研究活動情報

• 2023年, The 88th Annual Meeting of the Hokkaido Branch, the Japanese Society for Bacteriology, Outstanding performance award, Oral presentation
• 2017年, 13rd International Conference of Biology and Agriculture Innovation, Mongolian Young Scientist Association, Excellent Young Scientist, Oral Presentation
• 2011年, Oyu-Tolgoi and Rio-Tinto, Excellent Student Scholarship

• In silico characterization of chromosomally integrated blaCTX-M genes among clinical Enterobacteriaceae in Africa: insights from whole-genome analysis
  Misheck Shawa; Herman Chambaro; Harvey K. Kamboyi; Clement Sulwe; Joseph Y. Chizimu; Situmbeko J. Nasilele; Shohei Ogata; Mulemba Samutela; Tuvshinzaya Zorigt; Steward Mudenda; Manyando Simbotwe; Mwamba Nsofwa; Jedidiah Chanda; Freeman Chabala; Mike Nundwe; Joseph Ndebe; Msangwa Sinjani; Kyoko Hayashida; Naganori Nao; Roma Chilengi; Hirofumi Sawa; Yasuhiko Suzuki; Bernard Hang'ombe; Masahiro Kajihara; Hideaki Higashi
  Frontiers in Microbiology, 16, Frontiers Media SA, 2025年09月12日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Antimicrobial resistance (AMR) mediated by extended-spectrum β-lactamases (ESBLs) is a growing global concern, particularly among Enterobacteriaceae. The CTX-M-type ESBLs, encoded by the bla_CTX-M gene, are of significant public health importance due to their high prevalence and broad geographic distribution. Typically located on plasmids and often co-occurring with other AMR genes, bla_CTX-M contributes to multidrug resistance (MDR). However, increasing evidence suggests secondary chromosomal integration of bla_CTX-M, sometimes alongside other resistance determinants. The extent and implications of this mechanism remain poorly characterized, especially in Africa, where genomic surveillance is limited. In this study, we retrieved 295 chromosomal sequences of Enterobacteriaceae of African origin from the GenBank and performed in silico predictions of bla_CTX-M and other AMR genes. bla_CTX-M-carrying sequences were further characterized by in silico multilocus sequence typing and genome annotation. Chromosomal insertions were identified through alignment with reference genomes. Overall, 47 of 295 sequences (15.9%) harbored the bla_CTX-M gene, with the highest prevalence in Klebsiella pneumoniae (29/157, 18.5%), followed by Escherichia coli (13/72, 18.1%), Enterobacter spp. (4/38, 10.5%), and Shigella spp. (1/12, 8.3%). The most common allele was bla_CTX-M-15 (31/47, 66.0%), followed by bla_CTX-M-14 (12/47, 25.5%), bla_CTX-M-55 (3/47, 6.4%), and bla_CTX-M-27 (1/27, 3.7%). Co-occurrence of bla_CTX-M with additional AMR genes was frequently observed, with integration events often associated with mobile genetic elements such as ISEcp1 and IS26. Notably, strains from the same hospital setting were phylogenetically related and shared sequence types and AMR gene profiles, suggesting local clonal dissemination. These findings reveal a notable presence of chromosomally integrated bla_CTX-M among African Enterobacteriaceae, frequently in association with other resistance genes, thereby facilitating stable MDR propagation independent of plasmid maintenance. This evolutionary adaptation may have significant implications for the persistence and spread of MDR in clinical settings.
• Development of a PCR-dipstick DNA chromatography-based tool for the detection of CTX-M- and TEM-producing Escherichia coli and Klebsiella pneumoniae isolated from patients in Kafue and Katete districts of Zambia
  Misheck Shawa; Kyoko Hayashida; Naganori Nao; Atmika Paudel; Harvey Kamboyi; Herman Chambaro; Malala Mulavu; Cynthia Sipho Khumalo; Mike Nundwe; Tuvshinzaya Zorigt; Chinatsu Nakamura; Yongjin Qiu; Naoko Kawai; Maisa Kasanga; Joseph Chizimu; Lavel Moonga; Joseph Ndebe; Manyando Simbotwe; Shohei Ogata; Mulemba Samutela; Chie Nakajima; Roma Chilengi; Mable Mutengo; Masahiro Kajihara; Hirofumi Sawa; Bernard Hang’ombe; Yasuhiko Suzuki; Hideaki Higashi
  BMC Infectious Diseases, 25, 1, Springer Science and Business Media LLC, 2025年04月16日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）
• EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579
  Harvey K. Kamboyi; Atmika Paudel; Misheck Shawa; Misa Sugawara; Tuvshinzaya Zorigt; Joseph Y. Chizimu; Tomoe Kitao; Yoshikazu Furuta; Bernard M. Hang’ombe; Musso Munyeme; Hideaki Higashi
  BMC Microbiology, 24, 1, Springer Science and Business Media LLC, 2024年09月17日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）
• Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis
  Tuvshinzaya Zorigt; Yoshikazu Furuta; Atmika Paudel; Harvey Kakoma Kamboyi; Misheck Shawa; Mungunsar Chuluun; Misa Sugawara; Nyamdorj Enkhtsetseg; Jargalsaikhan Enkhtuya; Badgar Battsetseg; Musso Munyeme; Bernard M. Hang’ombe; Hideaki Higashi
  BMC Infectious Diseases, 24, 1, Springer Science and Business Media LLC, 2024年09月09日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）
• Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015–2020 in a national referral hospital of Zambia
  Misheck Shawa; Atmika Paudel; Herman Chambaro; Harvey Kamboyi; Ruth Nakazwe; Luke Alutuli; Tuvshinzaya Zorigt; Taona Sinyawa; Mulemba Samutela; Joseph Chizimu; Manyando Simbotwe; Kyoko Hayashida; Naganori Nao; Masahiro Kajihara; Yoshikazu Furuta; Yasuhiko Suzuki; Hirofumi Sawa; Bernard Hang’ombe; Hideaki Higashi
  PLOS ONE, 19, 4, e0302053, e0302053, Public Library of Science (PLoS), 2024年04月16日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11–0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.
• Complete Genome Sequences of 24 Strains of Bacillus cereus Isolated from Nosocomial Infection and Bacteremia Cases in Japan
  Yoshikazu Furuta; Mai Tsujinouchi; Misheck Shawa; Tuvshinzaya Zorigt; Yuya Miyajima; Atmika Paudel; Satowa Suzuki; Hideaki Higashi
  Microbiology Resource Announcements, 11, 4, American Society for Microbiology, 2022年04月21日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Bacillus cereus is mainly associated with foodborne illness but sometimes causes nosocomial infections. We previously reported that B. cereus strains of a specific sequence type, ST1420, were associated with nosocomial infection. Here, we determined the complete genome sequences of B. cereus strains isolated from nosocomial infection cases in Japanese hospitals.
• A GCDGC-specific DNA (cytosine-5) methyltransferase that methylates the GCWGC sequence on both strands and the GCSGC sequence on one strand
  Yoshikazu Furuta; Fumihito Miura; Takahiro Ichise; Shouta M. M. Nakayama; Yoshinori Ikenaka; Tuvshinzaya Zorigt; Mai Tsujinouchi; Mayumi Ishizuka; Takashi Ito; Hideaki Higashi
  PLOS ONE, 17, 3, e0265225, e0265225, Public Library of Science (PLoS), 2022年03月21日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, 5-Methylcytosine is one of the major epigenetic marks of DNA in living organisms. Some bacterial species possess DNA methyltransferases that modify cytosines on both strands to produce fully-methylated sites or on either strand to produce hemi-methylated sites. In this study, we characterized a DNA methyltransferase that produces two sequences with different methylation patterns: one methylated on both strands and another on one strand. M.BatI is the orphan DNA methyltransferase of Bacillus anthracis coded in one of the prophages on the chromosome. Analysis of M.BatI modified DNA by bisulfite sequencing revealed that the enzyme methylates the first cytosine in sequences of 5ʹ-GCAGC-3ʹ, 5ʹ-GCTGC-3ʹ, and 5ʹ-GCGGC-3ʹ, but not of 5ʹ-GCCGC-3ʹ. This resulted in the production of fully-methylated 5ʹ-GCWGC-3ʹ and hemi-methylated 5ʹ-GCSGC-3ʹ. M.BatI also showed toxicity when expressed in E. coli, which was caused by a mechanism other than DNA modification activity. Homologs of M.BatI were found in other Bacillus species on different prophage like regions, suggesting the spread of the gene by several different phages. The discovery of the DNA methyltransferase with unique modification target specificity suggested unrevealed diversity of target sequences of bacterial cytosine DNA methyltransferase.
• Clonal relationship between multidrug-resistant Escherichia coli ST69 from poultry and humans in Lusaka, Zambia
  Misheck Shawa; Yoshikazu Furuta; Atmika Paudel; O'Brian Kabunda; Evans Mulenga; Maron Mubanga; Harvey Kamboyi; Tuvshinzaya Zorigt; Herman Chambaro; Manyando Simbotwe; Bernard Hang'ombe; Hideaki Higashi
  FEMS Microbiology Letters, Oxford University Press (OUP), 2022年01月14日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Abstract
  
  Multidrug-resistant (MDR) Escherichia coli in food animals such as chickens is an emerging public health concern in Zambia. Additionally, the country's high demand for poultry products necessitates further investigation into the link between poultry and human MDR E. coli. Twenty cefotaxime-resistant E. coli isolates collected from poultry in Lusaka, Zambia, were screened for multidrug resistance and sequenced on MiSeq and MinION platforms. Genomes were assembled de novo and compared to 36 previously reported cefotaxime-resistant E. coli isolates from inpatients at the University Teaching Hospital, Lusaka. All (20/20, 100%) poultry isolates exhibited resistance to ampicillin, chloramphenicol, and doxycycline. Phylogenetic analysis and hierarchical clustering showed a high degree of genetic relatedness between E. coli O17:H18-ST69 from poultry and humans. The E. coli O17:H18-ST69 clone accounted for 4/20 (20%) poultry- and 9/36 (25%) human-associated isolates that shared two plasmids harboring 14 antimicrobial resistance (AMR) genes. However, comparison analysis showed that the isolates also had other AMR plasmids distinct for each niche. Our results suggested clonal transmission of MDR E. coli between poultry and humans, with the potential acquisition of niche-specific AMR plasmids. Thus, the control of MDR E. coli requires a One Health approach involving both human and animal health sectors.
• Risk factors and spatio-temporal patterns of livestock anthrax in Khuvsgul Province, Mongolia
  Tuvshinzaya Zorigt; Satoshi Ito; Norikazu Isoda; Yoshikazu Furuta; Misheck Shawa; Natsagdorj Norov; Baasansuren Lkham; Jargalsaikhan Enkhtuya; Hideaki Higashi
  PLOS ONE, 16, 11, e0260299, e0260299, Public Library of Science (PLoS), 2021年11月19日, ［査読有り］, ［筆頭著者］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Anthrax is a worldwide zoonotic disease. Anthrax has long been a public health and socio-economic issue in Mongolia. Presently, there is no spatial information on carcass burial sites as a potential hazard of future anthrax outbreaks and possible risk factors associated with anthrax occurrences in Mongolia. Here, we analyze retrospective data (1986–2015) on the disposal sites of livestock carcasses to describe historical spatio-temporal patterns of livestock anthrax in Khuvsgul Province, which showed the highest anthrax incidence rate in Mongolia. From the results of spatial mean and standard deviational ellipse analyses, we found that the anthrax spatial distribution in livestock did not change over the study period, indicating a localized source of exposure. The multi-distance spatial cluster analysis showed that carcass sites distributed in the study area are clustered. Using kernel density estimation analysis on carcass sites, we identified two anthrax hotspots in low-lying areas around the south and north regions. Notably, this study disclosed a new hotspot in the northern part that emerged in the last decade of the 30-year study period. The highest proportion of cases was recorded in cattle, whose prevalence per area was highest in six districts (i.e., Murun, Chandmani-Undur, Khatgal, Ikh-Uul, Tosontsengel, and Tsagaan-Uul), suggesting that vaccination should prioritize cattle in these districts. Furthermore, size of outbreaks was influenced by the annual summer mean air temperature of Khuvsgul Province, probably by affecting the permafrost freeze-thawing activity.
• Development of ELISA based on Bacillus anthracis capsule biosynthesis protein CapA for naturally acquired antibodies against anthrax
  Tuvshinzaya Zorigt; Yoshikazu Furuta; Manyando Simbotwe; Akihiro Ochi; Mai Tsujinouchi; Misheck Shawa; Tomoko Shimizu; Norikazu Isoda; Jargalsaikhan Enkhtuya; Hideaki Higashi
  PLOS ONE, 16, 10, e0258317, e0258317, Public Library of Science (PLoS), 2021年10月11日, ［査読有り］, ［筆頭著者］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteriumBacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) ofB.anthracisare mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen ofB.anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Usingin silicoanalyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains ofB.anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity ofB.anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.
• Direct Regulons of AtxA, the Master Virulence Regulator of Bacillus anthracis
  Yoshikazu Furuta; Cheng Cheng; Tuvshinzaya Zorigt; Atmika Paudel; Shun Izumi; Mai Tsujinouchi; Tomoko Shimizu; Wim G. Meijer; Hideaki Higashi
  mSystems, 6, 4, American Society for Microbiology, 2021年08月31日, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Bacillus anthracis is the Gram-positive bacterial species that causes anthrax. Anthrax is still prevalent in countries mainly in Asia and Africa, where it causes economic damage and remains a public health issue.
• Novel chromosomal insertions of ISEcp1-blaCTX-M-15 and diverse antimicrobial resistance genes in Zambian clinical isolates of Enterobacter cloacae and Escherichia coli
  Misheck Shawa; Yoshikazu Furuta; Gillan Mulenga; Maron Mubanga; Evans Mulenga; Tuvshinzaya Zorigt; Christone Kaile; Manyando Simbotwe; Atmika Paudel; Bernard Hang’ombe; Hideaki Higashi
  Antimicrobial Resistance & Infection Control, 10, 1, Springer Science and Business Media LLC, 2021年05月10日, ［査読有り］, ［国際共著］, ［国際誌］
  研究論文（学術雑誌）, Abstract
  
  Background
  
  The epidemiology of extended-spectrum β-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types.bla_CTX-Mgenes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given thatbla_CTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integratedbla_CTX-Mand the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements.
  
  Methods
  
  A total of 46 clinical isolates of cefotaxime-resistantEnterobacteriaceae(1Enterobacter cloacae, 9Klebsiella pneumoniae, and 36Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes,bla_CTX-Mgenes were categorized as either chromosomal or plasmid-borne.
  
  Results
  
  WGS-based genotyping identified 58 AMR genes, including fourbla_CTX-Malleles (i.e.,bla_CTX-M-14,bla_CTX-M-15,bla_CTX-M-27, andbla_CTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination ofbla_CTX-Mgenes. Out of 45bla_CTX-Mgene-carrying strains, 7 harbored the gene in their chromosome. In oneE. cloacaeand threeE. colistrains, chromosomalbla_CTX-M-15was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles.
  
  Conclusion
  
  Our study revealed the co-occurrence of ISEcp1-bla_CTX-M-15and multiple AMR genes on chromosomal insertions inE. cloacaeandE. coli, suggesting that ISEcp1may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among theseEnterobacteriaceaespecies.

• Development of a novel anthrax vaccine utilizing capsule biosynthesis protein CapA of Bacillus anthracis
  科学研究費助成事業
  2023年08月31日 - 2025年03月31日
  Zorigt Tuvshinzaya
  The aim of this study is to develop a novel anthrax vaccine using CapA, a protein pivotal in the capsule synthesis of B. anthracis.
  
  
  To prepare soluble antigen with high immunogenicity, we made constructs to express the full-length and partial peptides of CapA. The proteins were expressed in E. coli and their solubility and immunoreactivity were assessed via immunoblotting with sera from anthrax infected horse. We identified that the C-terminal of CapA is both soluble and immunoreactive, whereas the N-terminal is not. Besides, the full-length CapA is insoluble, making it less suitable for antigen preparation in vaccine development. Therefore, we focused on the C-terminus region of CapA and further expressed and purified it via affinity purification followed by cation exchange chromatography.
  日本学術振興会, 研究活動スタート支援, 北海道大学, 23K19460