CN109251899B - 耐药大肠杆菌噬菌体及其应用 - Google Patents
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Abstract
耐药大肠杆菌噬菌体及其应用。分类命名为Escherichia phage vB_EcoP‑EG1,保藏号为:CCTCC M 2018562,保藏日期为2018年8月21日。噬菌体Escherichia phage vB_EcoP‑EG1性质稳定,生命周期短,对宿主菌裂解能力强。基因组及蛋白质谱结果表明噬菌体Escherichia phage vB_EcoP‑EG1不编码毒性蛋白质。同时噬菌体Escherichia phage vB_EcoP‑EG1能够感染裂解多株临床随机分离得到的多耐药的大肠杆菌,尤其对尿道致病性大肠杆菌具有良好的杀伤效果。综上所述,Escherichia phage vB_EcoP‑EG1是噬菌体治疗的良好候选噬菌体,可以作为单独的制剂或者和其他噬菌体混合组成鸡尾酒制剂用于对抗临床多耐药性大肠杆菌导致的感染。
Description
技术领域
本发明属于微生物医学工程领域,具体涉及一株可以裂解临床分离的多株耐药大肠杆菌噬菌体及其应用。
背景技术
毒性噬菌体能够感染裂解细菌,在上个世纪初就被应用于杀灭病原菌。随后由于廉价有效的抗生素的发现并大规模应用而逐渐被忽视,只有少数东欧国家(如格鲁吉亚,俄国等)一直坚持采用噬菌体疗法。近年来由于耐药菌的不断出现,噬菌体再次受到人们重视,尤其最近十几年大量针对耐药菌的新的噬菌体被发现并广泛研究。很多体外体内实验证明噬菌体能够杀灭多种耐药菌,如铜绿假单胞菌、金黄色葡萄球菌、大肠杆菌和鲍曼不动杆菌等。最近一些临床成功案例也鼓舞人们采用噬菌体来治疗严重的耐药菌导致的感染,如美国加州大学圣地亚哥分校精神病学教授Tom Patterson感染了多耐药鲍曼不动杆菌引起败血症,在抗生素治疗无效的情况下,采用相应的噬菌体静脉注射及腹腔注射治疗最终成功战胜了感染,有力证明了噬菌体治疗的安全性及有效性,是抗生素治疗的有益补充。美国国立卫生研究院过敏与感染研究所已经把噬菌体列为对抗耐药菌的重要手段之一。抗生素的长期使用导致多耐药的大肠杆菌广泛出现,特别是能产生超广谱β-内酰胺酶和碳青霉烯酶的大肠杆菌,只有极少数最新抗生素有效,给临床治疗带来了极大的困难。随着新抗生素的研发周期不断变长,甚至面临着无药可用的窘境。寻找新的能杀灭多耐药大肠杆菌的替代疗法或补充疗法迫在眉睫。研究发现T4、T6和KEP10噬菌体在体外能够有效的杀灭临床分离得到的多耐药尿道致病性大肠杆菌,在小鼠尿路感染模型腹腔注射T4噬菌体或KEP10噬菌体可以抑制尿路感染症状降低小鼠的死亡率。T1噬菌体能感染裂解临床分离的大肠杆菌的耐药株,同时还能杀灭黏附在尿路上皮细胞上的多耐药的大肠杆菌。格鲁吉亚已经商品化的大肠杆菌噬菌体鸡尾酒制剂能较为有效的杀灭临床分离得到的多耐药的尿道致病性大肠杆菌。实验证明噬菌体还可以降解由大肠杆菌形成的生物膜。另外抗生素的长期使用会使粪便中携带的多耐药大肠杆菌数量升高,从而导致女性尿路感染的发生率提高10倍。动物实验表明噬菌体能够特异性的杀灭肠道污染的多重耐药的大肠杆菌,降低尿路感染风险,对正常菌群影响较小,符合现代精准治疗的需求。以上研究表明毒性噬菌体能够有效杀灭多耐药大肠杆菌。
发明内容
解决的技术问题:本发明提供一株新的裂解耐药大肠杆菌的噬菌体及其应用。
技术方案:大肠杆菌噬菌体,分类命名为Escherichia phage vB_EcoP-EG1,保藏号为:CCTCC M 2018562,保藏日期为2018年8月21日,保藏地址为湖北省武汉市武昌珞珈山,武汉大学中国典型培养物保藏中心。
所述的噬菌体在制备抗大肠杆菌的生物制剂中的应用。
所述的噬菌体在制备抗多耐药的大肠杆菌的生物制剂中的应用。
所述的噬菌体在多耐药的大肠杆菌细菌分型中的应用。
所述的噬菌体在表达蛋白或者运送药物的载体中的应用。
一种抗多耐药的大肠杆菌的生物制剂,包括所述的噬菌体。
有益效果:本发明Escherichia phage vB_EcoP-EG1噬菌体对多株临床分离得到的多耐药的大肠杆菌具有良好的杀伤效果。该噬菌体可以单独应用,或者作为有效组分和其他噬菌体组成鸡尾酒制剂,或者作为抗生素辅助制剂用于杀伤抑制多耐药的大肠杆菌。
附图说明
图1噬菌体的形态图,其中A.噬菌体Escherichia phage vB_EcoP-EG1在双层琼脂平板上形成的噬菌斑。B.噬菌体Escherichia phage vB_EcoP-EG1的电镜照片。
图2噬菌体Escherichia phage vB_EcoP-EG1的性质图,其中A.噬菌体Escherichia phage vB_EcoP-EG1在不同温度下的活性B.噬菌体Escherichia phage vB_EcoP-EG1在人的尿液中的稳定性C.噬菌体Escherichia phage vB_EcoP-EG1的生长曲线D.噬菌体Escherichia phage vB_EcoP-EG1对大肠杆菌的抑制效果实验。
图3噬菌体Escherichia phage vB_EcoP-EG1对大肠杆菌形成的生物膜的抑制效果图。
表1:Escherichia phage vB_EcoP-EG1的宿主谱分析
表2:噬菌体Escherichia phage vB_EcoP-EG1病毒颗粒的蛋白质组分的质谱分析
具体实施方式
下面结合具体实例对本发明的发明方法进行详细描述和说明。其内容是对本发明的解释而非限定本发明的保护范围。在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实施例1
1.噬菌体的初步分离和纯化
采集污水样品并处理:
利用大肠杆菌MG1655对南京废水中的强毒噬菌体进行分离富集。从南京市污染河道中取污水样本,将污水样品用0.45μm过滤器过滤除菌。取污水滤液1mL和MG1655过夜培养菌液500μL置于5mL液体LB培养基,200rpm 37℃恒温振荡培养24小时,富集噬菌体。取1mL培养液,14000g离心10分钟,取上清用0.45μm滤膜过滤。取10μL滤液,加入100μL过夜菌液(MG1655),再加入3.5mL上层琼脂(半固体LB培养基)混匀,铺于LB琼脂平板上(双层琼脂平板法),过夜培养后观察噬菌斑形成。初次分离的噬菌体大小、形态均不同,可能含有多种噬菌体。挑选单个噬菌斑开始第二轮扩增后铺双层琼脂平板形成噬菌斑,重复感染铺板循环直至得到大小、形态、分布均一的噬菌斑,即为得到单一纯化的噬菌体,然后将此噬菌体扩增并储存在4℃备用。可见该过夜培养后噬菌体在平板上形成的噬菌斑直径较大,约为13mm,对大肠杆菌裂解能力强(图1A)。
2.Escherichiaphage vB_EcoP-EG1噬菌体的超离纯化
大肠杆菌MG1655过夜培养液1:100转接到50mL液体LB培养基,培养到对数期加入噬菌体Escherichia phage vB_EcoP-EG1。侵染2小时后收集菌液,14000g 4℃离心10m,上清液通过0.45μm孔径过滤器,得到噬菌体粗提取物。将上清液进行超高速离心,106750g,4℃离心2小时(Beckman SW32转子,超速离心)。弃上清,加1mL SM buffer重新溶解噬菌体沉淀。重悬后噬菌体进行氯化铯密度梯度离心,210053g,4℃离心20h(Beckman,SW41转子)。离心结束后每层1mL将样品分层取出。噬菌体对应层样品加入SM buffer稀释后,加入到12mL超离管,210053g,4℃离心3h(Beckman SW41转子),沉淀噬菌体。离心后弃上清,稍干燥后加入100μL SM buffer溶解噬菌体并保存于4℃,即为纯化后的噬菌体Escherichia phagevB_EcoP-EG1。
3.噬菌体的形态学观察
纯化的噬菌体Escherichia phage vB_EcoP-EG1颗粒固定在带有碳涂布膜的铜格栅上(噬菌体滴度≥109PFU/mL)自然吸附约3min,用滤纸稍吸干后再用2%(W/V)磷钨酸负染2-3min,滤纸稍吸干,待干燥后采用FEI Tecnai G2Spirit Bio TWIN透射电子显微镜下进行观察拍照(图1B),电镜的工作电压为80kV。该噬菌体为短尾噬菌体,头部直径大约50nm,有一个10nm左右的尾。
4.噬菌体生物学特性分析实验
4.1噬菌体温度稳定性
采用恒温水浴法测定噬菌体在不同温度条件下的稳定性。在不同温度(4、25、37、45,50,55、60和65℃)下分别孵育含200μL(约1010PFU/mL)的等体积噬菌体。用双层琼脂平板法测定噬菌体效价。重复三次,并用均值表示(图2A)。Escherichia phage vB_EcoP-EG1噬菌体在4到50℃温度范围内能长时间保持稳定。
4.2噬菌体在人尿液中的稳定性
将噬菌体Escherichia phage vB_EcoP-EG1200μL(约1010pFU/mL)加入到10mL人尿液中,不同时间点(3h,6h,9h和24h)取样测定噬菌体的效价。重复三次,并用均值表示(图2B)。Escherichia phage vB_EcoP-EG1噬菌体在人的尿液中室温放置24小时滴度没有明显下降,该噬菌体在尿液中能长时间保持稳定,有利于通过导尿管膀胱灌注噬菌体进行局部的杀菌。
4.3噬菌体的一步生长曲线测定
大肠杆菌MG1655单克隆接种液体LB过夜培养,1:100转接培养至细菌数量达1×108CFU/mL。将菌液14000g,10min离心弃上清液,取得沉淀,向菌体沉淀加入1mL液体LB重悬,加噬菌体Escherichia phage vB_EcoP-EG1到大肠杆菌MG1655菌液中(MOI=10),37℃进行孵育培养10min,然后14000g 10min离心弃上清液,取得沉淀,洗两次后加入到25mL新鲜的液体LB培养基中,37℃200rpm进行恒温振荡培养,在不同时间点取出上清,采用双层琼脂平板法确定出噬菌体在不同时间点的数量。从一步生长曲线直接得到噬菌体的潜伏期和爆发期,通过将感染期细菌细胞的初始数量除以平台期噬菌体滴度来计算噬菌体的爆发量。Escherichia phage vB_EcoP-EG1的潜伏期为5分钟,爆发期为15分钟,平均每个宿主菌能产生152.5个子代噬菌体(图2C)。该噬菌体能够快速有效的裂解宿主菌,是噬菌体治疗的优良候选噬菌体。
4.4噬菌体细菌挑战实验
将大肠杆菌MG1655过夜菌液1:100转接至50mL液体LB培养基,在37℃下,200rpm进行恒温振荡培养至细菌数量约为1×108CFU/mL,在不同培养瓶中加入不同数量的噬菌体Escherichia phage vB_EcoP-EG1(MOI=0.1,1,10),通过在不同时间点测量OD600数值监测细菌生长情况。在对照样品中加入SM缓冲液代替噬菌体。在MOI为10时,Escherichiaphage vB_EcoP-EG1只需要30分钟就能完全裂解大肠杆菌,在MOI为0.1时60分钟能完全裂解大肠杆菌。在不同MOI条件下,Escherichia phage vB_EcoP-EG1都能有效快速彻底的杀灭宿主大肠杆菌(图2D)。
5.噬菌体宿主谱分析
采用点滴法测定噬菌体Escherichia phage vB_EcoP-EG1的宿主谱。将109个细菌细胞与融化上层琼脂混合,将混合物倒在固体琼脂上制备双层琼脂平板,凝固后,滴加10μL噬菌体悬浮液(约1010PFU/mL)到每个细菌菌株的平板上。吸附斑点后,将平板倒置并孵育24小时,37℃培养箱。观察平板表面是否有噬菌斑产生,根据形成空斑的透亮度评价其裂解细菌的能力(表1)。噬菌体Escherichia phage vB_EcoP-EG1能够感染37株临床耐药大肠杆菌中的13株,能够彻底裂解10株尿液来源的大肠杆菌,一株唾液来源的大肠杆菌,能部分裂解一株唾液来源的大肠杆菌和一株血液来源的大肠杆菌。Escherichia phage vB_EcoP-EG1能有效裂解21株尿液来源的多耐药大肠杆菌中的10株,对尿液来源的大肠杆菌裂解效果好。
6.噬菌体基因组及蛋白质谱分析。
Escherichia phage vB_EcoP-EG1噬菌体感染对数期大肠杆菌(MOI=10),侵染1.5小时后收集裂解液,14000g离心10分钟取上清液。加入DNase I和RNase A后放入37℃水浴2小时后加入10μL 20mg/mL蛋白酶K,55℃,15min。采用天根基因组提取试剂盒提取得到噬菌体Escherichia phage vB_EcoP-EG1的DNA。DNA样品采用Illumina HiSeq2500测序分析。最后验证后Escherichia phage vB_EcoP-EG1基因组总长度为39919bp,并含有139bp的末端重复序列。并把Escherichia phage vB_EcoP-EG1基因组全序列上传到NCBI网站,登陆号为MG488277。通过Blast比对分析表明Escherichia phage vB_EcoP-EG1噬菌体是一株全新的噬菌体,其和十几种噬菌体基因组相似性比较高(覆盖率50-84%,相似度75-91%),但是仍有很大的不同。尤其是尾丝蛋白(ORF47编码)和最相近的T7噬菌体的尾丝蛋白也只有58%的同源性。已知噬菌体的尾丝蛋白决定噬菌体的宿主范围,所以尾丝蛋白的序列不同也决定了噬菌体Escherichia phage vB_EcoP-EG1的宿主范围不同于其他大肠杆菌噬菌体。Escherichia phage vB_EcoP-EG1编码的51个蛋白中并没有发现溶源性相关蛋白,所以确证Escherichia phage vB_EcoP-EG1是毒性噬菌体。同时也没有发现Escherichia phagevB_EcoP-EG1编码对动植物细胞有害的毒性蛋白质,从而保证了其安全性和有效性。Escherichia phage vB_EcoP-EG1噬菌体全蛋白变性酶解后,采用液相色谱-串联质谱联用分析(LC-ESI MS/MS)整个噬菌体颗粒蛋白组分。质谱共得到29个蛋白质,检测到了12结构蛋白中有11个,同时还检测到一些酶类,同时还鉴定得到很多假定蛋白,但是并没有发现毒性蛋白,近一步确证了该噬菌体不表达毒性蛋白(表2)。
7.噬菌体对细菌生物膜形成的影响
用96孔酶标板结晶紫法:培养大肠杆菌临床分离株MG1655和多耐药菌株390G7到对数期,取出菌液倍比稀释,使菌浓度约为106CFU/mL,在96孔板中分别加入200μL菌液,放入37℃静置培养24小时。取出100μL菌液,并向设置的阳性孔分别加入含有噬菌体Escherichia phage vB_EcoP-EG1100μL(MOI=10),阴性对照孔加入100μL液体LB,再次放入37℃培养箱静置培养24小时。吸出培养液,用PBS洗三遍,再加入0.1%结晶紫200μL染色20min。弃染液,用PBS洗三遍后各孔加入200μL 33%冰醋酸,固定15min,酶标仪测得各孔OD550-590nm数值来确定菌膜的形成量。在图3中可以看加入噬菌体Escherichia phagevB_EcoP-EG1后,大肠杆菌MG1655和多耐药菌株390G7生物膜的形成量有明显的减少,说明噬菌体Escherichia phage vB_EcoP-EG1可以有效的抑制大肠杆菌生物膜的形成。Escherichia phage vB_EcoP-EG1有潜力应用于杀伤由于生物膜的存在导致的难治性的耐药大肠杆菌导致的感染。
表1.噬菌体Escherichia phage vB_EcoP-EG1的宿主谱
表2.Escherichia phage vB_EcoP-EG1病毒颗粒的质谱分析结果
Claims (1)
1.大肠杆菌噬菌体在多耐药的大肠杆菌细菌分型中的应用,所述的大肠杆菌噬菌体,分类命名为Escherichia phage vB_EcoP-EG1 ,保藏号为:CCTCC M 2018562,保藏日期为2018年8月21日。
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