CN105899078B - 生物学对抗福氏耐格里变形虫的方法，和包含原生动物Willaertia magna的消毒剂 - Google Patents
生物学对抗福氏耐格里变形虫的方法，和包含原生动物Willaertia magna的消毒剂 Download PDFInfo
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Abstract
本发明涉及对抗耐格里属和尤其是福氏耐格里变形虫种的增殖的方法，除了施加于人或动物体的治疗方法外，其特征在于它使用原生动物Willaertia magna，还涉及包含这种原生动物的消毒剂。
Description
技术领域
本发明涉及生物学控制耐格里(Naegleria)属和尤其是福氏耐格里变形虫(Naegleria fowleri(N.f.))的存在及其增殖的新方法。
背景技术
福氏耐格里变形虫是属于Vahlkampfiidae科的非寄生变形虫。这种变形虫在人类引起严重的病理性病症，非常幸运的是这种病症极其罕见(2007年检测到235例)：原发性变形虫脑膜炎(PAM)(Cervantes-Sandoval I,2008；Kemble SK,2012；TW,2010；Su MY,2013)。感染这些非寄生变形虫在用抗生素治疗的约一周和几周内具有灾难性预后(Su MY,2013)。对这种感染没有真正有效的治疗；非常幸运地，所述疾病是罕见的且要求一起具备引发PAM的特定条件。这些特定条件和所需要的接种物在现在仍然未知。然而，一些药物和抗生素似乎影响感染的进展，例如两性霉素B、利福平和咪康唑，其当组合时被证实对2个或3个个体有效。在1992年，文献报告仅7例在PAM后被证实幸存(Gautam PL,2012)且仅在从2岁到14岁的非常年轻的受试者中和治疗和感染留下或多或少大量神经学滞后效应的受试者中。因此存活率甚至低于埃博拉病毒感染。因此，监控和控制这种非寄生变形虫构成越来越重要的当务之急。
通常，众所周知福氏耐格里变形虫在环境中具有广泛分布(Martinez AJ,1997)，因为这种变形虫已从土壤、河水和湖水(Jamerson M,2009)或工业废水和生物膜(GoudotS,2012；S.A.Huws,2005)中分离，其特征在于它与其他非寄生变形虫共处。若干可能致病的细菌，包括嗜肺军团菌(Legionella pneumophila)，具有在非寄生变形虫中生存和复制的成熟机制(Huang SW,2010；De Jonckheere,2011)。此外，已表明核电站通过将河水再加热若干度极大地促进了福氏耐格里变形虫的发育。事实上，福氏耐格里变形虫是嗜热性的，其发育温度范围为25℃到45℃(Visvesvara GS,2007)。
EDF，其在法国运营超过11个核电站，它量化了与水中检测到的福氏耐格里变形虫的水平有关的风险。为了对该风险有更好的理解，EDF的研究和调查部门以及医学研究部门计算了当游泳时死于PAM的风险作为水中福氏耐格里变形虫的浓度的函数。该风险可以用下列方式分解：
1次游泳的风险＝当在水中游泳时吸入"n"个福氏耐格里变形虫的可能性，其中福氏耐格里变形虫以浓度"c"(每次游泳吸入10ml的水)存在
乘以
当已吸入"n"个福氏耐格里变形虫时的死亡概率(根据动物数据建模)。
当选择正常对数模型时，其提供最低估计，且完美符合实际数据(USA,Australia,New Zealand)，获得以下风险：
游泳水中N.f.的量n的浓度风险
1个福氏耐格里变形虫/升，风险＝10-8，即每1亿次游泳一次死亡
10个福氏耐格里变形虫/升，风险＝1.45x 10-7，即每7000000次游泳一次死亡
100个福氏耐格里变形虫/升，风险＝7.24x 10-6，即每140000次游泳一次死亡
1000个福氏耐格里变形虫/升，风险＝1.34x 10-3，即每746次游泳一次死亡
根据Conseil supérieur d’hygiène publique de France(CSHPF)[法国公共卫生高级委员会]的建议，超过100个福氏耐格里变形虫(N.f.)/升的极限值必须导致禁止游泳(尤其参见2004年5月4日CSHPF的意见，与2003年EDF在Bugey、Chooz、Dampierre、Golfech和Nogent的电力生产核电站(CNPE)上进行的使用单氯胺的抗变形虫处理的经验反馈有关)。
在本文中，完全意外地，发明人已经表明，变形虫属Willaertia magna(W.m.)消除非寄生福氏耐格里变形虫。这种杀生物作用通过已经证实的Willaertia magna对于其他细菌性试剂例如细菌嗜肺军团菌、假单胞菌属(Pseudomonas)和李斯特氏菌属(Listeria)的捕食能力来支持。
发明内容
因此本发明的主题首先是用于控制耐格里属和尤其是福氏耐格里变形虫的增殖的方法，其使用Willaertia属原生动物，优选Willaertia magna。根据本发明的方法不包括施加于人或动物体的治疗方法。在根据本发明的方法中，通常用Willaertia属原生动物，尤其是Willaertia magna种处理气流或液流。然而，它也可以是固体表面。
根据本发明的方法尤其用于消毒卫生用水或工业用水分配网络、工厂的冷却回路例如核型或空调网络。它可以实施用于控制水管中、或可以与人或动物食品接触或不接触的表面的生物膜的形成。
可以将原生动物直接添加到待处理的管或网络中循环的水或液体中。也可以将它们喷雾在待消毒的工业网络、烟囱、工厂中，和在待消毒的工业表面上，例如以水溶液诸如气溶胶的形式。
有利地，用于本发明上下文中的原生动物对应于2006年8月21日以保藏号PTA7824保藏于ATCC的菌株，或对应于2006年8月21日以保藏号PTA 7825保藏于ATCC的菌株，这两种菌株以CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE(CNRS)[French NationalCenter for Scientific Research]-3rue Michel Ange-75794PARIS CEDEX 16/France和UNIVERSITE LYON 1CLAUDE BERNARD[Lyon 1Claude Bernard University]-43Boulevarddu 11Novembre 1918-69622VILLEURBANNE Cedex/France的名义保藏。
对应于以保藏号PTA 7824保藏于ATCC的菌株，或对应于以保藏号PTA 7825保藏于ATCC的菌株的属于Willaertia属的原生动物是本发明的组成部分。所述保藏的菌株也描述于PCT国际申请WO2008/043969的公布说明书中。
因此这种原生动物可以用于消毒剂，尤其旨在消除福氏耐格里变形虫和控制福氏耐格里变形虫的增殖和污染。
此外，本发明的主题是包含Willaertia属原生动物，尤其是Willaertia magna的消毒剂。对应于以保藏号PTA 7824保藏于ATCC的菌株，或对应于以保藏号PTA 7825保藏于ATCC的菌株的原生动物将是优选的。有利地，根据本发明的消毒剂为例如在蒸馏水中的水溶液或悬浮液的形式。消毒剂可以是可喷雾形式，例如气溶胶或任何其他的施用方式。
发明人已经通过比较在Willaertia属，尤其是Willaertia magna存在和不存在的情况下福氏耐格里变形虫的复制证实了Willaertia属原生动物，尤其是Willaertia magna对福氏耐格里变形虫增殖的抑制活性。发明人还通过利用非寄生变形虫的另一个种-棘变形虫(Acanthamoeba)作为不引起福氏耐格里变形虫生长抑制的对照菌株证实了通过Willaertia magna属抑制这种福氏耐格里变形虫生长的独特性质。
考虑到不存在福氏耐格里变形虫的治愈性或预防性治疗的情况下，福氏耐格里变形虫对人的死亡率风险在小于一周内大于95％，本发明是控制这种变形虫瘟疫的重要科学进展，其对环境和人具有中性影响。事实上，EC条例No.1271/2008目前承认，Willaertiamagna菌株不在危险种类中，且没有提及任何危险或警告建议，这与目前使用的单氯胺不同。
此外,本发明涉及根据本发明和/或Willaertia属原生动物，尤其是Willaertiamagna的消毒剂，以及对应于以保藏号PTA 7824保藏于ATCC的菌株，或对应于以保藏号PTA7825保藏于ATCC的菌株的原生动物作为耐格里属的杀生物剂的用途。
以下实施例可以阐明本发明，但决不限制本发明。
具体实施方式
1.材料和方法
1.1所使用的菌株
-变形虫：所使用的菌株属于三个不同的变形虫种：
-福氏耐格里变形虫(ATCC 30809)；
-卡氏棘变形虫(Acanthamoeba castellanii(ATCC 30010))；
-Willaertia magna(以保藏号PTA 7824和PTA 7825保藏于ATCC的菌株)
将这三种菌株在10％胎牛血清存在下在SCGYEM培养基(血清酪蛋白葡萄糖酵母提取物培养基)上无菌培养，以每管3ml的比例分配到GREINERTM管。在维持中，每8-9天将营养体型亚培养。对于共培养，使用3到4天的亚培养以便正好在指数生长期中具备营养体。
SCGYEM培养基按照如下获得：
酪蛋白(MERCK,1.02244.010)10g
Na2HPO4 1.325g
KH2PO4 0.8g
葡萄糖 2.5g
酵母提取物(DIFCO 0127-17-9) 5g
蒸馏水 900ml
胎牛血清 100ml
将2.5ml NaOH(1N)、然后将Na2HPO4和KH2PO4添加到900ml蒸馏水中。在加热板上略微加热后，在磁力搅拌下逐渐添加酪蛋白。酪蛋白溶解后，加入葡萄糖和酵母提取物。
完全溶解后，将培养基在玻璃纤维(SARTORIUS SM 6513400)上、然后在1μm膜(WHATMAN 7190 004)上依次过滤。然后将培养基等分入玻璃瓶中。将瓶子在120℃下在高压灭菌器中灭菌20分钟。在最终使用和分配培养基之前，在层流净化罩下以最终容积10％的比例无菌添加胎牛血清。
1.2.制备Willaertia magna和福氏耐格里变形虫的储备悬浮液：
各初始变形虫悬浮液(储备悬浮液)的制备采用SCGYEM培养基中无菌培养的各变形虫的4个烧瓶(T 175ml)来进行。在指数生长期结束时收获福氏耐格里变形虫、卡氏棘变形虫和Willaertia magna的这些储备变形虫悬浮液，这通常在培养开始后4到5天获得。
为了增加所收获的变形虫的浓度，将烧瓶通过冰浴5到10分钟，然后人工搅拌，以尽可能多地收集变形虫。
合并这4个烧瓶的内容物以对在THOMA单元上获得的变形虫悬浮液计数([C]/ml)。
将变形虫悬浮液转移入50ml型管中以通过1500g离心10分钟来去除SCGYEM培养基。用无菌蒸馏水洗涤两次来冲洗第一次离心产生的变形虫沉淀，并在1500g离心10分钟。在2次洗涤结束时，将最终沉淀收集在40ml容积的无菌水中。
1.3.阐明Willaertia magna对福氏耐格里变形虫(菌株ATCC30809)的杀生物作用
在包含10ml无菌PAS(Page的变形虫盐水)培养基的T 25ml烧瓶中进行变形虫共培养。将变形虫共培养的烧瓶以预先在Malassez血细胞计数器上计数的无菌变形虫悬浮液的1×105个Willaertia magna/ml和1×105个福氏耐格里变形虫/ml的比例接种。通过将福氏耐格里变形虫/Willaertia magna之比固定为1来进行福氏耐格里变形虫对Willaertiamagna的侵染。将T 25ml烧瓶在30℃的孵育器中孵育。
PAS培养基按如下获得：
| 溶液2: | |||
| Na2HPO4(无水分子量:141.96) | 14.20g | Fluka 71629-100g | 0.2 |
| KH2PO4(无水分子量:136.09) | 13.60g | Sigma P5655 | 0.2 |
用0.22μm过滤器无菌过滤溶液1和2。为了获得1升PAS培养基：添加5.0ml溶液1和5.0ml溶液2，然后用无菌蒸馏水将容积调节至1升。
将Willaertia magna和福氏耐格里变形虫对照变形虫单培养在T25 ml烧瓶中在10ml灭菌PAS培养基中单独培养。
从预先在Malassez血细胞计数器上计数的无菌变形虫悬浮液，对于Willaertiamagna对照，将烧瓶以1×105个Willaertia magna/ml的比例接种，对于福氏耐格里变形虫对照，将烧瓶以1×105个福氏耐格里变形虫/ml的比例接种。将T 25ml烧瓶在30℃的孵育器中孵育。
各条件进行三次。Malassez单元上的各计数重复5次。实验在3个月时期内重复三(3)次。
共培养和对照烧瓶中福氏耐格里变形虫和Willaertia magna变形虫的命运以下列方式测定：
福氏耐格里变形虫侵扰后监控变形虫浓度120小时。在各时间间隔(在第3小时，然后每24小时)，将共培养和对照烧瓶取样并检查两种变形虫的细胞生长和其形态学和动态。对于各检查的烧瓶：
-在Malassez单元上直接计数变形虫。
-在120小时，由于联合使用MPN(最大可能数量)方法，低水平的福氏耐格里变形虫不再允许Malassez单元上的可靠计数。广泛用于计数变形虫的该方法具有更准确的优点、而且能将完整且活的变形虫与完整但死的变形虫区分开来。
2.结果
图1、2和3显示了共培养(Willaertia magna/福氏耐格里变形虫)和对照单培养变形虫的实验。
它们显示了与单培养中变形虫的各群体的发展相比，初始的Willaertia/耐格里属之比为1的共培养后，Willaertia magna和福氏耐格里变形虫的各群体的自发发展。
图4a和4b显示在Willaertia magna变形虫存在下，福氏耐格里变形虫随时间的生理状态。图4a对应于与Willaertia magna共培养的福氏耐格里变形虫的快速生理学降解的图像。图4b对应于阐明Willaertia magna对福氏耐格里变形虫的“死亡之吻”现象的图像。
图5显示共培养(卡氏棘变形虫/福氏耐格里变形虫)和对照单培养变形虫的实验。
在图1、2和3中，称为Willaertia magna的带有菱形点(◆)的曲线描述了无菌PAS培养基中单独的Willaertia magna的测量浓度。
称为福氏耐格里变形虫的带有方形点(■)的曲线描述了无菌PAS培养基中单独的福氏耐格里变形虫的测量浓度。
称为W+NF的带有三角形点(△)的曲线描述了无菌PAS培养基中与福氏耐格里变形虫共培养的Willaertia magna的测量浓度。
称为NF+W的带有叉形点(X)的曲线描述了无菌PAS培养基中与Willaertia magna共培养的福氏耐格里变形虫的测量浓度。
数据以在Malassez单元上计数的每毫升(ml)完整细胞的浓度来表示。
在图4a中，黑色箭头表示Willaertia magna的存在，白色箭头表示完整或破碎的福氏耐格里变形虫的存在。
在图4b中，黑色箭头表示Willaertia magna的存在，白色箭头表示福氏耐格里变形虫的存在。白色圆体现福氏耐格里变形虫细胞的轮廓。该“死亡之吻”现象已经在文献中(Berke G.Source Department of Cell Biology,1995)描述为允许粒酶透过的接触，其导致靶细胞的凋亡。
在图5中，由菱形点(◆)组成的曲线代表卡氏棘变形虫的单培养。
由三角形点(△)组成的曲线A+NF代表与福氏耐格里变形虫共培养的卡氏棘变形虫的浓度。
由方形点(■)组成的曲线NF代表福氏耐格里变形虫的单培养。
由叉形(X)组成的曲线NF+A代表与卡氏棘变形虫共培养的福氏耐格里变形虫的浓度。
2.1.Willaertia magna完全抑制福氏耐格里变形虫的生长
如表示与Willaertia magna共培养的福氏耐格里变形虫的发展的图1、2和3的叉形曲线所示，福氏耐格里变形虫群体曲线中在约24小时非常迅速地发生分离。这种福氏耐格里变形虫群体的降低仅在Willaertia magna存在下观察到。实验1、2和3(图1、2和3)中单培养的福氏耐格里变形虫的对照曲线(带有方形点的曲线)在24小时均正在生长。此外，单培养福氏耐格里变形虫的水平在24小时几乎加倍。
值得注意的是，显微镜观察早在24小时就证实了与Willaertia magna共培养的福氏耐格里变形虫的形态变化(图4)。福氏耐格里变形虫细胞的生理状态从伸展的营养体形式变为更圆的受应力形式，但与孢囊形式仍然不相似。福氏耐格里变形虫细胞的内部变得不透明，掩盖内部空泡(变形虫生长的标记)。Willaertia magna存在下福氏耐格里变形虫细胞的尺寸早在共培养的3小时比单培养中的尺寸小一半(图4a)。Willaertia magna变形虫的捕食方式基于吞噬作用。图4b表明了在最初24小时内类似于“死亡之吻”的抑制现象。Willaertia magna对福氏耐格里变形虫的吞噬作用确实发生了，但仅在共培养25小时之后通过在这些吞噬体中存在包含福氏耐格里变形虫的Willaertia magna来证实(数据未报告)。
进行的所有实验证实在120小时内完全消除福氏耐格里变形虫的最大抑制效果。Malassez单元在120小时的读数计数了存在且完整的福氏耐格里变形虫细胞，但不可以采用该计数区分死细胞和活细胞。NNA琼脂+大肠杆菌(E.coli)上的双倍读数可以计数仅来自营养体形式和/或来自孢囊形式的福氏耐格里变形虫活细胞。
下表显示通过Malassez单元上的读数获得的和采用MPN方法获得的变形虫(福氏耐格里变形虫)的浓度。
将在感染后120小时取样的来自Willaertia magna/福氏耐格里变形虫共培养的实验1、2和3(图1-3)的各烧瓶的等分试样接种在覆盖有一层大肠杆菌的NNA琼脂上。
各种类的浓度的测定通过计数每个皿中确定的前部数目(number of fronts)来测量，并显示于MPN表中以从其中推断变形虫浓度。
Nf＝福氏耐格里变形虫
W.m＝Willaertia magna
该表证实了NNA琼脂上不存在福氏耐格里变形虫前部，表明在120小时不存在活的孢囊或营养体形式的福氏耐格里变形虫。
发明人表明了Willaertia magna对耐格里属和尤其是福氏耐格里变形虫种的杀生物作用的不可预料的性质不与其他变形虫种共有。
如图5所示，与单培养中测量的这些种的浓度相比，变形虫属卡氏棘变形虫和福氏耐格里变形虫的共培养对两个种的各自浓度不造成任何影响。该图显示棘变形虫类型的另一种变形虫属在这两种变形虫的共培养期间对福氏耐格里变形虫群体的发展不存在相互作用。
在具有抑制耐格里属，尤其是福氏耐格里变形虫种的生长的特征方面，Willaertia magna是相当独一无二的。
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Claims (7)
1.用于控制耐格里属的增殖的方法，施加于人或动物体的治疗方法除外，其特征在于它使用对应于以保藏号PTA 7824保藏于ATCC的菌株，或对应于以保藏号PTA 7825保藏于ATCC的菌株的原生动物Willaertia magna。
2.根据权利要求1所述的方法，用于控制福氏耐格里变形虫的增殖。
3.根据权利要求1所述的方法，其特征在于用所述Willaertia magna菌株处理气流或液流或固体表面。
4.根据权利要求1所述的方法，其特征在于实施该方法以用于消毒卫生用水或工业用水分配网络、工业或核电站的冷却回路、空调网络或工业表面。
5.根据权利要求1所述的方法，其特征在于实施该方法以用于控制水管中、或可以与人或动物食品接触或不接触的表面的生物膜的形成。
6.包含原生动物Willaertia magna的消毒剂作为耐格里属的杀生物剂的用途，施加于人或动物体的治疗方法除外，其特征在于所述原生动物对应于以保藏号PTA 7824保藏于ATCC的菌株，或对应于以保藏号PTA 7825保藏于ATCC的菌株。
7.根据权利要求6所述的用途，其特征在于所述消毒剂为水溶液或悬浮液形式。
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| WO (1) | WO2015059411A1 (zh) |
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| FR3070004B1 (fr) * | 2017-08-10 | 2020-12-25 | Amoeba | Utilisation therapeutique ou non-therapeutique de protozoaires du genre willaertia comme fongistatique et/ou fongicide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5344838A (en) * | 1985-01-18 | 1994-09-06 | Cetylite Industries, Inc. | Sterilant composition |
| WO2013079722A1 (fr) * | 2011-12-02 | 2013-06-06 | Amoeba | Procédé de lutte biologique contre les listeria |
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| FR2906968B1 (fr) | 2006-10-12 | 2010-09-17 | Univ Claude Bernard Lyon | Nouveau procede de lutte biologique contre la proliferation de legionella pneumophila, et nouvel agent desinfectant contenant des protozoaires amibiens du genre willaertia |
| JP5137064B2 (ja) * | 2007-08-24 | 2013-02-06 | 学校法人玉川学園 | 自由生活性アメーバ防除剤 |
| FR2984081A1 (fr) * | 2011-12-20 | 2013-06-21 | Amoeba | Procede de lutte biologique contre les pseudomonas |
| FR2994980B1 (fr) * | 2012-09-05 | 2014-11-14 | Commissariat Energie Atomique | Procede de gazeification de charge de matiere carbonee, a rendement ameliore. |
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- 2014-10-22 EP EP14824878.4A patent/EP3060058B1/fr active Active
- 2014-10-22 JP JP2016525560A patent/JP6165337B2/ja active Active
- 2014-10-22 RU RU2016119629A patent/RU2649091C2/ru active
- 2014-10-22 US US15/030,844 patent/US9894903B2/en active Active
- 2014-10-22 CN CN201480058425.6A patent/CN105899078B/zh active Active
- 2014-10-22 CA CA2928127A patent/CA2928127C/fr active Active
- 2014-10-22 WO PCT/FR2014/052691 patent/WO2015059411A1/fr active Application Filing
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5344838A (en) * | 1985-01-18 | 1994-09-06 | Cetylite Industries, Inc. | Sterilant composition |
| WO2013079722A1 (fr) * | 2011-12-02 | 2013-06-06 | Amoeba | Procédé de lutte biologique contre les listeria |
Also Published As
| Publication number | Publication date |
|---|---|
| FR3012014B1 (fr) | 2016-07-22 |
| EP3060058A1 (fr) | 2016-08-31 |
| CN105899078A (zh) | 2016-08-24 |
| US20160249624A1 (en) | 2016-09-01 |
| EP3060058B1 (fr) | 2019-05-15 |
| RU2016119629A (ru) | 2017-11-28 |
| JP6165337B2 (ja) | 2017-07-19 |
| CA2928127C (fr) | 2018-08-21 |
| JP2016537330A (ja) | 2016-12-01 |
| ES2736023T3 (es) | 2019-12-23 |
| FR3012014A1 (fr) | 2015-04-24 |
| CA2928127A1 (fr) | 2015-04-30 |
| US9894903B2 (en) | 2018-02-20 |
| WO2015059411A1 (fr) | 2015-04-30 |
| RU2649091C2 (ru) | 2018-04-02 |
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