CN111458313A - 基于荧光d型氨基酸代谢标记的抗菌药敏试验检测方法 - Google Patents
基于荧光d型氨基酸代谢标记的抗菌药敏试验检测方法 Download PDFInfo
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Abstract
本发明涉及了一种基于荧光D型氨基酸代谢标记的抗菌药敏试验检测方法,其利用荧光D型氨基酸代谢标记物标记与抗生素溶液混合孵育后的菌液,以根据其荧光信号强度获得抗生素最小抑制浓度。
Description
技术领域
本发明涉及细菌代谢活性的评估方法以及抗菌药敏试验的检测方法,尤其是涉及基于荧光D型氨基酸代谢标记的抗菌药敏试验检测方法,用于快速评价或评测抗菌剂或细菌代谢活性,例如抗生素,对临床分离细菌菌株的抑菌效果,以指导临床用药。
背景技术
随着抗生素在医疗及养殖等行业中的广泛使用,尤其是广谱抗生素的大量使用,已导致细菌耐药问题日益凸显,甚至陆续出现了可以抵抗几乎所有抗生素的超级细菌。细菌耐药性的产生和传播已引起全世界的普遍关注,被世界卫生组织认为是21世纪最大的公共卫生安全问题之一。解决这一问题的关键是及时地合理使用抗生素,不误用、不滥用,即应根据患者感染致病菌的种类,有针对性且适量地使用抗生素,而且这也能为病人提供更有效的治疗。
目前最通用也是最有效的办法是通过抗菌药敏试验为临床上合理使用抗生素提供依据,信息包括致病菌对哪些抗生素敏感及相应的最小抑菌浓度 (minimum inhibitoryconcentration,MIC)。现有的抗菌药敏试验检测技术,如肉汤梯度稀释法、纸片扩散法、E-test法等,大多基于细菌是否生长增殖来评价抗生素的作用效果,通常需要较长时间的培养(一般大于8-16小时)以达到肉眼可分辨的菌量(浑浊度或抑菌圈),才能评价药物的作用效果,其最主要的缺点是耗时太长,延误对临床合理使用抗生素的指导。即使通过自动化设备来简化操作流程,现有的设备,如Vitek、BD Phoenix、Microscan Walk Away 等是以细菌浑浊度为检测指标,虽然仪器给出的MIC准确率有所提高,但仍受细菌分裂增殖需要较长培养时间的限制(一般8-24小时)[参考文献1]。而且这些基于细胞生长增殖的检测方法还存在一个问题,即在其所测得的MIC浓度下的细菌可能会处于一种不生长但依然有代谢活性(non-growing but metabolically active,NGMA)的状态,以此为依据进行给药,可能导致后期疾病复发[参考文献2]而反复用药。临床上,在尽可能短的时间内做出准确的诊断,并快速对症治疗,不管对细菌耐药问题的解决,还是对病患尤其是危重患者都有重大的意义。
针对现有抗菌药敏试验方法耗时长的问题,新兴的快速抗菌药敏试验检测技术也在不断涌现,如基于微流控的细菌显微成像技术、RNA检测法、原子力显微镜悬臂法以及基于细菌代谢的拉曼信号检测等方法。这些方法的检测机制各不相同,大体包括:细菌生长增殖、形态变化、转录组变化和代谢活性检测等,借助各种仪器设备可以在2-8小时内完成抗菌药敏试验,判断细菌对抗生素的敏感性,甚至定量地给出MIC。但这些方法仍存在大量不足之处。例如,基于微流控的细菌显微成像技术大部分可直接观察细菌在抗生素作用下的生长增殖或形态变化,是一个可视化的检测方法,更直观,且借助微流控技术可使检测所使用的样品和试剂量大大减少,但微流控技术尚未普及,通过显微成像进行判断的技术门槛高、推广难度大、设备成本高,而且基于细菌生长增殖或形态变化的检测机制,无法鉴别处于不生长但有代谢活性(NGMA)状态的细菌,易造成错误判断[参考文献3、4、5]。RNA检测法通过检测细菌在抗生素作用下转录子的变化来判断细菌对药物的敏感性,这种方法前期需要通过大量实验来积累数据并建立数据库,耗时费力,前期成本高,且该方法仅能判断细菌对抗生素是否敏感,而无法定量获得MIC[参考文献6、7]。原子力显微镜悬臂法虽然是通过显微观察、监测抗生素作用下细菌的运动情况来判断细菌的代谢活性(检测细菌的代谢活性可有效鉴别处于不生长但有代谢活性 (NGMA)状态的细菌,更准确,但该方法在检测时受各种因素的影响较大(如流动液体、细菌数量等)而无法准确反应细菌活性,且仪器设备极为复杂,对操作的技术要求特别高、检测通量低[参考文献8]。基于拉曼信号的方法也是通过检测细菌代谢活性来判断抗生素的作用效果,这种方法操作简单,测定时间短,但仍存在拉曼信号弱、对部分抗生素的MIC检测不准确、受样品纯度等背景干扰大、信噪比低、与现有仪器兼容性差(不如酶标仪、流式细胞仪等普遍)、价格较为昂贵等问题[参考文献9、10、11]。
发明内容
本发明提供一种基于荧光D型氨基酸代谢标记的细菌代谢活性的评估方法和抗菌药敏试验检测方法及其试剂盒,其具有快速、简便、灵敏、准确、直观的特点且与现有仪器兼容性好。
本发明一方面涉及的基于荧光D型氨基酸代谢标记的细菌代谢活性的评估方法,使用荧光D型氨基酸代谢标记物代谢标记待测细菌,以根据其荧光强度或荧光强度变化来评估生长过程中的细菌代谢活性。
本发明另一方面涉及的基于荧光D型氨基酸代谢标记的抗菌药敏试验检测方法,荧光D型氨基酸代谢标记物与抗生素溶液共同孵育菌液,检测获得与细菌生长速度和代谢活性正相关的荧光标记信号强度和/或实时检测其荧光信号变化,并根据分析其荧光信号强度获得待测抗生素最小抑制浓度。
本发明还涉及用于细菌代谢活性评估和/或抗菌药敏试验检测的试剂盒,其包含荧光D型氨基酸代谢标记物,和/或缓冲液、稀释液或载体或培养板、培养皿。
附图说明
图1是本发明实施方式中带有荧光基团的DAA探针的结构示意图;
图2是本发明实施方式中细菌代谢活性与FDAA的标记强度相关性数据; a为不同温度梯度下与大肠杆菌、枯草芽孢杆菌、沙门氏菌、地衣芽孢杆菌代谢活性相关的荧光标记强度数据;b为不同时间下地衣芽孢杆菌和大肠杆菌代谢活性相关的荧光标记强度数据。蓝色曲线(实心圆圈)为细菌的生长速率,可表示细菌的代谢活性;红色曲线(实心三角形)为被标记细菌的荧光强度;黑色曲线(横杠)为细菌生长曲线。红色曲线与蓝色曲线具有很好一致性,被标记细菌的荧光强度与细菌的生长速率一致,说明FDAA标记的细菌荧光强度可代表细菌的代谢活性。
图3是基于FDAA代谢标记的抗菌药敏试验快速检测方法的流程图;
图4是本申请具体实施方式中,苯唑西林(OX)对革兰氏阳性菌金黄色葡萄球菌(S.aureus)的高水平OX耐药菌株ST5、低水平OX耐药菌株ST59、 OX敏感菌株ST398的作用效果。
图5是本申请具体实施方式中,万古霉素(VA)对革兰氏阳性菌金黄色葡萄球菌(S.aureus)的高水平OX耐药菌株ST5、低水平OX耐药菌株ST59、 OX敏感菌株ST398的作用效果。
图6是本申请具体实施方式中,红霉素(E)对革兰氏阳性菌金黄色葡萄球菌(S.aureus)的高水平OX耐药菌株ST5、低水平OX耐药菌株ST59、 OX敏感菌株ST398的作用效果。
图7是本申请具体实施方式中,头孢吡肟(FEP)对革兰氏阴性菌大肠埃希菌(E.coli)的耐药菌株1113、敏感菌株1146的作用效果。
图8是本申请具体实施方式中,亚胺培南(IPM)对革兰氏阴性菌大肠埃希菌(E.coli)的耐药菌株1113、敏感菌株1146的作用效果。
图9是本申请具体实施方式中,左旋氧氟沙星(LEV)对革兰氏阴性菌大肠埃希菌(E.coli)的耐药菌株1113、敏感菌株1146的作用效果。
图10是本申请具体实施方式中,替加环素(TGC)对革兰氏阴性菌大肠埃希菌(E.coli)的耐药菌株1113、敏感菌株1146的作用效果。
具体实施方式
参考如下详细说明和示例性实施方式,应该理解的是,本申请不限于说明书阐述和附图所示的细节或方法。
本发明涉及的细菌代谢活性的评估方法,是基于荧光D型氨基酸代谢标记物标记菌体。其中,D型氨基酸(D-amino acid,DAA),尤其是D型丙氨酸,是一种存在于细菌肽聚糖中的五肽结构末端的特殊氨基酸,细菌在肽聚糖的合成和代谢过程中会在这一位置通过青霉素结合蛋白(penicillin binding proteins,PBPs)等酶类中的转肽酶结构域与邻近的肽段进行交联、水解(去掉最末端的D-丙氨酸)、替换(与周围环境中的其他D型氨基酸(DAA))等修饰[参考文献12]。某些青霉素结合蛋白对D-型丙氨酸侧链上带有的修饰基团有很高的容忍性,利用这一特点,可将侧链上带有各种荧光基团的DAA探针 (fluorescent D-amino acids,FDAAs,例如四甲基罗丹明(TAMRA)DAA荧光探针、带有FAM的DAA荧光探针、Cy5-DAA(其结构如图1所示))通过代谢标记的方式连接到细菌肽聚糖结构上[参考文献13]。肽聚糖结构广泛存在于各门类细菌中,故可以标记各类常见的细菌;同时肽聚糖的高周转速率使得DAA探针标记速度快、强度高。在本发明中,若细菌结合的FDAA越多,则荧光信号越强,表明细菌的代谢活性越强,因此,能够根据其荧光强度或荧光强度变化来评估生长过程中的细菌代谢活性。荧光强度变化可以由培养时间长短、温度不同、培养基、是否存在促菌生长剂或抑菌剂(如抗生素)等影响细菌代谢活性而引起。
本发明的具体实施方式中,将荧光D型氨基酸代谢标记物加入到待测细菌中培养以标记待测细菌,例如大肠杆菌、枯草芽孢杆菌、沙门氏菌、地衣芽孢杆菌。如图2-a所示,在不同温度下,大肠杆菌、枯草芽孢杆菌、沙门氏菌、地衣芽孢杆菌出现不同的代谢活性,37℃下相对于33℃、29℃和25℃具有更好的代谢活性,其荧光标记强度相应地高于其他温度,并且其荧光标记强度与温度引起的代谢活性呈正相关。图2-b所示,细菌的荧光标记强度与细菌的生长速率正相关,即随着细菌生长、繁殖速率改变,荧光标记强度也随之改变,反应了荧光D型氨基酸代谢标记物会在细菌生长代谢过程中标记到细菌细胞上,表明利用荧光D型氨基酸探针对细菌代谢活性标记具有可行性,其也具有较高的准确性和灵敏性。
某个菌株对某个抗生素敏感时,其代谢活性会受到相应的抑制,其生长速率也会受到抑制,进而相比不添加抗生素的细菌,其FDAA标记强度会相应下降。如果某个菌株对某个抗生素耐药,则其代谢活性不会受到抑制,进而相比不添加抗生素的细菌其FDAA标记强度不受影响。本发明涉及的D型氨基酸代谢标记的抗菌药敏试验检测方法,通过利用荧光D型氨基酸代谢标记物评估细菌代谢活性来判断抗生素的抗菌效果和/或细菌耐药性,其利用荧光D 型氨基酸代谢标记物标记细菌,此细菌可以是在与抗生素溶液混合孵育后,检测器荧光强度,并根据荧光信号强度获得抗生素最小抑制浓度。本发明的具体实施方式中,可以采用目前已知的荧光信号检测设备或装置,例如采用流式细胞技术检测荧光信号,所述的流式细胞技术(Flow Cytometry,FCM)是一种利用流式细胞仪对处在快速流动状态中的细胞或生物颗粒进行快速、灵敏、精确的多参数定量分析和分选的成熟技术。细胞或细胞上的荧光染料被激光激发后发射出荧光,荧光信号被检测器检测,经光电转换变为可被计算机识别的数字信号。荧光信号的强度代表了细菌细胞标记上的荧光染料数量。
本申请的具体实施方式中,基于荧光D型氨基酸代谢标记的抗菌药敏试验检测方法,包括如下步骤:一定浓度下的菌液与抗生素溶液混合孵育,加入荧光D型氨基酸代谢标记培养(一段时间后),检测样品的荧光强度和/或实时检测其荧光信号,本申请的某个具体实施方式中,菌液与抗生素溶液混合孵育以细菌进入(对数)生长期。本发明的某些具体实施方式中,采用肉汤培养基制成均匀菌液,例如采用阳离子调节肉汤培养基,菌液浓度可选地为OD600=0.2。某些具体实施方式中,抗生素溶液采用不同浓度,比如设置梯度浓度,例如采用0μg/mL、16μg/mL、32μg/mL、64μg/mL、128μg/mL、256μg/mL、 512μg/mL、1024μg/mL;0μg/mL、2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、 64μg/mL和/或128μg/mL;0μg/mL、0.0625μg/mL、0.125μg/mL、0.25μg/mL、 0.5μg/mL、1μg/mL、2μg/mL、4μg/mL和/或8μg/mL。某些具体实施方式中,孵育时间是为能够让细菌进入其生长期,例如1-3小时,更优选地为2h。本申请的某些具体实施方式中,荧光D型氨基酸探针为Cy5-DAA探针,采用的浓度可以标记细菌,例如探针终浓度为0.01-2mM,或者0.1-1mM,或者0.5mM。
本发明的具体实施方式中,基于荧光D型氨基酸代谢标记的抗菌药敏试验检测方法,可以用于革兰氏阳性菌、革兰氏阴性菌,例如革兰氏阴性菌包括但不限于肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、流感嗜血杆菌、产气肠杆菌、黏质沙雷菌、产酸克雷伯菌、大肠埃希菌(E.coli),革兰氏阳性菌包括但不限于金黄色葡萄球菌、肺炎链球菌、粪肠球菌、表皮葡萄球菌、头状葡萄球菌、咽峡炎链球菌,例如但不限于,金黄色葡萄球菌为高水平OX耐药菌株ST5、低水平OX耐药菌株ST59和OX敏感菌株ST398;大肠埃希菌为耐药菌株1113、敏感菌株1146。本发明能够针对的抗生素,例如包括但不限于青霉素类、头孢菌素类、巯青霉素烯类、氨基糖苷类、四环素类、酰胺醇类、大环内酯类、糖肽类、磺胺类、喹诺酮类、硝咪唑类。本申请涉及的荧光D型氨基酸代谢标记的抗菌药敏试验检测方法,可以用于不同作用机制的抗生素在亚抑菌浓度下对不同细菌代谢活性的检测,低浓度抗生素处理下细菌代谢活性的检测,抗生素对不同生长阶段细菌代谢活性的检测以及长时间低浓度抗生素作用后,细菌对相应抗生素的MIC改变的检测。
本发明的具体实施方式中用于细菌代谢活性评估和/或抗菌药敏试验检测的试剂盒,其包含荧光D型氨基酸代谢标记物,和\或缓冲液、稀释液或载体或培养板、培养皿。
如下结合具体实施例对本发明的技术方案做进一步描述。
实施例1
利用FDAA代谢标记方法检测苯唑西林(OX)、万古霉素(VA)、红霉素 (E)对革兰氏阳性菌金黄色葡萄球菌(S.aureus)的高水平OX耐药菌株ST5、低水平OX耐药菌株ST59、OX敏感菌株ST398的作用效果。
如附图3所示基于FDAA代谢标记的抗菌药敏试验快速检测方法的流程图,具体实验步骤如下:
1、挑取平板上培养过夜的待测菌落于生理盐水中充分混匀,制成均匀菌液,稀释到阳离子调节肉汤培养基(CAMHB)里,制成浓度为OD600=0.2 的菌液。
2、向96孔板分别加入45μl不同浓度的待测抗生素溶液(抗生素浓度梯度设置见表1)。
3、向96孔板中加入步骤1配好的菌液50μl,混匀,37℃静置避光孵育2h。
4、分别向每个孔中加入5μl 10mM的Cy5-DAA探针(终浓度0.5 mM,其结构见附图3),混匀后,继续避光培养0.5h。
5、将96孔板置于离心机中,4000rpm,5min离心,去上清。
6、加入200μl PBS溶液,吹打混匀。
7、将96孔板置于离心机中,4000rpm,5min离心,去上清。
8、加入200μl PBS溶液,吹打混匀,制成菌悬液。
9、使用流式细胞仪检测各样品的荧光强度。流式检测结果如附图4-5 所示。
表1.抗生素浓度梯度设置(终浓度,μg/mL)
从下表2可知,通过本发明(基于FDAA代谢标记的抗菌药敏试验快速检测方法)获得的革兰氏阳性菌的药敏试验结果与目前临床常用药敏试验设备Vetek 2获得的结果具有很好的一致性。
表2.FDAA代谢标记法与临床常用药敏试验设备Vetek 2的检测结果对比(革兰氏阳性菌)
注:S:敏感;R:耐药
实施例2
利用FDAA代谢标记方法检测头孢吡肟(FEP)、亚胺培南(IPM)、左旋氧氟沙星(LEV)、替加环素(TGC)对革兰氏阴性菌大肠埃希菌(E.coli)的耐药菌株1113、敏感菌株1146的作用效果。
具体实验步骤如下:
1、挑取平板上培养过夜的待测菌落于生理盐水中充分混匀,制成均匀菌液,稀释到阳离子调节肉汤培养基(CAMHB)里,制成浓度为 OD600=0.2的菌液。
2、向96孔板分别加入45μl不同浓度的待测抗生素溶液(抗生素浓度梯度设置见表3)。
3、向96孔板中加入步骤1配好的菌液50μl,混匀,37℃静置避光孵育2h。
4、分别向每个孔中加入5μl 10mM的Cy5-DAA探针(终浓度0.5 mM,其结构见附图3),混匀后,继续避光培养0.5h。
5、将96孔板置于离心机中,4000rpm,5min离心,去上清。
6、加入200μl PBS溶液,吹打混匀。
7、将96孔板置于离心机中,4000rpm,5min离心,去上清。
8、加入200μl PBS溶液,吹打混匀,制成菌悬液。
9、使用流式细胞仪检测各样品的荧光强度。流式检测结果如附图7-10 所示。
表3.抗生素浓度梯度设置(终浓度,μg/mL)
空白对照*:不加抗生素和探针。
如下表4所示,通过本发明(基于FDAA代谢标记的抗菌药敏试验快速检测方法)获得的革兰氏阴性菌的药敏试验结果与目前临床常用药敏试验设备Vetek 2获得的结果具有很好的一致性。
表4.FDAA代谢标记法与临床常用药敏试验设备Vetek 2的检测结果对比(革兰氏阴性菌)
注:S:敏感;R:耐药
本发明的技术方案可在短时间内实现细菌特异性标记荧光信号,通过目前已普遍被实验室、医疗机构使用的流式细胞仪检测细菌荧光信号强度,从而定量地评估细菌在不同抗生素种类、不同抗生素浓度作用下的药敏性,是一种快速(3-4h,改进之后可实现1.5-2.5小时给出MIC结果)、简便(操作简单且步骤少、无需使用特殊设备)、灵敏(荧光标记方法稳定、信号强、信噪比高)、准确(检测细菌的代谢活性可有效鉴别处于不生长但有代谢活性(NGMA) 状态的细菌)、直观(可视化好)且与现有仪器兼容性好(所用仪器少,仪器价格适中且已较为普遍)、易于推广的抗菌药敏试验快速检测方法。
参考文献:
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Claims (10)
1.细菌代谢活性的评估方法,其特征在于,使用荧光D型氨基酸(D-amino acid,DAA)代谢标记物代谢标记待测细菌,以根据其荧光强度或荧光强度变化来评估生长过程中的细菌代谢活性。
2.根据权利要求1所述的细菌代谢活性的评估方法,其特征在于,将荧光D型氨基酸代谢标记物加入到待测细菌中培养以标记待测细菌。
3.根据权利要求1或2所述的细菌代谢活性的评估方法,其特征在于,所述荧光D型氨基酸代谢标记物为D型丙氨酸荧光探针;优选地,所述荧光D型氨基酸代谢标记物包括四甲基罗丹明(TAMRA)DAA荧光探针、带有羧基荧光素(FAM)的DAA荧光探针、带有Cy5的DAA荧光探针。
4.抗菌药敏试验检测方法,其通过评估细菌代谢活性来判断抗生素的抗菌效果和/或细菌耐药性,其特征在于,荧光D型氨基酸代谢标记物与抗生素溶液共同孵育菌液,以检测获得与细菌生长速度和代谢活性正相关的荧光标记信号强度和/或实时检测其荧光信号变化,并根据分析其荧光信号强度获得待测抗生素最小抑制浓度。
5.根据权利要求4所述的抗菌药敏试验检测方法,其特征在于,其步骤包括:一定浓度下的菌液与抗生素溶液混合孵育以在细菌进入生长期后,加入荧光D型氨基酸代谢标记物共同培养。
6.根据权利要求4或5所述的抗菌药敏试验检测方法,其特征在于,所述抗生素溶液采用梯度浓度;所述荧光D型氨基酸代谢标记物的终浓度为0.01-2mM,优选地,所述荧光D型氨基酸代谢标记物的终浓度为0.1-1mM。
7.根据权利要求4或5所述的抗菌药敏试验检测方法,其特征在于,所述荧光D型氨基酸代谢标记物为D型丙氨酸荧光探针,优选地,所述荧光D型氨基酸代谢标记物包括四甲基罗丹明(TAMRA)DAA荧光探针、带有羧基荧光素(FAM)的DAA荧光探针、带有Cy5的DAA荧光探针;所述检测方法针对的细菌为革兰氏阳性菌、革兰氏阴性菌。
8.根据权利要求4-7任一项所述的抗菌药敏试验检测方法,其特征在于,所述抗生素包括青霉素类、头孢菌素类、巯青霉素烯类、氨基糖苷类、四环素类、酰胺醇类、大环内酯类、糖肽类、磺胺类、喹诺酮类或硝咪唑类。
9.用于细菌代谢活性评估和/或抗菌药敏试验检测的试剂盒,其包含荧光D型氨基酸代谢标记物,和/或缓冲液、稀释液或载体或培养板、培养皿。
10.如权利要求9所述的用于细菌代谢活性评估和/或抗菌药敏试验检测的试剂盒,其特征在于,所述荧光D型氨基酸代谢标记物包括四甲基罗丹明(TAMRA)DAA荧光探针、带有羧基荧光素(FAM)的DAA荧光探针、带有Cy5的DAA荧光探针。
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Probes | Papers in Press. First published August 19, 2004 as doi: 10.1373/clinchem. 2004.036723 |
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