CN115135332A - 艰难梭菌增殖抑制剂 - Google Patents
艰难梭菌增殖抑制剂 Download PDFInfo
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- CN115135332A CN115135332A CN202180015419.2A CN202180015419A CN115135332A CN 115135332 A CN115135332 A CN 115135332A CN 202180015419 A CN202180015419 A CN 202180015419A CN 115135332 A CN115135332 A CN 115135332A
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- Prior art keywords
- clostridium difficile
- enterococcus faecalis
- bacteria
- dead
- culture
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明提供艰难梭菌增殖抑制剂。艰难梭菌增殖抑制剂,其含有粪肠球菌的死菌。本发明也提供艰难梭菌增殖抑制用非口服药物制剂、杀菌剂、消毒剂、抗菌剂、除菌剂或清洗剂。
Description
技术领域
本发明涉及艰难梭菌增殖抑制剂。
背景技术
艰难梭菌感染(Clostridioides(Clostridium)difficile infection,CDI)大多由于由使用抗菌药等造成的消化道菌群的受损而发病。其大部分常使用抗菌药,并且年龄增长、有无基础疾病等为患病因素。
CDI大部分为胃肠道感染,症状多为腹泻、腹痛,有时也伴有发热、白细胞增多,如果发展为重症则有时也会因中毒性巨结肠、肠梗阻、消化道穿孔而致死。
已确认有在消化道带有艰难梭菌而不显现症状的“携带者”,在经常使用抗菌药的住院治疗患者中,这样的“携带者”较为多见。艰难梭菌与CDI患者及无症状携带者的粪便一起被***,并污染包括医疗工作者的手指在内的环境,从而作为医疗相关感染成为问题。因此,其为在需要进行如换尿布这样的***护理的老年人较多住院,且没有充分进行包括对抗菌药的合理使用的感染管理的医疗现场中常见的感染。但是,在滥用抗菌药、没有充分进行感染管理的医疗机构中,由于对CDI的关注及知识较低,因此没有适当诊断本疾病而表面上发生率较低的情况常被视为问题。
像这样,在医疗机构、老年人设施中,CDI处于容易慢性化的环境,且正在对采取感染预防对策的必要性进行讨论。
现有技术文献
非专利文献
非专利文献1
艰难梭菌感染的基础和临床,Modern media,56卷10号,2010
www.eiken.co.jp/modern_media/backnumber/pdf/MM1010_01.pdf
发明内容
发明要解决的问题
本发明的目的在于提供艰难梭菌增殖抑制剂。
用于解决问题的手段
本发明人等进行了深入研究,结果发现粪肠球菌(Enterococcus faecalis)的死菌能够抑制艰难梭菌的增殖,从而完成了本发明。
本发明的要点如下所述。
(1)艰难梭菌增殖抑制剂,其含有粪肠球菌的死菌。
(2)艰难梭菌增殖抑制用非口服药物制剂,其含有粪肠球菌的死菌。
(3)艰难梭菌增殖抑制用杀菌剂、消毒剂、抗菌剂、除菌剂或清洗剂,其含有粪肠球菌的死菌。
(4)外用药物,其含有粪肠球菌的死菌。
(5)卫生用品,其含有粪肠球菌的死菌。
发明效果
根据本发明,能够抑制艰难梭菌的增殖。
本说明书包含作为本申请的优先权的基础的日本专利申请特愿2020-43740的说明书和/或附图中记载的内容。
附图说明
[图1]示出实施例1的试验结果(活菌数(×103CFU/mL))。○:对照组,□:低浓度组(被测物质浓度:0.64mg/mL),△:高浓度组(被测物质浓度:64mg/mL)。和对照组比较时的显著差异(**:p<0.01)。
具体实施方式
以下,更详细地对本发明的实施方式进行说明。
本发明提供艰难梭菌增殖抑制剂,其含有粪肠球菌的死菌。本发明也提供使用粪肠球菌的死菌来抑制艰难梭菌的增殖的方法。另外,本发明提供抑制艰难梭菌的增殖的方法中所使用的药剂,其含有粪肠球菌的死菌。
粪肠球菌(Enterococcus faecalis)作为具有生物应答调节物质(BiologicalResponse Modifier,BRM)活性(药学志112:919-925 1992;药学志113:396-399 1992;动物临床医学3:11-20 1994)的乳球菌而为人所知。粪肠球菌EF-2001株可从日本Berumu株式会社(东京都千代田区永田町2-14-3)获得。
粪肠球菌-2001株可取自正常人的粪便,且具有下述性质。
为革兰氏阳性球菌。菌落外形(Trypto-Soya琼脂培养基,培养24小时):直径1.0mm,光滑,正圆形,白色菌落形态:球形至卵圆形(1.0×1.5μm),在液体培养基中常形成链。不形成芽胞。兼性厌氧。使葡萄糖发酵而产生乳酸(最终pH4.3)。不产气。过氧化氢酶阴性。在10~45℃增殖(最适为37℃)。在pH9.6、6.5%NaCl、40%胆汁中增殖。0.04%亚碲酸钾阳性。0.01%四唑鎓阳性。0.1%亚甲基蓝乳阳性。使精氨酸水解。使苦杏仁苷、纤维二糖、果糖、半乳糖、葡萄糖、甘油、乳糖、麦芽糖、甘露糖、甘露醇、核糖、水杨苷、蔗糖、松三糖、山梨糖醇发酵而产生酸。60℃、30分钟耐性。使酪蛋白、明胶消化。将酪氨酸脱羧成为酪胺。兰斯菲尔德(Lancefield)抗原分组D。GC%35.0±1.0%
粪肠球菌可以是死菌,也可以对菌体实施破坏处理(均质处理、酶处理、超声波处理等)、加热、干燥(冷冻干燥、喷雾干燥等)等处理。活菌可通过加热处理而成为死菌。可对粪肠球菌的死菌期待肠道免疫活化作用。菌体粒径可为0.05μm~50μm,优选为0.08~20μm,更优选为0.1~10μm。菌体可在和稀释剂混合后,添加糊料而制成颗粒状。稀释剂或糊料可从作为添加到食品或药物中的物质而被许可的材料中选择。
粪肠球菌的死菌对艰难梭菌的增殖抑制性地起作用。通过抑制艰难梭菌的增殖,可预防和/或治疗艰难梭菌感染。可将粪肠球菌的死菌作为非口服制剂的有效成分而用于药物。另外,可将粪肠球菌的死菌作为杀菌剂的有效成分而用于药物、准药品。可将粪肠球菌的死菌作为消毒剂的有效成分而用于医疗器械的杀菌、外用(人、动物的皮肤)。可将粪肠球菌的死菌作为抗菌剂或除菌剂的有效成分而用于卫生用品。此外,通过将粪肠球菌的死菌用于身体、器具、设备、设施等的清洗,可抑制艰难梭菌的增殖。因此,本发明提供艰难梭菌增殖抑制用杀菌剂、消毒剂、抗菌剂、除菌剂或清洗剂,其含有粪肠球菌的死菌。另外,本发明也提供外用药物,其含有粪肠球菌的死菌。并且,本发明也提供卫生用品,其含有粪肠球菌的死菌。
在本发明中,在使用粪肠球菌的死菌进行杀菌、消毒、除菌或清洗时,可将粪肠球菌的死菌应用于人、非人动物的身体表面(皮肤、粘膜等)。或者,可将粪肠球菌的死菌应用于人、非人动物的身体可能接触的器具、设备、设施等的表面,也可喷雾或散布到人、非人动物呼吸的空间。
可将含有粪肠球菌的死菌的杀菌剂、消毒剂、抗菌剂、除菌剂或清洗剂制剂化为喷雾剂(气雾剂、泵压喷雾剂)、贴剂(水性型糊剂、油性型膏剂等)、软膏剂、霜剂、凝胶剂、外用固体剂、外用液剂(搽剂、乳液剂等)等剂型,而用作外用药物(外用药物组合物)、准药品或卫生用品。
作为制剂的基剂,可以使用烃类(凡士林、液态石蜡等)、高级脂肪酸及其酯类(己二酸、肉豆蔻酸、棕榈酸、硬脂酸、它们的酯等)、蜡类(蜂蜡、羊毛脂等)、高级醇(鲸蜡醇、硬脂醇等)等油性成分、水、多元醇(甘油、1,3-丙二醇、1,2-丙二醇等)、低级醇(乙醇、异丙醇等)等水性成分、表面活性剂(硬脂酸甘油酯、山梨醇酐脂肪酸酯、聚氧乙烯醇醚、羧酸盐、硫酸酯盐等)、保存剂(对羟基苯甲酸酯类等)、抗氧化剂(亚硫酸氢钠、抗坏血酸等)、pH调节剂(柠檬酸水合物、乳酸、乙酸等)等。
粪肠球菌的死菌在制剂中的含量根据制剂的种类而有所不同,通常为0.001~100质量%,优选为0.01~100质量%。
粪肠球菌的死菌的使用量只要为能够确认到艰难梭菌的增殖抑制效果的量即可,根据剂型、应用部位、患者的年龄、体重、基础疾病的有无、种类等而有所不同,例如,在成人的情况下,每次的使用量可换算为粪肠球菌的死菌的量,对皮肤或鼻腔、口腔内粘膜以0.01~500mg/mL左右、优选为0.05~300mg/mL左右、更优选为0.1~100mg/mL左右的浓度,1天使用1次至多次(例如2次、3次、4次、5次左右)。在将粪肠球菌的死菌应用于人、非人动物以外(应用于例如器具、设备、设施等的表面、空间)时,也能够以和上述相同的使用量使用。
粪肠球菌的死菌也可添加到纱布、脱脂棉、酒精棉、棉棒、绷带、口罩、手套、创可贴、外科胶带、擦拭用品(毛巾、片材等)、湿巾布、湿毛巾、肥皂、***用凝固剂等中。
含有粪肠球菌的死菌的非口服药物制剂可以制剂化为外用剂[喷雾剂(气雾剂、泵压喷雾剂)、贴剂(水性型糊剂、油性型膏剂等)、软膏剂、霜剂、凝胶剂、外用固体剂、外用液剂(搽剂、乳液剂等)]、注射剂、输液、眼科用剂(滴眼剂、洗眼剂、眼软膏剂等)、栓剂等剂型,而用作药物。
作为外用剂(喷雾剂等)的基剂,可以使用烃类(凡士林、液态石蜡等)、高级脂肪酸及其酯类(己二酸、肉豆蔻酸、棕榈酸、硬脂酸、它们的酯等)、蜡类(蜂蜡、羊毛脂等)、高级醇(鲸蜡醇、硬脂醇等)等油性成分、水、多元醇(甘油、1,3-丙二醇、1,2-丙二醇等)、低级醇(乙醇、异丙醇等)等水性成分、表面活性剂(硬脂酸甘油酯、山梨醇酐脂肪酸酯、聚氧乙烯醇醚、羧酸盐、硫酸酯盐等)、保存剂(对羟基苯甲酸酯类等)、抗氧化剂(亚硫酸氢钠、抗坏血酸等)、pH调节剂(柠檬酸水合物、乳酸、乙酸等)等。外用剂可应用于皮肤、鼻腔或口腔粘膜。
注射剂及输液可以使用溶剂[水性溶剂(蒸馏水、生理盐水、林格氏溶液等)、非水性溶剂(乙醇、1,2-丙二醇、植物油等)]、增溶剂(谷氨酸、天冬氨酸、聚山梨糖醇酯80等)、保存剂(苯扎氯铵、苄索氯铵等)、稳定剂(亚硫酸盐、焦亚硫酸钠等)、乳化剂、悬浮剂(卵磷脂、单硬脂酸铝等)、缓冲剂(酸、碱、柠檬酸盐等)、着色剂等而进行制剂化。制剂可在制造的最终工序中杀菌,或者利用无菌操作法制造。另外,也可制造无菌的冷冻干燥品,并在其使用前溶解于无菌注射用蒸馏水或其他溶剂中而使用。注射剂及输液可应用到皮肤内或通过皮肤或粘膜而应用到体内。也可通过鼻腔而应用。作为施用法,可列举:皮内施用、皮下施用、肌内施用、静脉内施用、鼻腔内施用。
滴眼剂可以使用溶剂[水性溶剂(灭菌纯化水、生理盐水等)、非水性溶剂(乙醇、1,2-丙二醇、植物油等)]、缓冲液、等渗剂、保存剂(对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、苯扎氯铵、苄索氯铵等)、增稠剂(甲基纤维素、硫酸软骨素等)、悬浮剂(非离子性表面活性剂等)、着色剂等而进行制剂化。
洗眼剂可以使用生理盐水、硼酸溶液、苄索氯铵、缓冲液等制备。
眼软膏剂可以使用烷烃、液态石蜡等作为基剂,并根据需要添加保存剂、稳定剂等而进行制剂化。
栓剂可以使用基剂[油脂性基剂(可可脂、椰子油、棕榈仁油等)、水溶性基剂(甘油明胶、聚乙二醇等)]、表面活性剂(卵磷脂、胆固醇等)等而进行制剂化。
粪肠球菌的死菌在药物制剂中的含量根据制剂的种类而有所不同,通常为0.001~100质量%,优选为0.01~100质量%。
粪肠球菌的死菌的施用量只要为能够确认到艰难梭菌的增殖抑制效果的量即可,根据剂型、应用部位、患者的年龄、体重、基础疾病的有无、种类等而有所不同,例如,在成人的情况下,每次的施用量可换算为粪肠球菌的死菌的量,以0.01~500mg/mL左右、优选为0.05~300mg/mL左右、更优选为0.1~100mg/mL左右的浓度,1天施用1次至多次(例如2次、3次、4次、5次左右)。
另外,也可以通过将本发明的抗菌剂涂布于产品上或捏合到产品中,制造抗菌毛巾、抗菌塑料餐具产品、抗菌玩具类、抗菌PC相关产品、抗菌文具等。
实施例
以下结合实施例详细地对本发明进行说明,但本发明并不限定于这些实施例。
[实施例1]乳酸菌EF-2001株对于艰难梭菌的作用确认试验
概要
对乳酸菌对于艰难梭菌的作用进行研究。
相对于接种菌液1mL,以0.65mL的比例添加乳酸菌的低浓度液(1.63mg/mL)或高浓度液(163mg/mL),在乳酸菌的浓度为0.64mg/mL或64mg/mL的条件下对艰难梭菌进行厌氧培养。测定培养开始时、培养后12小时、24小时及48小时的活菌数。
在乳酸菌的低浓度(0.64mg/mL)及高浓度(64mg/mL)条件下,均在培养后12小时、24小时及48小时和对照组相比,确认到艰难梭菌的活菌数显著减少。由此推测乳酸菌对艰难梭菌的增殖抑制性地起作用。
材料及方法
1.被测物质
1.1.被测物质
名称:乳酸菌粉末EF-2001(日本Berumu株式会社)(经加热杀菌的粪肠球菌,直径为500nm=0.5μm)
性状:黄褐色粉末
保管条件:室温(18.0~28.0℃),遮光,防湿
保管场所:试验设施的被测物质保管室的保管库
2.施用样本
2.1.被测物质的制备方法
称量(电子天平:XP205DR,Mettler Toledo株式会社)必要量的乳酸菌粉末EF-2001,利用GAM肉汤(参照15.3.4.)以成为500mg/mL的方式制备。使用该500mg/mL溶液,利用GAM肉汤以成为163mg/mL及1.63mg/mL的方式稀释。由于乳酸菌粉末会沉淀,因此充分搅拌并悬浮。临用时制备。
3.病原微生物
3.1.使用菌株
艰难梭菌(Clostridioides(Clostridium)difficile)(ATCC43255,下文称为艰难梭菌(C.difficile))
3.2.保存条件
在超低温冰箱(设定温度:-80℃,MDF-394AT,三洋电机株式会社)中冻存至使用时。
3.3.试剂
(1)GAM琼脂培养基(日水制药株式会社)
(2)GAM肉汤培养基(日水制药株式会社)
(3)生理盐水(大塚制药工厂株式会社)
3.4.GAM肉汤的制备
称量GAM肉汤粉末29.5g,悬浮于注射用水(大塚制药工厂株式会社)500mL中,然后进行高压灭菌器(LSX-500,Tomy Seiko株式会社)处理(115℃,15分钟)。制备后在冷藏条件下保存。
3.5.前培养
将艰难梭菌(C.difficile)的保存菌株解冻,接种至GAM琼脂培养基中,放入填充有脱氧剂的厌氧袋中,在设定37℃的恒温器(ILE800,Yamato科学株式会社)中培养5天。培养后,将菌落挑起,加入GAM肉汤培养基中,放入填充有脱氧剂的厌氧袋中,在设定37℃的恒温器中培养2天。将培养后的培养液作为接种菌原液。
3.6.接种菌液的制备及活菌数测定
将利用GAM肉汤培养基将接种菌原液稀释10倍而成的溶液作为接种菌液。
关于活菌数确认,取接种菌液的一部分,用生理盐水适当稀释,涂抹到GAM琼脂培养基后,放入填充有脱氧剂的厌氧袋中,在设定37℃的恒温器中培养5天。利用手动菌落计数器(CC-1,Azwan株式会社)测量培养后的菌落数,并算出接种菌液1mL中所含的活菌数。其结果,接种菌液浓度为2.8×105CFU/mL。
使用后的剩余接种菌液在进行高压灭菌器(LSX-500,Tomy Seiko株式会社)处理(121℃,15分钟)后废弃。
4.试验方法
4.1.增殖抑制试验
4.1.1.试验组构成
显示浓度为混合后的最终浓度。
*:添加有GAM肉汤。
4.1.2.试验方法
相对于接种菌液1mL,分别以0.65mL的比例向试管中添加所制备的被检溶液(1.63mg/mL液及163mg/mL液),放入填充有脱氧剂的厌氧袋中,在设定37℃的恒温器中培养。在培养开始时、培养12、24及48小时后分别取出试管,取培养液的一部分,并适当稀释,将培养液原液、稀释液涂抹到CCFA培养基(日本Becton Dickinson株式会社),然后在设定37℃的恒温器中厌氧培养5天。利用手动菌落计数器测量培养后的菌落数,并算出活菌数。作为对照,添加GAM肉汤代替被检溶液。样本数设为5。
5.结果汇总
关于活菌数,算出平均值及标准误差。
对活菌数,以对照组相对于样本组,使用威尔科克森秩和检验(Wilcoxon ranksum test)进行显著差异检验。
显著性水平是将5%设为显著,并分别分成低于5%及低于1%进行表示。
需要说明的是,统计分析使用市售的统计程序(SAS***:SAS Institute Japan)。
试验成果
将试验结果示于表1、附录1-1~1-3及图1。
对照组的艰难梭菌的活菌数在培养开始时为360.0±75.9(×103CFU/mL),培养12小时后为3680.0±239.6(×103CFU/mL),培养24小时后为3880.0±475.8(×103CFU/mL),培养48小时后为33020.0±2267.5(×103CFU/mL)。
低浓度组的艰难梭菌的活菌数在培养开始时为306.0±22.7(×103CFU/mL),培养12小时后为33.2±4.9(×103CFU/mL),培养24小时后为27.1±6.7(×103CFU/mL),培养48小时后为15.7±4.9(×103CFU/mL)。和对照组相比,在培养后12小时、24小时及48小时确认到活菌数显著减少。
高浓度组的艰难梭菌的活菌数在培养开始时为300.0±22.1(×103CFU/mL),培养12小时后为12.0±1.1(×103CFU/mL),培养24小时后为8.4±3.4(×103CFU/mL),培养48小时后为5.7±3.2(×103CFU/mL)。和对照组相比,在培养后12小时、24小时及48小时确认到活菌数显著减少。
考察
通过经时性地测定活菌数来研究乳酸菌对于艰难梭菌的作用。
在乳酸菌的低浓度(0.64mg/mL)及高浓度(64mg/mL)条件下,均在培养后12小时、24小时及48小时和对照组相比,确认到艰难梭菌的活菌数显著减少。由此推测乳酸菌对艰难梭菌的增殖抑制性地起作用。
(表1)
表1.活菌数
各值表示平均值(×103CFU/mL)±S.E.
和对照组有显著差异(**:p<0.01)
(附录1-1~1-3)
附录1-1各组活菌数
对照组
单位:×103CFU/mL
附录1-2各组活菌数
低浓度组
单位:×103CFU/mL
和对照组有显著差异(**:p<0.01)
NS:和对照组没有显著差异
WL:利用威尔科克森检验进行分析
附录1-3各组活菌数
高浓度组
单位:×103CFU/mL
和对照组有显著差异(**:p<0.01)
NS:和对照组没有显著差异
WL:利用威尔科克森检验进行分析
[产品例1]除菌液、抗菌喷雾剂、除菌片
以下述配方制备除菌液。
(配方)
乳酸菌粉末EF-2001(日本Berumu株式会社)(经加热杀菌的粪肠球菌,直径为500nm=0.5μm)0.6质量%
甘油0.05质量%
癸酸单甘油酯0.50质量%
乙醇50质量%
纯化水余量
总量100质量%
将上述溶液填充到市售的泵压式喷雾容器中,制造抗菌喷雾剂。另外,将上述溶液浸泡到纱布或纸巾上而制造除菌片。
将本说明书所引用的所有刊物、专利及专利申请直接作为参考而并入本说明书中。
工业实用性
本发明可用于艰难梭菌感染的预防和/或治疗、杀菌、消毒、除菌、清洗等。
Claims (5)
1.艰难梭菌增殖抑制剂,其含有粪肠球菌的死菌。
2.艰难梭菌增殖抑制用非口服药物制剂,其含有粪肠球菌的死菌。
3.艰难梭菌增殖抑制用杀菌剂、消毒剂、抗菌剂、除菌剂或清洗剂,其含有粪肠球菌的死菌。
4.外用药物,其含有粪肠球菌的死菌。
5.卫生用品,其含有粪肠球菌的死菌。
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PCT/JP2021/008920 WO2021182377A1 (ja) | 2020-03-13 | 2021-03-08 | クロストリディオイデス ディフィシル菌増殖抑制剤 |
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JP4903559B2 (ja) * | 2004-03-16 | 2012-03-28 | コンビ株式会社 | 家畜・家禽類又は魚介類の感染防除剤 |
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CHOI ET AL.: "Heat-Killed Enterococcus faecalis EF-2001 Ameliorates Atopic Dermatitis in a Murine Model", NUTRIENTS, vol. 8, 5 March 2016 (2016-03-05), pages 1 - 11 * |
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MALAMOU ET AL.: "Inhibition of Clostridium difficile by faecal streptococci", J MED MICROBIOL, vol. 15, no. 4, 30 November 1982 (1982-11-30), pages 569 - 574 * |
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US20230144328A1 (en) | 2023-05-11 |
CA3171324A1 (en) | 2021-09-16 |
AU2021235383A1 (en) | 2022-11-10 |
KR20220154089A (ko) | 2022-11-21 |
JPWO2021182377A1 (zh) | 2021-09-16 |
EP4119150A1 (en) | 2023-01-18 |
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