University of the Ryukyus · Faculty of Medicine · 教授

Research keywords分子微生物学・細胞分子生物学・生化学

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• Differences in flaA gene sequences, swimming motility, and biofilm forming ability between clinical and environmental isolates of Aeromonas species2023 · The flagellin A gene (flaA) sequences, swimming motility, and biofilm forming ability were investigated in order to reveal the genetic and functional differences of flagella between clinical and environmental isolates of Aeromonas species. Twenty-eight clinical and 48 environmental strains of Aeromonas species isolated in Okinawa Prefecture of Japan were used in this study. The full-length flaA genes of these strains were sequenced and aligned, and a phylogenetic tree was constructed. In addition, swimming motility and biofilm forming ability were evaluated by conventional methods. Aeromonas veronii biovar sobria and A. hydrophila clearly divided into clinical and environmental strain clusters in the flaA phylogenetic classification, and the six and 13 specific amino acids respectively, of FlaA of both species were different in clinical and environmental strains. Furthermore, the flaA size of the clinical strain of A. veronii bv. sobria was mainly 909, 924, and 939 bp, and the size of A. hydrophila was 909 bp. The swimming motility of clinical isolates of both species was lower than the environmental isolates; however, the biofilm forming ability of the clinical isolates was high. Thus, the clinical isolates of A. veronii bv. sobria and A. hydrophila had different genetic and functional characteristics of flagellin than the environmental isolates. The characteristics of flagellin could serve as indicators to distinguish between clinical and environmental isolates of the both species. It may contribute to diagnosis of these diseases and the monitoring of clinical strain invasion into the natural environment.
• Comparison of species, virulence genes and clones of Aeromonas isolates from clinical specimens and well water in Okinawa Prefecture, Japan2021 · Miyagi K.
• A high-throughput sequencing determination method for upstream genetic structure (UGS) of ISEcp1-blaCTX-M transposition unit and application of the UGS to classification of bacterial isolates possessing blaCTX-M.2021 · INTRODUCTION: Because blaCTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of blaCTX-M could be a good indicator for classifying bacterial isolates harboring blaCTX-M in molecular epidemiology. However, determination of blaCTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of blaCTX-M location. METHODS: Extracted DNA samples obtained from 168 Escherichia coli isolates possessing blaCTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer. RESULTS: Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures. CONCLUSIONS: Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-blaCTX-M transposition unit was useful for classification of bacterial isolates harboring blaCTX-M.
• Analysis of the upstream genetic structures of the ISEcp1-bla CTX-M transposition units in Escherichia coli isolates carrying bla CTX-M obtained from the Indonesian and Vietnamese communities.2021 · Widyatama FS, Yagi N, Sarassari R, Shirakawa T, Le DT, Bui MHT, Kuntaman K, Hirai I

• コミュニティにおける薬剤耐性菌健康保菌の意義解明と薬剤耐性菌拡散封じ込めの試み2018 · 本来、健康に生活する人（以下、健常人）は薬剤耐性菌を保菌しているとは想定されていない。しかしながら東南アジアの健常人では第三世代セファロスポリンを分解し、これら細菌による感染症治療を困難にすることから臨床上重要視されている薬剤耐性菌である基質拡張型βラクタマーゼ（ESBL）産生菌を非常に高率に保菌している、いわゆる健康保菌の実態がある。この健康保菌はESBL産生菌のリザーバーとして機能している可能性もあることから、ESBL産生菌の拡散及び多剤耐性化における健康保菌の意義を明らかにする必要がある。本研究ではベトナム、ハノイ市郊外を調査地とし、指標菌としてのESBL産生大腸菌を収集し、収集した疫学情報と菌株の解析結果を総合的に解析し、薬剤耐性菌の拡散における健康保菌の意義を解明することを目的としている。 本年度の実績として以下の点が挙げられる。①ベトナムの調査地の各家屋の台所周辺及び、トイレ周辺から収集したESBL産生菌及び、当該家屋に居住する対象者から分離したESBL産生菌を用いた分子微生物学的解析を行った。その結果、トイレから分離されたESBL産生菌が当該家屋に居住する対象者から分離されたESBL産生菌と遺伝学的関連性を示した。この点からトイレを中心とした疫学的介入の可能性が想定された。②ベトナムの健常人から分離されたESBL産生菌の分子生物学的解析により、ベトナムの健常人が保菌するESBL産生菌のどの程度の遺伝学的多様性があるかについて解析した。その結果、ベトナムの健常人には１～複数の遺伝学的背景の異なるESBL産生菌が保菌されていることが明らかとなった。③2018年にベトナムの調査地に居住する対象者から新たにESBL産生菌の収集並びに聞き取り調査を行った。現在、得られたESBL産生菌の分子微生物学的性情の解析並びに、聞き取り調査の疫学的解析を進めている。 <BR>
• コミュニティにおける薬剤耐性菌健康保菌の意義解明と薬剤耐性菌拡散封じ込めの試み2017 · 基盤研究(B)
• 組換えファージによる肺炎クラミジアを標的とした新規遺伝子導入システムの開発2014 · 挑戦的萌芽研究
• 細胞周期チェックポイント制御に関わるヒトRad9の関与するシグナル伝達と機能制御

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