專利名稱：用于治療細菌感染的多價噬菌體制備方法
技術(shù)領(lǐng)域：
本發(fā)明涉及噬菌體的復(fù)價株系、獲得這些株系的方法及其在治療細菌感染中的應(yīng)用，特別是在那些有藥物抗性的細菌株系感染中的應(yīng)用。
背景技術(shù)：
噬菌體(phages)是一種多樣性的病毒，其生活史專一地與細菌細胞相關(guān)。噬菌體特征在于溶原性的或裂解性的生活史。作為抗菌劑，裂解性噬菌體尤其有用，該噬菌體當感染對它們敏感的細菌細胞后可在該細胞中復(fù)制，從而導致該細胞的完全破壞(通過裂解作用)及新噬菌體的釋放，新的噬菌體可攻擊和破壞其他的細菌細胞。該過程可發(fā)生于體外或體內(nèi)。
噬菌體的一個基本特征是其裂解活性的眾所周知的高特異性。該特性可應(yīng)用于如各種細菌的物種確定(噬菌體分類)(參見如專利說明GB 2285684、US 5824468和SU 543260以及國際專利通知WO 0100786和WO 0109370)。噬菌體的其他已知的應(yīng)用包括其作為分子生物學工具的應(yīng)用，如在特定的蛋白質(zhì)的表達和選擇中的應(yīng)用(如專利描述US6027930)和在滅菌和清除培養(yǎng)基中的應(yīng)用(如專利描述EP 0414304、EP 0290295和GB 2253859以及國際專利通知WO 9808944和WO9003122)。修飾的噬菌體用于疫苗的生產(chǎn)(如WO 9505454)。也使用了某些噬菌體來源的蛋白質(zhì)(如EP 0510907、US 5470573、WO9607329)。分離噬菌體和獲得噬菌體制備物的方法是眾所周知的且經(jīng)常進行完善(如GB 829266、CS 192212、RU 2109055)。
第二次世界大戰(zhàn)后在Georgia的Tbilisi微生物學和病毒學研究所(Institute of Microbiology and Virology of Tbilisi)已大規(guī)模地應(yīng)用了噬菌體治療。那里在細菌感染的治療和預(yù)防中應(yīng)用了各種噬菌體制備物的儲備。可用的數(shù)據(jù)顯示噬菌體治療的巨大有效性。相似的研究在波蘭已進行了超過25年。在弗羅茨瓦夫的波蘭科學院免疫學和試驗治療研究所的噬菌體實驗室(Bacteriophage Laboratoryof the Institute of Immunology and Experimental Therapy of thePolish Academy of Sciences in Wroclaw)，將噬菌體治療用于治療由抗藥形式的細菌和那些不對抗生素易感的細菌所導致的感染(參見Stefan lopek等人，Archivum Immunologiae et TherapiaeExperimentalis，1981，31，293；Stefan 等人，ArchivumImmunologiae et Therapiae Experimentalis，1983，31，267；Stefan 等人，Archivum Immunologiae et Therapiae Experimentalis，1984，32，317；Stefan 等人，Archivum Immunologiae etTherapiae Experimentalis，1985，33，219；Stefan 等人，Archivum Immunologiae et Therapiae Experimentalis，1985，33，241Stefan 等人，Archivum Immunologiae et TherapiaeExperimentalis，1987，35，569；Beata Weber-Dabrowska等人，Archivum Immunologiae et Therapiae Experimentalis，2000，48，31-37；Beata Weber-Dabrowska等人，Archivum Immunologiae etTherapiae Experimentalis，2000，48，547-551)。過去14年中在那里進行的噬菌體治療顯示噬菌體治療的高效性，該治療包括1473個具有不同組織和器官化膿性感染的患者。
在1289個病例中記錄到疾病癥狀的完全消除和康復(fù)。必須強調(diào)的是在至少一打左右的病例中，噬菌體治療呈現(xiàn)為消除致命性感染的唯一可能。進行的治療涉及單獨的患者。它由以下內(nèi)容組成a)從獲自患者的材料中分離的細菌株系的生長和鑒定，b)確定該株系對特定噬菌體的易感性及選擇對該株系顯示最高裂解活性的噬菌體，c)制備具有大量噬菌體顆粒的噬菌體溶菌產(chǎn)物，d)生產(chǎn)用于治療的無菌噬菌體制備物。
該程序相當昂貴、費力且費時。有時，從獲得研究材料起到獲得用于治療的完成的無菌噬菌體制備物要經(jīng)過7-10天。這種延遲在某些疾病狀況中太長。大規(guī)模的噬菌體治療不能用目前采用的程序進行。伴隨粘稠物阻塞癥的感染呈現(xiàn)為一個特別的問題。這是一種遺傳的全身疾病，該疾病由粘液分泌腺的功能異常構(gòu)成，這使得患者傾向于患慢性支氣管和肺病。分泌的粘液濃且粘，從而使其難由天然途徑(咳嗽)清除。粘液在支氣管中的累積和滯留以及在其清除中的損害造成了細菌感染的有利環(huán)境，從而導致慢性支氣管炎和肺炎。導致伴隨粘稠物阻塞癥感染的最危險的病原體是金黃色葡萄球菌(Staphylococcus aureus)和假單胞菌屬(Pseudomonas)的桿菌。這些微生物持久地復(fù)發(fā)感染導致呼吸系統(tǒng)中嚴重的病理學改變和早死。很少患者可幸存超過25年。用可獲得的抗生素對這些感染的治療造成了全世界范圍的治療問題，尤其是在剛過去的幾年中。因為絕大多數(shù)細菌株系顯示對所有抗生素包括最后手段的抗生素萬古霉素的抗性，所以對這些感染的抗生素治療已變得越來越低效了。因此，迫切需要向治療難治的和高度危險的細菌感染的醫(yī)學實踐中引入可替代的治療方法。
發(fā)明目的過去數(shù)年中細菌株系對所有抗生素的抗性已有廣泛的傳播，該抗生素包括最后手段的抗生素萬古霉素。結(jié)果，用抗生素治療由這些藥物抗性形式的細菌引起的感染是無效的。該狀況已造成了巨大的治療問題。因而，迫切需要向治療難治的細菌感染的醫(yī)學實踐中引入其他的治療方法。
考慮到上述目的，本發(fā)明致力獲得復(fù)價噬菌體株系，該噬菌體株系可用于生產(chǎn)在治療細菌感染中具有保證的效力的制備物，而無需對每一個病例單獨選擇噬菌體。在此，本發(fā)明的目的是提供允許獲得高純度的噬菌體制備物的途徑，該制備物尤其不含內(nèi)毒素。
本發(fā)明的一個特別的目的是獲得多價的噬菌體制備物，該制備物可有效應(yīng)用于治療與粘稠物阻塞癥相關(guān)的細菌感染，而無需每次單獨選擇噬菌體。
發(fā)明概述本發(fā)明提供獲得復(fù)價噬菌體株系的方法，通過該方法a)積聚足夠數(shù)目(n)的在給定區(qū)域隨機出現(xiàn)的確定屬的細菌病原體的不同株系，在此分離的細菌株系優(yōu)選為藥物抗性的，b)積聚足夠數(shù)目的對該給定屬的至少一個細菌株系特異的不同噬菌體株系，c)確定所積聚的噬菌體株系對每種所積聚的細菌株系的裂解活性，然后計算p值，該值為由給定的噬菌體裂解的菌株的數(shù)目與所有積聚的細菌株系的數(shù)目的比例。
d)對于結(jié)果所得的p值，檢驗n是否滿足條件n≥pqd2z1-α2]]>其中q＝1-pd是不大于0.1的常數(shù)，最適地不大于10％pz1-α是依賴于置信因子1-α的正態(tài)分布的隨機變量，該置信因子不小于0.95，e)選擇滿足在d)中限定的標準且具有不小于2/n的p值(優(yōu)選地不小于0.5)的噬菌體株系。
每一個分離的噬菌體的裂解活性范圍是關(guān)于在給定區(qū)域存在的病原體細菌株系的群體而確定的。為此，必須積聚足夠大的隨機選擇的藥物抗性細菌株系的集合。參考確立的群體大小為n個株系的細菌大樣本(在描述的例子中，對于在波蘭分離的假單胞菌屬和葡萄球菌屬的藥物抗性株系，n分別為845和880)，計算了成功頻率或由給定的噬菌體裂解的菌株株系的數(shù)目與所研究的株系的總數(shù)目的比例。人們認為該頻率是成功概率p。失敗概率則是q＝1-p。
關(guān)鍵的是選擇的樣本必須具有的大小n，從而可認為該樣本是給定屬株系的普通群體的代表(如假單胞菌屬或葡萄球菌屬)。為此，確定了下面使樣本具有代表性的標準P(|v-p|＜d)＝P(-d＜v-p＜d)＝1-α樣本頻率v和成功概率p之間的差的絕對值小于預(yù)先指定的值d的概率為1-α，這相應(yīng)于置信水平1-α。1-α的值通常設(shè)定為0.95或0.98。
樣本數(shù)目n應(yīng)該滿足條件n≥pqd2z1-α2]]>其中z1-α是正態(tài)分布的隨機變量。對于1-α＝0.95，z1-α＝1.96，而對于1-α＝0.98，z1-α＝2.33。
基于該標準和從所表征的噬菌體獲得的頻率p的值，通過給d和置信范圍1-α指定合理的值，可能證實用于確定給定噬菌體的裂解活性范圍的量級n及確定在指定的d和1-α的值時，對該n獲得的裂解活性對于給定屬的細菌株系的普通群體是否可認為是正確的。
噬菌體株系優(yōu)選在步驟b)中從源自環(huán)境的樣品中分離，通過使該樣品經(jīng)過孔徑為0.2-0.4μm的膜濾器、向濾液中添加培養(yǎng)基并使其與確定屬的細菌的液體培養(yǎng)基混合、使其在約37℃的溫度溫育約1小時、取出一部分懸浮液并將其涂于有固體培養(yǎng)基的平板上、于約37℃溫育2～24小時、分離單一禿斑周圍的培養(yǎng)基樣品、將其轉(zhuǎn)移到確定屬的細菌的液體培養(yǎng)基中并進行溫育直到培養(yǎng)物清澈，然后通過使溶菌產(chǎn)物經(jīng)過孔徑為0.2-0.4μm的膜濾器而獲得噬菌體制備物，在此優(yōu)選重復(fù)接種固體培養(yǎng)基并將禿斑再次分離5次。
將在步驟c)中的細菌株系優(yōu)選接種到固體培養(yǎng)基上，在該培養(yǎng)基上沉積了一部分在步驟b)中獲得的噬菌體制備物，于約37℃溫育約4小時，其后將溫度設(shè)定于約4℃進行2～24小時，借此噬菌體株系的裂解活性通過至少單個禿斑的出現(xiàn)而證實。
可對裂解物實施進一步的純化，特別關(guān)于內(nèi)毒素，在此使含有噬菌體的混合物與含有纖維素或其部分酯化的衍生物接觸，然后用可除去雜質(zhì)尤其是可除去內(nèi)毒素的溶液沖洗，其后將純化的噬菌體洗滌出來。內(nèi)毒素可有效地用水、非解離性物質(zhì)的溶液或可能緩沖的濃度不大于0.1M的鹽溶液進行洗脫。噬菌體級分也可有效地用非解離性物質(zhì)的溶液或任何緩沖液或可能緩沖的濃度大于0.05M的鹽溶液進行洗脫。同樣地，內(nèi)毒素和噬菌體的洗脫是在-25℃～+100℃之間的溫度中進行的。優(yōu)選地，內(nèi)毒素和噬菌體洗脫可用含有機溶劑的水性鹽溶液進行。該有機溶劑最好選自二甲基亞砜、二甲基甲酰胺、異丙醇和丙酮，且可使用以有機或無機酸部分酯化的纖維素作為基質(zhì)。用乙酸、硝酸、硫酸或磷酸部分酯化的纖維素作基質(zhì)，特別是其中0.01～5％的葡萄糖分子被酯化的纖維素可用作基質(zhì)，優(yōu)選其中0.25～1％的葡萄糖分子，更優(yōu)選0.5～1％的葡萄糖分子被酯化。
病原性細菌株系優(yōu)選為假單胞菌屬或葡萄球菌屬。
本發(fā)明的進一步的方面是治療或預(yù)防由細菌病原體導致的感染的藥物，該藥物含有活性試劑和可能的藥物學可接納的載體，從而該活性試劑包含對該屬的細菌特異的復(fù)價噬菌體株系且由本發(fā)明的方法獲得。
根據(jù)本發(fā)明，該藥物可具有如下特征，作為對葡萄球菌屬細菌特異的復(fù)價噬菌體株系，它含有至少一種株系的選自下述的噬菌體S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCMF/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)，優(yōu)選為噬菌體S1、S2和S4或S5。根據(jù)本發(fā)明的一個方面生產(chǎn)的該藥物用于治療或預(yù)防患粘稠物阻塞癥的人的感染，以及作為對葡萄球菌屬細菌特異的復(fù)價噬菌體株系，它含有至少一種選自F/00002、F/00004和F/00007的噬菌體株系。
根據(jù)本發(fā)明，該藥物可具有如下特征，作為對假單胞菌屬細菌特異的復(fù)價噬菌體株系，它含有至少一種株系的選自下述的噬菌體P1(PCM F/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCMF/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，最適為噬菌體P7、P20和P6。根據(jù)本發(fā)明的某些方面生產(chǎn)的該藥物用于治療或預(yù)防患粘稠物阻塞癥的人的感染，以及作為對假單胞菌屬細菌特異的復(fù)價噬菌體株系，它含有至少一個選自F/00010、F/00013和F/00018的噬菌體株系。
本發(fā)明進一步的目的是應(yīng)用根據(jù)本發(fā)明的方法獲得的復(fù)價噬菌體株系以開發(fā)治療或預(yù)防由細菌病原體導致的細菌感染的藥物。
對于生產(chǎn)治療或預(yù)防由葡萄球菌屬細菌導致的感染的藥物，有利的是應(yīng)用至少一種選自S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/0006)、S6(PCMF/00006)和S7(PCM F/00007)的噬菌體株系，最適為噬菌體S1、S2和S4或S5。對于制造治療或預(yù)防患粘稠物阻塞癥的人的由葡萄球菌屬細菌導致的感染的藥物，有利的是使用至少一種選自F/00002、F/00004和F/00007的噬菌體株系。
同樣地，對于制造治療或預(yù)防由假單胞菌屬細菌導致的感染的藥物，有利的是使用至少一種選自P1(PCM F/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCMF/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)的噬菌體株系，最適為噬菌體P7、P20和P6。對于制造治療或預(yù)防患粘稠物阻塞癥的人的由假單胞菌屬細菌導致的感染的藥物，有利的是使用至少一種選自F/00010、F/00013和F/00018的噬菌體株系。
本發(fā)明進一步的目的是對葡萄球菌屬細菌特異的復(fù)價噬菌體株系，該噬菌體株系選自S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCMF/00006)和S7(PCM F/00007)，最適選自S1、S2、S4和S5。
本發(fā)明一個目的也是對假單胞菌屬細菌特異的復(fù)價噬菌體株系，該噬菌體株系選自P1(PCM F/00008)、P2(PCM F/00009)、P3(PCMF/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，最適選自P7、P20和P6。
在研究中使用了我們自己的噬菌體集合，該噬菌體集合的活性是針對人的細菌感染中為最頻繁的病原學因子的細菌物種。所述的實施例涉及如下多價噬菌體，該噬菌體對葡萄球菌株系(金黃色葡萄球菌)包括其甲氧基苯青霉素抗性的株系起裂解作用，及對藍膿(blue pus)桿菌(銅綠假單胞菌(Pseudomonas aeruginosa))起裂解作用，該桿菌特別出現(xiàn)于那些患粘稠物阻塞癥的人中。這些物種當前是導致高死亡率的嚴重感染的原因。
本發(fā)明的優(yōu)點向治療中引入含有用本發(fā)明方法獲得的多價噬菌體的多價噬菌體制備物是在與細菌感染的斗爭中的一個主要發(fā)展，該細菌感染對于抗生素治療不易感。某些經(jīng)濟利益也與該新技術(shù)相關(guān)。它使得在原料成本中有顯著的減少并縮短了從收集用于鑒定的材料到獲得治療性噬菌體制備物之間的時間。因此，噬菌體制備物將可供患者即時使用，而不需患者等待單一的噬菌體制備物的生產(chǎn)。
包含用本發(fā)明的方法獲得的幾個多價噬菌體的裂解性噬菌體制備物的廣泛范圍從其在治療細菌感染中應(yīng)用的看來是非常重要的優(yōu)點。眾所周知，在一個細菌群體中，對噬菌體抗性的形式可以以約1×10-7的頻率出現(xiàn)。存在2個噬菌體時，該突變的概率為約1×10-14，且3個噬菌體時僅為1×10-21。利用這種安排，對噬菌體抗性的細菌株系的問題在實際上不存在。
包含多價噬菌體的示例性的藥物的極大的應(yīng)用性需要特別的強調(diào)。它們通常可在醫(yī)學實踐中應(yīng)用并允許有效操作有危險的細菌感染。同樣可能的是在外用的藥物的制備中應(yīng)用根據(jù)本發(fā)明獲得的噬菌體，從而將其應(yīng)用于如對皮膚病感染的治療和預(yù)防中。也已發(fā)現(xiàn)經(jīng)口給予根據(jù)本發(fā)明獲得的噬菌體溶菌產(chǎn)物可增加對將來可能的細菌感染的抗性。
在某些情況中，噬菌體治療可與抗生素治療結(jié)合使用。已顯示在細菌感染治療中使用的多價噬菌體制備物是高效的。在膿毒病的治療(88％的治愈率)和具有粘稠物阻塞癥的患者的感染的治療中觀察到了特別的效力。
附圖簡述

圖1顯示對噬菌體S1-S7獲得的統(tǒng)計學分析的結(jié)果。
圖2顯示對噬菌體P1-P20獲得的統(tǒng)計學分析的結(jié)果。
圖3顯示所選噬菌體株系對于從粘稠物阻塞癥患者中分離的屬于葡萄球菌屬的細菌株系的效率的統(tǒng)計學分析結(jié)果。
圖4顯示所選噬菌體株系對于從粘稠物阻塞癥患者中分離的屬于假單胞菌屬的細菌株系的效率的統(tǒng)計學分析結(jié)果。
為對本發(fā)明實質(zhì)有更好的理解，將其在下文通過實施例進行闡明。
實施例1.噬菌體分離葡萄球菌屬和假單胞菌屬的噬菌體從城市污水中分離。使該污水經(jīng)過孔大小為0.2-0.4μm的膜濾器，該膜濾器捕獲了細菌但允許噬菌體通過。從結(jié)果所得的濾液中，加入濃縮的液體培養(yǎng)基，并且將不同的濾液稀釋物與新鮮的細菌液體培養(yǎng)物混合(金黃色葡萄球菌或銅綠假單胞菌)。使樣品于37℃溫育1小時，其后用移液管從每一個樣品中吸取0.2ml的懸浮液并用玻璃棒涂于含有瓊脂培養(yǎng)基的平板上，然后將平板于37℃溫育過夜。如果尋找的噬菌體存在于用含有新鮮細菌培養(yǎng)物的適當?shù)臑V液稀釋物接種的平板上，那么則觀察到細菌的均一(云狀的)生長和分離的透明小區(qū)域，即所謂的禿斑，該斑點通常含有幾百萬個噬菌體顆粒。將單一的禿斑連同其周圍的瓊脂一起用鉑環(huán)切下，然后轉(zhuǎn)移到新鮮的細菌液體培養(yǎng)基上并進行溫育直到培養(yǎng)物清澈。在使其經(jīng)過孔大小為0.2-0.4μm的膜濾器后，濾液僅含有噬菌體，該膜濾器阻止了殘存于裂解物中的任何細菌。獲得的噬菌體的不同稀釋物對瓊脂培養(yǎng)基上各種細菌的接種和光禿斑點的再次分離進行了5次，這使得能夠獲得噬菌體的純系。
實施例2.確定金黃色葡萄球菌株系對與之特異的噬菌體微生物的易感性。
易感性用由Wahl描述的培養(yǎng)基進行確定。將具有該培養(yǎng)基的平板在37℃的溫度干燥30分鐘、用通過振蕩而混合新鮮的4-小時的金黃色葡萄球菌液體培養(yǎng)物覆蓋，將過量的懸浮液用移液管去除并再次于37℃干燥30分鐘。在一個分為6個部分的平板上，要研究的株系的易感性對6種不同但特定的噬菌體進行測定。將1滴1/10的噬菌體稀釋物置于每一個部分的表面。將平板在溫箱中于37℃溫育約4小時，其后將其置于冰箱中直到第二天。如果菌株對該確定的噬菌體具有高的易感性，那么在噬菌體引入到用細菌生長物均勻覆蓋的培養(yǎng)基中的位置即可見到透明的區(qū)域，這是細菌細胞的完全破壞(裂解作用)的結(jié)果。株系的較低的易感性由出現(xiàn)單一的光禿斑點來證實。
實施例3.確定假單胞菌屬株系對與之特異的噬菌體微生物的易感性。
確定敏感性的方法與在實施例2中描述的相似。盡管在該情況中，原培養(yǎng)基是有磷酸鹽緩沖液補充的瓊脂，且沉積了噬菌體的平板的溫育時間為5小時。
實施例4.選擇對于葡萄球菌屬和假單胞菌屬具有最廣的活性譜的噬菌體——獲得復(fù)價噬菌體株系。
噬菌體根據(jù)在實施例1中描述的方法分離。確定了從全波蘭分離的各種葡萄球菌屬菌種的845個藥物抗性臨床株系對來自特異于葡萄球菌屬的噬菌體集合的的噬菌體的易感性，以及不同假單胞菌屬菌種的880個藥物抗性臨床株系對來自特異于假單胞菌屬的噬菌體集合的噬菌體的易感性。
對兩組株系對各噬菌體的易感性的分析允許選擇具有出乎意料的高活性和廣譜裂解作用的噬菌體。這些噬菌體在表1和2中給出。
表1.保藏于波蘭弗羅茨瓦夫的波蘭微生物保藏所(PolishCollection of Microorganisms)(PCM)的對葡萄球菌屬有高裂解活性的噬菌體。
表2.保藏于波蘭弗羅茨瓦夫的波蘭微生物保藏所(PCM)的對假單胞菌屬有高裂解活性的噬菌體。
對噬菌體S1-S7和P1-P20的結(jié)果進行了統(tǒng)計學分析以確定對于具體的噬菌體所獲得的p值對于各個屬的細菌的普通群體是否可認為是正確的。該結(jié)果在圖1和2中給出。對大多數(shù)噬菌體計算的n值比研究樣品的細菌株系的數(shù)目小，這提示裂解譜的范圍至少對于在波蘭出現(xiàn)的臨床株系是正確的?？紤]到這些結(jié)果，可認為在表1和表2中列出的噬菌體是關(guān)于本發(fā)明的多價噬菌體。
出乎意料的是，在對葡萄球菌屬的噬菌體的情況中，7種活性最大的噬菌體中的3種即S1、S2和S4或S5顯示對95％的所研究的葡萄球菌屬株系的裂解活性。同樣地，對假單胞菌屬選擇的21種噬菌體中的3種即P7、P20和P6顯示對87％的所研究的葡萄球菌屬株系的裂解活性。
人們也可將遭受粘稠物阻塞癥的患者認為是“出現(xiàn)細菌感染的給定區(qū)域”，如在本發(fā)明的本質(zhì)中所定義的。在對該組患者中出現(xiàn)的病原體的研究中，使用了137個細菌株系，其中的84個鑒定為假單胞菌屬的而53個鑒定為金黃色葡萄球菌。所有的株系均自全波蘭的粘稠物阻塞癥患者的材料中分離。分析所研究的假單胞菌屬和金黃色葡萄球菌株系對噬菌體的易感性使得能夠選擇具有最高裂解活性和最寬的作用范圍的噬菌體。出乎意料的是，在假單胞菌屬的噬菌體的情況中，21種活性噬菌體中的3種，即F/00010、F/00013(原文有誤，譯者)和F/00018顯示對75％的假單胞菌屬株系的裂解活性(關(guān)于成功概率參見圖3，其中噬菌體3＝F/00010、噬菌體6＝F/00013且噬菌體11＝F/00018)。在對葡萄球菌屬選擇的噬菌體中，3種即F/00002、F/00004和F/00007顯示對98％的葡萄球菌屬株系的裂解活性(關(guān)于成功概率參見圖4，其中噬菌體1＝F/00002、噬菌體3＝F/00004且噬菌體4＝F/00007)。
實施例5.使對葡萄球菌屬的噬菌體和對革蘭氏陰性桿菌的噬菌體復(fù)制，從而獲得噬菌體溶菌產(chǎn)物。
將約5％的噬菌體溶菌產(chǎn)物添加到新鮮的4-小時的金黃色葡萄球菌培養(yǎng)物中(對數(shù)期)，該培養(yǎng)物培養(yǎng)于補充了葡萄糖的液體培養(yǎng)基中。通過振蕩使試管或瓶子混合并于37℃溫育3小時。在出現(xiàn)清澈的培養(yǎng)物(裂解)之后，使樣品冷藏至下一日。使裂解物通過孔大小為0.2-0.4μm的膜濾器以去除任何殘存的未裂解的細菌細胞?；畹氖删w顆粒的濃度通過Gratia的雙層方法確定。將不添加噬菌體溶菌產(chǎn)物的溫育的培養(yǎng)物用作對照。
復(fù)制對革蘭氏陰性桿菌(假單胞菌屬)的噬菌體。將蛋白胨水用于復(fù)制假單胞菌屬的噬菌體。將5％的噬菌體溶菌產(chǎn)物添加到新鮮的4-小時的銅綠假單胞菌培養(yǎng)物中，通過振蕩使試管或瓶子混合并于37℃溫育5小時。在培養(yǎng)物清澈之后，使樣品在冰箱中存放至下一日，其后使其通過孔大小如上的膜濾器進行過濾。溶菌液中活的噬菌體顆粒的數(shù)目用Gratia的方法確定。
無菌的液體噬菌體溶菌產(chǎn)物顯示持久的裂解活性。當冷藏時，它們可保持其活力達幾年，一些情況下超過10年。
實施例6.多價噬菌體制劑的生產(chǎn)及應(yīng)用。
由實施例5中描述的方法獲得的源自實施例4中所選的具有廣譜裂解活性的噬菌體的無菌液體溶菌物或其濃縮的或進一步純化的形式(參加下面的實施例)用于多價噬菌體藥物的制備中。
針對葡萄球菌屬的示例性多價噬菌體制劑是含有不同量的噬菌體S1、S2和S4或S5的裂解物或這些裂解物的衍生物的混合物。對于粘稠物阻塞癥患者的葡萄球菌屬引起的感染，應(yīng)該使用來自噬菌體F/00002、F/00004和F/00007的制劑。
針對假單胞菌屬的示例性多價噬菌體制劑是含有不同量的噬菌體P7、P20和P6的裂解物或這些裂解物的衍生物的混合物。對于粘稠物阻塞癥患者的假單胞菌屬引起的感染，應(yīng)該使用來自噬菌體F/00010、F/00013和F/00018的制劑。
該藥物在飯前30分鐘和預(yù)先中和胃液(如用Gel Aluminiphosphorici、純化的蘇打或VIchy水)之后以相當于10ml的裂解物的量每日3次經(jīng)口服給予。當直接向傷口應(yīng)用該制劑時，應(yīng)該在24小時內(nèi)進行3次。通過局部應(yīng)用，傷口不應(yīng)該用消毒劑清潔，這是因為這樣將導致噬菌體的失活。如果需要，傷口可用無菌的培養(yǎng)基或0.9％的NaCl生理溶液進行清潔。
在某些情況中，噬菌體治療可與抗生素治療結(jié)合應(yīng)用。在細菌感染的治療中所用的多價噬菌體制劑已顯示高的效力。在膿毒病的治療(88％的治愈率)和粘稠物阻塞癥患者的感染的治療中觀察到了特別的效力。
實施例7.噬菌體制備物的進一步純化，從含有噬菌體的混合物中去除內(nèi)毒素。
在幾個醫(yī)學應(yīng)用中或者歸因于給予的方法(如靜脈內(nèi)地)，需要高純度的噬菌體制備物，特別不含細菌內(nèi)毒素。根據(jù)本發(fā)明的方法，利用噬菌體對含有纖維素的基質(zhì)，或最適地其酯化的衍生物的親和力。在本實施例中使用一種可購得的纖維素的硫酸衍生物，該衍生物特征在于低水平的酯化(8μmole/ml凝膠)。將該基質(zhì)用于從含有噬菌體的混合物中去除內(nèi)毒素。在浸洗基質(zhì)和去除內(nèi)毒素之后，純化的下一步是洗脫吸附的噬菌體。
將1ml含有纖維素的硫酸衍生物的基質(zhì)加入到無菌層析柱中。通過用以70％乙醇浸泡的玻璃絲封住柱子的底部而防止基質(zhì)從柱子中的流出。
制備了下面的洗脫緩沖液緩沖液I0.01M磷酸鹽緩沖液，pH 7.6；緩沖液II含有1M氯化鈉的0.01M磷酸鹽緩沖液，pH 7.6。
進入洗脫液的組合物的鹽于145℃烘焙1小時。
溶液用蒸餾的無熱原的(apyrogenic)水制備。
層析前，用1ml基質(zhì)填充層析柱，該層析柱用5ml緩沖液I沖洗然后用5ml緩沖液II沖洗。
將柱子用10ml緩沖液I沖洗以備用于實際的層析。內(nèi)毒素的去除是通過用在Sepharose 4B上對濃縮的噬菌體溶菌產(chǎn)物Ps PCMF/00018進行分子篩程序獲得的大分子級分實現(xiàn)。
將0.2ml的噬菌體混合物轉(zhuǎn)移到含有1ml纖維素的硫酸衍生物基質(zhì)的層析柱中。獲取0.2ml體積的級分。第一個級分用3ml緩沖液I洗脫。在這些情況下，未結(jié)合的內(nèi)毒素從柱子中流出。第二個級分用緩沖液II洗脫。級分II含有純化的噬菌體。
級分中內(nèi)毒素單位的結(jié)果加到含有纖維素的硫酸衍生物基質(zhì)上的層析的材料含有2500單位的內(nèi)毒素/ml。用緩沖液I洗脫的級分含有600-1000單位的內(nèi)毒素/ml而用緩沖液II洗脫的級分含無內(nèi)毒素的噬菌體(約1單位/ml)。
噬菌體制備物中內(nèi)毒素的含量用美國Charleston的CharlesRiver Endosafe公司的凝膠方法確定。
以下布達佩斯條約對為專利程序進行的微生物保藏的國際認證各個菌株的保藏證明和存活證明BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposlt made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DF POSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut ofinternational depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DF POSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date ofthe original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNTTION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternationai depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
FormBP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOP THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or dnte of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPFST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date ofthe new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has beenrequested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date ofthe new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DF POSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the infomation has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status ofinternational depositary authority isconverted into a deposit under the Budapest Treaty， such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALPECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority ws acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of th test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATFMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last pege)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international deposiary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date ofthe transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the foliowing page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PUR POSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositay authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DE POSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international deposita ry authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a dcposit under the Budapest Treaty，such date is thc date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acqulred；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNTTION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negativeForrr BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or atansfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PUR POSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut ofinternationaldepositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
權(quán)利要求
1.獲得復(fù)價噬菌體株系的方法，該方法特征在于a)積聚足夠數(shù)目(n)的在給定區(qū)域隨機出現(xiàn)的特定物種的病原體細菌株系，在此分離的細菌株系最適為抗藥性株，b)積聚足夠數(shù)目的對至少一種給定種的細菌株系特異的噬菌體株系，c)確定所積聚的噬菌體株系對每種所積聚的細菌株系的裂解活性，然后估計p值，該值為由給定的噬菌體裂解的細菌株系的數(shù)目與所有積聚的細菌株系的數(shù)目的比例。d)從結(jié)果所得的p值，檢驗n是否滿足條件n≥pqd2z1-α2]]>其中q＝1-pd是不大于0.1的常數(shù)，最適地不大于10％pz1-α是依賴于置信因子1-α的正態(tài)分布的隨機變量，該置信因子不小于0.95e)選擇滿足d)中的標準且具有不小于2/n的p值，最適地不小于0.5的噬菌體株系。
2.權(quán)利要求1的方法，其中在步驟b)中噬菌體株系從源自環(huán)境的樣品中分離，通過使該樣品經(jīng)過孔大小為0.2-0.4μm的膜濾器、向獲得的濾液中添加培養(yǎng)基并使其與確定屬的細菌液體培養(yǎng)物混合、在約37℃的溫度溫育約1小時、取出一部分懸浮液并將其應(yīng)用于有固體培養(yǎng)基的平板、于約37℃溫育2～24小時、分離單個禿斑周圍的培養(yǎng)基樣品、將其轉(zhuǎn)移到該確定屬的細菌的液體培養(yǎng)物中并進行溫育直到培養(yǎng)物發(fā)亮，然后通過使溶菌產(chǎn)物經(jīng)過孔大小為0.2-0.4μm的膜濾器而獲得噬菌體產(chǎn)物，在此優(yōu)選固體培養(yǎng)基的接種和禿斑的再次分離操作重復(fù)5次。
3.權(quán)利要求1或2的方法，其中在步驟c)中所研究的細菌株系接種到各個固體培養(yǎng)基上，在該培養(yǎng)基上轉(zhuǎn)移有一部分在步驟b)中獲得的噬菌體制備物，于約37℃的溫度溫育約4小時，其后將其于約4℃的溫度放置約2～24小時，其后噬菌體株系的裂解活性通過至少單個禿斑的出現(xiàn)而證實。
4.權(quán)利要求1的方法，其中病原體細菌株系是葡萄球菌屬或假單胞菌屬的。
5.權(quán)利要求1或2的方法，該方法包含對溶菌產(chǎn)物的進一步純化，尤其是除內(nèi)毒素，在此使含有噬菌體的混合物與含有纖維素或其至少部分酯化的衍生物相接觸，然后用去除雜質(zhì)，尤其是內(nèi)毒素，的溶液洗滌，其后將純化的噬菌體洗脫。
6.權(quán)利要求5的方法，其中用水、非解離性物質(zhì)的溶液或濃度不大于0.1M的鹽溶液，尤其是緩沖的鹽溶液進行內(nèi)毒素的洗脫。
7.權(quán)利要求5的方法，其中用非解離性物質(zhì)的溶液，或任何緩沖液，或濃度大于0.05M的鹽溶液，尤其是緩沖的鹽溶液進行噬菌體的洗脫。
8.權(quán)利要求5的方法，其中內(nèi)毒素和噬菌體的洗脫在-25℃～100℃之間的溫度中進行。
9.權(quán)利要求5的方法，其中內(nèi)毒素和噬菌體的洗脫用含有機溶劑的水性鹽溶液進行。
10.權(quán)利要求9的方法，其中有機溶劑選自二甲基亞砜、二甲基甲酰胺、異丙醇和丙酮。
11.權(quán)利要求5的方法，其中用以有機或無機酸部分酯化的纖維素作為基質(zhì)。
12.權(quán)利要求5的方法，其中使用以乙酸、硝酸、硫酸或磷酸部分酯化的纖維素。
13.權(quán)利要求5的方法，其特征在于使用其中0.01％～5％的葡萄糖分子，尤其是0.25％～1％的葡萄糖分子被酯化的纖維素。
14.一種治療或預(yù)防由細菌病原體引起的感染的藥物，該藥物含有活性劑和可能的藥物學可接納的載體，其特征在于該活性劑包含對細菌特異的復(fù)價噬菌體株系，其屬于根據(jù)權(quán)利要求1-13的方法中獲得的類型。
15.權(quán)利要求14的藥物，其中對葡萄球菌屬細菌特異的所述復(fù)價噬菌體株系為至少一種選自下述的噬菌體株系S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)，尤其是噬菌體S1、S2和S4或S5。
16.權(quán)利要求14的藥物，其特征在于它用于治療或預(yù)防粘稠物阻塞癥患者中產(chǎn)生的感染，以及作為對葡萄球菌屬細菌特異的復(fù)價噬菌體株系，它含有至少一種選自F/00002、F/00004和F/00007的噬菌體株系。
17.權(quán)利要求14的藥物，其特征在于作為對假單胞菌屬細菌特異的復(fù)價噬菌體株系，它含有至少一種選自下述的噬菌體株系P1(PCMF/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，尤其是噬菌體P7、P20和P6。
18.根據(jù)權(quán)利要求14的藥物，其特征在于它用于治療或預(yù)防粘稠物阻塞癥患者中產(chǎn)生的感染，以及作為對假單胞菌屬細菌特異的復(fù)價噬菌體株系，它含有至少一種選自F/00010、F/00013和F/00018的噬菌體株系。
19.由根據(jù)權(quán)利要求1-13的方法獲得的復(fù)價噬菌體株系在生產(chǎn)用于治療或預(yù)防由病原體細菌引起的感染的藥物中的用途。
20.權(quán)利要求19的用途，其中為生產(chǎn)治療或預(yù)防由葡萄球菌屬細菌導致的感染的藥物，應(yīng)用至少一種選自S1(PCM F/00001)、S2(PCMF/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)的噬菌體株系，尤其是噬菌體S1、S2和S4或S5。
21.權(quán)利要求19的用途，其中為生產(chǎn)治療或預(yù)防粘稠物阻塞癥患者中由葡萄球菌屬細菌導致的感染的藥物，使用至少一種選自F/00002、F/00004和F/00007的噬菌體株系。
22.權(quán)利要求19的用途，其中為生產(chǎn)治療或預(yù)防由假單胞菌屬細菌引起的感染的藥物，應(yīng)用至少一種選自P1(PCM F/00008)、P2(PCMF/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)的噬菌體株系，尤其是噬菌體P7、P20和P6。
23.權(quán)利要求19的用途，其中為生產(chǎn)治療或預(yù)防粘稠物阻塞癥患者中由假單胞菌屬細菌引起的感染的藥物，使用至少一種選自F/00010、F/00013和F/00018的噬菌體株系。
24.對葡萄球菌屬細菌特異的復(fù)價噬菌體株系選自S1(PCMF/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)，尤其是選自S1、S2、S4和S5。
25.對假單胞菌屬細菌特異的復(fù)價噬菌體株系選自P1(PCMF/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，尤其是選自P7、P20和P6。
全文摘要
提供了噬菌體的復(fù)價株系、獲得這些株系的方法及其在治療細菌感染中的應(yīng)用，特別是針對那些抗藥性菌株，尤其是發(fā)生于粘稠物阻塞癥患者中的。
文檔編號A61P11/00GK1541101SQ02815833
公開日2004年10月27日 申請日期2002年7月18日 優(yōu)先權(quán)日2001年7月18日
發(fā)明者B·威必爾達布羅斯卡, B 威必爾達布羅斯卡, M·姆爾茲克, 瓤, A·格斯克, 箍, J·伯拉特恩斯克, 囟魎箍, M·魯薩克－澤拉首斯卡, 澤拉首斯卡, D·斯皮爾, ざ 申請人:波蘭科學院免疫和實驗治療研究所