JP2020031634A - バクテリオファージの保存のための組成物またはマトリックス - Google Patents
バクテリオファージの保存のための組成物またはマトリックス Download PDFInfo
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- JP2020031634A JP2020031634A JP2019152284A JP2019152284A JP2020031634A JP 2020031634 A JP2020031634 A JP 2020031634A JP 2019152284 A JP2019152284 A JP 2019152284A JP 2019152284 A JP2019152284 A JP 2019152284A JP 2020031634 A JP2020031634 A JP 2020031634A
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- bacteriophage
- cellulose
- nanofibril cellulose
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- A61L15/36—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing microorganisms
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
Description
組成物もしくはマトリックスを保存するステップ;
バクテリオファージを培養するステップ;
組成物もしくはマトリックスを輸送するステップ;および/または
組成物もしくはマトリックスから目的箇所もしくは対象にバクテリオファージを送達するステップ。目的箇所は、例えば、原核細胞培養物であってもよい。
本明細書に記載する1つ以上の実施形態による組成物もしくはマトリックス、または本明細書に記載する1つ以上の実施形態による配列の組成物もしくはマトリックスに原核細胞を添加するステップ、および
原核細胞を培養するのに適した条件で組成物、マトリックスまたは配列を維持し、それによってバクテリオファージが原核細胞に感染することを可能にするステップ。
以下の実施例では、「GrowDex」または「GrowDex(登録商標)」という用語は、ナノフィブリルセルロースヒドロゲルを指す。これらの実施例で使用されるGrowDexは、天然のナノフィブリルセルロースヒドロゲルである。
fRuSau02(完全な名称vB_SauM_fRuSau02;Leskinen et.al.Viruses 2017,9,258;doi:10.3390/v9090258に記載されている)
ΦEBHT.出典:DSMZ,Germany(Leibniz Institute DSMZ−German Collection of Microgranisms and Cell Cultures)
fHoEco02(完全な名称vB_EcoM_fHoEco02;Kiljunen et al.2018,Genome Announc 6:e00401−18;https://doi.org/10.1128/genomeA.00401−18))
fTu−Eco01(未公表、本発明者らのコレクションより、下水から分離)
黄色ブドウ球菌13KP(#5676):fRuSau02の宿主
黄色ブドウ球菌19A2(#6433);ΦEBHTの宿主
大腸菌123738(#5521)、fHoEco02の宿主
大腸菌123789(#5507)、fTu−Eco01の宿主
NFCヒドロゲルを、SM緩衝液または0.9%NaClのいずれかで、0(対照)、0.1、0.5、1.0および1.5%(w/w)のコンシステンシーに希釈した。希釈液の10μl滴を、96ウェルプレートのウェル上に分注した。細菌(大腸菌株123738(#5521)および123789(#5507)ならびに黄色ブドウ球菌株13KP(#5675)および19A2(#6433)の希釈一晩培養の200μlを、ウェル上に添加した。OD600を5時間追跡することにより細菌増殖をモニターした。
バクテリオファージをSM緩衝液または0.9%NaClのいずれかで1:100に希釈し、両方のファージ希釈液を0.1、0.5、1.0および1.5%(w/w)のコンシステンシーでGrowDexヒドロゲルと混合した。SM緩衝液および0.9%NaClに保存したファージを、対照として用いた。得られた組成物を4℃で約2時間保存した。組成物を、SM緩衝液中または0.9%NaCl中で、それぞれ連続希釈(10−4、10−5、10−6および10−7)として希釈した。希釈した組成物の滴を、ペトリ皿で寒天上で成長させた細菌のローン上に置いた。ファージの細菌感染能を観察した。
実験設定の概略を図3に示す。
ファージ−NFCヒドロゲル混合物の乾燥の効果を、他の点では実施例4と同様の方法でマルチウェルプレート上で試験したが、10μl滴を、プレートを4℃で保存する前に、RTで2時間、バイオセーフティキャビネット内で風乾させた。黄色ブドウ球菌株19A2に感染するΦEBHTの例示的な結果を図5に示す。24時間保存後、ファージは細菌成長を完全に阻害することができた。1週間および8週間の保存後、成長阻害は1%NFCに保存されたファージではあまり明白ではなかったが、0.5%組成物のファージおよびSM緩衝液ではなお明白であった。しかし、異なるファージは乾燥に対して異なった耐性を示し、その過程で不活性化されるファージもある。
長期保存の影響は、実施例4と同様の方法でマルチウェルプレート上で試験した。GrowDex濃度1.0%、0.5%および0%を用い、SM緩衝液で希釈した。組成物/マトリックスは以下のように保存した。
− 湿潤ペレット(96ウェルプレート10μl、ホイル/プラスチックシールドで覆う)
− 液体形態(2mlマイクロ遠心管に保存)
上記のように調製したマルチウェルプレート(1%GrowDex、4つのテストファージ(黄色ブドウ球菌に特異的なファージ2つ、大腸菌に特異的なファージ2つ)を郵便で送付し、輸送には約3日を要した。7日後に測定した。輸送および保存中、プレートは室温、+4℃および−15℃で一部保存した。
3種類の異なるヒドロゲル(GrowDex(登録商標)、Intrasite、およびPurilon(登録商標))の効果を、実施例2と同様の方法でマルチウェルプレート上で試験した。黄色ブドウ球菌株13KPの例示的結果を図8に示す。GrowDexおよびIntrasiteは細菌成長に影響を及ぼさなかったが、Purilonは明らかな成長阻害を示した。同様の結果が、試験した4種類の細菌株すべてについて得られた。
Claims (15)
- バクテリオファージおよびナノフィブリルセルロースまたはその誘導体を湿潤または乾燥状態で含む、組成物またはマトリックス。
- 前記組成物またはマトリックスが湿潤状態であり、ヒドロゲルまたは膜の形態である、請求項1に記載の組成物またはマトリックス。
- 前記組成物またはマトリックスが乾燥状態であり、任意に膜の形態である、請求項1または2に記載の組成物またはマトリックス。
- 前記バクテリオファージが、前記マトリックス中のバクテリオファージ粒子として2次元または3次元配置で少なくとも部分的に存在する、請求項1〜3のいずれか一項に記載の組成物またはマトリックス。
- 前記ナノフィブリルセルロースまたはその誘導体が、天然ナノフィブリルセルロースを含む、または天然ナノフィブリルセルロースである、請求項1〜4のいずれか一項に記載の組成物またはマトリックス。
- バクテリオファージを保存、培養、輸送および/または送達するための配列であって、固体支持体、および前記固体支持体上に配置された湿潤または乾燥状態でのナノフィブリルセルロースまたはその誘導体を含む組成物またはマトリックスを含む、配列。
- 固体支持体と、前記固体支持体上に配置された請求項1〜5のいずれか一項に記載の組成物またはマトリックスとを含む、配列。
- 前記固体支持体がマルチウェルプレートであり、前記組成物またはマトリックスが前記マルチウェルプレートの1つ以上のウェルに配置されている、請求項6または7に記載の配列。
- 前記配列が医療用多層製品であり、前記固体支持体が層の形態であり、前記組成物またはマトリックスが前記固体支持体上の層として配列されるか、または前記固体支持体内に含浸される、請求項6または7に記載の配列。
- バクテリオファージを貯蔵、培養、輸送および/または送達するための方法であって、前記方法が、前記バクテリオファージをナノフィブリルセルロースまたはその誘導体と混合するステップを含む、方法。
- 前記方法が、組成物またはマトリックスを乾燥するステップをさらに含む、請求項10に記載の方法。
- 請求項1〜5のいずれか一項に記載の組成物もしくはマトリックス、または請求項7〜9のいずれか一項に記載の配列を調製する方法であって、前記方法は、バクテリオファージをナノフィブリルセルロースまたはその誘導体と混合するステップを含む、方法。
- バクテリオファージを増殖させ、かつ/またはバクテリオファージが原核細胞に感染する能力を試験する方法であって、
請求項1〜5のいずれか一項に記載の組成物もしくはマトリックス、または請求項7〜9のいずれか一項に記載の配列の前記組成物もしくはマトリックスに原核細胞を添加するステップと、
原核細胞を培養するのに適した条件で前記組成物、マトリックスまたは配列を維持し、それによって前記バクテリオファージが前記原核細胞に感染することを可能にするステップと
を含む、方法。 - バクテリオファージを貯蔵、培養、輸送および/または送達するための、ナノフィブリルセルロースまたはその誘導体を含む組成物またはマトリックスの、湿潤または乾燥状態での使用。
- 治療に使用するための、請求項1〜5のいずれか一項に記載の組成物もしくはマトリックス、または請求項7〜9のいずれか一項に記載の配列。
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