CN116496994A - 一种耐甲氧西林金黄色葡萄球菌的噬菌体、组合物及其应用 - Google Patents
一种耐甲氧西林金黄色葡萄球菌的噬菌体、组合物及其应用 Download PDFInfo
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- CN116496994A CN116496994A CN202310170285.2A CN202310170285A CN116496994A CN 116496994 A CN116496994 A CN 116496994A CN 202310170285 A CN202310170285 A CN 202310170285A CN 116496994 A CN116496994 A CN 116496994A
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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Abstract
本发明提供了一种耐甲氧西林金黄色葡萄球菌的噬菌体、组合物及其应用,属于噬菌体技术领域。本发明提供的耐甲氧西林金黄色葡萄球菌的噬菌体为噬菌体MX‑6(保藏编号为CCTCCNO.M2023029)或噬菌体ML‑1337(保藏编号为CCTCCNO.M2023028)。本发明分离得到的耐甲氧西林金黄色葡萄球菌噬菌体,具有裂解谱宽、适应能力强、不含耐药基因和毒力基因、动物实验安全、无毒副作用的优势,对MRSA感染动物有一定的治疗效果。将本发明噬菌体与抗菌药联合使用,具有联合协同杀菌和/或抑菌能力,能够对多重耐药MRSA具有明显的杀菌和/或抑菌效果,且可降低抗菌药的最低抑菌浓度,减少抗菌药的使用量。
Description
技术领域
本发明属于噬菌体技术领域,尤其涉及一种耐甲氧西林金黄色葡萄球菌的噬菌体、组合物及其应用。
背景技术
金黄色葡萄球菌是一种重要的人畜共患致病菌,普遍分布在自然界(空气、水、灰尘、人和动物粪便中),是人类临床上主要的病原菌之一,能够引起人类轻度至重度甚至危及生命的感染。同时,金黄色葡萄球菌也是全世界食源性疾病的重要病原菌之一,它可以定植在动物的鼻孔、黏膜或皮肤等部位中,通过食物链,人们使用了被金黄色葡萄球菌感染的动物产品后会发生食物中毒,严重者可危害生命。
食品动物养殖过程中常用抗菌药来预防和治疗多种细菌性疾病,由于抗菌药的大量使用及不合理使用,不仅导致动物源性食品中抗菌药残留超标,引发食品安全问题,更重要的是,其还会导致家禽细菌耐药率的上升及耐药谱的不断扩大。自20世纪60年代以来,随着甲氧西林的临床使用,耐甲氧西林金黄色葡萄球菌(MRSA)就在亚洲、欧洲和北美等多个国家的动物源性食品中被检测到。研究显示,MRSA菌株除了对几乎所有的β-内酰胺类抗生素都具有耐药性外,同时对其他多种抗生素也具有高度耐药性,普遍具有较强的适应环境和定植能力,往往引起高感染率、高致死率,临床上几乎无特别有效的治疗药物。
自1917年发现噬菌体后,一直有利用它治疗疾病的报道,但由于其使用方法不如抗菌药方便,噬菌体的研发应用近乎停滞。近年来,在抗菌药滥用导致药物残留和超级细菌带来的威胁下,噬菌体的应用需求和研发骤然加速。噬菌体治疗有安全、无残留、特异性强等优点,但也存在一些局限性,如有报道噬菌体治疗会导致噬菌体抗性菌的出现。
发明内容
有鉴于此,本发明的目的在于提供一种裂解谱宽、适应能力强、不含耐药基因和毒力基因、无毒副作用、与抗菌药组合使用具有联合协同杀菌能力、对多重耐药MRSA具有明显杀菌效果的噬菌体及其应用。
本发明的另一目的在于提供一种既能降低抗菌药的最低抑菌浓度,又能减少抗菌药使用量的,对多重耐药MRSA具有明显杀菌效果的组合物及其应用。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种耐甲氧西林金黄色葡萄球菌的噬菌体,所述噬菌体为噬菌体MX-6或噬菌体ML-1337,所述噬菌体MX-6的保藏编号为CCTCCNO.M2023029,所述噬菌体ML-1337的保藏编号为CCTCC NO.M2023028。
优选的,所述噬菌体不含有耐药基因和毒力基因。
本发明还提供了一种具有杀菌和/或抑菌作用的组合物,所述组合物包括抗菌药和上述噬菌体。
优选的,所述抗菌药包括恩诺沙星或氨苄西林。
优选的,所述组合物中噬菌体的浓度为106PFU/mL。
优选的,所述组合物中抗菌药的浓度为1/2MIC及以下。
本发明还提供了一种上述噬菌体或上述组合物在制备抗耐甲氧西林金黄色葡萄球菌药物中的应用。
本发明还提供了一种上述噬菌体在防治食品耐甲氧西林金黄色葡萄球菌污染中的应用。
本发明的有益效果:
本发明分离得到的耐甲氧西林金黄色葡萄球菌噬菌体,具有裂解谱宽、适应能力强、不含耐药基因和毒力基因、动物实验安全、无毒副作用的优势,对MRSA感染动物有一定的治疗效果。将本发明噬菌体与抗菌药联合使用,具有联合协同杀菌能力,能够对多重耐药MRSA具有明显的杀菌效果,且可降低抗菌药的最低抑菌浓度(MIC),减少抗菌药的使用量。
生物保藏信息:
本发明金黄色葡萄球菌噬菌体(Staphylococcus aureusphage)MX-6,保藏在中国典型培养物保藏中心,地址为武汉市武昌区武汉大学,保藏编号为:CCTCC NO.M2023029,保藏日期为:2023年01月05日;
本发明金黄色葡萄球菌噬菌体(Staphylococcus aureusphage)ML-1337,保藏在中国典型培养物保藏中心,地址为武汉市武昌区武汉大学,保藏编号为:CCTCCNO.M2023028,保藏日期为:2023年01月05日。
附图说明
图1为噬菌体形态图,左图为MX-6噬菌体形态图,右图为ML-1337噬菌体形态图;
图2为噬菌体MX-6的热稳定性结果;
图3为噬菌体ML-1337的热稳定性结果;
图4为MX-6噬菌体的pH稳定性结果;
图5为ML-1337噬菌体的pH稳定性结果;
图6为MX-6噬菌体的一步生长曲线;
图7为ML-1337噬菌体的一步生长曲线;
图8为ML-1337噬菌体的核酸电泳结果;
图9为噬菌体MX-6基因组图谱;
图10为注射不同剂量ML-1337小鼠的存活情况;
图11为不同时间注射ML-1337小鼠的存活情况;
图12为连续多天注射ML-1337小鼠的存活情况;
图13为单剂量注射ML-1337对小鼠的治疗效果;
图14为噬菌体MX-6与恩诺沙星联合协同杀菌效果;
图15为噬菌体MX-6与氨苄西林联合协同杀菌效果;
图16为噬菌体ML-1337与恩诺沙星联合协同杀菌效果;
图17为噬菌体ML-1337与氨苄西林联合协同抑菌效果。
具体实施方式
本发明提供了一种耐甲氧西林金黄色葡萄球菌的噬菌体,所述噬菌体为噬菌体MX-6或噬菌体ML-1337,所述噬菌体MX-6的保藏编号为CCTCC NO.M2023029,所述噬菌体ML-1337的保藏编号为CCTCC NO.M2023028。
本发明提供的两株噬菌体MX-6、ML-1337裂解谱宽,能够裂解多种动物来源(鸡、猪、牛)的MRSA和金黄色葡萄球菌。经鉴定,本发明所述噬菌体为有尾噬菌体目短尾噬菌体科噬菌体,环境耐受能力强,在pH 3-11、温度30℃-50℃条件下效价稳定,最佳感染复数分别为1、0.01,无毒副作用,全基因组测序比对分析不含耐药基因和毒力基因等毒害因子。本发明提供的两株噬菌体可以单独使用,也可以与其余抗菌药组合使用,将本发明所述噬菌体与抗菌药联合应用时,能够降低抗生素的使用浓度,在国家倡导的兽用抗菌药减量使用方面发挥作用。
单独采用本发明噬菌体ML-1337作用于感染MRSA菌株的动物时,有治疗效果,最佳治疗剂量为1010PFU/只,最佳治疗时间为感染后1h内,连续服用7天,其动物存活率明显高于单次服用噬菌体。
本发明还提供了两种具有杀菌和/或抑菌作用的组合物,所述组合物包括抗菌药和上述噬菌体。
在本发明中,所述抗菌药优选的包括恩诺沙星或氨苄西林,本发明对于上述抗菌药的具体来源没有特殊限定,采用本领域常规市售产品均可。在本发明所述组合物1中,噬菌体MX-6的浓度优选为106PFU/mL,抗菌药的浓度优选为1/4MIC。本发明所述组合物1具有联合协同杀菌能力,噬菌体在上述最佳治疗量、抗菌药浓度的MIC减少4倍的情况下便能够完全杀灭多重耐药MRSA菌株。在本发明所述组合物2中,噬菌体ML-1337的浓度优选为106PFU/mL,恩诺沙星浓度降低至1/2MIC时二者具有显著的联合杀菌效果,与氨苄西林在1/4MIC和1/16MIC浓度下有着较好的联合抑菌效果。
本发明还提供了一种上述噬菌体或上述组合物在制备抗耐甲氧西林金黄色葡萄球菌药物中的应用。
本发明还提供了一种上述噬菌体在防治食品耐甲氧西林金黄色葡萄球菌污染中的应用。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
下述实施例中,如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
噬菌体的分离纯化与富集浓缩
1.1样品采集及处理
将来自不同养殖场的粪样用1×SM液体浸泡过夜,吸上清,10000r/min离心10min,用0.22μm一次性无菌滤器过滤除菌,得噬菌体原液,再取100μL处于对数生长期宿主菌MRA202109P0009(分离自山东省胶州市胶莱办事处孙希祥肉鸡养殖场)和NJP1337(分离自广西横县百兴奶牛场)分别于LB平板上均匀涂布,静置吸收,干燥后滴加10μL噬菌体原液,对照组滴加等量的生理盐水,待其干燥,于37℃温箱中培养18-20h,观察是否有空斑出现。
1.2噬菌体的分离纯化
用LB肉汤对噬菌体原液进行10倍梯度稀释,取每个梯度稀释液200μL分别与200μL培养至对数生长期的宿主菌混合于5ml离心管中,37℃静置孵育5min,混合液中加入3.6mL预热的0.6%LB半固体培养基,混匀后倒入1.5%LB固体培养基平板上,左右摇晃铺满整个板面,凝固后倒置,37℃过夜培养。挑取空斑溶解于SM缓冲液中,4℃静置过夜,重复纯化3-5次,直至空斑大小一致,纯化好的噬菌体加入SM缓冲液中4℃保存。
1.3噬菌体的富集浓缩
取100μL培养至对数生长期的宿主菌,加入5mLTSB和100μL噬菌体纯化液,放置于恒温摇床中,37℃,180r/min震荡过夜。次日将培养液取出,4℃,8000g离心10min,取上清加入100μL培养至对数生长期的宿主菌和5mLTSB,置于摇床中,37℃,180r/min培养过夜,重复3-5次,将离心后的上清液用0.22μm一次性无菌滤器过滤除菌,得噬菌体富集液。将噬菌体富集液与等体积40%甘油溶液混合,得到甘油终浓度为20%的保存液,-80℃保存。
实施例2
噬菌体的形态鉴定
吸取20μL噬菌体增殖液于铜网上,静置沉淀15min,用滤纸吸去多余液体,滴加15μL磷钨酸(PTA)染色5-10min后放在50℃烘灯下干燥5min,然后在透射电镜(TEM)下观察噬菌体形态,结果如图1所示。两株噬菌体均头部对称呈多面体状,MX-6、ML-1337直径分别40×40nm、60×60nm,尾巴长约15-20nm,结合噬菌体分类标准可知两株噬菌体均是有尾噬菌体目,短尾噬菌体科噬菌体。
实施例3
噬菌体效价的测定
将纯化好的噬菌体液用LB肉汤进行10倍梯度稀释,取10-2、10-4、10-6、10-8、10-10浓度的稀释液200μL分别与200μL培养至对数生长期的宿主菌MRA202109P0009、NJP1337混合于5mL离心管中,37℃静置孵育15min,混合液中加入4mL预热的0.6%LB半固体培养基,混匀后倒入1.5%LB固体培养基平板上,左右摇晃铺满整个版面,凝固后倒置37℃过夜培养,每个浓度做三个平行,重复三次,选取噬菌斑数量在30-300的浓度进行计数,并按公式进行噬菌体效价的计算。
计算公式:噬菌体效价(PFU/mL)=噬菌斑数/加样量×稀释倍数
结果显示,MX-6、ML-1337噬菌体效价分别为1.18×1010、2.5×109PFU/mL。
实施例4
噬菌体最佳感染复数(MOI)的测定
将宿主菌MRA202109P0009、NJP1337培养至对数生长期,按照感染复数为0.001、0.01、0.1、1、10的比例分别加入噬菌体和宿主菌,混匀,37℃,180r/min振荡培养8h,按照实施例3的方法,分别测定噬菌体效价,其中效价最高的MOI值即为最佳感染复数。
结果显示,MX-6、ML-1337噬菌体最佳感染复数分别为1、0.01。
实施例5
噬菌体热稳定性测定
取1mL噬菌体MX-6纯化液分别置于50℃、70℃、80℃、90℃,作用60min,在0、20、40、60min取样分别与宿主菌混合;取1mL噬菌体ML-1337为分别置于4℃、30℃、50℃、70℃、90℃,作用120min,在0、30、60、90、120min取样分别与宿主菌混合,按照实施例3,通过双层平板法测定效价。结果如图2和图3所示。不同温度作用一段时间后,噬菌体效价有着明显的变化。温度在50℃下,2株噬菌体效价基本不变,70℃、80℃、90℃作用20min后噬菌体效价均有明显下降。
实施例6
噬菌体pH稳定性测定
将LB肉汤配置成不同pH值(1-14)的液体培养基,按照体积1∶9的比例将噬菌体加入到不同pH值的LB液体培养基中(其中MX-6噬菌体所对应的LB液体培养基的pH分别为1-14,ML-1337噬菌体所对应的LB液体培养基的pH分别为2-11),37℃培养30min后取样,按照实施例3,通过双层平板法测定效价,每个pH值设置细菌对照。结果如图4和图5所示。2株噬菌体的pH值耐受范围较大,可在pH 3-11的环境中对宿主细菌产生较强的裂解作用。
实施例7
噬菌体裂解谱的测定
将金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌共计200株(牛源75株、猪源57株、鸡源68株,详见下表1)试验菌株培养至对数生长期(OD600≈0.7),取100μL滴加到TSA培养基上,用涂布棒涂布均匀,待表面干燥后倒置,标记试验组与对照组位置,分别滴加10μL噬菌体纯化液在试验组位置,阴性对照组滴加等量生理盐水,待液体吸收完全后,倒置放入37℃恒温培养箱,培养18-24h,观察是否出现空斑。结果如表1所示。
表1两株噬菌体对200株金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌的裂解情况
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注:裂解斑形态描述如下表示:“+++”透明、光滑;“++”半透明或有单菌落;“+”单个噬菌斑;“-”完全不裂解。
由表1可以看出,噬菌体ML-1337对牛源菌株裂解率为66.67%,对猪源菌株裂解率为94.74%,对鸡源菌株裂解率为52.94%,噬菌体MX-6对鸡源菌株裂解率为70.6%,对猪源菌株裂解率为96.49%,对牛源菌株裂解为65.33%。
实施例8
噬菌体的一步生长曲线测定
将宿主菌培养至对数生长期(OD600≈0.7),以MOI=0.1分别接种噬菌体,37℃静置孵育20min,然后6000r/min离心10min,弃上清,沉淀重悬于LB肉汤,置于摇床中37℃180r/min培养,同时开始计时,两种噬菌体分别在10-120min之间和0-120min之间,每10min取1mL于离心管中,10000r/min离心2min,取上清通过双层平板法测效价(具体方法同实施例3),以时间为横坐标,噬菌体效价为纵坐标绘制生长曲线。
结果如图6和图7所示,MX-6噬菌体的潜伏期为0min,爆发期为30min,平均裂解量为68PFU/cell;ML-1337噬菌体的潜伏期为20min,爆发期约为90min,平均裂解量为29PFU/cell。
实施例9
噬菌体毒害基因检测
利用病毒基因组提取试剂盒提取噬菌体基因组核酸,进行全基因组测序分析。结果如图8-9所示。
噬菌体ML-1337总核酸大小约15000bp。噬菌体ML-1337是由双链DNA编码的长尾噬菌体,基因组全长16927bp,GC%含量为41.39%。根据ORFfinder检索结果显示,ML-1337共编码83个大于75bp的ORFs,42个为正链,41个为负链。通过blast分析发现,其中73个ORFs具有编码基因功能,包括主要结构蛋白模块6个(ORF45、ORF46、ORF47、ORF49、ORF63和ORF71),DNA复制及组装模块5个(ORF4、ORF5、ORF35、ORF59、ORF72),裂解宿主模块2个(ORF48和ORF65),调控模块11个(ORF30、ORF31、ORF37、ORF56、ORF57、ORF58、ORF61、ORF68、ORF69、ORF74和ORF78),其他为假定蛋白(未知功能蛋白),并且在所有的基因模块中没有发现任何与耐药性、毒力因子有关的基因。
实施例10
噬菌体ML-1337对MRSA菌株感染小鼠治疗实验应用
MRSA最小致死量(MLD)测定
将MRSA菌株NJP1337培养至对数生长期,将菌液调整为104、106、108、1010CFU/mL四个不同浓度。取25只小鼠随机平均分为5组,即4组试验组和1组对照组,4组对照组分别注射上述不同剂量的MRSA菌株NJP1337,对照组则注射同等剂量的生理盐水,观察并记录小鼠死亡数量,确定能够导致小鼠100%死亡的菌液最小致死量(MLD)。
结果:单剂量腹腔注射1010CFU/只的MRSA菌株NJP1337能够引起试验小鼠100%的死亡,注射108CFU/只的小鼠死亡率达到40%,死亡小鼠肠管明显肿胀,肠壁变薄,肝脏肿胀淤血,因此确定NJP1337的最小致死量为1010CFU/mL。
噬菌体ML-1337安全性检测
将噬菌体ML-1337增殖并浓缩后加入SM缓冲液保存,取30只小鼠随机平均分成3组,即1组试验组和2组对照组,试验组注射最高效价(1010PFU/mL)(参照实验例3的方法)的噬菌体ML-1337,对照组1注射等量SM缓冲液,对照组2注射等量生理盐水,观察并记录小鼠生长状况,是否出现死亡情况等。
结果:试验组小鼠生长良好并全部存活,同条件下的注射生理盐水和SM缓冲液的对照组小鼠全部存活,剖检结果并未发现试验组小鼠与对照组小鼠脏器有任何异常变化,证明噬菌体ML-1337对小鼠无明显毒性作用。
不同剂量ML-1337对小鼠的保护率测定
将噬菌体ML-1337增殖并浓缩,得到最高效价1010PFU/mL,再用SM缓冲液稀释到4个不同浓度(104、106、108、1010PFU/mL)备用。取35只小鼠随机平均分成7组,即4组试验组和3组对照组。4组试验组注射最小致死量的NJP1337菌液1h后,分别注射不同剂量的噬菌体ML-1337,同等条件下设置噬菌体对照组、细菌对照组和生理盐水对照组,定时观察并记录小鼠死亡情况。
结果:4组试验组小鼠注射最小致死量的NJP1337菌液1h后,分别注射不同剂量的噬菌体ML-1337,小鼠的存活情况如图10所示,注射剂量为1010PFU/只的小鼠存活率为40%,而注射剂量为104、106和108PFU/只的3组小鼠分别在第2天、第4天和第6天全部死亡,同条件下的细菌对照组小鼠全部死亡,噬菌体对照组和生理盐水对照组小鼠生长良好并全部存活。
不同时间注射ML-1337对小鼠的保护率测定
取35只小鼠随机平均分成7组,即4组试验组和3组对照组。首先,向4组试验组小鼠注射最小致死量的MRSA菌株NJP1337,然后在不同的时间(0.5、1、2、6h)注射1010PFU/只的噬菌体ML-1337,剩余3组对照组分别注射细菌、噬菌体和生理盐水。
结果:4组试验组小鼠注射最小致死量的NJP1337菌液后,分别在不同时间注射1010PUF/只的噬菌体ML-1337,小鼠存活率如图11所示,注射菌液后0.5h和1h时注射ML-1337小鼠的存活率达到40%,而注射菌液2h后再注射ML-1337的试验组小鼠在4天内全部死亡,同条件下的细菌对照组小鼠在第三天全部死亡,噬菌体和生理盐水对照组小鼠生长良好并全部存活。
连续多天注射ML-1337对小鼠的保护率测定
将20只小鼠随机平均分成4组,即1组试验组和3组对照组,试验组小鼠注射最小致死量的MRSA菌株NJP1337,1h后使用1010PFU/mL的噬菌体ML-1337对其进行治疗,连续7天同一时间注射同等剂量的噬菌体,同等条件下设置噬菌体对照组、细菌对照组和生理盐水对照组,定时观察并记录小鼠死亡情况。
结果如图12所示:试验组小鼠注射最小致死量的NJP1337菌液后,连续7天同一时间使用1010PFU/只噬菌体ML-1337的小鼠存活率为60%,同条件下的细菌对照组小鼠全部死亡,噬菌体和生理盐水对照组小鼠生长良好并全部存活。
单剂量注射ML-1337对小鼠的治疗效果测定
取25只小鼠作为试验组,每只注射最小致死量的MRSA菌株NJP1337,然后在1h后注射1010PFU/mL/只的噬菌体ML-1337,观察小鼠死亡情况,并定期取小鼠血液测定噬菌体ML-1337的效价,在同等条件下设置细菌对照组、噬菌体对照组及生理盐水空白对照组。
结果如图13所示:试验组小鼠接种噬菌体ML-1337后,血液内噬菌体含量逐渐增高,1h时测得血液内噬菌体浓度为2.9×105PFU/mL,随后继续升高,至12h时达到最大,为6.33×107PFU/mL,随后浓度逐渐下降,在72h时降至6.83×104PFU/mL。
实施例11
体外联合杀菌应用
选取1株多重耐药ST9型MRSA菌株,采用微量肉汤法检测兽用抗菌药的最低抑菌浓度(MIC),结合上述金黄色葡萄球菌噬菌体最佳感染复数的测定结果,利用棋盘法设计抗菌药及噬菌体MX-6浓度(见表2),同时设抗菌药(恩诺沙星或氨苄西林)对照组、噬菌体对照组、阳性菌对照组、阴性对照组,将48孔板置于生长曲线仪37℃连续动态监测24小时,确定噬菌体与抗菌药是否具有联合协同作用。
表2棋盘法设计抗菌药及噬菌体浓度
1/32MIC+101 | 1/16MIC+101 | 1/8MIC+101 | 1/4MIC+101 | 1/2MIC+101 | MIC+101 | 1/8MIC | 1×101 |
1/32MIC+102 | 1/16MIC+102 | 1/8MIC+102 | 1/4MIC+102 | 1/2MIC+102 | MIC+102 | 1/4MIC | 1×102 |
1/32MIC+103 | 1/16MIC+103 | 1/8MIC+103 | 1/4MIC+103 | 1/2MIC+103 | MIC+103 | 1/2MIC | 1×103 |
1/32MIC+104 | 1/16MIC+104 | 1/8MIC+104 | 1/4MIC+104 | 1/2MIC+104 | MIC+104 | MIC | 1×104 |
1/32MIC+105 | 1/16MIC+105 | 1/8MIC+105 | 1/4MIC+105 | 1/2MIC+105 | MIC+105 | 2MIC | 1×105 |
1/32MIC+106 | 1/16MIC+106 | 1/8MIC+106 | 1/4MIC+106 | 1/2MIC+106 | MIC+106 | PC | NC |
细菌初始浓度设置为1×106CFU/mL,根据噬菌体MX-6的最佳感染复数,将噬菌体的初始浓度设置为1×106PFU/mL。实验分四组,空白对照组,抗菌药组(MIC、1/2MIC、1/4MIC、1/8MIC、1/16MIC),噬菌体组(1×106PFU/mL),联合应用组(噬菌体1×106PFU/mL+抗菌药MIC,噬菌体1×106PFU/mL+抗菌药1/2MIC,噬菌体1×106PFU/mL+抗菌药1/4MIC,噬菌体1×106PFU/mL+抗菌药1/8MIC,噬菌体1×106PFU/mL+抗菌药1/16MIC,),每隔4h测一次利用平板涂布计数法测得各组中细菌浓度,做三次重复,共监测20h,观察具有协同作用的联合应用组中抗生素及噬菌体的浓度。结果如图14、15所示。噬菌体ML-13337实验步骤同上,结果如图16、17所示。
结果发现,两种噬菌体与两种抗菌药均具有明显的协同作用,为了进一步明确抗菌药的浓度,通过菌落计数的方法连续监测发现,当噬菌体MX-6为106PFU/mL、恩诺沙星、氨苄西林浓度最低可降至1/4MIC时,组合物具有明显的联合杀菌效果。噬菌体ML-1337在106PFU/mL、恩诺沙星浓度降低至1/2MIC时二者具有显著的联合杀菌效果,而与氨苄西林在1/4MIC和1/16MIC浓度下有着较好的联合抑菌效果。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种耐甲氧西林金黄色葡萄球菌的噬菌体,其特征在于,所述噬菌体为噬菌体MX-6或噬菌体ML-1337,所述噬菌体MX-6的保藏编号为CCTCC NO.M2023029,所述噬菌体ML-1337的保藏编号为CCTCCNO.M2023028。
2.根据权利要求1所述的噬菌体,其特征在于,所述噬菌体不含有耐药基因和毒力基因。
3.一种具有杀菌和/或抑菌作用的组合物,其特征在于,所述组合物包括抗菌药和权利要求1所述噬菌体。
4.根据权利要求3所述的组合物,其特征在于,所述抗菌药包括恩诺沙星或氨苄西林。
5.根据权利要求3所述的组合物,其特征在于,所述组合物中噬菌体的浓度为106PFU/mL。
6.根据权利要求3所述的组合物,其特征在于,所述组合物中抗菌药的浓度为1/2MIC及以下。
7.权利要求1-2任意一项所述噬菌体或权利要求3-6任意一项所述组合物在制备抗耐甲氧西林金黄色葡萄球菌药物中的应用。
8.权利要求1-2任意一项所述噬菌体在防治食品耐甲氧西林金黄色葡萄球菌污染中的应用。
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