CN114431232A - 一种抑制葡萄球菌黄素合成的抗菌剂及其应用 - Google Patents
一种抑制葡萄球菌黄素合成的抗菌剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种抑制葡萄球菌黄素合成的抗菌剂及其应用,涉及微生物抗菌剂领域,该抗菌剂的组分包括百里酚和二甲基亚砜,其中,二甲基亚砜为溶剂。以及该抗菌剂在抑制金黄色葡萄球菌黄素合成中的应用。本发明将百里酚的二甲基亚砜溶液作为抑制葡萄球菌黄素合成的抗菌剂,在不影响菌株生长活力条件下抑制其葡萄球菌黄素合成,既防控耐甲氧西林金黄色葡萄球菌感染,又不引起金黄色葡萄球菌耐药性。
Description
技术领域
本发明涉及微生物抗菌剂领域,尤其涉及一种抑制葡萄球菌黄素合成的抗菌剂及其应用。
背景技术
食源性致病菌是威胁食品安全和公共卫生健康的重要危险因素之一,由其引发的食源性致病菌感染事件在世界范围内受到了广泛关注。金黄色葡萄球菌作为主要的革兰氏阳性致病菌之一,能够定植于人体鼻腔、皮肤等,在人体免疫力下降时诱发皮肤和软组织感染,进而发展成严重的菌血症和侵袭性疾病(血流感染、心内膜炎、败血症等)。值得注意的是,近年来,由于人们对抗生素药物的不合理使用,使得多重耐药金黄色葡萄球菌特别是耐甲氧西林金黄色葡萄球菌(Methicillin-Resistant Staphylococcus aureus,MRSA)出现并广泛流传于食品产业链和临床感染中。相较于金黄色葡萄球菌,MRSA菌株介导的临床感染症状更加难以治愈,成为威胁人类生命健康的重要危险因素之一。
传统的治疗MRSA菌株感染的方法主要基于使用抗生素类药物直接抑制菌株的生长活性,从而减轻菌株的感染症状。然而,这种方法往往容易诱导菌株产生耐受性及耐药性,从而导致抗生素类药物作用效力的降低,增加MRSA菌株临床感染的治疗难度及治疗成本。“抗毒力疗法”是近年来新兴的一种耐药菌感染治疗策略,其主要通过降低耐药菌株的毒力作用,从而减轻其对人体的感染症状,间接增强人体免疫系统对耐药菌株的免疫清除作用。同时,“抗毒力疗法”不会对致病菌产生直接的生存活力影响,所以不易诱导菌株耐药性的产生,显示出了较强的应用潜力。
中草药植物富含多种天然活性物质,同时具有分布广、作用稳定、毒副作用小等特点,已被作为重要的药用活性成分来源用于多种食源性致病菌的感染治疗中。百里酚又名5-甲基-2-异丙基苯酚,常温下为无色晶体或无色结晶粉末,天然存在于唇形科植物百里香草、麝香草、牛至草、香青兰、伞形科植物粗果芹种子中,已被作为香料原料、一般药物等获得广泛应用,然而百里酚作为治疗MRSA菌株感染的“抗毒力”药物,在不影响MRSA菌株生长活力的条件下有效抑制其葡萄球菌黄素合成的作用尚未见报道。
鉴于上述事实,本领域技术人员致力于发现能够抑制葡萄球菌黄素合成的活性成分,开发具有新作用机制的抗菌剂,既防控耐甲氧西林金黄色葡萄球菌感染,又不引起金黄色葡萄球菌耐药性。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是开发一种抑制葡萄球菌黄素合成的抗菌剂,既防控耐甲氧西林金黄色葡萄球菌感染,又不引起金黄色葡萄球菌耐药性。
为实现上述目的,本发明提供了一种抑制葡萄球菌黄素合成的抗菌剂,其组分包括百里酚和二甲基亚砜,其中,二甲基亚砜为溶剂。
进一步地,抗菌剂的最小抑菌浓度为170μg/mL。
进一步地,抗菌剂不影响菌株生长活力条件下抑制葡萄球菌黄素的浓度为50μg/mL。
本发明还提供了该抗菌剂在抑制金黄色葡萄球菌黄素合成中的应用。
进一步地,应用的方法包括以下步骤:
步骤1、配制抗菌剂,得到含有不同浓度的百里酚的抗菌剂;
步骤2、将步骤1得到的不同浓度的抗菌剂混合到金黄色葡萄球菌菌液中,并在37℃条件下进行第一共培养,得到第一共培养菌液,检测第一共培养菌液中金黄色葡萄球菌的生长曲线;得出不影响金黄色葡萄球菌生长活力的抗菌剂的亚抑制浓度;
步骤3、将浓度为步骤2得到的亚抑制浓度的抗菌剂混合到金黄色葡萄球菌菌液中,并在37℃条件下进行第二共培养,得到第二共培养菌液,使用甲醇提取第二共培养菌液,得到甲醇提取物;测定甲醇提取物中葡萄球菌黄素的含量。
进一步地,金黄色葡萄球菌为耐甲氧西林金黄色葡萄球菌标准菌株。
进一步地,金黄色葡萄球菌为耐甲氧西林金黄色葡萄球菌标准菌株ATCC BAA-1717。
进一步地,金黄色葡萄球菌菌液的培养步骤为:接种冻存的金黄色葡萄球菌在TSB培养基进行纯化培养,随后使用0.85%生理盐水洗涤重悬菌体,并调整菌液的OD600值至0.5,再使用TSB培养基稀释菌液1000倍。
进一步地,步骤1中抗菌剂的配制步骤还包括采用二甲基亚砜作溶剂,配制百里酚浓度为100mg/mL的的母液,再稀释至不同浓度。
进一步地,步骤2中第一共培养的时间为24小时,步骤3中第二共培养的时间为24小时。
进一步地,抗菌剂的亚抑制浓度为50μg/mL。
进一步地,应用方法步骤2中还包括以下步骤:利用生长曲线测定仪检测第一共培养菌液中金黄色葡萄球菌的生长曲线;得出不影响金黄色葡萄球菌生长活力的亚抑制条件下的抗菌剂中百里酚的浓度为50μg/mL;
进一步地,应用方法步骤3中还包括以下步骤:使用百里酚浓度为50μg/mL的抗菌剂与不含百里酚的对照试剂分别混合到金黄色葡萄球菌菌液中,并在37℃条件下进行第二共培养,分别得到第二共培养菌液,使用甲醇分别提取第二共培养菌液,得到甲醇提取物;分别测定甲醇提取物中葡萄球菌黄素的含量,计算葡萄球菌黄素合成抑制率。
进一步地,步骤3中不含百里酚的对照试剂为二甲基亚砜。
在本发明的较佳实施方式中,建立含有不同浓度的百里酚的抗菌剂作用下耐甲氧西林金黄色葡萄球菌的生长曲线图,并说明该抗菌剂的最小抑菌浓度和亚抑制浓度;
在本发明的另一较佳实施方式中,详细说明含有百里酚浓度为50μg/mL的抗菌剂作用下对耐甲氧西林金黄色葡萄球菌的葡萄球菌黄素合成的抑制率。
本发明将百里酚的二甲基亚砜溶液作为抑制葡萄球菌黄素合成的抗菌剂,产生意想不到的效果。技术效果为利用植物源天然活性成分百里酚在不影响菌株生长活力条件下抑制其葡萄球菌黄素合成,采用的“抗毒力疗法”策略缓解和解决金黄色葡萄球菌耐药性导致的日益严重的公共卫生安全隐患,为防控耐甲氧西林金黄色葡萄球菌感染提供新的方法,在食品安全、药品开发和公共卫生保障领域具有广泛的应用潜力。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例1的含有不同浓度的百里酚的抗菌剂作用下耐甲氧西林金黄色葡萄球菌的生长曲线图;
图2是本发明的另一个较佳实施例2的含有百里酚浓度为50μg/mL的抗菌剂作用下耐甲氧西林金黄色葡萄球菌的葡萄球菌黄素合成率。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
实施例1:含有不同浓度的百里酚的抗菌剂作用下耐甲氧西林金黄色葡萄球菌的生长曲线测定。
以耐甲氧西林金黄色葡萄球菌标准菌株ATCC BAA-1717为实验菌株,设置一系列浓度梯度的百里酚处理组,探究其对耐甲氧西林金黄色葡萄球菌生长活力的影响。
首先蘸取冻存于-80℃的上述菌液,将其划线接种至胰蛋白胨大豆琼脂(TSA)培养基上,放置于37℃恒温恒湿培养箱中培养12h。
挑取TSA培养基上的单菌落至胰蛋白胨大豆肉汤(TSB)中,放置于37℃、180r/min的摇床中培养6h。
吸取1mL上述菌液至1.5mL离心管中,在8000r/min的条件下离心1min,弃净上清后加入0.85%生理盐水吹打洗菌,混匀后再进行离心操作(8000r/min,1min),重复3次。
加入1mL 0.85%生理盐水重悬菌体,并将菌液的OD600值调节至0.5,再用TSB培养基稀释1000倍,备用。
用二甲基亚砜(DMSO)作溶剂,配制100mg/mL的百里酚母液,并使用DMSO将其分别稀释至17/16/15/10/9/8/7/6/5/4/3/2/1mg/mL。
为保证培养体系中DMSO不超过1%,于生长曲线板中每孔加入297μL上述菌液及3μL上述各浓度百里酚溶液,并利用生长曲线测定仪检测百里酚作用下MRSA菌株的生长曲线(37℃,24h,间隔30min)。实验结果如图1所示,抗菌剂中百里酚对MRSA标准菌株ATCC BAA-1717的最小抑菌浓度为170μg/mL,亚抑制浓度为50μg/mL。
实施例2:含有百里酚的抗菌剂对耐甲氧西林金黄色葡萄球菌黄素合成的抑制作用测试。
以耐甲氧西林金黄色葡萄球菌标准菌株ATCC BAA-1717为实验菌株,设置百里酚(0μg/mL)及百里酚(50μg/mL)两个处理组探究其对耐甲氧西林金黄色葡萄球菌黄素合成的影响。
首先蘸取冻存于-80℃的上述菌液,将其划线接种至胰蛋白胨大豆琼脂(TSA)培养基上,放置于37℃恒温恒湿培养箱中培养12h。
挑取TSA培养基上的单菌落至胰蛋白胨大豆肉汤(TSB)中,放置于37℃、180r/min的摇床中培养6h。
吸取1mL上述菌液至1.5mL离心管中,在8000r/min的条件下离心1min,弃净上清后加入0.85%生理盐水吹打洗菌,混匀后再进行离心操作(8000r/min,1min),重复3次;随后使用0.85%生理盐水调整菌液浓度至OD600=0.5。
使用DMSO配制浓度为5mg/mL的百里酚母液,随后吸取300μL该母液至30mL TSB中,充分混匀后再接种300μL上述菌液,置于37℃、180r/min条件下摇床培养24h;直接接种300μL上述菌液至30mL TSB中,置于37℃、180r/min条件下摇床培养24h。
将培养完成后的菌液转移至50mL离心管中,在8000r/min的条件下离心5min,弃净上清后加入0.85%生理盐水吹打洗菌,混匀后再进行离心操作(8000r/min,5min),重复3次。
向菌沉中加入5mL甲醇,充分吹打混匀,随后放入恒温水浴锅中于55℃下避光处理3h以完成葡萄球菌黄素的提取。
将50mL离心管在8000r/min的条件下离心10min,随后吸取200μL上清至96孔板内,并使用酶标仪检测OD462值。实验结果如图2所示,百里酚(50μg/mL)对耐甲氧西林金黄色葡萄球菌ATCC BAA-1717的黄素合成抑制率可达到90%以上。
总之,根据以上实验结果,亚抑制浓度的百里酚的抗菌剂能够有效抑制耐甲氧西林金黄色葡萄球菌的黄素合成。本发明将为防控耐甲氧西林金黄色葡萄球菌感染提供新的思路,在食品安全、药物开发和公共卫生领域具有广泛的应用潜力。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (10)
1.一种抑制葡萄球菌黄素合成的抗菌剂,其特征在于,所述抗菌剂的组分包括百里酚和二甲基亚砜,所述二甲基亚砜为溶剂。
2.如权利要求1所述的抗菌剂,其特征在于,所述抗菌剂最小抑菌浓度为170μg/mL。
3.如权利要求1所述的抗菌剂,其特征在于,所述抗菌剂不影响菌株生长活力条件下抑制葡萄球菌黄素的浓度为50μg/mL。
4.根据权利要求1-3所述的任一项抗菌剂在抑制金黄色葡萄球菌黄素合成中的应用。
5.如权利4所述的应用,其特征在于,所述应用的方法包括以下步骤:
步骤1、配制所述抗菌剂,得到含有不同浓度的百里酚的所述抗菌剂;
步骤2、将步骤1得到的不同浓度的所述抗菌剂混合到所述金黄色葡萄球菌菌液中,并在37℃条件下进行第一共培养,得到第一共培养菌液,检测所述第一共培养菌液中所述金黄色葡萄球菌的生长曲线;得出不影响所述金黄色葡萄球菌生长活力的所述抗菌剂的亚抑制浓度;
步骤3、将步骤2得到的亚抑制浓度的抗菌剂混合到所述金黄色葡萄球菌菌液中,并在37℃条件下进行第二共培养,得到第二共培养菌液,使用甲醇提取所述第二共培养菌液,得到甲醇提取物;测定所述甲醇提取物中葡萄球菌黄素的含量。
6.如权利5所述的应用,其特征在于,所述金黄色葡萄球菌为耐甲氧西林金黄色葡萄球菌标准菌株。
7.如权利5所述的应用,其特征在于,所述金黄色葡萄球菌菌液的培养步骤为:接种冻存的所述金黄色葡萄球菌在TSB培养基进行纯化培养,随后使用0.85%生理盐水洗涤重悬菌体,并调整所述菌液的OD600值至0.5,再使用所述TSB培养基稀释所述菌液1000倍。
8.如权利5所述的应用,其特征在于,所述步骤1中所述抗菌剂的配制步骤还包括采用二甲基亚砜作溶剂,配制所述百里酚浓度为100mg/mL的母液,再稀释至不同浓度。
9.如权利5所述的应用,其特征在于,所述步骤2中所述第一共培养的时间为24小时,所述步骤3中所述第二共培养的时间为24小时。
10.如权利5所述的应用,其特征在于,所述抗菌剂的亚抑制浓度为50μg/mL。
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CN114129547A (zh) * | 2021-11-04 | 2022-03-04 | 华南农业大学 | 香芹酚在提高耐甲氧西林金黄色葡萄球菌对β-内酰胺类抗生素敏感性方面的应用 |
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