to develop a microarray-based method for the detection of antibiotic-resistant Campylobacter in broiler farms to decrease the risk of contamination of chicken meat. METHODS AND RESULTS: A combination of DNA microarray and primer extension for rapid and simultaneous detection of fluoroquinolone- and macrolide-resistant Campylobacter jejuni/Campylobacter coli, termed Campylobacter Express Resistance Array (CAMERA), was used to analyse chicken caecal droppings. CAMERA assays could detect at least 105 colony forming units of C. jejuni/C. coli g-1 of chicken caecal contents spiked with C. jejuni/C. coli. To compare the CAMERA method and direct culturing method for screening antibiotic-resistant C. jejuni/C. coli in poultry farms, chicken caecal droppings obtained from 42 poultry houses were analysed using both methods. In total, 95.2% of the results (40/42 poultry houses) obtained using the CAMERA and culturing method were identical. In the remaining two poultry houses, the CAMERA could detect the prevalent strain of C. jejuni/C. coli based on results of the culturing method. CONCLUSIONS: The culturing method required >3 days to isolate and identify antibiotic-resistant C. jejuni/C. coli. In contrast, the CAMERA required only 6 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This method can facilitate quick screening and control of fluoroquinolone- and macrolide-resistant C. jejuni/C. coli in broiler farms.

Characterization of embB mutations involved in ethambutol resistance in multi-drug resistant Mycobacterium tuberculosis isolates in Zambia
Precious Bwalya, Eddie S. Solo, Joseph Y. Chizimu, Dipti Shrestha, Grace Mbulo, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Tuberculosis, 133, 2022年03月
研究論文（学術雑誌）, Background: Ethambutol (EMB) is an important anti-tuberculosis drug used in the management of multi-drug resistant tuberculosis (MDR-TB). Mutations in embB are the major mechanism of resistance. This study investigated embB mutations among MDR-TB isolates and analyzed their correlations with phenotypic drug susceptibility testing (DST) in Zambia. Method: A total of 132 MDR-TB isolates were collected from January 2014 to April 2017 and characterized using MGIT 960 systems, embB sequencing, and spoligotyping. Results: Out of 61 phenotypically EMB resistant isolates, 53 had mutations in embB. Among the 71 EMB susceptible isolates, 47 had embB mutations. Sensitivity of embB mutations was 86.9% while specificity was 33.8%. CAS1_Kili (SIT21) had high odds of having embB mutations, particularly, G918A (Met306eIl) (Odds ratio 16.7, p < 0.0001). Conclusion: Molecular EMB resistance testing by DNA sequencing can improve detection of EMB resistance among MDR-TB patients in Zambia. Additionally, CAS1_Kili was associated with embB amino acid substitution Met306Ile suggesting transmission. A detailed investigation to track and determine transmission hotspot area for MDR-TB could help optimize control strategies.

Broad-host-range IncW plasmid harbouring tet(X) in Escherichia coli isolated from pigs in Japan.
Masaru Usui, Akira Fukuda, Yasuhiko Suzuki, Chie Nakajima, Yutaka Tamura
Journal of global antimicrobial resistance, 28, 97, 101, 2022年03月, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: Tetracyclines are used in veterinary medicine for livestock. Tigecycline, a semisynthetic tetracycline derivative, is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. The prevalence of variants of the mobile tigecycline resistance gene tet(X) in livestock is increasing worldwide. However, the prevalence of Tet(X) among livestock in Japan is unclear. This study was conducted to clarify the prevalence of Tet(X) in pigs in Japan, focusing on isolation and molecular characterisation of plasmid-mediated tet(X)-positive Escherichia coli through retrospective analysis. METHODS: We retrospectively screened for tigecycline-resistant E. coli strains isolated from pigs. The tigecycline-resistant strain and tet(X)-harbouring plasmid were characterised. RESULTS: The IncW plasmid harbouring the tet(X) variant [previously named as tet(X6)] was detected in one E. coli isolate from pigs (0.8%; 1/120) in 2012. The tet(X) plasmid was transferable by conjugation to the E. coli ML4909 recipient strain. Some mobile genetic elements (TnAs3 and ISVsa3) were observed in the region surrounding tet(X). The tet(X)-harbouring plasmid shared a conserved backbone with IncW plasmid R388, which is a broad-host-range plasmid. CONCLUSION: The emergence and spread of tet(X) variants in Enterobacterales poses a public-health concern. To the best of our knowledge, this is the first report of the emergence of an IncW plasmid harbouring tet(X). Using tetracyclines in livestock exerts selective pressure on the tet(X) plasmid; therefore, prudent use of tetracyclines is required.

Characterization of embB mutations involved in ethambutol resistance in multi-drug resistant Mycobacterium tuberculosis isolates in Zambia.
Precious Bwalya, Eddie S Solo, Joseph Y Chizimu, Dipti Shrestha, Grace Mbulo, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Tuberculosis (Edinburgh, Scotland), 133, 102184, 102184, 2022年03月, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Ethambutol (EMB) is an important anti-tuberculosis drug used in the management of multi-drug resistant tuberculosis (MDR-TB). Mutations in embB are the major mechanism of resistance. This study investigated embB mutations among MDR-TB isolates and analyzed their correlations with phenotypic drug susceptibility testing (DST) in Zambia. METHOD: A total of 132 MDR-TB isolates were collected from January 2014 to April 2017 and characterized using MGIT 960 systems, embB sequencing, and spoligotyping. RESULTS: Out of 61 phenotypically EMB resistant isolates, 53 had mutations in embB. Among the 71 EMB susceptible isolates, 47 had embB mutations. Sensitivity of embB mutations was 86.9% while specificity was 33.8%. CAS1_Kili (SIT21) had high odds of having embB mutations, particularly, G918A (Met306eIl) (Odds ratio 16.7, p < 0.0001). CONCLUSION: Molecular EMB resistance testing by DNA sequencing can improve detection of EMB resistance among MDR-TB patients in Zambia. Additionally, CAS1_Kili was associated with embB amino acid substitution Met306Ile suggesting transmission. A detailed investigation to track and determine transmission hotspot area for MDR-TB could help optimize control strategies.

Enhancement of interleukin-2 production by bovine peripheral blood mononuclear cells treated with the combination of anti-programmed death-ligand 1 and cytotoxic T lymphocyte antigen 4 chimeric monoclonal antibodies.
Kei Watari, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
The Journal of veterinary medical science, 84, 1, 6, 15, 2022年01月07日, ［国内誌］
英語, 研究論文（学術雑誌）, Our previous studies demonstrate the therapeutic efficacy against bovine diseases of an anti-bovine programmed death-ligand 1 (PD-L1) chimeric antibody. In humans, PD-1 and PD-L1 antibodies are more effective when combined with an antibody targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and these combination therapies are therefore clinically used. Here we generated an anti-bovine CTLA-4 chimeric antibody (chAb) to enhance the therapeutic efficacy of the PD-L1 antibody. We further analyzed the effects of dual blockade of CTLA-4 and PD-1 pathways on T-cell responses. The established anti-bovine CTLA-4 chAb showed comparable blocking activity on the binding of bovine CTLA-4 to CD80 and CD86 as the anti-bovine CTLA-4 mouse monoclonal antibody. Anti-bovine CTLA-4 chAb also significantly increased IL-2 production from bovine peripheral blood mononuclear cells (PBMCs). Further, the combination of anti-CTLA-4 chAb with anti-PD-L1 chAb significantly upregulated IL-2 production by PBMCs. These results suggest that the combination of antibodies have higher potential to enhance immune responses against pathogens compared with single administration.

Conjugative IncHI2/HI2A plasmids harbouring mcr-9 in colistin-susceptible Escherichia coli isolated from diseased pigs in Japan.
Akira Fukuda, Hitomi Nakano, Yasuhiko Suzuki, Chie Nakajima, Masaru Usui
Access microbiology, 4, 11, acmi000454, 2022年, ［国際誌］
英語, 研究論文（学術雑誌）, Colistin is a last resort antimicrobial used for the treatment of gram-negative bacterial infections. Plasmid-mediated colistin resistance (mcr) genes are a cause of global concern, and, thus far, mcr-1-10 have been identified. In a previous study, we screened mcr-1-5 in Escherichia coli derived from diseased pigs in Japan and reported a high prevalence of mcr-1, -3 and -5. However, the previous report on the prevalence of mcr genes was inaccurate. In the present study, we aimed to clarify the prevalence of all reported variants of mcr in E. coli derived from the diseased pigs, which were previously screened for mcr-1-5. Additionally, we also characterized the mcr-9-positive E. coli , which was detected in this study. We screened mcr in 120 E. coli strains from diseased pigs and mcr-positive E. coli and an mcr-carrying plasmid were also characterized. One mcr-9-positive colistin-susceptible E. coli strain was detected (0.8 %). Plasmid-mediated mcr-9 was transferred to E. coli ML4909 as the recipient strain, and it was located on IncHI2/HI2A plasmid p387_L with other antimicrobial resistance genes (ARGs). The region harbouring ARGs including mcr-9, was similar to that on the Klebsiella pneumoniae chromosome harbouring mcr-9 isolated in Japan. mcr-3, -5 and -9 were detected (4.2 %) in colistin-susceptible strains. mcr-9 was found to be disseminated via the plasmid IncHI2/HI2A p387_L and transferred and inserted into chromosomes via a transposon. Our results suggest that mcr genes should be monitored regularly, regardless of their susceptibility to colistin.

Corrigendum: The TbD1 Locus Mediates a Hypoxia-Induced Copper Response in Mycobacterium bovis.
Ruoyao Ma, Damien Farrell, Gabriel Gonzalez, John A Browne, Chie Nakajima, Yasuhiko Suzuki, Stephen V Gordon
Frontiers in microbiology, 13, 947450, 947450, 2022年, ［国際誌］
英語, [This corrects the article DOI: 10.3389/fmicb.2022.817952.].

The TbD1 Locus Mediates a Hypoxia-Induced Copper Response in Mycobacterium bovis.
Ruoyao Ma, Damien Farrell, Gabriel Gonzalez, John A Browne, Chie Nakajima, Yasuhiko Suzuki, Stephen V Gordon
Frontiers in microbiology, 13, 817952, 817952, 2022年, ［国際誌］
英語, 研究論文（学術雑誌）, The Mycobacterium tuberculosis complex (MTBC) contains the causative agents of tuberculosis (TB) in mammals. The archetypal members of the MTBC, Mycobacterium tuberculosis and Mycobacterium bovis, cause human tuberculosis and bovine tuberculosis, respectively. Although M. tuberculosis and M. bovis share over 99.9% genome identity, they show distinct host adaptation for humans and animals; hence, while the molecular basis of host adaptation is encoded in their genomes, the mechanistic basis of host tropism is still unclear. Exploration of the in vitro phenotypic consequences of known genetic difference between M. bovis and M. tuberculosis offers one route to explore genotype-phenotype links that may play a role in host adaptation. The TbD1 ("Mycobacterium tuberculosis deletion 1 region") locus encompasses the mmpS6 and mmpL6 genes. TbD1 is absent in M. tuberculosis "modern" lineages (Lineages 2, 3, and 4) but present in "ancestral" M. tuberculosis (Lineages 1 and 7), Mycobacterium africanum lineages (Lineages 5 and 6), newly identified M. tuberculosis lineages (Lineages 8 and 9), and animal adapted strains, such as M. bovis. The function of TbD1 has previously been investigated in M. tuberculosis, where conflicting data has emerged on the role of TbD1 in sensitivity to oxidative stress, while the underlying mechanistic basis of such a phenotype is unclear. In this study, we aimed to shed further light on the role of the TbD1 locus by exploring its function in M. bovis. Toward this, we constructed an M. bovis TbD1 knockout (ΔTbD1) strain and conducted comparative transcriptomics to define global gene expression profiles of M. bovis wild-type (WT) and the ΔTbD1 strains under in vitro culture conditions (rolling and standing cultures). This analysis revealed differential induction of a hypoxia-driven copper response in WT and ΔTbD1 strains. In vitro phenotypic assays demonstrated that the deletion of TbD1 sensitized M. bovis to H2O2 and hypoxia-specific copper toxicity. Our study provides new information on the function of the TbD1 locus in M. bovis and its role in stress responses in the MTBC.

Genetic Diversity and Transmission of Multidrug-Resistant Mycobacterium tuberculosis strains in Lusaka, Zambia.
Joseph Yamweka Chizimu, Eddie Samuneti Solo, Precious Bwalya, Thoko Flav Kapalamula, Mwangala Lonah Akapelwa, Patrick Lungu, Dipti Shrestha, Yukari Fukushima, Victor Mukonka, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 114, 142, 150, 2022年01月, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVE: Zambia is among the 30 high tuberculosis burden countries in the world. Despite increasing reports of multidrug-resistant tuberculosis (MDR-TB) in routine surveillance, information on the transmission of MDR Mycobacterium tuberculosis strains is largely unknown. This study elucidated the genetic diversity and transmission of MDR M. tuberculosis strains in Lusaka, Zambia. METHODS: Eighty-five MDR M. tuberculosis samples collected from 2013 to 2017 at the University Teaching Hospital were used. Drug-resistance associated gene sequencing, spoligotyping, 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), and multiplex PCR for RD-Rio sub-lineage identification were applied. RESULTS: The identified clades were LAM (48%), CAS (29%), T (14%), X (6%) and Harlem (2%). Strains belonging to SITs 21/CAS1-Kili and 20/LAM1 formed the largest clonal complexes. Combined spoligotyping and 24 loci-MIRU-VNTR revealed 47 genotypic patterns with a clustering rate of 63%. Ninety-five percent of LAM strains belonged to the RD-Rio sub-lineage. CONCLUSION: The high clustering rate suggested that a large proportion of MDR-TB was due to recent transmission rather than the independent acquisition of MDR. This spread was attributed to clonal expansion of SIT21/CAS1-Kili and SIT20/LAM1 strains. Therefore, TB control programs recommending genotyping coupled with conventional epidemiological methods can guide measures for stopping the spread of MDR-TB.

Whole-Genome Sequencing Reveals Recent Transmission of Multidrug-Resistant Mycobacterium tuberculosis CAS1-Kili Strains in Lusaka, Zambia.
Joseph Yamweka Chizimu, Eddie Samuneti Solo, Precious Bwalya, Wimonrat Tanomsridachchai, Herman Chambaro, Misheck Shawa, Thoko Flav Kapalamula, Patrick Lungu, Yukari Fukushima, Victor Mukonka, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki
Antibiotics (Basel, Switzerland), 11, 1, 2021年12月28日, ［国際誌］
英語, 研究論文（学術雑誌）, Globally, tuberculosis (TB) is a major cause of death due to antimicrobial resistance. Mycobacterium tuberculosis CAS1-Kili strains that belong to lineage 3 (Central Asian Strain, CAS) were previously implicated in the spread of multidrug-resistant (MDR)-TB in Lusaka, Zambia. Thus, we investigated recent transmission of those strains by whole-genome sequencing (WGS) with Illumina MiSeq platform. Twelve MDR CAS1-Kili isolates clustered by traditional methods (MIRU-VNTR and spoligotyping) were used. A total of 92% (11/12) of isolates belonged to a cluster (≤12 SNPs) while 50% (6/12) were involved in recent transmission events, as they differed by ≤5 SNPs. All the isolates had KatG Ser315Thr (isoniazid resistance), EmbB Met306 substitutions (ethambutol resistance) and several kinds of rpoB mutations (rifampicin resistance). WGS also revealed compensatory mutations including a novel deletion in embA regulatory region (-35A > del). Several strains shared the same combinations of drug-resistance-associated mutations indicating transmission of MDR strains. Zambian strains belonged to the same clade as Tanzanian, Malawian and European strains, although most of those were pan-drug-susceptible. Hence, complimentary use of WGS to traditional epidemiological methods provides an in-depth insight on transmission and drug resistance patterns which can guide targeted control measures to stop the spread of MDR-TB.

Endobronchial Ultrasonography with a Guide Sheath Transbronchial Biopsy for Diagnosing Peripheral Pulmonary Lesions within or near Fibrotic Lesions in Patients with Interstitial Lung Disease.
Takayasu Ito, Shotaro Okachi, Tomoki Kimura, Kensuke Kataoka, Yasuhiko Suzuki, Fumie Kinoshita, Keiko Wakahara, Naozumi Hashimoto, Yasuhiro Kondoh
Cancers, 13, 22, 2021年11月17日, ［国際誌］
英語, 研究論文（学術雑誌）, In patients with interstitial lung disease (ILD), the most frequent locations of lung cancer are within or near fibrotic lesions. However, the diagnostic yield for peripheral pulmonary lesions (PPLs) within or near fibrotic lesions using endobronchial ultrasonography with a guide sheath transbronchial biopsy (EBUS-GS TBB) may be unsatisfactory compared to that for PPLs distant from fibrotic lesions because of the difficulty in reaching the lesions. Our objectives were to evaluate the yield for PPLs using EBUS-GS TBB according to the proximity of PPLs to fibrotic lesions and to determine factors affecting the yield for PPLs. We retrospectively investigated 323 consecutive lesions using EBUS-GS TBB between 1 November 2014 and 31 December 2016. We identified PPLs with ILD in such lesions. PPLs with ILD were divided into PPLs within or near fibrotic lesions which met the criterion of PPLs, and of fibrotic lesions overlapping each other (PPLs-FL) and those distant from fibrotic lesions, which met the criterion of PPLs and the area of fibrotic lesion not overlapping each other (PPLs-NFL). Of the 323 lesions, 55 were included (31 PPLs-FL and 24 PPLs-NFL). The diagnostic yield for PPLs-FL was significantly lower than for PPLs-NFL (45.2% vs. 83.3%, p = 0.004). Multivariate analysis revealed that PPLs-NFL (odds ratio (OR) = 7.509) and a probe position within the lesion (OR = 4.172) were significant factors affecting diagnostic yield. Lesion's positional relation to fibrotic lesions and the probe position were important factors affecting the successful diagnosis via EBUS-GS TBB in these patients.

Abnormal blood coagulation and kidney damage in aged hamsters infected with severe acute respiratory syndrome coronavirus 2
Marumi Ohno, Michihito Sasaki, Yasuko Orba, Toshiki Sekiya, Md Abdul Masum, Osamu Ichii, Tatsuya Sawamura, Akemi Kakino, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa, Masashi Shingai
Viruses, 13, 11, 2021年11月, ［国際誌］
英語, 研究論文（学術雑誌）, Systemic symptoms have often been observed in patients with coronavirus disease 2019 (COVID-19) in addition to pneumonia, however, the details are still unclear due to the lack of an appropriate animal model. In this study, we investigated and compared blood coagulation abnormalities and tissue damage between male Syrian hamsters of 9 (young) and over 36 (aged) weeks old after intranasal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite similar levels of viral replication and inflammatory responses in the lungs of both age groups, aged but not young hamsters showed significant prolongation of prothrombin time and prominent acute kidney damage. Moreover, aged hamsters demonstrated increased intravascular coagulation time-dependently in the lungs, suggesting that consumption of coagulation factors causes prothrombin time prolongation. Furthermore, proximal urinary tract damage and mesangial matrix expansion were observed in the kidneys of the aged hamsters at early and later disease stages, respectively. Given that the severity and mortality of COVID-19 are higher in elderly human patients, the effect of aging on pathogenesis needs to be understood and should be considered for the selection of animal models. We, thus, propose that the aged hamster is a good small animal model for COVID-19 research.

Characterization of Mutations Associated with Streptomycin Resistance in Multidrug-Resistant Mycobacterium tuberculosis in Zambia.
Precious Bwalya, Tomoyuki Yamaguchi, Eddie Samuneti Solo, Joseph Yamweka Chizimu, Grace Mbulo, Chie Nakajima, Yasuhiko Suzuki
Antibiotics (Basel, Switzerland), 10, 10, 2021年09月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Streptomycin (STR) is recommended for the management of multidrug-resistant tuberculosis (MDR-TB). Streptomycin resistance-conferring mutation types and frequency are shown to be influenced by genotypes of circulating strains in a population. This study aimed to characterize the mutations in MDR-TB isolates and examine their relationship with the genotypes in Zambia. A total of 138 MDR-TB isolates stored at the University Teaching Hospital Tuberculosis Reference Laboratory in Zambia were analyzed using spoligotyping and sequencing of STR resistance-associated genes. Streptomycin resistance was observed in 65.9% (91/138) of MDR-TB isolates. Mutations in rpsL, rrs, and gidB accounted for 33%, 12.1%, and 49.5%, respectively. Amino acid substitution K43R in rpsL was strongly associated with the CAS1_Kili genotype (p < 0.0001). The combination of three genes could predict 91.2% of STR resistance. Clustering of isolates based on resistance-conferring mutations and spoligotyping was observed. The clustering of isolates suggests that the increase in STR-resistant MDR-TB in Zambia is largely due to the spread of resistant strains from inadequate treatment. Therefore, rapid detection of STR resistance genetically is recommended before its use in MDR-TB treatment in Zambia.

Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.
Yuki Arimura, Yusuke Minato, Takayuki Wada, Masaaki Nakayama, Ayako Ryumon, Nao Hirata, Chie Nakajima, Yasuhiko Suzuki, Manabu Ato, Kazuo Kobayashi, Naoko Ohara, Seiji Iida, Naoya Ohara
Microbiology and immunology, 66, 1, 10, 14, 2021年09月21日, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. In this study, we assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. We successfully constructed a thyX deletion mutant strain from that strain, which reinforces the non-essentiality of thyX under a certain genetic background. This article is protected by copyright. All rights reserved.

Clonal/subclonal changes and accumulation of CTX-M-type β-lactamase genes in fluoroquinolone-resistant Escherichia coli ST131 and ST1193 strains isolated during the past 12 years, Japan.
Yukari Fukushima, Toyotaka Sato, Naoyuki Tsukamoto, Chie Nakajima, Yasuhiko Suzuki, Satoshi Takahashi, Shin-Ichi Yokota
Journal of global antimicrobial resistance, 27, 150, 155, 2021年09月10日, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: Fluoroquinolone (FQ)- and third-generation cephalosporin-resistant Escherichia coli are increasing in Japan. In the early 2000s, the FQ-resistant E. coli clone ST131 increased in clinical settings worldwide. It frequently produces extended-spectrum β-lactamases (ESBLs) such as CTX-M. This study aimed to explore the characteristics of FQ-resistant E. coli isolated in Japan during 2008-2009 and 2020. METHODS: We compared FQ-resistant E. coli clinical isolates from urine samples collected in 2020 (151 isolates) with a FQ-resistant E. coli collection isolated in 2008-2009 (42 isolates). Identification of E. coli ST131 clades and blaCTX-M were determined by multiplex PCR. Sequence types of non-ST131 isolates were determined by whole-genome sequencing. RESULTS: Although the prevalence of ST131 was comparable in 2020 (74.2%) and 2008-2009 (78.6%), the subclades differed during the two time periods (C1-nM27: 40.2% in 2008-2009 vs. 78.8% in 2020; C1-M27: 32.1% in 2008-2009 vs. 9.1% in 2020). The incidence of blaCTX-M among ST131 isolates increased from 27.3% in 2008-2009 to 64.3% in 2020. blaCTX-M was found in 80.6% and 93.8% of C1-M27 and C2 in 2020, respectively, and blaCTX-M possession in C1-nM27 increased from 19.2% in 2008-2009 to 40% in 2020. FQ-resistant ST1193 was detected only in 2020 (17.9% of 151 isolates, of which 14.8% possessed blaCTX-M). CONCLUSION: Increased resistance of E. coli to FQs and third-generation cephalosporins in Japan can be attributed to the accumulation of blaCTX-M in C1-nM27 and the increase of C1-M27 and C2 clades with high blaCTX-M possession, alongside the spread of ST1193.

Genome Sequences of Two Mycobacterium tuberculosis Isolates from Asian Elephants in Nepal.
Sarad Paudel, Evan P Brenner, Syeda A Hadi, Yasuhiko Suzuki, Chie Nakajima, Toshio Tsubota, Kamal Prasad Gairhe, Bhagwan Maharjan, Srinand Sreevatsan
Microbiology resource announcements, 10, 36, e0061421, 2021年09月09日, ［国際誌］
英語, 研究論文（学術雑誌）, This report describes the genome sequences of two Mycobacterium tuberculosis isolates, S1 and S3, recovered from Asian elephants in Nepal. These genome sequences will enhance our understanding of the genomic epidemiology of Mycobacterium tuberculosis in Asian elephants.

ウシCTLA-4ならびにPD-L1を標的とした抗体併用法による免疫増強効果の検討　　　　　　　　　　　　　　　
渡 慧, 今内 覚, 岡川 朋弘, 前川 直也, 村田 史郎, 鈴木 定彦, 大橋 和彦
日本獣医学会学術集会講演要旨集, 164回, [DIO, 4], (公社)日本獣医学会, 2021年09月
日本語

抗菌薬の繰り返し曝露がClostridioides difficileの性状に与える影響の解明　　　　　　　　　　　　　　　
鈴木 和総, 福田 昭, 中島 千絵, 鈴木 定彦, 臼井 優
日本獣医学会学術集会講演要旨集, 164回, [FO, 37], (公社)日本獣医学会, 2021年09月
日本語

Expression and functional analysis of swine PD-1/PD-L1 pathway(和訳中)　　　　　　　　　　　　　　　
Otgontuya Ganbaatar, 今内 覚, 岡川 朋弘, 野島 裕太郎, 前川 直也, 市川 世識, 小林 篤史, 芝原 友幸, 柳川 洋二郎, 鈴木 定彦, 村田 史郎, 大橋 和彦
日本獣医学会学術集会講演要旨集, 164回, [DIO, 3], (公社)日本獣医学会, 2021年09月
英語

ウシCTLA-4ならびにPD-L1を標的とした抗体併用法による免疫増強効果の検討　　　　　　　　　　　　　　　
渡 慧, 今内 覚, 岡川 朋弘, 前川 直也, 村田 史郎, 鈴木 定彦, 大橋 和彦
日本獣医学会学術集会講演要旨集, 164回, [DIO, 4], (公社)日本獣医学会, 2021年09月
日本語

焼成ホタテ貝殻粉末と石灰窒素による堆肥中の薬剤耐性菌制御の可能性　　　　　　　　　　　　　　　
江浪 誠俊, 福田 昭, 中島 千絵, 鈴木 定彦, 臼井 優
日本獣医学会学術集会講演要旨集, 164回, [FO, 15], (公社)日本獣医学会, 2021年09月
日本語

市販堆肥における薬剤耐性菌/耐性遺伝子の分布状況の解明　　　　　　　　　　　　　　　
小南 風佳, 福田 昭, 中島 千絵, 鈴木 定彦, 臼井 優
日本獣医学会学術集会講演要旨集, 164回, [FO, 35], (公社)日本獣医学会, 2021年09月
日本語

抗菌薬の繰り返し曝露がClostridioides difficileの性状に与える影響の解明　　　　　　　　　　　　　　　
鈴木 和総, 福田 昭, 中島 千絵, 鈴木 定彦, 臼井 優
日本獣医学会学術集会講演要旨集, 164回, [FO, 37], (公社)日本獣医学会, 2021年09月
日本語

Insights into the ancestry evolution of the Mycobacterium tuberculosis complex from analysis of Mycobacterium riyadhense.
Qingtian Guan, Musa Garbati, Sara Mfarrej, Talal AlMutairi, Thomas Laval, Albel Singh, Shamsudeen Fagbo, Alicia Smyth, John A Browne, Muhammad Amin urRahman, Alya Alruwaili, Anwar Hoosen, Conor J Meehan, Chie Nakajima, Yasuhiko Suzuki, Caroline Demangel, Apoorva Bhatt, Stephen V Gordon, Faisal AlAsmari, Arnab Pain
NAR genomics and bioinformatics, 3, 3, lqab070, 2021年09月, ［国際誌］
英語, 研究論文（学術雑誌）, Current evolutionary scenarios posit the emergence of Mycobacterium tuberculosis from an environmental saprophyte through a cumulative process of genome adaptation. Mycobacterium riyadhense, a related bacillus, is being increasingly isolated from human clinical cases with tuberculosis-like symptoms in various parts of the world. To elucidate the evolutionary relationship between M. riyadhense and other mycobacterial species, including members of the M. tuberculosis complex (MTBC), eight clinical isolates of M. riyadhense were sequenced and analyzed. We show, among other features, that M. riyadhense shares a large number of conserved orthologs with M. tuberculosis and shows the expansion of toxin/antitoxin pairs, PE/PPE family proteins compared with other non-tuberculous mycobacteria. We observed M. riyadhense lacks wecE gene which may result in the absence of lipooligosaccharides (LOS) IV. Comparative transcriptomic analysis of infected macrophages reveals genes encoding inducers of Type I IFN responses, such as cytosolic DNA sensors, were relatively less expressed by macrophages infected with M. riyadhense or M. kansasii, compared to BCG or M. tuberculosis. Overall, our work sheds new light on the evolution of M. riyadhense, its relationship to the MTBC, and its potential as a system for the study of mycobacterial virulence and pathogenesis.

Programmed death-ligand 1 expression in swine chronic infections and enhancement of interleukin-2 production via programmed death-1/programmed death-ligand 1 blockade.
Otgontuya Ganbaatar, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Naoya Maekawa, Yoshiki Ichikawa, Atsushi Kobayashi, Tomoyuki Shibahara, Yojiro Yanagawa, Hidetoshi Higuchi, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Immunity, inflammation and disease, 9, 4, 1573, 1583, 2021年08月20日, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: Chronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death-1 (PD-1). The inhibitory signal is produced by the interaction between PD-1 and its PD-ligand 1 (PD-L1) and impairs the effector functions of T cells. However, the expression dynamics of PD-L1 and the immunological functions of the PD-1/PD-L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD-L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD-1/PD-L1 pathway. METHODS: In the initial experiments, anti-bovine PD-L1 monoclonal antibodies (mAbs) were tested for cross-reactivity with swine PD-L1. Subsequently, immunohistochemical analysis was conducted using the anti-PD-L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti-PD-L1 mAb. RESULTS: Several anti-PD-L1 mAbs tested recognized swine PD-L1-expressing cells. The binding of swine PD-L1 protein to swine PD-1 was inhibited by some of these cross-reactive mAbs. In addition, immunohistochemical analysis revealed that PD-L1 was expressed at the site of infection in chronic infections of pigs. The PD-L1 blockade increased the production of interleukin-2 from swine PBMCs. CONCLUSIONS: These findings suggest that the PD-1/PD-L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways.

Prevalence and Multidrug-Resistance Salmonella in Swine Production Chain in a Central Province, Thailand.
Kaknokrat Chonsin, Ruchirada Changkwanyeun, Achiraya Siriphap, Apiradee Intarapuk, Watsawan Prapasawat, Kanjana Changkaew, Chaiwat Pulsrikan, Norikazu Isoda, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
Journal of food protection, 84, 12, 2174, 2184, 2021年08月19日, ［国際誌］
英語, 研究論文（学術雑誌）, Salmonella causes foodborne disease outbreaks worldwide and raises considerable concerns about public health and economic losses. To determine prevalence, serovar, antimicrobial resistance (AMR) patterns, and extended-spectrum beta-lactamase (ESBL) genes, the present cross-sectional study collected a total of 418 fecal, carcass (three slaughterhouses), pork and cutting board (four markets) samples from a province in central Thailand in 2017 and 2018. Results showed that 65.1% (272/418) of samples were positive for Salmonella. The percentage of Salmonella positive samples from markets (88.8%; 158/178) was significantly higher than those from slaughterhouses (47.5%; 114/240) ( P<0.05 ). In total, 1,030 isolates were identified; of these, 409 were assigned to 45 serovars with S. Rissen (20%; 82/409) being the most common. New serovars of Thai isolates, S. Cannstatt and S. Braubach, were identified in market and slaughterhouse samples, respectively. AMR of Salmonella isolates showed that 73.9% (133/180) of 19 different serovars exhibited multidrug resistance (MDR). Screening for ESBL production showed that 10.3% (41/399) of isolates were ESBL positive. ESBL-producing Salmonella isolates in market samples (75.6%; 31/41) were significantly higher than those in slaughterhouse samples (24.4%; 10/41) ( P<0.05 ). In market samples, 77.4% (24/31) were isolated from pork and 22.6% (7/31) from cutting boards. Nine ESBL-producing isolates carried single type ESBL genes bla TEM (9.8%; 4/41) or bla CTX-M (12.2%; 5/41), while 11 (26.8%) carried both bla TEM and bla CTX-M . No ESBL-producing Salmonella isolate carried the gene bla SHV . Results suggest that pigs, their flesh, and cutting boards could be reservoirs for widespread MDR, ESBL-producing Salmonella outbreak across the food chain.

Tuberculosis seroprevalence and comparison of hematology and biochemistry parameters between seropositive and seronegative captive Asian elephants of Nepal.
Jeewan Thapa, Susan K Mikota, Kamal Prasad Gairhe, Sarad Paudel, Dinesh Kumar Singh, Ishwari Prasad Dhakal, Chie Nakajima, Yasuhiko Suzuki
The Journal of veterinary medical science, 83, 8, 1278, 1283, 2021年08月12日, ［国内誌］
英語, 研究論文（学術雑誌）, We conducted a tuberculosis (TB) serosurveillance program of captive elephants in Nepal and compared hematology and biochemistry parameters between seropositive and seronegative elephants. A total of 153 elephants (male=20, female=133) from four national parks were tested for TB using the ElephantTB STAT-PAK® Assay (ChemBio Diagnostic Systems, Inc., Medford, NY, USA). The mean reported age for 138 elephants was 38.5 years (range 2-71 years). Seroprevalence for TB was 21.56% (33/153). The majority of seropositive elephants were female (n=30) and from Chitwan National Park (n=29). The occurrence of TB seropositive cases in other more remote national parks suggests TB may be widespread among the captive elephant population of Nepal. Hematology and biochemistry analyses were performed on 13 and 22 seropositive elephants, respectively and, nine elephants from a seronegative TB herd for comparison. Hematology parameters (hemoglobin, packed cell volume, platelet, white blood cells, and erythrocyte sedimentation rate) were comparable between the two groups. Total protein, globulin, and lactate dehydrogenase were significantly higher in seronegative elephants, and bilirubin was significantly higher in seropositive elephants whereas blood urea nitrogen, creatinine, glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST), glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT), gamma glutamyl transferase (GT), and albumin were not significantly different. The range of biochemical parameters that were significantly different between seropositive and seronegative elephants had narrow ranges. Thus, the potential of these parameters as a direct biomarker for TB diagnosis is limited based on the findings in this study. We recommend including blood parameters in future TB surveillance studies.

Drug-resistant Mycobacterium tuberculosis and its genotypes isolated from an outbreak in western Thailand.
Janisara Rudeeaneksin, Benjawan Phetsuksiri, Chie Nakajima, Supranee Bunchoo, Krairerk Suthum, Nattakan Tipkrua, Yukari Fukushima, Yasuhiko Suzuki
Transactions of the Royal Society of Tropical Medicine and Hygiene, 115, 8, 886, 895, 2021年08月02日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Multidrug-resistant TB (MDR-TB) outbreaks have occurred in the Thamaka district, Kanchanaburi province in Thailand. METHODS: Seventy-two isolates, which included 7% mono-, 30.6% MDR and extensively drug-resistant TB (XDR-TB), were genotyped by spoligotyping, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) and single nucleotide polymorphism genotyping, and their drug resistance was analysed. RESULTS: The spoligotyping results showed that Beijing spoligo-international type (SIT)1 was predominant (n=38; 52.8%) while the remaining were non-Beijing sublineages (n=34). The MIRU-VNTR analysis showed that Beijing isolates, most of which belonged to the modern type (n=37), formed 5 clusters and 13 individual patterns. In katG, only mutation Ser315Thr was identified. In rpoB, Ser531Leu was predominant, except for His526Arg and Leu533Pro, which were found in two isolates. A cluster of 14 Beijing strains contained these common mutations and shared the MIRU-VNTR genotype with isolates in the Thamaka district that had spread previously. Two U SIT523 isolates contained the mutations A1400G in rrs and Asp94Gly in gyrA genes, indicating a spread of XDR-TB. CONCLUSIONS: Most mutations were associated with drug resistance and the specific MDR Beijing and XDR-TB in U SIT523 isolates remain. This genotyping is a key tool for tracking TB transmission in the Thamaka district of Thailand.

Complete Genome Sequence of an mcr-10-Possessing Enterobacter roggenkampii Strain Isolated from a Dog in Japan.
Toyotaka Sato, Masaru Usui, Kazuki Harada, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota
Microbiology resource announcements, 10, 30, e0042621, 2021年07月29日, ［国際誌］
英語, 研究論文（学術雑誌）, The complete genome sequence of mcr-10-possessing Enterobacter roggenkampii En37, isolated from a dog in Japan, was determined. mcr-10 was located on a 70,277-bp IncFIB plasmid without any additional antimicrobial resistance genes.

Evaluation of IS1245 LAMP in Mycobacterium avium and the influence of host-related genetic diversity on its application.
Mwangala Lonah Akapelwa, Thoko Flav Kapalamula, Yuki Ouchi-Aizu, Bernard Mudenda Hang'ombe, Yukiko Nishiuchi, Stephen V Gordon, Eddie Samuneti Solo, Aki Tamaru, Tomoyasu Nishimura, Naoki Hasegawa, Kozo Morimoto, Yukari Fukushima, Yasuhiko Suzuki, Chie Nakajima
Diagnostic microbiology and infectious disease, 101, 4, 115494, 115494, 2021年07月21日, ［国際誌］
英語, 研究論文（学術雑誌）, Early detection and treatment are paramount for the timely control of Mycobacterium avium infections. Herein, we designed a LAMP assay targeting a widely used species-specific marker IS1245 for the rapid detection of M. avium and evaluated its applicability using human (n = 137) and pig (n = 91) M. avium isolates from Japan. The developed assay could detect as low as 1 genome copy of M. avium DNA within 30 minutes. All 91 (100%) M. avium isolates from pigs were detected positive while all other tested bacterial species were negative. Interestingly, among the 137 clinical M. avium isolates, 41 (30%) were undetectable with this LAMP assay as they lacked IS1245, the absence of which was revealed by PCR and whole-genome sequencing. These findings highlighted genotypic differences in M. avium strains from humans and pigs in Japan and how this diversity can influence the applicability of a detection tool across different geographic areas and hosts.

Complete Genome Sequence of an mcr-9-Possessing Enterobacter asburiae Strain Isolated from a Cat in Japan.
Toyotaka Sato, Masaru Usui, Kazuki Harada, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota
Microbiology resource announcements, 10, 26, e0028121, 2021年07月, ［国際誌］
英語, 研究論文（学術雑誌）, We report the complete genome sequence of the mcr-9-possessing strain Enterobacter asburiae En30, isolated from a cat in Japan. The genome sequence was obtained by using long- and short-read sequencing.

Interaction of Quinolones Carrying New R1 Group with Mycobacterium leprae DNA Gyrase.
Jong-Hoon Park, Tomoyuki Yamaguchi, Yuki Ouchi, Kentaro Koide, Ruttana Pachanon, Joseph Yamweka Chizimu, Shigetarou Mori, Hyun Kim, Tetsu Mukai, Chie Nakajima, Yasuhiko Suzuki
Microbial drug resistance (Larchmont, N.Y.), 27, 12, 1616, 1623, 2021年06月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.

Extracellular DNA of slow growers of mycobacteria and its contribution to biofilm formation and drug tolerance.
Aleksandr Ilinov, Akihito Nishiyama, Hiroki Namba, Yukari Fukushima, Hayato Takihara, Chie Nakajima, Anna Savitskaya, Gebremichal Gebretsadik, Mariko Hakamata, Yuriko Ozeki, Yoshitaka Tateishi, Shujiro Okuda, Yasuhiko Suzuki, Yuri S Vinnik, Sohkichi Matsumoto
Scientific reports, 11, 1, 10953, 10953, 2021年05月26日, ［国際誌］
英語, 研究論文（学術雑誌）, DNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria's eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.

Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.
Takuya Kodera, Tomoyuki Yamaguchi, Yukari Fukushima, Kumi Kobayashi, Yutaka Takarada, Joseph Yamweka Chizimu, Chie Nakajima, Eddie Samuneti Solo, Patrick Saili Lungu, Mitsuo Kawase, Yasuhiko Suzuki
Japanese journal of infectious diseases, 74, 3, 214, 219, 2021年05月24日, ［国内誌］
英語, 研究論文（学術雑誌）, Despite the availability of anti-tuberculosis drugs, the treatment of tuberculosis has been complicated by drug-resistant tuberculosis. The early detection of drug resistance makes early treatment possible. However, the available tools are mainly for rifampicin resistance detection, and the existing isoniazid resistance detection method is expensive, highly technical, and complicated, making it unsustainable for use in developing nations. This study aimed to develop a simple, rapid, and low-cost diagnostic kit for isoniazid-resistant tuberculosis using the single-stranded tag hybridization method to target an isoniazid resistance-conferring mutation. Specificity and sensitivity were assessed using DNA extracted from 49 isoniazid-resistant and 41 isoniazid-susceptible Mycobacterium tuberculosis clinical isolates cultured in mycobacterial growth indicator tubes. Positive signals were observed on mutant and wild-type lines with 100% sensitivity and specificity compared with Sanger sequencing results. In contrast, no positive signal was observed for non-tuberculosis mycobacteria. The detection limit of this method was 103 CFU or less. The STH-PAS system for isoniazid-resistant M. tuberculosis detection developed in this study offers a better alternative to conventional phenotypic isoniazid resistance determination, which will be of both clinical and epidemiological significance in resource-limited nations.

Rapid and simultaneous detection of fluoroquinolone- and macrolide-resistant Campylobacter jejuni/coli in retail chicken meat using CAMpylobacter Express Resistance Array (CAMERA)
Masaru Usui, Sho Tateno, Masanobu Onozaki, Naoaki Misawa, Yasuhiko Suzuki, Yutaka Tamura, Chie Nakajima
Food Control, 123, 107815, 107815, Elsevier BV, 2021年05月
研究論文（学術雑誌）

Genetic characterization of coliform bacterial isolates from environmental water in Thailand.
Risa Tsunoda, Masaru Usui, Chie Tagaki, Akira Fukuda, Chanchai Boonla, Wilai Anomasiri, Nop Sukpanyatham, Mwangala Lonah Akapelwa, Chie Nakajima, Yutaka Tamura, Yasuhiko Suzuki
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 27, 5, 722, 728, 2021年05月, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: In contrast to the study in other part of the world, information about characteristics of plasmids carrying antimicrobial resistance genes (ARGs) in Enterobacteriaceae derived from environmental water in tropical Asian countries including Thailand is limited. This study, therefore, aimed to gain insight into genetic information of antimicrobial resistance in environmental water in Thailand. METHODS: Coliform bacteria were isolated from environmental water collected at 20 locations in Thailand and identified. Then, susceptibility profiles to ampicillin, cefazoline, cefotaxime, kanamycin, ciprofloxacin, sulfamethoxazole, tetracycline, and nalidixic acid were assessed. In addition, antimicrobial resistant genes integrons, and replicon types were analyzed. And furthermore, plasmids carrying blaTEM and tetM were identified by S1-PFGE analysis and confirmed transmissibility by transconjugation experiments. RESULTS: In 130 coliform bacteria isolated, 89 were resistant to cefazoline while 41 isolates were susceptible. Cefazoline-resistant coliform bacteria were found to be significantly resistant to cefotaxime and tetracycline as compared to susceptible isolates. Hence, blaTEM and tetM correlating with β-lactam antibiotics and tetracycline, respectively, were analyzed found to co-localize on the IncFrepB plasmids in isolates from pig farms' wastewater by S1-PFGE analysis. And furthermore, transmissibility of the plasmids was confirmed. CONCLUSIONS: Results obtained in this study suggested that ARGs in coliform bacteria may have been spreading on the farm via IncFrepB plasmids. Hence, appropriate use of antimicrobials and good hygiene management on the farm are required to prevent the emergence and spread of resistant bacteria.

First report of Mycobacterium bovis in wild chacma baboons (Papio ursinus) at the human-wildlife interface area in Zambia.
David Squarre, Joseph Chizimu, Chie Nakajima, John B Muma, Bernard M Hang'ombe, Edgar Simulundu, Wizaso Mwasinga, Jackson Katampi, Paul Fandamu, Victor Mukonka, Yasuhiko Suzuki, Hirofumi Sawa, Hetron M Munang'andu, Griffin Shanungu, Herman M Chambaro, Musso Munyeme
Transboundary and emerging diseases, 69, 3, 1659, 1662, 2021年04月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium bovis (M. bovis) causes tuberculosis in mammals and is a major public health threat worldwide. While M. bovis has been reported in humans, domestic and wild ruminants at the human-wildlife-livestock interface area in Zambia, there is paucity of information on the role of primates as reservoir hosts. We screened seven wild chacma baboons (Papio ursinus) for tuberculosis at the human-wildlife interface area in Lochinvar National Park in the Kafue Flats, Zambia. Following necropsy, lung tissue and associated lymph nodes with tuberculous-like lesions collected from four adult male baboons were prepared for Mycobacterium culture. The isolates were initially typed using the Mycobacterium tuberculosis complex-discrimination multiplex PCR assay and further characterized by spoligotyping and 26-loci MIRU-VNTR. Mycobacteria were isolated from all four animals and identified as M. bovis by PCR. On Spoligotyping, all isolates belonged to SB 0120 spoligotype, which is similar to what was previously reported in humans, cattle and Kafue lechwe antelopes in Kafue Flats ecosystem. Furthermore, on MIRU-VNTR typing, the baboon isolates clustered with cattle and Kafue lechwe isolates from the same catchment area. This finding intimates probable cross-species transmission of M. bovis in the Kafue Flats ecosystem. Due to the close interaction of baboons and humans at interface areas in Zambia, our results have potential implications for public health. Equally, this finding raises concerns for conservation.

Molecular epidemiology of Mycobacterium bovis in central parts of Malawi.
Thoko Flav Kapalamula, Joseph Chizimu, Lawrence Belotindos, Mwangala Akapelwa, Dipti Shrestha, Mirriam Ethel Nyenje, Musso Munyeme, Bernard Mudenda Hang'ombe, Rajhab Sawasawa Mkakosya, Stephen V Gordon, Chie Nakajima, Yasuhiko Suzuki
Transboundary and emerging diseases, 69, 3, 1577, 1588, 2021年04月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine tuberculosis (bTB) is a neglected disease that affects cattle and humans. The burden of bTB is higher in developing countries as compared to industrialized countries. The reasons behind this discrepancy include the fact that bTB control measures, such as testing and slaughter of infected cattle and pasteurization of milk, are not usually practised in developing countries largely because of their high cost. To improve our understanding of bTB in developing countries, molecular typing studies are essential, in particular in terms of transmission dynamics, infection sources and knowledge of circulating strains of the principal causative agent, Mycobacterium bovis. In this study, we applied a suite of molecular typing techniques encompassing deletion analysis, spoligotyping and MIRU-VNTR to isolates recovered from samples collected during the routine post-mortem of cattle at the cold storage abattoir in Lilongwe, Malawi. Out of 63 isolates, 51 (81%) belonged to the European 1. M. bovis clonal complex. Spoligotyping identified 8 profiles, with SB0131 being the predominant type (56% of isolates). Spoligotypes SB0273 and SB0425 were identified in 14% and 13%, respectively, of the isolates. MIRU-VNTR showed a high discriminatory power of 0.959 and differentiated the 8 spoligotypes to 31 genotypes. The high diversity of M. bovis within the study area suggests the infection has been circulating in the area for a considerable period of time, likely facilitated by the lack of effective control measures. We also observed genetic similarities between isolates from Malawi (this study) to isolates described in previous studies in Zambia and Mozambique, suggesting transmission links in this region. The information provided by this study provides much needed evidence for the formulation of improved bTB control strategies.

Amino Acid Substitution Ser83Ile in GyrA of DNA Gyrases Confers High-Level Quinolone Resistance to Nontyphoidal Salmonella Without Loss of Supercoiling Activity.
Kentaro Koide, Lai Lai San, Ruttana Pachanon, Jong-Hoon Park, Yuki Ouchi, Siriporn Kongsoi, Fuangfa Utrarachkij, Chie Nakajima, Yasuhiko Suzuki
Microbial drug resistance (Larchmont, N.Y.), 27, 10, 1397, 1404, 2021年04月20日, ［国際誌］
英語, 研究論文（学術雑誌）, Aims: Quinolone-resistant nontyphoidal Salmonella having serine replaced by isoleucine at the 83rd amino acid in GyrA (GyrA-Ser83Ile) has recently been found in Asian countries. In this study, we aimed to examine the direct effect of substitution Ser83Ile on DNA gyrase activity and/or resistance to quinolones. Materials and Methods: Using 50% of the maximal inhibitory concentrations (IC50s) of quinolones, recombinant wild type (WT) and seven mutant DNA gyrases having amino acid substitutions, including Ser83Ile, were screened for enzymatic activity that causes supercoils in relaxed plasmid DNA and resistance to quinolones. Results: Little differences in supercoiling activity were observed between WT and mutant DNA gyrases. By contrast, the IC50s of ciprofloxacin and norfloxacin against GyrA-Ser83Ile/GyrB-WT were 11.6 and 73.3 μg/mL, respectively, which were the highest used against the DNA gyrases examined in this study. Conclusion: Ser83Ile in GyrA was shown to confer high-level quinolone resistance to DNA gyrases of nontyphoidal Salmonella, with no loss of supercoiling activity. Salmonella strain carrying GyrA with Ser83Ile may emerge under a high-concentration pressure of quinolones and easily spread even with no selection bias by quinolones. Hence, avoiding the overuse of quinolones is needed to prevent the spread of Salmonella with Ser83Ile in GyrA.

Effectiveness of Fluoroquinolones with Difluoropyridine Derivatives as R1 Groups on the Salmonella DNA Gyrase in the Presence and Absence of Plasmid-Encoded Quinolone Resistance Protein QnrB19.
Ruttana Pachanon, Kentaro Koide, Siriporn Kongsoi, Nami Ajima, Thoko Flav Kapalamula, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
Microbial drug resistance (Larchmont, N.Y.), 27, 10, 1412, 1419, 2021年04月09日, ［国際誌］
英語, 研究論文（学術雑誌）, Aims: WQ-3810 has strong inhibitory activity against Salmonella and other fluoroquinolone-resistant pathogens. The unique potentiality of this is attributed to 6-amino-3,5-difluoropyridine-2-yl at R1 group. The aim of this study was to examine WQ-3810 and its derivatives WQ-3334 and WQ-4065 as the new drug candidate for wild-type Salmonella and that carrying QnrB19. Materials and Methods: The half maximal inhibitory concentrations (IC50s) of WQ-3810, WQ-3334 (Br atom in place of methyl group at R8), and WQ-4065 (6-ethylamino-3,5-difluoropyridine-2-yl in place of 6-amino-3,5-difluoropyridine-2-yl group at R1) in the presence or absence of QnrB19 were assessed by in vitro DNA supercoiling assay utilizing recombinant DNA gyrase and QnrB19. Results: IC50s of WQ-3810, WQ-3334, and WQ-4065 against Salmonella DNA gyrase were 0.031 ± 0.003, 0.068 ± 0.016, and 0.72 ± 0.39 μg/mL, respectively, while QnrB19 increased IC50s of WQ-3810, WQ-3334, and WQ-4065 to 0.44 ± 0.05, 0.92 ± 0.34, and 9.16 ± 2.21 μg/mL, respectively. Conclusion: WQ-3810 and WQ-3334 showed stronger inhibitory activity against Salmonella Typhimurium DNA gyrases than WQ-4065 even in the presence of QnrB19. The results suggest that 6-amino-3,5-difluoropyridine-2-yl group at R1 is playing an important role and WQ-3810 and WQ-3334 to be good candidates for Salmonella carrying QnrB19.

Prevalence and Characterization of Quinolone-Resistance Determinants in Escherichia coli Isolated from Food-Producing Animals and Animal-Derived Food in the Philippines.
Lawrence Belotindos, Marvin Villanueva, Joel Miguel Jr, Precious Bwalya, Tetsuya Harada, Ryuji Kawahara, Chie Nakajima, Claro Mingala, Yasuhiko Suzuki
Antibiotics (Basel, Switzerland), 10, 4, 2021年04月09日, ［国際誌］
英語, 研究論文（学術雑誌）, Antimicrobial resistance to quinolones, which constitutes a threat to public health, has been increasing worldwide. In this study, we investigated the prevalence of quinolone-resistant determinants in Escherichia coli not susceptible to quinolones and isolated from food-producing animals and food derived from them, in the Philippines. A total of 791 E. coli strains were isolated in 56.4% of 601 beef, chicken, pork, egg, and milk samples, as well as environmental, cloacal, and rectal swab-collected samples from supermarkets, open markets, abattoirs, and poultry, swine, and buffalo farms. Using the disc diffusion method, it was determined that 78.6% and 55.4% of the isolates were resistant to at least one antimicrobial and multiple drugs, respectively. In 141 isolates not susceptible to quinolones, 115 (81.6%) harbored quinolone-resistant determinants and had mutations predominantly in the quinolone-resistance determining regions (QRDRs) of gyrA and parC. Plasmid-mediated, quinolone resistance (PMQR) and Qnr family (qnrA1, qnrB4, and qnrS1) genes were detected in all isolates. Forty-eight sequence types were identified in isolates harboring mutations in QRDR and/or PMQR genes by multilocus sequence typing analysis. Moreover, 26 isolates harboring mutations in QRDR and/or PMQR genes belonged mostly to phylogroup B1 and Enteroaggregative E. coli. In conclusion, a high prevalence of E. coli was found in food-producing animals and products derived from them, which could potentially spread high-risk clones harboring quinolone-resistance determinants.

Comparative genomic analysis of Mycobacterium intracellulare: implications for clinical taxonomic classification in pulmonary Mycobacterium avium-intracellulare complex disease.
Yoshitaka Tateishi, Yuriko Ozeki, Akihito Nishiyama, Mari Miki, Ryoji Maekura, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Sohkichi Matsumoto
BMC microbiology, 21, 1, 103, 103, 2021年04月06日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Mycobacterium intracellulare is a representative etiological agent of emerging pulmonary M. avium-intracellulare complex disease in the industrialized countries worldwide. The recent genome sequencing of clinical strains isolated from pulmonary M. avium-intracellulare complex disease has provided insight into the genomic characteristics of pathogenic mycobacteria, especially for M. avium; however, the genomic characteristics of M. intracellulare remain to be elucidated. RESULTS: In this study, we performed comparative genomic analysis of 55 M. intracellulare and related strains such as M. paraintracellulare (MP), M. indicus pranii (MIP) and M. yonogonense. Based on the average nucleotide identity, the clinical M. intracellulare strains were phylogenetically grouped in two clusters: (1) the typical M. intracellulare (TMI) group, including ATCC13950 and virulent M.i.27 and M.i.198 that we previously reported, and (2) the MP-MIP group. The alignment of the genomic regions was mostly preserved between groups. Plasmids were identified between groups and subgroups, including a plasmid common among some strains of the M.i.27 subgroup. Several genomic regions including those encoding factors involved in lipid metabolism (e.g., fadE3, fadE33), transporters (e.g., mce3), and type VII secretion system (genes of ESX-2 system) were shown to be hypermutated in the clinical strains. M. intracellulare was shown to be pan-genomic at the species and subspecies levels. The mce genes were specific to particular subspecies, suggesting that these genes may be helpful in discriminating virulence phenotypes between subspecies. CONCLUSIONS: Our data suggest that genomic diversity among M. intracellulare, M. paraintracellulare, M. indicus pranii and M. yonogonense remains at the subspecies or genovar levels and does not reach the species level. Genetic components such as mce genes revealed by the comparative genomic analysis could be the novel focus for further insight into the mechanism of human pathogenesis for M. intracellulare and related strains.

Evaluation of Xpert MTB/RIF with microscopy and culture for the diagnosis of tuberculosis in a referral laboratory in Nepal.
Bhagwan Maharjan, Jeewan Thapa, Dhirendra Kumar Shah, Bhabana Shrestha, Korkut Avsar, Yasuhiko Suzuki, Chie Nakajima
Japanese journal of infectious diseases, 74, 6, 517, 521, 2021年03月31日, ［国内誌］
英語, 研究論文（学術雑誌）, Sputum microscopy and Xpert MTB/RIF are the primary rapid diagnostic methods for tuberculosis (TB) in Nepal. Disagreements among Xpert, microscopy, and culture, for example, cases with Xpert positive and microscopy negative, were frequently observed in Nepal including in our reference laboratory. The objective of this study was to compare the effectiveness of Xpert with culture and microscopy for TB diagnosis in Nepal. A total of 125 TB suspected sputum samples were processed for Xpert, microscopy, and culture. The Xpert results when compared with culture showed 100% sensitivity and 97.4% specificity with an excellent agreement (kappa = 0.96), whereas microscopy showed the sensitivity and specificity of 43.2% and 98.7%, respectively, with a moderate agreement (kappa = 0.4). The sensitivity and specificity of microscopy, when compared with Xpert, were 43.5% and 100%, respectively. The majority of Xpert positive samples of a medium MTB detection and all samples of low and very low MTB detection were missed by microscopy. Our study showed that Xpert MTB/RIF is a reliable tool for the diagnosis and management of TB in Nepal. Because of its high cost and sustainability, alternative simple and rapid diagnostic methods with a similar efficiency would be helpful for TB control in Nepal.

The enhancement of Th1 immune response by anti-PD-L1 antibody in cattle infected with Mycobacterium avium subsp. paratuberculosis.
Yamato Sajiki, Satoru Konnai, Reiko Nagata, Satoko Kawaji, Hayato Nakamura, Sotaro Fujisawa, Tomohiro Okagawa, Naoya Maekawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
The Journal of veterinary medical science, 83, 2, 162, 166, 2021年02月25日, ［国内誌］
英語, 研究論文（学術雑誌）, Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne's disease.

Antimicrobial Resistance and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Slaughtered Pigs and Pork in the Central Region of Thailand.
Wimonrat Tanomsridachchai, Kanjana Changkaew, Ruchirada Changkwanyeun, Watsawan Prapasawat, Apiradee Intarapuk, Yukari Fukushima, Nattapong Yamasamit, Thoko Flav Kapalamula, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
Antibiotics (Basel, Switzerland), 10, 2, 2021年02月19日, ［国際誌］
英語, 研究論文（学術雑誌）, Methicillin-resistant Staphylococcus aureus (MRSA) have been a major public health concern in humans. Among MRSA, livestock-associated (LA)-MRSA strains have always been associated with exposure to livestock or their products and have emerged in different countries globally. Although studies have identified LA-MRSA from healthy pigs and pork in Thailand, prevalence in slaughtered pigs is still unknown. In addition, there are few reports on the epidemiology and molecular characteristics of LA-MRSA in Thailand. Hence, this is the first report investigating the epidemiology and molecular characteristics of MRSA in individual slaughtered pigs and pork in Thailand. A total of 204 nasal swab and 116 retailed pork samples were collected from three slaughterhouses and four fresh markets, respectively. Individual samples were used for screening for MRSA and obtained isolates were examined for drug- resistance profiling for 12 antimicrobial agents of 10 drug classes. In addition, SCCmec typing and multi-locus sequence typing were conducted to obtain genotype profiles. MRSA were isolated from 11 and 52 nasal swab and pork samples, respectively. The prevalence was significantly higher in the pork than in the nasal swab samples (p-value < 0.05). A high prevalence of ST9-SCCmecIX and ST398-SCCmecV with high-level antimicrobial resistance from markets and slaughterhouses indicated the spreading of MRSA with these genotypes in the Thai swine processing chains and suggested the need for further investigation to determine a control.

PD-L1 immunohistochemistry for canine cancers and clinical benefit of anti-PD-L1 antibody in dogs with pulmonary metastatic oral malignant melanoma.
Naoya Maekawa, Satoru Konnai, Maki Nishimura, Yumiko Kagawa, Satoshi Takagi, Kenji Hosoya, Hiroshi Ohta, Sangho Kim, Tomohiro Okagawa, Yusuke Izumi, Tatsuya Deguchi, Yukinari Kato, Satoshi Yamamoto, Keiichi Yamamoto, Mikihiro Toda, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
NPJ precision oncology, 5, 1, 10, 10, 2021年02月12日, ［国際誌］
英語, 研究論文（学術雑誌）, Immunotherapy targeting programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) represents promising treatments for human cancers. Our previous studies demonstrated PD-L1 overexpression in some canine cancers, and suggested the therapeutic potential of a canine chimeric anti-PD-L1 monoclonal antibody (c4G12). However, such evidence is scarce, limiting the clinical application in dogs. In the present report, canine PD-L1 expression was assessed in various cancer types, using a new anti-PD-L1 mAb, 6C11-3A11, and the safety and efficacy of c4G12 were explored in 29 dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1 expression was detected in most canine malignant cancers including OMM, and survival was significantly longer in the c4G12 treatment group (median 143 days) when compared to a historical control group (n = 15, median 54 days). In dogs with measurable disease (n = 13), one dog (7.7%) experienced a complete response. Treatment-related adverse events of any grade were observed in 15 dogs (51.7%). Here we show that PD-L1 is a promising target for cancer immunotherapy in dogs, and dogs could be a useful large animal model for human cancer research.

Characterization of Vibrio parahaemolyticus strains isolated from clinically asymptomatic seafood workers.
Kaknokrat Chonsin, Neunghatai Supha, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
FEMS microbiology letters, 368, 1, 2021年01月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Vibrio parahaemolyticus (VP) is a major cause of gastroenteritis outbreaks in Thailand and other countries due to the consumption of contaminated and undercooked seafood. However, there have been few reports of the molecular epidemiology of VP isolates from asymptomatic seafood handlers. Here, we report the phenotypic and genetic characterization of 61 VP isolates obtained from asymptomatic workers in two seafood-processing plants. We found 24 O:K serotypes, of which O11:KUT, O1:KUT and O3:KUT were the dominant serotypes. Analysis by PCR showed that 12 isolates harbored either tdh or trh genes with the potential to be pathogenic VP strains. The presence of T3SS2α and T3SS2β genes was correlated with the presence of tdh and trh, respectively. Four tdh+ isolates were positive for pandemic marker. In this study, VP isolates were commonly resistant to ampicillin, cephazolin, fosfomycin and novobiocin. Phylogenetic analysis of VP1680 loci in 35 isolates from 17 asymptomatic workers, 6 gastroenteritis patients, 7 environmental samples and 5 genomes from a database showed 22 different alleles. Gene VP1680 was conserved in tdh+ isolates and pandemic strains, while that of trh + isolates was diverse. Asymptomatic workers carrying VP were the most likely source of contamination, which raises concerns over food safety in seafood-processing plants.

Aerobic Composting and Anaerobic Digestion Decrease the Copy Numbers of Antibiotic-Resistant Genes and the Levels of Lactose-Degrading Enterobacteriaceae in Dairy Farms in Hokkaido, Japan.
Satoshi Katada, Akira Fukuda, Chie Nakajima, Yasuhiko Suzuki, Takashi Azuma, Ayaka Takei, Hideshige Takada, Eiryu Okamoto, Toshihide Kato, Yutaka Tamura, Masaru Usui
Frontiers in microbiology, 12, 737420, 737420, 2021年, ［国際誌］
英語, 研究論文（学術雑誌）, Efficient methods for decreasing the spread of antimicrobial resistance genes (ARGs) and transfer of antimicrobial-resistant bacteria (ARB) from livestock manure to humans are urgently needed. Aerobic composting (AC) or anaerobic digestion (AD) are widely used for manure treatment in Japanese dairy farms. To clarify the effects of AC and AD on antimicrobial resistance, the abundances of antimicrobial (tetracycline and cefazolin)-resistant lactose-degrading Enterobacteriaceae as indicator bacteria, copy numbers of ARGs (tetracycline resistance genes and β-lactamase coding genes), and concentrations of residual antimicrobials in dairy cow manure were determined before and after treatment. The concentration of tetracycline/cefazolin-resistant lactose-degrading Enterobacteriaceae was decreased over 1,000-fold by both AC and AD. ARGs such as tetA, tetB, and bla TEM were frequently detected and their copy numbers were significantly reduced by ∼1,000-fold by AD but not by AC. However, several ARG copies remained even after AD treatment. Although concentrations of the majority of residual antimicrobials were decreased by both AC and AD, oxytetracycline level was not decreased after treatment in most cases. In addition, 16S rRNA gene amplicon-based metagenomic analysis revealed that both treatments changed the bacterial community structure. These results suggest that both AC and AD could suppress the transmission of ARB, and AD could reduce ARG copy numbers in dairy cow manure.

環境からヒトの生活に侵入する病原微生物 人獣共通感染症としての結核と非定型抗酸菌症　　　　　　　　　　　　　　　
中島 千絵, 鈴木 定彦
日本化学療法学会雑誌, 69, 1, 50, 50, (公社)日本化学療法学会, 2021年01月
日本語

A TLR7 agonist activates bovine Th1 response and exerts antiviral activity against bovine leukemia virus.
Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Hayato Nakamura, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Developmental and comparative immunology, 114, 103847, 103847, 2021年01月, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine leukemia virus (BLV) infection is a bovine chronic infection caused by BLV, a member of the genus Deltaretrovirus. In this study, we examined the immunomodulatory effects of GS-9620, a toll-like receptor (TLR) 7 agonist, in cattle (Bos taurus) and its therapeutic potential for treating BLV infection. GS-9620 induced cytokine production in peripheral blood mononuclear cells (PBMCs) as well as CD80 expression in CD11c+ cells and increased CD69 and interferon (IFN)-γ expressions in T cells. Removing CD11c+ cells from PBMCs decreased CD69 expression in T cells in the presence of GS-9620. These results suggest that TLR7 agonism promotes T-cell activation via CD11c+ cells. Analyses using PBMCs from BLV-infected cattle revealed that TLR7 expression in CD11c+ cells was upregulated during late-stage BLV infection. Furthermore, GS-9620 increased IFN-γ and TNF-α production and inhibited syncytium formation in vitro, suggesting that GS-9620 may be used to treat BLV infection.

Characterization of Mycobacterium tuberculosis genotypes and their correlation to multidrug resistance in Lusaka, Zambia.
Eddie Samuneti Solo, Yasuhiko Suzuki, Trevor Kaile, Precious Bwalya, Patrick Lungu, Joseph Yamweka Chizimu, Yogendra Shah, Chie Nakajima
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 102, 489, 496, 2021年01月, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: The burden of multidrug-resistant tuberculosis (MDR-TB) has been reported to be increasing in Zambia. The reasons for the increase are still unclear. This study determined the diversity of Mycobacterium tuberculosis genotypes among isolates in Lusaka, the capital city, and investigated their association with MDR-TB. METHODS: Spoligotyping, large sequence polymorphism (LSP) analysis, and sequencing of MDR associated genes were performed on a total of 274 M. tuberculosis clinical isolates stored at the University Teaching Hospital from 2013 to 2017. Of these, 134 were MDR-TB while 126 were pan-susceptible. RESULTS: Spoligotyping showed the LAM family as the most predominant genotype (149/274, 54.4%) followed by the CAS family (44/274, 16.1%), T family (39/274, 14.2%), and minor proportions of X, S, Harleem, EAI and Beijing spoligofamilies were identified. Three M. bovis isolates were also observed. Among those, CAS1-Kili (SIT 21) and LAM1 (SIT 20) subfamilies showed a propensity for MDR-TB with p = 0.0001 and p = 0.001, respectively. CONCLUSIONS: This phenomenon might explain the future increase in the MDR-TB burden caused by specific lineages in Zambia. Therefore, it is recommended that the National TB control program in the country complements conventional control strategies with molecular analysis for monitoring and surveillance of MDR-TB epidemiology.

Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis.
Thoko Flav Kapalamula, Jeewan Thapa, Mwangala Lonah Akapelwa, Kyoko Hayashida, Stephen V Gordon, Bernard Mudenda Hang' Ombe, Musso Munyeme, Eddie Samuneti Solo, Precious Bwalya, Mirriam Ethel Nyenje, Aki Tamaru, Yasuhiko Suzuki, Chie Nakajima
PLoS neglected tropical diseases, 15, 1, e0008996, 2021年01月, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.

Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression.
Hiroto Takeuchi, Satoru Konnai, Naoya Maekawa, Satoshi Takagi, Hiroshi Ohta, Noboru Sasaki, Sangho Kim, Tomohiro Okagawa, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Frontiers in veterinary science, 8, 656715, 656715, 2021年, ［国際誌］
英語, 研究論文（学術雑誌）, Cancer cells can evade host immune systems via multiple mechanisms. Transforming growth factor beta 1 (TGF-β1) is an immunosuppressive cytokine that induces regulatory T cell (Tregs) differentiation and is involved in immune evasion mechanisms in cancer. The inhibition of the TGF-β1 signaling pathway can suppress cancer progression and metastasis through the modulation of anticancer immune responses. However, to best of our knowledge, no implementation of treatments targeting TGF-β1 has been reported in dog cancers. This study aimed to examine whether TGF-β1 is upregulated in canine cancers. We measured TGF-β1 concentrations in culture supernatants of canine melanoma cell lines and in serum samples from dogs with oral malignant melanoma. TGF-β1 production was observed in several cell lines, and serum TGF-β1 levels were elevated in dogs with oral malignant melanoma. Interestingly, the addition of recombinant TGF-β1 to canine peripheral blood mononuclear cell cultures decreased Th1 cytokine production and increased differentiation of CD4+CD25+Foxp3+ lymphocytes, suggesting that TGF-β1 is immunosuppressive in canine immune systems. We developed a decoy receptor for TGF-β, namely TGF-βRII-Ig, by identifying an open reading frame of the canine TGFBR2 gene. TGF-βRII-Ig was prepared as a recombinant fusion protein of the extracellular region of canine TGF-βRII and the Fc region of canine IgG-B. As expected, TGF-βRII-Ig bound to TGF-β1. In the presence of TGF-β1, the treatment with TGF-βRII-Ig increased Th1 cytokine production and decreased the differentiation of CD4+CD25+Foxp3+ lymphocytes. Our results suggest that TGF-βRII-Ig competitively inhibits the immunosuppressive effects of TGF-β1 and thereby activates immune responses. This study demonstrated the potential of TGF-βRII-Ig as a novel biologic for canine melanoma.

ERRATUM: Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.
Takuya Kodera, Tomoyuki Yamaguchi, Yukari Fukushima, Kumi Kobayashi, Yutaka Takarada, Joseph Yamweka Chizimu, Chie Nakajima, Eddie Samuneti Solo, Patrick Saili Lungu, Mitsuo Kawase, Yasuhiko Suzuki
Japanese journal of infectious diseases, 74, 4, 385, 385, 2021年, ［国内誌］
英語, 研究論文（学術雑誌）, Volume 74, no.3, p.214-219, 2021. Page 214, affiliation "1TBA Co., LTD, Sendai; 2Hokkaido University Research Center for Zoonosis Control, Sapporo; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia." should read "1TBA Co., LTD, Sendai, Japan; 2Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo, Japan; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia".

Emergence of the Novel Aminoglycoside Acetyltransferase Variant aac(6')-Ib-D179Y and Acquisition of Colistin Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae Due to a Disrupting Mutation in the DNA Repair Enzyme MutS.
Toyotaka Sato, Takayuki Wada, Suguru Nishijima, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Satoshi Takahashi, Shin-Ichi Yokota
mBio, 11, 6, 2020年12月22日, ［国際誌］
英語, 研究論文（学術雑誌）, Amikacin and colistin are effective against carbapenem-resistant Klebsiella pneumoniae In 2017, we successively isolated three carbapenem-resistant K. pneumoniae isolates (ST967) from a patient with chronic renal failure in Japan. The first (SMKP01, sputum, day 0) and second (SMKP02, blood, day 14) strains were resistant to most antimicrobials tested but still susceptible to amikacin (MICs of 4 and 0.5 mg/liter, respectively) and colistin (MIC of 0.5 mg/liter for both). The third strain (SMKP03, blood, day 51) was not susceptible to amikacin (MIC, 32 mg/liter), and its MIC for colistin varied (0.5 to 8 mg/liter). Whole-genome sequencing of SMKP01 revealed that 17 of 20 antimicrobial resistance genes, including qnrB91 (a novel qnrB2 variant) and aac(6')-Ib-cr, were located on an 86.9-kb IncFII-IncQ plasmid. The qnrB91 conferred greater fluoroquinolone resistance than qnrB2 SMKP03 aac(6')-Ib-cr that possessed a gene mutation that resulted in an R102W substitution, namely, aac(6')-Ib-D179Y, made a greater contribution to amikacin resistance than did aac(6')-Ib-cr SMKP03 harbored a nonsense mutation in mutS, which encodes a DNA repair enzyme. Introduction of this mutation into SMKP01 (SMKP01mutS A307T) resulted in a dramatic increase (>58-fold) in the frequency of spontaneous amikacin-resistant mutants relative to SMKP01, and the substantial mutants possessed aac(6')-Ib-D179Y SMKP01mutS A307T exhibited an unstable MIC for colistin (0.5 to 8 mg/liter). The results demonstrate that a disruptive mutation in MutS, arising during the clinical course of an infection, created a platform for the acquisition of amikacin nonsusceptibility and colistin heteroresistance in multidrug-resistant K. pneumoniae, mediated by the elevated frequency of spontaneous mutations.IMPORTANCE The emergence of multidrug resistance in pathogens such as Klebsiella pneumoniae is of great clinical concern. Antimicrobial resistance sometimes arises during the course of an infection. Although many studies have reported the emergence of antimicrobial resistance and novel antimicrobial resistance genes in the clinical isolates, the identity of the bacterial factor(s) that generate this emergence is still unclear. We report that a disruptive mutation in MutS, arising during the clinical course of an infection, created a context for the acquisition of colistin resistance and the emergence of a novel variant of the amikacin resistance gene in multidrug-resistant K. pneumoniae via an increase in the frequency of spontaneous mutation. This observation is important for understanding how K. pneumoniae develops multidrug resistance during infection and could potentially lead to new antimicrobial treatments for high-risk pathological microbes.

Enhanced Immunotherapeutic Efficacy of Anti-PD-L1 Antibody in Combination with an EP4 Antagonist.
Yamato Sajiki, Satoru Konnai, Zimeng Cai, Kensuke Takada, Tomohiro Okagawa, Naoya Maekawa, Sotaro Fujisawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
ImmunoHorizons, 4, 12, 837, 850, 2020年12月21日, ［国際誌］
英語, 研究論文（学術雑誌）, Combination treatment approaches are increasingly considered to overcome resistance to immunotherapy targeting immunoinhibitory molecules such as programmed death (PD)-1 and PD-ligand 1 (PD-L1). Previous studies have demonstrated that the therapeutic efficacy of anti-PD-L1 Abs is enhanced by combination treatment with cyclooxygenase-2 inhibitors, through downregulation of the immunosuppressive eicosanoid PGE2, although the underlying mechanism remains unclear. In this study, we show that serum PGE2 levels are upregulated after anti-PD-L1 Ab administration in a bovine model of immunotherapy and that PGE2 directly inhibits T cell activation via its receptor E prostanoid (EP) 4. Additionally, anti-PD-L1 Ab induces TNF-α production and TNF-α blockade reduces PGE2 production in the presence of anti-PD-L1 Ab, suggesting that anti-PD-L1 Ab-induced TNF-α impairs T cell activation by PGE2 upregulation. Our studies examining the therapeutic potential of the dual blockade of PD-L1 and EP4 in bovine and murine immune cells reveal that the dual blockade of PD-L1 and EP4 significantly enhances Th1 cytokine production in vitro. Finally, we show that the dual blockade decreases tumor volume and prolongs survival in mice inoculated with the murine lymphoma cell line EG7. Altogether, these results suggest that TNF-α induced by anti-PD-L1 Ab treatment is associated with T cell dysfunction via PGE2/EP4 pathway and that the dual blockade of PD-L1 and EP4 should be considered as a novel immunotherapy for cancer.

Sitafloxacin has a potent activity for eradication of extended spectrum β-lactamase-producing fluoroquinolone-resistant Escherichia coli forming intracellular bacterial communities in uroepithelial cells.
Yoshiki Hiyama, Toyotaka Sato, Satoshi Takahashi, Soh Yamamoto, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota, Naoya Masumori
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 26, 12, 1272, 1277, 2020年12月, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: Eradication of asymptomatic bacteriuria (ASB) before urological procedures is important to reduce the risk for infectious complications after surgery. However, the appropriate regimen for antimicrobial treatment has not been fully determined. We experienced continuous (over 10 months) isolation of extended spectrum β-lactamase (ESBL)-producing fluoroquinolone-resistant Escherichia coli from urine of an asymptomatic patient. The four isolates obtained (SMESC1 to 4) were international high-risk clones of O25b:H4-ST131-H30R, and originated from one strain, as revealed by the whole genome sequences. Although the patient received meropenem (MEPM) and fosfomycin (FOM), to which the strains were susceptible before the urological procedures, they could not be eradicated. METHODS: To explore the reason for the continuous isolation even after MEPM and FOM administration, antimicrobial killing of adherent and/or intracellular bacterial communities (IBC) formed by coculture of the E. coli cells and T24 bladder epithelial cells were examined. RESULTS: FOM and levofloxacin did not decrease viable E. coli cells compared with gentamicin. MEPM partly decreased them, and sitafloxacin (STFX) decreased them most potently. These observations indicate that E. coli can survive in the urinary tract under antimicrobial administration, and some antimicrobials such as FOM and MEPM cannot eradicate E. coli in uroepithelial cells. Adhesion on urinary epithelial cells and/or IBC formation might result in continuous isolation from the urinary tract and recurrence of ASB and urinary tract infections. CONCLUSIONS: The present study suggests that STFX is a promising optional agent for the eradication of ESBL-producing fluoroquinolone-resistant E. coli in the urinary tract before urological procedures.

Molecular analysis of streptomycin-resistance associating genes in Mycobacterium tuberculosis isolates from Nepal.
Dipti Shrestha, Bhagwan Maharjan, Nan Aye Thida Oo, Norikazu Isoda, Chie Nakajima, Yasuhiko Suzuki
Tuberculosis (Edinburgh, Scotland), 125, 101985, 101985, 2020年12月, ［国際誌］
英語, 研究論文（学術雑誌）, Mutation in rpsL (encoding ribosomal protein S12), rrs (encoding 16S ribosomal RNA) and gidB (encoding 7-methylguanosine methyltransferase) are associated with resistance to streptomycin (STR), which is used for the treatment of multi-drug resistant tuberculosis (MDR-TB) in Nepal. The aim of our study is to analyze the correlation between mutations in the target genes and STR-resistance in 197 Mycobacterium tuberculosis (MTB) isolates from Nepal. Mutations in rpsL was harbored by 65.9% of isolates, in which the most common mutation in rpsL is caused by K43R (58.8%) and were significantly associated with Beijing genotype (P < 0.001). About 13.2% of isolates harbored mutations in two highly mutable regions of rrs, the 530 loop and the 912 region. About 13.2% of gidB mutants do not show any mutation in rpsL and rrs, which might suggest the role of gidB mutations in STR-resistance in MTB. In addition, 5.6% of isolates do not show any mutations in three genes examined, suggesting the involvement of other mechanism in STR-resistance in MTB. Our findings can be implemented for the establishment of molecular STR-susceptibility testing, in which tuberculosis can be treated with appropriate drugs and can improve control strategies for DR-TB.

Interaction of the plasmid-encoded quinolone resistance protein QnrB19 with Salmonella Typhimurium DNA gyrase.
Ruttana Pachanon, Kentaro Koide, Siriporn Kongsoi, Chie Nakajima, Thoko Flav Kapalamula, Orasa Suthienkul, Yasuhiko Suzuki
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 26, 11, 1139, 1145, 2020年11月, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Plasmid-encoded quinolone resistance protein Qnr is an important factor in bacterial resistance to quinolones. Qnr interacts with DNA gyrase and reduces susceptibility to quinolones. The gene qnr likely spreads rapidly among Enterobacteriaceae via horizontal gene transfer. Though the vast amounts of epidemiological data are available, molecular details of the contribution of QnrB19, the predominant Qnr in Salmonella spp., to the acquisition of quinolone resistance has not yet been understood well. OBJECTIVE: We aimed to examine the role of QnrB19 in quinolone resistance acquisition using recombinant Salmonella Typhimurium DNA gyrases and QnrB19. MATERIALS AND METHODS: Recombinant QnrB19 was expressed in E. coli and purified by Ni-NTA agarose column chromatography. DNA supercoiling activities of recombinant Salmonella Typhimurium DNA gyrase were assessed with or without QnrB19 under the existence of three quinolones to measure IC50s, the concentration of each quinolone required for 50% inhibition in vitro. RESULTS: The IC50s of norfloxacin, ciprofloxacin and nalidixic acid against DNA gyrases were measured to be 0.30, 0.16 and 17.7 μg/mL, respectively. The addition of QnrB19 increased the IC50s of norfloxacin and ciprofloxacin to be 0.81 and 0.48 μg/mL, respectively, where no effect of QnrB19 was observed on the IC50 of nalidixic acid. CONCLUSION: QnrB19 was shown for the first time in vitro to have ability to grant non-classical quinolone resistance to S. Typhimurium DNA gyrase. Structural insight on quinolones in this study may contribute to investigate drugs useful for preventing the spread of plasmid carrying PMQR along with other factors associating with antimicrobial resistance in S. Typhimurium and other bacteria.

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography.
Yutaka Takarada, Takuya Kodera, Kumi Kobayashi, Chie Nakajima, Mitsuo Kawase, Yasuhiko Suzuki
Journal of microbiological methods, 177, 106062, 106062, 2020年10月, ［国際誌］
英語, 研究論文（学術雑誌）, Rapid and easy detection of nucleotide point mutations in bacterial pathogens associated with drug resistance is essential for the proper use of antimicrobials. Here, we developed a rapid and simple method for the detection of mutations using Loop-mediated isothermal amplification (LAMP) combined with the single-tag hybridization (STH) chromatographic printed array strips (PAS) method. This procedure is able to detect four mutations (C1349 T, A1295C, G1303 T, A1304 T) in Rifampicin Resistance Determining Region (RRDR) of rifampicin-resistant Mycobacterium tuberculosis (RR-TB), simultaneously. LAMP reactions contained a LAMP primer and eight allele-specific primers for each mutation. The allele-specific primers products were detected by nucleic acid chromatography using PAS. Four detection lines were detected there, one of which was detected at different positions depend on the wild type and the mutant type. We carried out the four mutations detection using 31 genomic DNA (2 A1295T, 1 G1303 T, 6 A1304 T, 22 C1349 T) from clinical isolate. The mutations have been confirmed by sequence analysis. The detection results were completely consistent with the sequence analysis. In the present study, four mutations could be detected, but only 60% of RR-TB could be detected with these four. It is expected that the detection rate will increase by adding more mutant primers. The combined LAMP and STH chromatographic PAS method is a simple and rapid method for detecting point mutations in clinical isolates as a point-of-care testing (POCT) technique. In addition, it does not require special equipment and can meet the demand in areas where drug-resistant bacteria are endemic, such as developing countries.

Elucidating the Structural Requirement of Uridylpeptide Antibiotics for Antibacterial Activity.
Yuma Terasawa, Chisato Sataka, Toyotaka Sato, Kazuki Yamamoto, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Akira Katsuyama, Takanori Matsumaru, Fumika Yakushiji, Shin-Ichi Yokota, Satoshi Ichikawa
Journal of medicinal chemistry, 63, 17, 9803, 9827, 2020年09月10日, ［国際誌］
英語, 研究論文（学術雑誌）, The synthesis and biological evaluation of analogues of uridylpeptide antibiotics were described, and the molecular interaction between the 3'-hydroxy analogue of mureidomycin A (3'-hydroxymureidomycin A) and its target enzyme, phospho-MurNAc-pentapeptide transferase (MraY), was analyzed in detail. The structure-activity relationship (SAR) involving MraY inhibition suggests that the side chain at the urea-dipeptide moiety does not affect the MraY inhibition. However, the anti-Pseudomonas aeruginosa activity is in great contrast and the urea-dipeptide motif is a key contributor. It is also suggested that the nucleoside peptide permease NppA1A2BCD is responsible for the transport of 3'-hydroxymureidomycin A into the cytoplasm. A systematic SAR analysis of the urea-dipeptide moiety of 3'-hydroxymureidomycin A was further conducted and the antibacterial activity was determined. This study provides a guide for the rational design of analogues based on uridylpeptide antibiotics.

Emergence of vancomycin- and teicoplanin-resistant Enterococcus faecium via vanD5-harbouring large genomic island.
Toyotaka Sato, Takayuki Wada, Masaaki Shinagawa, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Satoshi Takahashi, Shin-Ichi Yokota
The Journal of antimicrobial chemotherapy, 75, 9, 2411, 2415, 2020年06月25日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Treatment of VRE is of clinical concern. While certain numbers of vanD-type VRE have been isolated, only two vanD5-harbouring Enterococcus faecium isolates have been reported in Canada and Japan. METHODS: We report the isolation of vanD5-type E. faecium and the first ever determination of the whole-genome sequence to investigate the possible mechanisms of the acquisition of the vanD5 gene cluster in E. faecium. RESULTS: Two vanD5-harbouring vancomycin-resistant E. faecium were isolated from the skin (SMVRE19) and faeces (SMVRE20) of a patient with a skin ulcer in Japan. The isolates exhibited vancomycin and teicoplanin MIC values of 128 mg/L, whilst the previous isolates of vanD5-harbouring E. faecium were only resistant to vancomycin. SMVRE19 and SMVRE20 were clones related to ST18, which is also seen in vanA- and vanB-type VRE. These isolates harboured an insertion element, ISEfm1, in the ddl gene, similar to a previously described teicoplanin-resistant vanD3-type E. faecium. The vanD5 gene cluster was integrated into the SMVRE20 chromosome as a part of a large genomic island (approximately 127 kb), similar to other recently spreading vanD variants in the Netherlands. The genomic island shared the greatest similarity with a part of the Blautia coccoides genome sequence, except for the region surrounding the vanD gene cluster. CONCLUSIONS: This study reports that emergence of vancomycin- and teicoplanin-resistant vanD5-type E. faecium occurred via acquisition of the vanD5 cluster and ISEfm1 insertion into ddl. Considering the genetic similarity between the various VRE strains, the current study should serve as a warning against the spread of vanD5-type VRE.

WQ-3810 inhibits DNA gyrase activity in ofloxacin-resistant Mycobacterium leprae.
Jong-Hoon Park, Tomoyuki Yamaguchi, Yuki Ouchi, Kentaro Koide, Shigetarou Mori, Hyun Kim, Tetsu Mukai, Chie Nakajima, Yasuhiko Suzuki
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 26, 4, 335, 342, 2020年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. METHODOLOGY/PRINCIPAL FINDINGS: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. CONCLUSIONS/SIGNIFICANCE: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens.

Campylobacter upsaliensis isolated from a giant hepatic cyst.
Yasuo Ohkoshi, Toyotaka Sato, Hiromi Murabayashi, Kohei Sakai, Yasunari Takakuwa, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 26, 7, 752, 755, 2020年03月18日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Campylobacter upsaliensis is an enteropathogenic bacterium in animals, and is also rarely isolated from humans, where it can cause enteritis and bacteremia. This report describes the first case of isolation of C. upsaliensis from an infected giant hepatic cyst. This bacterium could not be cultured from abscess punctuate in a usual Campylobacter-selection medium (charcoal cefoperazone deoxycholate agar medium), because of high concentration of cefoperazone as a selection agent. It could not identified by matrix-assisted laser desorption ionization-time of flight mass spectrum. Rather, it was identified as C. upsaliensis by whole genome sequencing, including by multilocus sequence typing.

Mutations of rpoB, katG and inhA genes in multidrug-resistant Mycobacterium tuberculosis isolates from Zambia.
Eddie S Solo, Chie Nakajima, Trevor Kaile, Precious Bwalya, Grace Mbulo, Yukari Fukushima, Sylvia Chila, Nanthan Kapata, Yogendra Shah, Yasuhiko Suzuki
Journal of global antimicrobial resistance, 22, 302, 307, 2020年03月10日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: It is established that resistance to rifampicin (RIF) in 90% of the RIF resistantM. tuberculosis strains is attributable to point mutations in the rpoB gene while 50-95% of M. tuberculosis resistance to isoniazid (INH) is caused by mutations in katG gene. However, the patterns and frequencies of mutations vary with geographical differences. In Zambia, the genetic resistance of M. tuberculosis to RIF and INH were unreported before this study. METHODS: Using sequencing, we analyzed rpoB, katG and inhA genes for 99 multidrug-resistant (MDR) and 49 pan-susceptible M. tuberculosis isolates stored at a tuberculosis reference laboratory from 2013 to 2016 and compared with published profiles from other African countries. RESULTS: Ninety six percent (95/99) of the MDR-TB isolates carried mutations in bothrpoB and katG genes. No mutations were detected among the pan-susceptible isolates. rpoB gene codon 531 and katG gene codon 315 at 55.6 % (55/99) and 94.9 % (94/99) were the most altered among the RIF and INH resistant isolates, respectively. Distinctly, katG mutations were predominantly high among Zambian isolates (96%) compared to other countries in the region. CONCLUSION: The drug resistant associated mutations to RIF and INH circulating in Zambia are similar to what have been reported globally, therefore our data validates the applicability of molecular diagnostic tools in Zambia. However,katG mutations were predominantly high among M. tuberculosis isolates in this study compared to other regional countries and might disassociate cross-boundary transmission of MDR-TB from other African nations.

Comparison of Loop-Mediated Isothermal Amplification, Microscopy, Culture, and PCR for Diagnosis of Pulmonary Tuberculosis.
Benjawan Phetsuksiri, Janisara Rudeeaneksin, Sopa Srisungngam, Supranee Bunchoo, Wiphat Klayut, Chie Nakajima, Shigeyuki Hamada, Yasuhiko Suzuki
Japanese journal of infectious diseases, 73, 4, 272, 277, 2020年02月28日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Diagnosis tuberculosis (TB) in endemic countries is challenging due to high caseloads and limited resources. A simple, cost-effective diagnostic test for rapid detection of Mycobacterium tuberculosis in clinical specimens is in need. In Thailand, we evaluated the performance of in-house loop-mediated isothermal amplification (LAMP) targeting M. tuberculosis 16s rRNA gene for TB diagnosis. A total of 252 sputum samples from pulmonary TB suspects were analyzed. The sensitivity of LAMP was 99.04% (103/104; 95% confidential interval [CI]: 94.76-99.98%) and 72.73% (16/22; 95% CI: 49.78-89.27) for smear-positive and smear-negative samples with TB-culture positivity, respectively. LAMP detected 20.69% (24/116) of TB-culture negative samples but those all were positive by conventional PCR. The sensitivity of LAMP was higher than that of sputum microscopy while the performance of LAMP was similar to PCR. None of the samples positive for non-tuberculous mycobacteria by culture and PCR was positive by LAMP. Compared with TB culture, the positive predictive value (PPV), negative predictive value (NPV) and kappa coefficient of LAMP were 83.22%, 88.33% and 0.75 respectively. Based on the diagnostic performance, we proposed that this LAMP would be suitable as a potential diagnostic test for rapid TB diagnosis in resource-limited laboratory settings.

Contribution of β-lactamase and efflux pump overproduction to tazobactam-piperacillin resistance in clinical isolates of Escherichia coli.
Yuuki Suzuki, Toyotaka Sato, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Satoshi Takahashi, Shin-Ichi Yokota
International journal of antimicrobial agents, 55, 4, 105919, 105919, 2020年02月13日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: Tazobactam-piperacillin (TZP) is a mixture of a broad-spectrum penicillin and an irreversible β-lactamase inhibitor. TZP is effective against Gram-negative bacteria that produce extended-spectrum β-lactamases, and it is used as a first-line or second-line drug to treat serious infections. METHODS: This study identified three TZP-resistant and two TZP-intermediate strains among 514 clinical isolates of Escherichia coli. RESULTS: These five isolates possessed one or more β-lactamase genes, blaTEM-1, blaCTX-M-2, blaCTX-M-14, and/or blaCMY-8. The expression levels of β-lactamase genes and acrAB genes in the strains were examined by using real-time reverse transcription PCR. The total enzymatic piperacillin-degrading activity in cells was determined. Two TZP-resistance mechanisms were identified: hyperproduction of TEM-1 in the two resistant strains; and simultaneous high production of β-lactamase and efflux pump AcrAB in the two TZP-intermediate isolates. The latter are an international high-risk clone O25b:H4-ST131-H30R. CONCLUSION: TZP resistance is still rare in clinical isolates of E. coli. However, resistance can develop on high production and/or combinations of known antimicrobial resistance mechanisms in different ways.

In Vitro Derivation of Fluoroquinolone-Resistant Mutants from Multiple Lineages of Haemophilus influenzae and Identification of Mutations Associated with Fluoroquinolone Resistance.
Hiroyuki Honda, Toyotaka Sato, Masaaki Shinagawa, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Koji Kuronuma, Satoshi Takahashi, Hiroki Takahashi, Shin-Ichi Yokota
Antimicrobial agents and chemotherapy, 64, 2, 2020年01月27日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of β-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to β-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported. The current risk of fluoroquinolone resistance in H. influenzae (especially in high-BLNAR) has not yet been evaluated. Here, we examined the development of fluoroquinolone resistance in fluoroquinolone-susceptible clinical H. influenzae isolates in vitro during passaging in the presence of moxifloxacin (from 0.03 to 128 mg/liter). Twenty-nine isolates were examined. Seventeen isolates (58.6%) showed reduced moxifloxacin susceptibility, and 10 of these 17 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute breakpoint for moxifloxacin (MIC of >1 mg/liter) after repeat cultivation on moxifloxacin-containing agar. Seven of these ten isolates were high-BLNAR and represented multiple lineages. We identified 56 novel mutations in 45 genes induced during the development of fluoroquinolone resistance, except the defined quinolone resistance-determining regions (Ser84Leu and Asp88Tyr/Gly/Asn in GyrA and Gly82Asp, Ser84Arg, and Glu88Lys in ParC). Glu153Leu and ΔGlu606 in GyrA, Ser467Tyr and Glu469Asp in GyrB, and ompP2 mutations were novel mutations contributing to fluoroquinolone resistance in H. influenzae In conclusion, H. influenzae clinical isolates from multiple lineages can acquire fluoroquinolone resistance by multiple novel mutations. The higher rate of derivation of fluoroquinolone-resistant H. influenzae from high-BLNAR than β-lactamase-negative ampicillin-susceptible isolates (P = 0.01) raises the possibility of the emergence and spread of fluoroquinolone-resistant high-BLNAR in the clinical setting.

タイ国のブタとブタ肉から分離されたMRSAの分子的特性と抗菌薬耐性(Molecular characterization and antimicrobial resistance of MRSA from pigs and pork in Thailand)　　　　　　　　　　　　　　　
Tanomsridachchai Wimonrat, 中島 千絵, Changkaew Kanjana, Changkwanyeun Ruchirada, Prapasawat Watsawan, Intarapuk Apiradee, Yamasamit Nattapong, Suthienkul Orasa, 鈴木 定彦
日本細菌学雑誌, 75, 1, 60, 60, 日本細菌学会, 2020年01月
英語

フィリピンの食用動物から分離されたE.coliのキノロン耐性決定因子の検出(Detection of quinolone resistance determinants in E. coli from food animals in the Philippines)　　　　　　　　　　　　　　　
Belotindos Lawrence, Mingala Claro, Villanueva Marvin, 中島 千絵, 鈴木 定彦
日本細菌学雑誌, 75, 1, 64, 64, 日本細菌学会, 2020年01月
英語

病原性抗酸菌における細胞外DNAの存在と抗酸菌の生理におけるその役割(Existence of extracellular DNA in pathogenic mycobacteria and its role in mycobacterial physiology)　　　　　　　　　　　　　　　
イリノフ・アレクサンドル, シャバン・アミナ, 袴田 真理子, 西山 晃史, 尾関 百合子, 福島 由華里, 中島 千絵, 立石 善隆, 鈴木 定彦, 松本 壮吉
日本細菌学雑誌, 75, 1, 74, 74, 日本細菌学会, 2020年01月
英語

水平ゲノム転移を介したvanD5バンコマイシン耐性遺伝子を保有するEnterococcus faeciumの出現(Emergence of vanD5-type vancomycin-resistant Enterococcus faecium via horizontal genomic transfer)　　　　　　　　　　　　　　　
佐藤 豊孝, 和田 崇之, 福島 由華里, 中島 千絵, 鈴木 定彦, 高橋 聡, 横田 伸一
日本細菌学雑誌, 75, 1, 100, 100, 日本細菌学会, 2020年01月
英語

The performance of an in-house loop-mediated isothermal amplification for the rapid detection of Mycobacterium tuberculosis in sputum samples in comparison with Xpert MTB/RIF, microscopy and culture.
Benjawan Phetsuksiri, Wiphat Klayut, Janisara Rudeeaneksin, Sopa Srisungngam, Supranee Bunchoo, Sarawut Toonkomdang, Thanee Wongchai, Chie Nakajima, Yasuhiko Suzuki
Revista do Instituto de Medicina Tropical de Sao Paulo, 62, e36, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, Simple, low-cost and effective diagnostic tests for tuberculosis (TB) are needed especially in TB-high burden settings. The present study evaluated the performance of an in-house loop-mediated isothermal amplification (LAMP) for diagnosing TB by comparing it to Xpert MTB/RIF, microscopy and culture. In Thailand, a total of 204 excess sputum samples volume after the processing of cultures were used for Mycobacterium tuberculosis (MTB) detection by Xpert MTB/RIF and LAMP. Based on culture results as the gold standard, the overall sensitivity of LAMP and Xpert MTB/RIF were 82.1% (126/153; 95% confidential interval [CI]: 75.4-88.98%) and 86.9 % (133/153; 95% CI: 80.5-90.8%) respectively, and the specificity of both tests was 100% (51/51; 95% CI: 93.0-100.0%). In comparison with Xpert MTB/RIF, the sensitivity and specificity of LAMP were 94.7% (126/133; 95% CI: 89.5-97.9%), and 100.0% (73/73; 95% CI: 94.9-100.0%), respectively. The average threshold cycle (Ct) of Xpert MTB/RIF detection for positive and negative LAMP results was statistically different, of 18.4 and 27.0, respectively (p < 0.05). In comparison with the acid-fast staining technique, and analyzing LAMP and Xpert MTB/RIF in smear-negative/culture-positive specimens, there was an increase of the detection rate by 47.7% (21/44) and 54.6% (24/44). The diagnostic sensitivity and specificity of LAMP appeared to be comparable to those of Xpert MTB/RIF. We claim that this LAMP has potential to provide a sensitive diagnostic test for the rapid TB diagnosis. It allowed a fast detection of MTB before the cultures and it could be used in resource-limited laboratory settings.

Mesalazine-induced lung injury with severe respiratory failure successfully treated with steroids and non-invasive positive pressure ventilation.
Hajime Oi, Atsushi Suzuki, Yasuhiko Yamano, Toshiki Yokoyama, Toshiaki Matsuda, Kensuke Kataoka, Yasuhiko Suzuki, Tomoki Kimura, Yasuhiro Kondoh
Respiratory medicine case reports, 31, 101157, 101157, 2020年, ［国際誌］
英語, Drug-induced lung injury (DLI) has become more common because of the increasing number of therapeutic agents in use. Mesalazine, also known as 5-aminosalicylic acid (5-ASA), is one of the key drugs for the treatment of ulcerative colitis (UC). Although mesalazine-induced lung injury has been previously reported, few cases have included severe respiratory failure. In this report, we present a case of mesalazine-induced lung injury with severe respiratory failure, which was improved by discontinuation of mesalazine and introduction of corticosteroid therapy and ventilation support with non-invasive positive pressure ventilation (NPPV). We also review the previous literature on mesalazine-induced lung injury.

PD-L1 expression in equine malignant melanoma and functional effects of PD-L1 blockade.
Otgontuya Ganbaatar, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Naoya Maekawa, Erina Minato, Atsushi Kobayashi, Ryo Ando, Nobuya Sasaki, Daisuke Miyakoshi, Osamu Ichii, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
PloS one, 15, 11, e0234218, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on lymphocytes. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. In our previous studies, we have developed anti-bovine PD-L1 monoclonal antibodies (mAbs) and reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Furthermore, we found that blocking antibodies that target PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway for chronic equine diseases, including tumors, remains unclear. In this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 mAbs against EqPD-L1 using in vitro assays. In addition, we evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a considerable identity and similarity with homologs from non-primate species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Of note, immunohistochemistry confirmed the PD-L1 expression in EMM tumor tissues. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These findings showed that our anti-PD-L1 mAbs would be useful for analyzing the equine PD-1/PD-L1 pathway. Further research is warranted to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horses.

Properties of biopolymers detection with piezoelectric polymer biosensor in relaxation behavior process
Mariko Takeda, Kouki Yahagi, Takamichi Hirata, Takashi Kuroiwa, Chie Nakajima, Yasuhiko Suzuki, Fumio Munakata
IEEJ Transactions on Sensors and Micromachines, 140, 2, 43, 49, 2020年, ［査読有り］
研究論文（学術雑誌）, © 2020 The Institute of Electrical Engineers of Japan. The complex dielectric constant of a polymer piezoelectric sensor was measured to investigate the properties of biopolymers detection of piezoelectric polymer using relaxation behavior. From the measurement of the complex permittivity, relaxation of the polymer piezoelectric sensor was confirmed. In the properties of the detection of the polymer film, the real part (ε') of the relaxation process linearly shifted to the low frequency with loading the polymer films. This showed the same trend as the result that the frequency shifted to the low frequency with loading mass anticipated from Sauerbrey's equation. In the biopolymer detection, the relaxation was shifted to the low frequency with the fluorescently labeled avidin (the host material) immersion time. With accompanying fluorescently labeled biotin (the guest material) adsorption by host-guest reaction, the relaxation shifted to low frequency. It was considered that the biopolymer detection with the polymer piezoelectric sensor is possible by using relaxation behavior.

WQ-3810: A new fluoroquinolone with a high potential against fluoroquinolone-resistant Mycobacterium tuberculosis.
Yuki Ouchi, Tetsu Mukai, Kentaro Koide, Tomoyuki Yamaguchi, Jong-Hoon Park, Hyun Kim, Kazumasa Yokoyama, Aki Tamaru, Stephen V Gordon, Chie Nakajima, Yasuhiko Suzuki
Tuberculosis (Edinburgh, Scotland), 120, 101891, 101891, 2020年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro antimycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guérin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains.

Upregulation of PD-L1 expression by prostaglandin E2 and the enhancement of IFN-γ by anti-PD-L1 antibody combined with a COX-2 inhibitor in Mycoplasma bovis infection
Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K
Front Vet Sci, 7, 12, 12, 2020年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.

Clinical efficacy of the combined treatment of anti-PD-L1 rat-bovine chimeric antibody with a COX-2 inhibitor in calves infected with Mycoplasma bovis
Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Ishida M, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K
Jpn J Vet Res, 68, 2, 77, 90, 2020年, ［査読有り］
英語, 研究論文（学術雑誌）, Mycoplasma bovis (M. bovis) is a highly contagious pathogen and M. bovis-associated diseases, particularly pneumonia, occur predominantly as herd enzootics, causing considerable economic losses because of calf mortality, weight loss in surviving calves. In our previous studies, the programmed death-1 (PD-1)/ PD-ligand 1 (PD-L1) pathway and prostaglandin E2 (PGE2 ) were shown to be involved in the immunosuppression during chronic infectious diseases in cattle. In this study, the efficacy of dual blockade of the PD-1/PD-L1 pathway and PGE2 in M. bovis infection in vivo was investigated using anti-bovine PDL1 rat-bovine chimeric antibody, Boch4G12, and cyclooxygenase 2 (COX-2) inhibitor, meloxicam. The calves treated with Boch4G12 and meloxicam significantly enhanced M. bovis-specific IFN-γ response after the administration. On the other hand, IFN-γ response was not activated in the controls and cattle treated with meloxicam alone throughout the experimental period. Interestingly, bacterial loads in nasal discharge and bronchoalveolar lavage fluid among calves treated with Boch4G12 with or without meloxicam were significantly decreased. These results suggest that the combination of anti-PD-L1 antibody with a COX-2 inhibitor is a candidate for therapeutic applications in calves infected with M. bovis.

Acquisition of fluoroquinolone resistance leads to increased biofilm formation and pathogenicity in Campylobacter jejuni.
Matthew V X Whelan, Laura Ardill, Kentaro Koide, Chie Nakajima, Yasuhiko Suzuki, Jeremy C Simpson, Tadhg Ó Cróinín
Scientific reports, 9, 1, 18216, 18216, 2019年12月03日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The World Health Organization has listed C. jejuni as one of 12 microorganisms on a global priority list for antibiotic resistance due to a rapid increase in strains resistant to fluoroquinolone antibiotics. This fluoroquinolone resistance is conferred through a single point mutation in the QRDR region within the gyrA gene known to be involved in DNA supercoiling. We have previously revealed that changes in DNA supercoilikng play a major role in the regulation of virulence in C. jejuni with relaxation of DNA supercoiling associated with increased attachment to and invasion of human epithelial cells. The aim of this study was to investigate whether fluoroquinolone resistant strains of C. jejuni displayed altered supercoiling associated phenotypes. A panel of fluoroquinolone resistant mutants were derived and shown to have a greater ability to form viable biofilms under aerobic conditions, invade epithelial cells and promote virulence in the Galleria mellonella model of infection. We thus report for the first time that fluoroquinolone resistance in C. jejuni is associated with an increase in virulence and the ability to form viable biofilms in oxygen rich environments. These altered phenotypes likely play a critical role in the continued increase in fluoroquinolone resistance observed for this important pathogen.

WQ-3810 exerts high inhibitory effect on quinolone-resistant DNA gyrase of Salmonella Typhimurium.
Koide K, Kongsoi S, Nakajima C, Suzuki Y
Bioscience, biotechnology, and biochemistry, 83, 12, 2249, 2256, 2019年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The inhibitory effect of WQ-3810 on DNA gyrase was assayed to evaluate the potential of WQ-3810 as a candidate drug for the treatment of quinolone resistant Salmonella Typhymurium infection. The inhibitory effect of WQ-3810, ciprofloxacin and nalidixic acid was compared by accessing the drug concentration that halves the enzyme activity (IC50) of purified S. Typhimurium wildtype and mutant DNA gyrase with amino acid substitution at position 83 or/and 87 in subunit A (GyrA) causing quinolone resistance. As a result, WQ-3810 reduced the enzyme activity of both wildtype and mutant DNA gyrase at a lower concentration than ciprofloxacin and nalidixic acid. Remarkably, WQ-3810 showed a higher inhibitory effect on DNA gyrase with amino acid substitutions at position 87 than with that at position 83 in GyrA. This study revealed that WQ-3810 could be an effective therapeutic agent, especially against quinolone resistant Salmonella enterica having amino acid substitution at position 87.

Pyrimidine Analogues as a New Class of Gram-Positive Antibiotics, Mainly Targeting Thymineless-Death Related Proteins.
Oe C, Hayashi H, Hirata K, Kawaji K, Hashima F, Sasano M, Furuichi M, Usui E, Katsumi M, Suzuki Y, Nakajima C, Kaku M, Kodama EN
ACS infectious diseases, 6, 6, 1490, 1500, 2019年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Multidrug-resistant (MDR) bacteria are widespread throughout the world and pose an increasingly serious threat to human and animal health. Besides implementing strict measures to prevent improper antibiotic use, it remains essential that novel antibiotics must be developed. These antibiotics need to exert their activity via mechanisms different from those employed by currently approved antibiotics. In this study, we used several 5-fluorouracil (5-FU) analogues as chemical probes and investigated the potential of these pyrimidine analogues as antibacterial agents. Several 5-FU derivatives exerted potent activity against strains of Gram-positive cocci (GPC) that are susceptible or resistant toward approved antibiotics, without showing cross-resistance. Furthermore, we have provided evidence that the pyrimidine analogues exerted anti-GPC activity via thymineless death by inhibition of thymidylate synthetase (ThyA) and/or inhibition of RNA synthesis. Interestingly, whole genome resequencing of in vitro-selected, pyrimidine analogue-resistant Staphylococcus aureus mutants indicated that S. aureus strains with pyrimidine-analogue resistance induced an amino acid (AA) substitution, deletion, and/or insertion into thymineless-death related proteins except for ThyA, or enhanced the ThyA transcription level. Thus, S. aureus may avoid altering the ThyA function by introducing an AA substitution, suggesting that the pyrimidine analogues, which directly bind to ThyA without phosphorylation, may be more effective and show a higher genetic barrier than the pyrimidines that depend on phosphorylation for activity. The findings of this study may assist in the future development of a novel class of antibiotics for combating MDR GPC, including methicillin-resistant S. aureus and vancomycin-resistant Enterococci.

Insight into genetic diversity of Mycobacterium tuberculosis in Kandy, Sri Lanka reveals predominance of the Euro-American lineage.
Mendis C, Thevanesam V, Kumara A, Wickramasinghe S, Madegedara D, Gamage C, Gordon SV, Suzuki Y, Ratnatunga C, Nakajima C
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 87, 84, 91, 2019年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVE: Sri Lanka is a country where the molecular epidemiology of Mycobacterium tuberculosis (MTB) is poorly explored. Therefore, this study was performed to identify circulating lineages/sub-lineages of MTB and their transmission patterns. METHODS: DNA was extracted from 89 isolates of MTB collected during 2012 and 2013 from new pulmonary tuberculosis patients in Kandy, Sri Lanka and analyzed by spoligotyping, large sequence polymorphism (LSP), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, and drug resistance-associated gene sequencing. RESULTS: The predominant lineage was lineage 4 (Euro-American, 45.9%), followed by lineage 1 (Indo-Oceanic, 29.4%), lineage 2 (East-Asian, 23.5%), and lineage 3 (Central-Asian, 1.2%). Among 26 spoligotype patterns, eight were undesignated or new types and seven of these belonged to lineage 4. Undesignated lineage 4/SIT124 (n=2/8) and SIT3234 (n=8/8) clustered together based on 24-locus MIRU-VNTR typing. The dominant sub-lineage was Beijing/SIT1 (n=19), with the isoniazid resistance katG G944C mutation (Ser315Thr) detected in two of them. CONCLUSIONS: The population structure of MTB in Kandy, Sri Lanka was different from that in the South Asian region. The clonal expansion of locally evolved lineage 4/SIT3234 and detection of the pre-multidrug resistant Beijing isolates from new tuberculosis patients is alarming and will require continuous monitoring.

Immune inhibitory function of bovine CTLA-4 and the effects of its blockade in IFN-γ production
Watari K, Konnai S, Maekawa N, Okagawa T, Suzuki Y, Murata S, Ohashi K
BMC Vet Res, 15, 1, 380, 380, 2019年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is known as an immune inhibitory receptor that is expressed on activated effector T cells and regulatory T cells. When CTLA-4 binds to CD80 or CD86, immunoinhibitory signals are transmitted to retain a homeostasis of the immune response. Recent studies have reported that CTLA-4 is upregulated in chronic infections and malignant neoplasms, contributing to host immune dysfunction. On the other hand, the blockade of CTLA-4 and CD80 or CD86 binding by antibody restores the immune response against these diseases. In a previous report, we indicated that the expression of CTLA-4 was closely associated with disease progression in cattle infected with the bovine leukemia virus (BLV). In this study, we established an anti-bovine CTLA-4 antibody to confirm its immune enhancing effect. RESULTS: Bovine CTLA-4-Ig binds to bovine CD80 and CD86 expressing cells. Additionally, CD80 and CD86 bind to CTLA-4 expressing cells in an expression-dependent manner. Bovine CTLA-4-Ig significantly inhibited interferon-gamma (IFN-γ) production from bovine peripheral blood mononuclear cells (PBMCs) activated by Staphylococcus enterotoxin B (SEB). An established specific monoclonal antibody (mAb) for bovine CTLA-4 specifically recognized only with bovine CTLA-4, not CD28, and the antibody blocked the binding of CTLA-4-Ig to both CD80 and CD86 in a dose-dependent manner. The bovine CTLA-4 mAb significantly restored the inhibited IFN-γ production from the CTLA-4-Ig treated PBMCs. In addition, the CTLA-4 mAb significantly enhanced IFN-γ production from CTLA-4 expressing PBMCs activated by SEB. Finally, we examined whether a CTLA-4 blockade by CTLA-4 mAb could restore the immune reaction during chronic infection; the blockade assay was performed using PBMCs from BLV-infected cattle. The CTLA-4 blockade enhanced IFN-γ production from the PBMCs in response to BLV-antigens. CONCLUSIONS: Collectively, these results suggest that anti-bovine CTLA-4 antibody can reactivate lymphocyte functions and could be applied for a new therapy against refractory chronic diseases. Further investigation is required for future clinical applications.

Prostaglandin E2 induced immune exhaustion and enhancement of anti-viral effects by anti-PD-L1 antibody combined with COX-2 inhibitor in bovine leukemia virus infection
Sajiki Y, Konnai S, Okagawa T, Nishimori A, Maekawa N, Goto S, Watari K, Minato E, Kobayashi A, Kohara J, Yamada S, Kato Y, Takahashi H, Terasaki N, Takeda A, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K
J Immunol, 203, 5, 1313, 1324, 2019年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.

イヌ腫瘍組織およびイヌ腫瘍由来細胞株におけるイヌHER2の発現解析　　　　　　　　　　　　　　　
吉武 志江奈, 今内 覚, 前川 直也, 賀川 由美子, 西村 麻紀, 岡川 朋弘, 鈴木 定彦, 高木 哲, 中川 貴之, 村田 史郎, 大橋 和彦
日本獣医学会学術集会講演要旨集, 162回, 380, 380, (公社)日本獣医学会, 2019年08月
日本語

BLV感染症に対するCOX-2阻害剤と抗PD-L1抗体併用法の抗ウイルス効果の検討　　　　　　　　　　　　　　　
佐治木 大和, 今内 覚, 岡川 朋弘, 前川 直也, 後藤 伸也, 小原 潤子, 山田 慎二, 加藤 幸成, 鈴木 定彦, 村田 史郎, 大橋 和彦
日本獣医学会学術集会講演要旨集, 162回, 381, 381, (公社)日本獣医学会, 2019年08月
日本語

Mycoplasma bovis感染症に対する抗PD-L1キメラ抗体を用いた臨床試験　　　　　　　　　　　　　　　
後藤 伸也, 今内 覚, 平野 佑気, 小原 潤子, 岡川 朋弘, 前川 直也, 鈴木 定彦, 加藤 幸成, 村田 史郎, 大橋 和彦
日本獣医学会学術集会講演要旨集, 162回, 381, 381, (公社)日本獣医学会, 2019年08月
日本語

Differences in Warfarin Pharmacodynamics and Predictors of Response Among Three Racial Populations.
Minami Ohara, Yasuhiko Suzuki, Saki Shinohara, Inna Y Gong, Crystal L Schmerk, Rommel G Tirona, Ute I Schwarz, Ming-Shien Wen, Ming Ta Michael Lee, Kiyoshi Mihara, Edith A Nutescu, Minoli A Perera, Larisa H Cavallari, Richard B Kim, Harumi Takahashi
Clinical pharmacokinetics, 58, 8, 1077, 1089, 2019年08月, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Population differences in warfarin dosing requirement have been reported; however, unlike the pharmacokinetics (PK) of warfarin, the quantitative influences of pharmacodynamic (PD) factors on the anticoagulation response to warfarin in different ethnic populations are totally unknown. METHODS: Using population PK/PD analysis, we attempted to identify predictors of S-warfarin clearance [CL(S)] and half maximal effective concentration (EC50) to quantify racial differences in both PK and PD parameters, and to assess the contribution of these parameters to the international normalized ratio (INR) and over-anticoagulation response (INR ≥ 4) in a cohort of 309 White, Asian and African American patients. RESULTS: Similar to our previous findings, the median CL(S) was 30% lower in African American patients than Asian and White patients (169 vs. 243 and 234 mL/h, p < 0.01). EC50 showed a greater racial difference than CL(S) [1.03, 1.70 and 2.76 μg/mL for Asian, White and African American patients, respectively, p < 0.01). Significant predictors of INR included demographic/clinical (age, body weight, creatinine clearance and sex) and genotypic (CYP2C9*3,*8 and VKORC1 -1639G>A) factors, as well as African American ethnicity. In all three racial groups, genetic predictors of INR appeared to have greater influence than demographic/clinical predictors. Both CL(S) and EC50 contributed to the over-anticoagulation response to warfarin. Patients having VKORC1 -1639 G>A and/or factors associated with reduced CYP2C9 activity were more likely to have an INR ≥ 4. CONCLUSIONS: Although there were contrasting racial differences in CL(S) and EC50 that impacted on the INR, the racial difference in EC50 was greater than that for CL(S), thus explaining the higher warfarin requirement for African American patients.

Erratum to "Insight into multidrug-resistant Beijing genotype Mycobacterium tuberculosis isolates in Myanmar" [Int. J. Infect. Dis. 76 (November) (2018) 109-119].
San LL, Aye KS, Nan Aye TO, Shwe MM, Fukushima Y, Gordon SV, Suzuki Y, Nakajima C
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 85, 214, 214, 2019年08月, ［査読有り］, ［国際誌］
英語

DNA gyrase could be a crucial regulatory factor for growth and survival of Mycobacterium leprae.
Hyun Kim, Yasuo Fukutomi, Chie Nakajima, Youn Uck Kim, Shigetarou Mori, Keigo Shibayama, Noboru Nakata, Yasuhiko Suzuki
Scientific reports, 9, 1, 10815, 10815, 2019年07月25日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.
Hayashida K, Orba Y, Sequeira PC, Sugimoto C, Hall WW, Eshita Y, Suzuki Y, Runtuwene L, Brasil P, Calvet G, Rodrigues CDS, Santos CCD, Mares-Guia MAM, Yamagishi J, Filippis AMB, Sawa H
PLoS neglected tropical diseases, 13, 6, e0007480, 2019年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas.

Mixed Mycobacterium tuberculosis Lineage Infection in 2 Elephants, Nepal.
Paudel S, Nakajima C, Mikota SK, Gairhe KP, Maharjan B, Subedi S, Poudel A, Sashika M, Shimozuru M, Suzuki Y, Tsubota T
Emerging infectious diseases, 25, 5, 1031, 1032, 2019年05月, ［査読有り］, ［国際誌］
英語, Tuberculosis in elephants is primarily caused by Mycobacterium tuberculosis. We identified mixed M. tuberculosis lineage infection in 2 captive elephants in Nepal by using spoligotyping and large sequence polymorphism. One elephant was infected with Indo-Oceanic and East African-Indian (CAS-Delhi) lineages; the other was infected with Indo-Oceanic and East Asian (Beijing) lineages.

Prevalence of 16S rRNA methylases in Gram-negative bacteria derived from companion animals and livestock in Japan.
Usui M, Kajino A, Kon M, Fukuda A, Sato T, Shirakawa T, Kawanishi M, Harada K, Nakajima C, Suzuki Y, Tamura Y
The Journal of veterinary medical science, 81, 6, 874, 878, 2019年05月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The emergence and spread of aminoglycoside-resistant bacteria are a public health concern. The acquisition of the genes encoding 16S rRNA methylases, such as armA, rmtA, and rmtB, confers high-level resistance to aminoglycosides. However, the prevalence has not been well investigated in Japanese veterinary fields. To determine the prevalence of 16S rRNA methylases in animals, we detected 16S rRNA methylases genes in Gram-negative bacteria from animals. Here, we report the isolation of rmtB amd armA from two of the 446 Escherichia coli (0.5%) and one of the 103 Klebsiella spp. isolates (1.0%) from companion animals, respectively. However, none of the isolations were observed from 2445 E. coli isolates derived from livestock in Japan. The prevalence of 16S rRNA methylases in animals, especially in companion animals, should be carefully monitored in Japanese veterinary fields to avoid the spreading of the genes.

Evaluation of Susceptibilities to Carbapenems and Faropenem Against Cephalosporin-Resistant Neisseria gonorrhoeae Clinical Isolates with penA Mosaic Alleles.
Yoshiki Hiyama, Satoshi Takahashi, Toyotaka Sato, Masaaki Shinagawa, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Naoya Masumori, Shin-Ichi Yokota
Microbial drug resistance (Larchmont, N.Y.), 25, 3, 427, 433, 2019年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Neisseria gonorrhoeae is a principal pathogen for sexually transmitted infections, especially for male urethritis. Currently, the prevalence of multidrug resistance is increasing. Carbapenems are broad-spectrum antimicrobials that are widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria. However, susceptibility to carbapenems has not been well evaluated for cephalosporin-resistant N. gonorrhoeae isolates. In this study, we determined the susceptibility to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem against cephalosporin-resistant (resistant to cefixime, but susceptible to ceftriaxone) and cephalosporin-susceptible N. gonorrhoeae clinical isolates. The gene mutations associated with β-lactam resistance were evaluated. All cephalosporin-resistant N. gonorrhoeae isolates possessed mosaic mutation alleles in penA (NG-STAR penA-10.001, 27.001, or 108.001). They exhibited a low minimum inhibitory concentration (MIC) (≤0.125 mg/L) for meropenem and markedly high MICs (0.5-2 mg/L) for other carbapenems and faropenem. The strongest association was observed between the mosaic alleles in penA and decreased susceptibility to carbapenems and faropenem compared with mutations in mtrR, porB, and ponA. These results suggest that meropenem may serve as an alternative therapeutic agent for cephalosporin-resistant N. gonorrhoeae with a mosaic allele in penA, whereas other carbapenems and faropenem may be ineffective.

Zinc acetate potentiates the action of tosufloxacin Escherichia coli biofilm persisters.
Usui M, Yokoo H, Tamura Y, Nakajima C, Suzuki Y, Ghigo JM, Beloin C
Antimicrobial agents and chemotherapy, 63, 6, 2019年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Formation of bacterial biofilms is a major health threat due to their high levels of tolerance to multiple antibiotics and the presence of persisters responsible for infection relapses. We previously showed that a combination of starvation and induction of SOS response in biofilm led to increased levels of persisters and biofilm tolerance to fluoroquinolones. In this study, we hypothesized that inhibition of the SOS response may be an effective strategy to target biofilms and fluoroquinolone persister cells. We tested the survival of Escherichia coli biofilms to different classes of antibiotics in starved and nonstarved conditions and in the presence of zinc acetate, a SOS response inhibitor. We showed that zinc acetate potentiates, albeit moderately, the activity of fluoroquinolones against E. coli persisters in starved biofilms. The efficacy of zinc acetate to increase fluoroquinolone activity, particularly that of tosufloxacin, suggests that such a combination may be a potential strategy for treating biofilm-related bacterial infections.

Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapy.
Fujisawa S, Konnai S, Okagawa T, Maekawa N, Tanaka A, Suzuki Y, Murata S, Ohashi K
BMC veterinary research, 15, 1, 68, 68, 2019年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Refractory diseases, including bacterial infections, are causing huge economic losses in dairy farming. Despite efforts to prevent and treat those diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines can attenuate the acute and sever inflammation and may be a novel strategy for treatment. However, biological drugs targeting inflammatory cytokines have not been used in cattle. Therefore, in this study, bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were produced, and their anti-inflammatory functions were analyzed in vitro, to develop decoy receptors for bovine TNF-α. RESULTS: Both TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine fibroblast-derived cells (L929 cells) with TNF-α to induce cell death and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig suppressed TNF-α-induced cell death, significantly improving cell viability. In addition, cell death induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig's anti-inflammatory, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig and analyzed the gene expression and protein production of the inflammatory cytokines IL-1β and TNF-α. TNFR2-Ig significantly reduced the gene expression and protein production of these cytokines. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α. CONCLUSIONS: Collectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for inflammatory diseases, such as bovine clinical mastitis. Further investigation is required for future clinical application.

Nosocomial Mycobacterium tuberculosis transmission by brief casual contact identified using comparative genomics.
Seto J, Otani Y, Wada T, Suzuki Y, Ikeda T, Araki K, Mizuta K, Ahiko T
The Journal of hospital infection, 2019年01月, ［査読有り］
英語, 研究論文（学術雑誌）

Genetic diversity of Mycobacterium tuberculosis Central Asian Strain isolates from Nepal and comparison with neighboring countries.
Shah Y, Poudel A, Maharjan B, Thapa J, Yamaguchi T, Diab HM, Pandey BD, Solo E, Isoda N, Suzuki Y, Nakajima C
Transactions of the Royal Society of Tropical Medicine and Hygiene, 113, 4, 203, 211, 2019年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is an emerging threat for successful tuberculosis control worldwide. Central Asian Strain (CAS) has been reported as one of the dominant families contributing to MDR-TB in South Asia including Nepal, India and Pakistan. The aim of this study was to better understand the genetic characteristics of MDR-TB CAS family isolates circulating in Nepal and compare the results with neighboring countries. METHODS: A total of 145 MDR-TB CAS family isolates collected in Nepal from 2008 to 2013 were analyzed by spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analysis. In addition, we compared these data with published data from India and Pakistan to investigate a possible epidemiological link via construction of a minimum spanning tree (MST). RESULTS: Spoligotyping analysis exhibited CAS1_Delhi SIT26 (n=60) as the predominant lineage among the MDR-TB CAS family in all three countries. However, the combined analysis with spoligotyping and MIRU-VNTR further discriminated 60 isolates into 49 different types and 5 clusters. Each cluster was composed of 14 isolates with a clustering rate of 23.3%, suggesting ongoing transmissions. Based on MST data from neighboring countries, we elucidated an evolutionary relationship between the two countries, Nepal and India, which could be explained by their open border. CONCLUSION: This study identified the evolutionary relationships among MDR-TB CAS1_Delhi subfamily isolates from Nepal and those from neighboring countries.

【結核・非結核性抗酸菌症-エキスパートが教える 実臨床に役立つ最新知見】結核・非結核性抗酸菌症の基礎研究 結核菌の薬剤耐性獲得
山口 智之, 中島 千絵, 鈴木 定彦
呼吸器ジャーナル, 66, 4, 650, 656, (株)医学書院, 2018年11月
日本語, ＜文献概要＞Point ・抗結核薬の作用機序とその耐性獲得機構は,各薬剤によって異なる.・薬剤感受性試験には,培養を用いる方法と遺伝子診断を応用した迅速検査法がある.・薬剤耐性結核の発生防止には,耐性化のメカニズムと各検査法の原理を理解することが重要である.

Genetic diversity and distribution dynamics of multidrug-resistant Mycobacterium tuberculosis isolates in Nepal.
Maharjan B, Nakajima C, Isoda N, Thapa J, Poudel A, Shah Y, Yamaguchi T, Shrestha B, Hoffmann H, Avsar K, Shrestha A, Gordon SV, Suzuki Y
Scientific reports, 8, 1, 16634, 16634, 2018年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Multidrug-resistant tuberculosis (MDR-TB) is an emerging public health problem in Nepal. Despite the implementation of a successful TB control program in Nepal, notifications of MDR-TB are increasing, yet the reasons are unknown. The objective of this study was to understand the genetic diversity and epidemiological characteristics of MDR-Mycobacterium tuberculosis (MTB) isolates in Nepal. We isolated and genotyped 498 MDR-MTB isolates collected from April 2009 to March 2013 and analyzed the patients' background information. Our results showed that the lineage 2 (Beijing family) was the most predominant lineage (n = 241; 48.4%), followed by lineage 3 (n = 153, 30.7%). Lineage 4 was the third most prevalent (n = 73, 14.5%) followed by lineage 1 (n = 32, 6.4%). The lineages were significantly associated with geographic region, ethnic group, age and sex of patients. The Beijing genotype was found to have an important role in transmitting MDR-TB in Nepal and was significantly associated with the eastern region, mongoloid ethnic group and younger age group. We conclude that early diagnosis and treatment including molecular-epidemiological surveillance of MDR-TB cases will help to control transmission of MDR-TB in Nepal.

A case of laboratory cross-contamination of Mycobacterium tuberculosis identified using comparative genomics.
Seto J, Wada T, Suzuki Y, Ikeda T, Araki K, Umetsu Y, Ishikawa H, Mizuta K, Ahiko T
The international journal of tuberculosis and lung disease, 22, 10, 1239, 1242, 2018年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Loop-mediated isothermal amplification for rapid identification of Mycobacterium tuberculosis in comparison with immunochromatographic SD Bioline MPT64 Rapid^® in a high burden setting.
Phetsuksiri B, Rudeeaneksin J, Srisungngam S, Bunchoo S, Klayut W, Sangkitporn S, Nakajima C, Hamada S, Suzuki Y
Japanese journal of infectious diseases, 72, 2, 112, 114, 2018年10月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Loop-mediated isothermal amplification (LAMP) was assessed for rapid identification of Mycobacterium tuberculosis complex (MTC) in comparison with an immunochromatographic test (ICT) using SD Bioline Ag MPT64 Rapid®. One hundred and fifty-one MGIT cultures positive for acid-fast bacilli were tested for MTC. DNA was extracted from a small portion of culture samples by heat lysis and subjected to LAMP analysis. Of these, 144 were positive and 5 were negative by both tests. One culture that was ICT negative but was LAMP positive was confirmed to have a mutation in the mpt64 gene. The agreement was 98.68% (95% confidence interval [CI]: 94.80-99.77), and the kappa value was 0.83% (95% CI: 0.59-1.00). Good correlation results suggested that LAMP assay is a reliable molecular test for rapid identification of MTC and is practical for use in resource-limited, high burden settings.

Multiclonal Expansion and High Prevalence of β-Lactamase-Negative Haemophilus influenzae with High-Level Ampicillin Resistance in Japan and Susceptibility to Quinolones.
Honda H, Sato T, Shinagawa M, Fukushima Y, Nakajima C, Suzuki Y, Shiraishi T, Kuronuma K, Takahashi S, Takahashi H, Yokota SI
Antimicrobial agents and chemotherapy, 62, 9, 2018年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, β-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of β-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were β-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

Establishment of a mouse-tick infection model for Theileria orientalis and analysis of its transcriptome.
Hayashida K, Umemiya-Shirafuji R, Sivakumar T, Yamagishi J, Suzuki Y, Sugimoto C, Yokoyama N
International journal for parasitology, 2018年08月, ［査読有り］

Whole genome analysis of a multidrug-resistant Streptococcus pneumoniae isolate from a patient with invasive pneumococcal infection developing disseminated intravascular coagulation.
Yasuo Ohkoshi, Toyotaka Sato, Takayuki Wada, Yukari Fukushima, Hiromi Murabayashi, Yasunari Takakuwa, Kaoru Nishiyama, Hiroyuki Honda, Tsukasa Shiraishi, Koji Kuronuma, Hiroki Takahashi, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 24, 8, 674, 681, 2018年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Multidrug-resistant Streptococcus pneumoniae strains were isolated from blood and sputum of a patient with disseminated intravascular coagulation in Sapporo city, Japan. These antibiograms were only susceptible to vancomycin, linezolid, daptomycin, some carbapenems, and some fluoroquinolones. Identical antibiograms, serotypes (19F), and sequence types (ST10017) suggested a shared origin of these isolates. Only one ST10017 strain has been isolated in the same city in Japan previously (2014), and the 2014 isolate is still susceptible to macrolides. The whole genome of the blood-derived isolate was sequenced. The strain harbored resistance mutations in parC, gyrA, pbp1a, pbp2a, pbp2b, and pbp2x, and harbored the resistance genes, ermB and tetM. The nucleotide sequences of parC and pbp2x genes of strain MDRSPN001 were clearly different from those of other S. pneumoniae strains and were similar to those of oral streptococci strains. These findings suggest that strain MDRSPN001 has been rapidly and drastically evolving multidrug resistance by gene replacement and accumulation of genes originating from other strains, such as oral streptococci, Streptococcus mitis.

Phylogenetic uniqueness of Mycobacterium avium subspecies hominissuis isolated from an abnormal pulmonary bovine case.
Shiomi Yoshida, Tsubasa Araki, Tomohito Asai, Kazunari Tsuyuguchi, Kentaro Arikawa, Tomotada Iwamoto, Chie Nakajima, Yasuhiko Suzuki, Kenji Ohya, Tokuma Yanai, Takayuki Wada, Taro Yamamoto
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 62, 122, 129, 2018年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium avium subspecies hominissuis (MAH) is an important cause of infection in human pulmonary and swine intestinal cases. Although MAH is isolated from environmental sources frequently, infections of other animals have rarely been analysed. Recently, we detected granulomatous inflammation in bovine lung as an abnormal postmortem inspection case. To ascertain its genetic profile, we conducted a variable numbers of tandem repeats (VNTR) analysis and genomic characterization using deep sequencing. The VNTR type was a unique profile that differed from reported genotypes, but it was assigned within a broad genotypic complex of isolates from human patients and bathrooms. Genomic comparison with 116 registered genome sequences of the subspecies revealed that the strain was separate from five major genetic population groups proposed previously. Although the infection source remains unclear, its isolation from various resources such as animal infection cases should be elucidated more extensively to reveal its genetic diversity and ecological context.

Characterization of mutations conferring streptomycin resistance to multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar
Nan Aye Thida Oo, Lai Lai San, Jeewan Thapa, Khin Saw Aye, Wah Wah Aung, Chie Nakajima, Yasuhiko Suzuki
Tuberculosis, 111, 8, 13, Churchill Livingstone, 2018年07月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Numerous studies report that mutations of rpsL (encoding the S12 protein), rrs (encoding 16S rRNA) and gidB (encoding rRNA methyltransferase) are responsible for conferring resistance to streptomycin (STR), which is usually used in both multidrug-resistant tuberculosis (MDR-TB) treatments and re-treatments in Myanmar. The aim of this study was to explore the variation and frequency of mutations in rpsL, rrs and gidB in 141 STR-resistant MDR-TB isolates from Myanmar. Most isolates belonged to the Beijing genotype (105, 74.5%). Moreover, mutations in rpsL were identified in 69.5% (98/141) of the STR-resistant isolates, where the most prevalent (92.0%, 90/98) and significantly associated mutation with the Beijing genotype (P <
0.001) was Lys43Arg. Fifteen different mutations in gidB were found in 16.3% (23/141) of the isolates, and most of them were novel mutations. Moreover, based on our results, we suggest A276C nucleotide substitution in gidB as a phylogenetic marker for the Beijing family in Myanmar. Sequence analysis of rpsL, rrs and gidB with a sensitivity of 83.7% satisfactorily predicted STR resistance in Myanmar isolates. However, in 16.3% (23/141) of the isolates, none of the examined genes showed mutation. Hence, further studies are strongly recommended to elucidate other possible resistance mechanisms. The present findings may be useful in developing molecular STR susceptibility assays, which in turn could contribute to develop TB treatments and control strategies in Myanmar.

Antibacterial Activity of DC-159a Against Salmonella Typhimurium.
Koide K, Kongsoi S, Ouchi Y, Yamaguchi T, Nakajima C, Suzuki Y
Microbial drug resistance (Larchmont, N.Y.), 25, 1, 14, 22, MARY ANN LIEBERT, INC, 2018年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Quinolones show excellent antibacterial activity against Salmonella isolates. Recently, however, quinolone resistance has been increasing in bacteria. This study aimed to examine in vitro, and compare the activity of DC-159a against Salmonella enterica serovar Typhimurium with that of ciprofloxacin and nalidixic acid. Inhibitory effects of quinolones were assessed by the drug concentration needed to inhibit the supercoiling activity of recombinant DNA gyrases by 50% (IC50). Dilution methods were used to determine the minimum inhibitory concentration (MIC) of quinolones against two different strains, Salmonella Typhimurium and Salmonella Enteritidis. The IC(50)s of DC-159a against mutant DNA gyrases were much lower than those of nalidixic acid and ciprofloxacin. In particular, the IC50 of DC-159a against DNA gyrase with double mutation was less than 1/50 that of ciprofloxacin and nalidixic acid. MICs of DC-159a were higher than those of ciprofloxacin but lower than those of nalidixic acid. However, the estimated MICs of DC-159a against Salmonella strains with mutant DNA gyrase were lower than those of ciprofloxacin and nalidixic acid. Therefore, DC-159a can be suggested as an antibiotic candidate for treating salmonellosis caused by quinolone-resistant S. Typhimurium.

Cooperation of PD-1 and LAG-3 in the exhaustion of CD4+ and CD8+ T cells during bovine leukemia virus infection.
Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Junko Kohara, Yasuhiko Suzuki, Shinji Yamada, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
Veterinary research, 49, 1, 50, 50, 2018年06月19日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of PD-1+LAG-3+ heavily exhausted T cells among CD4+ and CD8+ T cells was higher in the blood of cattle with B-cell lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1 and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3 cooperatively mediate the functional exhaustion of CD4+ and CD8+ T cells and are associated with the development of B-cell lymphoma in BLV-infected cattle.

Insight into multidrug-resistant Beijing genotype Mycobacterium tuberculosis isolates in Myanmar.
San LL, Aye KS, Oo NAT, Shwe MM, Fukushima Y, Gordon SV, Suzuki Y, Nakajima C
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 76, 109, 119, 2018年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: Myanmar is a World Health Organization high tuberculosis (TB) burden country with a high multidrug-resistant (MDR)-TB burden. Of significance, a high prevalence of the Beijing genotype of Mycobacterium tuberculosis (MTB) among MDR-MTB has been reported previously. A detailed genetic characterization of TB clinical isolates was performed in order to explore whether there is an association between the prevalence of the Beijing MTB genotype and MDR-TB in Myanmar. METHODS: A total of 265 MDR-MTB clinical isolates collected in 2010 and 2012 were subjected to spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis, single nucleotide polymorphism (SNP) typing, and drug resistance-associated gene sequencing, including rpoC to detect potential compensatory evolution. RESULTS: Of the total MDR-MTB isolates, 79.2% (210/265) were of the Beijing genotype, the majority of which were the 'modern' subtype. Beijing genotype isolates were differentiated by 15-locus MIRU-VNTR and a high clustering rate (53.0%) was observed in the modern subtype. These MIRU-VNTR patterns were similar to Beijing genotype clones spreading across Russia and Central Asia. A high prevalence of katG Ser315Thr, and genetic evidence of extensive drug resistance (XDR) and pre-XDR and compensatory mutations in rpoC were observed among clustered isolates. CONCLUSIONS: MDR-MTB strains of the Beijing genotype might be spreading in Myanmar and present a major challenge to TB control in this country.

Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease.
Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Reiko Nagata, Satoko Kawaji, Yumiko Kagawa, Shinji Yamada, Yukinari Kato, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
Infection and immunity, 86, 5, 2018年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.

Genotypic characterization of pyrazinamide resistance in Mycobacterium tuberculosis isolated from Lusaka, Zambia
Precious Bwalya, Tomoyuki Yamaguchi, Georgina Mulundu, Chie Nakajima, Grace Mbulo, Eddie Samuneti Solo, Yukari Fukushima, Kunda Kasakwa, Yasuhiko Suzuki
Tuberculosis, 109, 117, 122, Churchill Livingstone, 2018年03月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Pyrazinamide forms a core part of treatment for all types of tuberculosis (TB) in Zambia. Due to challenges associated with pyrazinamide testing, little information is available to indicate the frequency of resistance to this drug in Zambia. To determine the frequency of pyrazinamide (PZA) resistance and its correlation with mutation in pncA in Mycobacterium tuberculosis isolated from patients in Lusaka, Zambia, BACTEC MGIT M960 was used for phenotypic PZA susceptibility testing while sequencing was used to determine resistance-conferring mutations in the pncA. Of the 131 isolates analyzed, 32 were phenotypically resistant to PZA. Among multidrug-resistant (MDR) M. tuberculosis isolates, the frequency of PZA resistance was 21 of 35 (58.3%). And 27 of 32 PZA resistant isolates had mutations in the pncA that seem to confer resistance. With BACTEC MGIT 960 as the reference standard, gene sequencing showed 84.4% sensitivity and 100% specificity. Nine new mutations were identified and the single nucleotide substitution T104G and C195T were the most frequent mutations. However, they were observed in both susceptible and resistant strains and indicating that they are non-resistance conferring mutations. This study has demonstrated that PZA susceptibility testing is necessary especially in patients suffering from MDR-TB as approximately half of the patients have PZA resistant TB. Similar studies will have to be carried out in other provinces to get an accurate estimate of PZA resistance in Zambia. Mutations in pncA were the major mechanism of PZA resistance with no involvement of rpsA and panD genes. However, the presence of mutations among phenotypically PZA susceptible M. tuberculosis isolates makes it challenging to independently use genotyping method for the determination of PZA resistance.

Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia
Z. Rahim, J. Thapa, Y. Fukushima, A. G. M. van der Zanden, S. V. Gordon, Y. Suzuki, C. Nakajima
TRANSBOUNDARY AND EMERGING DISEASES, 64, 6, 1965, 1969, WILEY, 2017年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M.orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M.orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M.orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M.orygis in South Asia.

Complete Genome Sequence of Multidrug-Resistant Streptococcus pneumoniae Serotype 19F Isolated from an Invasive Infection in Sapporo, Japan.
Toyotaka Sato, Yasuo Ohkoshi, Takayuki Wada, Yukari Fukushima, Hiromi Murabayashi, Yasunari Takakuwa, Kaoru Nishiyama, Tsukasa Shiraishi, Chie Nakajima, Yasuhiko Suzuki, Shin-Ichi Yokota
Genome announcements, 5, 44, 2017年11月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Invasive infection of multidrug-resistant Streptococcus pneumoniae is a serious clinical concern. Here, we report the complete genome sequence of a multidrug-resistant S. pneumoniae serotype 19F strain isolated from a patient with an invasive infection in Sapporo, Japan.

Quinolone Resistance Determinants of Clinical Salmonella Enteritidis in Thailand
Fuangfa Utrarachkij, Chie Nakajima, Ruchirada Changkwanyeun, Kanokrat Siripanichgon, Siriporn Kongsoi, Srirat Pornruangwong, Kanjana Changkaew, Risa Tsunoda, Yutaka Tamura, Orasa Suthienkul, Yasuhiko Suzuki
MICROBIAL DRUG RESISTANCE, 23, 7, 885, 894, MARY ANN LIEBERT, INC, 2017年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Salmonella Enteritidis has emerged as a global concern regarding quinolone resistance and invasive potential. Although quinolone-resistant S. Enteritidis has been observed with high frequency in Thailand, information on the mechanism of resistance acquisition is limited. To elucidate the mechanism, a total of 158 clinical isolates of nalidixic acid (NAL)-resistant S. Enteritidis were collected throughout Thailand, and the quinolone resistance determinants were investigated in the context of resistance levels to NAL, norfloxacin (NOR), and ciprofloxacin (CIP). The analysis of point mutations in type II topoisomerase genes and the detection of plasmid-mediated quinolone resistance genes showed that all but two harbored a gyrA mutation, the qnrS1 gene, or both. The most commonly affected codon in mutant gyrA was 87, followed by 83. Double codon mutation in gyrA was found in an isolate with high-level resistance to NAL, NOR, and CIP. A new mutation causing serine to isoleucine substitution at codon 83 was identified in eight isolates. In addition to eighteen qnrS1-carrying isolates showing nontypical quinolone resistance, one carrying both the qnrS1 gene and a gyrA mutation also showed a high level of resistance. Genotyping by multilocus variable number of tandem repeat analysis suggested a possible clonal expansion of NAL-resistant strains nationwide. Our data suggested that NAL-resistant isolates with single quinolone resistance determinant may potentially become fluoroquinolone resistant by acquiring secondary determinants. Restricted therapeutic and farming usage of quinolones is strongly recommended to prevent the emergence of fluoroquinolone-resistant isolates.

High diversity of multidrug-resistant Mycobacterium tuberculosis Central Asian Strain isolates in Nepal
Yogendra Shah, Bhagwan Maharjan, Jeewan Thapa, Ajay Poudel, Hassan Mahmoud Diab, Basu Dev Pandey, Eddie S. Solo, Norikazu Isoda, Yasuhiko Suzuki, Chie Nakajima
INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 63, 13, 20, ELSEVIER SCI LTD, 2017年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Objectives: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) poses a major public health problem in Nepal. Although it has been reported as one of the dominant genotypes of MTB in Nepal, little information on the Central Asian Strain (CAS) family is available, especially isolates related to multidrug resistance (MDR) cases. This study aimed to elucidate the genetic and epidemiological characteristics of MDR CAS isolates in Nepal.
Methods: A total of 145 MDR CAS isolates collected in Nepal from 2008 to 2013 were characterized by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis, and drug resistance-associated gene sequencing.
Results: Spoligotyping analysis showed CAS1_Delhi SIT26 as predominant (60/145, 41.4%). However, by combining spoligotyping and MIRU-VNTR typing, it was possible to successfully discriminate all 145 isolates into 116 different types including 18 clusters with 47 isolates (clustering rate 32.4%). About a half of these clustered isolates shared the same genetic and geographical characteristics with other isolates in each cluster, and some of them shared rare point mutations in rpoB that are thought to be associated with rifampicin resistance.
Conclusions: Although the data obtained show little evidence that large outbreaks of MDR-TB caused by the CAS family have occurred in Nepal, they strongly suggest several MDR-MTB transmission cases. (C) 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

ANTIMICROBIAL RESISTANCE, INTEGRON, VIRULENCE GENE, AND MULTILOCUS SEQUENCE TYPING OF &ITESCHERICHIA COLI&IT ISOLATES FROM POSTWEANING PIGLETS WITH AND WITHOUT DIARRHEA
Watsawan Prapasawat, Apiradee Intarapuk, Pornthip Chompook, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
SOUTHEAST ASIAN JOURNAL OF TROPICAL MEDICINE AND PUBLIC HEALTH, 48, 5, 1042, 1055, SOUTHEAST ASIAN MINISTERS EDUC ORGANIZATION, 2017年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Pathogenic Escherichia coli is a major cause of diarrhea in postweaning piglets. Virulence genes, antimicrobial resistance, integrons, and genetic diversity of E. coli were determined in 100 rectal swab samples collected from postweaning piglets with and without diarrhea (5-7 weeks of age) in a farm in a central province of Thailand. Of 246 E. coli isolates, 141 were positive for at least one virulence gene determined by multiplex PCR, the most commonly found from both groups of piglets being astA, while It, F4, F18, and F41 only from diarrheal piglets. More than 80% of E. coli isolates were resistant to 7 of 12 antimicrobial agents. One hundred and fifty-seven E. coli isolates carried class 1 and/or 2 integron(s). Integron-positive isolates are significantly associated with strains resistant to kanamycin, oxytetracycline, streptomycin, sulfamethoxazole/trimethoprim and tetracycline. Phylogenetic analysis by multilocus sequence typing revealed that the 31 representative E. coli isolates were genetically diverse, especially those from diarrheal piglets suggesting that E. coli from postweaning piglets were not derived from a single clone. Sequence type (ST)10, ST641 and ST1114 were most commonly found in both groups of piglets. No correlation was observed among ST, presence of integron and antimicrobial resistance. The study suggests that swines in a farm could be a reservoir and possible spread of diarrheagenic E. coli including strains with antimicrobial resistance genes.

Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis.
Goto S, Konnai S, Okagawa T, Nishimori A, Maekawa N, Gondaira S, Higuchi H, Koiwa M, Tajima M, Kohara J, Ogasawara S, Kato Y, Suzuki Y, Murata S, Ohashi K
Immunity, inflammation and disease, 5, 3, 355, 363, 2017年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.

Population Structure and Local Adaptation of MAC Lung Disease Agent Mycobacterium avium subsp hominissuis
Hirokazu Yano, Tomotada Lwamoto, Yukiko Nishiuchi, Chie Nakajima, Daria A. Starkova, Igor Mokrousov, Olga Narvskaya, Shiomi Yoshida, Kentaro Arikawa, Noriko Nakanishi, Ken Osaki, Ichiro Nakagawa, Manabu Ato, Yasuhiko Suzuki, Fumito Maruyama
GENOME BIOLOGY AND EVOLUTION, 9, 9, 2403, 2417, OXFORD UNIV PRESS, 2017年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial species responsible for chronic lung disease in humans. Despite increasing worldwide incidence, little is known about the genetic mechanisms behind the population evolution of MAH. To elucidate the local adaptation mechanisms of MAH, we assessed genetic population structure, the mutual homologous recombination, and gene content for 36 global MAH isolates, including 12 Japanese isolates sequenced in the present study. We identified five major MAH lineages and found that extensive mutual homologous recombination occurs among them. Two lineages (MahEastAsia1 and MahEastAsia2) were predominant in the Japanese isolates. We identified alleles unique to these two East Asian lineages in the loci responsible for trehalose biosynthesis (treS and mak) and in one mammalian cell entry operon, which presumably originated from as yet undiscovered mycobacterial lineages. Several genes and alleles unique to East Asian strains were located in the fragments introduced via recombination between East Asian lineages, suggesting implication of recombination in local adaptation. These patterns of MAH genomes are consistent with the signature of distribution conjugative transfer, a mode of sexual reproduction reported for other mycobacterial species.

Convenient PCR method for variable-number tandem-repeat typing of Mycobacterium tuberculosis clinical isolates.
Seto J, Wada T, Suzuki Y, Ikeda T, Mizuta K, Mitarai S, Ahiko T
Journal of microbiological methods, 139, 12, 14, 2017年08月, ［査読有り］
英語, 研究論文（学術雑誌）

A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma
Naoya Maekawa, Satoru Konnai, Satoshi Takagi, Yumiko Kagawa, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Tatsuya Deguchi, Chie Nakajima, Yukinari Kato, Keiichi Yamamoto, Hidetoshi Uemura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
SCIENTIFIC REPORTS, 7, 1, 8951, 8951, NATURE PUBLISHING GROUP, 2017年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.

A quantitative and efficient approach to select MIRU-VNTR loci based on accumulation of the percentage differences of strains for discriminating divergent Mycobacterium tuberculosis sublineages
Xin-Ling Pan, Chun-Lei Zhang, Chie Nakajima, Jin Fu, Chang-Xia Shao, Li-Na Zhao, Jia-Yi Cui, Na Jiao, Chang-Long Fan, Yasuhiko Suzuki, Toshio Hattori, Di Li, Hong Ling
EMERGING MICROBES & INFECTIONS, 6, 7, e68, NATURE PUBLISHING GROUP, 2017年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Although several optimal mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) loci have been suggested for genotyping homogenous Mycobacterium tuberculosis, including the Beijing genotype, a more efficient and convenient selection strategy for identifying optimal VNTR loci is needed. Here 281 M. tuberculosis isolates were analyzed. Beijing genotype and non-Beijing genotypes were identified, as well as Beijing sublineages, according to single nucleotide polymorphisms. A total of 22 MIRU-VNTR loci were used for genotyping. To efficiently select optimal MIRU-VNTR loci, we established accumulations of percentage differences (APDs) between the strains among the different genotypes. In addition, we constructed a minimum spanning tree for clustering analysis of the VNTR profiles. Our findings showed that eight MIRU-VNTR loci displayed disparities in h values of >= 0.2 between the Beijing genotype and non-Beijing genotype isolates. To efficiently discriminate Beijing and non-Beijing genotypes, an optimal VNTR set was established by adding loci with APDs ranging from 87.2% to 58.8%, resulting in the construction of a nine-locus set. We also found that QUB11a is a powerful locus for separating ST10s (including ST10, STF and STCH1) and ST22s (including ST22 and ST8) strains, whereas a combination of QUB11a, QUB4156, QUB18, Mtub21 and QUB26 could efficiently discriminate Beijing sublineages. Our findings suggested that two nine-locus sets were not only efficient for distinguishing the Beijing genotype from non-Beijing genotype strains, but were also suitable for sublineage genotyping with different discriminatory powers. These results indicate that APD represents a quantitative and efficient approach for selecting MIRU-VNTR loci to discriminate between divergent M. tuberculosis sublineages.

Evaluation of in-house loop-mediated isothermal amplification for tuberculosis diagnosis in comparison with Xpert MTB/RIF
Habeenzu C, Nakajima C, Solo E, Bwalya P, Kajino, K, Miller M, Kurosawa Y, Mudenda V, Kasonka L, Suzuki Y, Matsuba T
Journal of Infection in Developing Countries, 11, 6, 440, 444, 2017年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: To evaluate the diagnostic performances of an in-house loop-mediated isothermal amplification (LAMP) kit and the Xpert MTB/RIF test for the diagnosis of pulmonary tuberculosis in a resource-limited setting, this study was performed at the University Teaching Hospital, Ministry of Health, the Republic of Zambia. METHODOLOGY: Two hundred sputum specimens obtained from new tuberculosis (TB) suspects were used for the evaluation of the diagnostic performance of an in-house LAMP kit in comparison with the Xpert MTB/RIF kit. RESULTS: The sensitivity of in-house LAMP and Xpert MTB/RIF was 96.9% and 95.4% in smear-positive samples, 96.8% and 100% in smear-positive/culture-positive samples, and 39.1% and 73.9% in smear-negative/culture-positive samples, respectively. The specificity of in-house LAMP and MTB/RIF kits with culture was 96.5% and 94.5%, respectively. This indicated the superiority of the Xpert MTB/RIF kit; however, mechanical errors during sample processing and the insufficient quantity of samples by Xpert MTB/RIF kit occurred at 2.0% and 19.7%, respectively, comparing to the 100% accessibility of in-house LAMP. CONCLUSIONS: Considering the results obtained in this study together with the easy setup with much simpler equipment, such as an aluminum heat block or water bath, in in-house LAMP compared with real-time polymerase chain reaction equipment in Xpert MTB/RIF kit, the applicability of in-house LAMP for the screening of tuberculosis directly from sputum in resource-limited setting seemed to be high.

Anti-Bovine Programmed Death-1 Rat-Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle
Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Chie Nakajima, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
FRONTIERS IN IMMUNOLOGY, 8, 650, 650, FRONTIERS MEDIA SA, 2017年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat-bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4(+) T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.

Comparison of antibody responses against Mycobacterium tuberculosis antigen Rv0679c in tuberculosis patients from the endemic and non-endemic regions of the Beijing genotype: a case control study
Jingge Zhao, Takashi Matsuba, Xiaoyan Zhang, Susan Leano, Chie Nakajima, Haorile Chagan-Yasutan, Elizabeth Freda Telan, Yasuhiko Suzuki, Toshio Hattori
BMC INFECTIOUS DISEASES, 17, 1, 344, 344, BIOMED CENTRAL LTD, 2017年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Strains of the Beijing genotype of Mycobacterium tuberculosis (MTB) are reportedly associated with the virulence of tuberculosis (TB) infection, unfavorable outcomes of anti-TB treatment, and the global TB pandemic. Rv0679c, a hypothetical membrane protein related to host cell invasion, has a Beijing genotype-specific mutation at residue 142 (Asn142Lys). Antigenicity differences between Rv0679c-Asn142 (N-type) and Rv0679c-Lys142 (K-type) have been previously observed in mice antigen-antibody responses. However, the immune response to Rv0679c in humans remains unknown. Therefore, we aimed to investigate the anti-Rv0679c immune response in TB patients from the endemic and non-endemic regions of the Beijing MTB genotype.
Methods: We analyzed the Rv0679c-specific antibody responses in 84 subjects from the endemic region of the Beijing genotype MTB in China, including 45 pulmonary TB patients (C-PTB) and 39 healthy controls (C-HC), and 81 subjects from the Philippines (the endemic region of the non-Beijing genotype), including 51 pulmonary TB patients (P-PTB) and 30 healthy controls (P-HC). Anti-tuberculous-glycolipid (TBGL) antigen was used as the control antibody.
Results: TBGL IgG titers were higher in both C-PTB and P-PTB than those in their corresponding HC (C-PTB median 4.2, P-PTB median 11.2; C-PTB vs. P-PTB, p > 0.05), suggesting immune response comparability in PTB from two different countries. C-PTB showed a higher response compared to C-HC for anti-K-type IgG (53.3%) than anti-N-type IgG (6.67%); this response was not observed in P-PTB (both N-type and K-type 9.80%).
Conclusion: Dimorphic antigen Rv0679c was found to be associated with distinct immune response patterns, indicating the role of Beijing/non-Beijing genotype of MTB in stimulating specific responses in TB patients from the endemic region of Beijing MTB. Meanwhile, reactions to Rv0679c in patients and HC from non-endemic regions of the Beijing MTB may be caused by the response to the common epitope of Rv0679c N/K-type.

In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) ratbovine chimeric antibody against bovine leukemia virus infection
Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Yamato Sajiki, Yasuhiko Suzuki, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
PLOS ONE, 12, 4, e0174916, PUBLIC LIBRARY SCIENCE, 2017年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM(+) B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4(+) T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.

Survival and prevalence of Clostridium difficile in manure compost derived from pigs
Masaru Usui, Mayuko Kawakura, Nobuki Yoshizawa, Lai Lai San, Chie Nakajima, Yasuhiko Suzuki, Yutaka Tamura
ANAEROBE, 43, 15, 20, ELSEVIER SCI LTD, 2017年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Pigs, particularly piglets, have been identified as reservoir hosts of Clostridium difficile. To examine the survival ability of this pathogen in pig feces-based manure compost, C. difficile spores, which were prepared to contain as few vegetative cells as possible, were artificially inoculated into pig feces and incubated at different temperatures. While C. difficile survived in the feces incubated at temperatures below 37 C for over 30 days, cell numbers gradually decreased at thermophilic temperatures (over 55 C; p < 0.05). Next, to clarify the prevalence of C difficile in field manure compost, we isolated and characterized C difficile from the final products of manure compost products of 14 pig farms. A total of 11 C. difficile strains were isolated from 5 of 14 (36% positive rate) samples tested. Of these 11 strains, 82% were toxigenic, with ribotype 078 being the most prevalent. Thus, the application of composted manure to land therefore poses a possible risk of C. difficile transfer to the food chain. (C) 2016 Published by Elsevier Ltd.

Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis.　　　　　　　　　　　　　　　
Goto, S, Konnai, S, Okagawa, T, Nishimori, A, Maekawa, N, Gondaira, S, Higuchi, H, Koiwa, M, Tajima, M, Kohara, J, Ogasawara, S, Kato, Y, Suzuki, Y, Murata, S, Ohashi, K
Immunity, Inflammation and Disease, 10, 1, 9, 2017年, ［査読有り］
英語, 研究論文（学術雑誌）

Genotypic diversity of Mycobacterium tuberculosis strains in Myanmar
Thanda Tun, Khin Saw Aye, Wint Wint Nyunt, John A. Crump, Chie Nakajima, Yasuhiko Suzuki, Kyi Kyi Thinn, Gregory M. Cook, Htin Lin Aung
INFECTIOUS DISEASES, 49, 3, 237, 239, TAYLOR & FRANCIS LTD, 2017年, ［査読有り］, ［国際誌］
英語

Immunological Roles of Elevated Plasma Levels of Matricellular Proteins in Japanese Patients with Pulmonary Tuberculosis
Beata Shiratori, Jingge Zhao, Masao Okumura, Haorile Chagan-Yasutan, Hideki Yanai, Kazue Mizuno, Takashi Yoshiyama, Tadashi Idei, Yugo Ashino, Chie Nakajima, Yasuhiko Suzuki, Toshio Hattori
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 18, 1, MDPI AG, 2017年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Elevatedmatricellular proteins (MCPs), including osteopontin (OPN) and galectin-9 (Gal-9), were observed in the plasma of patients with Manila-type tuberculosis (TB) previously. Here, we quantified plasma OPN, Gal-9, and soluble CD44 (sCD44) by enzyme-linked immunosorbent assay (ELISA), and another 29 cytokines by Luminex assay in 36 patients with pulmonary TB, six subjects with latent tuberculosis (LTBI), and 19 healthy controls (HCs) from Japan for a better understanding of the roles of MCPs in TB. All TB subjects showed positive results of enzyme-linked immunospot assays (ELISPOTs). Spoligotyping showed that 20 out of 36 Mycobacterium tuberculosis (MTB) strains belong to the Beijing type. The levels of OPN, Gal-9, and sCD44 were higher in TB (positivity of 61.1%, 66.7%, and 63.9%, respectively) than in the HCs. Positive correlations between OPN and Gal-9, between OPN and sCD44, and negative correlation between OPN and ESAT-6-ELISPOT response, between chest X-ray severity score of cavitary TB and ESAT-6-ELISPOT response were observed. Instead of OPN, Gal-9, and sCD44, cytokines G-CSF, GM-CSF, IFN-alpha, IFN-gamma, IL-12p70, and IL-1RA levels were higher in Beijing MTB-infected patients. These findings suggest immunoregulatory, rather than inflammatory, effect of MCPs and can advance the understanding of the roles of MCPs in the context of TB pathology.

Collectin Kidney 1 Plays an Important Role in Innate Immunity against Streptococcus pneumoniae Infection
Insu Hwang, Kenichiro Mori, Katsuki Ohtani, Yasuyuki Matsuda, Nitai Roy, YounUck Kim, Yasuhiko Suzuki, Nobutaka Wakamiya
JOURNAL OF INNATE IMMUNITY, 9, 2, 217, 228, KARGER, 2017年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1(-/-)) mice and then performed a Streptococcus pneumoniae infection analysis. First, we found that recombinant human CL-K1 bound to S. pneumoniae in a calcium-dependent manner, and induced complement activation. CL-K1(-/-) mice sera formed less C3 deposition on S. pneumoniae. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on S. pneumoniae in infected lungs. CL-K1(-/-) mice revealed decreased phagocytosis of S. pneumoniae. Consequently, less S. pneumoniae clearance was observed in their lungs. CL-K1(-/-) mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by S. pneumoniae infection might cause higher CL-K1(-/-) mice lethality. Our results suggest that CL-K1 might play an important role in host protection against S. pneumoniae infection through the activation of the lectin complement pathway. (C) 2017 S. Karger AG, Basel

Difference in Antibody Responses to Mycobacterium tuberculosis Antigens in Japanese Tuberculosis Patients Infected with the Beijing/Non-Beijing Genotype
Jingge Zhao, Beata Shiratori, Masao Okumura, Hideki Yanai, Makoto Matsumoto, Chie Nakajima, Kazue Mizuno, Kenji Ono, Tetsuya Oda, Haorile Chagan-Yasutan, Yugo Ashino, Takashi Matsuba, Takashi Yoshiyama, Yasuhiko Suzuki, Toshio Hattori
JOURNAL OF IMMUNOLOGY RESEARCH, 2017, 4797856, 4797856, HINDAWI LTD, 2017年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The Beijing genotype Mycobacterium tuberculosis (MTB), notorious for its virulence and predisposition to relapse, could be identified by spoligotyping based on genetic heterogeneity. The plasma samples from 20 cases of Beijing and 16 cases of non-Beijing MTB infected individuals and 24 healthy controls (HCs) were collected, and antibodies against 11 antigens (Rv0679c142Asn, Rv0679c142Lys, Ag85B, Ag85A, ARC, TDM-M, TDM-K, HBHA, MDP-1, LAM, and TBGL) were measured by ELISA. Compared to the HCs, the MTB infected subjects showed higher titers of anti-Ag85B IgG (positivity 58.2%) and anti-ACR IgG (positivity 48.2%). Of note, anti-ACR IgG showed higher titer in Beijing MTB infected tuberculosis (TB) patients than in HC (Kruskal-Wallis test, p < 0.05), while the levels of anti-Ag85B, anti-TBGL, anti-TDM-K, and anti-TDM-M IgG were higher in non-Beijing TB patients than in HC. Moreover, anti-Ag85B IgG showed higher response in non-Beijing TB patients than in Beijing TB patients (p < 0.05; sensitivity, 76.9% versus 44.4%). The sensitivity and specificity analysis showed that 78.8% Beijing infected individuals were negative in anti-TBGL-IgG or/and anti-Ag85B-IgG, while 75.0% of those were positive in anti-TBGL-IgA or/and anti-ACR-IgG tests. These results indicate the possibility of developing antibody-based test to identify Beijing MTB.

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase
Tomoyuki Yamaguchi, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 81, 7, 1343, 1347, TAYLOR & FRANCIS LTD, 2017年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

A PCR-based survey of animal African trypanosomosis and selected piroplasm parasites of cattle and goats in Zambia
Simon Peter Musinguzi, Keisuke Suganuma, Masahito Asada, Dusit Laohasinnarong, Thillaiampalam Sivakumar, Naoaki Yokoyama, Boniface Namangala, Chihiro Sugimoto, Yasuhiko Suzuki, Xuenan Xuan, Noboru Inoue
JOURNAL OF VETERINARY MEDICAL SCIENCE, 78, 12, 1819, 1824, JAPAN SOC VET SCI, 2016年12月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, We screened cattle and goats from the districts of Chama, Monze and Mumbwa in Zambia for animal African trypanosomes, Babesia bigemina and Theileria parva using PCRs; 38.1% of the samples tested positive for at least one of the parasite species. The most common parasite was Trypanosoma vivax (19.8%). Its incidence was significantly higher in goats than in cattle, (P<0.05). B. bigemina was found in samples from all the three areas, making it the most widespread of the parasites in Zambia. Among the tested samples, 12.0% of the positive samples were mixed infections. There were significant differences in the infection rates of T. vivax (Mumbwa had a significantly higher infection rate [39.6%, P<0.0001]), Th. parva (Monze had the only cases [P<0.0004]) and B. bigemina (Monze had a significantly higher infection rate [40.5%, P<0.0001]). According to the hematocrit values, the packed cell volume (%) among the cattle with mixed infections was significantly lower than that of the other cattle. The presence of multiple parasite species and mixed infections among the Zambian cattle and goat populations is of both clinical and economic importance to livestock farming. The absence of trypanosomosis among the samples from Monze can be attributed to tsetse eradication efforts that took place around Lake Kariba. This shows that the prevention and control of these parasitic diseases can have a significant impact on the disease status, which can translate directly into the improvement of the livestock sector in Zambia.

Molecular epidemiology of pathogenic Leptospira spp. among large ruminants in the Philippines
Marvin A. Villanueva, Claro N. Mingala, Michelle M. Balbin, Chie Nakajima, Norikazu Isoda, Yasuhiko Suzuki, Nobuo Koizumi
JOURNAL OF VETERINARY MEDICAL SCIENCE, 78, 11, 1649, 1655, JAPAN SOC VET SCI, 2016年11月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The extent of Leptospira infection in large ruminants resulting to economic problems in livestock industry in a leptospirosis-endemic country like the Philippines has not been extensively explored. Therefore, we determined the prevalence and carrier status of leptospirosis in large ruminants using molecular techniques and assessed the risk factors of acquiring leptospirosis in these animals. Water buffalo and cattle urine samples (n=831) collected from 21 farms during 2013-2015 were subjected to flaB-nested PCR to detect pathogenic Leptospira spp. Leptospiral flaB was detected in both species with a detection rate of 16.1%. Leptospiral DNA was detected only in samples from animals managed in communal farms. Sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri. One farm contained Leptospira flaB sequences from all clusters identified in this study, suggesting this farm was the main source of leptospires for other farms. This study suggested that these large ruminants are infected with various pathogenic Leptospira species causing possible major economic loss in the livestock industry as well as potential Leptospira reservoirs that can transmit infection to humans and other animals in the Philippines.

Amino acid substitutions in GyrA affect quinolone susceptibility in Salmonella typhimurium
Siriporn Kongsoi, Ruchirada Changkwanyeun, Kazumasa Yokoyama, Chie Nakajima, Kanjana Changkaew, Orasa Suthienkul, Yasuhiko Suzuki
DRUG TESTING AND ANALYSIS, 8, 10, 1065, 1070, WILEY-BLACKWELL, 2016年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The prevalence of quinolone-resistant Salmonella has become a public health concern. Amino acid substitutions have generally been found within the quinolone resistance-determining region in subunit A of DNA gyrase (GyrA) of Salmonella Typhimurium. However, direct evidence of the contribution of these substitutions to quinolone resistance remains to be shown. To investigate the significance of amino acid substitutions in S. Typhimurium GyrA to quinolone resistance, we expressed recombinant wild-type (WT) and five mutant DNA gyrases in Escherichia coli and characterized them in vitro. WT and mutant DNA gyrases were reconstituted in vitro by mixing recombinant subunits A and B of DNA gyrase. The correlation between the amino acid substitutions and resistance to quinolones ciprofloxacin, levofloxacin, nalidixic acid, and sitafloxacin was assessed by quinolone-inhibited supercoiling assays. All mutant DNA gyrases showed reduced susceptibility to all quinolones when compared with WT DNA gyrases. DNA gyrase with a double amino acid substitution in GyrA, serine to phenylalanine at codon 83 and aspartic acid to asparagine at 87 (GyrA-S83F-D87N), exhibited the lowest quinolone susceptibility amongst all mutant DNA gyrases. The effectiveness of sitafloxacin was shown by the low inhibitory concentration required for mutant DNA gyrases, including the DNA gyrase with GyrA-S83F-D87N. We suggest sitafloxacin as a candidate drug for the treatment of salmonellosis caused by ciprofloxacin-resistant S. Typhimurium. Copyright (C) 2015 John Wiley & Sons, Ltd.

Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni
Ruchirada Changkwanyeun, Tomoyuki Yamaguchi, Siriporn Kongsoi, Kanjana Changkaew, Kazumasa Yokoyama, Hyun Kim, Orasa Suthienkul, Masaru Usui, Yutaka Tamura, Chie Nakajima, Yasuhiko Suzuki
DRUG TESTING AND ANALYSIS, 8, 10, 1071, 1076, WILEY, 2016年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright (C) 2016 John Wiley & Sons, Ltd.

Urine Levels of Defensin α1 Reflect Kidney Injury in Leptospirosis Patients.
Chagan-Yasutan H, Chen Y, Lacuesta TL, Leano PS, Iwasaki H, Hanan F, Taurustiati D, Ohmoto Y, Ashino Y, Saitoh H, Kiyomoto H, Suzuki Y, Telan FO, Hattori T
International journal of molecular sciences, 17, 10, MDPI AG, 2016年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Leptospirosis is a zoonotic disease whose severe forms are often accompanied by kidney dysfunction. In the present study, urinary markers were studied for potential prediction of disease severity. Urine samples from 135 patients with or without leptospirosis at San Lazaro Hospital, the Philippines, were analyzed. Urine levels of defensin alpha 1 (uDA1) were compared with those of neutrophil gelatinase-associated lipocalin (uNGAL) and N-acetyl-beta-D-glucosidase (uNAG). Serum creatinine (Cr) was used as a marker of kidney injury. The levels of uDA1/Cr, uNGAL/Cr, and uNAG/Cr were positive in 46%, 90%, and 80% of leptospirosis patients, and 69%, 70%, and 70% of non-leptospirosis patients, respectively. In leptospirosis patients, the correlation of uDA1/Cr, uNGAL/Cr and uNAG/Cr levels with serum Cr were r = 0.3 (p < 0.01), r = 0.29 (p < 0.01), and r = 0.02 (p = 0.81), respectively. uDA1/Cr levels were correlated with uNGAL/Cr levels (r = 0.49, p < 0.01) and uNAG/Cr levels (r = 0.47, p < 0.0001) in leptospirosis patients. These findings suggest that uDA1, uNGAL, and uNAG were elevated in leptospirosis patients and reflected various types of kidney damage. uDA1 and uNGAL can be used to track kidney injury in leptospirosis patients because of their correlation with the serum Cr level.

DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae
Tomoyuki Yamaguchi, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki
PLoS Neglected Tropical Diseases, 10, 9, e0005013, Public Library of Science, 2016年09月28日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Fluoroquinolones are a class of antibacterial agents used for leprosy treatment. Some new fluoroquinolones have been attracting interest due to their remarkable potency that is reportedly better than that of ofloxacin, the fluoroquinolone currently recommended for treatment of leprosy. For example, DC-159a, a recently developed 8-methoxy fluoroquinolone, has been found to be highly potent against various bacterial species. Nonetheless, the efficacy of DC-159a against Mycobacterium leprae is yet to be examined. Methodology/Principal Findings: To gather data that can support highly effective fluoroquinolones as candidates for new remedies for leprosy treatment, we conducted in vitro assays to assess and compare the inhibitory activities of DC-159a and two fluoroquinolones that are already known to be more effective against M. leprae than ofloxacin. The fluoroquinolone-inhibited DNA supercoiling assay using recombinant DNA gyrases of wild type and ofloxacin-resistant M. leprae revealed that inhibitory activities of DC-159a and sitafloxacin were at most 9.8- and 11.9-fold higher than moxifloxacin. Also the fluoroquinolone–mediated cleavage assay showed that potencies of those drugs were at most 13.5- and 9.8-fold higher than moxifloxacin. In addition, these two drugs retained their inhibitory activities even against DNA gyrases of ofloxacin-resistant M. leprae. Conclusions/Significance: The results indicated that DC-159a and sitafloxacin are more effective against wild type and mutant M. leprae DNA gyrases than moxifloxacin, suggesting that these antibacterial drugs can be good candidates that may supersede current fluoroquinolone remedies. DC-159a in particular is very promising because it is classified in a subgroup of fluoroquinolones that is known to be less likely to cause adverse effects. Our results implied that DC-159a is well worth further investigation to ascertain its in vivo effectiveness and clinical safety for humans.

The effects of administering lactic acid bacteria sealed in a capsule on the intestinal bacterial flora of cattle
Seyama Tomohiro, Hirayasu Hirofumi, Yoshida Gen, Ohnuma Aiko, Qiu Yongjin, Nakajima Chie, Kasai Koji, Suzuki Yasuhiko
JAPANESE JOURNAL OF VETERINARY RESEARCH, 64, 3, 197, 203, 2016年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, We examined the effects of encapsulated lactic acid bacteria administrated orally to lactating cattle on the intestinal flora. A dose of 3 X 10¹¹ colony forming unit (cfu) of freeze-dried Lactobacillus coryniformis subsp. torquens (JCM1099) encapsulated in an enteric capsule capable of bypassing the rumen was administered for seven days. DNA was extracted from feces 0 and 24 hr after daily administration. Metagenomic analysis showed an increasing trend of the alpha diversity, an index of the species diversity. Furthermore, principal component analysis of intestinal flora revealed that cattle could be differentiated by JCM1099 capsule and suspension administration via principal components 1, 2, and 3. We conclude that administration of encapsulated JCM1099 can alter the intestinal bacterial flora of cattle.

Development and evaluation of an interferon-γ release assay in Asian elephants (Elephas maximus).
Paudel S, Villanueva MA, Mikota SK, Nakajima C, Gairhe KP, Subedi S, Rayamajhi N, Sashika M, Shimozuru M, Matsuba T, Suzuki Y, Tsubota T
The Journal of veterinary medical science, 78, 7, 1117, 1121, JAPAN SOC VET SCI, 2016年07月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, We developed an interferon-gamma release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-gamma (rEIFN-gamma) as well as native interferon-gamma from the Asian elephants was performed using anti-elephant IFN-gamma rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-gamma rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-gamma in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.

Collectin CL-P1 utilizes C-reactive protein for complement activation
Nitai Roy, Katsuki Ohtani, Yasuyuki Matsuda, Kenichiro Mori, Insu Hwang, Yasuhiko Suzuki, Norimitsu Inoue, Nobutaka Wakamiya
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1860, 6, 1118, 1128, ELSEVIER SCIENCE BV, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown.
Methods: We prepared CHO/IdIA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1.
Results: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions.
General Significance: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction. (C) 2016 Elsevier B.V. All rights reserved.

Collectin CL-P1 utilizes C-reactive protein for complement activation.
Nitai Roy, Katsuki Ohtani, Yasuyuki Matsuda, Kenichiro Mori, Insu Hwang, Yasuhiko Suzuki, Norimitsu Inoue, Nobutaka Wakamiya
Biochimica et biophysica acta, 1860, 6, 1118, 28, 2016年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.

Immunohistochemical Analysis of PD-L1 Expression in Canine Malignant Cancers and PD-1 Expression on Lymphocytes in Canine Oral Melanoma
Naoya Maekawa, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
PLOS ONE, 11, 6, e0157176, PUBLIC LIBRARY SCIENCE, 2016年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs.

Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis
Takashi Matsuba, Umme Ruman Siddiqi, Toshio Hattori, Chie Nakajima, Jun Fujii, Yasuhiko Suzuki
FEMS MICROBIOLOGY LETTERS, 363, 10, OXFORD UNIV PRESS, 2016年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains.

Combined antibody and DNA detection for early diagnosis of leptospirosis after a disaster
Hiroko Iwasaki, Haorile Chagan-Yasutan, Prisca Susan A. Leano, Nobuo Koizumi, Chie Nakajima, Delsi Taurustiati, Firmanto Hanan, Talitha Lea Lacuesta, Yugo Ashino, Yasuhiko Suzuki, Nina G. Gloriani, Elizabeth Freda O. Telan, Toshio Hattori
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 84, 4, 287, 291, ELSEVIER SCIENCE INC, 2016年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Early diagnosis based on laboratory confirmation is essential for managing leptospirosis. This study investigated the effectiveness of a novel method of detecting leptospirosis that combines measurement of anti-Leptospira antibodies by the microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic test (ICT) and leptospiral DNA by loop-mediated isothermal amplification (LAMP) and real-time PCR in plasma and 2 types of urine pellets. Of 113 suspected cases, 68.1%, 76.1%, and 60.2% were positive by MAT, ELISA, and ICT, respectively. Real-time PCR using DNA purified from urine pellets collected by low-speed centrifugation yielded positive signals for patients in late acute as well as early phase who were positive by LAMP using plasma DNA or urine pellets. Among antibody-negative patients, 9.5% were positive by DNA detection. These findings indicate that the leptospirosis detection rate is increased by combining antibody and DNA detection, providing a new tool for timely diagnosis of infection. (C) 2016 Elsevier Inc. All rights reserved.

Genetic diversity and antimicrobial resistance pattern of Salmonella enterica serovar Enteritidis clinical isolates in Thailand
Fuangfa Utrarachkij, Chie Nakajima, Kanokrat Siripanichgon, Kanjana Changkaew, Yuwanda Thongpanich, Srirat Pornraungwong, Orasa Suthienkul, Yasuhiko Suzuki
JOURNAL OF INFECTION AND CHEMOTHERAPY, 22, 4, 209, 215, ELSEVIER SCIENCE BV, 2016年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Objective: To trace the history of antimicrobial resistance in Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) circulating in Thailand, we characterised clinical isolates obtained during 2004-2007.
Methods: Antimicrobial resistance profiles, multi-locus variable number tandem repeat analysis (MLVA) types and 3 representative virulence determinants (spvA, sodCI and sopE) were established from SE isolates (n = 192) collected from stool and blood of patients throughout Thailand during the period 2004-2007.
Results: Resistance was found in SE against 10 out of 11 antimicrobials studied. The highest resistance ratios were observed for nalidixic acid (83.2%), ciprofloxacin (51.1%) and ampicillin (50.5%), and 25.5% were multidrug resistant. Based on five polymorphic tandem repeat loci analysis, MLVA identified 20 distinct types with three closely related predominant types. A significant increase of AMP resistance from 2004 to 2006 was strongly correlated with that of a MLVA type, 5-5-11-7-3.
Conclusion: The usage of antimicrobials in human medicine or farm settings might act as selective pressures and cause the spread of resistant strains. Hence, a strict policy on antimicrobial usage needs to be implemented to achieve the control of resistant SE in Thailand. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Development of Point-of-Care Testing for Disaster-Related Infectious Diseases
Toshio Hattori, Haorile Chagan-Yasutan, Beata Shiratori, Shinichi Egawa, Takako Izumi, Toru Kubo, Chie Nakajima, Yasuhiko Suzuki, Toshiro Niki, Bachti Alisjahbana, Elizabeth Telan
TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, 238, 4, 287, 293, TOHOKU UNIV MEDICAL PRESS, 2016年04月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, After disaster, the victims lose their safe lives and are even exposed to nature where they could suffer from animal bites and vectors followed by suffering from zoonosis or vector-born diseases. Because of the urgent need for rapid and cheap diagnosis for infectious diseases after disaster, anonymous questionnaire clarified that leptospirosis, dengue, diarrhea, and cholera were recognized as common disaster-related infections in the Philippines, while diarrhea and pneumonia were more common in Indonesia. It should also be noted that infectious disease itself such as tuberculosis associated with acquired immune deficiency syndrome in South Africa is a disaster. Thus, the possible occurrence of similar situation in Asia should be prevented. We have conducted an international collaborative research in the Philippines and Indonesia on dengue virus, leptospira and mycobacterium tuberculosis (MTB) infectious diseases. Development of point-of-care testing for molecular diagnosis and disease severity was the principal purpose of the research. Loop-mediated isothermal amplification assay, which does not require a source of electricity, was developed for leptospirosis, dengue and MTB and has been proved to be useful where resource is limited. The plasma levels of matricellular proteins, including galectin-9 and osteopontin, were found to reflect the disease seventies in dengue virus and MTB infection, probably because matricellular proteins are one of the most functional extracellular proteins that are associated with inflammatory edema. The study on disaster-related infectious disease facilitates the international cooperation for development of point-of-care testing for tropical infectious diseases.

Genotypic characterization of multi-drug-resistant Mycobacterium tuberculosis isolates in Myanmar
Khin Saw Aye, Chie Nakajima, Tomoyuki Yamaguchi, Min Min Win, Mu Mu Shwe, Aye Aye Win, Thandar Lwin, Wint Wint Nyunt, Ti Ti, Yasuhiko Suzuki
JOURNAL OF INFECTION AND CHEMOTHERAPY, 22, 3, 174, 179, ELSEVIER SCIENCE BV, 2016年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The number of multi-drug-resistant tuberculosis (MDR-TB) cases is rising worldwide. As a countermeasure against this situation, the implementation of rapid molecular tests to identify MDR-TB would be effective. To develop such tests, information on the frequency and distribution of mutations associating with phenotypic drug resistance in Mycobacterium tuberculosis is required in each country. During 2010, the common mutations in the rpoB, katG and inhA of 178 phenotypically MDR M. tuberculosis isolates collected by the National Tuberculosis Control Program (NTP) in Myanmar were investigated by DNA sequencing. Mutations affecting the 81-bp rifampicin (RIF) resistance-determining region (RRDR) of the rpoB were identified in 127 of 178 isolates (71.3%). Two of the most frequently affected codons were 531 and 526, with percentages of 48.3% and 14.0% respectively. For isoniazid (INH) resistance, 114 of 178 MDR-TB isolates (64.0%) had mutations in the katG in which a mutation-conferring amino acid substitution at codon 315 from Ser to Thr was the most common. Mutations in the inhA regulatory region were also detected in 20 (11.2%) isolates, with the majority at position -15. Distinct mutation rate and pattern from surrounding countries might suggest that MDR-TB has developed and spread domestically in Myanmar. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Mycobacterium orygis-Associated Tuberculosis in Free-Ranging Rhinoceros, Nepal, 2015
Jeewan Thapa, Sarad Paudel, Amir Sadaula, Yogendra Shah, Bhagwan Maharjan, Gretchen E. Kaufman, Deborah McCauley, Kamal P. Gairhe, Toshio Tsubota, Yasuhiko Suzuki, Chie Nakajima
EMERGING INFECTIOUS DISEASES, 22, 3, 570, 572, CENTERS DISEASE CONTROL, 2016年03月, ［査読有り］, ［国際誌］
英語

Ungulate malaria parasites
Thomas J. Templeton, Masahito Asada, Montakan Jiratanh, Sohta A. Ishikawa, Sonthaya Tiawsirisup, Thillaiampalam Sivakumar, Boniface Namangala, Mika Takeda, Kingdao Mohkaew, Supawan Ngamjituea, Noboru Inoue, Chihiro Sugimoto, Yuji Inagaki, Yasuhiko Suzuki, Naoaki Yokoyama, Morakot Kaewthamasorn, Osamu Kaneko
SCIENTIFIC REPORTS, 6, 23230, 23230, NATURE PUBLISHING GROUP, 2016年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium.

Adenocarcinoma arising in urinary bladder endocervicosis
Masato Nakaguro, Toyonori Tsuzuki, Satoko Shimada, Tetsuro Taki, Mari Tsuchiyama, Atsuko Kitamura, Yasuhiko Suzuki, Yojiro Nakano, Kenzo Ono
PATHOLOGY INTERNATIONAL, 66, 2, 108, 113, WILEY-BLACKWELL, 2016年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Endocervicosis is a rare benign condition characterized by the presence of endocervical-type mucinous glands. Urinary bladder endocervicosis forms an elevated lesion in the posterior wall of the urinary bladder and is sometimes misdiagnosed as a malignant tumor clinically and pathologically. Herein we describe the first case of adenocarcinoma arising in urinary bladder endocervicosis. The patient, a 58-year-old woman, presented with asymptomatic hematuria. Cystoscopy revealed a nodular mass measuring 4cm in diameter in the posterior wall, and total cystectomy was performed. Histology revealed that the elevated lesion of the bladder wall was composed of haphazard proliferation of cystic glands lined by benign endocervical-type epithelium. An adenocarcinoma arose at the center of this endocervicosis. Mucin histochemistry revealed the presence of sulfomucin in both the endocervicosis and adenocarcinoma components. Immunohistochemically, the endocervicosis was positive for cytokeratin (CK) 7, AE1/AE3, CAM5.2, HBME1, CA19-9, and estrogen receptor (ER), and negative for CK20, CDX2, progesterone receptor (PR), MUC5AC, and -catenin. The adenocarcinoma showed similar immunohistochemical results, except for loss of ER expression and a slight increase in the ratio of Ki-67-positive cells. This case indicates that endocervicosis, known as a benign lesion, harbors the possibility of malignant transformation.

Serological investigation of Leptospira infection and its circulation in one intensive-type water buffalo farm in the Philippines
Marvin A. Villanueva, Claro N. Mingala, Nina G. Gloriani, Yasutake Yanagihara, Norikazu Isoda, Chie Nakajima, Yasuhiko Suzuki, Nobuo Koizumi
JAPANESE JOURNAL OF VETERINARY RESEARCH, 64, 1, 15, 24, HOKKAIDO UNIV, 2016年02月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Water buffalo is an indispensable livestock in the Philippines. Leptospirosis is a serious zoonosis that can be fatal to humans and cause reproductive problems in livestock. Leptospirosis has been reported in some countries where water buffaloes are commercially raised, highlighting the Leptospira prevalence in this farming system, but information on leptospirosis in water buffalo farms in the Philippines is limited. In this study, we collected blood samples from rats (n = 21), and water buffaloes (n = 170) from different groups and locations in one intensive-type buffalo farm in the Philippines. Serum was analyzed by microscopic agglutination test (MAT). Anti-Leptospira antibodies reacting with serogroups Canicola, Icterohaemorrhagiae and Pomona were found in sera of 30% tested rats, and 48% of water buffalo sera tested positive for at least one Leptospira strain, in which serogroups Mini, Hebdomadis, Tarassovi and Pyrogenes were predominantly agglutinated. The number of seropositive young water buffaloes (<1 year-old) was lower than that of older seropositive ones. Furthermore, sera from younger water buffaloes were reactive with single serotypes with low MAT titers, but older animals were reactive with multiple Leptospira strains with variable MAT titers. In addition, antibodies against serogroups Icterohaemorrhagiae and Pomona were detected in both animals. Finally, Leptospira infection was found associated with age and animal grouping, highlighting the impact of management in the persistence of leptospirosis at intensive-type buffalo farm settings in the Philippines. Further investigation and appropriate control strategies are required to prevent leptospirosis from causing risks to public health and economic losses to the water buffalo farming industry.

Detection of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Market-Ready Chickens in Zambia
K. Chishimba, B. M. Hang'Ombe, K. Muzandu, S. E. Mshana, M. I. Matee, C. Nakajima, Y. Suzuki
International Journal of Microbiology, 2016, Hindawi Limited, 2016年, ［査読有り］
英語, 研究論文（学術雑誌）, The frequent administering of antibiotics in the treatment of poultry diseases may contribute to emergence of antimicrobial-resistant strains. The objective of this study was to detect the presence of extended-spectrum β-lactamase- (ESBL-) producing Escherichia coli in poultry in Zambia. A total of 384 poultry samples were collected and analyzed for ESBL-producing Escherichia coli. The cultured E. coli isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction for detection of b l a CTX-M, b l a SHV, and b l a TEM genes. Overall 20.1%, 77/384, (95% CI
43.2-65.5%) of total samples analyzed contained ESBL-producing Escherichia coli. The antimicrobial sensitivity test revealed that 85.7% (66/77
CI: 75.7-92) of ESBL-producing E. coli isolates conferred resistance to beta-lactam and other antimicrobial agents. These results indicate that poultry is a potential reservoir for ESBL-producing Escherichia coli. The presence of ESBL-producing Escherichia coli in poultry destined for human consumption requires strengthening of the antibiotic administering policy. This is important as antibiotic administration in food animals is gaining momentum for improved animal productivity in developing countries such as Zambia.

Pathogenic lineage of mcr-negative colistin-resistant Escherichia coli, Japan, 2008-2015
Sato T, Fukuda A, Suzuki Y, Shiraishi T, Honda H, Shinagawa M, Yamamoto S, Ogasawara N, Usui M, Takahashi H, Takahashi S, Tamura Y, Yokota S
Emerging Infectioud Diseases, 22, 12, 2223, 2226, 2016年, ［査読有り］
英語, 研究論文（学術雑誌）

Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand
Kaknokrat Chonsin, Shigeaki Matsuda, Chonchanok Theethakaew, Toshio Kodama, Jiraphan Junjhon, Yasuhiko Suzuki, Orasa Suthienkul, Tetsuya Iida
FEMS MICROBIOLOGY LETTERS, 363, 2, fnv222, OXFORD UNIV PRESS, 2016年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms.

First insight into the genetic population structure of Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients in Egypt
Hassan Mahmoud Diab, Chie Nakajima, Saber A. Kotb, Alaa Mokhtar, Nagwa F. M. Khder, Ahmed S. A. Abdelaal, Azza Hegazy, Ajay Poudel, Yogendra Shah, Yasuhiko Suzuki
TUBERCULOSIS, 96, 13, 20, CHURCHILL LIVINGSTONE, 2016年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The present study aimed to assess the population structure of Mycobacterium tuberculosis (MTB) isolates from Egypt. A total of 230 MTB isolates were analysed using spoligotyping, large sequence polymorphism (LSPs), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and multi-locus sequence typing (MLST). The majority of isolates (93.0%) belonged to lineage 4, including 44.3, 13.4 and 10.8% of the ill-defined T clade, LAM and Haarlem families, respectively, and lineage 3 was identified in 7.0% of the isolates. MIRU-VNTRs typing allowed efficient discrimination of the spoligotype-defined clusters, including spoligo-international types (SIT) 53, 34, and 4, into 56 patterns, including 13 clusters and 43 unique patterns. A new SNP at position 311614 was identified in all six isolates to form the biggest MIRU-VNTR cluster, which suggested a recent clonal expansion. This SNP could possibly be used as a genetic marker for robust discriminations of Egyptian MTB isolates belonging to SIT53. The combination of spoligotyping, 12 MIRU-VNTRs loci and MLST provided insight into the genetic diversity and transmission dynamics of the Egyptian MTB genotypes and could be a key to implementation of effective control measures by public health authorities. (C) 2015 Elsevier Ltd. All rights reserved.

Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia
Nobuo Koizumi, Hidemasa Izumiya, Jung-Jung Mu, Zbigniew Arent, Shou Okano, Chie Nakajima, Yasuhiko Suzuki, Maki Mizutani Muto, Tsutomu Tanikawa, Kyle R. Taylor, Noriyuki Komatsu, Kumiko Yoshimatsu, Hoang Thi Thu Ha, Makoto Ohnishi
INFECTION GENETICS AND EVOLUTION, 36, 434, 440, ELSEVIER SCIENCE BV, 2015年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype-host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera. This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors. (C) 2015 The Authors. Published by Elsevier B.V.

No crucial amino acid changes in the predicted histo blood group antigen-binding sites of norovirus genotype GII.4 capsid between non-secretors and secretors origin might suggest an alternative route of infection or existence of coincidental molecules
Tomoko Yoda, Yasuhiko Suzuki, Ikuko Aoyama, Kenji Yamazaki, Shuji Nakata, Kazuo Takahashi
JOURNAL OF MEDICAL MICROBIOLOGY, 64, 12, 1544, 1547, SOC GENERAL MICROBIOLOGY, 2015年12月, ［査読有り］, ［国際誌］
英語

Diagnostic value of antibody responses to multiple antigens from Mycobacterium tuberculosis in active and latent tuberculosis
Muhammad Andrian Senoputra, Beata Shiratori, Fakhrial Mirwan Hasibuan, Raspati Cundarani Koesoemadinata, Lika Apriani, Yugo Ashino, Kenji Ono, Tetsuya Oda, Makoto Matsumoto, Yasuhiko Suzuki, Bachti Alisjahbana, Toshio Hattori
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 83, 3, 278, 285, ELSEVIER SCIENCE INC, 2015年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We investigated the antibody responses to 10 prospective Mycobacterium tuberculosis (MTB) antigens and evaluated their ability to discriminate between latent (LTBI) and active pulmonary tuberculosis (TB). Our results indicate that plasma levels of anti-alpha-crystallin (ACR), antilipoarabinomannan, anti-trehalose 6,6'-dimycolate, and anti-tubercular-glycolipid antigen antibodies were higher in patients with active TB, compared to those in the LTBI and control subjects. No differences in the antibodies were observed between the control and LTBI subjects. Antibodies against the glycolipid antigens could not distinguish between Mycobacterium avium complex (MAC)-negative TB patients and MAC-infected LTBI individuals. The most useful serological marker was antibodies to ACR, with MAC-negative TB patients having higher titers than those observed in MAC-positive LTBI and control subjects. Our data indicate that antibody to ACR is a promising target for the serological diagnosis of patients with active TB patients. When dealing with antiglycolipid antibodies, MAC coinfection should always be considered in serological studies. (C) 2015 Elsevier Inc. All rights reserved.

Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan
Ryuji Kawahara, Kazuko Seto, Masumi Taguchi, Chie Nakajima, Yuko Kumeda, Yasuhiko Suzuki
JOURNAL OF CLINICAL MICROBIOLOGY, 53, 9, 3035, 3038, AMER SOC MICROBIOLOGY, 2015年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored bla(CMY-2), a plasmid-mediated AmpC beta-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring bla(CMY-2).

Characterization of Salmonella Typhimurium DNA gyrase as a target of quinolones
Siriporn Kongsoi, Kazumasa Yokoyama, Apinun Suprasert, Fuangfa Utrarachkij, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
DRUG TESTING AND ANALYSIS, 7, 8, 714, 720, WILEY-BLACKWELL, 2015年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Quinolones exhibit good antibacterial activity against Salmonella spp. isolates and are often the choice of treatment for life-threatening salmonellosis due to multi-drug resistant strains. To assess the properties of quinolones, we performed an in vitro assay to study the antibacterial activities of quinolones against recombinant DNA gyrase. We expressed the S. Typhimurium DNA gyrase A (GyrA) and B (GyrB) subunits in Escherichia coli. GyrA and GyrB were obtained at high purity (>95%) by nickel-nitrilotriacetic acid agarose resin column chromatography as His-tagged 97-kDa and 89-kDa proteins, respectively. Both subunits were shown to reconstitute an ATP-dependent DNA supercoiling activity. Drug concentrations that suppressed DNA supercoiling by 50% (IC(50)s) or generated DNA cleavage by 25% (CC(25)s) demonstrated that quinolones highly active against S. Typhimurium DNA gyrase share a fluorine atom at C-6. The relationships between the minimum inhibitory concentrations (MICs), IC(50)s and CC(25)s were assessed by estimating a linear regression between two components. MICs measured against S. Typhimurium NBRC 13245 correlated better with IC(50)s (R=0.9988) than CC(25)s (R=0.9685). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test to identify quinolones with promising activity against S. Typhimurium. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and N-1 cyclopropyl substituents are desirable structural features in targeting S. Typhimurium gyrase. Copyright (c) 2014 John Wiley & Sons, Ltd.

Characterization of Campylobacter jejuni DNA gyrase as the target of quinolones
Ruchirada Changkwanyeun, Masaru Usui, Siriporn Kongsoi, Kazumasa Yokoyama, Hyun Kim, Orasa Suthienkul, Kanjana Changkaew, Chie Nakajima, Yutaka Tamura, Yasuhiko Suzuki
JOURNAL OF INFECTION AND CHEMOTHERAPY, 21, 8, 604, 609, ELSEVIER SCIENCE BV, 2015年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Quinolones have long been used as the first-line treatment for Campylobacter infections. However, an increased resistance to quinolones has raised public health concerns. The development of new quinolone-based antibiotics with high activity is critical for effective, as DNA gyrase, the target of quinolones, is an essential enzyme for bacterial growth in several mechanisms. The evaluation of antibiotic activity against Campylobacter jejuni largely relies on drug susceptibility tests, which require at least 2 days to produce results. Thus, an in vitro method for studying the activity of quinolones against the C. jejuni DNA gyrase is preferred. To identify potent quinolones, we investigated the interaction of C. jejuni DNA gyrase with a number of quinolones using recombinant subunits. The combination of purified subunits exhibited DNA supercoiling activity in an ATP dependent manner. Drug concentrations that inhibit DNA supercoiling by 50% (IC(50)s) of 10 different quinolones were estimated to range from 0.4 (sitafloxacin) to >100 mu g/mL (nalidixic acid). Sitafloxacin showed the highest inhibitory activity, and the analysis of the quinolone structure-activity relationship demonstrated that a fluorine atom at R-6 might play the important role in the inhibitory activity against C. jejuni gyrase. Measured quinolone IC50s correlated well with minimum inhibitory concentrations (R = 0.9943). These suggest that the in vitro supercoiling inhibition assay on purified recombinant C. jejuni DNA gyrase is a useful and predictive technique to monitor the antibacterial potency of quinolones. And furthermore, these data suggested that sitafloxacin might be a good candidate for clinical trials on campylobacteriosis. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Antimicrobial Resistance, Extended-Spectrum β-Lactamase Productivity, and Class 1 Integrons in Escherichia coli from Healthy Swine.
Changkaew K, Intarapuk A, Utrarachkij F, Nakajima C, Suthienkul O, Suzuki Y
Journal of food protection, 78, 8, 1442, 1450, INT ASSOC FOOD PROTECTION, 2015年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Administration of antimicrobials to food-producing animals increases the risk of higher antimicrobial resistance in the normal intestinal flora of these animals. The present cross-sectional study was conducted to investigate antimicrobial susceptibility and extended-spectrum beta-lactamase (ESBL) producing strains and to characterize class 1 integrons in Escherichia coil in healthy swine in Thailand. All 122 of the tested isolates had drug-resistant phenotypes. High resistance was found to ampicillin (98.4% of isolates), chloramphenicol (95.9%), gentamicin (78.7%), streptomycin (77.9%), tetracycline (74.6%), and cefotaxime (72.1%). Fifty-four (44.3%) of the E. coil isolates were confirmed as ESBL-producing strains. Among them, bla(CTX-M) (45 isolates) and bla(TEM) (41 isolates) were detected. Of the bla(CTX-M)-positive E. coli isolates, 37 carried the bla(CTX-M-1) cluster, 12 carried the bla(CTX-M-9) cluster, and 5 carried both clusters. Sequence analysis revealed bla(TEM-1), bla(TEM-135), and bla(TEM-175) in 38, 2, and 1 isolate, respectively. Eighty-seven (71%) of the 122isolates carried class 1 integrons, and eight distinct drug-resistance gene cassettes with seven different integron profiles were identified in 43 of these isolates. Gene cassettes were associated with resistance to aminoglycosides (aadAl, aadA2, aadA22, or aadA23), trimethoprim (dfrA5, dfrAl2, or dftA17), and lincosamide (linF). Genes encoding beta-lactamases were not found in class 1 integrons. This study is the first to report ESBL-producing E. coli with a class 1 integron carrying the linF gene cassette in swine in Thailand. Our findings confirm that swine can be a reservoir of ESBL-producing E. coli harboring class 1 integrons, which may become a potential health risk if these integrons are transmitted to humans. Intensive analyses of animal, human, and environmental isolates are needed to control the spread of ESBL-producing E. coil strains.

Molecular characterization of Mycobacterium orygis isolates from wild animals of Nepal
Jeewan Thapa, Chie Nakajima, Bhagwan Maharjan, Ajay Poudel, Yasuhiko Suzuki
JAPANESE JOURNAL OF VETERINARY RESEARCH, 63, 3, 151, 158, HOKKAIDO UNIV, 2015年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Mycobacterium orygis, a new member of the Mycobacterium tuberculosis complex, was isolated from a captive spotted deer (Axis axis) and a blue bull (Boselaphus tragocamelus) in Nepal. Analyses by spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing, region of difference and single nucleotide polymorphism of genes gyrB, mmpL6, TbD1, PPE55 and Rv2042c confirmed the isolates as M. orygis. Moreover, analyses by spoligotyping (SIT587) as well as MIRU-VNTR showed that the isolates shared a similar pattern with many reported isolates. From previous and the present studies, it can be inferred that South Asia is one of the endemic regions for M. orygis. Further investigation including a larger sample size and different host interaction will help to understand the ecology and epidemiology of M. orygis in Nepal.

A case of Manila type Mycobacterium tuberculosis infection in Japan.
Usami O, Nakajima C, Endo S, Inomata S, Kanamori H, Hirakata Y, Uchiyama B, Kaku M, Suzuki Y, Hattori T
Clinical case reports, 3, 7, 622, 625, 2015年07月, ［査読有り］, ［国際誌］
英語, A 76-year-old Japanese woman contracted a Mycobacterium tuberculosis (TB, Manila type) infection in Japan, despite never having traveled. However, her son was treated for TB in the Philippines 3 years before he stayed at her house. Spoligotyping allows us to identify the TB genotype and identify the route of infection.

Molecular epidemiology of pathogenic Leptospira spp. in the straw-colored fruit bat (Eidolon helvum) migrating to Zambia from the Democratic Republic of Congo
Hirohito Ogawa, Nobuo Koizumi, Aiko Ohnuma, Alisheke Mutemwa, Bernard M. Hang'ombe, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa
INFECTION GENETICS AND EVOLUTION, 32, 143, 147, ELSEVIER SCIENCE BV, 2015年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The role played by bats as a potential source of transmission of Leptospira spp. to humans is poorly understood, despite various pathogenic Leptospira spp. being identified in these mammals. Here, we investigated the prevalence and diversity of pathogenic Leptospira spp. that infect the straw-colored fruit bat (Eidolon helvum). We captured this bat species, which is widely distributed in Africa, in Zambia during 2008-2013. We detected the flagellin B gene (flaB) from pathogenic Leptospira spp. in kidney samples from 79 of 529 E. helvum (14.9%) bats. Phylogenetic analysis of 70 flaB fragments amplified from E. helvum samples and previously reported sequences, revealed that 12 of the fragments grouped with Leptospira borgpetersenii and Leptospira kirschneri; however, the remaining 58 flaB fragments appeared not to be associated with any reported species. Additionally, the 16S ribosomal RNA gene (rrs) amplified from 27 randomly chosen flaB-positive samples was compared with previously reported sequences, including bat-derived Leptospira spp. All 27 rrs fragments clustered into a pathogenic group. Eight fragments were located in unique branches, the other 19 fragments were closely related to Leptospira spp. detected in bats. These results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that Leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. Our study indicates that pathogenic Leptospira spp. in E. helvum in Zambia have unique genotypes. (C) 2015 The Authors. Published by Elsevier B.V.

New Aspects of Collectin Functions
Katsuki Ohtani, Yasuhiko Suzuki, Nobutaka Wakamiya
Glycoscience: Biology and Medicine, 1029, 1036, Springer Japan, 2015年01月01日, ［査読有り］
英語, 論文集(書籍)内論文, Collectin is a very unique protein, which has two characteristic domains: A collagen-like sequence and carbohydrate recognition domain (CRD). It is considered as a receptor recognizing a pathogen with associated molecular patterns (PAMPs) that play an important role in innate immunity. Recently, it was demonstrated that several collectins can not only activate the complement pathway but are also involved in various biological functions different from a host defense. In this chapter, we focus on “complement activation-related collectins,” consisting of mannan-binding lectin (MBL), complement 1q (C1q), collectin kidney 1 (CL-K1), and collectin placenta 1 (CL-P1), and summarize and discuss various new aspects of their functions.

Molecular identification of non-tuberculous mycobacteria isolated from clinical specimens in Zambia
Grace Mwikuma, Geoffry Kwenda, Bernard M. Hang'ombe, Edgar Simulundu, Trevor Kaile, Selestine Nzala, Seter Siziya, Yasuhiko Suzuki
ANNALS OF CLINICAL MICROBIOLOGY AND ANTIMICROBIALS, 14, 1, 1, BIOMED CENTRAL LTD, 2015年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: The emergence of Acquired Immunodeficiency Syndrome has highlighted the increased incidence and importance of the disease caused by Non-tuberculous Mycobacteria (NTM). While disease due to M. avium-intracellulare complex is apparently common throughout the world, other Non-tuberculous mycobacterial species have been isolated from both immunocompromised and immunocompetent individuals. The increasing number of infections caused by these organisms has made it clinically important to quickly identify mycobacterial species. The diagnosis of a pathogenic versus a non-pathogenic species not only has epidemiological implications but is also relevant to the demands of patient management. Since antibiotic treatment varies according to the species encountered, species identification would reduce the burden of some of these emerging opportunistic pathogens especially in immunocompromised patients and improve their quality of life.
Findings: A total of 91 NTM suspected isolates from four regions of Zambia were included in the study. These isolates were identified using the sequence analysis of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Mycobacteria.
Fifty-four of the 91 (59%) isolates were identified as NTM and these included M. intracellulare (27.8%), M. lentiflavum (16.7%), M. avium (14.8%), M. fortuitum (7.4%), M. gordonae (7.4%), M. kumamotonense (3.7%), M. indicus pranii (3.7%), M. peregrinum (3.7%), M. elephantis (1.85%), M. flavescens (1.85%), M. asiaticum (1.85%), M. bouchedurhonense (1.85%), M. chimaera (1.85%), M. europaeum (1.85%), M. neourum (1.85%), M. nonchromogenicum (1.5%).
Conclusion: The study has shown that DNA sequencing of the ITS region may be useful in the preliminary identification of NTM species. All species identified in this study were potentially pathogenic.

Evaluation of Anti-TBGL Antibody in the Diagnosis of Tuberculosis Patients in China
Jingge Zhao, Zhaoqin Zhu, Xiaoyan Zhang, Yasuhiko Suzuki, Haorile Chagan-Yasutan, Haili Chen, Yanmin Wan, Jianqing Xu, Yugo Ashino, Toshio Hattori
JOURNAL OF IMMUNOLOGY RESEARCH, 2015, 834749, 834749, HINDAWI PUBLISHING CORP, 2015年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Tuberculous glycolipid (TBGL) is a component of the Mycobacterium tuberculosis cell wall, and anti-TBGL antibodies are used for serodiagnosis of tuberculosis. Anti-TBGL IgG and IgA levels were measured in 45 pulmonary TB patients (PTB), 26 extrapulmonary TB patients (ETB), 16 AIDS-TB patients, and 58 healthy controls (HC) including 39 health care workers (HW) and 19 newly enrolled students (ST). Anti-TBGL IgG measurements yielded 68.9% and 46.2% sensitivity in PTB and ETB, respectively, and 81.0% specificity. However, anti-TBGL IgA measurements were significantly less sensitive in detecting ETB than PTB (15.4% versus 46.7% sensitivity) but showed up to 89.7% specificity. Samples from AIDS-TB patients exhibited low reaction of anti-TBGL IgG and IgA with 6.3% and 12.5% sensitivity, respectively. Unlike anti-lipoarabinomannan (LAM) IgG that was found to elevate in sputum smearpositive subjects, anti-TBGL IgG and IgA elevated in those with cavitation and bronchiectasis, respectively. Anti-TBGL IgG in cavitary TB yielded 78.2% sensitivity compared to 57.1% in those otherwise. Meanwhile, higher anti-TBGL IgA titers were observed in HW than in ST, and increasing anti-TBGL IgG titers were observed in HW on follow-up. Therefore, higher anti-TBGL antibody titers are present in patients presenting cavities and bronchiectasis and subjects under TB exposure risk.

Scavenger receptor CL-P1 mainly utilizes a collagen-like domain to uptake microbes and modified LDL
Kenichiro Mori, Katsuki Ohtani, SeongJae Jang, YounUck Kim, Insu Hwang, Nitai Roy, Yasuyuki Matsuda, Yasuhiko Suzuki, Nobutaka Wakamiya
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1840, 12, 3345, 3356, ELSEVIER SCIENCE BV, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD.
Methods: We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively.
Results: CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL.
Conclusions: CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL.
General significance: We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body. (C) 2014 Elsevier B.V. All rights reserved.

Scavenger receptor CL-P1 mainly utilizes a collagen-like domain to uptake microbes and modified LDL.
Kenichiro Mori, Katsuki Ohtani, SeongJae Jang, YounUck Kim, Insu Hwang, Nitai Roy, Yasuyuki Matsuda, Yasuhiko Suzuki, Nobutaka Wakamiya
Biochimica et biophysica acta, 1840, 12, 3345, 56, 12, 2014年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. METHODS: We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. RESULTS: CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. CONCLUSIONS: CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. GENERAL SIGNIFICANCE: We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2 mu 2
Jang SeongJae, Ohtani Katsuki, Fukuoh Atsushi, Mori Kenichiro, Yoshizaki Takayuki, Kitamoto Noritoshi, Kim YounUck, Suzuki Yasuhiko, Wakamiya Nobutaka
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1840, 11, 3226, 3237, 2014年11月, ［査読有り］

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2μ2.
Jang S, Ohtani K, Fukuoh A, Mori K, Yoshizaki T, Kitamoto N, Kim Y, Suzuki Y, Wakamiya N
Biochimica et biophysica acta, 1840, 11, 3226, 37, 11, 2014年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated. METHODS: We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in μ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out. RESULTS: We identified μ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2μ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules. CONCLUSIONS: Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2μ2. GENERAL SIGNIFICANCE: This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.

Dominant modern sublineages and a new modern sublineage of Mycobacterium tuberculosis Beijing family clinical isolates in Heilongjiang Province, China
Di Li, Cai-Bo Dong, Jia-Yi Cui, Chie Nakajima, Chun-Lei Zhang, Xin-Ling Pan, Gao-Xiang Sun, En-Yu Dai, Yasuhiko Suzuki, Min Zhuang, Hong Ling
INFECTION GENETICS AND EVOLUTION, 27, 294, 299, ELSEVIER SCIENCE BV, 2014年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium tuberculosis Beijing family includes a variety of sublineages. Knowledge of the distribution of a certain sublineage of the Beijing family may help to understand the mechanisms of its rapid spread and to establish an association between a certain genotype and the disease outcome. We have previously found that M. tuberculosis Beijing family clinical isolates represent approximately 90% of the clinical isolates from Heilongjiang Province, China. To clarify the distribution of M. tuberculosis Beijing family sublineages in Heilongjiang Province, China and to investigate the regularity rule for their evolution, we examined single nucleotide polymorphisms (SNPs) of 250 M. tuberculosis Beijing family clinical isolates using 10 SNP loci that have been identified as appropriate for defining Beijing sublineages. After determining the sequence type (ST) of each isolate, the sublineages of all M. tuberculosis Beijing family isolates were determined, and phylogenetic analysis was performed. We found that 9 out of the 10 SNP loci displayed polymorphisms, but locus 1548149 did not. In total, 92.8% of the isolates in Heilongjiang Province are modern sublineages. ST10 is the most prevalent sublineage (ST10 and ST22 accounted for 63.2% and 23.6% of all the Beijing family isolates, respectively). A new ST, accounting for 4% of the Beijing family isolates in this area, was found for the first time. Each new ST isolate showed a unique VNTR pattern, and none were clustered. The present findings suggest that controlling the spread of these modern sublineages is important in Heilongjiang Province and in China. (c) 2014 Published by Elsevier B.V.

The Babesia bovis gene and promoter model: an update from full-length EST analysis.
Yamagishi J, Wakaguri H, Yokoyama N, Yamashita R, Suzuki Y, Xuan X, Igarashi I
BMC genomics, 15, 678, 2014年08月, ［査読有り］

Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytes
Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
IMMUNOLOGY, 142, 4, 551, 561, WILEY-BLACKWELL, 2014年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-gamma (IFN-gamma) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-gamma production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.

Characterization of Antibiotic Resistance in Escherichia coli Isolated from Shrimps and Their Environment
Kanjana Changkaew, Fuangfa Utrarachkij, Kanokrat Siripanichgon, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
JOURNAL OF FOOD PROTECTION, 77, 8, 1394, 1401, INT ASSOC FOOD PROTECTION, 2014年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Antimicrobial resistance in bacteria associated with food and water is a global concern. To survey the risk, 312 Escherichia coli isolates from shrimp farms and markets in Thailand were examined for susceptibility to 10 antimicrobials. The results showed that 17.6% of isolates (55 of 312) were resistant to at least one of the tested drugs, and high resistance rates were observed to tetracycline (14.4%; 45 of 312), ampicillin (8.0%; 25 of 312), and trimethroprim (6.7%; 21 of 312); 29.1% (16 of 55) were multidrug resistant. PCR assay of the tet(A), tet(B), tet(C), tet(D), tet(E), and tet(G) genes detected one or more of these genes in 47 of the 55 resistant isolates. Among these genes, tet(A) (69.1%; 38 of 55) was the most common followed by tet(B) (56.4%; 31 of 55) and tet(C) (3.6%; 2 of 55). The resistant isolates were further investigated for class 1 integrons. Of the 55 resistant isolates, 16 carried class 1 integrons and 7 carried gene cassettes encoding trimethoprim resistance (dfrA12 or dfrA17) and aminoglycosides resistance (aadA2 or aadA5). Two class 1 integrons, In54 (dfrA17-aadA5) and In27 (dfrA12-orfF-aadA2), were found in four and three isolates, respectively. These results indicate a risk of drug-resistant E. coli contamination in shrimp farms and selling places. The occurrence of multidrug-resistant E. coli carrying tet genes and class 1 integrons indicates an urgent need to monitor the emergence of drug-resistant E. coli to control the dissemination of drug-resistant strains and the further spread of resistance genes to other pathogenic bacteria.

Identification and characterization of bovine programmed death-ligand 2
Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Chie Nakajima, Yasuhiko Suzuki, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
MICROBIOLOGY AND IMMUNOLOGY, 58, 7, 388, 397, WILEY-BLACKWELL, 2014年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-gamma production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-gamma production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-gamma production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases.

Functional analysis of the receptor binding domain of SARS coronavirus S1 region and its monoclonal antibody
Hyun Kim, Yeongjin Hong, Keigo Shibayama, Yasuhiko Suzuki, Nobutaka Wakamiya, Youn Uck Kim
GENES & GENOMICS, 36, 3, 387, 397, SPRINGER, 2014年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Severe acute respiratory syndrome (SARS) is caused by the SARS coronavirus (CoV). The spike protein of SARS-CoV consists of S1 and S2 domains, which are responsible for virus binding and fusion, respectively. The receptor-binding domain (RBD) positioned in S1 can specifically bind to angiotensin-converting enzyme 2 (ACE2) on target cells, and ACE2 regulates the balance between vasoconstrictors and vasodilators within the heart and kidneys. Here, a recombinant fusion protein containing 193-amino acid RBD (residues 318-510) and glutathione S-transferase were prepared for binding to target cells. Additionally, monoclonal RBD antibodies were prepared to confirm RBD binding to target cells through ACE2. We first confirmed that ACE2 was expressed in various mouse cells such as heart, lungs, spleen, liver, intestine, and kidneys using a commercial ACE2 polyclonal antibody. We also confirmed that the mouse fibroblast (NIH3T3) and human embryonic kidney cell lines (HEK293) expressed ACE2. We finally demonstrated that recombinant RBD bound to ACE2 on these cells using a cellular enzyme-linked immunosorbent assay and immunoassay. These results can be applied for future research to treat ACE2-related diseases and SARS.

Identification and Characterization of a Trypanosoma congolense 46 kDa Protein as a Candidate Serodiagnostic Antigen
Mo Zhou, Keisuke Suganuma, Ngasaman Ruttayaporn, Thu-Thuy Nguyen, Shino Yamasaki, Ikuo Igarashi, Shin-ichiro Kawazu, Yasuhiko Suzuki, Noboru Inoue
JOURNAL OF VETERINARY MEDICAL SCIENCE, 76, 6, 799, 806, JAPAN SOC VET SCI, 2014年06月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Trypanosoma congolense is a major livestock pathogen in Africa, causing large economic losses with serious effects on animal health. Reliable serodiagnostic tests are therefore urgently needed to control T congolense infection. In this study, we have identified one T congolense protein as a new candidate serodiagnostic antigen. The 46.4 kDa protein (TcP46, Gene ID: TcIL3000.0.25950) is expressed 5.36 times higher in metacyclic forms than epimastigote forms. The complete nucleotide sequences of TcP46 contained an open reading frame of 1,218 bp. Southern blot analysis indicated that at least two copies of the TcP46 gene were tandemly-arranged in the T congolense genome. The recombinant TcP46 (rTcP46) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Western blot analysis and confocal laser scanning microscopy revealed that the native TcP46 protein is expressed in the cytoplasm during all life-cycle stages of the parasite. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on rTcP46 detected the specific antibodies as early as 8 days post-infection from mice experimentally infected with T congolense. No cross-reactivity was observed in the rTcP46-based ELISA against serum samples from cattle experimentally infected with Babesia bigemina, B. bovis and Anaplasma marginale. These results suggest that rTcP46 could be used as a serodiagnostic antigen for T congolense infection.

Expression of PD-L1 on canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade.
Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K
PloS one, 9, 6, e98415, 6, 2014年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the "exhausted'' status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1-expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-gamma production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed.

Protective Efficacy of Passive Immunization with Monoclonal Antibodies in Animal Models of H5N1 Highly Pathogenic Avian Influenza Virus Infection
Yasushi Itoh, Reiko Yoshida, Shintaro Shichinohe, Megumi Higuchi, Hirohito Ishigaki, Misako Nakayama, Van Loi Pham, Hideaki Ishida, Mitsutaka Kitano, Masahiko Arikata, Naoko Kitagawa, Yachiyo Mitsuishi, Kazumasa Ogasawara, Hideaki Tsuchiya, Takahiro Hiono, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Mutsumi Ito, Le Quynh Mai, Yoshihiro Kawaoka, Hiroko Miyamoto, Mari Ishijima, Manabu Igarashi, Yasuhiko Suzuki, Ayato Takada
PLOS PATHOGENS, 10, 6, e1004192, PUBLIC LIBRARY SCIENCE, 2014年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.

Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal.
Paudel S, Mikota SK, Nakajima C, Gairhe KP, Maharjan B, Thapa J, Poudel A, Shimozuru M, Suzuki Y, Tsubota T
Tuberculosis (Edinburgh, Scotland), 94, 3, 287, 292, 3, 2014年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. (C) 2014 Elsevier Ltd. All rights reserved.

Diagnostic value of the recombinant tandem repeat antigen TeGM6-4r for surra in water buffaloes
Thu-Thuy Nguyen, Mo Zhou, Ngasaman Ruttayaporn, Quoc Doanh Nguyen, Viet Khong Nguyen, Yasuyuki Goto, Yasuhiko Suzuki, Shin-ichiro Kawazu, Noboru Inoue
VETERINARY PARASITOLOGY, 201, 1-2, 18, 23, ELSEVIER SCIENCE BV, 2014年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Trypanosoma evansi infection, or surra, is currently affecting various species of animals, especially water buffaloes. Since diagnosis is an important aspect of surra control, development of novel diagnostic antigens is of interest to implement and improve the currently utilized methods. Our study evaluated the tandem repeat antigen TeGM6-4r in T. evansi antibody detection in water buffaloes. TeGM6-4r-based ELISA was performed with 20 positive and 8 negative controls and 484 field samples from water buffaloes in Northern Vietnam. To examine cross-reactivity, sera from Japanese cattle that had been experimentally infected with Theileria orientalis (n=10), Babesia bovis (n=3), Babesia bigemina (n = 7) and Trypanosoma theileri (n=59) were included in the study. The sensitivity of the test was 80%. TeGM6-4r did not react with Theileria or Babesia infected sera, however it showed cross reactivity with 11/59 T. theileri infected samples. The reference test, CATT/T. evansi also reacted with 3/59 T. theileri infected sera. The lysate antigen-based ELISA reacted with 4/59 T. theileri, 9/10 Theileria and 3/10 Babesia infected sera. In contrast, TeGM6-4r-based ELISA was 86.3% sensitive and 58.3% specific in the screening of field samples. The average seroprevalence of T. evansi infection among water buffaloes in Northern Vietnam was 27.1% by CATT/T. evansi and 53.7% by TeGM6-4r. Seroprevalence in the five surveyed provinces ranged from 17.4% to 39.8% in the reference test, and 47.3% to 67.3% in the recombinant antigen based test. The finding indicated that the disease is still widely endemic in the area and that surveillance programs need to be carried out regularly to better control surra. We proposed TeGM6-4r as a useful serodiagnostic antigen for the detection and epidemiological surveillance of T. evansi infection among water buffaloes. (C) 2014 Elsevier B.V. All rights reserved.

Bacterial Diversity in Sea Ice from the Southern Ocean and the Sea of Okhotsk　　　　　　　　　　　　　　　
Okubo, T, Tosaka, Y, Sato, T, Usui, M, Nakajima, C, Suzuki, Y, Imura, S, Tamura, Y
Journal of Applied & Environmental Microbiology, 2, 6, 266, 272, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）

Elevated OPN, IP-10, and Neutrophilia in Loop-Mediated Isothermal Amplification Confirmed Tuberculosis Patients
Beata Shiratori, Susan Leano, Chie Nakajima, Haorile Chagan-Yasutan, Toshiro Niki, Yugo Ashino, Yasuhiko Suzuki, Elisabeth Telan, Toshio Hattori
MEDIATORS OF INFLAMMATION, 2014, 513263, 513263, HINDAWI PUBLISHING CORPORATION, 2014年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Tuberculosis (TB) is the second most common cause of death from infectious diseases and results in high socioeconomic losses to many countries. Proper diagnosis is the first step in TB eradication. To develop a rapid, simple, and accurate diagnostic TB test and to characterize the prevalence of Mycobacterium tuberculosis (MTB) genotypes and immune profiles of TB patients, a total of 37 TB patients and 30 healthy control (HC) from Metro Manila were enrolled. Loop-mediated isothermal amplification (LAMP) reliably detected MTB infection. Manila genotype was identified by spoligotyping method in all TB patients. Osteopontin (OPN), interferon-gamma-induced protein 10 kDa (IP-10), and neutrophil counts were found to reflect the acute stage of MTB infection. The sensitivity and specificity were 94.6% and 93.3%, respectively, for both OPN and IP-10, and they were 83.8% and 78.6%, respectively, for neutrophils. The combination of OPN, IP-10, neutrophil count, IL-6, IL-8, TNF-alpha, MCP-1, platelets, galectin-9, and leukocyte count correctly identifies all the HC and 96.3% of TB patients. LAMP method may serve as a rapid, supportive method in addition to time-consuming culture methods. OPN, IP-10, and neutrophil counts are useful in detecting MTB infection and may have utility in monitoring the course of the disease.

What hansen’s disease research learned from tuberculosis research: From molecular biological aspect
Yasuhiko Suzuki, Tomoyuki Yamaguchi, Hyun Kim, Kazumasa Yokoyama, Chie Nakajima
Japanese Journal of Leprosy, 83, 3, 131, 137, Japanese Leprosy Association, 2014年, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, As for the Mycobacterium leprae which is a causative agent of Hansen’s disease, many studies had been done since it was identified in 1873. However, those studies, at the same time, experienced many struggles because of the difficulty of culture of M. leprae on the artificial growth media. Hence, the study of Hansen’s disease progressed by taking the knowledge from the study of tuberculosis caused by the bacteria belonging to the same genus, genus Mycobacterium. For instance, the knowledge of mutations in specific genes responsible for rifampicin-and quinolone-resistance in M. tuberculosis led the elucidation of drug-resistant acquisition mechanism of M. leprae. Similarly, it is necessary for the researcher of Hansen’s disease to get important information from the latest topic of the tuberculosis study and utilize them to the study of the disease.

Intra-subspecies sequence variability of the MACPPE12 gene in Mycobacterium avium subsp hominissuis
Tomotada Iwamoto, Kentaro Arikawa, Chie Nakajima, Noriko Nakanishi, Yukiko Nishiuchi, Shiomi Yoshida, Aki Tamaru, Yutaka Tamura, Yoshihiko Hoshino, Heekyung Yoo, Young Kil Park, Hajime Saito, Yasuhiko Suzuki
INFECTION GENETICS AND EVOLUTION, 21, 479, 483, ELSEVIER SCIENCE BV, 2014年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The PE (Pro-Glu) and PPE (Pro-Pro-Glu) multigene families are unique to mycobacteria, and are highly expanded in the pathogenic members of this genus. We determined the intra-subspecies genetic variability of the MACPPE12 gene, which is a specific PPE gene in Mycobacterium avium subsp. hominissuis (MAH), using 334 MAH isolates obtained from different isolation sources (222 human isolates, 145 Japanese and 77 Korean; 37 bathroom isolates; and 75 pig isolates). In total, 31 single-nucleotide polymorphisms (SNPs), which consisted of 16 synonymous SNPs and 15 nonsynonymous SNPs, were determined through comparison with the MACPPE12 gene sequence of MAH strain 104 as a reference. As the result, the 334 MAH isolates were classified into 19 and 13 different sequevars at the nucleic acid level (NA types) and amino acid level (AA types), respectively. Among the 13 AA types, only one type, the AA02 type, presented various NA types (7 different types) with synonymous SNPs, whereas all other AA types had a one-to-one correspondence with the NA types. This finding suggests that AA02 is a longer discernible lineage than the other AA types. Therefore, AA02 was classified as an ancestral type of the MACPPE12 gene, whereas the other AA types were classified as modern types. The ubiquitous presence of AA02 in all of the isolation sources and all different sequevars classified by the hsp65 genotype further supports this classification. In contrast to the ancestral type, the modern types showed remarkable differences in distribution between human isolates and pig isolates, and between Japanese isolates and Korean isolates. Divergence of the MACPPE12 gene may thus be a good indicator to characterize MAH strains in certain areas and/or hosts. (C) 2013 Elsevier B.V. All rights reserved.

Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification
Yukiko Nishiuchi, Aki Tamaru, Yasuhiko Suzuki, Seigo Kitada, Ryoji Maekura, Yoshitaka Tateishi, Mamiko Niki, Hisashi Ogura, Sohkichi Matsumoto
JOURNAL OF WATER AND HEALTH, 12, 2, 211, 219, IWA PUBLISHING, 2014年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA (R) elute card,DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.

Expression, immunolocalization and serodiagnostic value of Tc38630 protein from Trypanosoma congolense
Kennedy Miyoro Mochabo, Mo Zhou, Keisuke Suganuma, Shin-Ichiro Kawazu, Yasuhiko Suzuki, Noboru Inoue
Parasitology Research, 112, 9, 3357, 3363, 2013年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Animal African trypanosomosis is a serious constraint to livestock sector development in sub-Saharan Africa. The disease, mainly caused by Trypanosoma congolense, has a limitation in its diagnosis and treatment. There is urgent need for a simple, rapid detection technique to replace the few available serological tests that are of variable sensitivity and specificity. Currently, there is a promising use of recombinant proteins to improve on the trypanosome lysate to detect antibodies. In this respect, we have identified a stage-specific gene that is relatively highly expressed in metacyclic and blood trypomastigotes of T. congolense. According to previously obtained differential protein expression data, the gene TcIL3000.0.38630 (1,236 bp) is by 8.5 times more expressed in metacyclic and blood trypomastigotes than in procyclic trypomastigotes and epimastigotes. The same stage specific expression pattern was shown in Western blot analysis. In addition, in confocal laser scanning microscopy the Tc38630 protein was present in the cytosol and on the cell surface of metacyclic and blood trypomastigotes. Through bioinformatics, the Tc38630 had N-terminal signal sequence, hydrophilic extracellular domain, single transmembrane alpha-helix and short cytoplasmic domain, which is characteristic of the Trypanosoma brucei invariant surface glycoprotein. However, unlike T. brucei invariant surface glycoprotein, the Tc38630 existed as a single copy gene with a probable allelic polymorphism at the Nar I restriction site. The recombinant Tc38630-based ELISA detected antibodies against Tc38630 as early as 7 days post infection in experimentally infected mouse model. Taken together, our results suggest that the Tc38630 is a novel potential diagnostic antigen of Animal African trypanosomosis. © 2013 Springer-Verlag Berlin Heidelberg.

Preliminary investigation of trypanosomosis in exotic dog breeds from Zambia’s Luangwa and Zambezi valleys using LAMP
Namangala B, Oparaocha E, Kajino K, Hayashida K, Moonga L, Inoue N, Suzuki Y, Sugimoto C
Am J Trop Med Hygine, 89, 1, 116, 118, AMER SOC TROP MED & HYGIENE, 2013年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Canine African trypanosomosis (CAT) is rarely reported in the literature. In this preliminary study, we evaluated the performance of loop-mediated isothermal amplification (LAMP) against microscopy to detect CAT in six exotic dog breeds naturally infected with trypanosomes from Zambia's South Luangwa National Park and Chiawa Game Management Area. To our knowledge, this is the first report of CAT in Zambia. The patients exhibited a variety of aspecific clinical signs. The LAMP did not only confirm all six parasitologically positive CAT cases detected passively between April 2010 and January 2012, but was also critical in trypanosome speciation. According to LAMP, the majority of the dogs had monolytic infections with either Trypanosoma congolense or Trypanosoma brucei rhodesiense. The LAMP is thus a potential simple and cost-effective tool for trypanosome diagnosis in endemic regions. The rare report of zoonotic trypanosomes in dogs in Zambia has public health implications and justifies further investigations of CAT.

Tumor budding as a useful prognostic marker in T1-stage squamous cell carcinoma of the esophagus.
Hitoshi Teramoto, Masahiko Koike, Chie Tanaka, Suguru Yamada, Goro Nakayama, Tsutomu Fujii, Hiroyuki Sugimoto, Michitaka Fujiwara, Yasuhiko Suzuki, Yasuhiro Kodera
Journal of surgical oncology, 108, 1, 42, 6, 2013年07月, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Establishing a new prognostic factor for early-stage cancer may seem difficult due to the small number of disease-specific deaths. Tumor budding has been recognized as a useful microscopic finding reflecting biological activity of the tumor. METHODS: Tumor budding stand for isolated single cancer cells and cell clusters scattered beyond the tumor margin at the invasive front. It was searched for in the resected esophagus with T1 squamous cell carcinoma (SCC), and the correlation between the tumor budding, patient survival, and various pathologic factors were analyzed to verify whether tumor budding is a prognostic factor in superficial esophageal cancer. RESULTS: Seventy-nine patients undergoing curative esophagectomy were assigned to frequent (n = 29) and rare (n = 50) groups according to the microscopically observed frequency of tumor budding in the tumor. Three-year survival rates after esophagectomy were 48.8% for the frequent group and 94.5% for the rare group. Multivariate analysis using the Cox proportional hazards model identified this morphological variable as a significant independent prognostic factor. CONCLUSIONS: Tumor budding reflects the biological activity of the tumor and may be a useful prognostic indicator even in early-stage SCC of esophagus.

Whole-genome sequencing of Theileria parva strains provides insight into parasite migration and diversification in the African continent.
Hayashida K, Abe T, Weir W, Nakao R, Ito K, Kajino K, Suzuki Y, Jongejan F, Geysen D, Sugimoto C
DNA research : an international journal for rapid publication of reports on genes and genomes, 20, 3, 209, 220, 2013年06月, ［査読有り］

Applicability of In-House Loop-Mediated Isothermal Amplification for Rapid Identification of Mycobacterium tuberculosis Complex Grown on Solid Media
Benjawan Phetsuksiri, Janisara Rudeeaneksin, Sopa Srisungngam, Supranee Bunchoo, Dhanida Roienthong, Tetsu Mukai, Chie Nakajima, Shigeyuki Hamada, Yasuhiko Suzuki
JAPANESE JOURNAL OF INFECTIOUS DISEASES, 66, 3, 249, 251, NATL INST INFECTIOUS DISEASES, 2013年05月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, A simple, rapid, and low-cost identification method is required in tuberculosis high-burden countries. We report the applicability of in-house loop-mediated isothermal amplification (LAMP) targeting 16S ribosomal RNA for the rapid identification of Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media. Eighty acid-fast staining-positive clinical isolates were selected and used to evaluate the LAMP assay in comparison with polymerase chain reaction and conventional culture-based tests. The LAMP assay identified 60 M. tuberculosis isolates from 80 clinical isolates using simple heat-extracted DNA directly from the colony suspension. The results were in complete agreement with those obtained using the other methods, and the utility of the direct LAMP assay from a colony was demonstrated. The LAMP assay appears to be a practical and low-cost method that can be used for the rapid identification of M. tuberculosis isolates and suitable for endemic low-resource settings.

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi.
Seto J, Suzuki Y, Otani K, Qiu Y, Nakao R, Sugimoto C, Abiko C
Microbiology and immunology, 57, 2, 111, 117, 2, 2013年02月, ［査読有り］
英語

Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro
Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
Veterinary Research, 44, 1, 59, 59, 1, 2013年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1+ CD4+ T cells in blood and lymph node and PD-1 + CD8+ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.© 2013 Ikebuchi et al.
licensee BioMed Central Ltd.

Simple multiplex PCR assay for identification of Beijing family Mycobacterium tuberculosis isolates with a lineage-specific mutation in Rv0679c
Chie Nakajima, Aki Tamaru, Zeaur Rahim, Ajay Poudel, Bhagwan Maharjan, Khin Saw Aye, Hong Ling, Toshio Hattori, Tomotada Iwamoto, Yukari Fukushima, Haruka Suzuki, Yasuhiko Suzuki, Takashi Matsuba
Journal of Clinical Microbiology, 51, 7, 2025, 2032, American Society for Microbiology, 2013年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to host cell invasion. Here, we conducted a sequence analysis of the Rv0679c gene using clinical isolates and found that a single nucleotide polymorphism, C to G at position 426, can be observed only in the isolates that are identified as members of the Beijing genotype family. Here, we developed a simple multiplex PCR assay to detect this point mutation and applied it to 619 clinical isolates. The method successfully distinguished Beijing lineage clones from non-Beijing strains with 100% accuracy. This simple, quick, and cost-effective multiplex PCR assay can be used for a survey or for monitoring the prevalence of Beijing genotype M. tuberculosis strains. © 2013, American Society for Microbiology.

Characterization of extensively drug-resistant Mycobacterium tuberculosis in Nepal
Ajay Poudel, Bhagwan Maharjan, Chie Nakajima, Yukari Fukushima, Basu D. Pandey, Antje Beneke, Yasuhiko Suzuki
TUBERCULOSIS, 93, 1, 84, 88, CHURCHILL LIVINGSTONE, 2013年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global control of TB. Although molecular characterization of drug resistance-associated mutations in multidrug-resistant isolates in Nepal has been made, mutations in XDR isolates and their genotypes have not been reported previously. In this study, we identified and characterized 13 XDR Mycobacterium tuberculosis isolates from clinical isolates in Nepal. The most prevalent mutations involved in rifampicin, isoniazid, ofloxacin, and kanamycin/capreomycin resistance were Ser531Leu in rpoB gene (92.3%), Ser315Thr in katG gene (92.3%), Asp94Gly in gyrA gene (53.9%) and A1400G in rrs gene (61.5%), respectively. Spoligotyping and multilocus sequence typing revealed that 69% belonged to Beijing family, especially modern types. Further typing with 26-loci variable number of tandem repeats suggested the current spread of XDR M. tuberculosis. Our result highlights the need to reinforce the TB policy in Nepal with regard to control and detection strategies. (C) 2012 Elsevier Ltd. All rights reserved.

Revisiting a medical case of "stinging" in the human oral cavity caused by ingestion of raw squid (Cephalopoda: Teuthida): new data on the functioning of squid's spermatophores
Jose Eduardo A. R. Marian, Yukihiro Shiraki, Kumi Kawai, Sawako Kojima, Yasuhiko Suzuki, Kenzo Ono
ZOOMORPHOLOGY, 131, 4, 293, 301, SPRINGER, 2012年12月
英語, 研究論文（学術雑誌）, Male squid produce intricate spermatophores that, when transferred to the female, undergo the spermatophoric reaction, a complex process of evagination that leads to the attachment of the spermatangium, that is, the everted spermatophore containing the sperm mass. While this process is still not completely understood, the medical literature includes several reports of "oral stinging" (i.e., punctured wounds in the human oral cavity) following consumption of raw male squid, which contains undischarged spermatophores able to inflict such wounds. Here, we revisit a recent medical report of oral stinging by Shiraki et al. (Pathol Int 61:749-751, 2011), providing an in-depth reanalysis of their histological biopsies and revealing vital information on the functioning of squid spermatophores. The morphology of the spermatangia attached within the oral cavity is similar to the condition found in spermatangia naturally attached to female squids. The spermatangia were able to superficially puncture the superficial layers of the oral stratified squamous epithelium, and numerous, minute stellate particles from the squid spermatophore were found adhered to the oral epithelium. These findings corroborate previous hypotheses on the functioning of squid spermatophores, namely that spermatophore attachment generally involves tissue scarification, and that stellate particles play a vital role in the attachment process. Moreover, spermatophore attachment is confirmed to be autonomous (i.e., performed by the spermatophore itself) in another squid species (possibly a loliginid), and the results strongly indicate that the attachment mechanism is not dependent upon a specialized epithelium, nor a mate's specific chemical stimulus. From the pathological point of view, the best prophylactic measure at present is the removal of the internal organs of the raw squid prior to its consumption.

Molecular mechanism of rifampicin and isoniazid resistance in Mycobacterium tuberculosis from Bangladesh
Zeaur Rahim, Chie Nakajima, Rubhana Raqib, Khalequ Zaman, Hubert P. Endtz, Adri G. M. van der Zanden, Yasuhiko Suzuki
TUBERCULOSIS, 92, 6, 529, 534, CHURCHILL LIVINGSTONE, 2012年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Despite having 100% coverage of directly observed treatment short-course, multi drug-resistant (MDR) tuberculosis (TB) is still increasing in Bangladesh. Early detection of MDR-TB by rapid molecular test and early initiation of treatment will effectively stop this trend. To develop rapid diagnostic tools, molecular characterization of genes conferring Mycobacterium tuberculosis resistance to rifampicin (RIF) and isoniazid (INH) will be required. Hence, this study elucidated the molecular mechanism RIF and INH resistance in 218 MDR strains from hospitalized (n = 161) and non-hospitalized (n = 57) TB patients in Bangladesh. Mutations in rpoB gene were detected in 207 (95.0%) with majority at codon 531 (52.3%). Mutations in katG or inhA or both were detected in 206 (94.5%) with majority at codon 315 of katG (83.9%). It was noteworthy that a novel C to T mutation at position 34 and G to A mutations at position -47 in inhA regulatory region were found, respectively, in combination with mutation at codon 315 of katG. This is the first comprehensive molecular analysis of rpoB and katG genes and inhA regulatory regions of MDR isolates from Bangladesh. This study provides basic data for the construction of low cost tailor-made molecular system for rapid diagnosis of MDR-TB in Bangladesh. (C) 2012 Elsevier Ltd. All rights reserved.

Mycobacterium bovis infection at the interface between domestic and wild animals in Zambia
Mudenda B. Hang'ombe, Musso Munyeme, Chie Nakajima, Yukari Fukushima, Haruka Suzuki, Wigganson Matandiko, Akihiro Ishii, Aaron S. Mweene, Yasuhiko Suzuki
BMC VETERINARY RESEARCH, 8, 221, 221, BIOMED CENTRAL LTD, 2012年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important.
Results: A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia.
Conclusions: These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host.

Heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity
Peter S. Lee, Reiko Yoshida, Damian C. Ekiert, Naoki Sakai, Yasuhiko Suzuki, Ayato Takada, Ian A. Wilson
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 109, 42, 17040, 17045, NATL ACAD SCIENCES, 2012年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes.

Impact of Amino Acid Substitutions in B Subunit of DNA Gyrase in Mycobacterium leprae on Fluoroquinolone Resistance
Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki
PLoS Neglected Tropical Diseases, 6, 10, e1838, 10, 2012年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Ofloxacin is a fluoroquinolone (FQ) used for the treatment of leprosy. FQs are known to interact with both A and B subunits of DNA gyrase and inhibit supercoiling activity of this enzyme. Mutations conferring FQ resistance have been reported to be found only in the gene encoding A subunit of this enzyme (gyrA) of M. leprae, although there are many reports on the FQ resistance-associated mutation in gyrB in other bacteria, including M. tuberculosis, a bacterial species in the same genus as M. leprae. Methodology/Principal Findings: To reveal the possible contribution of mutations in gyrB to FQ resistance in M. leprae, we examined the inhibitory activity of FQs against recombinant DNA gyrases with amino acid substitutions at position 464, 502 and 504, equivalent to position 461, 499 and 501 in M. tuberculosis, which are reported to contribute to reduced sensitivity to FQ. The FQ-inhibited supercoiling assay and FQ-induced cleavage assay demonstrated the important roles of these amino acid substitutions in reduced sensitivity to FQ with marked influence by amino acid substitution, especially at position 502. Additionally, effectiveness of sitafloxacin, a FQ, to mutant DNA gyrases was revealed by low inhibitory concentration of this FQ. Significance: Data obtained in this study suggested the possible emergence of FQ-resistant M. leprae with mutations in gyrB and the necessity of analyzing both gyrA and gyrB for an FQ susceptibility test. In addition, potential use of sitafloxacin for the treatment of problematic cases of leprosy by FQ resistant M. leprae was suggested. © 2012 Yokoyama et al.

Impact of Amino Acid Substitutions in B Subunit of DNA Gyrase in Mycobacterium leprae on Fluoroquinolone Resistance
Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki
PLOS NEGLECTED TROPICAL DISEASES, 6, 10, PUBLIC LIBRARY SCIENCE, 2012年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Ofloxacin is a fluoroquinolone (FQ) used for the treatment of leprosy. FQs are known to interact with both A and B subunits of DNA gyrase and inhibit supercoiling activity of this enzyme. Mutations conferring FQ resistance have been reported to be found only in the gene encoding A subunit of this enzyme (gyrA) of M. leprae, although there are many reports on the FQ resistance-associated mutation in gyrB in other bacteria, including M. tuberculosis, a bacterial species in the same genus as M. leprae.
Methodology/Principal Findings: To reveal the possible contribution of mutations in gyrB to FQ resistance in M. leprae, we examined the inhibitory activity of FQs against recombinant DNA gyrases with amino acid substitutions at position 464, 502 and 504, equivalent to position 461, 499 and 501 in M. tuberculosis, which are reported to contribute to reduced sensitivity to FQ. The FQ-inhibited supercoiling assay and FQ-induced cleavage assay demonstrated the important roles of these amino acid substitutions in reduced sensitivity to FQ with marked influence by amino acid substitution, especially at position 502. Additionally, effectiveness of sitafloxacin, a FQ, to mutant DNA gyrases was revealed by low inhibitory concentration of this FQ.
Significance: Data obtained in this study suggested the possible emergence of FQ-resistant M. leprae with mutations in gyrB and the necessity of analyzing both gyrA and gyrB for an FQ susceptibility test. In addition, potential use of sitafloxacin for the treatment of problematic cases of leprosy by FQ resistant M. leprae was suggested.

Pulmonary tuberculosis in patients with diabetes mellitus in Bangladesh
Zeaur Rahim, Mst. Sabiha Banu Momi, Sajal K. Saha, K. Zaman, Khwaja Nazim Uddin, S. N. A. Ashraf Jamil, Nazmun Nahar, A. K. Azad Khan, E. A. W. D. Cooreman, Motiuddin Ahmed, A. G. M. van der Zanden, Chie Nakajima, Yasuhiko Suzuki
INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE, 16, 8, 1132, 1133, INT UNION AGAINST TUBERCULOSIS LUNG DISEASE (I U A T L D), 2012年08月, ［査読有り］, ［国際誌］
英語

Dominant Incidence of Multidrug and Extensively Drug-Resistant Specific Mycobacterium tuberculosis Clones in Osaka Prefecture, Japan
Aki Tamaru, Chie Nakajima, Takayuki Wada, Yajun Wang, Manabu Inoue, Ryuji Kawahara, Ryoji Maekura, Yuriko Ozeki, Hisashi Ogura, Kazuo Kobayashi, Yasuhiko Suzuki, Sohkichi Matsumoto
PLOS ONE, 7, 8, e42505, PUBLIC LIBRARY SCIENCE, 2012年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Infection and transmission of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb) and extensively drug-resistant M. tuberculosis (XDR-Mtb) is a serious health problem. We analyzed a total of 1,110 Mtb isolates in Osaka Prefecture and neighboring areas from April 2000 to March 2009. A total of 89 MDR-Mtb were identified, 36 (48.5%) of which were determined to be XDR-Mtb. Among the 89 MDR-Mtb isolates, 24 (27.0%) phylogenetically distributed into six clusters based on mycobacterial interspersed repetitive units-various number of tandem repeats (MIRU-VNTR) typing. Among these six clusters, the MIRU-VNTR patterns of four (OM-V02, OM-V03, OM-V04, and OM-V06) were only found for MDR-Mtb. Further analysis revealed that all isolates belonging to OM-V02 and OM-V03, and two isolates from OM-V04 were clonal. Importantly such genotypes were not observed for drug-sensitive isolates. These suggest that few but transmissible clones can transmit after acquiring multidrug resistance and colonize even in a country with a developed, well-organized healthcare system.

Rapid Identification of Mycobacterium tuberculosis in BACTEC MGIT960 Cultures by In-House Loop-Medicated Isothermal Amplification
Janisara Rudeeaneksin, Supranee Bunchoo, Sopa Srisungngam, Pathom Sawanpanyalert, Sawet Chamnangrom, Atipa Kamolwat, Porntip Thanasripakdeekul, Tooru Taniguchi, Chie Nakajima, Yasuhiko Suzuki, Benjawan Phetsuksiri
JAPANESE JOURNAL OF INFECTIOUS DISEASES, 65, 4, 306, 311, NATL INST INFECTIOUS DISEASES, 2012年07月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Definitive diagnosis of tuberculosis (TB) by conventional culture, followed by bacterial identification based on biochemical tests is time-consuming and tedious. Simple loop-mediated isothermal amplification (LAMP) specific for Mycobacterium tuberculosis complex, targeting the M. tuberculosis 16S ribosomal RNA gene, termed TB-LAMP, was evaluated as an alternative for rapid culture confirmation. TB-LAMP was assessed for its ability to detect M. tuberculosis complex in BACTEC MGIT 960-positive cultures. Of the 103 cultures evaluated, 100 were identified to contain M. tuberculosis complex by TB-LAMP and had concordant results with standard biochemical tests of niacin accumulation, nitrate reductase, lack of heat-stable catalase, and susceptibility to para-nitrobenzoic acid. These results indicate that TB-LAMP in combination with BACTEC MOLT 960 is a specific, reliable, and technically feasible method for rapid and accurate identification of M. tuberculosis complex.

Genetic diversity of Mycobacterium avium subsp hominissuis strains isolated from humans, pigs, and human living environment
Tomotada Iwamoto, Chie Nakajima, Yukiko Nishiuchi, Tomoko Kato, Shiomi Yoshida, Noriko Nakanishi, Aki Tamaru, Yutaka Tamura, Yasuhiko Suzuki, Masao Nasu
INFECTION GENETICS AND EVOLUTION, 12, 4, 846, 852, ELSEVIER SCIENCE BV, 2012年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mycobacterium avium subsp. hominissuis (MAH) strains are genetically diverse and cause infections in pigs and humans. To elucidate the geographical and host-dependent variations in the genetic diversity of MAH, we performed variable numbers of tandem repeat (VNTR) analysis targeting 19 loci for MAH samples from humans (n = 146), bathroom environments (n = 37), and pigs (n = 75) in Japan; these data were then compared with previously reported VNTR data from other countries. The minimum spanning tree (MST) and the multi-dimensional scaling (MDS) analyses based on the VNTR data indicated a high degree of genetic relatedness between isolates from humans and bathrooms in Japan, but a low degree of similarity with the isolates from France and Finland. Moreover, the comparison showed a higher similarity of isolates from Japanese pigs with those from French humans and pigs and Finnish humans and pigs than with other isolates from humans and bathrooms in Japan. The singularity of the Japanese MAH was characterized as the prevalence of hsp65 sequevar code 15 and ISMav6 for the human and bathroom isolates; however, none of the isolates obtained from the pigs belonged to the code 15 or possessed ISMav6. The genetic diversity of MAH and its regional variations imply a possible regional or local specific source of infection and route of transmission of MAH for humans. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

A New Loop-Mediated Isothermal Amplification Method for Rapid, Simple, and Sensitive Detection of Leptospira spp. in Urine
Nobuo Koizumi, Chie Nakajima, Tsunehito Harunari, Tsutomu Tanikawa, Toshihiro Tokiwa, Eriko Uchimura, Tokujiro Furuya, Claro Niegos Mingala, Marvin Ardeza Villanueva, Makoto Ohnishi, Yasuhiko Suzuki
JOURNAL OF CLINICAL MICROBIOLOGY, 50, 6, 2072, 2074, AMER SOC MICROBIOLOGY, 2012年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.

Quinolone-Induced Upregulation of Osteopontin Gene Promoter Activity in Human Lung Epithelial Cell Line A549
Beata Shiratori, Jing Zhang, Osamu Usami, Haorile Chagan-Yasutan, Yasuhiko Suzuki, Chie Nakajima, Toshimitsu Uede, Toshio Hattori
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 56, 6, 2868, 2872, AMER SOC MICROBIOLOGY, 2012年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Quinolones, in addition to their antibacterial activities, act as immunomodulators. Osteopontin (OPN), a member of the extracellular matrix proteins, was found to play a role in the immune and inflammatory response. We found that quinolones significantly enhanced OPN secretion, namely, garenoxacin (220%), moxifloxacin (62%), gatifloxacin (82%), sparfloxacin, (79%), and sitafloxacin (60%). Enhancement of OPN secretion was shown to be due to the effect of quinolones on the OPN gene promoter activity. We also examined the role of quinolones on apoptosis and found that sparfloxacin decreased the late apoptosis of A549 cells, but garenoxacin did not show the antiapoptotic effect. The antiapoptotic effects of quinolones do not appear to be associated with OPN elevation.

Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolated in Nepal
Ajay Poudel, Chie Nakajima, Yukari Fukushima, Haruka Suzuki, Basu Dev Pandey, Bhagwan Maharjan, Yasuhiko Suzuki
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 56, 6, 2831, 2836, AMER SOC MICROBIOLOGY, 2012年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIP-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.

Sensitivities of ciprofloxacin-resistant Mycobacterium tuberculosis clinical isolates to fluoroquinolones: role of mutant DNA gyrase subunits in drug resistance
Yasuhiko Suzuki, Chie Nakajima, Aki Tamaru, Hyun Kim, Takashi Matsuba, Hajime Saito
INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, 39, 5, 435, 439, ELSEVIER SCIENCE BV, 2012年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Minimum inhibitory concentrations of sitafloxacin, gatifloxacin, moxifloxacin, sparfloxacin, levofloxacin and ciprofloxacin against 59 ciprofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Japan were determined. The isolates were most susceptible to sitafloxacin and gatifloxacin. To understand better the basis for drug resistance, nucleotide sequences encoding the gyrA and gyrB quinolone resistance-determining region were determined. Predicted amino acid sequences revealed distinct mutational patterns likely to be responsible for fluoroquinolone resistance. Double gyrA mutations as well as mutations in both gyrA and gyrB correlated with increased resistance to all fluoroquinolones. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

A Rapid Loop-Mediated Isothermal Amplification Assay Targeting hspX for the Detection of Mycobacterium tuberculosis Complex
Aixiao Bi, Chie Nakajima, Yukari Fukushima, Aki Tamaru, Isamu Sugawara, Akio Kimura, Ryuji Kawahara, Zhongyi Hu, Yasuhiko Suzuki
JAPANESE JOURNAL OF INFECTIOUS DISEASES, 65, 3, 247, 251, NATL INST INFECTIOUS DISEASES, 2012年05月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, A rapid, simple, and low-cost diagnostic tool for tuberculosis (TB) detection is urgently needed in countries with a high TB burden. Here, we report a novel loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene for the rapid detection of Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti. The specificity of this assay was evaluated using 4 reference strains of Mycobacterium tuberculosis complex (MTC), 22 species of non-tuberculous mycobacteria (NTM), 7 non-mycobacterial species, and 50 clinical M. tuberculosis isolates. All the reference MTC strains and M. tuberculosis clinical isolates were successfully detected by this method, and there were no false-positive results with NTM or non-mycobacterial species, which demonstrates the high specificity of this assay for MTC. The detection limit was 10 copies of MTC genome within 27 min, and the detection speed of this assay was higher than that of any other isothermal methods reported so far. Because of its speed, simplicity, sensitivity, specificity, and inexpensiveness, the TB hspX LAMP assay is a potential gene diagnostic method for TB detection in developing countries with a high TB burden.

Distinct Clinical Features in Nontuberculous Mycobacterial Disease with or without Latent Tuberculosis Infection
Umme Ruman Siddiqi, Haorile Chagan-Yasutan, Chie Nakajima, Hiroki Saitoh, Yugo Ashino, Osamu Usami, Beata Shiratori, Motoki Usuzawa, Yasuhiko Suzuki, Toshio Hattori
TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, 226, 4, 313, 319, TOHOKU UNIV MEDICAL PRESS, 2012年04月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Nontuberculous mycobacteria (NTM) diseases are in the face of a progressive increase even in immune-competent subjects, and the clinical features of NTM diseases are heterogenous. The decision to institute treatment of the patients should be made after a period of follow up, because therapy is often prolonged, and frequently ineffective. The reasons why some patients develop severe NTM diseases are not clear. Here we observed the involvement of latent tuberculosis infection (LTBI) in clinical and laboratory features of NTM diseases. We evaluated various tuberculosis-related inflammatory markers including osteopontin (OPN), pentraxin-3 (PTX-3), and soluble IL-2 receptor (sIL-2R) in NTM infected patients with or without LTBI. Eight NTM and 5 tuberculosis (TB) patients, and 5 healthy subjects were enrolled. Polymerase Chain Reaction (PCR) analysis confirmed the absence of tuberculosis specific gene (RD1 region), among clinical isolates from NTM patients. Interferon-gamma (IFN-gamma) release assay (IGRA) using Early Secreted Antigenic Target-6 (ESAT-6) and CFP-10, the RD1-encoded protein, was employed for determining LTBI. IGRA was positive in 4/8 NTM (NTM with LTBI, 50%) and 5/5 TB patients. Only 2 of 4 NTM with LTBI were under chemotherapy among all NTM patients, and others were followed up. The plasma levels of OPN, PTX3 and sIL-2R were significantly higher in NTM patients with LTBI than in those without LTBI (P < 0.05). The two patients under therapy showed the highest OPN levels that persisted after treatment. The increased inflammatory levels in NTM patients with LTBI indicate enhanced inflammatory reaction. Extensive therapy may be necessary in such patients.

Protective Efficacy of Neutralizing Monoclonal Antibodies in a Nonhuman Primate Model of Ebola Hemorrhagic Fever
Andrea Marzi, Reiko Yoshida, Hiroko Miyamoto, Mari Ishijima, Yasuhiko Suzuki, Megumi Higuchi, Yukie Matsuyama, Manabu Igarashi, Eri Nakayama, Makoto Kuroda, Masayuki Saijo, Friederike Feldmann, Douglas Brining, Heinz Feldmann, Ayato Takada
PLOS ONE, 7, 4, e36192, PUBLIC LIBRARY SCIENCE, 2012年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.

Possible horizontal transmission of Salmonella via reusable egg trays in Thailand
Fuangfa Utrarachkij, Srirat Pornraungwong, Kanokrat Siripanichgon, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 154, 1-2, 73, 78, ELSEVIER SCIENCE BV, 2012年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Salmonella contamination of eggshells, egg contents, reusable egg trays, and various environmental samples was assessed. Although the overall Salmonella contamination rate from egg farms was low (3.2%), over a quarter (26.7%) of egg trays from farms and more than one third (36.7%) of trays from the market were contaminated. Salmonella strains isolated from reusable egg trays were analyzed by serotyping, antimicrobial susceptibility test and Xbal pulsed-field gel electrophoresis (PFGE) typing. Five serovars (S. Braenderup, S. Emek, S. Weltevreden, S. Stanley, and S. Derby) were isolated, and half of the strains assessed were found to be resistant to one or more of the six antimicrobial agents examined. The overall resistance rates to nalidixic acid, trimethoprim-sulfamethoxazole, tetracycline, and ampicillin were 40.7%, 36.0%, 26.7% and 3.5%, respectively. The PFGE types were matched against sample location and drug resistance. S. Braenderup PFGE type A2 (susceptible to all tested drugs) was isolated from all sample sites; PFGE type A2 (resistant to nalidixic acid) was isolated from Farm C and the market. S. Braenderup PFGE type A1 (resistant to four drugs) was isolated from Farms A and C. S. Weltevreden PFGE type C3 (susceptible to all tested drugs) was isolated from Farms A and B and type C4 (susceptible to all tested drugs) was isolated from Farm A and the market. The distribution of the related genotypes and resistance patterns of Salmonella in egg farms and the market indicate drug-resistant strains of Salmonella may be spread on reusable egg trays. (C) 2011 Elsevier B.V. All rights reserved.

Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae
Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 56, 2, 697, 702, AMER SOC MICROBIOLOGY, 2012年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Amino acid substitutions at position 89 or 91 in GyrA of fluoroquinolone-resistant Mycobacterium leprae clinical isolates have been reported. In contrast, those at position 94 in M. tuberculosis, equivalent to position 95 in M. leprae, have been identified most frequently. To verify the possible contribution of amino acid substitutions at position 95 in M. leprae to fluoroquinolone resistance, we conducted an in vitro assay using wild-type and mutant recombinant DNA gyrases. Fluoroquinolone-mediated supercoiling activity inhibition assay and DNA cleavage assay revealed the potent contribution of an amino acid substitution of Asp to Gly or Asn at position 95 to fluoroquinolone resistance. These results suggested the possible future emergence of quinolone-resistant M. leprae isolates with these amino acid substitutions and the usefulness of detecting these mutations for the rapid identification of fluoroquinolone resistance in leprosy.

Characterization of Toxoplasma gondii 5’ UTR with encyclopedic TSS information
Yamagishi J, Watanabe J, Goo YK, Masatani T, Suzuki Y, Xuan X
The Journal of Parasitology, 98, 2, 445, 447, American Society of Parasitologists, 2012年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The 5′ UTR is widely involved in gene expression via post-transcriptional regulation. However, a detailed profile of the 5′ UTR for Toxoplasma gondii has not yet been demonstrated. To investigate the issue, we compared the predicted open reading frames (ORFs) and transcription start sites (TSSs) of T. gondii obtained by TSS-seq, a method that enables analysis of encyclopedic TSSs with next-generation sequencers. As a result, it was demonstrated that the mode length of the 5′ UTR is between 120 and 140 nucleotides (nts) when a subset of genes with predicted signal peptides was examined. However, when genes without the signal peptide were examined, the length was extended to approximately 600 nts. Because additional information on the predicted signal peptide generates increased reliability to the 5′ end estimation of each ORF, we believe that the former value was more reliable as a representative of the 5′ UTR length of T. gondii. The discrepancy suggests that current predictions of the 5′ end of the ORF were less accurate and considerably more discordant with the natural status. The 5′ untranslated region (5′ UTR) is defined as that between the 5′ end of the transcripts and just in front of a start codon of an ORF. Therefore, the 5′ UTR does not contain any information for a protein sequence; however, it is involved in the control of protein expression via the modulation of translational efficiency (Kozak, 1991b; Hughes, 2006).http://www.journalofparasitology.org/doi/abs/10.1645/GE-2864.1

Comparison of human blood concentrations of collectin kidney 1 and mannan-binding lectin
Takayuki Yoshizaki, Katsuki Ohtani, Wataru Motomura, Seong-Jae Jang, Ken-ichiro Mori, Noritoshi Kitamoto, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
JOURNAL OF BIOCHEMISTRY, 151, 1, 57, 64, OXFORD UNIV PRESS, 2012年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mannan-binding lectin (MBL) was first discovered as a collectin in animal blood, and was shown to have such unique characteristics as a collage-like domain and a carbohydrate recognition domain. We recently identified human collectin kidney 1 (CL-K1, COLEC11) from a human kidney cDNA library. To quantitate the CL-K1 concentration in blood, we developed several polyclonal and monoclonal antibodies using recombinant human CL-K1 in CHO cells and the CL-K1 fragment in Escherichia coli. Using these antibodies, we established a sandwich enzyme-linked immunosorbent assay (ELISA) system. The concentration of CL-K1 in human plasma was 0.34 +/- 0.13 mu g/ml and that in MBL was 1.72 +/- 1.51 mu g/ml. Concentrations of MBL are often low due to its single nucleotide polymorphisms (SNPs) which seem to be related to an opsonic defect. However, no low concentrations of CL-K1 were observed on testing over two hundred blood samples. We also found that the blood concentration of CL-K1 was not dependent on gender or age and did not correlate completely with that of MBL. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of CL-K1 in humans.

Influence of Lineage-Specific Amino Acid Dimorphisms in GyrA on Mycobacterium tuberculosis Resistance to Fluoroquinolones
Hyun Kim, Chie Nakajima, Youn Uck Kim, Kazumasa Yokoyama, Yasuhiko Suzuki
JAPANESE JOURNAL OF INFECTIOUS DISEASES, 65, 1, 72, 74, NATL INST INFECTIOUS DISEASES, 2012年01月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, We conducted in vitro DNA supercoiling assays, utilizing recombinant DNA gyrases, to elucidate the influence of the lineage-specific serine or threonine residue at position 95 of GyrA on fluoroquinolone resistance in Mycobacterium tuberculosis. There was little effect of the GyrA-Ala74Ser amino acid substitution on activity of the GyrA-Ser95 gyrase, while activity of the GyrA-Asp94Gly-Ser95 gyrase was reduced. These findings were in striking contrast to previous reports analyzing GyrA with Thr95 and suggest an important impact of the amino acid in the development of fluoroquinolone resistance.

Frequent Detection of Anti-Tubercular-Glycolipid-IgG and -IgA Antibodies in Healthcare Workers with Latent Tuberculosis Infection in the Philippines
Umme Ruman Siddiqi, Prisca Susan A. Leano, Haorile Chagan-Yasutan, Beata Shiratori, Hiroki Saitoh, Yugo Ashino, Yasuhiko Suzuki, Toshio Hattori, Elizabeth Freda O. Telan
CLINICAL & DEVELOPMENTAL IMMUNOLOGY, 2012, 610707, 610707, HINDAWI PUBLISHING CORPORATION, 2012年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Anti-tubercular-glycolipid-IgG (TBGL-IgG) and -IgA (TBGL-IgA) antibodies, and the QuantiFERON-TB Gold test (QFT) were compared in healthcare workers (HCWs, n = 31) and asymptomatic human immunodeficiency virus-carriers (HIV-AC, n = 56) in Manila. In HCWs, 48%, 51%, and 19% were positive in QFT, TBGL-IgG, and -IgA, respectively. The TBGL-IgG positivity was significantly higher (P = 0.02) in QFT-positive than QFT-negative HCWs. Both TBGL-IgG- and -IgA-positive cases were only found in QFT-positive HCWs (27%). The plasma IFN-gamma levels positively correlated with TBGL-IgA titers (r = 0.74, P = 0.005), but not TBGL-IgG titers in this group, indicating that mucosal immunity is involved in LTBI in immunocompetent individuals. The QFT positivity in HIV-AC was 31% in those with CD4+ cell counts >350/mu L and 12.5% in low CD4 group (<350/mu L). 59 % and 29% were positive for TBGL-IgG and -IgA, respectively, in HIV-AC, but no association was found between QFT and TBGL assays. TBGL-IgG-positive rates in QFT-positive and QFT-negative HIV-AC were 61% and 58%, and those of TBGL-IgA were 23% and 30%, respectively. The titers of TBGL-IgA were associated with serum IgA (P = 0.02) in HIV-AC. Elevations of TBGL-IgG and -IgA were related to latent tuberculosis infection in HCWs, but careful interpretation is necessary in HIV-AC.

Biological Functions of the Novel Collectins CL-L1, CL-K1, and CL-P1
Katsuki Ohtani, Yasuhiko Suzuki, Nobutaka Wakamiya
JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY, 2012, 493945, 493945, HINDAWI PUBLISHING CORPORATION, 2012年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Collectins are characterized by a collagen-like sequence and a carbohydrate recognition domain and are members of the vertebrate C-type lectin superfamily. Recently, "novel collectins", different from "classical collectins" consisting of mannan-binding lectin (MBL) and surfactant proteins A and D (SP-A and SP-D), have been found by reverse genetics. These "novel collectins" consist of collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1), and collectin placenta 1 (CL-P1) and are encoded by three separate genes. Experimental findings on human and animal collectins have shown that both novel collectins and classical collectins play an important role in innate immunity. Based on our recent results and those of others, in this paper, we summarize the new biological functions of these novel collectins in embryonic morphogenesis and development.

Comparative genome analysis of three eukaryotic parasites with differing abilities to transform leukocytes reveals key mediators of theileria-induced leukocyte transformation
Hayashida K, Hara Y, Abe T, Chisato Yamasaki C.Y, Toyoda A, Kosuge T, Suzuki Y, Sato Y, Kawashima S, Katayama T, Wakaguri H, Noboru Inoue N.I, Keiichi Homma K.H, Tada-Umezaki M, Yagi Y, Yasuyuki Fujii Y.F, Takuya Habara T.H, Kanehisa M, Hidemi Watanabe H, W, Ito K, Gojobori T, Hideaki Sugawara H.S, Imanishi T, Weir W, Gardner M, Arnab Pain A, P, Shiels B, Hattori M, Nene V, Sugimoto C
mBio, 3, 5, 2012年, ［査読有り］

Molecular cloning and functional analysis of scavenger receptor zebrafish CL-P1.
Mitsuko Fukuda, Katsuki Ohtani, Seong-Jae Jang, Takayuki Yoshizaki, Ken-Ichiro Mori, Wataru Motomura, Itsuro Yoshida, Yasuhiko Suzuki, Yutaka Kohgo, Nobutaka Wakamiya
Biochimica et biophysica acta, 1810, 12, 1150, 9, 12, 2011年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Scavenger receptors are generally expressed in macrophages and vascular endothelial cells and some scavenger receptors are thought to contribute to the development of atherosclerosis. METHODS: We cloned the cDNA of a zebrafish CL-P1 (collectin placenta 1) and performed a knockdown study using its antisense morpholino oligonucleotides (MO). RESULTS: Zebrafish CL-P1 (zCL-P1) is 51% identical to human CL-P1 in its amino acid sequence. Microbes and OxLDL bound to zCL-P1 cDNA transfected cells. zCL-P1 mRNA expression gradually increased after 6hours post-fertilization (hpf), reached its highest level at 24hpf, and then decreased, which is similar to the gene expression pattern of Tie-2. The knockdown of zCL-P1 led to an increase in the number of zebrafish embryos with severe morphological abnormalities such as short body lengths and defects in the dorsal aorta at 48hpf. Simultaneous injection of both MO and synthetic zCL-P1 or zVEGF mRNA rescued the abnormal phenotype. CONCLUSIONS: In vivo knockdown study shows that zCL-P1 is implicated in vasculogenesis and those of our in vitro study support its role as a scavenger receptor. GENERAL SIGNIFICANCE: These results suggest that zCL-P1 might be essential for vasculogenesis during the early embryonic phase in bone fish.

Stinging in the oral cavity caused by ingestion of the sperm bags of a squid: a case report.
Yukihiro Shiraki, Kumi Kawai, Sawako Kojima, Yasuhiko Suzuki, Kenzo Ono
Pathology international, 61, 12, 749, 51, 2011年12月, ［国際誌］
英語, 研究論文（学術雑誌）, We present a case of stinging in the oral cavity caused by ingestion of the sperm bags of a squid. The patient experienced severe pain in her oral cavity immediately after eating raw squid. When she was examined at our hospital, we found that several small whitish spindle-shaped stings were stuck to the mucous membrane of the hard palate. A biopsy was performed, and the whitish stings were removed as well. We also performed a histological examination of the remaining part of the raw squid brought by the patient. The biopsy showed that the sperm bags of the squid had thrust into the squamous epithelium of the patient. The remaining part of the raw squid consisted of the testis and the sperm bags. After removal of all stings, the pain reduced, and the wound healed in due course. Larva migrans and anisakiasis are infections known to be caused by consumption of raw seafood. Although the condition reported here is relatively rare, doctors should also keep this condition in mind for patients reporting pain after eating raw seafood.

Molecular cloning and functional analysis of scavenger receptor zebrafish CL-P1
Fukuda Mitsuko, Ohtani Katsuki, Jang Seong-Jae, Yoshizaki Takayuki, Mori Ken-ichiro, Motomura Wataru, Yoshida Itsuro, Suzuki Yasuhiko, Kohgo Yutaka, Wakamiya Nobutaka
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1810, 12, 1150, 1159, 2011年12月, ［査読有り］

The induction of human CL-P1 expression in hypoxia/reoxygenation culture condition and rat CL-P1 after ischemic/reperfusion treatment.
Satoshi Koyama, Katsuki Ohtani, Jun Fukuzawa, Naoyuki Yao, Mitsuko Fukuda, Seong-Jae Jang, Naoyuki Hasebe, Kenjiro Kikuchi, Hiroyuki Itabe, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
Biochimica et biophysica acta, 1810, 9, 836, 42, 9, 2011年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Oxidative stress-induced endothelial dysfunction and oxidized low-density lipoprotein (LDL) might play a key role in the pathogenesis of atherosclerosis. We recently identified a vascular endothelial scavenger receptor, collectin placenta 1 (CL-P1), which acts as a receptor for oxidized LDL as well as for microbes. METHODS: We demonstrate how hypoxic and oxidative stress induced CL-P1 expression and compared their effects with the expression of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), an endothelial scavenger receptor expressed by oxidative stress. RESULTS: Hypoxia/reoxygenation induced CL-P1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs). The expression of LOX-1 mRNA in these cells peaked slightly at 24 h, while the expression of CL-P1 had an onset at 72 h and was sustained for 120 h after reoxygenation. Furthermore, the exposure of rat carotid artery endothelium to ischemia/reperfusion increased the maximal CL-P1 mRNA expression at 72 h and expression of its protein peaked at 7 days after this treatment. We demonstrate that CL-P1 up-regulation is induced in vitro and in vivo by oxidative stress. GENERAL SIGNIFICANCE: The inducible expression of CL-P1 by oxidative stress might play a crucial role in endothelial dysfunction or chronic activation leading to the pathogenesis of atherosclerosis.

The induction of human CL-P1 expression in hypoxia/reoxygenation culture condition and rat CL-P1 after ischemic/reperfusion treatment
Satoshi Koyama, Katsuki Ohtani, Jun Fukuzawa, Naoyuki Yao, Mitsuko Fukuda, Seong-Jae Jang, Naoyuki Hasebe, Kenjiro Kikuchi, Hiroyuki Itabe, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1810, 9, 836, 842, ELSEVIER SCIENCE BV, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Oxidative stress-induced endothelial dysfunction and oxidized low-density lipoprotein (LDL) might play a key role in the pathogenesis of atherosclerosis. We recently identified a vascular endothelial scavenger receptor, collectin placenta 1 (CL-Pi), which acts as a receptor for oxidized LDL as well as for microbes.
Methods: We demonstrate how hypoxic and oxidative stress induced CL-Pi expression and compared their effects with the expression of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), an endothelial scavenger receptor expressed by oxidative stress.
Results: Hypoxia/reoxygenation induced CL-P1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs). The expression of LOX-1 mRNA in these cells peaked slightly at 24 h, while the expression of CL-P1 had an onset at 72 h and was sustained for 120 h after reoxygenation. Furthermore, the exposure of rat carotid artery endothelium to ischemia/reperfusion increased the maximal CL-P1 mRNA expression at 72 h and expression of its protein peaked at 7 days after this treatment. We demonstrate that CL-P1 up-regulation is induced in vitro and in vivo by oxidative stress.
General significance: The inducible expression of CL-P1 by oxidative stress might play a crucial role in endothelial dysfunction or chronic activation leading to the pathogenesis of atherosclerosis. (C) 2011 Elsevier B.V. All rights reserved.

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance
Hyun Kim, Chie Nakajima, Kazumasa Yokoyama, Zeaur Rahim, Youn Uck Kim, Hiroki Oguri, Yasuhiko Suzuki
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 55, 8, 3661, 3667, AMER SOC MICROBIOLOGY, 2011年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.

A case of autoimmune hepatitis with Graves' disease treated by propylthiouracil.
Ikuko Sato, Taku Tsunekawa, Yuri Shinohara, Yuichiro Nishio, Yuko Shimizu, Yasuhiko Suzuki, Shuko Yoshioka
Nagoya journal of medical science, 73, 3-4, 205, 9, 2011年08月, ［国内誌］
英語, 研究論文（学術雑誌）, A 58-year-old woman was referred to our hospital because of liver dysfunction. Her serum levels of AST (619 IU/l) and ALT (603 IU/l) had increased. Histological findings in the liver biopsy were compatible to autoimmune hepatitis (AIH), and the diagnosis of AIH was confirmed by the diagnostic criteria. She was admitted to a nearby hospital 3 years ago, and diagnosed with Graves' disease. She received methimazole (MMI) at first, which was discontinued due to liver injury in one month, then propylthiouracil (PTU) was administered. One year later, transaminase increased and was decreased by stopping PTU administration. PTU was restarted after her transaminase decreased, but a recurrence of hepatotoxicity was observed, and she was referred to our hospital. Oral prednisolone decreased liver function immediately. In this case, PTU-induced liver injury was suspected as a possible trigger of AIH. While PTU remains a commonly used drug in the treatment of hyperthyroidism, severe liver injury is reported in some cases. If liver injury is observed in patients treated with PTU, rechallenge is not recommended in order to avoid severe hepatotoxicity.

Involvement of TRP channels in the signal transduction of bradykinin in human osteoblasts.
Yasuhiko Suzuki, Daisuke Kodama, Shigemi Goto, Akifumi Togari
Biochemical and biophysical research communications, 410, 2, 317, 21, 2011年07月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Bradykinin (BK), a mediator of pain and inflammation, is involved in bone metabolism. We have previously reported that BK increased the synthesis of interleukin-6 and prostaglandin E(2) via phosphorylation of ERK1/2 in human osteoblasts, SaM-1. In the present study, we investigated the signal transduction pathway of BK focusing on intracellular Ca(2+) kinetics in SaM-1 cells. Bath-applied BK increased intracellular Ca(2+) concentration through the activation of B(2) receptors. Removal of extracellular Ca(2+) attenuated the effects of BK. Additionally, thapsigargin, endoplasmic reticulum Ca(2+) pump inhibitor, completely inhibited BK-induced increase of intracellular Ca(2+). These results suggested that bath-applied BK activated store-operated Ca(2+) channels (SOCCs) following Ca(2+) store depletion via B(2) receptor. Although the molecular components of SOCCs have yet to be conclusively identified in all cell types, recent studies demonstrated that transient receptor potential canonical (TRPC) channels are candidates for them. TRPC1, TRPC3, TRPC4 and TRPC6 were expressed in SaM-1 cells and inhibitors of TRP channel, 2-aminoethoxydiphenyl borate, GdCl(3), LaCl(3) and flufenamic acid, inhibited the effects of BK. These findings suggested that BK activated SOCCs and induced Ca(2+) influx via B(2) receptor in human osteoblasts. Molecular components of the SOCCs are suggested to be TRPC channels.

Gene regulation function of the three specificity protein-1 (Sp1) within the human collectin placenta-1 proximal promoter
Youn Uck Kim, Katsuki Ohtani, Kenichiro Mori, Seong Jae Jang, Yasuhiko Suzuki, Nobutaka Wakamiya
GENES & GENOMICS, 33, 3, 273, 281, SPRINGER, 2011年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Scavenger receptors including collection-placenta 1 (CL-P1) are cell surface glycoproteins capable of binding to oxidized low density lipoprotein (oxLDL), which are expressed on endothelial cells, macrophages, and smooth muscle cells. We cloned and characterized a 750 base-pair fragment containing the 5'-flanking region of the human CL-P1 gene using a human genomic library. The fragment obtained from the translational start site, which contained three specificity protein-1 (Sp1) sites upstream of transcriptional start site and putative binding sites for granulocyte chemotactic factor and early growth response factor-1, was ligated to the pGL4.10-basic vector, and promoter activity was confirmed by transfection studies. Luciferase and electrophoretic gel motility shift assays revealed that three Sp1 binding sites showed specific DNA-protein binding. Deletion and mutation analyses showed that the region comprising the three Sp1 sites was required for CL-P1 proximal promoter activity in human umbilical vein endothelial cells, Hs683 cells, and SK-LMS cells. Furthermore, the third Sp1 (-28/-20) binding site was regulated through some factor, not the Sp1 protein, by hydrogen peroxide stimulation.

Genotypes and Characteristics of Clustering and Drug Susceptibility of Mycobacterium tuberculosis Isolates Collected in Heilongjiang Province, China
Juan Wang, Yan Liu, Chun-Lei Zhang, Bin-Ying Ji, Liu-Zhuo Zhang, Yong-Zhen Shao, Shui-Lian Jiang, Yasuhiko Suzuki, Chie Nakajima, Chang-Long Fan, Yuan-Ping Ma, Geng-Wen Tian, Toshio Hattori, Hong Ling
JOURNAL OF CLINICAL MICROBIOLOGY, 49, 4, 1354, 1362, AMER SOC MICROBIOLOGY, 2011年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, For the last decade China has occupied second place, after India, among the top five countries with high burdens of tuberculosis (TB). Heilongjiang Province is located in northeastern China. The prevalence of drug-resistant TB in Heilongjiang Province is higher than the average level in China. To determine the transmission characteristics of Mycobacterium tuberculosis strains isolated in this area and their genetic relationships, especially among the Beijing family strains, we investigated their genotypes. From May 2007 to October 2008, 200 M. tuberculosis isolates from patients presenting pulmonary TB were analyzed by molecular typing using PCR-based methods: spacer-oligonucleotide typing (spoligotyping), Beijing family-specific PCR (detection of the deletion of region of difference 105 [RD105]), and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Different combinations of MIRU-VNTR loci were evaluated to define the genotypes and clustering characteristics of the local strains. We found that Beijing family strains represented 89.5% of the isolates studied. However, the rates of multidrug-resistant (MDR) M. tuberculosis among Beijing and non-Beijing family strains were not statistically different. The 15-locus set is considered the optimal MIRU-VNTR locus combination for analyzing the M. tuberculosis strains epidemic in this area, while the 10-locus set is an ideal set for first-line molecular typing. We found that the clustering rate of all the M. tuberculosis isolates analyzed was 10.0% using the 15-locus set typing. We conclude that the Beijing family genotype is predominant and that highly epidemic TB and MDR TB are less likely associated with the active transmission of M. tuberculosis in the study area.

Rapid detection of Mycobacterium tuberculosis complex in Cattle and Lechwe (Kobus leche kafuensis) at the slaughter house.　　　　　　　　　　　　　　　
Hang’ombe M B, Nakajima C, Ishii A, Fukushima Y, Munyeme M, Matandiko W, Mweene A S, Suzuki Y
Vet. Sci. Dev., 1, e5, 2011年, ［査読有り］
英語, 研究論文（学術雑誌）

Possible Mode of Emergence for Drug-Resistant Leprosy Is Revealed by an Analysis of Samples from Mexico
Masanori Matsuoka, Yasuhiko Suzuki, Iris Estrada Garcia, Mary Fafutis-Morris, Alberto Vargas-Gonzalez, Cristina Carreno-Martinez, Yukari Fukushima, Clue Nakajima
JAPANESE JOURNAL OF INFECTIOUS DISEASES, 63, 6, 412, 416, NATL INST INFECTIOUS DISEASES, 2010年11月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Mexico is a country with sporadic leprosy cases, and the reemergence of drug resistance is a concern In this study, molecular analysis of Mycobacterium leprae was employed to clarify the spread of drug-resistant leprosy Thus, drug resistance-determining regions in the folP1, rpoB, and gyrA genes, which are associated with resistance to dapsone, rifampicin, and ofloxacin, respectively, were analyzed by direct sequencing of the PCR product No mutations in the folP1 gene were observed in any of the 72 slit skin samples obtained from 38 patients, although two samples carrying a mutation at codon 425 in the rpoB gene, which confers resistance to rifampicin, a key component of multidrug therapy, were identified In addition, a mutation at codon 91 in the gyrA gene, which correlates with ofloxacin resistance, was found in one sample These results demonstrate the existence of rifampicin- and ofloxacin-resistant leprosy Interestingly, wild-type and mutant sequences in the gyrA gene were found to coexist in one clinical sample In addition, all three drug resistance-related mutations were found in only one of the two earlobes of the patients concerned, suggesting a possible pathway for the spread of drug-resistant M leprae

Occurrence and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus pseudintermedius in an Academic Veterinary Hospital
Kanako Ishihara, Natsumi Shimokubo, Akie Sakagami, Hiroshi Ueno, Yasukazu Muramatsu, Tsuyoshi Kadosawa, Chie Yanagisawa, Hideaki Hanaki, Chie Nakajima, Yasuhiko Suzuki, Yutaka Tamura
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 76, 15, 5165, 5174, AMER SOC MICROBIOLOGY, 2010年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.

A Novel Method for the Purification of DNA by Capturing Nucleic Acid and Magnesium Complexes on Non-Woven Fabric Filters under Alkaline Conditions for the Gene Diagnosis of Tuberculosis by Loop-Mediated Isothermal Amplification (LAMP)
Tadashi Fukasawa, Naozumi Oda, Yasunao Wada, Aki Tamaru, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki
JAPANESE JOURNAL OF INFECTIOUS DISEASES, 63, 4, 246, 250, NATL INST INFECTIOUS DISEASES, 2010年07月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg2+) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg2+ complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg2+ complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg2+ was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.

Identification of Mycobacterium tuberculosis clinical isolates in Bangladesh by a species distinguishable multiplex PCR
Chie Nakajima, Zeaur Rahim, Yukari Fukushima, Isamu Sugawara, Adri G. M. van der Zanden, Aki Tamaru, Yasuhiko Suzuki
BMC INFECTIOUS DISEASES, 10, 118, 118, BIOMED CENTRAL LTD, 2010年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Species identification of isolates belonging to the Mycobacterium tuberculosis complex (MTC) seems to be important for the appropriate treatment of patients, since M. bovis is naturally resistant to a first line anti-tuberculosis (TB) drug, pyrazinamide, while most of the other MTC members are susceptible to this antimicrobial agent. A simple and low-cost differentiation method was needed in higher TB burden countries, such as Bangladesh, where the prevalence of M. bovis among people or cattle has not been investigated.
Methods: Genetic regions cfp32, RD9 and RD12 were chosen as targets for a species distinguishable multiplex PCR and the system was evaluated with twenty reference strains of mycobacterial species including non-tubercular mycobacteria (NTM). A total of 350 clinical MTC isolates obtained in Bangladesh were then analyzed with this multiplex PCR.
Results: All of the MTC reference strains gave expected banding patterns and no non-specific amplifications were observed in the NTM strains. Out of 350 clinical isolates examined by this method, 347 (99.1%) were positive for all of the cfp32, RD9 and RD12 and determined as M. tuberculosis. Two isolates lacked cfp32 PCR product and one lacked RD12, however, those three samples were further examined and identified as M. tuberculosis by the sequence analyses of hsp65 and gyrB.
Conclusions: The MTC-discrimination multiplex PCR (MTCD-MPCR) developed in this study showed high specificity and was thought to be very useful as a routine test because of its simplicity. In the current survey, all the 350 MTC isolates obtained from Bangladesh TB patients were determined as M. tuberculosis and no other MTC were detected. This result suggested the general TB treatment regimen including pyrazinamide to be the first choice in Bangladesh.

A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples
Kazuhisa Okada, Siriporn Chantaroj, Tooru Taniguchi, Yasuhiko Suzuki, Amonrattana Roobthaisong, Orapim Puiprom, Takeshi Honda, Pathom Sawanpanyalert
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 66, 2, 135, 139, ELSEVIER SCIENCE INC, 2010年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the call gene. The detection limits of the method were 5 fg of purified genomic DNA/reaction and 0.54 CFU/reaction. The method was applied to rectal swab samples from cholera patients and healthy volunteers (19 subjects each) and yielded the same results as the "gold standard" culture method, while the polymerase chain reaction-based method failed to detect V cholerae in 8 of the positive samples. Direct application of this LAMP method without precultivation enabled the rapid detection of 5 asymptomatic carriers from rectal swabs of 21 household contacts of cholera patients. This LAMP method could be a sensitive, specific, inexpensive, and rapid detection tool for V cholerae carrying the ctxA gene in the clinical laboratory and in the field. (C) 2010 Elsevier Inc. All rights reserved.

Development of novel humanized anti-CD20 antibodies based on affinity constant and epitope
Susumu Uchiyama, Yasuhiko Suzuki, Kentaro Otake, Masami Yokoyama, Mitsuo Ohta, Shuichi Aikawa, Midori Komatsu, Tetsuji Sawada, Yoshitoyo Kagami, Yasuo Morishima, Kiichi Fukui
CANCER SCIENCE, 101, 1, 201, 209, WILEY-BLACKWELL PUBLISHING, INC, 2010年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We describe novel humanized anti-CD20 monoclonal antibodies (mAbs) developed for therapeutic use on the basis of their physicochemical properties and cellular cytotoxicity. A distinct correlation between apparent dissociation constants (K(d)) and apoptotic activity for eight murine anti-CD20 mAbs (OUBM1-OUBM8) and previously-developed murine anti-CD20 mAbs enabled us to categorize anti-CD20 mAbs into two groups. Group A mAbs had lower K(d) values and did not induce definite apoptosis, while Group B mAbs had greater K(d) values and did induce definite apoptosis. A murine version mAb of rituximab, 2B8, belongs to Group B. An epitope analysis showed that the epitope of two murine mAbs, OUBM3 and OUBM6, differed from that of 2B8 or 2F2 (ofatumumab). Two mAbs, OUBM3 from Group A and OUBM6 from Group B, were selected and humanized. As expected, the humanized OUBM3 with the lower K(d) did not induce apoptosis, while the humanized OUBM6 (hOUBM6) with the greater K(d) did. Both hOUBM3 and hOUBM6 induced highly-effective, complement-dependent cytotoxicity and antibody-dependent, cell-mediated cytotoxicity against Burkitt's and follicular lymphomas. Importantly, hOUBM6 exhibited cellular cytotoxicity against diffuse, large B cells that are less effectively depleted by rituximab and also exhibited effective cytotoxicity against tumor cells from human CD20(+) leukemia and lymphoma patients. These results suggest the potential impact of the further development of our anti-CD20 mAbs. Our study shows that the selection of mAbs based on their physicochemical parameters, followed by the biological activity assessment for the selected mAbs, is a rational and efficient approach for pharmaceutical mAb development. (Cancer Sci 2009).

Application of a Modified Loop-Mediated Isothermal Amplification Kit for Detecting Norovirus Genogroups I and II
Tomoko Yoda, Yasuhiko Suzuki, Kenji Yamazaki, Naomi Sakon, Masashi Kanki, Tetsuo Kase, Kazuo Takahashi, Kiyoshi Inoue
JOURNAL OF MEDICAL VIROLOGY, 81, 12, 2072, 2078, WILEY-LISS, 2009年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Norovirus is a major etiologic agent in worldwide outbreaks of gastroenteritis associated with food as well as person-to-person transmission. The ubiquitous nature of Norovirus necessitates simple and rapid detection methods with high accuracy and sensitivity. To this end, several investigators have evaluated the usefulness of commercial reverse-transcription loop-mediated isothermal amplification (RT-LAMP) kits for detecting Norovirus genogroups I (GI) and II (GII). In previous studies, the conventional Loopamp kit for Norovirus GII showed a relatively high detection rate, while that for Norovirus GI showed a relatively low detection rate. In the present study, clinical Norovirus specimens were used to compare the detection rate of a modified Loopamp kit for Norovirus GI with the rates of the conventional Loopamp kit for Norovirus GI and an "in-house" RT-LAMP GI primer set, methods which had a high detection rate. Results from the present study showed that the modified Loopamp kit for Norovirus GI had a higher detection rate for two viral genotypes (GI.3, GI.11). On comparison with an "in-house" GII primer set using genotype GII.4 viruses circulating recently, the detection rate by the Loopamp kit for Norovirus GII was found to be higher, with a 98% detection rate. These results indicate the applicability of the modified LAMP kit for GI and the conventional LAMP kit for GII for detection of Noroviruses in clinical samples. J. Med. Virol. 81:2072-2078, 2009. (C) 2009 Wiley-Liss, Inc.

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (Diospyros kaki Thunb.) fruit.
Takashi Akagi, Ayako Ikegami, Yasuhiko Suzuki, Junya Yoshida, Masahiko Yamada, Akihiko Sato, Keizo Yonemori
Planta, 230, 5, 899, 915, 2009年10月, ［国際誌］
英語, 研究論文（学術雑誌）, Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3'5' hydroxylase (F3'5'H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3' hydroxylation (F3'H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3'5'H to F3'H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3'5'H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.

Loop-Mediated Isothermal Amplification (LAMP) for the Direct Detection of Human Pulmonary Infections with Environmental (Nontuberculosis) Mycobacteria
Bal Ram Adhikari, Basu Dev Pandey, Prakash Ghimire, Bhawana Shrestha, Manoj Khadka, Tomoko Yoda, Yasuhiko Suzuki
JAPANESE JOURNAL OF INFECTIOUS DISEASES, 62, 3, 212, 214, NATL INST INFECTIOUS DISEASES, 2009年05月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Most first-line anti-tuberculosis drugs have less in vitro activity against atypical mycobacteria. Loop-mediated isothermal amplification (LAMP) was used for the rapid diagnosis of mycobacterial species. The sensitivity of LAMP was 96.1% (49/51) in smear-positive and culture-positive sputum samples and 85.0% (17/20) in smear-negative and culture-positive samples. Of the 77 total LAMP-positive samples, 75 (97.4%) were identified as Mycobacterium tuberculosis and 2 (2.6%) as M. intracellulare. One of the M. intracellulare-infected cases was identified in a patient with suspected mycobacteriosis and another was found in a follow-up patient.

Regulator of complement activation (RCA) gene cluster in Xenopus tropicalis.
Oshiumi H, Suzuki Y, Matsumoto M, Seya T
Immunogenetics, 61, 5, 371, 384, 5, 2009年05月, ［査読有り］

Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum
Ajay Poudel, B. D. Pandey, B. Lekhak, B. Rijal, B. R. Sapkota, Y. Suzuki
Kathmandu University Medical Journal, 7, 26, 109, 114, 2009年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. Objectives: The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Materials and methods: Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis. Result: Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (χ2=5. 33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. Conclusion: As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal.

Epithelial inclusion cyst arising in an intramammary lymph node: case report with cytologic findings.
Masato Nakaguro, Yasuhiko Suzuki, Shu Ichihara, Tadao K Kobayashi, Kenzo Ono
Diagnostic cytopathology, 37, 3, 199, 202, 2009年03月, ［国際誌］
英語, 研究論文（学術雑誌）, The occurrence of epithelial inclusion cysts (EIC) in axillary lymph nodes is a rare but well recognized entity, arising either from direct implantation or from embryonal rests. Theoretically, EIC can occur in intramammary lymph nodes, but there has been only one prior report of such a lesion. Here, we describe a case of an EIC arising in an intramammary lymph node of a 37-year-old woman. This report focuses on the FNA cytologic features of this lesion and its differential diagnoses. On FNA, the EIC arising in an intramammary lymph node was characterized by mature lymphocytes, squamous epithelial cells, and keratinizing material. The presence of squamous cells can lead to the erroneous diagnosis of more common breast lesions, such as squamous cell carcinoma or metaplastic carcinoma. Contrary to these more sinister diagnoses, EIC arising in an intramammary lymph node is a benign condition. As this rare lesion sometimes mimics a neoplasm both clinically and radiographically, awareness of this entity is important to prevent over treatment.

Scavenger Receptor Collectin Placenta 1 (CL-P1) Predominantly Mediates Zymosan Phagocytosis by Human Vascular Endothelial Cells
SeongJae Jang, Katsuki Ohtani, Atsushi Fukuoh, Takayuki Yoshizaki, Mitsuko Fukuda, Wataru Motomura, Kenichiro Mori, Jun Fukuzawa, Noritoshi Kitamoto, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
JOURNAL OF BIOLOGICAL CHEMISTRY, 284, 6, 3956, 3965, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2009年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan
A. Edagawa, A. Kimura, H. Doi, H. Tanaka, K. Tomioka, K. Sakabe, C. Nakajima, Y. Suzuki
JOURNAL OF APPLIED MICROBIOLOGY, 105, 6, 2104, 2114, WILEY-BLACKWELL, 2008年12月
英語, 研究論文（学術雑誌）, To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems.
Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20.0%) water samples from 17 (42.5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real-time PCR (from 1.7 x 10(5) to 2.6 x 10(11) cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0.05).
Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems.
More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.

A novel method for simple detection of mutations conferring drug resistance in Mycobacterium leprae, based on a DNA microarray, and its applicability in developing countries
Masanori Matsuoka, Khin Saw Aye, Kyaw Kyaw, Esterlina Virtudes Tan, Ma Victoria Balagon, Paul Saunderson, Robert Gelber, Masanao Makino, Chie Nakajima, Yasuhiko Suzuki
JOURNAL OF MEDICAL MICROBIOLOGY, 57, 10, 1213, 1219, SOC GENERAL MICROBIOLOGY, 2008年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A simple method to detect mutations in the genome of Mycobacterium leprae that confer resistance to key drugs for leprosy was exploited on the basis of a reverse hybridization system. A series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB and gyrA genes for dapsone, rifampicin and ofloxacin resistance, respectively, were selected and fixed on a glass slide as capture probes, to develop a DNA microarray termed the leprosy drug susceptibility-DNA microarray (LDS-DA). Mutations in clinical isolates of M. leprae were successfully identified by the LDS-DA. Feasibility studies were conducted to evaluate the performance of the LDS-DA in two developing countries, Myanmar and the Philippines. The high concordance of the results obtained by this method with the results of nucleotide sequencing strongly supports the applicability of the LDS-DA as a drug susceptibility test in place of sequencing, a time-consuming and costly procedure. This is a rapid and simple method for the simultaneous susceptibility testing of three front-line drugs for leprosy, and solves the problems of previously reported methods.

Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients
Basu Dev Pandey, Ajay Poudel, Tomoko Yoda, Aki Tamaru, Naozumi Oda, Yukari Fukushima, Binod Lekhak, Basista Risal, Bishnu Acharya, Bishwa Sapkota, Chie Nakajima, Tooru Taniguchi, Benjawan Phetsuksiri, Yasuhiko Suzuki
JOURNAL OF MEDICAL MICROBIOLOGY, 57, 4, 439, 443, SOC GENERAL MICROBIOLOGY, 2008年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 % respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

Immunolocalization of a novel collectin CL-K1 in murine tissues
Wataru Motomura, Takayuki Yoshizaki, Katsuki Ohtani, Toshikatsu Okumura, Mituko Fukuda, Jun Fukuzawa, Kenichiro Mori, Seong-Jae Jang, Naoki Nomura, Itsuro Yoshida, Yasuhiko Suzuki, Yutaka Kohgo, Nobutaka Wakamiya
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, 56, 3, 243, 252, HISTOCHEMICAL SOC INC, 2008年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We have recently identified a novel collectin, CL-K1, that may play a role in innate immunity as a member of the collectin family. In this study using mice, we investigated the tissue distribution of CL-K1 for better understanding of its pathophysiological relevance. Real-time PCR analyses demonstrated that CL-K1 mRNA was expressed in all tissues tested. Immunohistochemical analyses demonstrated that CL-K1 was expressed in proximal tubules of kidney, in mucosa of the gastrointestinal tract, and in bronchial glands of bronchioles similar to the localization of SP-A and SP-D in these pulmonary structures. Immunohistochemistry also showed that CL-K1 was highly expressed in hepatocytes around the central which suggests that murine CL-K1 may be mainly produced in the liver and veins in liver, secreted into the blood stream as is human CL-K1. CL-K1 was especially detected in vascular smooth muscle in several types of tissues. In addition, it was also expressed in intestinal Paneth cells, in mesangial cells of kidney, in pancreatic islet D cells, and in neurons of the brain. It is of interest that this profile of CL-K1 expression is unique among the collectins. Together these histological findings may be useful for understanding the biological function of this novel collectin.

Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken Toll-like receptor 2 subfamily
Megumi Higuchi, Aya Matsuo, Masashi Shingai, Kyoko Shida, Akihiro Ishii, Kenji Funami, Yasuhiko Suzuki, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa Seya
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, 32, 2, 147, 155, PERGAMON-ELSEVIER SCIENCE LTD, 2008年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappa B reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2 kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pamm3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappa B in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappa B. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events. (C) 2007 Elsevier Ltd. All rights reserved.

Streptococcus mutans由来sucrose phosphorylaseの熱安定性に関する研究
高本 鉄兵, 深田 はるみ, 久保 亜希子, 鈴木 定彦, 北村 進一
Journal of Applied Glycoscience Supplement, 2008, 108, 108, 日本応用糖質科学会, 2008年
日本語

Recombinant interleukin-12 and interleukin-18 antitumor therapy in a guinea-pig hepatoma cell implant model
Ikuo Shiratori, Yasuhiko Suzuki, Hiroyuki Oshiumi, Nasim A. Begum, Takashi Ebihara, Misako Matsumoto, Kaoru Hazeki, Ken Kodama, Yasuo Kashiwazaki, Tsukasa Seya
CANCER SCIENCE, 98, 12, 1936, 1942, BLACKWELL PUBLISHING, 2007年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Interleukin (IL)-12 and IL-18 are secreted by myeloid cells activated with adjuvants such as Bacillus Calmette-Guerin (BCG) cell wall. They induce T-helper 1 polarization in the host immune system and upregulate production of lymphocyte interferon-gamma, which leads to the induction of an antitumor gene program. It has been reported that humans have an immune system that more closely resembles that of the guinea pig in adjuvant-response features rather than the mouse system, which prevents the mouse results being extrapolated to human immunotherapy. Here we have constructed a tumor-implant system in guinea pigs to evaluate the antitumor potential of guinea pig IL-12 (gpIL-12) and guinea pig IL-18 (gpIL-18). Purified recombinant gpIL-12 and gpIL-18 were prepared and applied intraperitoneally to tumor-bearing (line 10 hepatoma) guinea pigs as the basis of the adjuvant immunotherapy. Intraperitoneal administration of gpIL-12 and gpIL-18 led to retardation of primary tumor growth and suppression of lymph-node metastasis in tumor-bearing guinea pigs. The permissible range of IL-12 appeared wider in guinea pigs than in mice. Even at an IL-12 dose higher than that in mice, there was no evidence of side-effects until day 26, when the guinea pigs were killed. gpIL-18 augmented the antitumor effect of gpIL-12 but exerted less ability to suppress lymph-node metastasis. The effects of gpIL-12 and gpIL-18 on the tumors implanted in guinea pigs will encourage us to use IL-12- and IL-18-inducible adjuvants for immunotherapy in human patients with solid cancer.

Intracranial lesion of Erdheim-Chester disease.
Satoko Shimada, Kenzo Ono, Yoshio Hashizume, Masato Nakaguro, Yasuhiko Suzuki, Naoyoshi Mori
Human pathology, 38, 6, 950, 1, 2007年06月, ［国際誌］
英語

Tumor prone phenotype of mice deficient in a novel apoptosis-inducing gene, drs
Yukihiro Tambe, Atsuko Yoshioka-Yamashita, Ken-ichi Mukaisho, Seiki Haraguchi, Tokuhiro Chano, Takahiro Isono, Takao Kawai, Yasuhiko Suzuki, Ryoji Kushima, Takanori Hattori, Motohito Goto, Shuichi Yamada, Makoto Kiso, Yumiko Saga, Hirokazu Inoue
CARCINOGENESIS, 28, 4, 777, 784, OXFORD UNIV PRESS, 2007年04月, ［国際誌］
英語, 研究論文（学術雑誌）, The drs gene was originally isolated as a suppressor of v-src transformation. Expression of drs mRNA is markedly downregulated in a variety of human cancer cell lines and tissues, suggesting the potential role of this gene as a tumor suppressor. Previously, we found that Drs protein associates with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein in the endoplasmic reticulum, and sequentially activates caspases to induce apoptosis in human cancer cells without involvement of the mitochondria. In this study, we investigated the tumor suppressor function of drs and the correlation between Drs-mediated apoptosis and tumor suppression by generating a gene-knockout (KO) mouse. Between 7 and 12 months after birth, malignant tumors including lymphomas, lung adenocarcinomas and hepatomas were generated in about 30% of the drs KO mice, whereas no tumors were found in any of the wild-type mice during the same period of time. drs KO embryonic fibroblasts also showed enhanced sensitivity to transformation by v-src oncogene. Reintroduction of drs into a tumor cell line derived from the tumor of a drs KO mouse led to the suppression of tumor formation in nude mice, which was accompanied by enhanced apoptosis and the activation of caspase-9 and -3. Furthermore, introduction of drs into this cell line enhanced sensitivity to apoptosis mediated by caspase-3, -9 and -12 under low serum culture conditions. The present results thus indicate that drs contributes to the suppression of malignant tumor formation, and this suppression is closely correlated with drs-mediated apoptosis.

Association of the Rv0679c protein with lipids and carbohydrates in Mycobacterium tuberculosis/Mycobacterium bovis BCG
Takashi Matsuba, Yasuhiko Suzuki, Yoshinori Tanaka
ARCHIVES OF MICROBIOLOGY, 187, 4, 297, 311, SPRINGER, 2007年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The Rv0679c gene in Mycobacterium tuberculosis H37Rv encodes a protein with a predicted molecular mass of 16,586 Da consisting of 165 amino acids which contains a putative N-terminal signal sequence and a consensus lipoprotein-processing motif. Globomycin treatment, Triton X-114 separation and mass spectrometry analyses clarified a property of the Rv0679c protein as a lipoprotein. In addition, trifluoromethanesulphonic acid treatment of the lysate revealed an association of the recombinant Rv0679c protein with carbohydrates. The Rv0679c protein homolog of Mycobacterium bovis BCG was also expressed as the protein associated with lipids and carbohydrates. In Western blot analysis, each of the protein homolog and Lipoarabinomannan (LAM) was detected as a similar pattern by anti-Rv0679c and anti-LAM antibodies, respectively. Interestingly, the Rv0679c protein was detected in commercially available LAM purified from M. tuberculosis. Inhibition assay of LAM synthesis in M. bovis BCG by ethambutol showed an altered migration pattern of the Rv0679c protein to low molecular mass similar to that of LAM. The results suggest that the Rv0679c protein exists as a tight complex with LAM in M. tuberculosis/M. bovis BCG.

Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses
Tomoko Yoda, Yasuhiko Suzuki, Kenji Yamazaki, Naomi Sakon, Masashi Kanki, Ikuko Aoyama, Teizo Tsukamoto
JOURNAL OF MEDICAL VIROLOGY, 79, 3, 326, 334, WILEY, 2007年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range.

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria
Susumu Okamoto, Aki Tamaru, Chie Nakajima, Kenji Nishimura, Yukinori Tanaka, Shinji Tokuyama, Yasuhiko Suzuki, Kozo Ochi
MOLECULAR MICROBIOLOGY, 63, 4, 1096, 1106, WILEY-BLACKWELL, 2007年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.

Anti-influenza A virus activities of mannan-binding lectins and bovine conglutinin.
Takao Kawai, Tetsuo Kase, Yasuhiko Suzuki, Souji Eda, Takashi Sakamoto, Katsuki Ohtani, Nobutaka Wakamiya
The Journal of veterinary medical science, 69, 2, 221, 4, 2, 2007年02月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Mannan-binding lectin (MBL) and bovine conglutinin (BKg) belong to the collectin family, which is involved in first-line host defense against various infectious agents. We have previously reported that human MBL inhibited type A influenza viral hemagglutination, infection and spreading to adjacent cells without complement activation. In this study, we investigated the direct antiviral activities of bovine MBL, rabbit MBL and BKg. All collectins used in this study inhibited viral infectivity and hemagglutination at concentrations of 0.02-0.3 microg/ml. They also demonstrated inhibitory activity against viral spreading. Like human MBL, bovine MBL and BKg showed antiviral activities at their physiological concentrations. These results suggest that mammalian MBLs and BKg may inhibit the spread of influenza A virus through the bloodstream.

Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction
Rungrat Nintasen, Fuangfa Utrarachkij, Kanokrat Siripanichgon, Adisak Bhumiratana, Yasuhiko Suzuki, Orasa Suthienkul
MICROBIOLOGY AND IMMUNOLOGY, 51, 8, 777, 785, CENTER ACADEMIC PUBL JAPAN, 2007年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (inip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3'proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.

Titanium and zirconium complexes with non-salicylaldimine-type imine-phenoxy chelate ligands: syntheses, structures, and ethylene-polymerization behavior.
Yasuhiko Suzuki, Hidetsugu Tanaka, Toshiyuki Oshiki, Kazuhiko Takai, Terunori Fujita
Chemistry, an Asian journal, 1, 6, 878, 87, 2006年12月18日, ［国際誌］
英語, 研究論文（学術雑誌）, New Ti and Zr complexes that bear imine-phenoxy chelate ligands, [{2,4-di-tBu-6-(RCH=N)-C6H4O}2MCl2] (1: M = Ti, R = Ph; 2: M = Ti, R = C6F5; 3: M = Zr, R = Ph; 4: M = Zr, R = C6F5), were synthesized and investigated as precatalysts for ethylene polymerization. 1H NMR spectroscopy suggests that these complexes exist as mixtures of structural isomers. X-ray crystallographic analysis of the adduct 1HCl reveals that it exists as a zwitterionic complex in which H and Cl are situated in close proximity to one of the imine nitrogen atoms and the central metal, respectively. The X-ray molecular structure also indicates that one imine phenoxy group with the syn C=N configuration functions as a bidentate ligand, whereas the other, of the anti C=N form, acts as a monodentate phenoxy ligand. Although Zr complexes 3 and 4 with methylaluminoxane (MAO) or [Ph3C]+ [B(C6F5)4]-/AliBu3 displayed moderate activity, the Ti congeners 1 and 2, in association with an appropriate activator, catalyzed ethylene polymerization with high efficiency. Upon activation with MAO at 25 degrees C, 2 displayed a very high activity of 19900 (kg PE) (mol Ti)(-1) h(-1), which is comparable to that for [Cp2TiCl2] and [Cp2ZrCl2], although increasing the polymerization temperature did result in a marked decrease in activity. Complex 2 contains a C6F5 group on the imine nitrogen atom and mediated nonliving-type polymerization, unlike the corresponding salicylaldimine-type complex. Conversely, with [Ph3C]+ [B(C6F5)4]-/AliBu3 activation, 1 exhibited enhanced activity as the temperature was increased (25-75 degrees C) and maintained very high activity for 60 min at 75 degrees C (18740 (kg PE) (mol Ti)(-1) h(-1)). 1H NMR spectroscopic studies of the reaction suggest that this thermally robust catalyst system generates an amine-phenoxy complex as the catalytically active species. The combinations 1/[Ph3C]+ [B(C6F5)4]-/AliBu3 and 2/MAO also worked as high-activity catalysts for the copolymerization of ethylene and propylene.

Hafnocene catalysts for selective propylene oligomerization: efficient synthesis of 4-methyl-1-pentene by beta-methyl transfer.
Yasuhiko Suzuki, Takahiro Yasumoto, Kazushi Mashima, Jun Okuda
Journal of the American Chemical Society, 128, 39, 13017, 25, 2006年10月04日, ［国際誌］
英語, 研究論文（学術雑誌）, A series of hafnocene complexes (eta5-C5Me4R1)(eta5-C5Me4R2)HfCl2 with [R1, R2] = [H, H] (1), [Me, H] (2), [Me, Me] (3), [Et, Me] (4), [ (i)Pr, Me] (5), [SiMe(3), Me] (6), [ (t)Bu, Me] (7), [ (n)Bu, Me] (8), [ (i)Bu, Me] (9), [Et, Et] (10), [ (n)Bu, (n)Bu] (11), [ (i)Bu, (i)Bu] (12) was tested as catalyst precursors for propylene oligomerization. Upon activation with methylaluminoxane or [Ph(3)C][B(C(6)F(5))(4)]/Al(i)Bu(3), complexes 2-4 and 8-12 catalyzed the dimerization of propylene to produce 4-methyl-1-pentene with selectivities ranging from 23.9 to 61.6 wt % in the product mixture. The selectivity was dependent on the nature of the substituents R(1) and R(2), with the highest value found for (eta5-C5Me4(i)Bu)2HfCl2 (12). Rapid deactivation was observed for 5-7, whereas (eta5-C5Me4H)2HfCl2 (1) polymerized propylene. 4-Methyl-1-pentene is proposed to form by repeated 1,2-insertion of propylene into the hafnocene methyl cation, followed by selective beta-methyl elimination. Detailed analysis of the byproduct distribution (isobutene, 1-pentene, 2-methyl-1-pentene, 2,4-dimethyl-1-pentene, 4-methyl-1-heptene, 4,6-dimethyl-1-heptene), determined by gas chromatography, was performed with the aid of a stochastic simulation involving rate constants for the propagation by insertion, beta-hydride elimination, and beta-methyl elimination. The rate of termination is dependent on the structure of the growing chain of the active species as well as on the bulkiness of the cyclopentadienyl ligands. The selectivity highly depends on the reaction conditions (pressure, temperature, concentration of methylaluminoxane). The rates of beta-methyl elimination leading to 4-methyl-1-pentene were proportional to propylene pressure for 2-4 and 8-10 but practically independent from propylene pressure for the sterically bulkier derivatives 11-12.

Expression and characterization of the genes encoding azoreductases from Bacillus subtilis and Geobacillus stearothermophilus
Wataru Sugiura, Tomoko Yoda, Takashi Matsuba, Yoshinori Tanaka, Yasuhiko Suzuki
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70, 7, 1655, 1665, TAYLOR & FRANCIS LTD, 2006年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Azoreductases have been characterized as enzymes that can decolorize azo dyes by reducing azo groups. In this study, genes encoding proteins having homology with theazoreductase gene of Bacillus sp. OY1-2 were obtained from Bacillus subtilis ATCC6633, B. subtilis ISW1214, and Geobacillus stearotherophilus IF013737 by polymerase chain reaction. All three genes encoded proteins with 174 amino acids. The deduced amino acid sequences of azoreductase homologs from B. subtilis ISW1214,B-.. subtilis ATCC6633, and G. stearotherophilus IF013737 showed similarity of 53.3, 53.9, and 53.3 % respectively to that of Bacillus sp. OY1-2. All three genes were expressed in Escherichia coli, and were characterized as having the decolorizing activity of azo dyes in a beta-NADPH dependent manner. The transformation of several azo dyes into colorless compounds by recombinant enzymes was demonstrated to have distinct substrate specificity from that of azoreductase from Bacillus sp. OY1-2.

Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant
T Ichinohe, Watanabe, I, E Tao, S Ito, A Kawaguchi, S Tamura, H Takahashi, H Sawa, M Moriyama, J Chiba, K Komase, Y Suzuki, T Kurata, T Sata, H Hasegawa
JOURNAL OF MEDICAL VIROLOGY, 78, 7, 954, 963, WILEY-LISS, 2006年07月, ［査読有り］
英語, 研究論文（学術雑誌）, A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.

Identification and characterization of a novel human collectin CL-K1
Hiroyuki Keshi, Takashi Sakamoto, Takao Kawai, Katsuki Ohtani, Tsuyoshi Katoh, Seong-Jae Jang, Wataru Motomura, Takayuki Yoshizaki, Mitsuko Fukuda, Satoshi Koyama, Jun Fukuzawa, Atsushi Fukuoh, Itsuro Yoshida, Yasuhiko Suzuki, Nobutaka Wakamiya
MICROBIOLOGY AND IMMUNOLOGY, 50, 12, 1001, 1013, CENTER ACADEMIC PUBL JAPAN, 2006年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca2+-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding, character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.

紫外線吸収剤及びその関連化合物によるエストロゲン様作用
松本比佐志, 足立伸一, 鈴木定彦
薬学雑誌, 125, 8, 643, 652, 2005年08月, ［国内誌］
日本語, 研究論文（学術雑誌）, The estrogenic activities of ultraviolet absorbers and their related compounds were investigated using MCF-7 cell proliferation assay. Nine of 33 chemicals (benzophenone, 2,4-dihydroxybenzophenone, 2,2′,4,4′- tetrahydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, 2,2′-dihydroxy- 4,4′-dimethoxybenzophenone, 4-hydroxybenzophenone, 3-(4-methylbenzylidene) camphor, ethyl 2-cyano-3,3-diphenylacrylate (etocrylene) and 2-ethylhexyl-2-cyano-3,3-diphenylacrylate (octocrylene)) were positive compared with the vehicle control. Benzhydrol, ethyl cinnamate and 2,2′-dihydroxy- 4-methoxybenzophenone were weakly active. When each xenoestrogen was added to the cells along with ICI 182780, an estrogen receptor (ER) antagonist, the cell growth was reduced according to its doses. Therefore, the cell proliferation was suggested to generate through ER. Most of these chemicals were also positive using CHOOSER assay, a new method of testing estrogenic activity of xenoestrogen. Each xenoestrogen was also confirmed to bind to ERα and ERβ using a human ER competitive binding assay against 17β-estradiol. The concentration order of the strength of its inhibitory effect using both ERα and ERβ was similar to that of MCF-7 cell proliferation assay, except for benzyl 4-hydroxybenzoate (B4HB). B4HB showed a stronger activity on CHOOSER assay and the competitive binding assay using both ERα and ERβ, although there was no activity observed on MCF-7 cell proliferation assay. Our findings were to detect the estrogenic activity of etocrylene and octocrylene in vitro, in addition to confirming the activities of some ultraviolet absorbers as previously reported. © 2005 The Pharmaceutical Society of Japan.

Estrogenic activity of ultraviolet absorbers and the related compounds
H Matsumoto, S Adachi, Y Suzuki
YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN, 125, 8, 643, 652, PHARMACEUTICAL SOC JAPAN, 2005年08月, ［査読有り］
日本語, 研究論文（学術雑誌）, The estrogenic activities of ultraviolet absorbers and their related compounds were investigated using MCF-7 cell proliferation assay. Nine of 33 chemicals (benzophenone, 2,4-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 2 -hydroxy-4-methoxybenzophenone, 2,2'-dihydroxy-4,4'-dimethoxybenzophenone, 4-hydroxybenzophenone, 3-(4-methylbenzylidene) camphor, ethyl 2-cyano-3,3-diphenylacrylate (etocrylene) and 2-ethylhexyl-2-cyano-3,3-diphenylacrylate (octocrylene)) were positive compared with the vehicle control. Benzhydrol, ethyl cinnamate and 2,2'-dihydroxy-4-methoxybenzophenone were weakly active. When each xenoestrogen was added to the cells along with ICI 182780, an estrogen receptor (ER) antagonist, the cell growth was reduced according to its doses. Therefore, the cell proliferation was suggested to generate through ER. Most of these chemicals were also positive using CHOOSER assay, a new method of testing estrogenic activity of xenoestrogen. Each xenoestrogen was also confirmed to bind to ER alpha and ER beta using a human ER competitive binding assay against 17 beta-estradiol. The concentration order of the strength of its inhibitory effect using both ER alpha and ER beta was similar to that of MCF-7 cell proliferation assay, except for benzyl 4-hydroxybenzoate (B4HB). B4HB showed a stronger activity on CHOOSER assay and the competitive binding assay using both ER alpha and ER beta, although there was no activity observed on MCF-7 cell proliferation assay. Our findings were to detect the estrogenic activity of etocrylene and octocrylene in vitro, in addition to confirming the activities of some ultraviolet absorbers as previously reported.

Bisphenol A in ambient air particulates responsible for the proliferation of MCF-7 human breast cancer cells and its concentration changes over 6 months
H Matsumoto, S Adachi, Y Suzuki
ARCHIVES OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY, 48, 4, 459, 466, SPRINGER, 2005年05月
英語, 研究論文（学術雑誌）, To survey the estrogenic activity of the organic extracts from particulate matter of urban ambient outdoor air, samples were collected on glass fiber filters using a high-volume air sampler on the rooftop of our institute for 6 months (six filters/month). After extracting the organic materials and separating them into three fractions, i.e., acidic, neutral, and basic, we applied a cell-growth assay using MCF-7 human breast cancer cells to the original extract and the extracts of the fractions. Only the extract in the acidic fraction showed cell proliferation activity in a dose-response manner. To survey the chemical(s) responsible for the activity, a gas chromatography/ mass spectrometry (GC/MS) analysis was conducted after silylating the extract. The presence of bisphenol A (BPA) was confirmed, because the retention times and the MS fragment patterns between the silylated derivative of a component in the sample and that of BPA itself were the same. By using a GC/ MS-SIM (selective ion monitoring) technique, the average value was found to be 0.51 ng/m(3) of air (range: 0.02 similar to 1.92 ng/m(3) of air). The trend of the residual levels in air particulates showed seasonal variation, increasing from autumn to winter and decreasing from winter to spring. The only exception was that the value in January was lower than those in December and February. Considering the content of BPA in the extract of the acidic fraction and the strength of the activities with the extract and BPA itself, the estrogenic activity due to BPA in the fraction seemed to decrease. In spite of this decline, the possibility remains that the estrogenic activity mainly originated from BPA.

Characterization of multidrug-resistance phenotypes and genotypes of Escherichia coli strains isolated from swine from an abattoir in Osaka, Japan
Y Kumai, Y Suzuki, Y Tanaka, K Shima, RK Bhadra, S Yamasaki, K Kuroda, G Endo
EPIDEMIOLOGY AND INFECTION, 133, 1, 59, 70, CAMBRIDGE UNIV PRESS, 2005年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A total of 455 highly tetracycline-resistant Escherichia coli strains were isolated from 84 healthy swine from abattoirs and it was found that 56.9, 43.1, 22.2, 15.4, 2.6 and 1.5 % of strains were resistant to chloramphenicol, ampicillin, kanamycin, trimethoprim-sulphamethoxazole, ofloxacin and gentamicin respectively. Interestingly, E. coli strains isolated from certain finisher hog groups exhibited resistance against 2-7 antimicrobials, but strains isolated from multiparous sow groups in each herd were resistant to only 2-4 antimicrobial agents. When randomly selected 108 tetracycline-resistant isolates were tested for the presence of resistance genes, the following genes tet(A) (n = 6), tet(B) (n = 95), tet(D) (n = 1) or both tet(A) and tet(B) (n = 6) were found to be distributed among them. Furthermore, 52 isolates carried the integrase 1 gene and 24 strains gave live different PCR amplicon profiles using primers from the variable region of integron. Extensive nucleotide sequence analyses of these amplicons revealed the presence of dhfrI, dhfrXII, dfr17, aadA, aadA2, aadA5, aadA21, aacA4 and catB3 genes which code for different antibacterial resistance proteins.

新しいアッセイ用哺乳類細胞(CHOOSER)を用いた簡便，高感度で再現性の良いエストロゲン様活性測定方法の開発
足立伸一, 山本康次, 織田肇, 小野芳朗, 鈴木定彦
水環境学会誌, 28, 7, 451, 456, 公益社団法人 日本水環境学会, 2005年
日本語, Several in vitro assays have been developed to assess the activity of estrogenic substances. Recently, a yeast two-hybrid screen or a MCF-7 (human breast cancer cell) screen has been adopted mainly for screening chemicals and environmental estrogenic activity. However a yeast two-hybrid screen does not assay using an animal cell. MCF-7 screen is a complicated assay and the cell used proliferates depending on estrogen, thus obstructing a precise assay for estrogenic activity. In addition both screens need long incubation period (4-6 days) for a highly sensitive assay. The CHOOSER is a Chinese hamster ovary (CHO) cell transformed with the gene encoding the human estrogen receptor (ER α) and an estrogen responsive promoter linked to a reporter gene which was developed by Suzuki et. al. at Osaka prefectural institute of public health. When the reporter gene is activated by estrogenic substances, is expressed, producing alkaline phosphatase which is secreted into the medium. The CHOOSER can assay high sensitively (under 10-11M as 17 β-estradiol) at any time in a short incubation period (2 days). The number of the CHOOSER can be counted precisely because a CHO is dissociated the aggregates easily with trypsin and form a single cell suspension. And the CHOOSER can proliferate in the medium without estrogen, therefore it is possible to assay for estrogenic activity more proliferate in the medium without estrogen, therefore it is possible to assay for estrogenic activity more precisely. The CHOOSER assay will be a useful prescreening method for estrogenic activity before in vivo assay.

Malignant mesothelioma of the tunica vaginalis testis: a case with a predominant sarcomatous component.
Satoko Shimada, Kenzo Ono, Yasuhiko Suzuki, Naoyoshi Mori
Pathology international, 54, 12, 930, 4, 2004年12月, ［国際誌］
英語, 研究論文（学術雑誌）, We present a case of malignant mesothelioma (MM) of the tunica vaginalis testis. A 64-year-old man was referred for an operation on a right hydrocele that later proved to be a tumor during surgery. The tumor was malignant with a biphasic pattern of epithelial and sarcomatous components. The latter component was predominant. Cuboidal or columnar cells formed irregular tubular structures in the epithelial component. In contrast, spindle-shaped or polygonal cells formed intricate structures with stromal connective tissues in the sarcomatous component. Immunohistochemical staining revealed that the tumor was mesothelial in origin and positive for cytokeratin, vimentin, HBME-1 antigen and calretinin. In general, MM occur in the pleura or peritoneum; those originating in the tunica vaginalis testis are very rare and represent less than 5% of all MM. In addition, MM in the tissues usually consist primarily of an epithelial component. According to previous reports tumors with a predominant sarcomatous component are extremely rare. In general, a sarcomatous component predicts poor prognosis and our case does, in fact, deteriorate over time. Our case suggests that despite its low incidence, MM must be considered when a case is diagnosed as hydrocele testicle.

Identification of the Cryptosporidium isolate from chickens in Japan by sequence analyses
A Kimura, Y Suzuki, T Matsui
JOURNAL OF VETERINARY MEDICAL SCIENCE, 66, 7, 879, 881, JAPAN SOC VET SCI, 2004年07月, ［国内誌］
英語, 研究論文（学術雑誌）, The Cryptsosporidium isolate from chickens in Japan by Itakura et al. is not yet accurately identified because of several discrepancies in phenotypic features. We attempted to identify this isolate by analyzing the partial sequences of the 18S rRNA, COWP and HSP70 genes. The chicken isolate showed nearly 100% homology in each gene with C. baileyi, but less than 91 % homology with C. meleagridis. In addition, these genes were classified into the same cluster with C. baileyi other than C. meleagridis by phylogenetic analysis. From these results, the Cryptosporidium isolate from chickens in Japan is considered to be one of the strains of C. baileyi.

Choroidal neovascularization is provided by bone marrow cells.
Minoru Tomita, Haruhiko Yamada, Yasushi Adachi, Yunze Cui, Eri Yamada, Akiko Higuchi, Keizo Minamino, Yasuhiko Suzuki, Miyo Matsumura, Susumu Ikehara
Stem cells (Dayton, Ohio), 22, 1, 21, 6, 2004年, ［国際誌］
英語, 研究論文（学術雑誌）, Choroidal neovascularization (CNV) is a known cause of age-related macular degeneration (ARMD). Moreover, the most common cause of blindness in the elderly in advanced countries is ARMD with CNV. It has recently been shown that bone marrow cells (BMCs) can differentiate into various cell lineages in vitro and in vivo. Adults maintain a reservoir of hematopoietic stem cells included in BMCs that can enter the circulation to reach various organs in need of regeneration. It has recently been reported that endothelial progenitor cells (EPCs) included in BMCs are associated with neovascularization. We examine the role of BMCs in CNV using a model of CNV in adult mice. Using methods consisting of fractionated irradiation (6.0 Gy x 2) followed by bone marrow transplantation (BMT), adult mice were engrafted with whole BMCs isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP). Three months after BMT, we confirmed that the hematopoietic cells in the recipients had been completely replaced with donor cells. We then carried out laser photocoagulation to induce CNV in chimeric mice (donor cells >95%). Two weeks after the laser photocoagulation, by which time CNV had occurred, immunohistochemical examination was carried out. The vascular wall cells of the CNV expressed both EGFP and CD31. These findings indicate that newly developed blood vessels in the CNV are derived from the BMCs and suggest that the inhibition of EPC mobilization from the bone marrow to the eyes could be a new approach to the fundamental treatment of CNV in ARMD.

Characterization of clinical isolates of Mycobacterium tuberculosis resistant to drugs and detection of RpoB mutation in multidrug-resistant tuberculosis in the Philippines
DMD Agdamag, S Kageyama, R Solante, AS Espantaleon, JCE Sangco, Y Suzuki
INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE, 7, 11, 1104, 1108, INT UNION AGAINST TUBERCULOSIS LUNG DISEASE (I U A T L D), 2003年11月
英語, 研究論文（学術雑誌）, SETTING: Retrospective study of Mycobacterium tuberculosis isolates at the STD/AIDS Cooperative Central Laboratory, Philippines.
OBJECTIVE: To describe patterns of M. tuberculosis resistance against first-line anti-tuberculosis drugs, and to analyze the rpoB gene codon mutation of rifampicin (RMP) resistant isolates and correlate genotypic and phenotypic patterns.
DESIGN: One hundred and sixty-four M. tuberculosis complex isolates were retrieved for phenotypic analysis; 89 were resistant to any anti-tuberculosis drug and 50 were RMP-resistant, whereas 48 were multidrug-resistant (MDR). Of these 48, only 33 were available for genotypic analysis of the rpoB gene.
RESULTS: Most drug-resistant isolates were phenotypically resistant to isoniazid (INH) (93%), and the probability of an RMP-resistant isolate becoming MDR was 96%. In 33 MDR isolates, 13 types of mutations in nine independent codons were identified; the most frequently mutated codons were S531L (61%) and G510H (15%), which were present in 76% (25/33) of the isolates. S531L was noted in 85.7% of the RMP + INH + SM resistant isolates, while only 80% of the isolates with INH + RMP, EMB + SM resistance showed this mutation.
CONCLUSION: The high probability of RMP isolates being MDR suggests that genetic analysis of RMP resistance is useful in detecting MDR-TB. Worldwide accumulation of findings on circulating MDR-TB strains provides indispensable information about the re-emergence of TB.

Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice
K Suzuki, Y Suzuki, S Kitamura
JOURNAL OF EXPERIMENTAL BOTANY, 54, 389, 1997, 1999, OXFORD UNIV PRESS, 2003年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-D-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-D-glucuronic acid to UDP-D-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots.

Precise characterization of norovirus (Norwalk-like virus)-specific monoclonal antibodies with broad reactivity
T Yoda, Y Suzuki, Y Terano, K Yamazaki, N Sakon, T Kuzuguchi, H Oda, T Tsukamoto
JOURNAL OF CLINICAL MICROBIOLOGY, 41, 6, 2367, 2371, AMER SOC MICROBIOLOGY, 2003年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.

DNAマイクロアレイを用いた臨床分離結核菌の薬剤耐性判定　　　　　　　　　　　　　　　
吉川陽子, 市原竜生, 鈴木定
臨床微生物迅速診断研究会誌, 14, 45, 50, 2003年

Haplotype analysis of the human collectin placenta 1 (hCL-P1) gene
H Ohmori, Y Makita, M Funamizu, S Chiba, K Ohtani, Y Suzuki, N Wakamiya, A Hata
JOURNAL OF HUMAN GENETICS, 48, 2, 82, 85, SPRINGER-VERLAG TOKYO, 2003年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Collectins are a family of C-type lectins found in vertebrates. These proteins have four regions, a relatively short N-terminal region, a collagen-like region, an alphahelical coiled coil, and a carbohydrate recognition domain. Collectins are involved in host defense through their ability to bind carbohydrate antigens on microorganisms. Type A scavenger receptors are classical-type scavenger receptors that also have collagen-like domains. We previously described a new scavenger receptor, collectin from placenta [collectin placenta 1 (CL-P1)]. CL-P1 is a type II membrane protein with all four regions. We found that CL-P1 can bind and phagocytize both bacteria and yeast. In addition to that, it reacts with oxidized low-density lipoprotein (LDL) but not with acetylated LDL. These results suggest that CL-P1 might play important roles in host defenses and/or atherosclerosis formation. One rational strategy to study the role of CL-P1 in these pathological conditions would be to perform a haplotype association study using human samples. As a first step for this strategy, we analyzed the haplotype structure of the CL-P1 gene. By sequencing the CL-P1 gene in ten Japanese volunteers, we identified five single-nucleotide polymorphisms (SNPs) with a minor allele frequency of at least 29%. To obtain SNPs in the 5'-upstream region of the gene, we screened a total of 20 SNPs described in the database and finally picked up one SNP for the present study. Thus, a total of six SNPs, one in the 5'-upstream region, two in intron 2, one in exon 5, and two in exon 6, were used to analyze the haplotype structure of the gene, with DNAs derived from 54 individuals (108 alleles). The analysis revealed that only two of six SNPs showed significant linkage disequilibrium (r(2) > 0.5) with each other. This haplotype information may be useful in disease-association studies in which a contribution of the CL-P1 gene has been suspected, especially in immunological disturbance or atherosclerosis. Two SNPs in exon 6, both leading to amino acid substitutions, could be candidates for influencing disease susceptibility.

[Detection of drug-resistant Mycobacterium tuberculosis isolates using DNA microarray].
Yoko Yoshikawa, Tatsuo Ichihara, Yasuhiko Suzuki
Rinsho Biseibutsu Jinsoku Shindan Kenkyukai shi = JARMAM : Journal of the Association for Rapid Method and Automation in Microbiology, 14, 1, 45, 50, 2003年, ［国内誌］
日本語, 研究論文（学術雑誌）, Rapid identification of drug-resistant strains of Mycobacterium tuberculosis is an important problem to adequate patient treatment. However, current clinical assays for determining antibiotic susceptibility in M. tuberculosis require many weeks to complete due to the slow growth of the bacilli. We have developed simple and rapid drug susceptibility test using DNA microarray "Oligoarray TB," that allows the detection of rifampicin (RFP), isoniazide (INH), kanamycin (KM), streptomycin (SM), and ethambutol (EM)-resistant strains within 6 h with DNA extracted directly from sputum or cultured cells. The genes related to drug-resistance, results of detection using Oligoarray TB, comparative evaluation with media, and sequencing analysis of strains generating discrepant results in testing for INH-resistant will be discussed in this presentation.

Molecular cloning of mouse collectin liver 1
T Kawai, Y Suzuki, S Eda, T Kase, K Ohtani, Y Sakai, H Keshi, A Fukuoh, T Sakamoto, M Nozaki, NG Copeland, NA Jenkins, N Wakamiya
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66, 10, 2134, 2145, TAYLOR & FRANCIS LTD, 2002年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish.

Isolation of a novel mouse variant of the drs tumor suppressor gene
T Kawai, Y Suzuki, A Yamashita, H Inoue
CANCER LETTERS, 183, 1, 79, 86, ELSEVIER SCI IRELAND LTD, 2002年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The drs gene was isolated as a transformation suppressor against the v-src oncogene. Drs protein has a transmembrane domain and three consensus repeats (CRs) called Sushi motifs in the extracellular domain. The dry gene also has the ability to suppress anchorage-independent growth of human cancer cell lines. In this paper, we report the isolation of a novel variant cDNA of mouse drs (mDRS-2) containing two CRs, in addition to a Mouse homolog of drs (mDRS-1) containing three CRs. We investigated the Suppressor function of these mDRS cDNAs in human cancer cells and found that the lack of one CR is critical for suppression of anchorage-independent growth by drs. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Molecular cloning and characterization of chick sialoprotein associated with cones and rods, a developmentally regulated glycoprotein of interphotoreceptor matrix.
Masahiro Zako, Masayoshi Iwaki, Masahiko Yoneda, Osamu Miyaishi, Jinsong Zhao, Yasuhiko Suzuki, Makoto Takeuchi, Goichiro Miyake, Hiroshi Ikagawa, Koji Kimata
The Journal of biological chemistry, 277, 28, 25592, 600, 2002年07月12日, ［国際誌］
英語, 研究論文（学術雑誌）, MY-174 is an IgM class monoclonal antibody originally established against chick PG-M/versican. The antibody specifically stains the photoreceptor layer, where we recently reported an absence of PG-M/versican. In this study, we re-characterized the antibody and identified the molecule that reacts to MY-174 at the photoreceptor layer. Immunohistochemistry localized the antigen to the matrix surrounding photoreceptors. A variety of glycosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylated N- and O-linked glycoconjugates having a molecular mass of more than 30-kDa. The peptide sequences obtained from purified MY-174 antigen showed we had sequenced a full-length cDNA with an open reading frame of 2787 base pairs, encoding a polypeptide of 928 amino acids, with 56 and 54% identities to human and mouse sialoprotein associated with cones and rods (SPACRs), respectively, and with the structural features observed in SPACRs. The specific sialylated O-glycoconjugates here are involved in the epitope structure for MY-174. SPACR first appeared by embryonic days 15-16, and expression increased with developmental age, paralleling the adhesion between neural retina and retinal pigment epithelium. Thus, we concluded that the MY-174 antigen at the photoreceptor layer, a developmentally regulated glycoprotein, is identical to chick SPACR and may be involved in a novel system mediating adhesion between neural retina and retinal pigment epithelium.

Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice
T Shimada, K Inoue, Y Suzuki, T Kawai, E Azuma, T Nakajima, M Shindo, K Kurose, A Sugie, Y Yamagishi, Y Fujii-Kuriyama, M Hashimoto
CARCINOGENESIS, 23, 7, 1199, 1207, OXFORD UNIV PRESS, 2002年07月
英語, 研究論文（学術雑誌）, Arylhydrocarbon receptor knock-out, AhR(-/-), mice have recently been shown to be rather resistant to benzo[a]pyrene (B[a]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[a]P to reactive metabolites (Y.Shimizu, Y.Nakatsuru, M.Ichinose, Y.Takahashi, H.Kume, J.Mimura, Y.Fujii-Kuriyama and T.Ishikawa (2000) Proc. Natl Acad. Sci. USA, 97, 779-782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[a]P, plays a role in the B[a]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(-/-) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[a]P, 7,12-dimethylbenz[a]anthracene, dibenz[a,l]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[b]fluoranthene, and benzo[a]anthracene and by a co-planar PCB congener 3,4,3',4'-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[e]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(+/-/+/-) mice. In AhR(-/-) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylations and of mutagenic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[a]P and related carcinogens causing initiation of cancers in mice.

Rapid detection of pyrazinamide-resistant Mycobacterium tuberculosis by a PCR-based in vitro system
Y Suzuki, A Suzuki, A Tamaru, C Katsukawa, H Oda
JOURNAL OF CLINICAL MICROBIOLOGY, 40, 2, 501, 507, AMER SOC MICROBIOLOGY, 2002年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterialpyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5' ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.

Mannose-binding lectin and the prognosis of fulminant hepatic failure caused by HBV infection
Y Hakozaki, M Yoshiba, K Sekiyama, E Seike, J Iwamoto, K Mitani, M Mine, T Morizane, K Ohtani, Y Suzuki, N Wakamiya
LIVER, 22, 1, 29, 34, BLACKWELL MUNKSGAARD, 2002年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background/Aims: The mannose-binding lectin (MBL) gene was reported to play an important role in determining the clinical outcome of persistent hepatitis B virus (HBV) infection. We investigated serum MBL concentrations and MBL gene mutations to determine whether they were related to the prognosis of patients with fulminant hepatic failure (FHF) caused by HBV infection. Methods: We investigated serum MBL concentrations and MBL gene mutations in 43 HBV-infected Japanese patients with FHF and 260 HBsAg-negative healthy controls. Serum MBL concentrations were measured by an enzyme-linked immunosorbent assay, and mutations in the MBL gene were analysed by nested PCR and direct DNA sequencing. Results: Only a mutation in codon 54 of the MBL gene was found. The frequency of this mutation in nonsurvivors (40%, 8/20) was higher than in survivors (13%, 3/23), and the difference was slightly significant (p = 0.043). The H allele frequency in survivors (70.5%, 31/44) was higher than in nonsurvivors (39.5%, 15/38) (p = 0.0048). Because of these factors the mean serum MBL concentration in survivors, 1.61 mug/ml (range 0.3-3.86), was significantly higher than in nonsurvivors, 0.79 mug/ml (range 0.04-1.51) (p < 0.0001). The likelihood ratio for nonsurvival was 0 for over 2.0 mug/ml, 0.67 for 1.0-2.0 mug/ml, and 2.24 for 0-1.0 mug/ml. Conclusions: The mutation in codon 54 of the MBL gene tended to be higher in nonsurvivors than in survivors. The H allele frequency (high producing allele in H/Y) in survivors was higher than that in nonsurvivors. High levels of serum MBL correlated with the survival of patients with FHF due to HBV infection. Serum MBL may be useful as a predictive factor for the survival of patients with FHF caused by HBV.

Identification of human mannose binding lectin (MBL) recognition sites for novel inhibitory antibodies.
Hui Zhao, Nobutaka Wakamiya, Yasuhiko Suzuki, Matthew T Hamonko, Gregory L Stahl
Hybridoma and hybridomics, 21, 1, 25, 36, 1, 2002年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mannose binding lectin (MBL) binding initiates activation of the lectin complement pathway. Recent studies from our laboratory have demonstrated that MBL-dependent complement activation mediates cellular injury following oxidative stress in vivo and in vitro. A panel of novel inhibitory monoclonal antibodies (MAbs) against MBL (e.g., MAb 3F8, 2A9, and hMBL1.2) has been developed that inhibit MBL binding and lectin pathway activation. Here, we further characterized the interactions of these MAbs and their Fab fragments to MBL. Whole MAbs or their Fab fragments bound to MBL with relatively high affinity. Fab fragments of 3F8 were functionally effective in inhibiting MBL-dependent complement activation, however, steric hindrance of MAb 2A9 was essential for inhibition of MBL-dependent complement activation. We identified the hinge region, and residues EDCVLLL within the carbohydrate recognition domain of MBL as the recognition sites for MAb 3F8 and 2A9, respectively. The interaction of MAbs (e.g., 3F8 and 2A9) to MBL was dependent on the conformation of their recognition sites. These findings demonstrate that MBL binding can be inhibited by at least two separate and independent mechanisms.

The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells
K Ohtani, Y Suzuki, S Eda, T Kawai, T Kase, H Keshi, Y Sakai, A Fukuoh, T Sakamoto, H Itabe, T Suzutani, M Gasawara, Yoshida, I, N Wakamiya
JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 47, 44222, 44228, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2001年11月
英語, 研究論文（学術雑誌）, Collectins are a family of C-type lectins that have collagen-like sequences and carbohydrate recognition domains (CRD). They are involved in host defense through their ability to bind to carbohydrate antigens of microorganisms. The scavenger receptors type A and MARCO are classical type scavenger receptors that have internal collagen-like domains. Here we describe a new scavenger receptor that is a membrane-type collectin from placenta (collectin placenta 1 (CL-P1)), which has a typical collectin collagen-like domain and a CRD. The cDNA has an insert of about 2.2 kilobases coding for a protein containing 742 amino acid residues. The deduced amino acid sequence shows that CL-P1 is a type II membrane protein, has a coiled-coil region, a collagen-like domain, and a CRD. It resembles type A scavenger receptors because the scavenger receptor cysteine-rich domain is replaced by a CRD. Northern analyses, reverse transcription-polymerase chain reaction, and immunohistochemistry show that CL-P1 is expressed in vascular endothelial cells but not in macrophages. By immunoblotting and flow cytometry CL-P1 appears to be a membrane glycoprotein of about 140 kDa in human umbilical vein or arterial endothelial cells, placental membrane extracts, and CL-P1 transfected Chinese hamster ovary cells. We found that CL-P1 can bind and phagocytose not only bacteria (Escherichia coli and Staphylococcus aureus) but also yeast (Saccharomyces cerevisiae). Furthermore, it reacts with oxidized low density lipoprotein (OxLDL) but not with acetylated LDL (AcLDL). These binding activities are inhibited by polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate) and OxLDL but not by polycationic ligands (polyadenylic acid or polycytidylic acid), LDL, or AcLDL. These results indicate that CL-P1 might play important roles in host defenses that are different from those of soluble collectins in innate immunity.

Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coli expressed capsid protein and the reactivity of two broadly reactive monoclonal antibodies generated against GII capsid towards GI recombinant fragments
Tomoko Yoda, Yoshitake Terano, Yasuhiko Suzuki, Kenji Yamazaki, Isao Oishi, Tsuyoshi Kuzuguchi, Hiroyoshi Kawamoto, Etsuko Utagawa, Koichi Takino, Hajime Oda, Tadayoshi Shibata
BMC Microbiology, 1, 1, 1, 11, BioMed Central Ltd., 2001年10月08日
英語, 研究論文（学術雑誌）, Background: Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein. Results: In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody. Conclusion: The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.

Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall
S Tsuji, J Uehori, M Matsumoto, Y Suzuki, A Matsuhisa, K Toyoshima, T Seya
JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 26, 23456, 23463, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2001年06月
英語, 研究論文（学術雑誌）, Galactofuranosyl residues are present in various microorganisms but not in mammals, In this study, we identified a human lectin binding to galactofuranosyl residues and named this protein human intelectin (hIntL). The mature hIntL was a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit was a 120-kDa homotrimer in which 40-kDa polypeptides mere bridged by disulfide bonds. The hIntL gene was spilt into 8 exons on chromosome 1q21.3, and hIntL mRNA was expressed in the heart, small intestine, colon, and thymus, hIntL showed high levels of homology with mouse intelectin, Xenopus laevis cortical granule lectin/oocyte lectin, lamprey serum lectin, and ascidian galactose-specific lectin, These homologues commonly contained no carbohydrate recognition domain, which is a characteristic of C-type lectins, although some of them have been reported as Ca2+-dependent lectins. Recombinant hIntL revealed affinities to D-pentoses and a D-galactofuranosyl residue in the presence of Ca2+, and recognized the bacterial arabinogalactan of Nocardia containing D-galactofuranosyl residues. These results suggested that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in the recognition of bacteria-specific components in the host.

Mannose-binding lectin gene: polymorphisms in Japanese patients with systemic lupus erythematosus, rheumatoid arthritis and Sjogren’s syndrome
A Tsutsumi, K Sasaki, N Wakamiya, K Ichikawa, T Atsumi, K Ohtani, Y Suzuki, T Koike, T Sumida
Genes Immun., 2, 2, 99, 104, NATURE PUBLISHING GROUP, 2001年04月
英語, 研究論文（学術雑誌）, Mannose-binding lectin (MBL) is a key element of the innate immunity, with a structure similar to complement C1q. Serum MBL levels are greatly affected by the polymorphisms of the MBL gene. In particular, codon 54 mutation of the MBL gene results in a significant reduction of serum MEL. To determine whether polymorphism of the MBL gene is associated with occurrence of systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjogren's syndrome in the Japanese population, we analyzed the MBL gene polymophisms of these patients and controls, by polymerase chain reaction-restriction fragment length polymorphism methods. We found that patients studied had a significantly higher frequency of having homozygous codon 54 mutation compared to controls. In particular patients with SLE or Sjogren's syndrome showed higher probabilities of being homozygous for this mutation. Among subjects with the same genotype, SLE patients tended to have higher serum MBL concentration than controls. Analysis of the promotor region suggested that SLE patients heterozygous for the codon 54 mutation have a higher probability of having a low producing haplotype for the gene without the codon 54 mutation. We conclude that persons homozygous for codon 54 mutation of the MBL gene may be prone to occurrence of autoimmune disorders including SLE, in the Japanese. MBL may have protective effects on occurrence and progression of SLE.

Recombinant expression of human mannan-binding lectin
T Vorup-Jensen, ES Sorensen, UB Jensen, W Schwaeble, T Kawasaki, Y Ma, K Uemura, N Wakamiya, Y Suzuki, TG Jensen, K Takahashi, RAB Ezekowitz, S Thiel, JC Jensenius
INTERNATIONAL IMMUNOPHARMACOLOGY, 1, 4, 677, 687, ELSEVIER SCIENCE BV, 2001年04月
英語, 研究論文（学術雑誌）, Mannan-binding lectin (MBL) constitutes an important part of the innate immune defend by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MEL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography. (C) 2001 Elsevier Science B.V. All rights reserved.

Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp OY1-2 isolated from soil
Y Suzuki, T Yoda, A Ruhul, W Sugiura
JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 12, 9059, 9065, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2001年03月
英語, 研究論文（学術雑誌）, Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp, OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH, A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp, OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp, OY1-2, In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.

Molecular cloning of CL-P1 gene
N Wakamiya, Y Suzuki
SEIKAGAKU, 73, 3, 205, 208, JAPANESE BIOCHEMICAL SOC, 2001年03月, ［査読有り］
日本語, 研究論文（学術雑誌）

Usefulness of self ligation mediated polymerase chain reaction: a rapid method for fingerprinting in molecular epidemiology of tuberculosis.
アミン ルフル, 鈴木 定彦, 高鳥毛 敏雄, 多田羅 浩三, 白倉 良太
Kekkaku, 76, 1, 9, 18, JAPANESE SOCIETY FOR TUBERCULOSIS, 2001年
英語, 結核菌の中に挿入されているDNA断片IS6110をプローブとしたRFLP分析が結核の疫学や診断の強力な手段の1つとして利用されている。しかし, RFLP分析を行うにはDNA量が1 micro-gram以上必要であり, 検査結果が出るまでに2日を要する難点がある。そこで, われわれはこの不便さを克服するためにPCRの技術を利用した新たな検査方法であるSelf Ligation Mediated Polymerase Chain Reaction (SL-PCR) 法を開発した。この方法は, IS6110の内側から外側向きに配列した1対のプライマーを用いるものである。DNA断片のIS6110を制限酵素Sau 3A Iを使って分解し, PCR法を使って増幅させ, 結核菌の染色体のDNAと結合させる方法である。この方法を用いることにより, 結核菌の株を8時間以内で識別することが可能となる。今後, 結核患者の接触者調査や結核蔓延状況の解明などのスクリーニング検査法としても活用しうる方法である。

日本人におけるMBL(mannan-binding lectin)遺伝子変異と血中濃度の検討
芥子 宏行, 大谷 克城, 坂本 隆志, 岸 雄一郎, 荒木 宏昌, 鈴木 定彦, 若宮 伸隆
医学のあゆみ, 194, 12, 957, 958, 医歯薬出版(株), 2000年09月, ［査読有り］
日本語, 研究論文（学術雑誌）, 健常日本人497例におけるMBL遺伝子変異と血中濃度の測定により,MBLの遺伝子多型はコドン54のみにみられ,その頻度は野生型70.8%,ヘテロ変異型22.5%,ホモ変異型6.7%であった.本ホモ変異型の比率は欧米の比率に比べて高値であるが,血中濃度とgenotypeが十分相関しており,今後,MBLの関与が考えられる各種疾患群との比較において日本人のMBL遺伝子多型と血中濃度の一つのスタンダードになりうる

Mannose-binding lectin polymorphisms in patients with hepatitis C virus infection
K Sasaki, A Tsutsumi, N Wakamiya, K Ohtani, Y Suzuki, Y Watanabe, N Nakayama, T Koike
SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, 35, 9, 960, 965, TAYLOR & FRANCIS AS, 2000年09月
英語, 研究論文（学術雑誌）, Background: Persistent infection with hepatitis C virus (HCV) leads to liver cirrhosis (LC) and often to Liver cancer. Little is known about host factors that determine the variable natural history. Mannose-binding lectin (MBL) is an important constituent of the innate immune system. In white patients there is an association between codon 52 mutation of the MBL gene and persistent hepatitis B virus (HBV) infection. To determine whether MBL gene polymorphisms affect the course of HCV infection, we investigated the association between MBL gene polymorphisms and HCV infection in Japanese subjects. Methods: Fifty-two HCV-infected Japanese patients (8 with chronic inactive hepatitis (CIH), 31 with chronic active hepatitis (CAH), 13 with LC) and 50 normal controls were studied. MBL gene mutations were determined by means of polymerase chain reaction and restriction fragment length polymorphism analyses. Results: Codon 52 and codon 57 mutations were absent in all subjects. Homozygous mutation in codon 54 was present in one (0.9%) patient. Heterozygous codon 54 mutation was present in 17 (32%) of the 52 patients and in 21 (41%) of the controls. No significant difference in the frequency of codon 54 mutation was observed between patient and control groups. However, although no significant relationship was observed between MBL polymorphisms and the levels of HCV RNA, all patients with heterozygous or homozygous codon 54 mutations had CAH or LC. In contrast, 8 of the 34 patients without codon 54 mutation remained at CIH. (P = 0.0405). Conclusion: MBL may be one of the factors that influence the course of HCV infection.

Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection
T Yoda, Y Terano, A Shimada, Y Suzuki, K Yamazaki, N Sakon, Oishi, I, ET Utagawa, Y Okuno, T Shibata
JOURNAL OF MEDICAL VIROLOGY, 60, 4, 475, 481, WILEY-LISS, 2000年04月
英語, 研究論文（学術雑誌）, The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant: NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA rests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WE. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test. J. Med. Virol. 60:475-481, 2000. (C) 2000 Wiley-Liss, Inc.

A comparative analysis of the antigenic, structural, and functional properties of three different preparations of recombinant human interleukin-18
S Kikkawa, K Shida, H Okamura, NA Begum, M Matsumoto, S Tsuji, M Nomura, Y Suzuki, K Toyoshima, T Seya
JOURNAL OF INTERFERON AND CYTOKINE RESEARCH, 20, 2, 179, 185, MARY ANN LIEBERT INC PUBL, 2000年02月
英語, 研究論文（学術雑誌）, We compared the structural and functional properties of three recombinant human interleukin-18 (rIL-18) preparations, commercially available (Pep rIL-18) and prepared in our laboratory (active and inactive, according to their ability to potentiate IL-12-mediated interferon-gamma [IFN-gamma] induction in lymphocytes). All three preparations showed multimer formation on SDS-PAGE/immunoblotting using monoclonal antibodies (mAb) against the inactive form of rIL-18. In contrast, only the 18-kDa bands were recognized in each sample by mAb against the active form of rIL-18, The amounts of multimers and the 18-kDa moiety of Pep rIL-18 resembled those of the inactive rather than the active form. Likewise, the reaction profile of Pep rIL-18 toward mAb was very similar to that of inactive but not active rIL-18 on sandwich ELISA. Pep rIL-18 potentiated IFN-gamma-inducing activity together with IL-12, but its potency was 100-fold less than that of the active rIL-18, and excess doses were required for its activity. The inactive rIL-18 showed virtually no IFN-gamma-inducing ability, but when reduced and reconstituted, it inhibited the IFN-gamma-inducing activitly of active rIL-18. These results suggest that there are two categories of recombinant IL-18 that are structurally, functionally, and antigenically different, and the mAb 125-2H and 21 can discriminate these two IL-18 populations by recognizing the epitopes specfically expressed on active and inactive IL-18, respectively.

Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli
T Yoda, Y Terano, Y Suzuki, K Yamazaki, Oishi, I, E Utagawa, A Shimada, S Matsuura, M Nakajima, T Shibata
MICROBIOLOGY AND IMMUNOLOGY, 44, 11, 905, 914, CENTER ACADEMIC PUBL JAPAN, 2000年
英語, 研究論文（学術雑誌）, The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli, All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions, Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548), Furthermore, two MAbs (1B4 and 1F6) reacted in WE with three purified NV strains (genogroup II) derived from patients' stool samples, It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.

The 3’-UT of the ubiquitous mRNA of human CD46 confers selective suppression of protein production in murine cells
SHIDA K, NOMURA M, MATSUMOTO M, SUZUKI Y, TOYOSHIMA K, SEYA T
Eur J Immunol, 29, 11, 3603, 3608, WILEY-V C H VERLAG GMBH, 1999年11月
英語, 研究論文（学術雑誌）, Mice express CD46 protein and its approximately 1.5-kb mRNA only in the testicular germ cells, unlike primates and pigs which ubiquitously express CD46 and its approximately 4 kb mRNA. Human CD46 is not well expressed in transgenic mice carrying human CD46 cDNA. To analyze the mechanism of regulation of human CD46 expression in mouse cells, we cloned the long (ubiquitous similar to 4 kb, L-form) and short (similar to 1.5 kb, S-form) forms of human CD46 cDNA whose size difference is due to a stretch of the 3'-UT. Transfection of either cDNA resulted in marked S-form-dependent protein generation in all mouse cell lines tested. in contrast, there were virtually no differences in protein synthesis between S- and L-form cDNA in the simian and swine cell lines. Quantitative mRNA analyses and luciferase reporter gene assays suggested that one major cause of this interspecies discrepancy is transcriptional regulation, i.e. selective suppression of the 4-kb mRNA leading to low levels of protein synthesis. Although other mechanisms such as mRNA stability and translational regulation may lead to the low expression levels of L-form-derived CD46 in mice, the silencer activity in the L-form 3'-UT appears to function in human CD46 transcriptional regulation in mice.

RFLP分析による結核小規模感染事例の疫学的研究
田丸亜貴, 鈴木定彦
結核, 74, 7, 555, 561, 1999年07月, ［査読有り］
日本語, 研究論文（学術雑誌）, Restriction fragment length polymorphism (RFLP) analysis was performed with 13 events considered as microepidemics of tuberculosis occurred in Osaka prefecture from July 1995 to July 1998. In 7 out of 13 events, isolates from patients involved in each event showed identical RFLP patterns, hence these events were verified as microepidemics. Out of 7 microepidemics, three were intrafamily infection, the other three occurred in work places and the remaining one was in a school occurred among students. The total number of patients who were involved in microepidemics was 19 and their ages distributed in the wide range from 18 to 69 years old, but 11 were at twenties or forties. The total delay in case-finding was 2 months in the shortest case, and more than 14 months in the longest. In the remaining 6 of 13 events, the isolates from patients were showed different RFLP patterns, although they were suspected as microepidemics. Furthermore, in the 2 microepidemics, one isolate showed different RFLP pattern from the other isolates involved in each events. These facts suggest that there could be many overlooked sources of tuberculosis infection in Osaka.

Human mannan-binding lectin inhibits the infection of influenza A virus without complement
T Kase, Y Suzuki, T Kawai, T Sakamoto, K Ohtani, S Eda, A Maeda, Y Okuno, T Kurimura, N Wakamiya
IMMUNOLOGY, 97, 3, 385, 392, BLACKWELL SCIENCE LTD, 1999年07月
英語, 研究論文（学術雑誌）, Mannan-binding lectin (MBL) is a C-type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagenlike sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro-organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV-A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti-human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non-damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.

Antibody-independent classical complement pathway activation and homologous C3 deposition in xeroderma pigmentosum cell lines
M Kurita, M Matsumoto, S Tsuji, M Kawakami, Y Suzuki, H Hayashi, K Toyoshima, T Seya
CLINICAL AND EXPERIMENTAL IMMUNOLOGY, 116, 3, 547, 553, BLACKWELL SCIENCE LTD, 1999年06月
英語, 研究論文（学術雑誌）, Of human malignantly transformed cell lines, xeroderma pigmentosum (XP) cell lines were found to be highly susceptible to homologous complement (C): cells were opsonized by C3 fragments on incubation with diluted normal human serum. C3 fragment deposition on XP cells was Ca2+-dependent and occurred on live cells but not UV-irradiated apoptotic cells. (Ca2+ is required for activation of the classical C pathway via C1q and the lactin pathway via mannose binding lectin (MBL), and the surface of apoptotic cells usually activates the alternative C pathway.) In this study we tested which of the pathways participates in XP cell C3 deposition. In seven cell lines that allowed C3 deposition (i), C1q was shown to be essential but MBL played no role in C activation, (ii) Cls but not MASP bound XP cells for activation, (iii) no antibodies recognizing XP cells were required for homologous C3 deposition, and (iv) the alternative pathway barely participated in C3 deposition. Furthermore, the levels of C-regulatory proteins for host cell protection against C, decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), were found to be relatively low in almost all XP cell lines compared with normal cells. These results indicate that XP cells activate the classical C pathway in an antibody-independent manner through the expression of a molecule which directly attracts C1q in a C-activating form, and that relatively low levels of DAF and MCP on XP cells facilitate effective C3 deposition. The possible relationship between the pathogenesis of XP and our findings is discussed.

Molecular cloning of a novel human collectin from liver (CL-L1)
K Ohtani, Y Suzuki, S Eda, T Kawai, T Kase, H Yamazaki, T Shimada, H Keshi, Y Sakai, A Fukuoh, T Sakamoto, N Wakamiya
JOURNAL OF BIOLOGICAL CHEMISTRY, 274, 19, 13681, 13689, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1999年05月
英語, 研究論文（学術雑誌）, Collectins are a C-lectin family with collagen-like sequences and carbohydrate recognition domains. These proteins can bind to carbohydrate antigens of microorganisms and inhibit their infection by direct neutralization and agglutination, the activation of complement through the lectin pathway, and opsonization by collectin receptors, Here we report the cloning of a cDNA encoding human collectin from liver (CL-L1 (collectin liver 1)) that has typical collectin structural characteristics, consisting of an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain. The cDNA has an insert of 831 base pairs coding for a protein of 277 amino acid residues. The deduced amino acid sequence shows that this collectin has a unique repeat of four lysine residues in its C-terminal area. Northern blot, Western blot, and reverse tp anscription-polymerase chain reaction analyses showed that CL-L1 is present mainly in liver as a cytosolic protein and at low levels in placenta. More sensitive analyses by reverse transcription-polymerase chain reactions showed that most tissues (except skeletal muscle) have CL-L1 mRNA Zoo-blot analysis indicated that CL-L1 is limited to mammals and birds. A chromosomal localization study indicated that the CL-L1 gene localizes to chromosome 8q23-q24.1, different from chromosome 10 of other human collectin genes. Expression studies of fusion proteins lacking the collagen and N-terminal domains produced in Escherichia coli affirmed that CL-L1 binds mannose weakly. CL-L1 and recombinant CL-L1 fusion proteins do not bind to mannan columns. Analysis of the phylogenetic tree of CL-LI and other collectins indicated that CL-L1 belongs to a fourth subfamily of collectins following the mannan-binding protein, surfactant protein A, and surfactant protein D subfamilies including bovine conglutinin and collectin-43 (CL-43), These findings indicate that CL-LI may be involved in different biological functions.

大阪府下で採取された結核臨床分離株のRFLP分析　　　　　　　　　　　　　　　
田丸 亜貴, 勝川 千尋, 鈴木 定彦
結核, 74, 3, 322, 322, (一社)日本結核・非結核性抗酸菌症学会, 1999年03月
日本語

臨床分離株を用いた結核菌感受性PZA液体培地法の検討　　　　　　　　　　　　　　　
中曽根 智恵, 石田 智恵子, 高嶋 哲也, 田丸 亜貴, 鈴木 定彦, 勝川 千尋
結核, 74, 3, 327, 327, (一社)日本結核・非結核性抗酸菌症学会, 1999年03月
日本語

血清マンナン結合蛋白質のハンセン病における役割に関する研究
鈴木 定彦, 勝川 千尋, 田丸 亜貴, Ruhul Amin, 牧野 正直, 若宮 伸隆
日本ハンセン病学会雑誌, 68, 1, 50, 50, 日本ハンセン病学会, 1999年03月, ［査読有り］
日本語

Preparation of recombinant alpha2 antigen of M. leprae in E. coli and the application for sero-diagnosis of leprosy.　　　　　　　　　　　　　　　
Yin Y, Suzuki Y, Makino M, Wu Q, Hou W
Chin. Med. Sci. J., 14, 106, 1999年, ［査読有り］
中国語, 研究論文（学術雑誌）

Gene cloning and expression of Mycobacterium leprae alpha 2 antigen.　　　　　　　　　　　　　　　
Yin Y, Suzuki Y, Makino M, Wu Q
Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 21, 62, 68, 1999年, ［査読有り］
中国語, 研究論文（学術雑誌）

High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells
K Ohtani, Y Suzuki, S Eda, T Kawai, T Kase, H Keshi, Y Sakai, S Yamamoto, T Sakamoto, N Wakamiya
JOURNAL OF IMMUNOLOGICAL METHODS, 222, 1-2, 135, 144, ELSEVIER SCIENCE BV, 1999年01月
英語, 研究論文（学術雑誌）, We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MEL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 mu g/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MEL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans. (C) 1999 Elsevier Science B.V. All rights reserved.

Characterization of truncated human mannan-binding protein (MBP) expressed in Escherichia coli
S Eda, Y Suzuki, T Kawai, K Ohtani, T Kase, T Sakamoto, N Wakamiya
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 62, 7, 1326, 1331, TAYLOR & FRANCIS LTD, 1998年07月
英語, 研究論文（学術雑誌）, Mannan-binding protein (MBP) is a calcium-dependent mammalian serum lectin important in first-line host defense. MBP belongs to the collectin family, which is characterized by an NH2-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain (CRD). We have expressed a recombinant human MBP, consisting of the short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain, and the CRD, in Escherichia coli. The truncated MBP was capable of forming trimers by association of the neck domain and could bind sugar with a specificity similar to that of the native form. Results of hemagglutination inhibition (HI) assay of influenza A virus showed that the truncated MBP inhibited hemagglutination less strongly, although the native MBP induced the HI phenomenon. These results suggest that an oligomeric structure is an advantage for MBP to have full biological activity against influenza A virus.

Comparative studies on the catalytic roles of cytochrome P450 2C9 and its Cys- and Leu-variants in the oxidation of Warfarin, flurbiprofen, and diclofenac by human liver microsomes
H Yamazaki, K Inoue, K Chiba, N Ozawa, T Kawai, Y Suzuki, JA Goldstein, FP Guengerich, T Shimada
BIOCHEMICAL PHARMACOLOGY, 56, 2, 243, 251, PERGAMON-ELSEVIER SCIENCE LTD, 1998年07月
英語, 研究論文（学術雑誌）, S-Warfarin 7-hydroxylation, S-flurbiprofen 4'-hydroxylation, and diclofenac 4'-hydroxylation activities were determined in Liver microsomes of 30 humans of which 19 were wild-type (Arg144.Ile359), 8 were heterozygous Cys (Cys144.Ile359), and 3 were heterozygous Leu (Arg144.Leu359) allelic variants of the cytochrome P450 2C9 (CYP2C9) gene. All of the human samples examined contained P450 protein(s) immunoreactive with anti-CYP2C9 antibodies in liver microsomes. Individuals with the Cys144 allele of CYP2C9 had similar, but slightly lower, activities for the oxidations of these substrates than those of wild-type CYP2C9. One of the three human samples heterozygous for the Leu359 allele had very low V-max and high K-m values for the oxidation of three substrates examined, while the other two individuals gave kinetic parameters comparable to those seen in the wild-type and Cys144 CYP2C9. Reverse transcriptase-polymerase chain reaction analysis, however, showed that all of the three human samples with the heterozygous Leu359 variant were found to express both Ile359 and Leu359 variants at relatively similar extents in liver RNA of three humans. These results suggest that the Cys144 variant of CYP2C9 catalyzes the CYP2C9 substrates at rates comparative to, but slightly lower than, those of wild-type CYP2C9, while the Leu359-allelic variant has slower rates for the oxidation of these drug substrates. Activities for the oxidation-of these CYP2C9 substrates in humans with heterozygous Leu359 allele is likely to be dependent on the levels of expression of each of the wild- and Leu-variants in the livers. However, one of the humans with a heterozygous Leu allele was found to have very low activities towards the oxidation of CYP2C9 substrates. The basis of this defect in catalytic functions towards CYP2C9 substrates is unknown. (C) 1998 Elsevier Science Inc.

Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene
Y Suzuki, C Katsukawa, A Tamaru, C Abe, M Makino, Y Mizuguchi, H Taniguchi
JOURNAL OF CLINICAL MICROBIOLOGY, 36, 5, 1220, 1225, AMER SOC MICROBIOLOGY, 1998年05月
英語, 研究論文（学術雑誌）, In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, greater than or equal to 200 mu g/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 as found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.

Post-translational modification and intracellular localization of a splice product of CD46 cloned from human testis: role of the intracellular domains in O-glycosylation
T Hara, Y Suzuki, T Nakazawa, H Nishimura, S Nagasawa, M Nishiguchi, M Matsumoto, M Hatanaka, M Kitamura, T Seya
IMMUNOLOGY, 93, 4, 546, 555, BLACKWELL SCIENCE LTD, 1998年04月
英語, 研究論文（学術雑誌）, We obtained a unique CD46 cDNA, STc/CY4, from the human testis, the predicted amino acid sequence of which suggested the presence of a novel isoform of CD46. This message was present predominantly in the testis, and the predicted isoform possessed a short (11 amino acids) transmembrane section (TM) and an unidentified cytoplasmic tail (CT). When expressed in Chinese hamster ovary (CHO) cells, this CD46 isoform underwent no O-glycosylation and was mostly retained in the endoplasmic reticulum, This unusual behaviour of the new isoform was due in part to the short TM and the unusual sequences of the CY. The molecular mass of this isoform was 42 000, approximately 20 000 smaller than conventional CD46. These properties of the STc/CY4 isoform were similar to those of sperm CD46. The only difference between sperm CD46 and the STc/CY4 isoform expressed on CHO cells was that only the latter possessed N-linked sugars of high mannose types, Since the STc/CY4 isoform may behave like sperm CD46 in cellular localization and post-translational modification, studies of sperm-egg interassociation were performed using hamster eggs and CHO cell clones expressing various isoforms including the STc/CY4. Rosette formation was seen most effectively between hamster eggs and STc/CY4-expressing CHO cells. These results infer that O-gycosylation perturbs CD46-mediated sperm-binding to eggs and thus sperm CD46 lacking O-linked sugars can serve as an adhesion molecule, The possible role of CD46 in fertilization and the structural differences between sperm and conventional CD46 are discussed.

Molecular and biological characterization of rabbit mannan-binding protein (MBP)
T Kawai, Y Suzuki, S Eda, K Ohtani, T Kase, T Sakamoto, H Uemura, N Wakamiya
GLYCOBIOLOGY, 8, 3, 237, 244, OXFORD UNIV PRESS, 1998年03月
英語, 研究論文（学術雑誌）, Mannan-binding protein (MBP) is a member of the collectin family of protein, There are two types of MBP, MBP-A and MBP-C, which were found in rodent (rats and mice), rhesus monkey, and cynomolgus monkey, while chimpanzee and human have only one MBP, It was considered that the loss of one MBP gene occurred during hominoid evolution, In this article two rabbit MBP, a liver and serum MBP, were characterized biologically and genetically, Analyses by SDS-PAGE under reduced condition and their amino acid sequences of both MBPs showed that they have a same molecular weight of 32 kDa and their amino acid sequences were identical, A serum MBP has a higher ability to activate complement than does a liver MBP; however, a liver MBP inhibits hemagglutination by influenza virus as strongly as a serum MBP does, cDNA clones encoding the rabbit MBP were isolated from a rabbit cDNA liver library using whole cDNA of mouse MBP-C as a probe. The cDNA carried an insert of 744 bp coding for a protein of 247 acid residues with a signal peptide of 22 residues. The deduced amino acid sequence of the cDNA was identical to that of amino acid sequences of the 32 kDa proteins determined here, Northern blot analysis showed that mRNA transcripts of about 0.9 and 3.0 kb were expressed only in the liver. The analysis of the phylogenetic tree of rabbit and bovine MBPs and other collectins indicates that the loss of MBP gene occurred not only during hominoid evolution but also at some points after the separation of birds and mammals.

Regulation of promoter and intron enhancer activity in immunoglobulin heavy chain genes through late stage of B cell development　　　　　　　　　　　　　　　
Microbiol . Immunol, 42, 399, 405, 1998年

Characterization of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan
E Katsukawa, A Tamaru, Y Miyata, C Abe, M Makino, Y Suzuki
JOURNAL OF APPLIED MICROBIOLOGY, 83, 5, 634, 640, BLACKWELL SCIENCE LTD, 1997年11月
英語, 研究論文（学術雑誌）, Mutations in the rpsL and rrs genes associated with streptomycin resistance in Mycobacterium tuberculosis clinically isolated in Japan were characterized. The rpsL genes of 172 clinical isolates were amplified by PCR and classified into two groups on the basis of MboII restriction digestion, Thirty-three out of 54 (61.1%) streptomycin-highly resistant isolates (MIC > 200 mu g ml(-1)) were not digested by MboII. By contrast, the remaining 21 of 54 (38.9%) streptomycin-highly resistant isolates, all of 41 isolates with streptomycin resistance at a lower level (20 mu g ml(-1) < MIC less than or equal to 200 mu g ml(-1)), and all of 77 streptomycin-sensitive isolates, were restricted, Thus, all isolates resistant for MboII digestion showed a high level of resistance to streptomycin. Subsequently, the sequence for the rpsL and rrs genes from the 46 isolates were analysed, Eighteen out of 19 (94.7%) streptomycin-highly resistant isolates carried a mutation in any rpsL gene at position 43 or 88, or the rrs gene; 10 out of 17 (58.8%) streptomycin-resistant isolates at a lower level were confirmed to exhibit the mutation of either the mutated rpsL gene at position 88, or the rrs gene, In the total 36 streptomycin-resistant isolates, the mutation of the rpsL or rrs gene was observed in 28 streptomycin-resistant isolates, corresponding to 77.8%, whereas none of the streptomycin-sensitive isolates had mutations in either the rpsL or rrs gene.

Characterization of recombinant bovine conglutinin expressed in a mammalian cell
Y Suzuki, S Eda, T Kawai, K Ohtani, T Kase, T Sakamoto, N Wakamiya
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 238, 3, 856, 860, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1997年09月
英語, 研究論文（学術雑誌）, We describe here the successful expression of recombinant bovine conglutinin in CHO cells as well as its physical rind biological characteristics. Geneticin-resistant transformants harboring bovine conglutinin cDNA in the expression vector pNOW/CMV-A were screened by Western blot analysis for secretion into media of recombinant conglutinin. A four-day amplification of the transgene with increasing concentrations of methotrexate resulted in a dose-dependent increase in the production of recombinant conglutinin to a final concentration of 18.6 mu g/ml of media. Recombinant conglutinin purified from this media by affinity column chromatography on mannan-agarose had a migration pattern similar to that of native conglutinin on polyacrylamide gel electrophoresis under reducing, nonreducing, and native conditions. The recombinant conglutinin exhibited sugar binding, conglutination, hemagglutination inhibition, and neutralization of influenza A virus, activities engaged in by the native conglutinin. This is the first report describing a high level of expression of a serum cruciform collectin with the full range of biological activity. (C) 1997 Academic Press.

Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: The existence of genes possibly involved in chloroplast division
T Wakasugi, T Nagai, M Kapoor, M Sugita, M Ito, S Ito, J Tsudzuki, K Nakashima, T Tsudzuki, Y Suzuki, A Hamada, T Ohta, A Inamura, K Yoshinaga, M Sugiura
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 94, 11, 5967, 5972, NATL ACAD SCIENCES, 1997年05月
英語, 研究論文（学術雑誌）, The complete nucleotide sequence of the chloroplast genome (150,613 bp) from the unicellular green alga Chlorella vulgaris C-27 has been determined, The genome contains no large inverted repeat and has one copy of rRNA gene cluster consisting of 16S, 23S, and 5S rRNA genes, It contains 31 tRNA genes, of which the tRNA(Leu)(GAG) gene has not been found in land plant chloroplast DNAs analyzed so far, Sixty-nine protein genes and eight ORFs conserved with those found in land plant chloroplasts have also been found, The most striking is the existence of two adjacent genes homologous to bacterial genes involved in cell division, minD and minE, which are arranged in the same order in Escherichia coli, This finding suggests that the mechanism of chloroplast division is similar to bacterial division, Other than minD and minE homologues, genes encoding ribosomal proteins L5, L12, L19, and S9 (rpl5, rpl12, rpl19, and rps9); a chlorophyll biosynthesis Mg chelating subunit (chlI); and elongation factor EF-Tu (tufA), which have not been reported from land plant chloroplast DNAs, are present in this genome, However, many of the new chloroplast genes recently found in red and brown algae have not been found in C. vulgaris. Furthermore, this algal species possesses two long ORFs related to ycf1 and ycf2 that are exclusively found in land plants, These observations suggest that C. vulgaris is closer to land plants than to red and brown algae.

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor
C Hashimoto, H Masuda, M Ayaki, Y Suzuki, A Uenaka, T Seya, J Miyoshi, K Takahashi, Y Inui
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1355, 3, 205, 208, ELSEVIER SCIENCE BV, 1997年03月
英語, 研究論文（学術雑誌）, A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.

Swine influenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium
T Suzuki, G Horiike, Y Yamazaki, K Kawabe, H Masuda, D Miyamoto, M Matsuda, SI Nishimura, T Yamagata, T Ito, H Kida, Y Kawaoka, Y Suzuki
FEBS LETTERS, 404, 2-3, 192, 196, ELSEVIER SCIENCE BV, 1997年03月, ［査読有り］
英語, 研究論文（学術雑誌）, We determined the ratio of N-glycolylneuraminic acid (Neu5Gc) to N-acetylneuraminic acid (Neu5Ac) in swine respiratory epithelia by fluorometric high-performance liquid chromatography, and examined the binding specificity of swine influenza virus strains for gangliosides containing different molecular species of sialic acid (Neu5Ac and Neu5Gc), and for bovine erythrocyte sialoglycoprotein 2 (GP-2) containing Neu5Ge as its predominate sialic acid (96% of total sialic acids), The presence of Neu5Gc, which had not been detected in human tracheal epithelia, and Neu5Ac in swine tracheal epithelia was observed in a 1:1 ratio, The swine influenza virus H1 and H3 isolates tested, except for A/swine/Iowa/15/30 (H1N1), displayed a marked binding ability for sialylsugar chains containing Neu5Gc compared with that of the human influenza virus strains, These results suggest that swine influenza viruses recognize sialylsugar chains containing the molecular species of sialic acid present predominantly in the swine tracheal epithelium. (C) 1997 Federation of European Biochemical Societies.

Cloning and characterization of a cDNA encoding bovine mannan-binding protein
T Kawai, Y Suzuki, S Eda, K Ohtani, T Kase, Y Fujinaga, T Sakamoto, T Kurimura, N Wakamiya
GENE, 186, 2, 161, 165, ELSEVIER SCIENCE BV, 1997年02月
英語, 研究論文（学術雑誌）, To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE, The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.

Recombinant human surfactant protein D, lacking the N-terminal and collagenouse domains, has less bacterial agglutinating activity but is able to inhibit haemagglutination by influenze A virus　　　　　　　　　　　　　　　
Biochem. J., 323, 393, 399, 1997年

Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species
T Ito, Y Suzuki, L Mitnaul, A Vines, H Kida, Y Kawaoka
VIROLOGY, 227, 2, 493, 499, ACADEMIC PRESS INC ELSEVIER SCIENCE, 1997年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Despite their uniform ability to bind to oligosaccharide-containing terminal sialic acids, influenza A viruses show differences in receptor specificity. To test whether agglutination of erythrocytes from different animal species could be used to assess the receptor specificity of influenza A viruses, we determined the agglutinating activities of a range of virus strains, including those with known receptor specificities, using erythrocytes from seven animal species. All equine and avian viruses, including those known to recognize N-acetyl and N-glycolyl sialic acid linked to galactose by the alpha 2,3 linkage (NeuAc alpha 2,3Gal and NeuGc alpha 2,3Gal), agglutinated erythrocytes from all of the animal species tested (chickens, ducks, guinea pigs, humans, sheep, horses, and cows). The human viruses, including those known to preferentially recognize NeuAc alpha 2,6Gal, agglutinated all but the horse and cow erythrocytes. Fluorescence-activated cell sorting analysis of erythrocytes using linkage-specific lectins [Sambucus nigra agglutinin for sialic acid (SA)alpha 2,6Gal and Maackia amurensis agglutinin for SA alpha 2,3Gal] showed that both cow and horse erythrocytes contain a large amount of SA alpha 2,3Gal-, but virtually no SA2,6Gal-specific lectin-reactive oligosaccharides on the cell surface, while human and chicken erythrocytes contained both types of oligosaccharides. Considering that the majority (>93%) of sialic acid in horse and cow erythrocytes is of the N-glycolyl type, our results suggest that viruses able to agglutinate these erythrocytes (i.e., avian and equine viruses) recognize NeuGc alpha 2,3Gal. These findings also show that agglutinating assays with erythrocytes from different animal species would be useful in characterizing the receptor specificity of influenza A viruses. (C) 1997 Academic Press

Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus
S Eda, Y Suzuki, T Kase, T Kawai, K Ohtani, T Sakamoto, T Kurimura, N Wakamiya
BIOCHEMICAL JOURNAL, 316, 316, 43, 48, PORTLAND PRESS, 1996年05月
英語, 研究論文（学術雑誌）, Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.

High expression of membrane cofactor protein of complement (CD46) in human leukaemia cell lines: Implication of an alternatively spliced form containing the STA domain in CD46 up-regulation
T Hara, Y Suzuki, T Semba, M Hatanaka, M Matsumoto, T Seya
SCANDINAVIAN JOURNAL OF IMMUNOLOGY, 42, 6, 581, 590, BLACKWELL SCIENCE LTD, 1995年12月
英語, 研究論文（学術雑誌）, Human membrane cofactor protein (MCP, CD46) is a receptor for the measles virus and serves as a complement regulator which protects host cells from autologous complement attack. MCP is highly polymorphic due to a variety of mRNA splice products. The levels of MCP expression on T and myeloid cell lines are usually two-eightfold higher than those on their normal counterparts, whereas Burkitt's lymphoma B cell lines express less MCP than B cell lineages carrying no EB virus. The molecule has a Ser/Thr-rich (ST) domain adjacent to the functional domain, namely short consensus repeats (SCR). The ST domain and a cytoplasmic tail (CYT) contribute to the MCP polymorphism. The ST domain is encoded by three exons (A, B and C) and major ST isoforms are STABC, STBC and STC. The authors investigated the relationship between the expression levels and isoform usage of MCP by flow cytometry using specific antibodies against STA and STC, by reverse transcriptase-polymerase chain reaction (RT-PCR) with size markers for each splice variant, and by RT-PCR/Southern blotting using a specific probe for STA. The results were (1) the profiles of mean shifts of myeloid and T cell lines were STC < STA on flow cytometry while those of B cell lines and normal blood cells were STA < STC; (2) all cell lines tested by RT-PCR expressed the messages for the isoforms STBC/CYT1, STC/CYT1, STBC/CYT2, and STC/CYT2. The band for STABC/CYT2 overlapped that for STC/CYT1, and the band for STABC/CYT1 was marginal in all cell lines examined; (3) semi-quantitative analysis of the STABC isoforms by Southern blotting indicated the presence of high levels of the STABC messages in myeloid and T-cell lines in comparison with B lymphoid cells and normal leucocytes. Thus, the quantity of MCP expressed parallels the STABC message level, which is up-regulated in T and myeloid leukaemia cell lines.

わが国のリファンピシン耐性臨床分離結核菌におけるrpoB遺伝子の変異
鈴木 定彦, 勝川 千尋, 井上 清
感染症学雑誌, 69, 4, 413, 419, (一社)日本感染症学会, 1995年04月
英語, 小川培地上に生育した結核菌標準株4株(H37Ra株,H37Rv株,青山B株,ATCC35416株),リファンピシン耐性臨床分離結核菌株47株および感受性臨床分離結核菌株17株より抽出したDNAを鋳型としてPCRを行い411塩基対のrpoB遺伝子断片を増幅させた。ストレプトアビジン化ダイナビーズを用いた直接塩基配列決定法によりこのDNA断片の塩基配列を決定し,相互に比較した結果,この遺伝子領域の変異とリファンピシン耐性の間に非常に高い相関が見られた。リファンピシン耐性株47株中44株において変異が検出された。これに対して,感受性株17株および標準株4株においては,まったく変異が検出されなかった

Mutations in rpoB gene of rifampicin resistant clinical isolates of Mycobacterium tuberculosis in Japan　　　　　　　　　　　　　　　
J. Jpn. Assoc. Infect. Dis., 69, 413, 41, 1995年

FLUORESCENCE IN-SITU HYBRIDIZATION ANALYSIS OF CHROMOSOMAL LOCALIZATION OF 3 HUMAN CYTOCHROME-P450-2C GENES (CYP2C8, 2C9, AND 2C10) AT 10Q24.1
K INOUE, J INAZAWA, Y SUZUKI, T SHIMADA, H YAMAZAKI, FP GUENGERICH, T ABE
JAPANESE JOURNAL OF HUMAN GENETICS, 39, 3, 337, 343, TOKYO MEDICAL DENTAL UNIV, 1994年09月
英語, 研究論文（学術雑誌）, Chromosomal localization of three human cytochrome P450 genes belonging to the CYP2C subfamily (CYP2C8, 2C9, and 2C10) was identified by fluorescence in situ hybridization (FISH). An original MP-8 clone was used as a DNA probe for the assignment of the CYP2C10 gene, while two cDMA probes, a 1.37 kb fragment of CYP2C8 and a 1.19 kb fragment of CYP2C9, were obtained after amplifying the predicted fragments (MP-20 and MP-4 clones, respectively) by polymerase chain reaction using a single human liver cDNA library. The results showed that three human CYP2C8, 2C9, and 2C10 cDNAs were located at the same subchromosomal region, 10q24.1.

Comparizon of somatic mutation frequency among immunoglobulin genes
MOTOYAMA N, MIWA T, SUZUKI Y, OKADA H, AZUMA T
J. Exp. Med., 179, 2, 395, 403, 1994年

CLONING AND SEQUENCING OF A CDNA CODING FOR BOVINE CONGLUTININ
Y SUZUKI, YP YIN, M MAKINO, T KURIMURA, N WAKAMIYA
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 191, 2, 335, 342, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1993年03月
英語, 研究論文（学術雑誌）

INDUCIBILITY OF PROTEIN-REACTIVE ANTIBODIES BY PEPTIDE IMMUNIZATION - COMPARISON OF 3 EPITOPE PEPTIDES OF HEN EGG-WHITE LYSOZYME
J SEKI, XH WANG, A OTA, Y SUZUKI, N SAKATO, H FUJIO
JOURNAL OF BIOCHEMISTRY, 111, 2, 259, 264, JAPANESE BIOCHEMICAL SOC, 1992年02月
英語, 研究論文（学術雑誌）, Three epitope peptides of hen egg-white lysozyme (HEL) were tested for ability to induce antibodies reactive with native HEL. Each peptide was coupled to bovine gamma-globulin (B-gamma-G) and 4 rabbits were immunized with each peptide-B-gamma-G conjugate in complete Freund's adjuvant. The mean association constants (K0s) of HEL-reactive antibodies (HEL-R-Abs) from each immunizing group to [H-3] acetyl HEL or to [H-3] acetyl-peptide were measured in solution by a double antibody method. Only peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) induced high-affinity antibodies to HEL (K0 = 2.5 x 10(6)-2.3 x 10(7) M-1) among the three epitope peptides tested. The association constants of anti-peptide loop I.II to [H-3]acetyl peptide loop I.II were always one to two orders of magnitude higher than those to HEL. In addition, 50 to 80% of the anti-peptide loop I.II antibodies were reactive with native HEL. The specificity of anti-peptide loop I.II was directed to a conformational feature of the peptide rather than to native HEL and reactivity of the antibody to HEL was interpreted as a kind of cross-reaction. The HEL-R-Abs from anti-Ploop I.II antisera also manifested neutralizing activities against the enzymic activity of HEL when Micrococcus luteus was used as the substrate.

ANALYSIS OF THE PROMOTER REGION IN THE RIBOSOMAL-RNA OPERON FROM MYCOBACTERIUM-BOVIS BCG
Y SUZUKI, A NAGATA, T YAMADA
ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY, 60, 1, 7, 11, KLUWER ACADEMIC PUBL, 1991年07月
英語, 研究論文（学術雑誌）, At least two transcriptional initiation sites were observed in rRNA operon of Mycobacterium bovis BCG at approximately -187 and -265. The former transcriptional signal was recognized by Escherichia coli RNA polymerase, whereas the later was not.

HUMAN BRAIN PROSTAGLANDIN-D SYNTHASE HAS BEEN EVOLUTIONARILY DIFFERENTIATED FROM LIPOPHILIC-LIGAND CARRIER PROTEINS
A NAGATA, Y SUZUKI, M IGARASHI, N EGUCHI, H TOH, Y URADE, O HAYAISHI
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 88, 9, 4020, 4024, NATL ACAD SCIENCES, 1991年05月
英語, 研究論文（学術雑誌）, cDNAs for glutathione-independent prostaglandin D synthase were isolated from cDNA libraries of human brain. The longest cDNA insert was 837 base pairs long and contained a coding region of 570 base pairs corresponding to 190 amino acid residues with a calculated M(r) of 21,016. Between two cDNA inserts isolated from the two different libraries, nucleotide substitutions were observed at 16 positions, including conservative amino acid substitutions at 2 positions and nonconservative substitutions at 5 positions, indicating genetic heterogeneity of this enzyme in humans. The computer-assisted homology search revealed that the enzyme is a member of the lipocalin superfamily, comprising secretory hydrophobic molecule transporters, showing the greatest homology (28.8-29.4% identity; 51.3-53.1% similarity) to alpha-1-microglobulin among the members of this superfamily. In a phylogenetic tree of the superfamily, this enzyme, alpha-1-microglobulin, and the gamma-chain of the complement component C8 form a cluster separate from the other 14 members. The two distinctive characteristics of glutathione-independent prostaglandin D synthase, as compared to the other members of this superfamily, are its enzymatic properties and its association with membranes that were probably acquired after evolutionary divergence of the two lipocalins. Based on the observed sequence homology, the tertiary structure of the enzyme was deduced to consist of an eight-stranded anti-parallel beta-barrel forming a hydrophobic pocket. Furthermore, the Cys-65 residue in the pocket, which is conserved only in the human and rat enzymes but not in other lipocalins, was considered to be a putative active site of the enzyme.

ACTIVATION OF IDIOTYPE-SPECIFIC CD4+ T-CELL LINE - CELLULAR PROCESSING OF EXOGENOUS SELF-IMMUNOGLOBULIN
N SAKATO, P RUGDECH, T YODA, A OTA, Y ZHAO, M SEMMA, Y SUZUKI, H FUJIO
IMMUNOLOGY, 71, 2, 153, 157, BLACKWELL SCIENCE LTD, 1990年10月
英語, 研究論文（学術雑誌）

Primary Structure of Rat Mrain Prostaglandin D Synthetase Deduced from cDNA Sequence　　　　　　　　　　　　　　　
J. Biol Che., 264, 1041, 1045, 1989年

Primary Structure of Rat Brain Prostaglandin D Synthetase Deduced from the cDNA Sequence
YOSHIHIRO URADE, AKIHISA NAGATA, YASUHIKO SUZUKI, YUTAKA FUJII, OSAMU HAYAISHI
Annals of the New York Academy of Sciences, 559, 1, 491, 493, 1989年, ［査読有り］
英語, 研究論文（学術雑誌）

COMPLETE NUCLEOTIDE-SEQUENCE OF THE 16S RIBOSOMAL-RNA GENE OF MYCOBACTERIUM-BOVIS BCG
Y SUZUKI, A NAGATA, Y ONO, T YAMADA
JOURNAL OF BACTERIOLOGY, 170, 6, 2886, 2889, AMER SOC MICROBIOLOGY, 1988年06月
英語

MOLECULAR-CLONING AND CHARACTERIZATION OF AN RIBOSOMAL-RNA OPERON IN STREPTOMYCES-LIVIDANS TK21
Y SUZUKI, Y ONO, A NAGATA, T YAMADA
JOURNAL OF BACTERIOLOGY, 170, 4, 1631, 1636, AMER SOC MICROBIOLOGY, 1988年04月
英語, 研究論文（学術雑誌）

The nucieotide sequence of 16S rRNA gene from Streptomyces lividans Tk21
Yasuhiko Suzuki, Takeshi Yamada
Nucleic Acids Research, 16, 1, 370, 1988年01月11日
英語, 研究論文（学術雑誌）

Mycobacterium phlei のリボソームRNA遺伝子の解析
鈴木定彦, 山田毅
結核, 63, 1, 1, 3, 1988年, ［査読有り］
英語, 研究論文（学術雑誌）, The number of rRNA genes in Mycobacterium phlei was determined by means of Southern hybridization analysis. The results indicated that M. phlei possesses at least and probably 2 sets of rRNA genes. Based on the results together with our previous reports on Mycobacterium bovis BCG and Mycobacterium lepraemurium, the relationship between slow growth and rRNA genes was discussed. © 1988, JAPANESE SOCIETY FOR TUBERCULOSIS. All rights reserved.

STUDY ON RIBOSOMAL-RNA GENES IN MYCOBACTERIUM-SMEGMATIS
Y SUZUKI, T YAMADA
MICROBIOLOGY AND IMMUNOLOGY, 32, 12, 1259, 1262, CENTER ACADEMIC PUBL JAPAN, 1988年
英語

Chlorella chloroplast DNA seguence containing a gene for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase and a part of a possible gene for the beta’s subunit of RNA polymerase
K YOSHINAGA, T OHTA, Y SUZUKI, M SUGIURA
Plant Mol. Biol, 10, 3, 245, 250, KLUWER ACADEMIC PUBL, 1988年
英語, 研究論文（学術雑誌）

THE NUMBER OF RIBOSOMAL-RNA GENES IN MYCOBACTERIUM-LEPRAEMURIUM
Y SUZUKI, T MORI, Y MIYATA, T YAMADA
FEMS MICROBIOLOGY LETTERS, 44, 1, 73, 76, ELSEVIER SCIENCE BV, 1987年09月
英語, 研究論文（学術雑誌）

ORGANIZATION OF RIBOSOMAL-RNA GENES IN MYCOBACTERIUM-BOVIS BCG
Y SUZUKI, K YOSHINAGA, Y ONO, A NAGATA, T YAMADA
JOURNAL OF BACTERIOLOGY, 169, 2, 839, 843, AMER SOC MICROBIOLOGY, 1987年02月
英語, 研究論文（学術雑誌）

ALTERATION OF RIBOSOMES AND RNA-POLYMERASE IN DRUG-RESISTANT CLINICAL ISOLATES OF MYCOBACTERIUM-TUBERCULOSIS
T YAMADA, A NAGATA, Y ONO, Y SUZUKI, T YAMANOUCHI
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 27, 6, 921, 924, AMER SOC MICROBIOLOGY, 1985年
英語, 研究論文（学術雑誌）

RAPID PREPARATION OF PLASMID BY GEL-PERMEATION CHROMATOGRAPHY ON TOYOPEARL HW75S
K YOSHINAGA, Y SUZUKI
AGRICULTURAL AND BIOLOGICAL CHEMISTRY, 47, 4, 919, 920, JAPAN SOC BIOSCI BIOTECHN AGROCHEM, 1983年
英語

世界の結核研究の動向(40)結核菌の薬剤耐性と遺伝子変異について
中島千絵, 鈴木定彦, 複十字, 415, 2024年

比較ゲノム解析による乳牛由来黄色ブドウ球菌の国際的な拡散状況の解明
大川佳穂, 福田昭, 米山裕, 戸部隆太, 中島千絵, 中島千絵, 鈴木定彦, 鈴木定彦, 臼井優, 北海道獣医師会雑誌, 68, 8, 2024年

伴侶動物由来メチシリン耐性Staphylococcus pseudintermediusの現状と人獣共通感染症の可能性
盛田佐和, 福田昭, 岡村幸一, 小松忠人, 露木勇三, 鳥居花衣, 佐々木潤平, 鈴木定彦, 中島千絵, 臼井優, 動物用抗菌剤研究会報, 45, 2023年

鼻疽菌検出のための新規dry LAMP法の開発
中瀬満, THAPA Jeewan, BATBAATAR Vanaabaatar, KHURTSBAATAR Ochirbat, ENKHTUUL Batchuluun, UNENBAT Jugderkhorloo, 中島千絵, 鈴木定彦, 木村享史, 日本獣医学会学術集会講演要旨集, 166th, 2023年

免疫チェックポイント分子Programmed death-ligand1(PD-L1)を標的とした抗体薬による犬の鼻腔内腺癌に対する免疫療法の検討
多田佳史, 前川直也, 今内覚, 細谷謙次, 大脇稜, 竹内寛人, 賀川由美子, 高木哲, 高木哲, 鈴木定彦, 鈴木定彦, 岡川朋弘, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 166th, 2023年

低温調理における耐熱性細菌・非耐熱性細菌の死滅条件の解明
中野瞳, 福田昭, 中島千絵, 鈴木定彦, 臼井優, 北海道獣医師会雑誌, 66, 8, 2022年

ブタPD-1/PD-L1経路の発現と機能解析【JST・京大機械翻訳】
OTGONTUYA Ganbaatar, 今内覚, 岡川朋弘, 野島裕太郎, 前川直也, 市川世識, 小林篤史, 芝原友幸, 柳川洋二郎, 鈴木定彦, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021年

動物用抗体医薬品の開発および臨床研究
今内 覚, 岡川 朋弘, 前川 直也, 中島 千絵, 鈴木 定彦, 山本 啓一, 戸田 幹洋, 村田 史郎, 大橋 和彦, JVPA digest : 日本動物用医薬品協会会報, 67, 1, 10, 2020年02月
日本動物薬事協会, 日本語

等温増幅とDNAクロマトグラフィーによるリファンピシン耐性結核菌の多重変異検出方の開発
宝田裕, 小寺拓也, 小林久美, 中島千絵, 中島千絵, 川瀬三雄, 鈴木定彦, 鈴木定彦, 日本分子生物学会年会プログラム・要旨集(Web), 43rd, 2020年

Molecular characterization and antimicrobial resistance of MRSA from pigs and pork in Thailand(和訳中)　　　　　　　　　　　　　　　
Tanomsridachchai Wimonrat, 中島 千絵, Changkaew Kanjana, Changkwanyeun Ruchirada, Prapasawat Watsawan, Intarapuk Apiradee, Yamasamit Nattapong, Suthienkul Orasa, 鈴木 定彦, 日本細菌学雑誌, 75, 1, 60, 60, 2020年01月
日本細菌学会, 英語

Detection of quinolone resistance determinants in E. coli from food animals in the Philippines(和訳中)　　　　　　　　　　　　　　　
Belotindos Lawrence, Mingala Claro, Villanueva Marvin, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 75, 1, 64, 64, 2020年01月
日本細菌学会, 英語

Existence of extracellular DNA in pathogenic mycobacteria and its role in mycobacterial physiology(和訳中)　　　　　　　　　　　　　　　
イリノフ・アレクサンドル, シャバン・アミナ, 袴田 真理子, 西山 晃史, 尾関 百合子, 福島 由華里, 中島 千絵, 立石 善隆, 鈴木 定彦, 松本 壮吉, 日本細菌学雑誌, 75, 1, 74, 74, 2020年01月
日本細菌学会, 英語

Emergence of vanD5-type vancomycin-resistant Enterococcus faecium via horizontal genomic transfer(和訳中)　　　　　　　　　　　　　　　
佐藤 豊孝, 和田 崇之, 福島 由華里, 中島 千絵, 鈴木 定彦, 高橋 聡, 横田 伸一, 日本細菌学雑誌, 75, 1, 100, 100, 2020年01月
日本細菌学会, 英語

NADPH再生系を内包するハイブリッド型アゾレダクターゼの開発
堀内正隆, 永田崇, 片平正人, 小橋川敬博, 鈴木定彦, 落合正則, 第42回日本分子生物学会年会, 福岡国際会議場(福岡)2019.12.3-6, 42nd, 2019年12月
日本語, 研究発表ペーパー・要旨（全国大会，その他学術会議）

免疫チェックポイント分子Programmed death‐ligand 1(PD‐L1)を標的とする抗体薬による免疫療法が奏功した肺転移のある口腔内悪性黒色腫の犬の1例
竹内寛人, 前川直也, 今内覚, 高木哲, 細谷謙次, 賀川由美子, 岡川朋弘, 鈴木定彦, 鈴木定彦, 山本啓一, 山本啓一, 村田史郎, 大橋和彦, 北海道獣医師会雑誌, 63, 8, 343, 343, 2019年08月09日
(公社)北海道獣医師会, 日本語

Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl green loop-mediated isothermal amplification (LAMP) method
Jeewan Thapa, Bhagwan Maharjan, Meena Malla, Yukari Fukushima, Ajay Poudel, Basu Dev Pandey, Kyoko Hyashida, Stephen V. Gordon, Chie Nakajima, Yasuhiko Suzuki, Tuberculosis, 117, 1, 6, 2019年07月01日, ［国際誌］
© 2019 Elsevier Ltd The purpose of this study was to develop a simple visual methyl green (MeG) based dry loop-mediated isothermal amplification (LAMP) method for early detection of Mycobacterium tuberculosis (MTB) from clinical samples. We identified MeG as an indicator of a positive LAMP reaction, where a positive reaction gave a blue-green color while a negative reaction was colorless. The MeG MTB-LAMP system was further simplified by drying all reagents for ease of use, and was then validated for its ability to diagnose TB directly using Nepalese clinical samples. We evaluated the dry MeG MTB-LAMP with 69 new TB suspected samples from patients that did not have a confirmed history of TB treatment and found the sensitivity in culture positive samples as 92.8% (13/14) and specificity in culture negative samples as 96.3% (53/55). Our LAMP system has the potential to be a point of care test for early diagnosis of active TB in developing countries., 英語

非結核性抗酸菌(MAC及びM.kansasii)の新規核酸増幅検査法の開発
橋本章司, 新井剛, 高田宏宗, 韓由紀, 田村嘉孝, 永井崇之, 松井謹, 小野原健一, 吉多仁子, 中島千絵, 鈴木定彦, 結核, 94, 3, 311, 2019年03月15日
日本語

One Healthアプローチを通じた細菌性感染症の制御 病原性微生物の特性に関する新規分析法の開発(Development of new methods for the characterization of bacterial pathogens)　　　　　　　　　　　　　　　
中島 千絵, 鈴木 定彦, Thapa Jeewan, 日本細菌学雑誌, 74, 1, 5, 5, 2019年03月
日本細菌学会, 英語

スリランカ、Kandyにおける結核菌の集団構成 欧米系統の優勢(Population structure of Mycobacterium tuberculosis in Kandy, Sri Lanka: Dominance of Euro-American lineage)　　　　　　　　　　　　　　　
Mendis Charitha, Ratnatunga Champa, Thevanesam Vasanthi, Kumara Athula, Wickramasinghe Susiji, Madagedara Dushantha, Gamage Chandika, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 45, 45, 2019年03月
日本細菌学会, 英語

タイ国の屠殺場と市場から分離されたMRSAの分子的分析(Molecular analysis of MRSA isolates from slaughterhouses and markets in Thailand)　　　　　　　　　　　　　　　
Tanomsridachchai Wimonrat, Changkaew Kanjana, Changkwanyeun Ruchirada, 中島 千絵, Suthienkul Orasa, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 45, 45, 2019年03月
日本細菌学会, 英語

ループ仲介等温増幅法を用いた人畜共通感染性結核の迅速検出法(Rapid detection of zoonotic tuberculosis using Loop mediated isothermal amplification)　　　　　　　　　　　　　　　
Kapalamula Thoko, Thapa Jeewan, 中島 千絵, Akapelwa Mwangala, Gordon Stephen V, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 67, 67, 2019年03月
日本細菌学会, 英語

ループ仲介等温増幅法を用いたMycobacterium aviumの迅速検出ツールの開発(Development of a rapid detection tool for Mycobacterium avium using Loop-mediated isothermal Amplification)　　　　　　　　　　　　　　　
Akapelwa Mwangala, Kapalamula Thoko, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 68, 68, 2019年03月
日本細菌学会, 英語

ザンビア、Lusakaで分離された結核菌のpyrazinamide耐性の分析(Determination of pyrazinamide resistance in Mycobacterium tuberculosis isolated from Lusaka, Zambia)　　　　　　　　　　　　　　　
Bwalya Precious, Yamaguchi Tomoyuki, Mulundu Georgina, 中島 千絵, Mbulo Grace, Solo Eddie, Fukushima Yukari, Kasakwa Kunda, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 79, 79, 2019年03月
日本細菌学会, 英語

バイオフィルム中における大腸菌persister制御のためのトスフロキサシンとSOS反応阻害剤の可能性　　　　　　　　　　　　　　　
臼井 優, 横尾 勇人, 田村 豊, 中島 千絵, 鈴木 定彦, Jean-Marc Ghigo, Beloin Christophe, 日本細菌学雑誌, 74, 1, 80, 80, 2019年03月
日本細菌学会, 日本語

WQ-3810はMycobacterium lepraeのDNAジャイレースに対して強力な阻害活性を示した(WQ-3810 showed strong inhibitory activity against Mycobacterium leprae DNA gyrase)　　　　　　　　　　　　　　　
Park JongHoon, 山口 智之, 大内 勇樹, Kim Hyun, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 82, 82, 2019年03月
日本細菌学会, 英語

SOS応答を標的とした薬剤耐性制御物質としてのプロテアーゼ阻害剤の可能性　　　　　　　　　　　　　　　
横尾 勇人, 臼井 優, 鈴木 定彦, 中島 千絵, 田村 豊, 日本細菌学雑誌, 74, 1, 99, 99, 2019年03月
日本細菌学会, 日本語

プラスミドにコードされるキノロン耐性タンパク質QnrB19とサルモネラDNAジャイレースの相互作用(Interaction of the plasmid-encoded quinolone resistance protein QnrB19 with salmonella DNA gyrases)　　　　　　　　　　　　　　　
Pachanon Ruttana, 小出 健太郎, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 99, 99, 2019年03月
日本細菌学会, 英語

タイ水圏環境中から分離された多剤耐性大腸菌の遺伝子学的特徴(Genomic characterization of multidrug resistant E. coli in environmental water in Thailand)　　　　　　　　　　　　　　　
角田 梨紗, 中島 千絵, 臼井 優, 田村 豊, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 100, 100, 2019年03月
日本細菌学会, 英語

キノロン耐性結核菌におけるFitness Costおよびその補完機構(Fitness Cost and Compensatory Mechanism in Fluoroquinolone Resistance Mycobacterium tuberculosis)　　　　　　　　　　　　　　　
大内 勇樹, 向井 徹, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 101, 101, 2019年03月
日本細菌学会, 英語

フィリピンの食用動物から分離されたE.coliとSalmonellaにおけるキノロン耐性の決定因子(Quinolone Resistance Determinants in E. coli and Salmonella from Food animals in the Philippines)　　　　　　　　　　　　　　　
Belotindos Lawrence, Mingala Claro, Villanueva Marvin, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 129, 129, 2019年03月
日本細菌学会, 英語

Mycobacterium lepraeのDNAジャイレースの機能的分析と細菌増殖と生存における役割(Functional analysis of Mycobacterium leprae DNA gyrase and its role in bacterial growth and survival)　　　　　　　　　　　　　　　
金 玄, 福富 康夫, 中島 千絵, Kim Youn Uck, 森 茂太郎, 柴山 恵吾, 中田 登, 鈴木 定彦, 日本細菌学雑誌, 74, 1, 121, 121, 2019年03月
日本細菌学会, 英語

免疫学的解析を基盤とした動物用創薬研究　　　　　　　　　　　　　　　
今内 覚, 岡川朋弘, 前川直也, 中島千絵, 鈴木定彦, 山本啓一, 戸田幹洋, 村田史郎, 大橋和彦, 臨床獣医, 37, 3, 37, 41, 2019年03月, ［招待有り］
(株)緑書房, 日本語, 記事・総説・解説・論説等（学術雑誌）

バイオフィルム中における大腸菌persister制御のためのトスフロキサシンとSOS反応阻害剤の可能性
臼井優, 横尾勇人, 田村豊, 中島千絵, 鈴木定彦, JEAN-MARC Ghigo, BELOIN Christophe, 日本細菌学雑誌(Web), 74, 1, 2019年

SOS応答を標的とした薬剤耐性制御物質としてのプロテアーゼ阻害剤の可能性
横尾勇人, 臼井優, 鈴木定彦, 中島千絵, 田村豊, 日本細菌学雑誌(Web), 74, 1, 2019年

「人獣共通感染症研究―ワンヘルスの取り組みと動物実験の役割―」(II)ワンヘルスの理念と人獣共通感染症制圧への取り組み:ザンビアでの活動
鈴木定彦, Labio 21, 75, 14‐18, 2019年01月01日
日本語

結核・非結核性抗酸菌症―エキスパートが教える 実臨床に役立つ最新知見―結核・非結核性抗酸菌症の基礎研究 結核菌の薬剤耐性獲得
山口智之, 中島千絵, 鈴木定彦, 呼吸器ジャーナル, 66, 4, 650‐656, 667, 2018年11月01日
＜文献概要＞Point ・virulenceとhost defenseとのせめぎ合いの観点から,非結核性抗酸菌は結核菌よりもvirulenceが弱いにもかかわらず,免疫能がほぼ正常な宿主で感染症となりうる.・非結核性抗酸菌に対する免疫は,マクロファージとTh1細胞を中心とした細胞性免疫が主体である.・非結核性抗酸菌は,通性細胞内寄生菌で,マクロファージ殺菌エフェクターに抵抗性である., (株)医学書院, 日本語

動物難治性疾病に対する創薬研究
今内 覚, 岡川朋弘, 前川直也, 中島千絵, 鈴木定彦, 山本啓一, 戸田幹洋, 村田史郎, 大橋和彦, MPアグロジャーナル, 35, 10, 22, 25, 2018年10月, ［招待有り］
MPアグロ, 日本語, 記事・総説・解説・論説等（学術雑誌）

ウシCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)の機能解析および慢性感染症における発現解析
渡慧, 今内覚, 岡川朋弘, 前川直也, 後藤伸也, 佐治木大和, 村田史郎, 鈴木定彦, 大橋和彦, 日本獣医学会学術集会講演要旨集, 161st, 340, 340, 2018年08月21日
(公社)日本獣医学会, 日本語

肺転移のある悪性黒色腫罹患犬に対するPD‐L1を標的とした抗体薬の治療効果
前川直也, 今内覚, 高木哲, 細谷謙次, 賀川由美子, 岡川朋弘, 和泉雄介, 出口辰弥, 鈴木定彦, 山本啓一, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 161st, 429, 429, 2018年08月21日
(公社)日本獣医学会, 日本語

イヌCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)を標的としたバイオ医薬品の開発に向けた基礎的検討
石原悠太郎, 今内覚, 岡川朋弘, 前川直也, 鈴木定彦, 大田寛, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 161st, 430, 430, 2018年08月21日
(公社)日本獣医学会, 日本語

Mycoplasma bovis感染症における免疫抑制機序の解析
後藤伸也, 今内覚, 岡川朋弘, 前川直也, 佐治木大和, 渡慧, 樋口豪紀, 小岩政照, 田島誉士, 鈴木定彦, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 161st, 342, 342, 2018年08月21日
(公社)日本獣医学会, 日本語

チミジル酸合成酵素遺伝子過剰発現によるBCGの増殖速度の促進とその機序の解明
大原直也, 大原直也, 和田崇之, 阿戸学, 鈴木定彦, 結核, 93, 4, 290, 2018年04月15日
日本語

ポリマー圧電素子を用いた病原体特異的分子検出用バイオセンサの開発―分子識別素子の設計―
武田真理子, 平田孝道, 黒岩崇, 中島千絵, 鈴木定彦, 宗像文男, Chemical Sensors, 34, Supplement A, 37‐39, 39, 2018年03月09日
電気化学会化学センサ研究会, 日本語

ポリマー圧電素子を用いた病原体特異的分子検出用バイオセンサの開発―検知用バイオセンサの試作と評価―
武田真理子, 平田孝道, 黒岩崇, 中島千絵, 鈴木定彦, 宗像文男, 電気化学会大会講演要旨集(CD-ROM), 85th, ROMBUNNO.PS‐93, 2018年02月23日
日本語

ポリマー圧電素子を用いた病原体特異的分子検出用バイオセンサの開発―分子識別素子の設計―
武田真理子, 平田孝道, 黒岩崇, 中島千絵, 鈴木定彦, 宗像文男, 電気化学会大会講演要旨集(CD-ROM), 85th, ROMBUNNO.1B13, 2018年02月23日
日本語

ミャンマーにおける北京型結核菌の考察(Insight into the Beijing genotype Mycobacterium tuberculosis in Myanmar)　　　　　　　　　　　　　　　
Lai Lai San, Nan Aye Thida Oo, Wah Wah Aung, Khin Saw Aye, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 73, 1, 50, 50, 2018年02月
日本細菌学会, 英語

GyrAに新規アミノ酸置換を有するSalmonella enteritidisの全ゲノムシークエンス分析(Whole genome sequencing of Salmonella Enteritidis having a novel amino acid substitution on GyrA)　　　　　　　　　　　　　　　
小出 健太郎, Utrarachkil Fuangfa, Suthienkul Orasa, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 73, 1, 51, 51, 2018年02月
日本細菌学会, 英語

ミャンマーで分離された結核菌における薬剤耐性関連変異の同定(Detection of drug-resistant associated mutations in Mycobacterium tuberculosis isolates of Myanmar)　　　　　　　　　　　　　　　
Nan Aye Thida Oo, Lai Lai San, Khin Saw Aye, Wah Wah Aung, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 73, 1, 55, 55, 2018年02月
日本細菌学会, 英語

メチルグリーンを用いたループ仲介等温増幅法による結核の診断(Diagnosis of tuberculosis by methyl green based loop-mediated isothermal amplification method)　　　　　　　　　　　　　　　
Thapa Jeewan, Maharjan Bhagwan, 福島 由華里, Malla Meena, Poudel Ajay, Pandey Basu Dev, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 73, 1, 64, 64, 2018年02月
日本細菌学会, 英語

BCGの増殖率促進に関与する遺伝子の調査(Investigation of genes involved in promoting the growth rate of BCG)　　　　　　　　　　　　　　　
竜門 亜矢子, 中山 真彰, 和田 崇之, 橘 理人, 阿戸 学, 中島 千絵, 鈴木 定彦, 小崎 弘貴, 大原 直也, 日本細菌学雑誌, 73, 1, 68, 68, 2018年02月
日本細菌学会, 日本語

Ofloxancin耐性Mycobacterium lepraeのDNAジャイレースに対するWQ-3810の阻害活性(Inhibitory activity of WQ-3810 against DNA gyrases of ofloxacin-resistant Mycobacterium leprae)　　　　　　　　　　　　　　　
Park JongHoon, 山口 智之, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 73, 1, 136, 136, 2018年02月
日本細菌学会, 英語

タイの水圏環境における薬剤耐性と残留抗菌薬濃度との関係　　　　　　　　　　　　　　　
角田 梨紗, 臼井 優, 高田 秀重, 中島 千絵, 鈴木 定彦, 田村 豊, 日本細菌学雑誌, 73, 1, 139, 139, 2018年02月
日本細菌学会, 日本語

フルオロキノロン耐性で非PGG3 Mycobacterium tuberculosisのDNAジャイレースの特徴(Characterization of DNA gyrase in fluoroquinolone-resistant non-PGG3 Mycobacterium tuberculosis)　　　　　　　　　　　　　　　
大内 勇樹, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 73, 1, 139, 139, 2018年02月
日本細菌学会, 英語

BCGの増殖速度の促進における遺伝子の調査
竜門亜矢子, 中山真彰, 中山真彰, 和田崇之, 橘理人, 阿戸学, 中島千絵, 鈴木定彦, 小崎弘貴, 大原直也, 大原直也, 日本細菌学雑誌(Web), 73, 1, 2018年

タイの水圏環境における薬剤耐性と残留抗菌薬濃度との関係
角田梨紗, 臼井優, 高田秀重, 中島千絵, 鈴木定彦, 田村豊, 日本細菌学雑誌(Web), 73, 1, 2018年

NADPH再生系を伴ったアゾレダクターゼによるアゾ染料分解反応のNMR解析
堀内正隆, 永田崇, 片平正人, 小橋川敬博, 鈴木定彦, 落合正則, 日本分子生物学会年会プログラム・要旨集(Web), 41st, ROMBUNNO.3P‐0752 (WEB ONLY), 2018年
日本語

牛マイコプラズマ感染症における免疫抑制機序の解明
後藤伸也, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 樋口豪紀, 小岩政照, 田島誉士, 小原潤子, 加藤幸成, 鈴木定彦, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 160th, 370, 370, 2017年08月30日
(公社)日本獣医学会, 日本語

Multiplex PCR法による鶏肉からの薬剤耐性カンピロバクターの迅速検出法
松井有優, 臼井優, 中島千絵, 小野崎正修, 鈴木定彦, 田村豊, 日本獣医学会学術集会講演要旨集, 160th, 415, 2017年08月30日
日本語

PD‐L1を標的としたイヌ用キメラ抗体医薬の作製と悪性黒色腫および未分化肉腫罹患犬における抗腫瘍効果の検討
前川直也, 今内覚, 高木哲, 賀川由美子, 岡川朋弘, 西森朝美, 池渕良洋, 和泉雄介, 出口辰弥, 加藤幸成, 鈴木定彦, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 160th, 460, 460, 2017年08月30日
(公社)日本獣医学会, 日本語

BLV感染症をモデルとした抗ウシPD‐L1キメラ抗体の臨床応用試験
西森朝美, 今内覚, 岡川朋弘, 前川直也, 池渕良洋, 後藤伸也, 佐治木大和, 鈴木定彦, 小原潤子, 小笠原諭, 加藤幸成, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 160th, 370, 370, 2017年08月30日
(公社)日本獣医学会, 日本語

Novel Mycobacterium avium Complex Species Isolated From Black Wildebeest (Connochaetes gnou) in South Africa
P. N. Kabongo-Kayoka, C. L. Obi, C. Nakajima, Y. Suzuki, T. Hattori, J. N. Eloff, J. Wright, N. Mbelle, L. J. McGaw, TRANSBOUNDARY AND EMERGING DISEASES, 64, 3, 929, 937, 2017年06月
A study was undertaken to isolate and characterize Mycobacterium species from black wildebeest suspected of being infected with tuberculosis in South Africa. This led to the discovery of a new Mycobacterium avium complex species, provisionally referred to as the Gnou isolate from black wildebeest (Connochaetes gnou). Sixteen samples from nine black wildebeest were processed for Mycobacterium isolation. Following decontamination, samples were incubated in an ordinary incubator at 37 degrees C on Lowenstein-Jensen slants and in liquid medium tubes using the BACTEC MGIT 960 system, respectively. Identification of the isolate was carried out by standard biochemical tests and using the line probe assay from the GenoType((R)) CM/AS kit (Hain Lifescience GmbH, Nehren, Germany). The DNA extract was also analysed using gene sequencing. Partial gene sequencing and analysis of 16S rRNA gene, and 16S-23S rRNA (ITS), rpoB and hsp65 and phylogenetic analyses by searching GenBank using the BLAST algorithm were conducted. Phylogenetic trees were constructed using four methods, namely Bayesian inference, maximum likelihood, maximum parsimony and neighbour-joining methods. The isolate was identified as Mycobacterium intracellulare using the GenoType((R)) CM/AS kit and as Mycobacterium avium complex (MAC) by gene sequencing. The gene sequence targeting all the genes, ITS, 16S rRNA, rpoB and hsp65 and phylogenetic analyses indicated that this isolate presented a nucleotide sequence different from all currently published sequences, and its position was far enough from other MAC species to suggest that it might be a new species., WILEY, 英語

らい菌のキノロン系抗菌薬耐性の獲得に伴うDNAジャイレース活性変化の解析
鈴木定彦, 山口智之, 中島千絵, Japanese Journal of Leprosy, 86, 1, 50, 2017年04月22日
日本語

ポリマー圧電素子を用いたDNA検出用バイオセンサの検知特性
武田真理子, 矢萩洸貴, 杉山龍男, 平田孝道, 中島千絵, 鈴木定彦, 宗像文男, Chemical Sensors, 33, Supplement A, 58‐60, 60, 2017年03月25日
電気化学会化学センサ研究会, 日本語

ポリマー圧電素子を用いたDNA検出用バイオセンサの検知特性
武田真理子, 矢萩洸貴, 杉山龍男, 平田孝道, 中島千絵, 鈴木定彦, 宗像文男, 電気化学会大会講演要旨集(CD-ROM), 84th, ROMBUNNO.2U03, 60, 2017年03月17日
電気化学会化学センサ研究会, 日本語

忘れてはいけない人獣共通感染症 8.結核と非結核性抗酸菌症
鈴木定彦, 中島千絵, 鈴木定彦, 中島千絵, 化学療法の領域, 33, 3, 411‐418, 2017年02月25日
日本語

ヨーネ菌組換え抗原タンパクに対する実験感染牛の血清抗体反応性　　　　　　　　　　　　　　　
松葉 隆司, 永田 礼子, 川治 聡子, 中島 千絵, 鈴木 定彦, 藤井 潤, 日本細菌学雑誌, 72, 1, 117, 117, 2017年02月
日本細菌学会, 日本語

Impact of T95S and D668G in GyrA of Mycobacterium tuberculosis on compensatory evolution(和訳中)　　　　　　　　　　　　　　　
大内 勇樹, 小出 健太郎, 山口 智之, 朴 鐘勲, 金 玄, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 72, 1, 131, 131, 2017年02月
日本細菌学会, 英語

Antibacterial activity of WQ-3810, a novel fluoroquinolone, against Salmonella Typhimurium(和訳中)　　　　　　　　　　　　　　　
小出 健太郎, コングソイ・シリポン, 大内 勇樹, 朴 鐘勲, 山口 智之, チャンクワイエン・ルチラダ, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 72, 1, 135, 135, 2017年02月
日本細菌学会, 英語

らい菌及び結核菌由来DNAジャイレースの性状解析　　　　　　　　　　　　　　　
金 玄, 福富 康夫, 中島 千絵, 松岡 正典, 森 茂太郎, 柴山 恵吾, 鈴木 定彦, 日本細菌学雑誌, 72, 1, 136, 136, 2017年02月
日本細菌学会, 日本語

Analysis of streptomycin-resistance associating genes in Mycobacterium tuberculosis in Nepal(和訳中)　　　　　　　　　　　　　　　
シュレスタ・ディプティ, マハージャン・バグワン, ウーナン・エーティダ, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 72, 1, 141, 141, 2017年02月
日本細菌学会, 英語

Molecular diversity of Mycobacterium tuberculosis in Kandy, Sri Lanka: Insight to Beijing genotype(和訳中)　　　　　　　　　　　　　　　
メンディス・チャリサ, Ratnatunga Champa, Thevanesam Vasanthi, Kumara Athula, Wickramasinghe Susiji, Madegedara Dushantha, Gamage Chandika, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 72, 1, 167, 167, 2017年02月
日本細菌学会, 英語

ヨーネ菌組換え抗原タンパクに対する実験感染牛の血清抗体反応性　　　　　　　　　　　　　　　
松葉 隆司, 永田 礼子, 川治 聡子, 中島 千絵, 鈴木 定彦, 藤井 潤, 日本細菌学雑誌, 72, 1, 117, 117, 2017年02月
日本細菌学会, 日本語

らい菌及び結核菌由来DNAジャイレースの性状解析　　　　　　　　　　　　　　　
金 玄, 福富 康夫, 中島 千絵, 松岡 正典, 森 茂太郎, 柴山 恵吾, 鈴木 定彦, 日本細菌学雑誌, 72, 1, 136, 136, 2017年02月
日本細菌学会, 日本語

ヨーネ菌組換え抗原タンパクに対する実験感染牛の血清抗体反応性
松葉隆司, 永田礼子, 川治聡子, 中島千絵, 鈴木定彦, 藤井潤, 日本細菌学雑誌(Web), 72, 1, 117(J‐STAGE), 2017年
日本語

らい菌及び結核菌由来DNAジャイレースの性状解析
KIM Hyun, 福富康夫, 中島千絵, 松岡正典, 森茂太郎, 柴山恵吾, 鈴木定彦, 日本細菌学雑誌(Web), 72, 1, 136(J‐STAGE), 2017年
日本語

種内相互組み換えによって促進される鳥型結核菌の地域適応
矢野大和, 丸山史人, 西内由紀子, 中川一路, 中島千絵, 鈴木定彦, 岩本朋忠, 日本ゲノム微生物学会年会要旨集, 11th, 57, 2017年
日本語

人獣共通感染症としての結核
中島千絵, 鈴木定彦, 獣医疫学雑誌, 20, 2, 101‐104, 104, 2016年12月20日
人獣共通感染症としての観点から人型結核菌(M. tuberculosis)を見た場合，状況は上述の人における様相とはかなり異なる。毎年1千万人前後の新患を出し，世界人口の1/3が既に結核菌に感染していると言われているが，この人における重度の蔓延状態と比べると，動物におけるM. tuberculosisによる感染報告は恐ろしく少ない。霊長類や一部の例外を除いて，患者と密な接触のあった動物(牛，犬等)において稀に報告がある程度である。例外的にエチオピアにおいて牛群の集団感染が報告されているが，これはこの地域特有の人と牛との濃厚な接触習慣により，個々の牛が人から感染を受けているものと考えられる。これらに対して，大きな例外となるのが人に飼育されているアジアゾウである。最初はアメリカのサーカスや動物園で飼育されているゾウで報告され，その後，タイやネパール等のアジアゾウ本来の分布域内の飼育施設において多数の感染例が報告される様になった。我々がネパールの国立公園において行った調査では，これらのゾウ感染株はM. tuberculosisであり，ネパール患者から得られる株特有の遺伝子上の変異を持っていたことから，感染源は人間で，恐らくは日々密接な係りを持っている飼育係と考えられた。何故，アジアゾウのみから多数のM. tuberculosisによる結核発症例が報告されるのかは不明だが，菌の遅い増殖速度を考えると，動物のM. tuberculosisに対する感受性等の要因の他に，ゾウが人間並みに長命であることも関与しているかもしれない。以上より，M. tuberculosisは基本的に人の結核起因菌であり，高度に人に順化していると考えられる。世界の牛結核発生状況は人結核と趣が異なり，人結核がほとんど見られなくなった西欧諸国やニュージーランド等で高度の蔓延が観察される。また，今一つ異なる特長は，世界的に蔓延している菌株の遺伝的特徴が非常に良く似ている点である(M. tuberculosisでは地域によって多様性が見られる)。有名な例はEulと呼ばれる株で，この菌株の特徴を持った菌がアメリカ大陸やオーストラリア，ニュージーランド，南アフリカなどで分離される。型別情報からこれらの起源を辿ると，元々はイギリスやアイルランドで蔓延していた株であることが判る。これは，近代以降にこれらの国から牛が，主として大英帝国関連の国々へ輸出された名残であると推測される。南ア以外のアフリカ諸国では，また別のタイプの牛結核菌が分離されるが，これらも元を辿るとフランス，オランダ等の由来であることが示唆され，アフリカの牛結核はほぼヨーロッパが起源となっている。, 獣医疫学会, 日本語

Collectin CL-P1 is involved in C-reactive protein-mediated complement activation
Nitai Roy, Katsuki Ohtani, Yasuyuki Matsuda, Kenichiro Mori, Insu Hwang, Yasuhiko Suzuki, Norimitsu Inoue, Nobutaka Wakamiya, IMMUNOBIOLOGY, 221, 10, 1215, 1215, 2016年10月
ELSEVIER GMBH, URBAN & FISCHER VERLAG, 英語, 研究発表ペーパー・要旨（国際会議）

マイクロアレイ技術を応用した薬剤耐性カンピロバクターの迅速検出法(CAMERA法)
臼井 優, 舘野 翔, 田勢 準也, 田村 豊, 中島 千絵, 鈴木 定彦, 小野崎 正修, 大曽根 司郎, グリーンテクノ情報, 12, 2, 14, 17, 2016年09月
NPO法人グリーンテクノバンク, 日本語

ウシCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)の機能解析と特異抗体の樹立
渡慧, 今内覚, 前川直也, 岡川朋弘, 西森朝美, 鈴木定彦, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 159th, 365, 365, 2016年08月30日
(公社)日本獣医学会, 日本語

家畜における免疫チェックポイント関連因子の遺伝子比較解析とウシ用抗体を用いた動物種横断的研究
野島裕太郎, 今内覚, 西森朝美, 前川直也, 岡川朋弘, 森康行, 賀川由美子, 鈴木定彦, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 159th, 364, 364, 2016年08月30日
(公社)日本獣医学会, 日本語

ウシ腫瘍壊死因子(TNF‐α)デコイレセプターによる新規抗炎症剤の開発
藤澤宗太郎, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 村田史郎, 鈴木定彦, 大橋和彦, 日本獣医学会学術集会講演要旨集, 159th, 364, 364, 2016年08月30日
(公社)日本獣医学会, 日本語

ウシ用腸溶性生菌剤カプセルが腸内細菌相に与える影響
瀬山智博, 平康博章, 吉田弦, 大沼愛子, 邱永晋, 中島千絵, 笠井浩司, 鈴木定彦, 日本畜産学会大会講演要旨, 121st, 237, 2016年03月27日
日本語

感染症 現状の問題点と未来への展望 結核―人獣共通感染症の観点から
鈴木定彦, 中島千絵, 臨床と微生物, 43, 2, 169‐173, 2016年03月25日
日本語

BCG thyXを用いた抗酸菌のPAS耐性機序の解析
大原直也, 大原直也, 阿戸学, 鈴木定彦, 小林和夫, 結核, 91, 3, 325, 325, 2016年03月15日
(一社)日本結核・非結核性抗酸菌症学会, 日本語

Mycobacterium aviumの系統分岐に伴う大規模なゲノム構造の変化　　　　　　　　　　　　　　　
岩本 朋忠, 矢野 大和, 西内 由紀子, 有川 健太郎, 中島 千絵, 鈴木 定彦, 丸山 史人, 日本細菌学雑誌, 71, 1, 58, 58, 2016年02月
日本細菌学会, 日本語

ミャンマーで単離された北京型多剤耐性結核菌株のMIRU-VNTRを用いた判別法(MIRU-VNTR Typing of Beijing MDR-TB strains from Myanmar)　　　　　　　　　　　　　　　
Lai Lai San, Nan Aye Thida Oo, Wah Wah Aung, Khin Saw Aye, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 71, 1, 59, 59, 2016年02月
日本細菌学会, 英語

ミャンマーで単離されたストレプトマイシン耐性結核菌における変異の同定(Detection of mutations in streptomycin resistance Mycobacterium tuberculosis isolates from Myanmar)　　　　　　　　　　　　　　　
Nan Aye Thida Oo, Lai Lai San, Khin Saw Aye, Wah Wah Aung, Wah Wah, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 71, 1, 105, 105, 2016年02月
日本細菌学会, 英語

Mycobacterium lepraeのDNAジャイレースに対する新規の8-メトキシフルオロキノロンの阻害活性(Inhibitory activities of a new 8-methoxy fluoroquinolone against DNA gyrase of Mycobacterium leprae)　　　　　　　　　　　　　　　
山口 智之, 中島 千絵, 鈴木 定彦, 日本細菌学雑誌, 71, 1, 140, 140, 2016年02月
日本細菌学会, 英語

抗酸菌のPAS耐性機序の解明 BCG thyX欠損株および過剰発現株の作製　　　　　　　　　　　　　　　
有村 友紀, 中山 真彰, 妹尾 昌紀, 田川 淳平, 阿戸 学, 中島 千絵, 鈴木 定彦, 中山 浩次, 小林 和夫, 大原 直也, 日本細菌学雑誌, 71, 1, 142, 142, 2016年02月
日本細菌学会, 日本語

南アジアはMycobacterium orygisの風土病地域である(South Asia is an endemic region for Mycobacterium orygis)　　　　　　　　　　　　　　　
Thapa Jeewan, 中島 千絵, Rahim Zeaur, Paudel Sarad, Shah Yogendra, Maharian Bhagwan, Poudel Ajay, Gairhe Kamal Prasad, 坪田 敏男, 鈴木 定彦, 日本細菌学雑誌, 71, 1, 159, 159, 2016年02月
日本細菌学会, 英語

東アジアの小型哺乳動物から分離されたレプトスピラの反復配列多型解析法による分子型別
小泉信夫, 泉谷秀昌, 慕蓉蓉, 岡野祥, 中島千絵, 鈴木定彦, 谷川力, 小松謙之, 吉松組子, 武藤(水谷)麻紀, 武藤(水谷)麻紀, 大西真, レプトスピラ・シンポジウム, 53rd, 5, 2016年
日本語

ザンビアにおける結核対策への貢献
鈴木定彦, 鈴木定彦, 日本熱帯医学会大会プログラム抄録集, 56th, 30, 2015年12月04日
日本語

CAMERA法による野外サンプル(鶏肉及び鶏糞便)からの薬剤耐性カンピロバクターの迅速検出法
臼井優, 中島千絵, 舘野翔, 田勢準也, 小野崎正修, 大曾根司朗, 鈴木定彦, 田村豊, 日本獣医学会学術集会講演要旨集, 158th, 363, 2015年08月30日
日本語

新興・再興感染症と感染症対策 C.細菌 結核 終わらない脅威とその対策
鈴木定彦, 中島千絵, 生体の科学, 66, 4, 335, 338, 2015年08月15日
日本語

らい菌のDNAジャイレースに対するキノロン系抗菌薬の阻害活性試験とその結果から考える結合様式
山口智之, 中島千絵, 鈴木定彦, Jpn J Lepr, 84, 1, 26, 2015年05月25日
日本語

Inquisition of DNA gyrase genes on fluoroquinolone resistant clinical Mycobacterium avium isolates
MENDA Conscillioah, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 191, 2015年02月25日
英語

Detection of loop-mediated isothermal amplification reaction by non-fluorescent leuco dyes
THAPA Jeewan, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 200, 2015年02月25日
英語

Extended-spectrum beta-lactamases in Escherichia coli isolated from swine in Thailand
CHANGKAEW Kanjana, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 190, 2015年02月25日
英語

Diversity of MIRU-VNTR among Mycobacterium tuberculosis Central Asian family isolates from Nepal
SHAH Yogendra, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 208, 2015年02月25日
英語

Virulence factors and class 1 integrons in Vibrio parahaemolyticus from shrimp farming environment
CHANGKWANYEUN Ruchirada, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 190, 2015年02月25日
英語

The selection of ciprofloxacin resistance in Salmonella Typhimurium exposed residue level in vitro
KONGSOI Siriporn, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 190, 2015年02月25日
英語

Alteration opf Mycobacterium leprae DNA gyrase activity by mutations conferring quinolone resistance
YAMAGUCHI Tomoyuki, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 191, 2015年02月25日
英語

Understanding the circulation of Leptospira among water buffaloes in an intensive farm setting
VILLANUEVA Marvin, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 70, 1, 208, 2015年02月25日
英語

DNAアレイを応用した迅速・簡便な薬剤耐性カンピロバクター検出法の開発
中島千絵, 臼井優, 田村豊, 鈴木定彦, 日本細菌学雑誌, 70, 1, 202, 2015年02月25日
日本語

抗酸菌におけるパラアミノサリチル酸に対する新たな耐性機序の可能性
ZHAO Na, 中山真彰, 関塚剛史, 黒田誠, 本田尚子, 阿戸学, 中島千絵, 鈴木定彦, 大原直也, 日本細菌学雑誌, 70, 1, 193, 2015年02月25日
日本語

結核菌Rv0679cタンパク機能の解析
松葉隆司, 中島千絵, 藤井潤, 鈴木定彦, 日本細菌学雑誌, 70, 1, 182, 2015年02月25日
日本語

ハンセン病の予防法及び診断・治療法の開発・普及に関する研究 薬剤耐性獲得機構解明とその迅速感受性試験法開発への応用
鈴木定彦, 中島千絵, ハンセン病の予防法及び診断・治療法の開発・普及に関する研究 平成26年度 総括・分担研究報告書, 13, 17, 2015年
日本語

GRP‐tag化酵素を利用したアゾ染料分解システムの構築
堀内正隆, 鈴木定彦, 落合正則, 日本生化学会大会(Web), 88th, 2P0907 (WEB ONLY), 2015年
日本語

多剤耐性結核の分子疫学的解析,診断・治療法の開発に関する研究 スリランカ,ネパール,バングラデシュ,ミャンマーおよびタイとの結核研究ネットワーク活用による多剤耐性結核の分子疫学解析
鈴木定彦, 多剤耐性結核の分子疫学的解析、診断・治療法の開発に関する研究 平成26年度 委託業務成果報告書, 44‐46, 2015年
日本語

H5N1インフルエンザウイルスに対する中和抗体を用いた受動免疫の感染防御効果
吉田玲子, 伊藤靖, 七戸新太郎, 小笠原一誠, 日尾野隆大, 岡松正敏, 迫田義博, 喜田宏, LE Mai Quynh, 河岡義裕, 五十嵐学, 鈴木定彦, 高田礼人, 日本ウイルス学会学術集会プログラム・抄録集, 62nd, 234, 2014年10月31日
日本語

結核研究からハンセン病研究へ:分子生物学的見地から
鈴木定彦, Jpn J Lepr, 83, 2, 16, 2014年07月25日
日本語

らい菌にキノロン系抗菌薬耐性を与える酵素内アミノ酸置換がもたらすDNAジャイレース活性低下の解析
山口智之, 中島千絵, 鈴木定彦, Jpn J Lepr, 83, 2, 47, 2014年07月25日
日本語

Mycobacterium tuberculosis complex genotypic lineages circulating in Egyptian population
DIAB Hassan, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 69, 1, 144, 2014年02月25日
英語

High levels of OPN, IP-10 and neutrophilia in LAMP confirmed TB patients in Manila
SHIRATORI Beata, SHIRATORI Beata, LEANO Susan, NAKAJIMA Chie, CHAGAN‐YASUTAN Haorile, NIKI Toshiro, SUZUKI Yasuhiko, TELAN Elisabeth, HATTORI Toshio, 日本細菌学雑誌, 69, 1, 140, 2014年02月25日
英語

Tuberculosis caused by Mycobacterium orygis in dairy cattle and captured monkeys in Bangladesh
NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 69, 1, 141, 2014年02月25日
英語

Development of loop-mediated isothermal amplification method for detection of Legionella pneumophila
CHANGKAEW Kanjana, SUZUKI Yasuhiko, NAKAJIMA Chie, 日本細菌学雑誌, 69, 1, 201, 2014年02月25日
英語

Isolation and characterization of Mycobacterium orygis from a deer in Nepal
THAPA Jeewan, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 69, 1, 141, 2014年02月25日
英語

Impact of amino acid substitutions in GyrA affects quinolone resistance in Salmonella Typhimurium
KONGSOI Siriporn, SUZUKI Yasuhiko, NAKAJIMA Chie, 日本細菌学雑誌, 69, 1, 196, 2014年02月25日
英語

Impact of mutations in DNA gyrase genes on fluoroquinolone resistance in Campylobacter jejuni
CHANGKWANYEUN Ruchirada, KIM Hyun, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 69, 1, 196, 2014年02月25日
英語

Epidemiological study of leptospirosis among water buffalo (Bubalus bubalis) in the Philippines
VILLANUEVA Marvin, KOIZUMI Nobuo, SUZUKI Yasuhiko, NAKAJIMA Chie, 日本細菌学雑誌, 69, 1, 240, 2014年02月25日
英語

Genotypic characterization of multidrug-resistant Mycobacterium tuberculosis isolated in Myanmar
SUZUKI Yasuhiko, NAKAJIMA Chie, 日本細菌学雑誌, 69, 1, 199, 2014年02月25日
英語

結核診断用LAMP法の改良
松葉隆司, 中島千絵, 鈴木定彦, 日本細菌学雑誌, 69, 1, 202, 2014年02月25日
日本語

結核菌DNAジャイレースにおけるキノロン耐性決定領域外に見出されたアミノ酸置換のキノロン剤耐性への影響(Clarification of correlation between mutants DNA GyrA and quinolone resistance in M. tuberculosis)　　　　　　　　　　　　　　　
金 玄, 横山 和正, 中島 千絵, 森 茂太郎, 柴山 恵吾, 鈴木 定彦, 日本細菌学雑誌, 69, 1, 199, 199, 2014年02月
日本細菌学会, 英語

サハラ以南アフリカにおけるエイズ・結核研究ネットワークの構築に関する研究
鈴木定彦, 中島千絵, サハラ以南アフリカにおけるエイズ・結核研究ネットワーク構築に関する研究 平成25年度 総括・分担研究報告書, 6, 8, 2014年
日本語

結核研究からハンセン病研究へ:分子生物学的見地から
鈴木定彦, 山口智之, KIM Hyun, 横山和正, 中島千絵, Jpn J Lepr, 83, 3, 131, 137, 2014年
As for the Mycobacterium leprae which is a causative agent of Hansen’s disease, many studies had been done since it was identified in 1873. However, those studies, at the same time, experienced many struggles because of the difficulty of culture of M. leprae on the artificial growth media. Hence, the study of Hansen’s disease progressed by taking the knowledge from the study of tuberculosis caused by the bacteria belonging to the same genus, genus Mycobacterium. For instance, the knowledge of mutations in specific genes responsible for rifampicin-and quinolone-resistance in M. tuberculosis led the elucidation of drug-resistant acquisition mechanism of M. leprae. Similarly, it is necessary for the researcher of Hansen’s disease to get important information from the latest topic of the tuberculosis study and utilize them to the study of the disease., Japanese Leprosy Association, 日本語

ハンセン病の予防法及び診断・治療法の開発・普及に関する研究 薬剤耐性獲得機構解明とその迅速感受性試験法開発への応用に関する研究
鈴木定彦, 中島千絵, ハンセン病の予防法及び診断・治療法の開発・普及に関する研究 平成25年度 総括・分担研究報告書, 15, 18, 2014年
日本語

レプトスピラ症患者の血漿および尿中のバイオマーカーの解析
ホルロ, 中島千絵, 小泉信夫, 鈴木定彦, 服部俊夫, レプトスピラ・シンポジウム, 51st, 10, 2014年
日本語

細菌の進化から考える抗菌薬耐性 10.結核菌の薬剤耐性
鈴木定彦, 中島千絵, 化学療法の領域, 30, 1, 113, 121, 2013年12月25日
日本語

Collectin CL-P1 is an endocytosis and phagocytosis receptor using clathrin-dependent endocytosis pathway and clathrin-independent phagocytosis pathway
N. Wakamiya, S. Jang, A. Fukuoh, K. Mori, Y. Suzuki, K. Ohtani, MOLECULAR IMMUNOLOGY, 56, 3, 301, 302, 2013年12月
PERGAMON-ELSEVIER SCIENCE LTD, 英語, 研究発表ペーパー・要旨（国際会議）

病原体特異的等温遺伝子増幅(LAMP)法によるピロリン酸の発生量をモニターするポータブルバイオセンサーの開発
矢萩洸貴, 中島千絵, 黒岩崇, 杉山龍男, 鈴木定彦, 宗像文男, 電気学会ケミカルセンサ研究会資料, CHS-13, 1-15, 71, 74, 2013年08月08日
日本語

コレクチンCL-K1血中濃度測定ELISAシステムの樹立と日本人における正常値について
吉崎 隆之, 大谷 克城, 本村 亘, 張 成宰, 北元 憲利, 森 健一郎, 吉田 逸朗, 鈴木 定彦, 若宮 伸隆, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 88, 2, 96, 96, 2013年04月01日
著者最終原稿版, 日本語

結核菌DNAジャイレース上の菌系統特異的アミノ酸多型のキノロン剤耐性への影響
金玄, 横山和正, 中島千絵, 鈴木定彦, Jpn J Lepr, 82, 1/2, 22, 22, 2013年04月01日
日本語

結核菌DNAジャイレースにおけるキノロン耐性決定領域外に見出されたアミノ酸置換のキノロン剤耐性への影響
金玄, 横山和正, 中島千絵, 鈴木定彦, Jpn J Lepr, 82, 1/2, 24, 24, 2013年04月01日
日本語

Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis from Asian Countries
NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 68, 1, 217, 2013年02月25日
英語

Inhibition activity of quinolones against DNA gyrase of Campylobacter jejuni
CHANGKWANYEUN Ruchirada, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 68, 1, 191, 2013年02月25日
英語

Amino acid substitutions in DNA gyrase B subunit confer Quinolone resistance on Mycobacterium leprae
SUZUKI Yasuhiko, KIM Hyun, MUKAI Tetsu, NAKAJIMA Chie, 日本細菌学雑誌, 68, 1, 191, 2013年02月25日
英語

Antimicrobial Resistance and Class 1 Integrons in Escherichia coli Isolated from Shrimp Environment
CHANGKAEW Kanjana, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 68, 1, 195, 2013年02月25日
英語

Salmonella Typhimurium DNA Gyrase and Its Interaction with Quinolones
KONGSOI Siriporn, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 68, 1, 190, 2013年02月25日
英語

結核の革新的な診断・治療及び対策の強化に関する研究 多様な研究シーズを想定した結核菌臨床分離株のゲノム情報集積
和田崇之, 鈴木定彦, 中島千絵, 結核の革新的な診断・治療及び対策の強化に関する研究 平成24年度 総括・研究分担報告書, 106, 111, 2013年
日本語

ヒトMAC症由来Mycobacterium avium subsp.hominissuisの遺伝的多様性とゲノム構造解析
岩本朋忠, 中西典子, 中島千絵, 有川健太郎, 鈴木定彦, 日本ゲノム微生物学会年会要旨集, 7th, 51, 2013年
日本語

サハラ以南アフリカにおけるエイズ・結核研究ネットワークの構築に関する研究
鈴木定彦, 中島千絵, サハラ以南アフリカにおけるエイズ・結核研究ネットワーク構築に関する研究 平成24年度 総括・分担研究報告書, 15, 18, 2013年
日本語

ハンセン病の予防法及び診断・治療法の開発・普及に関する研究 薬剤耐性獲得機構解明とその迅速感受性試験法開発への応用
鈴木定彦, 松岡正典, 中島千絵, ハンセン病の予防法及び診断・治療法の開発・普及に関する研究 平成24年度 総括・分担研究報告書, 15, 20, 2013年
日本語

国際共同基盤研究に応用する抗酸菌感染症研究の整備 薬剤耐性結核菌の迅速検出法
鈴木定彦, 中島千絵, 国際共同基盤研究に応用する抗酸菌感染症研究の整備 平成24年度 総括・分担研究報告書, 17, 22, 2013年
日本語

組織におけるコレクチンCL-K1の生化学的検討
大谷 克城, 本村 亘, 吉崎 隆之, 森 健一郎, 松田 泰幸, 黄 仁秀, ニタイ・ロイ, 吉田 逸朗, 鈴木 定彦, 若宮 伸隆, 日本生化学会大会プログラム・講演要旨集, 85回, 3T16, 07, 2012年12月
(公社)日本生化学会, 日本語

サハラ以南アフリカにおけるエイズ・結核研究ネットワーク構築の試み
服部俊夫, 鈴木定彦, 山岡昇司, 井戸栄治, 一瀬休生, 仲宗根正, 久保亨, 臼澤基紀, 垣本和宏, 福本学, 児玉栄一, 日本国際保健医療学会学術大会プログラム・抄録集, 27th, 123, 2012年11月02日
日本語

アカゲザルモデルを用いたエボラ出血熱に対する中和モノクローナル抗体の防御効果
吉田玲子, MARZI Andrea, 宮本洋子, 石島麻理, 鈴木定彦, 五十嵐学, 中山絵里, 黒田誠, FELDMANN Heinz, 高田礼人, 日本ウイルス学会学術集会プログラム・抄録集, 60th, 199, 2012年10月31日
日本語

コレクチンCL‐K1のマウスおよびヒト組織における発現検討
大谷克城, 本村亘, 吉崎隆之, 森健一郎, 松田泰幸, HWANG Insu, CHANDRA Roy, 吉田逸朗, 鈴木定彦, 若宮伸隆, 日本糖質学会年会要旨集, 31st, 123, 2012年08月31日
日本語

ウシの免疫抑制受容体PD‐1に対するモノクローナル抗体の作製および免疫活性化能の検討
池渕良洋, 今内覚, 岡川朋弘, 横山和正, 中島千絵, 鈴木定彦, 村田史郎, 大橋和彦, 日本獣医学会学術集会講演要旨集, 154th, 233, 2012年08月31日
日本語

ゼブラフィッシュCL-P1の遺伝子クローニングとその機能解析
福田 光子, 大谷 克城, 張 成宰, 吉崎 隆之, 森 健一郎, 本村 亘, 吉田 逸朗, 鈴木 定彦, 高後 裕, 若宮 伸隆, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 87, 4, 178, 178, 2012年08月01日
日本語

コレクチンCL-K1の組織における発現検討
大谷 克城, 本村 亘, 吉崎 隆之, 森 健一郎, 松田 泰幸, 黄 仁秀, ロイ・ニタイ, 吉田 逸朗, 鈴木 定彦, 若宮 伸隆, 補体シンポジウム講演集, 49, 23, 23, 2012年08月
(一社)日本補体学会, 日本語

らい菌のDNAジャイレースの性状解析
金玄, 横山和正, 中島千絵, 松岡正典, 向井徹, 福富康夫, 鈴木定彦, Jpn J Lepr, 81, 1/2, 46, 2012年04月01日
日本語

らい菌のDNAジャイレースBサブユニット上のアミノ酸置換とニューキノロン耐性
横山和正, 金玄, 中島千絵, 松岡正典, 向井徹, 鈴木定彦, Jpn J Lepr, 81, 1/2, 48, 2012年04月01日
日本語

らい菌のDNAジャイレースAサブユニット上のアミノ酸置換とニューキノロン耐性
横山和正, 金玄, 中島千絵, 松岡正典, 向井徹, 鈴木定彦, Jpn J Lepr, 81, 1/2, 47, 2012年04月01日
日本語

A New Loop-Mediated Isothermal Amplification Method for Detection of Leptospira spp. in Urine
KOIZUMI Nobuo, OHNISHI Makoto, NAKAJIMA Chie, SUZUKI Yasuhiko, 日本細菌学雑誌, 67, 1, 126, 2012年02月25日
英語

人獣共通感染拡症としての結核
鈴木定彦, 日本獣医師会獣医学術学会年次大会講演要旨集, 2011, 146, 147, 2012年01月20日
日本語

海外から輸入される多剤耐性結核に関する研究 黒竜江省で収集した結核菌の分子疫学的解析
服部俊夫, 凌虹, 鈴木定彦, 中島千絵, 海外から輸入される多剤耐性結核に関する研究 平成23年度 総括・分担研究報告書, 88, 90, 2012年
日本語

サハラ以南アフリカにおけるエイズ・結核研究ネットワークの構築に関する研究 ザンビアにおける結核菌の分子疫学的解析
鈴木定彦, 中島千絵, サハラ以南アフリカにおけるエイズ・結核研究ネットワーク構築に関する研究 平成23年度 総括・分担研究報告書, 6, 8, 2012年
日本語

Loop‐mediated isothermal amplification法による尿からの簡便なレプトスピラ検出法
小泉信夫, 中島千絵, 春成常仁, 谷川力, 常盤俊大, 内村江利子, 古谷徳次郎, MINGALA Claro Niegos, VILLANUEVA Marvin Ardeza, 大西真, 鈴木定彦, レプトスピラ・シンポジウム, 49th, 7, 2012年
日本語

人獣共通感染症の克服に向けて 結核
鈴木定彦, 松葉隆司, 中島千絵, 最新医学, 66, 12, 2689, 2696, 2011年12月10日
日本語

CORRELATION BETWEEN BEIJING GENOTYPE FAMILY AND THE ENVELOPE PROTEIN IN MYCOBACTERIUM TUBERCULOSIS
MATSUBA Takashi, NAKAJIMA Chie, TAMARU Aki, SIDDIQI Umme R, HATTORI Toshio, RAHIM Zeaur, POUDEL Ajay, PANDEY Basu D, PANDEY Basu D, SUZUKI Yasuhiko, 日本細菌学雑誌, 66, 2-3, 426, 2011年10月31日
英語

コレクチンCL-K1のヒト血中濃度
大谷 克城, 吉崎 隆之, 森 健一郎, 本村 亘, 松田 泰幸, 黄 仁秀, 吉田 逸朗, 金 然旭, 鈴木 定彦, 若宮 伸隆, 補体シンポジウム講演集, 48, 31, 31, 2011年09月
(一社)日本補体学会, 日本語

コレクチンCL-K1の機能について
大谷 克城, 吉崎 隆之, 森 健一郎, 本村 亘, 黄 仁秀, 吉田 逸朗, 金 然旭, 鈴木 定彦, 若宮 伸隆, 日本生化学会大会プログラム・講演要旨集, 84回, 4T8a, 11, 2011年09月
(公社)日本生化学会, 日本語

鳥取大学におけるバイオハザード対策用クラスIIキャビネットの維持管理とその発展途上国への応用
松葉隆司, 甲斐政親, 飯塚昌, 鈴木定彦, 空気清浄, 49, 2, 53, 58, 2011年07月31日
日本空気清浄協会, 日本語

コレクチンCL‐P1のリガンド認識ドメインについての解析
森健一郎, 大谷克城, JANG SeongJae, KIM YounUc, 本村亘, HWANG Insu, 吉田逸朗, 鈴木定彦, 若宮伸隆, 日本糖質学会年会要旨集, 30th, 73, 2011年06月27日
日本語

コレクチンCL‐K1の糖鎖特異性と機能解析
大谷克城, 吉崎隆之, 森健一郎, 本村亘, HWANG Insu, 吉田逸朗, KIM YounUck, 鈴木定彦, 若宮伸隆, 日本糖質学会年会要旨集, 30th, 31, 2011年06月27日
日本語

結核菌のニューキノロン高度耐性獲得機構
金玄, 横山和正, 中島千絵, 鈴木定彦, Jpn J Lepr, 80, 2, 151, 2011年04月01日
日本語

新たな等温増幅法による結核菌群菌の検出
中島千絵, 鈴木定彦, 結核, 86, 3, 343, 2011年03月15日
日本語

オホーツクと南極海で採取した流氷における重金属濃度と金属耐性遺伝子の検出
能田淳, 登坂唯香, 中島千絵, 大久保寅彦, 石原加奈子, 鈴木定彦, 伊村智, 田村豊, 日本獣医学会学術集会講演要旨集, 151st, 248, 2011年03月01日
日本語

輸入感染症としての多剤耐性結核の対策・制御に関する研究 黒竜江省の結核菌解析に関する研究
服部俊夫, 凌虹, 鈴木定彦, 輸入感染症としての多剤耐性結核の対策・制御に関する研究 平成22年度 総括・分担研究報告書, 81, 85, 2011年
日本語

輸入感染症としての多剤耐性結核の対策・制御に関する研究[VI]潜在性結核の検出にかかわるT細胞機能と黒竜江省の結核菌解析
服部俊夫, 凌虹, 鈴木定彦, 輸入感染症としての多剤耐性結核の対策・制御に関する研究 平成20-22年度 総合研究報告書, 96, 101, 2011年
日本語

国際共同基盤研究に応用する抗酸菌感染症研究の整備 薬剤耐性結核菌の迅速検出法
鈴木定彦, 中島千絵, 国際共同基盤研究に応用する抗酸菌感染症研究の整備 平成22年度 総括・分担研究報告書, 21, 25, 2011年
日本語

コレクチンCL‐K1の機能とヒト血中濃度について
大谷克城, 吉崎隆之, 森健一郎, 本村亘, 松田泰幸, HWANG Insu, 吉田逸朗, KIM Younuck, 鈴木定彦, 若宮伸隆, 日本分子生物学会年会プログラム・要旨集(Web), 34th, WEB ONLY 2T8A-2, 2011年
日本語

らい菌および結核菌のニューキノロン耐性獲得機構と耐性菌迅速鑑別法
KIM Hyun, 鈴木晴香, 松岡正典, 松葉隆司, 横山和正, 中島千絵, 鈴木定彦, Jpn J Lepr, 80, 1, 17, 27, 2011年, ［国内誌］
Drugs included in new-quinolone are used for the treatment of leprosy with single lesion. These drugs are also known to be effective drugs for the treatment of multi-drug resistant M. tuberculosis. Recent emergence of new-quinolone resistant M. leprae and M. tuberculosis enforced the urgent elucidation of the mode of emergence of new-quinolone resistant strains. In this review, new-quinolone drugs, their mode of action and mechanism of acquisition of resistance by M. leprae and M. tuberculosis were explained. And rapid differentiation methods for resistant bacilli were also introduced., 1, 英語

[Envelope structure and components of Mycobacterium tuberculosis].
Matsuba T, Nakajima C, Suzuki Y, Nihon saikingaku zasshi. Japanese journal of bacteriology, 65, 3, 355, 368, 2010年12月, ［査読有り］, ［国内誌］
2-4, 日本語

地球規模課題対応国際科学技術協力事業 結核及びトリパノソーマ症の診断法と治療薬開発
鈴木定彦, 飯塚昌, 大栗博毅, 杉本千尋, 環境科学会誌, 23, 6, 522, 529, 2010年11月30日
環境科学会, 日本語

コレクチンCL-K1の機能解析
大谷 克城, 吉崎 隆之, 森 健一郎, 本村 亘, 吉田 逸朗, 鈴木 定彦, 若宮 伸隆, 補体シンポジウム講演集, 47, 22, 22, 2010年09月
(一社)日本補体学会, 日本語

結核菌のニューキノロン高度耐性獲得機構
金玄, 中島千絵, 鈴木定彦, Jpn J Lepr, 79, 2, 140, 2010年04月01日
日本語

人獣共通感染症としての結核
鈴木定彦, 松葉隆司, 中島千絵, 結核, 85, 2, 69, 78, 2010年02月15日
日本語

人獣共通感染症としての結核
鈴木 定彦, 松葉 隆司, 中島 千絵, 結核, 85, 2, 79, 86, 2010年02月
(一社)日本結核病学会, 日本語

新規コレクチンの発見
大谷 克城, 鈴木 定彦, 若宮 伸隆, 旭川医科大学研究フォーラム, 10, 1, 2, 12, 2010年02月
コレクチンは、コラーゲン様ドメインを内部構造にもつ、C型レクチンのスーパーファミリーであり、自然免疫に重要な役割を果たすと考えられている。我々は、従来の生化学的アプローチでは得ることができなかった新しいコレクチンファミリー(新規コレクチン)を、リバースジェネティックスを活用したアプローチにより、初めてクローニングすることに成功した。本稿では、3つの新規コレクチンのクローニングと構造および機能に関する最新の知見を紹介する。(著者抄録), 旭川医科大学, 日本語

ＤＮＡマイクロアレイを用いた薬剤耐性らい菌の簡易検出法の創出と、その開発途上国における有用性
松岡正典, 鈴木定彦, 牧野正直, 日本ハンセン病学会誌, 79, 3, 257, 261, 2010年
The simple method to detect mutations conferring resistant to dapsone, rifampicin, and quinolone was exploited in Mycobacterium. leprae on the basis of reverse DNA hybridization with capture probe fixed to the glass slide. Mutations were discriminated by a series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB, and gyrA genes of M. leprae. The method was transferred to two laboratories in developing countries. The results obtained with the kit at those laboratories were highly concordant with results of sequencing. The method is feasible for the testing by local person in areas with high prevalence of leprosy., 英語

南極およびオホーツク海で採取した流氷における構成菌種の同定と薬剤耐性遺伝子の検出
登坂唯香, 中島千絵, 大久保寅彦, 石原加奈子, 鈴木定彦, 伊村智, 田村豊, 極域科学・宙空圏・気水圏・生物・地学シンポジウム講演予稿集(CD-ROM), 2010, ROMBUNNO.PB23, 2010年
日本語

輸入感染症としての多剤耐性結核の対策・制御に関する研究 非結核性抗酸菌症患者の潜伏結核感染 ハルピンの薬剤耐性結核の起源
服部俊夫, 凌虹, 鈴木定彦, 輸入感染症としての多剤耐性結核の対策・制御に関する研究 平成21年度 総括・分担研究報告書, 82, 87, 2010年
日本語

DNAマイクロアレイを用いた薬剤耐性らい菌の簡易検出法の創出と,その開発途上国における有用性
松岡正典, 鈴木定彦, 牧野正直, Jpn J Lepr, 79, 3, 257, 261, 2010年
The simple method to detect mutations conferring resistant to dapsone, rifampicin, and quinolone was exploited in Mycobacterium. leprae on the basis of reverse DNA hybridization with capture probe fixed to the glass slide. Mutations were discriminated by a series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB, and gyrA genes of M. leprae. The method was transferred to two laboratories in developing countries. The results obtained with the kit at those laboratories were highly concordant with results of sequencing. The method is feasible for the testing by local person in areas with high prevalence of leprosy., 英語

Zoonotic aspects of tuberculosis caused by mycobacterium bovis
Yasuhiko Suzuki, Takashi Matsuba, Chie Nakajima, Kekkaku, 85, 2, 79, 86, 2010年, ［査読有り］, ［国内誌］
Pathogens transmitting between the environment, wildlife, livestock and humans are major health concerns for human and domestic animal and in addition, for the sustainability of agriculture and the conservation of wildlife. Among pathogens causing zoonosis, Genus Mycobacterium including Mycobacterium tuberculosis, M. bovis, M. avium is thought to be important. The most important bacteria as an etiological agent of zoonosis in Genus Mycobacterium is M. bovis. M. bovis is the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, domestic livestock, non-human primates and humans. The reservoirs of M. bovis in wildlife have their own role as sources of infection in humans and domestic animals and have their health impact on humans. The approaches for the control and management of M. bovis infections are also discussed in this review., 2, 日本語

A novel method for simple detection of mutations conferring drug resistance in Mycobacterium leprae, based on a DNA microarray, and its applicability in developing countries
Masanori Matsuoka, Yasuhiko Suzuki, Masanao Makino, Japanese Journal of Leprosy, 79, 3, 257, 261, 2010年, ［査読有り］, ［国内誌］
The simple method to detect mutations conferring resistant to dapsone, rifampicin, and quinolone was exploited in Mycobacterium. leprae on the basis of reverse DNA hybridization with capture probe fixed to the glass slide. Mutations were discriminated by a series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB, and gyrA genes of M. leprae. The method was transferred to two laboratories in developing countries. The results obtained with the kit at those laboratories were highly concordant with results of sequencing. The method is feasible for the testing by local person in areas with high prevalence of leprosy., 3, 英語

バングラデシュにおいて猿から分離された結核菌群菌
中島千絵, 鈴木定彦, 感染症学雑誌, 83, 5, 631, 2009年09月20日
日本語

バングラデシュの多剤耐性結核菌における耐性に関与する遺伝子変異
鈴木定彦, 中島千絵, 田丸亜貴, 感染症学雑誌, 83, 5, 631, 2009年09月20日
日本語

スカベンジャー受容体コレクチンCL-P1のリガンド認識ドメインの解析
森 健一郎, 大谷 克城, 張 成宰, 金 然旭, 本村 亘, 孫 啓輝, 吉田 逸朗, 鈴木 定彦, 若宮 伸隆, 日本生化学会大会プログラム・講演要旨集, 82回, 4T20p, 12, 2009年09月
(公社)日本生化学会, 日本語

ドメイン欠損CL-P1のリガンド結合解析
森 健一郎, 大谷 克城, 張 成宰, 金 然旭, 本村 亘, 孫 啓輝, 吉田 逸朗, 鈴木 定彦, 若宮 伸隆, 補体シンポジウム講演集, 46, 29, 30, 2009年08月
(一社)日本補体学会, 日本語

人獣共通感染症としての結核
鈴木定彦, 結核, 84, 5, 281, 2009年05月15日
日本語

肺Mycobacteirum avium complex症患者由来M.aviumと豚由来M.aviumの血清型比較
西内由紀子, 田栗貴博, 松本壮吉, 立石善隆, 北田清悟, 田丸亜貴, 鈴木定彦, 前倉亮治, 結核, 84, 5, 409, 2009年05月15日
日本語

MLPA法による多剤耐性結核菌の検出
田丸亜貴, 河原隆二, 松本壮吉, 鈴木定彦, 結核, 84, 5, 397, 2009年05月15日
日本語

結核と非結核性抗酸菌症診療の新展開 6.超多剤耐性結核菌における耐性化のメカニズムと耐性化防止対策
鈴木定彦, 中島千絵, 福島由華里, 田丸亜貴, 松葉隆司, 化学療法の領域, 25, 4, 612, 620, 2009年03月25日
日本語

生活環境に分布するMycobacterium avium complexと臨床分離株の多型解析
西内由紀子, 松本壮吉, 立石善隆, 鈴木定彦, 日本細菌学雑誌, 64, 1, 96, 2009年02月20日
日本語

超多剤耐性結核菌における耐性のメカニズムと耐性化防止対策　　　　　　　　　　　　　　　
鈴木定彦, 中島千絵, 福島由華里, 田丸亜貴, 松葉隆司, 化学療法の領域, 25, 612, 620, 2009年

コレクチンCL‐P1のリガンド認識ドメインの解析
森健一郎, 大谷克城, JANG SeongJae, KIM YounUck, 本村亘, SUN QiHui, 吉田逸朗, 鈴木定彦, 若宮伸隆, 日本分子生物学会年会講演要旨集, 32nd, Vol.1, 74, 2009年
日本語

特徴ある感染症対策―Post‐exposure Prophylaxis:PEPを中心に 曝露後発症対策(Post‐exposure Prophylaxis:PEP)結核(化学予防)
鈴木定彦, 中島千絵, 福島由華里, 田丸亜貴, 松葉隆司, 臨床と微生物, 35, 6, 669, 675, 2008年11月25日
日本語

マルチプレックスPCR法を用いたバングラデシュにおける結核菌群菌の浸淫度調査
中島千絵, 鈴木定彦, 感染症学雑誌, 82, 6, 723, 724, 2008年11月20日
日本語

人及び豚の抗酸菌感染症に由来するMycobacterium aviumの遺伝学的特長の比較
中島千絵, 田村豊, 田丸亜貴, 鈴木定彦, 感染症学雑誌, 82, 6, 767, 2008年11月20日
日本語

動物病院におけるメチシリン耐性黄色ブドウ球菌の伝播
田村豊, 村松康和, 中島千絵, 柳沢千恵, 鈴木定彦, 花木秀明, 感染症学雑誌, 82, 6, 669, 2008年11月20日
日本語

【自然免疫と生体防御レクチン】新規コレクチンCL-L1,CL-P1,CL-K1の構造と機能
鈴木 定彦, 大谷 克城, 張 成宰, 本村 亘, 若宮 伸隆, 臨床検査, 52, 8, 861, 869, 2008年08月
コレクチンは動物界において幅広く保存されているコラーゲン様構造を有する動物C型レクチンであり,自然免疫のキープレイヤーとして活躍していることが知られている.最近,従来の遺伝子クローニング戦略では得ることができなかった新しいコネクチンファミリー蛋白質(新規コレクチン)が,リバースジェネティックスを活用したアプローチによりヒト組織からクローニングされた.本稿では,新規コレクチンのクローニングと構造および機能に関する最新の知見を紹介する.(著者抄録), (株)医学書院, 日本語

Streptococus mutans由来sucrose phosphorylaseの熱安定性に関する研究
高本鉄兵, 深田はるみ, 久保亜希子, 鈴木定彦, 北村進一, J Appl Glycosci, 55, Suppl., 51, 2008年07月20日
日本語

結核の化学予防　　　　　　　　　　　　　　　
鈴木定彦, 中島千絵, 福島由華里, 田丸亜貴, 松葉隆司, 臨床と微生物, 25, 669, 675, 2008年

Characterization of a CD4-independent clinical HIV-1 that can efficiently infect human hepatocytes through CXCR4.
Xiao P, Usami O, Suzuki Y, Ling H, Shimizu N, Hoshino H, Zhuang M, Ashino Y, Gu H, Hattori T, AIDS, 22, 1749, 1759, 2008年

Anti-rabies antibodies in Japanese volunteers immunized with imported vaccine.　　　　　　　　　　　　　　　
Usuzawa m, Changa-Yasutan H, Jitthiang P, Promjunyakul W, Medina P, Demetria C, Miranda ME, Suzuki Y, Oshitani H, Hattori T, Southeast Asian Journal of Tropical Medicine and Public Health, 39, suppl 1, 68, 72, 2008年

【胎盤】ヒト胎盤コレクチン1
大谷 克城, 鈴木 定彦, 若宮 伸隆, 臨床検査, 51, 13, 1714, 1718, 2007年12月
(株)医学書院, 日本語

生体防御レクチンである新規コレクチンファミリーの発見とその役割
若宮 伸隆, 大谷 克城, 坂本 隆志, 芥子 宏行, 福應 温, 張 成宰, 吉崎 隆之, 福田 光子, 小山 聡, 福澤 純, 本村 亘, 吉田 逸朗, 鈴木 定彦, 補体シンポジウム講演集, 44, 15, 16, 2007年08月
(一社)日本補体学会, 日本語

ホスホリラーゼによるアミロース合成機構の解析
藪内梨世, 久保亜希子, 鈴木定彦, 鷹羽武史, 北村進一, J Appl Glycosci, 54, Suppl., 37, 2007年07月20日
日本語

尾索動物ホヤのコレクチン遺伝子の検索と分子進化
大谷克城, 安住薫, 鈴木定彦, JANG Seong‐Jae, 吉崎隆之, 森健一郎, 本村亘, 福澤純, 吉田逸朗, 若宮伸隆, 日本糖質学会年会要旨集, 27th, 73, 2007年07月10日
日本語

Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses (vol 79, pg 326, 2007)
T. Yoda, Y. Suzuki, K. Yamazaki, N. Sakon, M. Kanki, Aoyama, I, T. Tsukamoto, JOURNAL OF MEDICAL VIROLOGY, 79, 6, 863, 863, 2007年06月
WILEY-LISS, 英語, その他

rsmG変異によるストレプトマイシン耐性機構の解明および臨床分離結核菌における本変異の重要性
岡本晋, 田丸亜貴, 中島千絵, 西村賢治, 田中幸徳, 徳山真治, 鈴木定彦, 越智幸三, 日本放線菌学会大会講演要旨集, 22nd, 45, 2007年05月31日
日本語

肺Mycobacterium avium complex(MAC)症患者の家庭浴室内におけるMACの検出
西内由紀子, 田丸亜貴, 北田清悟, 田栗貴博, 前倉亮治, 松本壮吉, 鈴木定彦, 結核, 82, 4, 439, 2007年04月15日
日本語

薬剤耐性らい菌の簡易検出法の開発途上国への移転とそれによる耐性菌の伝播調査
松岡正典, 鈴木定彦, TAN Esterlina, AYE Khin Saw, Jpn J Lepr, 76, 2, 141, 2007年04月01日
日本語

抗生物質耐性菌のその獲得メカニズムを解明
越智幸三, 岡本晋, 西村賢治, 鈴木定彦, 田丸亜貴, 中島千恵, 田中幸徳, 徳山真治, 食品試験研究成果情報, 19, 40, 41, 2007年03月31日
日本語

家庭浴室のMycobacterium avium complexの検出―培養法,PCR法およびLAMP法の比較―
西内由紀子, 松本壮吉, 鈴木定彦, 日本細菌学雑誌, 62, 1, 178, 178, 2007年02月25日
日本語

尾索動物ホヤのコレクチン遺伝子群の検索と遺伝子進化について
大谷克城, 安住薫, 鈴木定彦, JANG Seong‐Jae, 吉崎隆之, 森健一郎, 本村亘, 福澤純, 吉田逸朗, 若宮伸隆, 生化学, 4P-0018, 2007年
日本語

ウイルス感染と宿主応答・ウイルス側の要因 ヒトPBMC移植NOD/SCIDマウスを用いたin vivoにおけるマンノースバインディングレクチンの抗HIV活性の評価
堀端 重男, 大谷 克城, 坂本 隆志, 岸 雄一郎, 木佐木 博, 鈴木 定彦, 駒野 淳, 山本 直樹, 若宮 伸隆, 本多 三男, 日本免疫学会総会・学術集会記録, 36, 202, 202, 2006年11月
(NPO)日本免疫学会, 日本語

多剤耐性結核の現状と今後 6.多剤耐性結核の菌検出法の進歩
鈴木定彦, 田丸亜貴, 中島千絵, 西原みづき, 福島由華里, 松葉隆司, 化学療法の領域, 22, 11, 1705, 1713, 2006年10月25日
日本語

M.lepraeの迅速薬剤感受性試験法
鈴木定彦, 松岡正典, Jpn J Lepr, 75, 2, 102, 102, 2006年04月01日
日本語

新規ヒト化抗すい癌ムチン抗体の作製とその基礎的解析
木村健二郎, 沢田鉄二, 小松みどり, 六車一哉, 西原承浩, 山下好人, 八代正和, 山田靖哉, 加藤保之, 平川弘聖, 鈴木定彦, 日本外科学会雑誌, 107, 2, 563, 563, 2006年03月05日
一般社団法人日本外科学会, 日本語

【TLR以外のパターン認識レセプターとその役割】コレクチンとその免疫応答における役割
大谷 克城, 鈴木 定彦, 若宮 伸隆, 臨床免疫, 45, 3, 266, 274, 2006年03月
(有)科学評論社, 日本語

テトラサイクリンを用いた発現制御ベクターによる結核菌糖タンパクの発現
松葉隆司, 鈴木定彦, 田中吉紀, 日本細菌学雑誌, 61, 1, 85, 2006年02月25日
日本語

コレクチンとその免疫応答における役割　　　　　　　　　　　　　　　
大谷克城, 鈴木定彦, 若宮伸隆, 臨床免疫, 45, 266, 274, 2006年

多剤耐性結核菌検出法の進歩　　　　　　　　　　　　　　　
鈴木定彦, 田丸亜貴, 中島千絵, 西原みづき, 福島由華里, 松葉隆司, 化学療法の領域, 22, 1705, 1713, 2006年

抗酸菌感染の国際的対応への貢献を目指した基盤に関する研究 結核菌の同定,耐性検査へのマイクロアレイの応用に関する研究―DNAマイクロアレイを用いた結核菌の分子疫学的解析法の構築―
鈴木定彦, 抗酸菌感染の国際的対応への貢献を目指した基盤に関する研究 平成17年度 総括・分担研究報告書, 65, 69, 2006年
日本語

DNAマイクロアレイを用いたMycobacterium lepraeの迅速薬剤感受性試験法
鈴木定彦, 松岡正典, Jpn J Lepr, 75, 3, 271, 277, 2006年, ［国内誌］
Antibiotic susceptibility test of Mycobacterium leprae still relies on the time consuming methods based on the growth of M. leprae in the mouse footpad. Thus, the establishment of a rapid, simple and reliable method for the detection of drug-resistant M. leprae is one of the most urgent subjects in the treatment of leprosy patients. Recently, many data on the mutation of specific genes correlating with drug resistance have been accumulated. Application of these data permit the establishment of new gene diagnostic methods for drug susceptibility test of leprosy. In this paper, the method using the low density oligonucleotide array that enables the detection of base substitutions involved in resistance against anti-leprosy drugs on a single platform was discussed. The low density oligonucleotide array described in this paper will open the new perspectives in terms of patient management for leprosy with low cost requirement., 3, 中国語

大気浮遊粉じん中のビスフェノールAと多環芳香族炭化水素との濃度相関
松本比佐志, 鈴木定彦, 大阪府立公衆衛生研究所研究報告, 43, 7, 16, 2005年11月
大阪府立公衆衛生研究所, 日本語

Mannose binding lectinによるHIV-1複製の非特異的抑制効果
滝澤 万里, 大谷 克城, 岸 雄一郎, 坂本 高志, 鈴木 定彦, 若宮 伸隆, 本多 三男, 日本免疫学会総会・学術集会記録, 35, 182, 182, 2005年11月
(NPO)日本免疫学会, 日本語

新しいアッセイ用ほ乳類細胞(CHOOSER)を用いた簡便,高感度で再現性の良いエストロゲン様活性測定方法の開発
足立伸一, 山本康次, 織田肇, 小野芳朗, 鈴木定彦, 水環境学会誌, 28, 7, 451, 456, 2005年07月10日
Several in vitro assays have been developed to assess the activity of estrogenic substances. Recently, a yeast two-hybrid screen or a MCF-7 (human breast cancer cell) screen has been adopted mainly for screening chemicals and environmental estrogenic activity. However a yeast two-hybrid screen does not assay using an animal cell. MCF-7 screen is a complicated assay and the cell used proliferates depending on estrogen, thus obstructing a precise assay for estrogenic activity. In addition both screens need long incubation period (4-6 days) for a highly sensitive assay. The CHOOSER is a Chinese hamster ovary (CHO) cell transformed with the gene encoding the human estrogen receptor (ER α) and an estrogen responsive promoter linked to a reporter gene which was developed by Suzuki et. al. at Osaka prefectural institute of public health. When the reporter gene is activated by estrogenic substances, is expressed, producing alkaline phosphatase which is secreted into the medium. The CHOOSER can assay high sensitively (under 10-11M as 17 β-estradiol) at any time in a short incubation period (2 days). The number of the CHOOSER can be counted precisely because a CHO is dissociated the aggregates easily with trypsin and form a single cell suspension. And the CHOOSER can proliferate in the medium without estrogen, therefore it is possible to assay for estrogenic activity more proliferate in the medium without estrogen, therefore it is possible to assay for estrogenic activity more precisely. The CHOOSER assay will be a useful prescreening method for estrogenic activity before in vivo assay., 公益社団法人 日本水環境学会, 日本語

薬剤耐性らい菌の遺伝子変異およびその簡易検出法の開発
松岡正典, 張良ふん, 鈴木定彦, 吉川陽子, Jpn J Lepr, 74, 2, 121, 121, 2005年04月01日
日本語

不織布核酸精製キットを用いたLAMP法によるかくたんからの抗酸菌検出
田丸亜貴, 小田直純, 深沢忠, 鈴木定彦, 和田恭直, 結核, 80, 3, 322, 2005年03月15日
日本語

結核菌Rv0679cタンパクの局在と性状解析
松葉隆司, 鈴木定彦, 田中吉紀, 日本細菌学雑誌, 60, 1, 132, 2005年02月25日
日本語

新しい感染症検査法の発展と動向 2. 薬剤耐性結核菌のDNAマイクロアレイ検出
鈴木定彦, 吉川陽子, 田丸亜貴, 田中吉紀, 化学療法の領域, 21, 3, 334, 342, 2005年02月25日
日本語

薬剤耐性結核菌のDNAマイクロアレイ検出　　　　　　　　　　　　　　　
鈴木定彦, 吉川陽子, 田丸亜貴, 田中吉紀, 化学療法の領域, 21, 334, 342, 2005年

新規コレクチンCL-K1(Colec11)の生体内局在と分子生物学的解析
芥子 宏行, 坂本 隆志, 大谷 克城, 鈴木 定彦, 若宮 伸隆, 日本免疫学会総会・学術集会記録, 34, 153, 153, 2004年11月
(NPO)日本免疫学会, 日本語

紫外線吸収剤及びその関連化合物によるエストロゲン様作用
松本比佐志, 足立伸一, 鈴木定彦, 日本食品化学学会総会・学術大会講演要旨集, 10th, 66, 2004年06月17日
日本語

結核菌のキノロン系抗生物質に対する交差耐性とDNAジャイレース遺伝子上の変異の相関
鈴木定彦, 松葉隆司, 斎藤肇, 田中吉紀, 日本細菌学雑誌, 59, 1, 114, 2004年02月25日
日本語

臨床分離株間で一アミノ酸置換を有する結核菌タンパクに対するモノクローナル抗体作出
松葉隆司, 鈴木定彦, 田中吉紀, 日本細菌学雑誌, 59, 1, 157, 2004年02月25日
日本語

創薬のための生体機能解析に関する研究 レクチン機能を利用した血管における生体防御システムの解明と創薬への応用
若宮伸隆, 本多三男, 鈴木定彦, 板部洋之, 岸雄一郎, 創薬等ヒューマンサイエンス研究総合研究報告書 平成13-15年度 第2分野 創薬のための生体機能解析に関する研究, 85, 89, 2004年
日本語

創薬のための生体機能解析に関する研究 レクチン機能を利用した血管における生体防御システムの解明と創薬への応用
若宮伸隆, 本多三男, 鈴木定彦, 板部洋之, 岸雄一郎, 創薬等ヒューマンサイエンス研究重点研究報告書 平成15年度 第2分野 創薬のための生体機能解析に関する研究, 67, 73, 2004年
日本語

遺伝子を標的とした感染症の迅速検査法の進歩 DNAマイクロアレイを用いた臨床分離結核菌の薬剤耐性判定
吉川陽子, 市原竜生, 鈴木定彦, JARMAM, 14, 1, 45, 50, 2003年12月25日
日本語

Characterization of clinical isolates of Mycobacterium tuberculosis resistant to drugs and detection of RpoB mutation in multidrug-resistant tuberculosis in the Philippines
DMD Agdamag, S Kageyama, R Solante, AS Espantaleon, JCE Sangco, Y Suzuki, INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE, 7, 11, 1104, 1108, 2003年11月
SETTING: Retrospective study of Mycobacterium tuberculosis isolates at the STD/AIDS Cooperative Central Laboratory, Philippines.
OBJECTIVE: To describe patterns of M. tuberculosis resistance against first-line anti-tuberculosis drugs, and to analyze the rpoB gene codon mutation of rifampicin (RMP) resistant isolates and correlate genotypic and phenotypic patterns.
DESIGN: One hundred and sixty-four M. tuberculosis complex isolates were retrieved for phenotypic analysis; 89 were resistant to any anti-tuberculosis drug and 50 were RMP-resistant, whereas 48 were multidrug-resistant (MDR). Of these 48, only 33 were available for genotypic analysis of the rpoB gene.
RESULTS: Most drug-resistant isolates were phenotypically resistant to isoniazid (INH) (93%), and the probability of an RMP-resistant isolate becoming MDR was 96%. In 33 MDR isolates, 13 types of mutations in nine independent codons were identified; the most frequently mutated codons were S531L (61%) and G510H (15%), which were present in 76% (25/33) of the isolates. S531L was noted in 85.7% of the RMP + INH + SM resistant isolates, while only 80% of the isolates with INH + RMP, EMB + SM resistance showed this mutation.
CONCLUSION: The high probability of RMP isolates being MDR suggests that genetic analysis of RMP resistance is useful in detecting MDR-TB. Worldwide accumulation of findings on circulating MDR-TB strains provides indispensable information about the re-emergence of TB., INT UNION AGAINST TUBERCULOSIS LUNG DISEASE (I U A T L D), 英語

好アルカリ菌Bacillus clausiiからの溶菌酵素遺伝子のクローニングと発現
井川聡, 増井昭彦, 鈴木定彦, 藤原信明, 日本生物工学会大会講演要旨集, 2003, 58, 58, 2003年08月25日
日本生物工学会, 日本語

Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice
K Suzuki, Y Suzuki, S Kitamura, JOURNAL OF EXPERIMENTAL BOTANY, 54, 389, 1997, 1999, 2003年08月
A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-D-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-D-glucuronic acid to UDP-D-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots., OXFORD UNIV PRESS, 英語

コレクチンと生体防御
鈴木定彦, 加瀬哲男, 大阪府立公衆衛生研究所ニュース, 21, 2, 3, 2003年06月06日
日本語

Precise characterization of norovirus (Norwalk-like virus)-specific monoclonal antibodies with broad reactivity
T Yoda, Y Suzuki, Y Terano, K Yamazaki, N Sakon, T Kuzuguchi, H Oda, T Tsukamoto, JOURNAL OF CLINICAL MICROBIOLOGY, 41, 6, 2367, 2371, 2003年06月
We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains., AMER SOC MICROBIOLOGY, 英語

ニワトリ由来Cryptosporidium sp.の分子生物学的手法による種の同定
木村明生, 鈴木定彦, 松井利博, 日本獣医学会学術集会講演要旨集, 135th, 91, 2003年03月25日
日本語

イネのUDP‐グルクロン酸デカルボキシラーゼのクローニングと発現解析
鈴木潔, 鈴木定彦, 北村進一, 日本農芸化学会大会講演要旨集, 2003, 134, 2003年03月05日
日本語

CL-P1, a Newly Cloned Endothelial Cell Specific Scavenger Receptor
Fukuzawa Jun, Osaki Junzo, Saito Tetsuya, Baljinnyam Erdene, Sakuragi Hitoshi, Yao Naoyuki, Haneda Takashi, Hasebe Naoyuki, Kikuchi Kenjiro, Itabe Hiroyuki, Takeuchi Masayoshi, Koyama Satoshi, Koge Keiichi, Ohtani Katsuki, Fukuoh Atsushi, Suzuki Yasuhiko, Wakamiya Nobutaka, Circulation journal : official journal of the Japanese Circulation Society, 67, 114, 114, 2003年03月01日
社団法人日本循環器学会, 英語

Haplotype analysis of the human collectin placenta 1 (hCL-P1) gene
H Ohmori, Y Makita, M Funamizu, S Chiba, K Ohtani, Y Suzuki, N Wakamiya, A Hata, JOURNAL OF HUMAN GENETICS, 48, 2, 82, 85, 2003年
Collectins are a family of C-type lectins found in vertebrates. These proteins have four regions, a relatively short N-terminal region, a collagen-like region, an alphahelical coiled coil, and a carbohydrate recognition domain. Collectins are involved in host defense through their ability to bind carbohydrate antigens on microorganisms. Type A scavenger receptors are classical-type scavenger receptors that also have collagen-like domains. We previously described a new scavenger receptor, collectin from placenta [collectin placenta 1 (CL-P1)]. CL-P1 is a type II membrane protein with all four regions. We found that CL-P1 can bind and phagocytize both bacteria and yeast. In addition to that, it reacts with oxidized low-density lipoprotein (LDL) but not with acetylated LDL. These results suggest that CL-P1 might play important roles in host defenses and/or atherosclerosis formation. One rational strategy to study the role of CL-P1 in these pathological conditions would be to perform a haplotype association study using human samples. As a first step for this strategy, we analyzed the haplotype structure of the CL-P1 gene. By sequencing the CL-P1 gene in ten Japanese volunteers, we identified five single-nucleotide polymorphisms (SNPs) with a minor allele frequency of at least 29%. To obtain SNPs in the 5'-upstream region of the gene, we screened a total of 20 SNPs described in the database and finally picked up one SNP for the present study. Thus, a total of six SNPs, one in the 5'-upstream region, two in intron 2, one in exon 5, and two in exon 6, were used to analyze the haplotype structure of the gene, with DNAs derived from 54 individuals (108 alleles). The analysis revealed that only two of six SNPs showed significant linkage disequilibrium (r(2) > 0.5) with each other. This haplotype information may be useful in disease-association studies in which a contribution of the CL-P1 gene has been suspected, especially in immunological disturbance or atherosclerosis. Two SNPs in exon 6, both leading to amino acid substitutions, could be candidates for influencing disease susceptibility., SPRINGER-VERLAG TOKYO, 英語

レクチン機能を利用した血管における生体防御システムの解明と創薬への応用
若宮伸隆, 本多三男, 鈴木定彦, 板部洋之, 岸雄一郎, 創薬等ヒューマンサイエンス研究重点研究報告書 平成14年度 第2分野 創薬のための生体機能解析に関する研究, 72, 76, 2003年
日本語

Molecular cloning of mouse collectin liver 1
T Kawai, Y Suzuki, S Eda, T Kase, K Ohtani, Y Sakai, H Keshi, A Fukuoh, T Sakamoto, M Nozaki, NG Copeland, NA Jenkins, N Wakamiya, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66, 10, 2134, 2145, 2002年10月
Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish., TAYLOR & FRANCIS LTD, 英語

Isolation of a novel mouse variant of the drs tumor suppressor gene
T Kawai, Y Suzuki, A Yamashita, H Inoue, CANCER LETTERS, 183, 1, 79, 86, 2002年09月
The drs gene was isolated as a transformation suppressor against the v-src oncogene. Drs protein has a transmembrane domain and three consensus repeats (CRs) called Sushi motifs in the extracellular domain. The dry gene also has the ability to suppress anchorage-independent growth of human cancer cell lines. In this paper, we report the isolation of a novel variant cDNA of mouse drs (mDRS-2) containing two CRs, in addition to a Mouse homolog of drs (mDRS-1) containing three CRs. We investigated the Suppressor function of these mDRS cDNAs in human cancer cells and found that the lack of one CR is critical for suppression of anchorage-independent growth by drs. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved., ELSEVIER SCI IRELAND LTD, 英語

心血管疾患と炎症 新しくクローニングされた内皮特異的スカベンジャー受容体CL-P1　　　　　　　　　　　　　　　
福澤 純, 小山 聡, 高下 圭一, 長谷部 直幸, 菊池 健次郎, 大谷 克城, 福應 温, 鈴木 定彦, 若宮 伸隆, Journal of Cardiology, 40, Suppl.1, 91, 91, 2002年08月
(一社)日本心臓病学会, 日本語

血管内皮特異的スガベンジャー受容体CL-P1の細胞内領域結合分子の解析
福應 温, 大谷 克城, 坂本 隆志, 芥子 宏行, 鈴木 定彦, 福澤 純, 小山 聡, 高下 圭一, 小笠原 正洋, 吉田 逸朗, 若宮 伸隆, 生化学, 74, 8, 1097, 1097, 2002年08月
(公社)日本生化学会, 日本語

血管内皮に発現するスカベンジャー受容体様コレクチンCL‐P1のクローニングと機能の解析
大谷克城, 鈴木定彦, 坂本隆志, 江田宗司, 河合高生, 福応温, 芥子宏行, 若宮伸隆, 日本糖質学会年会要旨集, 23rd, 87, 2002年07月25日
日本語

新規コレクチン遺伝子群のクローニングとその分子進化について
鈴木定彦, 大谷克城, 坂本隆志, 江田宗司, 河合高生, 福応温, 芥子宏行, 若宮伸隆, 日本糖質学会年会要旨集, 23rd, 46, 2002年07月25日
日本語

Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice
T Shimada, K Inoue, Y Suzuki, T Kawai, E Azuma, T Nakajima, M Shindo, K Kurose, A Sugie, Y Yamagishi, Y Fujii-Kuriyama, M Hashimoto, CARCINOGENESIS, 23, 7, 1199, 1207, 2002年07月
Arylhydrocarbon receptor knock-out, AhR(-/-), mice have recently been shown to be rather resistant to benzo[a]pyrene (B[a]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[a]P to reactive metabolites (Y.Shimizu, Y.Nakatsuru, M.Ichinose, Y.Takahashi, H.Kume, J.Mimura, Y.Fujii-Kuriyama and T.Ishikawa (2000) Proc. Natl Acad. Sci. USA, 97, 779-782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[a]P, plays a role in the B[a]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(-/-) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[a]P, 7,12-dimethylbenz[a]anthracene, dibenz[a,l]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[b]fluoranthene, and benzo[a]anthracene and by a co-planar PCB congener 3,4,3',4'-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[e]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(+/-/+/-) mice. In AhR(-/-) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylations and of mutagenic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[a]P and related carcinogens causing initiation of cancers in mice., OXFORD UNIV PRESS, 英語

結核菌青山B株全染色体コスミド整列クローンライブラリーの作成 IS6110と本株の特徴
陵本功子, 中嶋一彦, 下山孝, 鈴木定彦, 田村俊秀, 結核, 77, 3, 262, 2002年03月15日
日本語

大気浮遊粉じん中のビスフェノールAの検出と多環芳香族炭化水素との相関
松本比佐志, 鈴木定彦, 日本薬学会年会要旨集, 122nd, 3, 187, 2002年03月05日
日本語

熱帯熱マラリア原虫の赤血球侵入に対する動物血清レクチンの阻害効果
大西義博, 木村明生, 鈴木定彦, 西村和彦, 日本寄生虫学会大会プログラム・抄録集, 71st, 85, 2002年03月01日
日本語

蛍光抗体法スライド上でのCryptosporidium parvumオーシスト同定の試み
木村明生, 鈴木定彦, 楠原康弘, 木俣勲, 井関基弘, 日本寄生虫学会大会プログラム・抄録集, 71st, 158, 2002年03月01日
日本語

Identification of human mannose binding lectin (MBL) recognition sites for novel inhibitory antibodies.
Hui Zhao, Nobutaka Wakamiya, Yasuhiko Suzuki, Matthew T Hamonko, Gregory L Stahl, Hybridoma and hybridomics, 21, 1, 25, 36, 2002年02月, ［国際誌］
Mannose binding lectin (MBL) binding initiates activation of the lectin complement pathway. Recent studies from our laboratory have demonstrated that MBL-dependent complement activation mediates cellular injury following oxidative stress in vivo and in vitro. A panel of novel inhibitory monoclonal antibodies (MAbs) against MBL (e.g., MAb 3F8, 2A9, and hMBL1.2) has been developed that inhibit MBL binding and lectin pathway activation. Here, we further characterized the interactions of these MAbs and their Fab fragments to MBL. Whole MAbs or their Fab fragments bound to MBL with relatively high affinity. Fab fragments of 3F8 were functionally effective in inhibiting MBL-dependent complement activation, however, steric hindrance of MAb 2A9 was essential for inhibition of MBL-dependent complement activation. We identified the hinge region, and residues EDCVLLL within the carbohydrate recognition domain of MBL as the recognition sites for MAb 3F8 and 2A9, respectively. The interaction of MAbs (e.g., 3F8 and 2A9) to MBL was dependent on the conformation of their recognition sites. These findings demonstrate that MBL binding can be inhibited by at least two separate and independent mechanisms., 英語

Rapid detection of pyrazinamide-resistant Mycobacterium tuberculosis by a PCR-based in vitro system
Y Suzuki, A Suzuki, A Tamaru, C Katsukawa, H Oda, JOURNAL OF CLINICAL MICROBIOLOGY, 40, 2, 501, 507, 2002年02月
Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterialpyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5' ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis., AMER SOC MICROBIOLOGY, 英語

若宮伸隆、鈴木定彦. MBLとその機能を生理学的意義　　　　　　　　　　　　　　　
臨床病理中四会誌, 17, 2, 12, 2002年

Mannose-binding lectin and the prognosis of fulminant hepatic failure caused by HBV infection
Yukiya Hakozaki, Makoto Yoshiba, Kazuhiko Sekiyama, Eiji Seike, Junichi Iwamoto, Keiji Mitani, Masafumi Mine, Toshio Morizane, Katsuki Ohtani, Yasuhiko Suzuki, Nobutaka Wakamiya, Liver, 22, 1, 29, 34, 2002年
Background/Aims: The mannose-binding lectin (MBL) gene was reported to play an important role in determining the clinical outcome of persistent hepatitis B virus (HBV) infection. We investigated serum MBL concentrations and MBL gene mutations to determine whether they were related to the prognosis of patients with fulminant hepatic failure (FHF) caused by HBV infection. Methods: We investigated serum MBL concentrations and MBL gene mutations in 43 HBV-infected Japanese patients with FHF and 260 HBsAg-negative healthy controls. Serum MBL concentrations were measured by an enzyme-linked immunosorbent assay, and mutations in the MBL gene were analysed by nested PCR and direct DNA sequencing. Results: Only a mutation in codon 54 of the MBL gene was found. The frequency of this mutation in nonsurvivors (40%, 8/20) was higher than in survivors (13%, 3/23), and the difference was slightly significant (p = 0.043). The H allele frequency in survivors (70.5%, 31/44) was higher than in nonsurvivors (39.5%, 15/38) (p = 0.0048). Because of these factors the mean serum MBL concentration in survivors, 1.61 μg/ml (range 0.3-3.86), was significantly higher than in nonsurvivors, 0.79 μg/ml (range 0.04-1.51) (p<
0.0001). The likelihood ratio for nonsurvival was 0 for over 2.0 μg/ml, 0.67 for 1.0-2.0 μg/ml, and 2.24 for 0-1.0 μg/ml. Conclusions: The mutation in codon 54 of the MBL gene tended to be higher in nonsurvivors than in survivors. The H allele frequency (high producing allele in H/Y) in survivors was higher than that in nonsurvivors. High levels of serum MBL correlated with the survival of patients with FHF due to HBV infection. Serum MBL may be useful as a predictive factor for the survival of patients with FHF caused by HBV., 英語

レクチン機能を利用した血管における生体防御システムの解明と創薬への応用
若宮伸隆, 鈴木定彦, 板部洋之, 本多三男, 岸雄一郎, 創薬等ヒューマンサイエンス研究重点研究報告書 平成13年度 第2分野 創薬のための生体機能解析に関する研究, 74, 83, 2002年
日本語

感染防御とコレクチンファミリー
鈴木定彦, 若宮伸隆, Annual Review 免疫, 2002, 217, 225, 2002年
中外医学社, 日本語

MCF‐7細胞増殖を誘発する大気浮遊粉じん中のビスフェノールA
松本比佐志, 鈴木定彦, 日本内分泌かく乱化学物質学会研究発表会要旨集, 4th, 129, 2001年12月
日本語

The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells
K Ohtani, Y Suzuki, S Eda, T Kawai, T Kase, H Keshi, Y Sakai, A Fukuoh, T Sakamoto, H Itabe, T Suzutani, M Gasawara, Yoshida, I, N Wakamiya, JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 47, 44222, 44228, 2001年11月
Collectins are a family of C-type lectins that have collagen-like sequences and carbohydrate recognition domains (CRD). They are involved in host defense through their ability to bind to carbohydrate antigens of microorganisms. The scavenger receptors type A and MARCO are classical type scavenger receptors that have internal collagen-like domains. Here we describe a new scavenger receptor that is a membrane-type collectin from placenta (collectin placenta 1 (CL-P1)), which has a typical collectin collagen-like domain and a CRD. The cDNA has an insert of about 2.2 kilobases coding for a protein containing 742 amino acid residues. The deduced amino acid sequence shows that CL-P1 is a type II membrane protein, has a coiled-coil region, a collagen-like domain, and a CRD. It resembles type A scavenger receptors because the scavenger receptor cysteine-rich domain is replaced by a CRD. Northern analyses, reverse transcription-polymerase chain reaction, and immunohistochemistry show that CL-P1 is expressed in vascular endothelial cells but not in macrophages. By immunoblotting and flow cytometry CL-P1 appears to be a membrane glycoprotein of about 140 kDa in human umbilical vein or arterial endothelial cells, placental membrane extracts, and CL-P1 transfected Chinese hamster ovary cells. We found that CL-P1 can bind and phagocytose not only bacteria (Escherichia coli and Staphylococcus aureus) but also yeast (Saccharomyces cerevisiae). Furthermore, it reacts with oxidized low density lipoprotein (OxLDL) but not with acetylated LDL (AcLDL). These binding activities are inhibited by polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate) and OxLDL but not by polycationic ligands (polyadenylic acid or polycytidylic acid), LDL, or AcLDL. These results indicate that CL-P1 might play important roles in host defenses that are different from those of soluble collectins in innate immunity., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 英語

腎臓由来新規コレクチン(CL‐K1)のクローニング
芥子宏行, 河合高生, 鈴木定彦, 大谷克城, 福応温, 若宮伸隆, 日本免疫学会総会・学術集会記録, 31, 233, 2001年10月31日
日本語

新規膜型コレクチンCL‐P1のクローニングと機能解析
大谷克城, 鈴木定彦, 坂本隆志, 芥子宏行, 福応温, 若宮伸隆, 日本免疫学会総会・学術集会記録, 31, 234, 2001年10月31日
日本語

Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coli expressed capsid protein and the reactivity of two broadly reactive monoclonal antibodies generated against GII capsid towards GI recombinant fragments
Tomoko Yoda, Yoshitake Terano, Yasuhiko Suzuki, Kenji Yamazaki, Isao Oishi, Tsuyoshi Kuzuguchi, Hiroyoshi Kawamoto, Etsuko Utagawa, Koichi Takino, Hajime Oda, Tadayoshi Shibata, BMC Microbiology, 1, 1, 1, 11, 2001年10月08日
Background: Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein. Results: In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody. Conclusion: The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material., BioMed Central Ltd., 英語

Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall
S Tsuji, J Uehori, M Matsumoto, Y Suzuki, A Matsuhisa, K Toyoshima, T Seya, JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 26, 23456, 23463, 2001年06月
Galactofuranosyl residues are present in various microorganisms but not in mammals, In this study, we identified a human lectin binding to galactofuranosyl residues and named this protein human intelectin (hIntL). The mature hIntL was a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit was a 120-kDa homotrimer in which 40-kDa polypeptides mere bridged by disulfide bonds. The hIntL gene was spilt into 8 exons on chromosome 1q21.3, and hIntL mRNA was expressed in the heart, small intestine, colon, and thymus, hIntL showed high levels of homology with mouse intelectin, Xenopus laevis cortical granule lectin/oocyte lectin, lamprey serum lectin, and ascidian galactose-specific lectin, These homologues commonly contained no carbohydrate recognition domain, which is a characteristic of C-type lectins, although some of them have been reported as Ca2+-dependent lectins. Recombinant hIntL revealed affinities to D-pentoses and a D-galactofuranosyl residue in the presence of Ca2+, and recognized the bacterial arabinogalactan of Nocardia containing D-galactofuranosyl residues. These results suggested that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in the recognition of bacteria-specific components in the host., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 英語

Mannose-binding lectin gene: polymorphisms in Japanese patients with systemic lupus erythematosus, rheumatoid arthritis and Sjogren's syndrome
A Tsutsumi, K Sasaki, N Wakamiya, K Ichikawa, T Atsumi, K Ohtani, Y Suzuki, T Koike, T Sumida, GENES AND IMMUNITY, 2, 2, 99, 104, 2001年04月
Mannose-binding lectin (MBL) is a key element of the innate immunity, with a structure similar to complement C1q. Serum MBL levels are greatly affected by the polymorphisms of the MBL gene. In particular, codon 54 mutation of the MBL gene results in a significant reduction of serum MEL. To determine whether polymorphism of the MBL gene is associated with occurrence of systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjogren's syndrome in the Japanese population, we analyzed the MBL gene polymophisms of these patients and controls, by polymerase chain reaction-restriction fragment length polymorphism methods. We found that patients studied had a significantly higher frequency of having homozygous codon 54 mutation compared to controls. In particular patients with SLE or Sjogren's syndrome showed higher probabilities of being homozygous for this mutation. Among subjects with the same genotype, SLE patients tended to have higher serum MBL concentration than controls. Analysis of the promotor region suggested that SLE patients heterozygous for the codon 54 mutation have a higher probability of having a low producing haplotype for the gene without the codon 54 mutation. We conclude that persons homozygous for codon 54 mutation of the MBL gene may be prone to occurrence of autoimmune disorders including SLE, in the Japanese. MBL may have protective effects on occurrence and progression of SLE., NATURE PUBLISHING GROUP, 英語

Recombinant expression of human mannan-binding lectin
T Vorup-Jensen, ES Sorensen, UB Jensen, W Schwaeble, T Kawasaki, Y Ma, K Uemura, N Wakamiya, Y Suzuki, TG Jensen, K Takahashi, RAB Ezekowitz, S Thiel, JC Jensenius, INTERNATIONAL IMMUNOPHARMACOLOGY, 1, 4, 677, 687, 2001年04月
Mannan-binding lectin (MBL) constitutes an important part of the innate immune defend by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MEL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography. (C) 2001 Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV, 英語

耐性菌シリーズ 4 結核菌の薬剤耐性
鈴木定彦, 鈴木文, 田丸亜貴, 勝川千尋, 感染症, 31, 2, 77-83,75-76, 83, 2001年03月20日
アステラス製薬(株), 日本語

IS1245をプローブとしたMycobacterium aviumのRFLP分析
田丸亜貴, 鈴木定彦, 阿野裕美, 吉多仁子, 結核, 76, 3, 328, 2001年03月15日
日本語

DNAチップ技術を用いた結核菌分子疫学的解析法の開発
鈴木定彦, 田丸亜貴, 結核, 76, 3, 328, 2001年03月15日
日本語

Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp OY1-2 isolated from soil
Y Suzuki, T Yoda, A Ruhul, W Sugiura, JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 12, 9059, 9065, 2001年03月
Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp, OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH, A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp, OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp, OY1-2, In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 英語

【スカベンジャー受容体ファミリーと異物排除の分子機構】新規膜型コレクチンは,血管内皮に存在し,スカベンジャー様機能をもつ
若宮 伸隆, 鈴木 定彦, 生化学, 73, 3, 205, 208, 2001年03月
(公社)日本生化学会, 日本語

微生物の薬剤耐性 細菌の化学療法剤と薬剤耐性 結核菌
鈴木定彦, 鈴木文, 田丸亜貴, 勝川千尋, 臨床と微生物, 28, 1, 35, 41, 2001年01月25日
日本語

鈴木定彦、若宮伸隆 感染防御とコレクチンファミリーAnnual Review　　　　　　　　　　　　　　　
免疫, 2002, 217, 225, 2001年

若宮伸隆、鈴木定彦 膜型コレクチンは血管内皮に存在しスカベンジャーレセプター様の機能を持つ　　　　　　　　　　　　　　　
生化学, 73, 205, 208, 2001年

鈴木定彦、田丸亜貴、鈴木文、勝川千尋 結核菌の薬剤耐性　　　　　　　　　　　　　　　
感染症, 31, 75, 83, 2001年

鈴木定彦、田丸亜貴、鈴木文、勝川千尋 結核菌の化学療法剤と薬剤耐性　　　　　　　　　　　　　　　
臨床と微生物, 28, 35, 41, 2001年

Expression of glycosylated human interferon-beta in high levels in Chinese Hamster Ovary (CHO) cells　　　　　　　　　　　　　　　
J. Biol. Macromolec, 1, 12, 18, 2001年

Comparison of self-ligation mediated polymerase chain reaction and mixed-linker polymerase chain reaction in molecular epidemiology of tuberculosis　　　　　　　　　　　　　　　
Kekkaku, 76, 9, 18, 2001年

ハンセン氏病における動物血清レクチンの感染防止効果について (ヒューマンサイエンス振興財団S)
若宮伸隆, WISNU I M, 岸雄一郎, 本多三男, 鈴木定彦, 創薬等ヒューマンサイエンス研究 平成12年度 国際共同研究事業研究報告書, 78, 85, 2001年
日本語

DNAマイクロアレイを使った結核菌薬剤耐性遺伝子の迅速検出
市原竜生, 鈴木定彦, 日本臨床微生物学雑誌, 10, 4, 93, 95, 2000年12月25日
日本臨床微生物学会, 日本語

結核 分子遺伝学からのアプローチ
長谷川好規, 阿部千代治, 飯沼由嗣, 鈴木定彦, 高橋光良, 水口康雄, 結核, 75, 12, 725, 728, 2000年12月15日
Recent progress of molecular genetics has been providing tools for new approaches to disease treatment and diagnosis of Mycobacterium tuberculosis. In 1998, Cole et al. reported the complete genome sequence of Mycobacterium tuberculosis. The new informa tion will provide us the knowledge and understanding of the biology of Mycobacterium tuberculosis. Further, it will provide us new conception of diagnosis and treatment of the disease. Four topics were selected in this symposium. Dr. Iinuma reviewed and prospected the clinical utility of nucleic acid amplification methods of Mycobacterium tuberculosis. Dr. Suzuki reviewed the molecular mechanism of acquired resistance to anti-TB drugs and reported the early detection of genetic mutation by new designed DNA tip method. Dr. Takahashi reviewed the method of molecular epidemiology and genetic elements as a tool for strain differentiation of tuberculosis. Dr. Mizuguchi interpreted the essential feature of mycobacterial genome maps, and genes and their biological activity. He also reviewed the importance and the utility of the complete genome sequence of tuberculosis in asso ciation with pathogenecity. These topics were summarized in this report, based on the symposium of "Molecular genetic approaches to Mycobacterium tuberculosis " in the 75 thannual meeting of the Japanese Society for Tuberculosis., JAPANESE SOCIETY FOR TUBERCULOSIS, 日本語

膜型コレクチンCL‐P1は血管内皮に発現し,スカベンジャーレセプター様の機能を示す
大谷克城, 鈴木定彦, 坂本隆志, 芥子宏行, 福応温, 若宮伸隆, 日本分子生物学会年会プログラム・講演要旨集, 23rd, 311, 2000年11月25日
日本語

結核の現状と課題 結核菌の薬剤耐性と関与遺伝子
鈴木定彦, 鈴木文, 田丸亜貴, 勝川千尋, Pharma Med, 18, 10, 47, 53, 2000年10月10日
(株)メディカルレビュー社, 日本語

Recognition of N-glycolylneuraminic acid linked to galactose by the alpha 2,3 linkage is associated with intestinal replication of influenza A virus in ducks
T Ito, Y Suzuki, T Suzuki, A Takada, T Horimoto, K Wells, H Kida, K Otsuki, M Kiso, H Ishida, Y Kawaoka, JOURNAL OF VIROLOGY, 74, 19, 9300, 9305, 2000年10月
The hemagglutinin (IU) of H3 human influenza viruses does not support viral replication in duck intestine despite its avian origin. A Leu-to-Gin mutation at position 226 and a Ser-to-Gly mutation at position 228 in the HA of human A/Udorn/307/72 (H3N2) permit a reassortant virus [human Udorn HA, with all other genes from A/mallard/New York/6750/78 (H2N2)] to replicate in ducks. To understand the molecular basis of this change in host range restriction, we investigated the receptor specificity of duck influenza viruses as well as of human-duck virus reassortants. The results indicate that the recognition of a glycoconjugate moiety possessing N-glycolneuramic acid (NeuGc) linked to galactose by the alpha 2,3 linkage (NeuGc alpha 2,3Gal) is associated with viral replication in duck intestine. Immunofluorescence assays with NeuGc alpha 2,3Gal-specific antiserum detected this moiety primarily on the crypt epithelial cells of duck colon. Such recognition, together with biochemical evidence of NeuGc in crypt cells, correlated exactly with the ability of the virus to replicate in duck colon. These results suggest that recognition of the NeuGc alpha 2,3-Gal moiety plays an important role in the enterotropism of avian influenza viruses., AMER SOC MICROBIOLOGY, 英語

結核菌の薬剤耐性獲得機序の解析と迅速診断への展開
鈴木定彦, 田丸亜貴, RUHUL A, 勝川千尋, 防菌防ばい, 28, 9, 561, 573, 2000年09月10日
日本防菌防黴学会, 日本語

Mannose-binding lectin polymorphisms in patients with hepatitis C virus infection
K Sasaki, A Tsutsumi, N Wakamiya, K Ohtani, Y Suzuki, Y Watanabe, N Nakayama, T Koike, SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, 35, 9, 960, 965, 2000年09月
Background: Persistent infection with hepatitis C virus (HCV) leads to liver cirrhosis (LC) and often to Liver cancer. Little is known about host factors that determine the variable natural history. Mannose-binding lectin (MBL) is an important constituent of the innate immune system. In white patients there is an association between codon 52 mutation of the MBL gene and persistent hepatitis B virus (HBV) infection. To determine whether MBL gene polymorphisms affect the course of HCV infection, we investigated the association between MBL gene polymorphisms and HCV infection in Japanese subjects. Methods: Fifty-two HCV-infected Japanese patients (8 with chronic inactive hepatitis (CIH), 31 with chronic active hepatitis (CAH), 13 with LC) and 50 normal controls were studied. MBL gene mutations were determined by means of polymerase chain reaction and restriction fragment length polymorphism analyses. Results: Codon 52 and codon 57 mutations were absent in all subjects. Homozygous mutation in codon 54 was present in one (0.9%) patient. Heterozygous codon 54 mutation was present in 17 (32%) of the 52 patients and in 21 (41%) of the controls. No significant difference in the frequency of codon 54 mutation was observed between patient and control groups. However, although no significant relationship was observed between MBL polymorphisms and the levels of HCV RNA, all patients with heterozygous or homozygous codon 54 mutations had CAH or LC. In contrast, 8 of the 34 patients without codon 54 mutation remained at CIH. (P = 0.0405). Conclusion: MBL may be one of the factors that influence the course of HCV infection., TAYLOR & FRANCIS AS, 英語

日本人におけるMBL(mannan-binding lectin)遺伝子変異と血中濃度の検討
芥子 宏行, 大谷 克城, 坂本 隆志, 岸 雄一郎, 荒木 宏昌, 鈴木 定彦, 若宮 伸隆, 医学のあゆみ, 194, 12, 957, 958, 2000年09月
健常日本人497例におけるMBL遺伝子変異と血中濃度の測定により,MBLの遺伝子多型はコドン54のみにみられ,その頻度は野生型70.8%,ヘテロ変異型22.5%,ホモ変異型6.7%であった.本ホモ変異型の比率は欧米の比率に比べて高値であるが,血中濃度とgenotypeが十分相関しており,今後,MBLの関与が考えられる各種疾患群との比較において日本人のMBL遺伝子多型と血中濃度の一つのスタンダードになりうる, 医歯薬出版(株), 日本語

薬剤耐性結核菌の分子遺伝学的解析とその応用
鈴木定彦, 薬剤耐性菌シンポジウム講演記録, 29th, 24, 27, 2000年08月24日
日本語

DNAチップによる結核菌の耐性診断
鈴木定彦, 市原竜生, 田丸亜貴, RUHUL A, 勝川千尋, 牧野正直, 阿部千代治, Bio Ind, 17, 5, 36, 44, 2000年05月12日
シ-エムシ-, 日本語

Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection
T Yoda, Y Terano, A Shimada, Y Suzuki, K Yamazaki, N Sakon, Oishi, I, ET Utagawa, Y Okuno, T Shibata, JOURNAL OF MEDICAL VIROLOGY, 60, 4, 475, 481, 2000年04月
The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant: NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA rests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WE. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test. J. Med. Virol. 60:475-481, 2000. (C) 2000 Wiley-Liss, Inc., WILEY-LISS, 英語

【自然免疫を担う生体防御レクチン】生体防御レクチンとしてのコレクチンファミリー
若宮 伸隆, 鈴木 定彦, 蛋白質・核酸・酵素, 45, 5, 655, 663, 2000年04月
共立出版(株), 日本語

大阪府における病院内結核患者同時発生事例のRFLP解析
田丸亜貴, 鈴木定彦, 結核, 75, 3, 292, 2000年03月15日
日本語

結核菌の薬剤耐性に関与する遺伝子の解析と迅速診断への応用
鈴木定彦, 結核, 75, 3, 225, 225, 2000年03月15日
日本語

結核菌青山B株整列クローンライブラリーの作成
陵本功子, 鈴木定彦, 下山孝, 田村俊秀, 結核, 75, 3, 329, 2000年03月15日
日本語

A comparative analysis of the antigenic, structural, and functional properties of three different preparations of recombinant human interleukin-18
S Kikkawa, K Shida, H Okamura, NA Begum, M Matsumoto, S Tsuji, M Nomura, Y Suzuki, K Toyoshima, T Seya, JOURNAL OF INTERFERON AND CYTOKINE RESEARCH, 20, 2, 179, 185, 2000年02月
We compared the structural and functional properties of three recombinant human interleukin-18 (rIL-18) preparations, commercially available (Pep rIL-18) and prepared in our laboratory (active and inactive, according to their ability to potentiate IL-12-mediat