JP6232115B2 - 微生物が潜在的に抗菌剤化合物に対して耐性を有するか否かを決定するための方法 - Google Patents
微生物が潜在的に抗菌剤化合物に対して耐性を有するか否かを決定するための方法 Download PDFInfo
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Description
(a) 前記抗菌剤化合物、その酵素的改変生成物、またはその改変酵素の基質化合物の参照マススペクトルを提供する工程と、
(b) 微生物、その細胞溶解物、またはその培地上清を、水性液中の前記抗菌剤化合物または前記基質化合物に曝露させることにより、曝露済みサンプルを提供する工程と、
(c) 前記曝露済みサンプルのマススペクトルを取得する工程と、
(d) 工程(c)で取得した前記マススペクトルを、工程(a)の前記参照マススペクトルと比較する工程と、
(e) 前記曝露の後に、前記抗菌剤化合物、または前記基質化合物の改変が起こったか否かを、前記比較から決定し、かつ前記改変が観察されたときに前記微生物が前記抗菌剤化合物に対して潜在的に耐性を有することを確認する工程と、を含み、
前記微生物、前記その細胞溶解物、または前記その培地上清を、前記水性液中の前記抗菌剤化合物または前記基質化合物に曝露する工程は、質量分析用サンプル支持体上で行われ、前記サンプルは、マトリックス支援レーザー脱離イオン化質量分析(MALDI−MS)のためのマススペクトル用サンプルを生成するために前記サンプル支持体上で乾燥されることを特徴とする方法を提供する。
a) 微生物を溶解させるための溶解緩衝液、
b) 少なくとも1つの抗菌剤化合物、または抗菌化合物改変酵素の基質、および
c) MALDIマトリックス材料、
前記部品キットは更に次のものを含む:
d) 前記の少なくとも1つの抗菌剤化合物または基質を担うキャリヤ、前記キャリヤは所望により、使い捨てのマススペクトル用サンプル支持体の形態である。
− 少なくとも1つの抗菌剤化合物、または抗菌化合物改変酵素の基質、
− 微生物、その細胞溶解物、またはその培地上清を、水性液中で少なくとも1つの抗菌剤化合物に曝露させるための容器であって、好ましくは、前記少なくとも1つの基質化合物が前記容器内に提供される、
− 前記微生物を溶解させるための溶解緩衝液、
− MALDIマトリックス材料、
− 質量分析装置、
− 抗菌剤化合物、その酵素的改変生成物、その分子標的、またはその改変酵素の基質化合物の参照マススペクトル、
− マススペクトル用サンプル支持体、
所望により更に次のものを含む:
− 液体取扱いのための自動化ピペッター、
− 前記プログラムがコンピュータ上で作動するときに、上述の本発明の方法の全工程を実施するためのコンピュータプログラムコード手段を含むコンピュータプログラム、例えば、結果解釈のためのアルゴリズムや、インタフェースソフトウェア、エキスパートシステムソフトウェアを含む。
用語「抗生物質」および「抗生物質化合物」は本明細書において互換可能な語として使用され、本明細書において、細菌の生存可能性を低減させるか、あるいは細菌の成長または繁殖を阻害する、化合物または組成物を指す。「成長または繁殖を阻害する」とは、世代周期が少なくとも2倍に増加し、好ましくは少なくとも10倍に増加し、好ましくは少なくとも100倍に増加し、最も好ましくは無期限に全細胞死亡となることを意味する。本開示に使用されるとき、抗生物質はさらに、抗菌剤、細菌発育阻止剤、または殺菌剤を含むことが意図される。本発明の態様に有用な抗生物質の非制限的な例としては、ペニシリン、セファロスポリン、アミノグリコシド、スルホンアミド、マクロライド、テトラサイクリン、リンコサミド、キノロン、クロラムフェニコール、グリコペプチド、メトロニダゾール、リファムピン、イソニアジド、スペクチノマイシン、葉酸阻害剤、スルファメトキサゾール等が挙げられる。
本発明は、微生物の抗生物質耐性を特徴付けるための方法を提供する。この方法の第一工程は、耐性を特徴付ける抗生物質、またはその好適な擬似基質、または抗生物質化合物の分子標的の、1つ以上の参照マススペクトルを得ることである。参照スペクトルは、サンプルの分析に使用される任意のマススペクトル(MS)技法を用いて生成することができる。好ましいMS技法はMALDI−MSである。
1.サンプルをMS装置に搭載する。所望により(MALDIと呼ばれる特殊な形態のMSの場合)マトリックスと合わせたサンプルを、マススペクトルサンプル支持体に塗布し、溶媒を蒸発させてその支持体上のサンプルまたは混合物を乾燥させる。
2.さまざまな方法のうち1つ(例えば電子ビームを衝突させる方法)によってサンプルの組成物をイオン化し、これにより荷電粒子(イオン)が形成される。
3.電場により陽イオンを加速させる。
4.イオンが電磁場を通過する際のイオンの動きの詳細情報に基づいて粒子の質量電荷比(m/z)を計算する。
5.イオンを検出する。このイオンは、工程4で、m/zに従って分類されている。
Claims (12)
- 微生物が潜在的に抗菌剤化合物に対して耐性を有するか否かを決定するための方法であって、
(a) 前記抗菌剤化合物、その酵素的改変生成物、またはその改変酵素の基質化合物の参照マススペクトルを提供する工程と、
(b) 微生物、その細胞溶解物、またはその培地上清を、水性液中の前記抗菌剤化合物または前記基質化合物に曝露させることにより、曝露済みサンプルを提供する工程と、
(c) 前記曝露済みサンプルのマススペクトルを取得する工程と、
(d) 工程(c)で取得した前記マススペクトルを、工程(a)の前記参照マススペクトルと比較する工程と、
(e) 前記曝露の後に、前記抗菌剤化合物、または前記基質化合物の改変が起こったか否かを、前記比較から決定し、かつ前記改変が観察されたときに前記微生物が前記抗菌剤化合物に対して潜在的に耐性を有することを確認する工程と、を含み、
前記微生物、前記その細胞溶解物、または前記その培地上清を、前記水性液中の前記抗菌剤化合物または前記基質化合物に曝露する工程は、質量分析用サンプル支持体上で行われ、前記サンプルは、マトリックス支援レーザー脱離イオン化質量分析(MALDI−MS)のためのマススペクトル用サンプルを生成するために前記サンプル支持体上で乾燥されることを特徴とする方法。 - 前記改変には、前記抗菌剤化合物または前記基質化合物の酵素的不活性化または酵素的分解が含まれることを特徴とする請求項1に記載の方法。
- 前記酵素的分解が、ベータラクタマーゼによる分解によるものであることを特徴とする請求項2に記載の方法。
- 前記微生物は、クレブシエラ・ニューモニエ(Klebsiella pneumoniae)、大腸菌(Escherichia coli)、クレブシエラ・オキシトカ(Klebsiella oxytoca)およびプロテウス・ミラビリス(Proteus mirabilis)から選択される、基質特異性拡張型ベータラクタマーゼ(ESBL)産生が疑われる微生物であることを特徴とする請求項1乃至3のいずれか1項に記載の方法。
- 前記抗菌剤化合物はベータラクタム抗生物質であることを特徴とする請求項1乃至4のいずれか1項に記載の方法。
- 前記ベータラクタム抗生物質は、ペニシリン、セファロスポリン、セファマイシン、カルバペネム、セフタジジム、セフォタキシム、セフトリアキソン、セフポドキシム、およびアズトレオナムからなる群から選択されることを特徴とする請求項5に記載の方法。
- 前記マススペクトルは、MALDIトリプル四重極質量分析、MALDI−TOF質量分析、及びMALDI−FT−ICR質量分析の1つを使用して取得されることを特徴とする請求項1乃至6のいずれか1項に記載の方法。
- 前記工程(b)において、前記微生物は、前記サンプルにおいて、前記微生物に由来する1つ以上の構造的生体分子または代謝生成物を定量することによって定量されることを特徴とする請求項1乃至7のいずれか1項に記載の方法。
- 前記抗菌剤化合物のみまたは前記基質化合物のみのレベルの減少、または 反応生成物のみのレベルの増加は、質量分析によって測定され、前記改変の指標として用いられることを特徴とする請求項1乃至8のいずれか1項に記載の方法。
- 前記抗菌剤化合物または前記基質化合物のレベル、及び反応生成物のレベルは、質量分析によって測定され、生成物対基質の比は前記改変の指標として用いられることを特徴とする請求項1乃至8のいずれか1項に記載の方法。
- 前記曝露済みサンプルは、前記微生物の、抗菌剤化合物曝露済み未精製細胞分解物であることを特徴とする請求項1乃至10のいずれか1項に記載の方法。
- 前記曝露済みサンプルは、ベータラクタム抗生物質耐性の特徴付が行われる微生物を含んでいることが疑われる、ヒトまたは動物被検体の体液または体内組織サンプルであることを特徴とする請求項1乃至10のいずれか1項に記載の方法。
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