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Clostridioides difficileの分子疫学、病原性関連遺伝子と臨床背景の研究

岡田, 雄大 東京大学 DOI:10.15083/0002006996

2023.03.24

概要

[課程-2]
審査の結果の要旨
氏名 岡田 雄大
本研究の前半では、日本で過去に 1 報のみと数少ない Clostridioides difficile (C. difficile)の
multilocus sequence typing (MLST)による分子疫学と病原遺伝子の関係の研究についての研
究を行うとともに、既報では検証されていない toxin enzyme immunoassay(EIA)検査に関連
する微生物学的及び臨床的な考察を加えて解析した。後半では、C. difficile の accessory
gene regulator (agr) loci の in silico 及び in vitro の解析により、agr の多様性の検証や実験室
株・臨床株における亜型の検証や分子疫学との関連について解析した。その結果、以下の
知見が得られた。
1. MLST による分子疫学と病原遺伝子 tcdA/B, cdtA/B, tcdC の有無の検証では、2013-2014
年に東京大学医学部附属病院で検出された C. difficile 計 105 株のうち、74 の毒素産生
株では ST17, ST81, ST2, ST54, ST8, ST3, ST37, ST53 が多い一方で、毒素非産生株では
ST109, ST15, ST100 が多かった。中国や韓国で多いとされる toxin A 欠損の毒素産生株
ST81, ST37 が多かった一方で、北米やヨーロッパでの流行が問題となった高病原性株
が属する ST1 や ST11 に代表される cdtA/B 陽性の株が 4.8%と非常に少なかった。
2. 74 の毒素産生株が検出された便検体の C. difficile toxin EIA 検査結果 (陽性または陰性)
と臨床背景や病原遺伝子の関連についての検証を行ったところ、38 例の EIA 陰性症例
と比べ、36 例の EIA 陽性症例は有意に年齢が高く、慢性腎臓病の有病率や β ラクタム
系抗菌薬・プロトンポンプ阻害薬への曝露率が有意に高かった一方で、病原遺伝子に
関しては、EIA の陽性と cdtA/B、tcdC 欠損、tcdA 欠損変異の存在の有意な相関が見ら
れなかった。
3. C. difficile の毒素産生に必要と報告されている Quorum sensing(QS)関連遺伝子である
agr を対象とし、核酸・アミノ酸配列について in silico 解析を行ったところ、従来から
報告されている 2 種類の agr loci のうち、agrB と agrD のみから形成される agr1 は遺
伝的多様性が低く GenBank 上の C. difficile の Whole genome sequences の大半が保有す
ることが判明した。一方で、GenBank の登録菌株の中では、agrB, agrD, agrC, agrA の 4
つの遺伝子から構成される agr2 を保有しない菌株が多いだけでなく、agr2 の中でも互
いに 80%程度の核酸配列の相同性を示す 2 種類の亜型があることが判明した。本研究
でこれらを各々agr2R 及び agr2M と命名した。GenBank のデータベース上で agr1,
agr2R, agr2M を持つ各々の whole genome sequence(WGS)から抽出した agr の核酸配列に
対して ORF 解析を施行し推定されたアミノ酸配列の多様性を見たところ、frameshift
に伴う stop codon による配列の途絶が推定されるパターンを含めると Agr1_D: 2 種類、

Agr1_B: 10 種類、Agr2R_D: 2 種類、Agr2R_B: 7 種類、Agr2R_C: 6 種類、Agr2R_A: 3 種
類、Agr2M_D: 1 種類、Agr2M_B: 2 種類、Agr2M_C: 3 種類、Agr2M_A: 3 種類となっ
た。
4. C. difficile agr の in silico 解析結果を踏まえ、東大病院の 2013-2014 年の計 133 の C.
difficile 株と標準株 CD630 と R20291 に対して agr1 及び agr2R/2M の保有パターンにつ
いて PCR 及び DNA sequencing で解析したところ、agr1 のみの株が 34、agr1+agr2R が
61, agr1+agr2M が 26 あり、agr loci を持たない菌株が 2 つあった。
5. MLST に基づく分子系統樹解析を最尤法で施行したところ、agr1+agr2R は Clade1 と
Clade 2 に、agr1+agr2M は Clade 4 に主に見られ、Clade 1 の中で agr2R を持つ株と持
たない株の明確な分岐点は系統樹上では指摘できなかった。2 つの agr loci を持たない
菌株は Clade C-Ⅰ及び Clade C-Ⅲに属した。
6. 毒素遺伝子を保有する臨床株において toxin EIA 検査と agr 亜型の保有パターンには明
確な相関は示されず、分子系統解析でも高病原性株や近縁株のみに分布する亜型は指
摘されなかった。
以上、本研究の前半では C. difficile の MLST による分子疫学解析でアジア圏からの報告
と同様に Clade 1 についで Clade 4 の菌株が多い傾向が示され、また本研究の菌株において
は toxin EIA 検査陽性と cdtA/B、tcdC 欠損、tcdA 欠損変異の相関がないことが示された。
今後、国・地域・施設レベルで高病原性株の増加を含めた分子疫学や臨床像の変化を知る
際に土台となる貴重な情報が本研究により得られたと考える。
本研究の後半では、agr2 の中の 2 つのグループ agr2R と agr2M を発見し、agr 亜型毎の
アミノ酸配列の多様性を示した上で、臨床株における agr1, agr2R, agr2M の実在を分子疫
学と関連付けて示した。本研究で示された agr の多様性は、agr の機能的な研究を通じ C.
difficile infection の治療薬の開発に役立ちうる上、C. difficile の進化の過程の解明に役立つ
可能性があると考える。
よって本論文は博士(医学 )の学位請求論文として合格と認められる。

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