研究者データベース

中島 千絵(ナカジマ チエ)
人獣共通感染症国際共同研究所 バイオリソース部門
教授

基本情報

所属

  • 人獣共通感染症国際共同研究所 バイオリソース部門

職名

  • 教授

学位

  • 博士(獣医学)(北海道大学)

ホームページURL

J-Global ID

研究キーワード

  • 多剤耐性   結核菌群菌   遺伝子変異   

研究分野

  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 医療管理学、医療系社会学
  • ライフサイエンス / 衛生学、公衆衛生学分野:実験系を含まない
  • ライフサイエンス / 衛生学、公衆衛生学分野:実験系を含む
  • ライフサイエンス / 衛生学、公衆衛生学分野:実験系を含まない
  • ライフサイエンス / 衛生学、公衆衛生学分野:実験系を含む
  • ライフサイエンス / 医療管理学、医療系社会学

職歴

  • 2006年04月 - 現在 北海道大学 人獣共通感染症リサーチセンター
  • - 2000年04月 財団法人 畜産生物科学安全研究所

研究活動情報

論文

  • 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 2021年04月26日 
    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 was 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.
  • 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 2021年04月26日 
    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 practiced 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.
  • 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.) 2021年04月20日 
    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.
  • Ruttana Pachanon, Kentaro Koide, Siriporn Kongsoi, Nami Ajima, Thoko Flav Kapalamula, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
    Microbial drug resistance (Larchmont, N.Y.) 2021年04月09日 
    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.
  • 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 
    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.
  • 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 
    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.
  • Bhagwan Maharjan, Jeewan Thapa, Dhirendra Kumar Shah, Bhabana Shrestha, Korkut Avsar, Yasuhiko Suzuki, Chie Nakajima
    Japanese journal of infectious diseases 2021年03月31日 
    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.
  • 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 
    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.
  • 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 
    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.
  • 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 2021年01月16日 
    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.
  • 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 
    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.
  • 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 
    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 (SMKP01mutSA307T) 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 SMKP01mutSA307T 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.
  • 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 2020年12月15日 
    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.
  • Kaknokrat Chonsin, Neunghatai Supha, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
    FEMS microbiology letters 2020年12月15日 
    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 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, six gastroenteritis patients, seven environmental samples and five genomes from a database showed 22 different alleles. Gene VP1680 was conserved in tdh+ isolates and pandemic strains, 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.
  • 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 [査読有り][通常論文]
     
    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.
  • Dipti Shrestha, Bhagwan Maharjan, Nan Aye Thida Oo, Norikazu Isoda, Chie Nakajima, Yasuhiko Suzuki
    Tuberculosis (Edinburgh, Scotland) 125 101985 - 101985 2020年12月 [査読有り][通常論文]
     
    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.
  • 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 [査読有り][通常論文]
     
    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.
  • 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 2020年10月30日 
    Despite the availability of anti-tuberculosis drugs, treatment of tuberculosis has been complicated by drug resistant tuberculosis. Early detection of drug resistance make early appropriate treatment possible. However, the available tools are mainly for rifampicin resistance and the existing isoniazid resistance detection method is expensive, highly technical and complicated to maintain to make them unsustainable for use in developing nations. This study aimed to develop a simple, rapid and low-cost diagnostic kit for isoniazid resistant tuberculosis using single-stranded tag hybridization method targeting an isoniazid resistance conferring mutation. Specificity and sensitivity were assessed by utilizing DNA extracted from 49 isoniazid resistant and 41 susceptible Mycobacterium tuberculosis clinical isolates cultured in Mycobacterial Growth Indicator Tubes. Positive signals were observed at mutant and wildtype lines with the sensitivity and specificity of 100% comparing to sanger sequencing results. In contrast, no positive signal was observed with non-tuberculosis mycobacteria. And the detection limit of this method was supposed to be 103 CFU or less. The STH-PAS method for isoniazid resistant M. tuberculosis detection developed in this study offers a better alternative for conventional phenotypic isoniazid resistance determination which will be of both clinical and epidemiological significance in resource limited nations.
  • 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 2020年10月12日 [査読有り][通常論文]
     
    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.
  • Yutaka Takarada, Takuya Kodera, Kumi Kobayashi, Chie Nakajima, Mitsuo Kawase, Yasuhiko Suzuki
    Journal of microbiological methods 177 106062 - 106062 2020年10月 [査読有り][通常論文]
     
    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.
  • 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 [査読有り][通常論文]
     
    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.
  • 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09月01日 [査読有り][通常論文]
     
    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.
  • 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09月 [査読有り][通常論文]
     
    OBJECTIVES: It is established that resistance to rifampicin (RIF) in 90% of RIF-resistant Mycobacterium tuberculosis isolates is attributable to point mutations in the rpoB gene, whilst 50-95% of M. tuberculosis resistance to isoniazid (INH) is caused by mutations in the katG gene. However, the patterns and frequencies of mutations vary by geographical region. In Zambia, the genetic mechanisms of resistance of M. tuberculosis to RIF and INH were unreported before this study. METHODS: Using gene sequencing, the rpoB, katG and inhA genes of 99 multidrug-resistant M. tuberculosis (MDR-TB) and 49 pan-susceptible M. tuberculosis isolates stored at a tuberculosis reference laboratory from 2013 to 2016 were analysed and were compared with published profiles from other African countries. RESULTS: Of the 99 MDR-TB isolates, 95 (96.0%) carried mutations in both rpoB and katG. No mutations were detected among the pan-susceptible isolates. The most common mutations among RIF- and INH-resistant isolates were in codon 531 of the rpoB gene (55.6%; 55/99) and codon 315 of the katG gene (94.9%; 94/99), respectively. Distinctly, katG mutations were predominantly high among Zambian isolates (96.0%) compared with other countries in the region. CONCLUSION: Resistance-associated mutations to RIF and INH circulating in Zambia are similar to those reported globally, therefore these data validate the applicability of molecular diagnostic tools in Zambia. However, katG mutations were predominantly high among M. tuberculosis isolates in this study compared with other regional countries and might distinguish cross-boundary transmission of MDR-TB from other African nations.
  • Benjawan Phetsuksiri, Janisara Rudeeaneksin, Sopa Srisungngam, Supranee Bunchoo, Wiphat Klayut, Chie Nakajima, Shigeyugi Hamada, Yasuhiko Suzuki
    Japanese journal of infectious diseases 73 4 272 - 277 2020年07月22日 [査読有り][通常論文]
     
    The diagnosis of tuberculosis (TB) in endemic countries is challenging due to high caseloads and limited resources. A simple and cost-effective diagnostic test for the rapid detection of Mycobacterium tuberculosis (M. tuberculosis) in clinical specimens is crucially needed. We evaluated the performance of an in-house assay based on loop-mediated isothermal amplification (LAMP) targeting the M. tuberculosis 16S ribosomal RNA (rRNA) gene for the diagnosis of TB in Thailand. A total of 252 sputum samples from suspected cases of pulmonary TB were analyzed. The sensitivity of LAMP was 99.04% (103/104; 95% confidence interval [CI]: 94.76-9.98%) 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 all those were positive by conventional polymerase chain reaction (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 were positive by LAMP. Compared to 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 propose that LAMP would be suitable as a potential diagnostic test for rapid TB diagnosis in resource-limited laboratory settings.
  • 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07月 [査読有り][通常論文]
     
    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.
  • Chihiro Oe, Hironori Hayashi, Kazushige Hirata, Kumi Kawaji, Fusako Hashima, Mina Sasano, Maaya Furuichi, Emiko Usui, Makoto Katsumi, Yasuhiko Suzuki, Chie Nakajima, Mitsuo Kaku, Eiichi N Kodama
    ACS infectious diseases 6 6 1490 - 1500 2020年06月12日 [査読有り][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • Yuuki Suzuki, Toyotaka Sato, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki, Satoshi Takahashi, Shin-Ichi Yokota
    International journal of antimicrobial agents 105919 - 105919 2020年02月13日 [査読有り][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • 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 [査読有り][通常論文]
     
    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.
  • 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][通常論文]
     
    © 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.
  • 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][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • Koide K, Kongsoi S, Nakajima C, Suzuki Y
    Bioscience, biotechnology, and biochemistry 83 12 2249 - 2256 2019年12月 [査読有り][通常論文]
  • 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][通常論文]
  • 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  2019年08月 [査読有り][通常論文]
  • 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][通常論文]
     
    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.
  • 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][通常論文]
  • 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 2019年05月 [査読有り][通常論文]
  • 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04月 [査読有り][通常論文]
  • 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][通常論文]
     
    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.
  • 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][通常論文]
  • 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  2018年11月 [査読有り][通常論文]
  • Phetsuksiri B, Rudeeaneksin J, Srisungngam S, Bunchoo S, Klayut W, Sangkitporn S, Nakajima C, Hamada S, Suzuki Y
    Japanese journal of infectious diseases 2018年10月 [査読有り][通常論文]
  • 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][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • Nan Aye Thida Oo, Lai Lai San, Jeewan Thapa, Khin Saw Aye, Wah Wah Aung, Chie Nakajima, Yasuhiko Suzuki
    Tuberculosis 111 8 - 13 2018年07月01日 [査読有り][通常論文]
     
    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.
  • Koide K, Kongsoi S, Ouchi Y, Yamaguchi T, Nakajima C, Suzuki Y
    Microbial drug resistance (Larchmont, N.Y.) 25 1 14 - 22 2018年07月 [査読有り][通常論文]
  • 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 2018年06月 [査読有り][通常論文]
  • 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][通常論文]
     
    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.
  • Precious Bwalya, Tomoyuki Yamaguchi, Georgina Mulundu, Chie Nakajima, Grace Mbulo, Eddie Samuneti Solo, Yukari Fukushima, Kunda Kasakwa, Yasuhiko Suzuki
    Tuberculosis 109 117 - 122 2018年03月01日 [査読有り][通常論文]
     
    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.
  • 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 2017年12月 [査読有り][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • 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 2017年10月 [査読有り][通常論文]
     
    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.
  • 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 2017年10月 [査読有り][通常論文]
     
    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.
  • 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 2017年09月 [査読有り][通常論文]
     
    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.
  • 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 2017年09月 [査読有り][通常論文]
     
    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.
  • 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  2017年08月 [査読有り][通常論文]
     
    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.
  • 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  2017年07月 [査読有り][通常論文]
     
    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.
  • 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  2017年06月 [査読有り][通常論文]
     
    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.
  • Charity Habeenzu, Chie Nakajima, Eddie Solo, Precious Bwalya, Kiichi Kajino, Mari Miller, Youichi Kurosawa, Victor Mudenda, Lackson Kasonka, Yasuhiko Suzuki, Takashi Matsuba
    JOURNAL OF INFECTION IN DEVELOPING COUNTRIES 11 6 440 - 444 2017年06月 [査読有り][通常論文]
     
    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.
  • 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  2017年05月 [査読有り][通常論文]
     
    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.
  • Masaru Usui, Mayuko Kawakura, Nobuki Yoshizawa, Lai Lai San, Chie Nakajima, Yasuhiko Suzuki, Yutaka Tamura
    ANAEROBE 43 15 - 20 2017年02月 [査読有り][通常論文]
     
    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.
  • 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 2017年01月 [査読有り][通常論文]
     
    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.
  • 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  2017年 [査読有り][通常論文]
     
    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.
  • Tomoyuki Yamaguchi, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 7 1343 - 1347 2017年 [査読有り][通常論文]
     
    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.
  • 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 2017年 [査読有り][通常論文]
  • 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 2016年11月 [査読有り][通常論文]
     
    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.
  • 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 2016年10月 [査読有り][通常論文]
     
    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.
  • Siriporn Kongsoi, Ruchirada Changkwanyeun, Kazumasa Yokoyama, Chie Nakajima, Kanjana Changkaew, Orasa Suthienkul, Yasuhiko Suzuki
    DRUG TESTING AND ANALYSIS 8 10 1065 - 1070 2016年10月 [査読有り][通常論文]
     
    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.
  • Tomoyuki Yamaguchi, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki
    PLoS Neglected Tropical Diseases 10 9 e0005013  2016年09月28日 [査読有り][通常論文]
     
    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.
  • Tomoyuki Yamaguchi, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki
    PLOS NEGLECTED TROPICAL DISEASES 10 9 2016年09月 [査読有り][通常論文]
     
    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.
  • Tomohiro Seyama, Hirofumi Hirayasu, Gen Yoshida, Aiko Ohnuma, Yongjin Qiu, Chie Nakajima, Koji Kasai, Yasuhiko Suzuki
    JAPANESE JOURNAL OF VETERINARY RESEARCH 64 3 197 - 203 2016年08月 [査読有り][通常論文]
     
    We examined the effects of encapsulated lactic acid bacteria administrated orally to lactating cattle on the intestinal flora. A dose of 3 x 10(11) 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.
  • 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 / the Japanese Society of Veterinary Science 78 7 1117 - 1121 2016年07月 [査読有り][通常論文]
     
    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.
  • 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  2016年06月 [査読有り][通常論文]
     
    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.
  • Takashi Matsuba, Umme Ruman Siddiqi, Toshio Hattori, Chie Nakajima, Jun Fujii, Yasuhiko Suzuki
    FEMS MICROBIOLOGY LETTERS 363 10 2016年05月 [査読有り][通常論文]
     
    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.
  • 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 2016年04月 [査読有り][通常論文]
     
    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.
  • 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 2016年04月 [査読有り][通常論文]
     
    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.
  • 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 2016年04月 [査読有り][通常論文]
     
    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.
  • 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 2016年03月 [査読有り][通常論文]
  • 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 2016年03月 [査読有り][通常論文]
     
    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.
  • 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 2016年02月 [査読有り][通常論文]
     
    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.
  • K. Chishimba, B. M. Hang'Ombe, K. Muzandu, S. E. Mshana, M. I. Matee, C. Nakajima, Y. Suzuki
    International Journal of Microbiology 2016 2016年 [査読有り][通常論文]
     
    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.
  • 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 2016年01月 [査読有り][通常論文]
     
    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.
  • 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 2015年12月 [査読有り][通常論文]
     
    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.
  • Ryuji Kawahara, Kazuko Seto, Masumi Taguchi, Chie Nakajima, Yuko Kumeda, Yasuhiko Suzuki
    JOURNAL OF CLINICAL MICROBIOLOGY 53 9 3035 - 3038 2015年09月 [査読有り][通常論文]
     
    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).
  • Jeewan Thapa, Chie Nakajima, Bhagwan Maharjan, Ajay Poudel, Yasuhiko Suzuki
    JAPANESE JOURNAL OF VETERINARY RESEARCH 63 3 151 - 158 2015年08月 [査読有り][通常論文]
     
    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.
  • 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 2015年08月 [査読有り][通常論文]
     
    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.
  • Changkaew K, Intarapuk A, Utrarachkij F, Nakajima C, Suthienkul O, Suzuki Y
    Journal of food protection 78 8 1442 - 1450 2015年08月 [査読有り][通常論文]
     
    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.
  • Siriporn Kongsoi, Kazumasa Yokoyama, Apinun Suprasert, Fuangfa Utrarachkij, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
    DRUG TESTING AND ANALYSIS 7 8 714 - 720 2015年08月 [査読有り][通常論文]
     
    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.
  • 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][通常論文]
  • 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10月 [査読有り][通常論文]
  • 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 2014年10月 [査読有り][通常論文]
     
    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.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    IMMUNOLOGY 142 4 551 - 561 2014年08月 [査読有り][通常論文]
     
    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.
  • Kanjana Changkaew, Fuangfa Utrarachkij, Kanokrat Siripanichgon, Chie Nakajima, Orasa Suthienkul, Yasuhiko Suzuki
    JOURNAL OF FOOD PROTECTION 77 8 1394 - 1401 2014年08月 [査読有り][通常論文]
     
    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.
  • 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 2014年07月 [査読有り][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • 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][通常論文]
     
    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.
  • 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 2014年01月 [査読有り][通常論文]
     
    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.
  • Beata Shiratori, Susan Leano, Chie Nakajima, Haorile Chagan-Yasutan, Toshiro Niki, Yugo Ashino, Yasuhiko Suzuki, Elisabeth Telan, Toshio Hattori
    MEDIATORS OF INFLAMMATION 2014 513263  2014年 [査読有り][通常論文]
     
    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.
  • Yasuhiko Suzuki, Tomoyuki Yamaguchi, Hyun Kim, Kazumasa Yokoyama, Chie Nakajima
    Japanese Journal of Leprosy 83 3 131 - 137 2014年 [査読有り][通常論文]
     
    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.
  • 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 2013年05月 [査読有り][通常論文]
     
    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.
  • 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 2013年 [査読有り][通常論文]
     
    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.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Veterinary Research 44 1 59  2013年 [査読有り][通常論文]
     
    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.
  • Ajay Poudel, Bhagwan Maharjan, Chie Nakajima, Yukari Fukushima, Basu D. Pandey, Antje Beneke, Yasuhiko Suzuki
    TUBERCULOSIS 93 1 84 - 88 2013年01月 [査読有り][通常論文]
     
    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.
  • Zeaur Rahim, Chie Nakajima, Rubhana Raqib, Khalequ Zaman, Hubert P. Endtz, Adri G. M. van der Zanden, Yasuhiko Suzuki
    TUBERCULOSIS 92 6 529 - 534 2012年11月 [査読有り][通常論文]
     
    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.
  • 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  2012年11月 [査読有り][通常論文]
     
    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.
  • Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki
    PLoS Neglected Tropical Diseases 6 10 e1838  2012年10月 [査読有り][通常論文]
     
    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.
  • Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki
    PLOS NEGLECTED TROPICAL DISEASES 6 10 2012年10月 [査読有り][通常論文]
     
    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.
  • 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 2012年08月 [査読有り][通常論文]
  • 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  2012年08月 [査読有り][通常論文]
     
    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.
  • 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 2012年07月 [査読有り][通常論文]
     
    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.
  • 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 2012年06月 [査読有り][通常論文]
     
    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.
  • 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 2012年06月 [査読有り][通常論文]
     
    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.
  • 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 2012年06月 [査読有り][通常論文]
     
    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.
  • Ajay Poudel, Chie Nakajima, Yukari Fukushima, Haruka Suzuki, Basu Dev Pandey, Bhagwan Maharjan, Yasuhiko Suzuki
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 56 6 2831 - 2836 2012年06月 [査読有り][通常論文]
     
    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.
  • Yasuhiko Suzuki, Chie Nakajima, Aki Tamaru, Hyun Kim, Takashi Matsuba, Hajime Saito
    INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS 39 5 435 - 439 2012年05月 [査読有り][通常論文]
     
    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.
  • 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 2012年05月 [査読有り][通常論文]
     
    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.
  • 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 2012年04月 [査読有り][通常論文]
     
    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.
  • Fuangfa Utrarachkij, Srirat Pornraungwong, Kanokrat Siripanichgon, Chie Nakajima, Yasuhiko Suzuki, Orasa Suthienkul
    INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 154 1-2 73 - 78 2012年03月 [査読有り][通常論文]
     
    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.
  • Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 56 2 697 - 702 2012年02月 [査読有り][通常論文]
     
    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.
  • Hyun Kim, Chie Nakajima, Youn Uck Kim, Kazumasa Yokoyama, Yasuhiko Suzuki
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 1 72 - 74 2012年01月 [査読有り][通常論文]
     
    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.
  • Hyun Kim, Chie Nakajima, Kazumasa Yokoyama, Zeaur Rahim, Youn Uck Kim, Hiroki Oguri, Yasuhiko Suzuki
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 8 3661 - 3667 2011年08月 [査読有り][通常論文]
     
    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.
  • 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 2011年04月 [査読有り][通常論文]
     
    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.
  • Hyun Kim, Haruka Suzuki, Masanori Matsuoka, Takashi Matsuba, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki
    Japanese Journal of Leprosy 80 1 17 - 27 2011年 [査読有り][通常論文]
     
    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.
  • 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][通常論文]
  • Matsuba T, Nakajima C, Suzuki Y
    Nihon saikingaku zasshi. Japanese journal of bacteriology 65 3 355 - 368 2-4 2010年12月 [査読有り][通常論文]
  • Masanori Matsuoka, Yasuhiko Suzuki, Iris Estrada Garcia, Mary Fafutis-Morris, Alberto Vargas-Gonzalez, Cristina Carreno-Martinez, Yukari Fukushima, Chie Nakajima
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 63 6 412 - 416 2010年11月 [査読有り][通常論文]
     
    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
  • 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 2010年08月 [査読有り][通常論文]
     
    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.
  • Tadashi Fukasawa, Naozumi Oda, Yasunao Wada, Aki Tamaru, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 63 4 246 - 250 2010年07月 [査読有り][通常論文]
     
    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.
  • Chie Nakajima, Zeaur Rahim, Yukari Fukushima, Isamu Sugawara, Adri G. M. van der Zanden, Aki Tamaru, Yasuhiko Suzuki
    BMC INFECTIOUS DISEASES 10 118  2010年05月 [査読有り][通常論文]
     
    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.
  • Yasuhiko Suzuki, Takashi Matsuba, Chie Nakajima
    Kekkaku 85 2 79 - 86 2010年 [査読有り][通常論文]
     
    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.
  • Saiki Imamura, Itabajara da Silva Vaz, Satoru Konnai, Shinji Yamada, Chie Nakajima, Misao Onuma, Kazuhiko Ohashi
    EXPERIMENTAL AND APPLIED ACAROLOGY 48 4 345 - 358 2009年08月 [査読有り][通常論文]
     
    We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick's defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.
  • Satoru Konnai, Chie Nakajima, Saiki Imamura, Shinji Yamada, Hideto Nishikado, Michi Kodama, Misao Onuma, Kazuhiko Ohashi
    IMMUNOLOGY 126 2 209 - 219 2009年02月 [査読有り][通常論文]
     
    Previously, a putative immunosuppressant-coding gene was identified from a complementary DNA library derived from the salivary glands of partially-fed Haemaphysalis longicornis. Using real-time polymerase chain reaction, the gene was shown to be predominantly expressed during blood feeding with the site of expression being mainly in the salivary glands; this was confirmed by Western blotting analysis. To investigate the function of this novel protein, in this study, we examined the proliferative responses of bovine mononuclear cells and murine splenic cells as well as the expression of profiles of several cytokines in these cells in the presence of the recombinant protein (H. longicornis-derived 36 000 molecular weight protein: rHL-p36). The addition of rHL-p36 at the beginning of the 72 hr cultivation period clearly inhibited proliferation of several mitogen-stimulated cells in a dose-dependent manner, with concomitantly significant down-regulation of messenger RNA levels for interleukin-2. The inhibitory response could be abrogated by blockage of HL-p36 with antibody, suggesting the direct involvement of rHL-p36 in the cell proliferation. Furthermore, the proliferative response of splenocytes isolated from rHL-p36-inoculated mice was significantly lower than for those from control mice, suggesting that rHL-p36 could also directly suppress immune responses in vivo. Interestingly, microarray analysis of the splenocytes showed that the expression of several immunomodulating genes was down-regulated by rHL-p36 inoculation. In conclusion, these results suggest that HL-p36 is an immunosuppressor that might play an important role in the modulation of host immune responses.
  • A. Edagawa, A. Kimura, H. Doi, H. Tanaka, K. Tomioka, K. Sakabe, C. Nakajima, Y. Suzuki
    JOURNAL OF APPLIED MICROBIOLOGY 105 6 2104 - 2114 2008年12月 [査読有り][通常論文]
     
    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.
  • 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 2008年10月 [査読有り][通常論文]
     
    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.
  • Saiki Imamura, Satoru Konnai, Itabajara da Silva Vaz Junior, Shinji Yamada, Chie Nakajima, Yuko Ito, Tomoko Tajima, Jun Yasuda, Martin Simuunza, Misao Onuma, Kazuhiko Ohashi
    JAPANESE JOURNAL OF VETERINARY RESEARCH 56 2 85 - 98 2008年08月 [査読有り][通常論文]
     
    Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36-kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM36 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8 % for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8 % in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T parva-infected ticks fed on immunized cattle, the occurrence of T parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.
  • 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 2008年04月 [査読有り][通常論文]
     
    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.
  • Susumu Okamoto, Aki Tamaru, Chie Nakajima, Kenji Nishimura, Yukinori Tanaka, Shinji Tokuyama, Yasuhiko Suzuki, Kozo Ochi
    MOLECULAR MICROBIOLOGY 63 4 1096 - 1106 2007年02月 [査読有り][通常論文]
     
    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.
  • Satoru Konnai, Saiki Imamura, Chie Nakajima, William Harold Witola, Shinji Yamada, Martin Simuunza, Andrew Nambota, Jun Yasuda, Kazuhiko Ohashi, Misao Onuma
    ACTA TROPICA 99 1 34 - 41 2006年08月 [査読有り][通常論文]
     
    In order to investigate the transmission dynamics of Theileria parva (T parva) by the brown ear tick, Rhipicephalus appendiculatus (R. appendiculatus), under experimental conditions, detection of T parva in ticks and cattle was performed by a quantitative real-time PCR assay. A calf inoculated with a T parva mixture became PCR-positive for T parva infection on day 8 post-inoculation, and subsequently, nymphal ticks were introduced and maintained to feed on the infected calf for 6 days. Engorged nymphs were collected daily and allowed to molt into adults, and overall, 70.8% (121/171) of the adult ticks acquired the T parva infection. Furthermore, the T parva infection rate in ticks under field conditions was monitored by real-time PCR in R. appendiculatus ticks collected from a traditionally managed pastoral land of Zambia, on which Sanga breed cattle are traditionally reared and the area has endemic East Coast fever (ECF). A total of 70 cattle were randomly selected in the same area and 67 (95.7%) were found to be serologically positive for R. appendiculatus tick antigen (RIM36). Twenty-nine (43.3%) of the 67 serologically positive cattle were real-time PCR-positive for T parva, although no piroplasms could be detected in the blood smears. Unexpectedly, out of 614 R. appendiculatus nymphal and adult ticks collected by flagging vegetation, 4.1% were positive for T parva DNA. However, since the rate of transmission of T parva from infected cattle to ticks and vice versa and the serological evidence of exposure to R. appendiculatus ticks in naturally exposed cattle were relatively high, it would be wise in such a case to consider vector control as well as vaccination against ECF as control measures. (c) 2006 Elsevier B.V. All rights reserved.
  • C Nakajima, S Imamura, S Konnai, S Yamada, H Nishikado, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 5 447 - 452 2006年05月 [査読有り][通常論文]
     
    A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein. designated chimadanin. Which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.
  • C Nakajima, ID Vaz, S Imamura, S Konnai, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 67 11 1127 - 1131 2005年11月 [査読有り][通常論文]
     
    A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.
  • HM Pham, C Nakajima, K Ohashi, M Onuma
    JOURNAL OF CLINICAL MICROBIOLOGY 43 4 1646 - 1650 2005年04月 [査読有り][通常論文]
     
    We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.
  • ID Vaz, S Imamura, C Nakajima, FC de Cardoso, CAS Ferreira, G Renard, A Masuda, K Ohashi, N Onuma
    VETERINARY PARASITOLOGY 127 2 147 - 155 2005年01月 [査読有り][通常論文]
     
    The nucleotide and deduced amino acid sequences of the actins from ticks, Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus, have been determined. Nucleotide sequence analysis showed open reading frames of 1128-nucleotide-long encoding proteins of 376 amino acids with a predicted molecular weight of 41.82 kDa each. Comparison between the nucleic acid and deduced amino acid sequences as well as structural and phylogenetic analyses of thew genes confirmed the high similarity among actins from ticks in comparison to other species. (C) 2004 Elsevier B.V. All rights reserved.
  • T Sakogawa, S Kataoka, A Okayama, C Nakajima, T Hayama
    APIDOLOGIE 30 1 75 - 75 1999年01月 [査読有り][通常論文]
  • C Nakajima, T Sakogawa, A Okayama, A Nakamura, T Hayama
    Journal of veterinary pharmacology and therapeutics 21 4 269 - 73 1998年08月 [査読有り][通常論文]
     
    Disposition of mirosamicin, a macrolide antibiotic, to honeybee adults, larvae, honey and royal jelly in the beehive after in-feed administration to adult bees was studied. Treatment was initiated at the end of July when the availability of natural pollen and nectar was poor. The drug was mixed with pollen-substitute paste and administered to honeybee colonies continuously for a week at a dosage of 200 mg/hive/week. High distributions in adult bees, jelly, larvae and a relatively low distribution in honey, of mirosamicin were observed. One day dosing of microsamin in sucrose syrup, a nectar substitute, resulted in a very high and long lasting residue in honey. Both royal and worker jelly, secreted from the jelly glands of adult bees, are acidic, so that a high distribution of a basic drug, such as mirosamicin, in jelly can be expected. This mechanism was considered to be responsible for a high concentration of mirosamicin in honeybee larvae, the host of Paenibacillus-larvae infection (American foulbrood), as primary larval food is jelly.

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    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 [査読無し][通常論文]
  • 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 2020年01月 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
     
    © 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.
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 今内覚, 岡川朋弘, 前川直也, 中島千絵, 中島千絵, 鈴木定彦, 鈴木定彦, 山本啓一, 山本啓一, 戸田幹洋, 戸田幹洋, 村田史郎, 大橋和彦 臨床獣医 37 (3) 37‐41 2019年 [査読無し][通常論文]
  • 中島千絵 動物用抗菌剤研究会報 (40) 6‐7 2018年12月25日 [査読無し][通常論文]
  • 【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 2018年11月 [査読無し][通常論文]
     
    <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.
  • 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 [査読無し][招待有り]
  • 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 2018年03月09日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
     
    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.
  • 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 [査読無し][通常論文]
  • 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 2017年03月25日 [査読無し][通常論文]
  • 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 2017年03月17日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 中島千絵, 鈴木定彦 獣医疫学雑誌 20 (2) 101‐104 2016年12月20日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 鈴木定彦, 中島千絵 臨床と微生物 43 (2) 169‐173 2016年03月25日 [査読無し][通常論文]
  • BCG thyXを用いた抗酸菌のPAS耐性機序の解析
    大原 直也, 阿戸 学, 鈴木 定彦, 小林 和夫, 有村 友紀, 中山 真彰, 中島 千絵, 妹尾 昌紀, 田川 淳平, 中山 浩次 結核 91 (3) 325 -325 2016年03月 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 鈴木 定彦, 中島 千絵 生体の科学 66 (4) 335 -338 2015年07月 [査読無し][通常論文]
  • 山口智之, 中島千絵, 鈴木定彦 Jpn J Lepr 84 (1) 26 2015年05月25日 [査読無し][通常論文]
  • MENDA Conscillioah, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 191 2015年02月25日 [査読無し][通常論文]
  • THAPA Jeewan, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 200 2015年02月25日 [査読無し][通常論文]
  • CHANGKAEW Kanjana, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 190 2015年02月25日 [査読無し][通常論文]
  • SHAH Yogendra, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 208 2015年02月25日 [査読無し][通常論文]
  • CHANGKWANYEUN Ruchirada, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 190 2015年02月25日 [査読無し][通常論文]
  • KONGSOI Siriporn, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 190 2015年02月25日 [査読無し][通常論文]
  • YAMAGUCHI Tomoyuki, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 191 2015年02月25日 [査読無し][通常論文]
  • VILLANUEVA Marvin, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 70 (1) 208 2015年02月25日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 山口智之, 中島千絵, 鈴木定彦 Jpn J Lepr 83 (2) 47 2014年07月25日 [査読無し][通常論文]
  • DIAB Hassan, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 69 (1) 144 2014年02月25日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 69 (1) 141 2014年02月25日 [査読無し][通常論文]
  • CHANGKAEW Kanjana, SUZUKI Yasuhiko, NAKAJIMA Chie 日本細菌学雑誌 69 (1) 201 2014年02月25日 [査読無し][通常論文]
  • THAPA Jeewan, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 69 (1) 141 2014年02月25日 [査読無し][通常論文]
  • KONGSOI Siriporn, SUZUKI Yasuhiko, NAKAJIMA Chie 日本細菌学雑誌 69 (1) 196 2014年02月25日 [査読無し][通常論文]
  • CHANGKWANYEUN Ruchirada, KIM Hyun, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 69 (1) 196 2014年02月25日 [査読無し][通常論文]
  • VILLANUEVA Marvin, KOIZUMI Nobuo, SUZUKI Yasuhiko, NAKAJIMA Chie 日本細菌学雑誌 69 (1) 240 2014年02月25日 [査読無し][通常論文]
  • SUZUKI Yasuhiko, NAKAJIMA Chie 日本細菌学雑誌 69 (1) 199 2014年02月25日 [査読無し][通常論文]
  • 松葉隆司, 松葉隆司, 中島千絵, 中島千絵, 鈴木定彦, 鈴木定彦 日本細菌学雑誌 69 (1) 202 2014年02月25日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
     
    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.
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 2013年05月 [査読無し][通常論文]
     
    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.
  • 金玄, 横山和正, 中島千絵, 鈴木定彦 Jpn J Lepr 82 (1/2) 22 2013年04月01日 [査読無し][通常論文]
  • 金玄, 横山和正, 中島千絵, 鈴木定彦 Jpn J Lepr 82 (1/2) 24 2013年04月01日 [査読無し][通常論文]
  • NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 68 (1) 217 2013年02月25日 [査読無し][通常論文]
  • CHANGKWANYEUN Ruchirada, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 68 (1) 191 2013年02月25日 [査読無し][通常論文]
  • SUZUKI Yasuhiko, KIM Hyun, MUKAI Tetsu, NAKAJIMA Chie 日本細菌学雑誌 68 (1) 191 2013年02月25日 [査読無し][通常論文]
  • CHANGKAEW Kanjana, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 68 (1) 195 2013年02月25日 [査読無し][通常論文]
  • KONGSOI Siriporn, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 68 (1) 190 2013年02月25日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 2013年 [査読無し][通常論文]
     
    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.
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 2012年07月 [査読無し][通常論文]
     
    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.
  • Ajay Poudel, Chie Nakajima, Yukari Fukushima, Haruka Suzuki, Basu Dev Pandey, Bhagwan Maharjan, Yasuhiko Suzuki ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 56 (6) 2831 -2836 2012年06月 [査読無し][通常論文]
     
    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.
  • 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 2012年06月 [査読無し][通常論文]
     
    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.
  • 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 2012年06月 [査読無し][通常論文]
     
    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.
  • 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 2012年05月 [査読無し][通常論文]
     
    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.
  • 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 2012年04月 [査読無し][通常論文]
     
    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.
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • KOIZUMI Nobuo, OHNISHI Makoto, NAKAJIMA Chie, SUZUKI Yasuhiko 日本細菌学雑誌 67 (1) 126 2012年02月25日 [査読無し][通常論文]
  • Kazumasa Yokoyama, Hyun Kim, Tetsu Mukai, Masanori Matsuoka, Chie Nakajima, Yasuhiko Suzuki ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 56 (2) 697 -702 2012年02月 [査読無し][通常論文]
     
    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.
  • Hyun Kim, Chie Nakajima, Youn Uck Kim, Kazumasa Yokoyama, Yasuhiko Suzuki JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 (1) 72 -74 2012年01月 [査読無し][通常論文]
     
    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.
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 鈴木 定彦, 松葉 隆司, 中島 千絵 最新医学 66 (12) 2689 -2696 2011年12月 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • Hyun Kim, Chie Nakajima, Kazumasa Yokoyama, Zeaur Rahim, Youn Uck Kim, Hiroki Oguri, Yasuhiko Suzuki ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 (8) 3661 -3667 2011年08月 [査読無し][通常論文]
     
    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.
  • 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 [査読無し][通常論文]
  • 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 2011年04月 [査読無し][通常論文]
     
    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.
  • 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 [査読無し][通常論文]
  • 中島千絵, 鈴木定彦 結核 86 (3) 343 2011年03月15日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 金 玄, 鈴木 晴香, 松岡 正典, 松葉 隆司, 横山 和正, 中島 千絵, 鈴木 定彦 日本ハンセン病学会雑誌 = Japanese journal of leprosy 80 (1) 17 -27 2011年 [査読無し][通常論文]
     
    ハンセン病においてニューキノロン薬は、単一病巣例治療の重要な選択薬剤となっている。一方、増加傾向を見せ大きな問題となっている多剤耐性結核菌に対する有効な治療薬としても本薬剤は注目を集めている。近年になって、ニューキノロン薬に対して耐性を示すらい菌や結核菌が頻繁に報告されるようになり、耐性獲得機構の解明が急務となっている。本総説では、ニューキノロン薬に関して概説し、その作用機序とらい菌および結核菌に於けるニューキノロン薬耐性獲得機構について解説するとともに迅速なニューキノロン薬耐性菌鑑別法についても紹介した。
  • 松葉 隆司, 中島 千絵, 鈴木 定彦 日本細菌学雑誌 65 (3) 355 -368 2010年12月24日 [査読無し][通常論文]
     
    細菌は属や種による莢膜やべん毛の有無などを含む詳細な構造の違いはあるが,菌体表層を覆うエンベロープ構造の違いに由来する染色性を指標に,グラム陰性菌と陽性菌に大別されてきた。両者の構造上の大きな違いは,グラム陰性菌には外膜と内膜(細胞質膜),ペリプラズム間隙およびリポ多糖体(LPS)があり,グラム陽性菌には外膜,ペリプラズム間隙やLPSがなくペプチドグリカン量が多いこととされている。しかしながら,グラム染色性により分類しにくい結核菌を含むマイコバクテリウム属細菌については,グラム陰性菌と類似したエンベロープ構造を有する可能性が長年指摘されてきた。近年,電子顕微鏡装置や試料作製技術の進歩と化学構造解析研究の発展に伴い,より詳細な結核菌のエンベロープ構造が明らかとされている。一方で,分子生物学的手法を用いて結核菌エンベロープ各成分の生合成経路,機能や免疫生物活性に関する知見も積み重ねられてきている。本稿では,結核菌エンベロープ構造や成分について最近の知見を中心に,著者らの成績も含めて概説したい。
  • Masanori Matsuoka, Yasuhiko Suzuki, Iris Estrada Garcia, Mary Fafutis-Morris, Alberto Vargas-Gonzalez, Cristina Carreno-Martinez, Yukari Fukushima, Chie Nakajima JAPANESE JOURNAL OF INFECTIOUS DISEASES 63 (6) 412 -416 2010年11月 [査読無し][通常論文]
     
    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
  • 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 2010年08月 [査読無し][通常論文]
     
    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.
  • Tadashi Fukasawa, Naozumi Oda, Yasunao Wada, Aki Tamaru, Yukari Fukushima, Chie Nakajima, Yasuhiko Suzuki JAPANESE JOURNAL OF INFECTIOUS DISEASES 63 (4) 246 -250 2010年07月 [査読無し][通常論文]
     
    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.
  • 鈴木 定彦, 松葉 隆司, 中島 千絵 結核 85 (2) 79 -86 2010年02月15日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 中島千絵, 鈴木定彦 感染症学雑誌 83 (5) 631 2009年09月20日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 中島千絵, 鈴木定彦 感染症学雑誌 82 (6) 723-724 2008年11月20日 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • Saiki Imamura, Satoru Konnai, Itabajara da Silva Vaz Junior, Shinji Yamada, Chie Nakajima, Yuko Ito, Tomoko Tajima, Jun Yasuda, Martin Simuunza, Misao Onuma, Kazuhiko Ohashi JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (2) 85 -98 2008年08月 [査読無し][通常論文]
     
    Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36-kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM36 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8 % for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8 % in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T parva-infected ticks fed on immunized cattle, the occurrence of T parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.
  • 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 [査読無し][通常論文]
  • 岡本晋, 田丸亜貴, 中島千絵, 西村賢治, 西村賢治, 田中幸徳, 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 [査読無し][通常論文]
  • 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 [査読無し][通常論文]
  • 中島千絵, 小沼操 獣医畜産新報 (1022) 735 -742 2006年09月01日 [査読無し][通常論文]
  • 中島 千絵, 小沼 操 獣医畜産新報 59 (9) 735 -742 2006年09月 [査読無し][通常論文]
  • 中島 千絵, 今村 彩貴, 今内 覚, 山田 慎二, 西門 秀人, 大橋 和彦, 小沼 操 The journal of veterinary medical science 68 (5) 447 -452 2006年05月25日 [査読無し][通常論文]
     
    吸血中のフタトゲチマダニ(Haemaphysalis longicornis)の唾液腺に由来するcDNAライブラリーより,トロンビン阻害作用を持つ蛋白質をコードする遺伝子を同定した.この蛋白質(chimadaninと命名)は93アミノ酸残基より成り,分泌シグナルを持つことから分泌蛋白質であることが推測された.相同性検索において既知の蛋白質あるいは保存ドメイン配列データベースより類似のものは検出されず,新規な蛋白質配列であると判断された.また,ダニの吸血段階及び臓器別に遺伝子の発現を調べたところ,吸血開始後の主として唾液腺において発現することが示唆された.推定成熟ペプチド部の塩基配列を大腸菌に組み込んで発現・精製し,リコンビナント蛋白質の機能を調べたところ,血液凝固系の検査である活性化部分トロンボプラスチン時間及びプロトロンビン時間の両試験において,濃度依存的に明らかな凝固時間延長作用を示した.また,トロンビンによるペプチド基質の分解を濃度依存的に抑制したため,トロンビンの酵素活性阻害作用を持った血液凝固抑制蛋白質であることが示唆された.この蛋白質は,ダニの吸血において重要な働きをしている可能性があると考えられる.
  • C Nakajima, S Imamura, S Konnai, S Yamada, H Nishikado, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 68 (5) 447 -452 2006年05月 [査読無し][通常論文]
     
    A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein. designated chimadanin. Which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.
  • 中島 千絵, da Silva Vaz Jr. Itabajara, 今村 彩貴, 今内 覚, 大橋 和彦, 小沼 操 The journal of veterinary medical science 67 (11) 1127 -1131 2005年11月25日 [査読無し][通常論文]
     
    吸血中のフタトゲチマダニ(Haemaphysalis longicornis)より唾液腺を採取してcDNAライブラリーを作成し, ランダムに選んだ発現タンパク遺伝子の配列をblastx (NCBI)等のプログラムを用いて解析した.得られた633配列はその相同性によって, 同一あるいはごく近縁の遺伝子由来と思われる213配列に集約された.全633配列のうち, blastxを用いた相同性検索により発現タンパクの機能予測がなされたものは全体の36%であり, 残りの64%は未知の遺伝子であった.機能が予測されたタンパクの大半は細胞の生存に必要なハウスキーピングタンパクであったが, 血液凝固阻害因子類似の蛋白を含めたプロテアーゼインヒビターやメタロプロテアーゼ, 他のダニで分離されている免疫抑制物質に類似したタンパク遺伝子等も同定された.これらのタンパクはダニの吸血に際し何らかの重要な役割を演じている可能性があり, 新たな抗ダニワクチン抗原の候補となりうると考えられる.
  • C Nakajima, ID Vaz, S Imamura, S Konnai, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (11) 1127 -1131 2005年11月 [査読無し][通常論文]
     
    A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.
  • 今村彩貴, BONIFACE Mamangala, 今内覚, 中島千絵, MWASE Tembo, 田島朋子, 安田準, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 140th 84 2005年08月31日 [査読無し][通常論文]
  • 伊藤裕子, 今内覚, 今村彩貴, 中島千絵, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 140th 79 2005年08月31日 [査読無し][通常論文]
  • 今村彩貴, 今内覚, 中島千絵, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 140th 78 2005年08月31日 [査読無し][通常論文]
  • 今内覚, 今村彩貴, 中島千絵, SIMUNZA Martin, AMOS Chota, CHEMBENSOFU Mwelwa, ANDOREU Nambota, 安田準, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 140th 84 2005年08月31日 [査読無し][通常論文]
  • 小沼操, 今村彩貴, 中島千絵 獣医畜産新報 (998) 753-757 2004年09月01日 [査読無し][通常論文]
  • 中島千絵, 今村彩貴, 今内覚, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 138th 65 2004年08月01日 [査読無し][通常論文]
  • 小沼操, 今村彩貴, 中島千絵 日本獣医学会学術集会講演要旨集 137th 82 2004年03月01日 [査読無し][通常論文]
  • 迫川朋子, 片岡重明, 片岡敦子, 中島千絵, 吐山豊秋 ミツバチ科学 19 (4) 145-148 1998年10月 [査読無し][通常論文]
  • 中島千絵 日本獣医学会学術集会講演要旨集 126th 21 1998年08月 [査読無し][通常論文]
  • 岡山 敦子, 追川 朋子, 中島 千絵, 吐山 豊秋 The journal of veterinary medical science 59 (10) 953 -954 1997年10月25日 [査読無し][通常論文]
     
    アメリカ腐蛆病菌野外分離株を用い, その湿性芽胞および乾燥芽胞に対する各種消毒剤の殺菌効果について検討した. 使用消毒剤は, グルタルアルデヒド, 次亜塩素酸ナトリウム, ポビドンヨード, エチレンオキサイドガス, グルコン酸クロルヘキシジンおよび塩化ジデシルジメチルアンモニウムとした. その中でグルタルアルデヒドの効果が最も強力かつ速効性であった.
  • 中島 千絵, 岡山 敦子, 迫川 朋子, 中村 晃, 吐山 豊秋 The journal of veterinary medical science 59 (9) 765 -767 1997年09月25日 [査読無し][通常論文]
     
    アンピシリン (ABPC) はアメリカ腐蛆病 (AFB) の原因菌であるPaenibacillus larvaeに対するin vitroにおける抗菌活性が非常に高い薬物である. そこで, 実際に蜜蜂の群に投与してその巣箱内における動態を観察し, 防除薬としての効果を評価した. 薬は蔗糖液あるいは花粉代替え飼料 (ペースト) に30mg/群の割合で添加し, 1日間投与した. 蔗糖液への添加投与ではハチミツ中への移行が大きく, 投与後14日まで残留検出限界 (0.01ppm) を上回った濃度が持続した. ペーストへの添加投与ではハチミツへの移行は小さく投与後14日には残留検出限界以下となったが, AFBの標的である若齢幼虫やその餌であるゼリー中への移行も小さかった. これはABPCの体内移行性が乏しく, 酸性物質であるためにゼリー中に分泌されにくいこと, またゼリー中で不活化されることによるものと思われた. 腐蛆病菌に感染感受性をもつのは孵化後2日目までの若齢幼虫であり, この間の幼虫は成虫が分泌するゼリーしか摂食しない. 従って, この時期の幼虫へ薬を効果的に投与するためには, 成虫に摂取された薬がゼリー中に分泌され, しかも安定であることが重要である. 以上より, ABPCの腐蛆病に対する防除効果は小さいと考えられた.
  • 吐山 豊秋, 岡山 敦子, 中島 千絵, 迫川 朋子, 片岡 重明, 中村 晃 日本獣医師会雑誌 = Journal of the Japan Veterinary Medical Association 50 (8) 429 -437 1997年08月20日 [査読無し][通常論文]
  • 吐山豊秋, 岡山敦子, 中島千絵, 迫川朋子, 片岡重明, 中村晃 日本獣医師会雑誌 50 (8) 429-437 1997年08月 [査読無し][通常論文]
  • 中島千絵, 岡山敦子, 迫川朋子, 中村晃, 吐山豊秋 日本獣医学会学術集会講演要旨集 123rd 72 1997年03月 [査読無し][通常論文]
  • 迫川朋子, 片岡重明, 岡山敦子, 中島千絵, 吐山豊秋 日本獣医学会学術集会講演要旨集 123rd 169 1997年03月 [査読無し][通常論文]
  • 大口 克志, 高原 ひろみ, 松本 真里子, 中島 千絵, 城戸 靖雅 食品衛生学雑誌 37 (5) 319 -324 1996年10月05日 [査読無し][通常論文]
     
    スペクチノマイシン (SPCM) のp-ニトロフェニルヒドラジン (p-NPH) プレカラム誘導体化HPLCによる残留分析法を検討した. 試料 (筋肉, 脂肪, 肝臓, 腎臓, 小腸, 鶏皮膚, 血漿) をEDTA-2Na飽和10%トリクロロ酢酸で抽出し, Sep-pak tC18カートリッジでクリーンアップした後, SPCMをp-NPHで誘導体化し, HPLC (カラム: TSK-gel ODS 80TM, 移動相: MeCN-水-酢酸 (73:20:0.5, v/v/v), UV (420nm)) により分析した. 鶏及び豚組織への添加回収率は, それぞれ73~97%及び75~87%, 検出限界は0.05μg (力価)/gであった. ブロイラー及び豚にSPCMを投与し, 開発した分析法により臓器・組織中のSPCM残留の経時的推移を調査した.
  • 岡山敦子, 迫川朋子, 中島千絵, 吐山豊秋 日本獣医学会学術集会講演要旨集 122nd 197 1996年08月 [査読無し][通常論文]
  • 岡山 敦子, 迫川 朋子, 中島 千絵, 吐山 豊秋 The journal of veterinary medical science 58 (5) 439 -441 1996年05月25日 [査読無し][通常論文]
     
    1987年4月から1994年10月にかけて, 日本国内のアメリカ腐蛆病発症蜂群より分離した28株および性状検査用対照株として用いたATCC基準株1株の合計29株のBacillus larvaeについて生物性状を調べ, ほぼ基準株と一致する結果を得た. また, 各種抗菌剤の最小発育阻止濃度を測定したところ, B.larvaeはペニシリン系のアンピシリン, マクロライド系のミロサマイシンおよびリンコサミド系のリンコマイシンに対して高い感受性を示した.
  • 中村 晃, 杉本 忠美, 岡山 敦子, 迫川 朋子, 中島 千絵 ミツバチ科学 17 (2) 61 -66 1996年04月20日 [査読無し][通常論文]
  • 中島千絵, 岡山敦子, 迫川朋子, 中村晃, 吐山豊秋 日本獣医学会学術集会講演要旨集 121st 65 1996年03月 [査読無し][通常論文]
  • 迫川朋子, 岡山敦子, 中島千絵, 片岡重明, 吐山豊秋 日本獣医学会学術集会講演要旨集 121st 148 1996年03月 [査読無し][通常論文]
  • 岡山敦子, 迫川朋子, 中島千絵, 吐山豊秋 日本獣医学会学術集会講演要旨集 120th 102 1995年09月 [査読無し][通常論文]
  • 岡山敦子, 山本譲, 長沢朋子, 中島千絵, 吐山豊秋 日本獣医学会学術集会講演要旨集 119th 128 1995年03月 [査読無し][通常論文]
  • 伊東正吾, 曽根勝, 鈴木邦夫, 和泉屋公一, 望月洋, 筒井敏彦, 中島千絵, 小笠晃, 中原達夫 Anim Sci Technol 65 (9) 834-841 1994年09月 [査読無し][通常論文]
  • 福田苗美, 小田憲司, 西田由美, 芳賀知也子, 中島千絵, 長沢朋子, 氏政雄揮, 片江宏巳, 吐山豊秋 日本獣医学会学術集会講演要旨集 118th 73 1994年08月 [査読無し][通常論文]
  • 小田憲司, 福田苗美, 西田由美, 芳賀知也子, 中島千絵, 長沢朋子, 氏政雄揮, 片江宏巳, 吐山豊秋 日本獣医学会学術集会講演要旨集 117th 87 1994年03月 [査読無し][通常論文]
  • 高原ひろみ, 岡田真里子, 大口克志, 中島千絵, 城戸靖雅 日本食品衛生学会学術講演会講演要旨集 65th 63 1993年 [査読無し][通常論文]
  • 中島千絵, 吐山豊秋 家畜診療 (319) 43-50 1990年01月 [査読無し][通常論文]

特許

共同研究・競争的資金等の研究課題

  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2015年04月 -2019年03月 
    代表者 : 和田 崇之, 柳井 徳磨, 吉田 志緒美, 鈴木 定彦, 中島 千絵, 斑目 広郎, 今城 雅之
     
    抗酸菌属は多様な自然環境に生存する一方,病原菌としての側面を持ち,ヒトを含めた様々な動物に感染して病変を起こす.本課題では,国内外における野生動物・捕獲動物に関して抗酸菌症の調査研究を行い,原因菌のゲノム解読に基づく遺伝子解析を行った.国際連携としては,台湾との連携の下,新規Mycobacterium marinum検出法を確立し,飼育動物例における菌種同定を行った.飼育動物,家畜動物,伴侶動物の症例において,それぞれ異なる抗酸菌種の分離培養と比較ゲノム解析を行い,個々の菌種および分離株における遺伝的多様性について,新たな知見を蓄積することに成功した.
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 中島 千絵, 鈴木 定彦
     
    結核菌の多剤耐性化は、薬剤の作用分子をコードする遺伝子上に順次変異が蓄積されて行くことによって生じるが、特定の菌株でより耐性化や耐性菌による二次感染が起こりやすいことが知られている。結核高蔓延国であるミャンマー、ネパール、タイにおいて得られた多剤耐性結核菌株の遺伝子解析を行ったところ、北京型のモダンタイプと呼ばれる系統の割合が薬剤耐性の度合いと共に上昇し、また、イソニアジド耐性に係るKatG315の変異等、特定の遺伝子変異を持つ株が蔓延している傾向にあった。これらの特徴は感染性の高い多剤耐性結核菌を早期に特定するためのマーカーとして使用可能であると考えられ、簡易検出用のPCR法の開発に至った。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2012年04月 -2016年03月 
    代表者 : 鈴木 定彦, 中島 千絵
     
    多剤、並びに超多剤耐性結核の調査が不十分と考えられるネパール、バングラデシュ、ミャンマー、タイおよびフィリピンにおいて結核あるいは結核様症状を呈したヒトより総計2,208株の抗酸菌を分離して当該国において菌株バンクとして保存した。さらに、これらの菌株からDNAを抽出して、当該国、並びに日本においてDNAバンクとして保存した。 日本に輸入したDNAを対象として遺伝子型を決定するとともに薬剤耐性関連遺伝子変異の解析を実施した結果、ネパールおよびタイにおいては、多剤、並びに超多剤耐性結核の多くがBeijing型結核菌により引き起こされ、急速な伝播動態を示している事が明らかとなった。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 中島 千絵, 鈴木 定彦
     
    広い宿主域を持つと考えられる新たな結核菌群菌Mycobacterium orygisを、バングラデシュ及びネパールの複数の動物種の肺結核病変から分離した。その遺伝学的解析により、この飛沫感染を起こすと考えられる結核菌群菌が、これらの国々の野生動物の間で保持されている可能性が示唆された。また、ネパールにおいてゾウから分離されたヒト型結核菌の遺伝子解析を行ったところ、トランスポゾンの活性化によると考えられるゲノムの小規模な構造変化が観察された。これらの異種動物感染例から得られた菌株の遺伝学的解析により、結核菌群菌の宿主指向性に関与するゲノム上の領域が同定できる可能性がある。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2009年 -2011年 
    代表者 : 鈴木 定彦, 中島 千絵, 松葉 隆司
     
    多剤および超多剤耐性結核の調査が不十分と考えられるアジアの国々のうち、特にミャンマー、バングラデシュならびにネパールに焦点を絞り、臨床分離結核菌株を収集し、リファンピシンならびにイソニアジド耐性と遺伝子変異の関係を調査した。ミャンマーの多剤耐性結核菌のrpoB遺伝子変異ならびkatG又はinhA遺伝子変異保有割合はそれぞれ71.3ならびに72.5%であった。一方、バングラでシュにおいてはrpoB遺伝子変異ならびkatG又はinhA遺伝子変異保有割合はそれぞれ95.0ならびに94.5%であり、ネパールにおいてはそれぞれ97.2ならびに93.8%であった。また、バングラデシュにおいて多剤耐性結核菌218株中7株、ネパールにおいては多剤耐性結核菌109株中13株の超多剤耐性結核菌を見出した。これらの超多剤耐性結核菌全てにおいてカナマイシン耐性rrsならびにgyrAまたはgyrB遺伝子上に変異が見られた。薬剤耐性と遺伝子変異の相関は国ごとに異なるものであり、遺伝子変異分析を基盤とする結核菌薬剤感受性試験を実施するにあたっては事前にその相関を調査する必要があるものと結論された。また、今回の調査により超多剤耐性結核菌の存在がバングラデシュならびにネパールおいて明らかになったことから、これらの伝播動態調査が急務と考えられた。

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