CN117286112A - 一种鲍曼不动杆菌噬菌体及其应用 - Google Patents
一种鲍曼不动杆菌噬菌体及其应用 Download PDFInfo
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Abstract
本发明公开了一种鲍曼不动杆菌噬菌体及其应用，涉及生物技术领域。分离出了一株有裂解活性的新型噬菌体PhaR5，保藏号为：CCTCC NO：M 2023181，该噬菌体可以裂解多株具有荚膜的多重耐药鲍曼不动杆菌。本发明的噬菌体可被用于制备喷雾，用于人体体表和医院病房的环境消毒。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种鲍曼不动杆菌噬菌体及其应用。
背景技术
鲍曼不动杆菌(Acinetobacter baumannii，Ab)属非发酵、氧化酶阴性、触酶阳性的革兰氏阴性菌，广泛存在于自然界的水、土壤和医院环境，是医院获得性感染的主要条件致病菌之一，可引起住院病人的呼吸道、泌尿道和伤口感染，也是引起烧伤感染的病原菌。该菌在医院环境中分布很广且可以长期存活，有很强的环境适应性，极易造成危重患者的感染，常从被感染患者的血、尿、脓液及呼吸道分泌物等标本中分离出，在非发酵菌中感染仅次于铜绿假单胞菌。且对干燥和消毒剂均有较强的抵抗性，导致其在医院的感染病房内很难被清除，容易引发细菌性的皮肤伤口感染、泌尿系统感染，甚至败血症等；对于重症感染和免疫力低下的患者有较高的致病率和致死率，从而增加了患者的住院成本和时间。为了治疗细菌性的感染，抗生素的大量使用暂时抑制了细菌的生长，但长期的滥用和误用导致耐药细菌甚至超级细菌的不断涌现，严重威胁人类的健康；特别是对碳青霉烯类药物耐药的鲍曼不动杆菌的出现，迫使感染科医生选择使用多粘菌素。多粘菌素发现于上世纪的日本，由于其具有较强的肾毒性和神经毒性，早期只用于农业和畜牧业，但多重耐药的严峻形势迫使临床上开始使用多粘菌素。已有研究报道，随着多粘菌素的使用，医院内不断发现对多粘菌素耐药的鲍曼不动杆菌。探索靶向多重耐药菌的新途径将会增加对抗细菌感染的“工具箱”。
噬菌体(Bacteriophage or Phage)也称为细菌的病毒，数量多，分布广泛，且具有很强的宿主菌特异性。噬菌体根据生命周期的不同，分为裂解性和温和性。裂解性噬菌体通过吸附到宿主菌表面的受体，将噬菌体遗传物质注射到细菌胞质内，使用宿主菌的代谢和合成系统来合成子代噬菌体，最后裂解宿主菌来释放子代噬菌体。温和性噬菌体则将其DNA整合到宿主菌的染色体或者质粒上形成前噬菌体，赋予宿主菌新的表型，如：毒力增强(毒力基因)或对抗生素的耐药性(耐药基因)，温和性噬菌体一般不会裂解宿主菌，只有当宿主菌面临不利的环境时，前噬菌体会被诱导出来从而裂解宿主菌。与抗生素相比，噬菌体特异性强，不会影响人体的正常菌群，而且噬菌体杀菌的机制不受细菌耐药性的影响，对抗生素不敏感的多重耐药菌可以被噬菌体裂解，因此，裂解性噬菌体可以被用于噬菌体治疗，从而可以缓解多重耐药菌引发的严峻耐药形势。
分离自临床的鲍曼不动杆菌多具有较厚的荚膜，荚膜形成的物理屏障阻碍了药物和靶位点的识别和结合，难以发挥杀菌的作用，所以，以荚膜为代表的毒力因子增加了鲍曼不动杆菌的难治性。如果噬菌体可以裂解荚膜，一方面有利于噬菌体成功侵染病原菌，同时，荚膜的瓦解会增加抗生素对病原菌的杀伤作用，有利于噬菌体与抗生素的联用；而且荚膜的瓦解增强了机体对病原菌的免疫识别，机体的免疫系统就能轻而易举地杀灭病原菌。
发明内容
本发明从河水中分离出了一株新型的裂解性鲍曼不动杆菌噬菌体，其对具有荚膜的多重耐药鲍曼不动杆菌有杀伤作用，通过对其生物学特征的探索和分析，发现其有较好的医疗用途。
本发明提供了一株新型裂解性鲍曼不动杆菌噬菌体，命名为PhaR5，该噬菌体能裂解多重耐药鲍曼不动杆菌。同时提供该噬菌体的相关医用途径，可以将该噬菌体制作成喷雾，用于重症监护病房的环境消毒，以降低院内鲍曼不动杆菌的感染率。
本发明的技术方案具体如下：
本发明提供了一种鲍曼不动杆菌噬菌体，命名为鲍曼不动杆菌噬菌体PhaR5，本发明的噬菌体PhaR5是从浙江省海宁市的河水中采集并分离。于2023年2月23日保存在中国典型培养物保藏中心(地址：湖北省武汉市武昌区八一路299号)，保藏号为：CCTCC NO：M2023181。
本发明还提供了所述的鲍曼不动杆菌噬菌体在防治鲍曼不动杆菌污染方面的应用。
本发明还提供了所述的鲍曼不动杆菌噬菌体在制备用于预防或治疗由鲍曼不动杆菌引起的感染性疾病药物中的应用。
该噬菌体能裂解具有荚膜多重耐药鲍曼不动杆菌，所述具有荚膜的多重耐药鲍曼不动杆菌为鲍曼不动杆菌XH666或鲍曼不动杆菌XH762，两株菌均对碳青霉烯类药物、氨基糖苷类药物和多粘菌素不敏感。
本发明还提供了一种鲍曼不动杆菌抑制剂，活性成分包含所述的鲍曼不动杆菌噬菌体。
所述鲍曼不动杆菌为鲍曼不动杆菌XH666或鲍曼不动杆菌XH762。
所述鲍曼不动杆菌抑制剂的剂型为液剂或固体剂。
本发明还提供了一种鲍曼不动杆菌的防治方法，将所述的鲍曼不动杆菌噬菌体、所述的鲍曼不动杆菌抑制剂喷洒在防治区域。
本发明的有益效果：
本发明提供了一株有裂解活性的新型噬菌体PhaR5，保藏号为：CCTCC NO：M2023181，该噬菌体可以裂解多株具有荚膜的多重耐药鲍曼不动杆菌。本发明的噬菌体可被用于制备喷雾，用于人体体表和医院病房的环境消毒。
附图说明
图1为噬菌体PhaR5在多重耐药鲍曼不动杆菌XH666菌苔上形成的噬菌斑。
图2为噬菌体PhaR5透射电镜图。
图3为噬菌体PhaR5酸碱度敏感性测试结果图。
图4为噬菌体PhaR5温度敏感性测试结果图。
图5为噬菌体PhaR5对多重耐药鲍曼不动杆菌XH666和XH762的生长抑制作用图。
图6为多重耐药鲍曼不动杆菌XH666与XH762分别与噬菌体PhaR5共培养的生长动力曲线图。
具体实施方式
实施例中使用的鲍曼不动杆菌XH666是2015年3月份分离自浙江大学附属邵逸夫医院，属于鲍曼不动杆菌。鲍曼不动杆菌XH762于2013年8月份分离自浙江大学附属第一医院。XH666和XH762在医院检验科经过质谱鉴定的，是鲍曼不动杆菌，而且经过药敏测试(根据美国临床实验室标准化协会(CLSI)推荐的药敏测试)，都是多重耐药鲍曼不动杆菌。
实施例1：噬菌体PhaR5的分离纯化
从浙江省海宁市采集500mL河水并进行低速的离心(4200rpm，15分钟)以沉淀较大的尘埃颗粒，取离心后的河水上清，并使用一次性的滤器(滤膜孔径为0.45μm)进行抽滤，从而过滤掉上清中的细菌。以多重耐药鲍曼不动杆菌XH666为宿主菌，向2mL 2×MHB培养基(北京索莱宝科技有限公司)中加入在100μL MHB培养基中过夜培养的鲍曼不动杆菌XH666，实验组加入2mL过滤后的河水上清，对照组加入2mL无菌去离子水，将两组在37℃，200rpm培养5小时，培养期间用肉眼或者分光光度计检测培养液的浑浊度或光密度值OD600，如果实验组的光密度值比对照组要低，则取2mL实验组培养液进行离心(4200rpm，15分钟)取上清，然后取10μL上清进行双层琼脂平板法获得肉眼可见的噬菌斑。用枪头吸取单个的噬菌斑并在1mL的MHB培养基中吹打均匀后，进行离心(4200rpm，3分钟)后取10μL上清进行双层琼脂平板法获得肉眼可见的噬菌斑，再次用枪头吸取单个的噬菌斑并重复上述操作，直至出现大小和形态一致的噬菌斑(如图1所示)，然后向双层琼脂平板内加入适量体积的MHB，在摇床上(室温，20rpm，5小时)缓慢溶解噬菌斑中的噬菌体，在收集MHB后离心(4200rpm，15分钟)取上清，最后为了去除宿主菌将上清进行滤膜(滤膜孔径为0.45μm)过滤，此即为纯化后的单株噬菌体，命名为PhaR5。保藏在中国典型培养物保藏中心，保藏单位地址：中国·武汉·武汉大学，保藏号：CCTCC NO：M 2023181，分类命名：Acinetobacter baumanniiphage，保藏日期为2023年02月23日。
实施例2：噬菌体PhaR5透射电镜观察
采用双层琼脂平板法扩增实施例1中纯化的噬菌体PhaR5，在37℃温箱内培养3小时，选择噬菌斑互联成网络的光滑平板，该平板有最高的噬菌体滴度，然后向该平板内加入2mL的MHB，在摇床上(室温，20rpm，5小时)缓慢溶解噬菌斑中的噬菌体，在收集MHB后离心(4200rpm，15分钟)取上清，再将上清进行滤膜(滤膜孔径为0.45μm)过滤，将滤液进行离心(4℃，13800rpm，1小时)后，用枪头吸取掉多余的上清，仅保留底部白色的部分，最后加入100μL的超纯水，此即为用于透射电镜观察用的噬菌体溶液。取适量噬菌体溶液在铜网表面，用2％醋酸铀进行负染后置于透射电镜下观察。结果如图2所示，该噬菌体属于肌尾噬菌体，和大肠杆菌噬菌体T4的形态一致。
实施例3：噬菌体PhaR5基因组的提取与二代测序
用病毒基因组提取试剂盒(购买自北京博迈德基因技术有限公司)对实施例1中纯化的噬菌体PhaR5进行基因组的提取，提取后的基因组用适量超纯水溶解并检测浓度，满足二代测序的浓度和总量要求后，送至天津诺禾致源生物信息科技有限公司进行全基因组测序。并将测序结果上传到NCBI数据库，GenBank号是OL763420.1。
实施例4：噬菌体PhaR5的冻干
实施例1中纯化的噬菌体PhaR5与0.5M的蔗糖以1∶3体积比混合，并置于液氮中速冻，然后置于冻干机中冻干过夜。
将噬菌体PhaR5的冻干粉末在室温下放置若干天，然后比较噬菌体PhaR5在冻干前、冻干后、以及冻干后室温放置第1天、第4天、第7天、第14天和第30天的滴度。结果发现，冻干之后的噬菌体更容易储存于携带，而且冻干后放置30天也不会影响滴度。
实施例5：噬菌体PhaR5的物理化学稳定性
向有着不同pH值(pH分别为1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0)的SM缓冲液中分别加入等量实施例1中纯化的噬菌体PhaR5，37℃孵育60分钟，然后检测噬菌体的滴度，获得噬菌体对不同酸碱度的敏感性测试结果。测试结果如图3所示，强酸环境不利于噬菌体的稳定，而在pH 6-8的条件下，噬菌体表现稳定。
将含有实施例1中纯化的噬菌体PhaR5的SM缓冲液分别置于7种不同的温度(-80℃、-20℃、4℃、25℃、37℃、42℃、60℃)下孵育60分钟，然后检测噬菌体PhaR5的滴度。检测结果如图4所示，噬菌体在低温下很稳定，滴度几乎没有降低，随着温度的升高，特别是到60℃，噬菌体滴度下降好几个数量级，说明高温不适宜该噬菌体的保存与使用。
实施例6：噬菌体PhaR5对多重耐药鲍曼不动杆菌的生长抑制作用
将实施例1中纯化的噬菌体PhaR5与鲍曼不动杆菌XH666、鲍曼不动杆菌XH762在MHB培养基中振荡共培养(按照噬菌体与细菌的MOI为1来共培养噬菌体与细菌)3小时后，观察菌液浑浊情况，以保藏号为ATCC 17978的菌株(简称ATCC 17978菌株)和噬菌体PhaR5作为对照组(ATCC 17978菌株为噬菌体PhaR5的非敏感对照菌株，其生长不受噬菌体的影响)。MOI(multiplicity ofinfection)即感染复数，指的是感染时病毒与细胞数量的比值。
结果如图5所示，将噬菌体分别与这三种菌(MOI为1)在液体培养基振荡培养3小时后(200rpm/min)，肉眼即可观察菌液的浑浊度。发现对噬菌体不敏感的ATCC 17978的菌液浑浊，生长不受噬菌体的影响；而XH666和XH762的菌液变得透明，并无明显的细菌生长，说明细菌的生长受到噬菌体的抑制作用。
实施例7：多重耐药鲍曼不动杆菌与噬菌体PhaR5共培养的生长动力曲线
先将过夜培养的多重耐药鲍曼不动杆菌XH666与鲍曼不动杆菌XH762的菌液按照1∶100接种到新鲜的液体培养基中，振荡培养到OD600值为0.6，然后按照MOI为1加入噬菌体PhaR5，继续振荡共培养8小时，每1小时使用酶标仪检测OD600来获取菌株的生长动力曲线。以保藏号为ATCC 17978的菌株作为对照菌株，保藏号为ATCC 17978的菌株为噬菌体PhaR5的非敏感菌株，其生长不受噬菌体的影响。结果如图6所示，对噬菌体不敏感的ATCC 17978的OD600总体呈上升趋势，说明菌的生长没有受到抑制；而对噬菌体敏感的XH666和XH762的OD600呈下降趋势，说明噬菌体对其有很强的生长抑制作用，而且在长达8小时的共培养周期内，并未出现明显的噬菌体抗性突变菌株。
Claims (8)
1.一种鲍曼不动杆菌噬菌体，其特征在于，命名为鲍曼不动杆菌噬菌体PhaR5，保藏号为：CCTCC NO：M 2023181。
2.权利要求1所述的鲍曼不动杆菌噬菌体在防治鲍曼不动杆菌污染方面的应用。
3.权利要求1所述的鲍曼不动杆菌噬菌体在制备用于预防或治疗由鲍曼不动杆菌引起的感染性疾病药物中的应用。
4.如权利要求3或4所述的应用，其特征在于，所述鲍曼不动杆菌为鲍曼不动杆菌XH666或鲍曼不动杆菌XH762。
5.一种鲍曼不动杆菌抑制剂，其特征在于，活性成分包含权利要求1所述的鲍曼不动杆菌噬菌体。
6.如权利要求5所述的鲍曼不动杆菌抑制剂，其特征在于，所述鲍曼不动杆菌为鲍曼不动杆菌XH666或鲍曼不动杆菌XH762。
7.如权利要求5所述的鲍曼不动杆菌抑制剂，其特征在于，所述鲍曼不动杆菌抑制剂的剂型为液剂或固体剂。
8.一种鲍曼不动杆菌的防治方法，其特征在于，将权利要求1所述的鲍曼不动杆菌噬菌体、权利要求5～7任一所述的鲍曼不动杆菌抑制剂喷洒在防治区域。
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