CN112353820A - 新型st型crpa菌株的应用 - Google Patents
新型st型crpa菌株的应用 Download PDFInfo
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Abstract
本发明涉及临床致病微生物技术领域，公开了新型ST型CRPA菌株在制备CRPA临床感染疾病的药物中的应用；还公开了新型ST型CRPA菌株在制备CRPA临床感染疾病的动物模型中的应用。本发明首次获得新型ST型CRPA菌株，研究结果显示新型ST型CRPA菌株的毒力强，其毒力与碳青霉烯类耐药无明显相关性，其对双碳青霉烯敏感性差，在临床疾病中存在隐患，值得临床重视，为CRPA临床感染疾病的治疗和研究提供了更多的选择和可能性。
Description
技术领域
本发明涉及临床致病微生物技术领域，具体涉及新型ST型CRPA菌株的应用。
背景技术
铜绿假单胞菌是医院获得性感染中非常重要的条件致病菌之一，近年来，临床菌株对多种抗菌药物的耐药性不断升级，并在世界范围内广为扩散，日益增加的感染流行已经对人类健康构成了巨大威胁。作为重要的机会性感染病原菌，铜绿假单胞菌可引起一系列的院内感染，包括脓毒血症、肺炎、尿路感染和皮肤软组织感染等，也是肺囊性纤维化患者慢性感染的一个主要原因。一些多重耐药或泛耐药菌株已被证实对常用抗菌药物产生耐药，如β内酰胺类、氨基糖苷类、氟喹诺酮类和多黏菌素类。碳青霉烯类因其对细菌青霉素结合蛋白亲和性高，对超广谱β内酰胺酶稳定及对细菌外膜的穿透性强，被作为抗多重耐药革兰阴性菌包括铜绿假单胞菌感染的重要抗生素，也是治疗耐药铜绿假单胞菌的一线药物，但是随着碳青霉烯类在临床的广泛应用，耐碳青霉烯铜绿假单胞菌(CarbapenemResistant Pseudomonas aeruginosa，CRPA)也不断增多，所致感染治疗棘手，对临床感染患者构成巨大威胁，给临床治疗带来了巨大挑战。2017年，WHO将耐碳青霉烯铜绿假单胞菌列为需要紧急关注和应对的多重耐药菌。
综上所述，加深对CRPA菌株及其分型的认识是研究CRPA临床感染疾病的重要前提，可为提高CRPA临床感染诊治与医院感染防控能力提供更多的可能性。
发明内容
基于以上问题，本发明提供新型ST型CRPA菌株的应用，本发明为CRPA临床感染疾病的治疗和研究提供了更多的选择和可能性。
为解决以上技术问题，本发明提供了新型ST型CRPA菌株在制备CRPA临床感染疾病的药物中的应用，所述新型ST型CRPA菌株为ST-7490型CRPA菌株，其分类命名为铜绿假单胞菌ST7490(Pseudomonas aeruginosa ST7490)，保藏编号为CCTCC NO：M2020537，保藏于中国典型培养物保藏中心(CCTCC)，保藏地址：中国武汉武汉大学；保藏日期为：2020年9月24日。
进一步的，所述药物具有如下作用中的一种或多种：(1)降低新型ST型CRPA菌株毒性的作用；(2)具有降低或扭转新型ST型CRPA菌株耐药性的作用；(3)具有抑制或杀灭新型ST型CRPA菌株的作用。
为解决以上技术问题，本发明还提供了新型ST型CRPA菌株在制备CRPA临床感染疾病的动物模型中的应用。
与现有技术相比，本发明的有益效果是：本发明首次获得新型ST型CRPA菌株，研究结果显示新型ST型CRPA菌株的毒力强，其毒力与碳青霉烯类耐药无明显相关性，其对双碳青霉烯敏感性差，在临床疾病中存在隐患，值得临床重视，为CRPA临床感染疾病的治疗和研究提供了更多的选择和可能性。
附图说明
图1为本实施例的MAlDI-TOF-MS鉴定结果图；
图2为本实施例的七个铜绿假单胞菌新ST型结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例：
下面通过实施例对本发明进行具体的描述，有必要在此指出的是以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制。下述实施例中，文中未注明具体条件的实验方法，通常按照常规条件，或按照制造商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。
本实施例使用的质控菌株为质控菌株铜绿假单胞菌Psedomonas aeruginosaATCC 27853，野生型的铜绿假单胞菌PAO1由四川大学国家重点实验室提供，均购自美国标准培养物中心(American type culture center，ATCC)，由四川大学华西医院感染性疾病中心实验室保存。本实施例的实验菌株保存在发明人所在医院感染管理科，菌株来源于四川大学华西医院2017年1月至2017年12月从临床各科室各类临床标本中分离所得，并经Vitek II全自动细菌鉴定系统初步判断为CRPA。每位患者仅收集一株菌，如存在多次培养为同一种菌，则以临床第1次分离的耐碳青霉烯铜绿假单胞菌菌株为实验菌株，剔除同一患者的重复菌株。将实验菌株复苏后，常规培养、分离提纯并经细菌鉴定后，用甘油肉汤保菌冰箱存储。
经含美洛培南(4ug/ml)显色平板孵育后，筛选显示为蓝色或黄色的菌落，作为耐耐碳青霉烯类铜绿假单胞菌菌株，每个单菌落在LB平板上经过二次转种增菌与分离纯化，收集纯菌落，煮沸法提取DNA，通过gyrB引物扩增PCR产物，产物及测序引物送测序。PCR扩增产物经过纯化测序，数据经Seqman软件程序(DNAstar软件)转换后，获得所需gyrB基因序列。在NCBI网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)数据库中，进行DNA碱基序列比对，再次对分离纯化后的菌株进行菌种鉴定，MAlDI-TOF-MS鉴定结果见附图1。经过分离提纯，获得2017年1月至2017年12月期间各类临床标本中分离的CRPA共57株。
本实施例采用微量肉汤稀释法对上述57株目标铜绿假单胞菌菌株进行体外药敏试验，测定其对临床常用抗菌药物的最低抑菌浓度(minimal inhibitoryconcentrations，MICs)。菌悬液用MH肉汤进行1:100(V/V)稀释(此时含菌量约为106CFU/ml)，将备用的细菌悬液在加入96孔板之前再次使用MH肉汤再稀释10倍，即相对于比浊时按照1:1000稀释，使每孔的菌液最终浓度为5×105CFU/ml加入在96孔板相应含有不同浓度的孔洞。将抗菌药物加入溶液中配制成1024μg/ml的浓度，作为母液备用，。将制备好的抗菌药物母液按照对倍稀释法稀释，可得到在不同浓度的抗菌药物对倍稀释工作液。使用到的各种抗菌药物的配制溶液如下：
(1)美罗培南、阿米卡星、庆大霉素、妥布霉素、头孢吡肟、左氧氟沙星、氨曲南、哌拉西林/他唑巴坦：水；
(2)头孢他啶：水，溶剂：Na2CO3；
(3)环丙沙星：水加少量冰醋酸；
(4)亚胺培南：磷酸盐缓冲液，PH 7.2，0.01mol/L。
根据《2016年CLSI铜绿假单胞菌抑菌圈直径及MIC解释标准》：肉汤稀释法，接种液采用0.5号麦氏标准，孵育16-20h，质控菌株为铜绿假单胞菌ATCC27853。57株CRPA体外药敏结果见表1，结果显示本实施例分离的57株CRPA菌株对碳青霉烯类属于低水平耐药，美罗培南及亚胺培南MIC50均为32ug/ml。多数CRPA对氨基糖苷类、氟喹诺酮较敏感，但对临床常用的β内酰胺类，包括酶抑制剂复合制剂如哌拉西林他唑巴坦、第三代头孢菌素，单环类抗生素氨曲南均呈现明显耐药。
表1常用抗菌药物对57株CRPA的MIC值(μg/ml)
*本研究重点关注对碳青霉烯类耐药，其他抗生素MIC最高浓度梯度在耐药水平以上。
接着又用铜绿假单胞菌ATCC27853作为标准菌株进行了上述57株CRPA菌株的外排泵抑制实验，CRPA菌株外排泵抑制实验结果见表2，57株CRPA中除1株本身对美洛培南敏感而对亚胺培南耐药外，有49株(87.5％)对美罗培南耐药的CRPA，通过外排泵抑制实验，美罗培南联合外排泵抑制剂PaβN(50ug/ml)后其MIC值下降≥4倍，美罗培南联合外排泵抑制剂PaβN(50ug/ml)对7株CRPA菌株的MIC下降＜4倍，由此可见，在49株对美罗培南耐药的CRPA菌株中存在外排泵耐药机制，占87.5％。
表2 CRPA菌株外排泵抑制实验结果
见表3、表4、表5、表6和表7，发明人在后续实验研究中发现57株CRPA菌株中，30株存在基因突变(包括碱基点突变、插入、缺失框移等)提前出现终止密码子，导致OprD氨基酸表达中断，氨基酸明显缩短或分段表达可明显改变OprD通道蛋白的完整性，从而介导对碳青霉烯类耐药，其他27株CRPA的OprD通道蛋白的氨基酸改变无明显中断，保持OprD通道蛋白的完整性，但这些菌株表达的OprD蛋白的氨基酸出现明显缩短或氨基酸缺失，插入、过度表达导致氨基酸末端延长等。且上述57株CRPA中，未发现有携带A类碳青霉烯酶基因的blaKPC、blaGES，B类碳青霉烯酶基因中，4株携带blaImp-45，1株携带blaVim-2，未发现携带blaSPM、bla GIM，blaNDM、blaFIM型金属β内酰胺酶基因，8株携带D类blaOXA-50碳青霉烯酶基因。上述57株CRPA不仅携带碳青霉烯酶基因，同时也携带有多种其他类抗菌药物耐药基因，菌株的临床表型可为多重耐药、泛耐药，从而导致现有药物治疗失败。另外，57株CRPA的MexX外排泵基因均阳性，未检测到MexA、MexB、MexC、MexD等外排泵基因，推测MexX型外排泵与我院CRPA对美罗培南的耐药有关。
表3 57株CRPA的OPRD通道蛋白编码基因分析结果
注：因075002和075021对亚胺培南敏感，故其OprD与标准菌株PAO1一致，无需分析。
表4 57株CRPA与PAO1的OprD氨基酸比对结果
表5 57株CRPA携带氨基糖苷类耐药的相关基因结果
表6 57株CRPA携带β-内酰胺类耐药相关基因
其余PER、VEB、MOX、FOX、CMY、MIR等β内酰胺酶基因均为阴性。
表7 57株CRPA携带氟喹诺酮耐药相关基因
本实施例对上述57株CRPA菌株进行了多位点序列分型(multilocus sequencetyping,MLST)研究，研究结果见表8和附图2，上述57株CRPA菌株中，首次发现7个新型ST型CRPA菌株，分别是ST-2965、ST-2966，ST-2967，ST-2968，ST-2969，ST-2972，ST-7490。
表8 57株CRPA菌株MLST分型结果
本实施例通过大蜡螟幼虫造模的方式研究上述7个新型ST型CRPA菌株毒力并与临床发现的高、中、低碳青酶烯耐药CRPA菌株及敏感菌株进行了毒力比较。选用的高耐药的CRPA菌株2株(075011、075033)，一般耐药菌株1株(075040)，敏感菌株1株，编号为6017。
大蜡螟感染耐碳青霉烯的铜绿假单胞体内试验如下：
(1)菌液制备
用新的ST型CRPA菌株7株，高耐药的CRPA菌株2株，一般耐药菌株1株，敏感菌株1株，编号为6017，分别接种于LB琼脂平板上，37℃过夜孵化培养；根据比浊仪器的显示，挑取一定数量的单菌落，将细菌溶解于配制好的无菌的PBS溶液中，混匀后比浊浓度到3，然后采用10倍稀释法分别稀释10倍、102倍、103倍、104倍、105倍、106倍，然后每个浓度分别吸取三个10μl菌液，将10ul的含菌菌液呈三角形样点样至琼脂平板，37℃孵育过夜，采用平板计数法计数以确认活菌数量，以三个菌液形成的单个菌落数量的平均值来估算出原液及不同稀释倍数下菌液浓度；
(2)蜡螟幼虫的选择及分组
大蜡螟幼虫均购自天津柳林养殖有限责任公司，大蜡螟幼虫的发货及使用前均由纸箱避光，加水湿化；幼虫在成茧纸壳中分离至9cm的塑料皿，选择虫体长度在3cm左右，尽量在外观上长度一致，虫体颜色蜡黄，选择质量在300mg±20mg的幼虫作为研究对象；活力判断以将幼虫背翻，能够迅速复原的虫体为正常，当虫体颜色发黑或者出现黑点或者不能迅速由背翻复原判断为活力欠佳不作为研究对象，将活力足，虫体质量、长度基本一致的大蜡螟幼虫的塑料皿放入纸箱避光、加湿后37℃孵箱过夜，确保幼虫在过夜的过程中不出现自然死亡，过夜后虫体死亡或活力不佳不入组；每个菌株按照配制的浓度梯度从高到低(麦氏比浊至3的原液、稀释10倍、102倍、103倍、104倍、105倍、106倍)需要7组幼虫，每个浓度下需要大蜡螟幼虫16条；如两组间死亡幼虫差距较大，则增加一次对半稀释倍数；另外需要设置空白对照组及仅注射PBS作为对照组；
(3)细菌接种大蜡螟幼虫
用左手将幼虫固定，右手持25ul的玻璃注射器对准幼虫腹部倒数第二对足任意一边足跟处作为穿刺点，用25μL微量注射器动作轻柔的抽取各稀释浓度菌液，防止空气在注射菌液是进入虫体腹腔，抽吸菌液的量为10μL，缓慢进针，当有落空感时即意味着针尖已经位于幼虫腹腔内，轻柔缓慢的的注入注射器内的10ul菌液；为模拟人体内状态及温度，将接种好菌液后的幼虫放置在纸箱中避光、加湿；将纸箱安置在恒温37℃孵箱中；如出现虫体发黑无活动的则认为幼虫已死亡；
注射菌液死亡的虫体判断，如虫体发黑则认为已经死亡；结果判断，前期预实验结果表明在观察期96小时内，临床菌株毒力导致的虫体死亡数量变化较大，在96小时之后的时间，虫体的死亡几乎没有变化，因此将临床菌株的细菌毒力实验的观察终点设置在96小时；
(4)大蜡螟幼虫感染铜绿假单胞菌的模型
将临床分离提纯的CRPA菌株复苏后接种于琼脂平板上，37℃过夜孵化增菌；根据比浊需要挑取单菌落溶解于事先配好的无菌的PBS溶液中，混匀后应用麦氏比浊仪比浊至3成原液，采用10倍稀释法应用PBS溶液分别稀释10倍、102倍、103倍、104倍、105倍、106倍，并分别在各稀释浓度下取10ul点样至琼脂平板37度孵育过夜培养，三重复点样，然后选择三重复生成的单菌落平板计数，以平均数来估算出原液及不同倍数下菌液浓度，菌株编号同前。
将每一株菌按照稀释浓度梯度从原液由高到低注射到大蜡螟幼虫腹腔，每组16条，设置的空白对照组及注射PBS对照组无一条幼虫死亡，故结果可信，详细结果见表9。
表9临床新ST型CRPA菌株不同稀释浓度下菌落计数的估算结果(cfu/ml)
按照以上确定的稀释浓度梯度进行菌液量的计算，取相同浓度梯度，即确保在同一梯度的菌量下观察大蜡螟幼虫的死亡情况。从大蜡螟幼虫实验来看，每一个CRPA菌株，随着注射至大蜡螟幼虫的浓度升高，大蜡螟幼虫的死亡率呈逐渐上升的趋势。在原液10^8CFU/ml浓度下72小时所有大蜡螟幼虫全部死亡。新的ST型菌株，075006(ST-2967)、075022(ST-2966)在106CFU/ml72小时全部死亡，075015(ST-2968)、075056(ST-7490)在104CFU/ml72小时全部死亡，075016(ST-2969)在105CFU/ml72小时全部死亡，075025(ST-2972)、075037(ST-29657)在108CFU/ml 72小时全部死亡，高耐药的菌株075011在浓度为107CFU/ml72小时全部死亡，075033在105CFU/ml 72小时全部死亡，敏感菌株在107CFU/ml 72小时全部死亡。
观察72小时，PBS对照组及空白对照组无一条幼虫死亡，结果可信可靠。结果见表10。
表10新ST型、高耐药、低耐药、普通耐药CRPA菌株的毒力实验结果
双碳青霉烯类联合实验结果见表11、表12和表13，结果显示：本院耐碳青霉烯铜绿假单胞菌(CRPA)，对单一的碳青霉烯类的美罗培南，亚胺培南耐药结果相似，临床菌株对上述两种临床常见药物的耐药率均＞90％，而对比阿培南的耐药率为63.2％，显示比阿培南对CRPA的敏感性高于常见的碳青霉烯类亚胺培南和美罗培南；同样美洛培南、亚胺培南对临床CRPA菌株MIC50，MIC90一致，但比阿培南，较上述两种碳青霉烯类的MIC90低于一个浓度梯度。联合药敏显示，在亚胺培南、美罗培南、比阿培南的三种药物两两组合中，均能够明显降低CRPA的耐药性，从美罗培南、亚胺培南、比阿培南单药耐药率的91.2％，91.12％，63.2％，分别减低到美罗培南+亚胺培南的35.2％，美罗培南+比阿培南的17.6％，亚胺培南+比阿培南组的7.1％，其中，耐药率下降最为明显的是亚胺培南+比阿培南组，
表11两种碳青霉烯类药物联合抗CRPA的MIC值(ug/ml)
IMP亚安培南MEM美罗培南BIM比阿培南
表12 57株CRPA对碳青霉烯类两两联合体外药敏结果
表13双碳青霉烯联合抗CRPA的FIC指数及抗菌活性变化
上述实验显示新型ST型CRPA菌株075015(ST-2968)、075056(ST-7490)的毒力最强，075016(ST-2969)次之，075022(ST-2966)在本研究中排在第三位，以上毒力排名前三位的新型ST型CRPA菌株需要临床与基础研究给予更多的关注。另外，上述结果中值得注意的是，新型ST型CRPA菌株中的ST-7490型(075056)CRPA菌株，三种碳青霉烯组合的双碳青霉烯方案，均不能将MIC降至耐药折点以下，提示该新型ST-7490对双碳青霉烯敏感性差，存在隐患，值得临床重视。
本实施例的新型ST型CRPA菌株中的ST-7490型(075056)CRPA菌株即为分类命名为为铜绿假单胞菌ST7490(Pseudomonas aeruginosa ST7490)，保藏编号为CCTCC NO：M2020537，保藏于中国典型培养物保藏中心(CCTCC)，保藏地址：中国武汉武汉大学；保藏日期为：2020年9月24日。
ST-7490型CRPA菌株可应用于制备CRPA临床感染疾病的药物中，制备的药物具有如下作用中的一种或多种：(1)降低新型ST型CRPA菌株毒性的作用；(2)具有降低或扭转新型ST型CRPA菌株耐药性的作用；(3)具有抑制或杀灭新型ST型CRPA菌株的作用。ST-7490型CRPA菌株还可应用于制备CRPA临床感染疾病的动物模型中。
如上即为本发明的实施例。上述实施例以及实施例中的具体参数仅是为了清楚表述发明验证过程，并非用以限制本发明的专利保护范围，本发明的专利保护范围仍然以其权利要求书为准，凡是运用本发明的说明书及附图内容所作的等同结构变化，同理均应包含在本发明的保护范围内。
Claims (3)
1.新型ST型CRPA菌株在制备CRPA临床感染疾病的药物中的应用，其特征在于，所述新型ST型CRPA菌株为ST-7490型CRPA菌株，其分类命名为铜绿假单胞菌ST7490(Pseudomonasaeruginosa ST7490)，保藏编号为CCTCC NO：M2020537。
2.根据权利要求1所述的应用，其特征在于，所述药物具有如下作用中的一种或多种：(1)降低新型ST型CRPA菌株毒性的作用；(2)具有降低或扭转新型ST型CRPA菌株耐药性的作用；(3)具有抑制或杀灭新型ST型CRPA菌株的作用。
3.权利要求1或2所述的新型ST型CRPA菌株在制备CRPA临床感染疾病的动物模型中的应用。
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