CN114019055A - 评价细菌氨基糖苷类抗生素耐药效应的试剂盒及其应用 - Google Patents
评价细菌氨基糖苷类抗生素耐药效应的试剂盒及其应用 Download PDFInfo
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- CN114019055A CN114019055A CN202111318212.0A CN202111318212A CN114019055A CN 114019055 A CN114019055 A CN 114019055A CN 202111318212 A CN202111318212 A CN 202111318212A CN 114019055 A CN114019055 A CN 114019055A
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- glycerol
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- oleic acid
- phosphate
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Abstract
本发明涉及甘油、甘油‑3‑磷酸、棕榈油酸和油酸在制备用于评价细菌氨基糖苷类抗生素耐药效应的试剂盒和检测方法的新应用。应用本发明的试剂和方法可检测细菌中甘油、甘油‑3‑磷酸、棕榈油酸和油酸的含量。本发明进一步提供了采用气相色谱‑质谱方法检测细菌中甘油、甘油‑3‑磷酸、棕榈油酸和油酸含量的方法,并提出基于以上四种代谢物中任一种或一种以上代谢物含量信息,可评价细菌氨基糖苷类抗生素耐药效应。本发明的试剂盒和检测方法具有操作简单、快速、高灵敏度、特异性、稳定性和重复性的特点,易于推广。
Description
技术领域
本发明涉及甘油、甘油-3-磷酸、棕榈油酸和油酸在制备用于评价细菌氨基糖苷类抗生素耐药效应的试剂或试剂盒和检测方法的新应用,属于分析化学、环境化学、药物化学、生物化学领域。
技术背景
抗生素作为杀菌剂、生长和(或)质量促进剂,被广泛使用于人、动物、农业和养殖业,并且,其使用量还在快速增长。抗生素的使用使得其原型物及其代谢物被大量地排放到环境中,尤其是水环境,例如:地表水、地下水、海水、污水及沉积物。微生物对抗生素产生耐药是抗生素使用及其在环境蓄积的严重后果之一,并已威胁到人和生态系统的健康。值得关注的是,微生物抗生素耐药广泛存在于人和环境中,并且由于微生物种类繁多、传代时间短等特点,微生物耐药易于传播。因而,微生物耐药机理研究具有紧迫性。
众多研究表明,代谢重编程可调控抗生素药效。基于气相色谱-质谱技术的代谢组学发现,葡萄糖和丙氨酸在卡那霉素耐药的迟缓爱德华氏菌中降低。随后的机理研究表明,外源葡萄糖和(或)丙氨酸以乙酰辅酶A形式进入三羧酸循环,并激活该通路中的代谢酶,以用于相关代谢物合成;此外,用三羧酸循环代谢物激活该通路,促进还原型烟酰胺腺嘌呤二核苷酸和质子动力形成及抗生素摄取,从而恢复抗生素耐药菌对抗生素的敏感性。研究表明,在压力诱导的抗生素耐受条件下(如:缺氧,低pH、铁限制、感染),甘油三酯在结核杆菌及相关的环境细菌中蓄积。敲除tgs1基因,过表达citA基因和14C标记乙酸示踪实验表明,甘油三酯合成可从三羧酸循环及其它促生长的通路中夺走碳源,从而抑制细菌生长及抗生素药效;而扰乱上述代谢转换则无法抑制压力条件下的细菌生长,并可在感染条件下,使细菌重新对抗生素敏感。此外,激活糖酵解及能产生活性氧的通路(如:呼吸链)、核苷酸氧化、抑制糖异生、补充一些外源氨基酸(如:甘氨酸、丝氨酸、缬氨酸和丙氨酸)均可提高抗生素药效。以上数据表明,重编程代谢是提高抗生素药效的有效方式。
细菌可通过基因突变、抗性基因的表达和转移、和(或)表型适应获得抗生素耐药效应,该效应会诱发代谢紊乱,进而降低相关生理效率。然而,细菌补偿、减小甚至消除抗生素耐药相关代谢负荷的能力很强,从而使得耐药菌能保持稳定并存活。研究细菌如何获得抗生素耐药,发现可用于诊断细菌是否产生抗生素耐药的潜在标志物,将有利于更有效地使用抗生素,预防并减少抗生素及其耐药带来的人及生态系统健康危害。
油酸和棕榈油酸是生物体内主要的游离脂肪酸,并且是其它大分子脂质的主要组成成分。甘油和甘油-3-磷酸可为甘油酯、磷脂等大分子脂质合成提供甘油骨架,并可进入三羧酸循环,进一步转化成其它代谢物(如:有机酸、氨基酸)。目前尚未发现将甘油、甘油-3-磷酸、棕榈油酸和(或)油酸用于评价细菌氨基糖苷类抗生素耐药效应的报道。常用的光谱技术基于特征官能团的光谱信号检测化合物,因而,基于光谱技术的试剂盒方法,通常检测的是具有相同官能团化合物混合物,无法分辨具有相同官能团的化合物,如:同系物、官能团位置异构体、顺反异构体。因而,无法区分棕榈油酸、油酸与其它脂肪酸。众所周知,色谱-质谱技术对化合物的分辨率远高于光谱技术。气相色谱-质谱技术能准确区分脂肪酸的碳链长度、不饱和碳键的位置、数量和顺反异构,其在脂肪酸不饱和键位置和顺反异构体的区分上,强于液相色谱-质谱技术。此外,由于甘油与甘油-3-磷酸具有强极性,在常用的反相液相色谱柱上保留弱,与其它强极性代谢物和溶剂在死时间附近流出,因而在反相液相色谱-质谱检测上通常会出现峰形差、离子抑制效应强等效应,从而影响定性与定量结果。然而,甘油与甘油-3-磷酸经衍生化后,可在毛细管柱上得到很好的保留,从而在质谱上得到有效的检测。其次,气相色谱-质谱采集的数据经由ChromaTOF(LECO Cor.,USA)软件进行重叠峰去卷积后,可消除目标代谢物的干扰信号,并提取目标代谢物的特征离子,从而实现代谢物高灵敏、高特异性的定性和定量分析。
基于当前的研究现状及技术特点,本发明采用气相色谱-质谱技术检测甘油、甘油-3-磷酸、棕榈油酸和油酸,该方法具有易于操作、高灵敏度、特异性和稳定性的特点。本发明采集庆大霉素敏感和耐药的大肠杆菌代谢轮廓后,筛选出甘油、甘油-3-磷酸、棕榈油酸和油酸中任一种或一种以上代谢物作为标志物,实现在制备用于评价细菌氨基糖苷类抗生素耐药效应的试剂盒及其检测的新用途,筛选出的标志物具有优良诊断灵敏度和特异性。目前尚未发现将甘油、甘油-3-磷酸、棕榈油酸和(或)油酸用于评价细菌氨基糖苷类抗生素耐药效应的报道。
发明内容
本发明目的是针对当前脂肪酸和强极性代谢物检测存在的低灵敏度、低特异性等问题,提供一种小分子代谢物试剂盒在评价细菌氨基糖苷类抗生素耐药效应的应用,亦提供了可同时检测上述小分子代谢物的方法,实现了非极性和强极性代谢物的同时检测。
甘油、甘油-3-磷酸、棕榈油酸和(或)油酸在制备用于评价细菌氨基糖苷类抗生素耐药效应的试剂或试剂盒中的应用。
所述的试剂或试剂盒用于检测细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸的含量。
所述的试剂或试剂盒为采用气相色谱-质谱技术检测细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸含量的试剂的组合。
一种用于评价细菌氨基糖苷类抗生素耐药效应的试剂或试剂盒,包括:
①标准品:甘油、甘油-3-磷酸、棕榈油酸和油酸,所述标准品分别用于细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸的定性;
②80-100%(v/v)甲醇/水提取液:所述提取液用于提取细菌代谢物;
③衍生化试剂:甲氧胺吡啶溶液(10-20mg/mL)、N-甲基-N-(三甲基硅基)三氟乙酰胺。
细菌为革兰氏阴性菌。
氨基糖苷类抗生素包括:链霉素,庆大霉素,妥布霉素,奈替米星,阿米卡星,等。
为实现上述目的,本发明以大肠杆菌为模型,以庆大霉素为例,采用如下技术方案:
1.庆大霉素耐药大肠杆菌筛选:用庆大霉素连续处理大肠杆菌,直至产生耐药效应(其最小抑制浓度MIC值为对照的4倍及以上)后,保存于-80℃冰箱中备用。
2.培养庆大霉素敏感和耐药的大肠杆菌至所需的量后,收集大肠杆菌样本。
3.采用气相色谱-质谱技术采集大肠杆菌代谢轮廓后,筛选差异代谢物,筛选参数包括差异水平p值、变化倍数、诊断灵敏度、特异性和AUC(受试者工作特征曲线下面积)值等。
4.大肠杆菌庆大霉素耐药效应的评价:甘油、甘油-3-磷酸、棕榈油酸和油酸在庆大霉素耐药大肠杆菌中显著高于对照(庆大霉素敏感大肠杆菌)(p<0.05,two-tailedMann-Whitney U test);将甘油、甘油-3-磷酸、棕榈油酸和油酸分别作为标志物,各自代入SPSS软件,进行二元逻辑回归模块分析,构建二元逻辑回归模型,得出常数项、标志物在回归模型中的系数,从而获得二元回归方程;由二元回归方程,即可得各个样本的分类预测概率。构建的二元逻辑回归模型为:样本分类预测概率=1/[1+e-(c+K*a)];其中,c为常数项;a为标志物含量;K为标志物在方程中的系数。
以甘油为标志物,并将其含量代入二元逻辑回归模型分析,可得二元逻辑回归方程1:样本分类预测概率=1/[1+e-(-12.2177528224161+0.0000758608097666215*a)];其中,a表示甘油的含量。
以甘油-3-磷酸为标志物,并将其含量代入二元逻辑回归模型分析,可得二元逻辑回归方程2:样本分类预测概率=1/[1+e-(-12.6611212286704+0.0000162482475918412*a)];其中,a表示甘油-3-磷酸的含量。
以棕榈油酸为标志物,并将其含量代入二元逻辑回归模型分析,可得二元逻辑回归方程3:样本分类预测概率=1/[1+e-(-13.8577241693595+0.000913993147713712*a)];其中,a表示棕榈油酸的含量。
以油酸为标志物,并将其含量代入二元逻辑回归模型分析,可得二元逻辑回归方程4:样本分类预测概率=1/[1+e-(-14.3237726151905+0.000506491803675298*a)];其中,a表示油酸的含量。
将样本分类预测概率的临界值设为0.5,若样本分类预测概率值<0.5,则大肠杆菌无庆大霉素耐药;若样本分类预测概率值≥0.5,则大肠杆菌产生了庆大霉素耐药。
5.大肠杆菌庆大霉素耐药效应评价的诊断性能分析:以样本分类预测概率值为变量,进行受试者工作特征曲线分析,评价庆大霉素耐药效应的诊断性能,若灵敏度和特异性均≥80.0%,且AUC≥0.8,则诊断性能优良。
6.评价系统所包括的装置:检测仪器为气相色谱-质谱联用仪;色谱柱为DB-5MS毛细管柱,柱长30m,膜厚0.25μm,内径250μm。
7.确定试剂盒组成:
⑴标准品:甘油、甘油-3-磷酸、棕榈油酸和油酸,分别用于细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸的定性。
⑵提取液:80%甲醇水溶液(v/v),用于细菌代谢物提取。
⑶衍生化试剂:①20mg/mL甲氧胺吡啶溶液,用于羰基和醛基的肟化反应,以防止链状糖环化,从而阻止不同构象的糖之间相互转化;此外,避免α-酮酸脱羧、酮基转化成烯醇。②N-甲基-N-(三甲基硅基)三氟乙酰胺(for GC derivatization,≥98.5%),用于氨基、羧基、羟基等含活泼氢代谢物的硅烷化反应,生成挥发性或半挥发性、并具有更强稳定性的衍生物,以利于气相色谱-质谱检测。
⑷仪器分析条件:
①气相色谱条件:进样量1μL,进样口温度300℃,分流比10:1。载气为高纯氦气,采用恒流模式,线速度为40cm/s。色谱柱为DB-5MS毛细管色谱柱。色谱柱升温程序:70℃保持3min后,以5℃/min的速率升至300℃,保持10min。
②质谱条件:传输线与离子源温度各设为280℃和230℃。电离方式为电子轰击,电离电压70eV。采用全扫描模式采集质谱信号,扫描范围:33-600m/z。溶剂切割时间5.3min,质量扫描速率5张谱图/s。检测器电压与调谐电压相同。
⑸质谱数据处理:原始质谱数据以NetCDF格式导入R2.3.11软件后,应用XCMS程序进行峰匹配和积分,获得大肠杆菌样本中所有离子峰的面积和保留时间。采用ChromaTOF软件对质谱数据进行重叠峰去卷积及谱库(Wiley,Fiehn和NIST11)检索和匹配后,获得各个代谢物的特征离子,并对代谢物进行鉴定,最后,根据标样的保留指数、保留时间和质谱碎片特征信息,进一步确认定性结果。甘油、甘油-3-磷酸、棕榈油酸和油酸的特征离子(m/z)分别为218,357,117和117,对应的保留时间(min)分别为14.10,26.07,31.40和35.03。
8.采用大肠杆菌样本测试本发明的应用效果。分别以甘油、甘油-3-磷酸、棕榈油酸和油酸为标志物,基于大肠杆菌中甘油、甘油-3-磷酸、棕榈油酸和油酸的含量,均可正确识别庆大霉素敏感和耐药的样本,正确率均为100.0%,最优灵敏度和特异性均可达100.0%,AUC均为1.0,表明甘油、甘油-3-磷酸、棕榈油酸和油酸分别作为标志物,均可正确识别大肠杆菌庆大霉素耐药效应,并且具有优越的诊断性能(图6和7)。
本发明的主要优点在于:
①由于常用光谱技术检测的是特征官能团信号,其难以区分具有相同官能团的同系物和同分异构体,因而,光谱检测脂肪酸通常检测的是脂肪酸混合物,其对脂肪酸的分辨能力远低于色谱-质谱技术。气相色谱-质谱技术能准确区分脂肪酸的碳链长度、不饱和碳键的位置、数量和顺反异构,其在脂肪酸不饱和键位置和顺反异构的区分上,强于液相色谱-质谱技术。此外,由于甘油与甘油-3-磷酸具有强极性,在常用的反相液相色谱柱上保留弱,与其它强极性代谢物和溶剂在死时间附近流出,因而,在反相液相色谱-质谱检测上通常会出现峰形差、离子抑制效应强等效应,从而影响定性与定量结果。然而,甘油与甘油-3-磷酸经衍生化后,可在毛细管柱上得到很好的保留,从而在质谱上得到高效的检测。其次,气相色谱-质谱采集的质谱数据经由ChromaTOF软件对重叠峰去卷积后,可消除目标代谢物的干扰信号,并提取目标代谢物的特征离子,从而实现目标代谢物高灵敏、高特异性的定性和定量分析。
②本发明提供了一种基于气相色谱-质谱技术的细菌氨基糖苷类抗生素耐药效应的检测试剂盒,可通过检测细菌样本中甘油、甘油-3-磷酸、棕榈油酸和(或)油酸含量,准确评价细菌氨基糖苷类抗生素耐药效应。本发明采集细菌样本进行检测,具有周期短、操作简单、高灵敏度、高特异性、高稳定性和高重复性等特点,是现行检测方法的有效补充,并可为抗生素耐药机理研究、抗生素耐药诊断和评估、抗生素高效使用、抗生素生态效应评估等提供有效分析方法。
附图说明
图1:庆大霉素耐药大肠杆菌相关表型。S:庆大霉素敏感的大肠杆菌;R:庆大霉素耐药的大肠杆菌。每组3个生物学重复。(A)庆大霉素耐药大肠杆菌的MIC;(B)庆大霉素耐药大肠杆菌的生长曲线。*:p<0.05,**:p<0.01,two-tailed independent samples t-test。
图2:主成分分析中样本分布图。QC:质量控制样本;S:庆大霉素敏感的大肠杆菌;R:庆大霉素耐药的大肠杆菌。QC,S和R组各有3,6和6个生物学重复。
图3:QC样本中离子含量相对标准偏差分布图。
图4:甘油、甘油-3-磷酸、棕榈油酸和油酸在庆大霉素耐药大肠杆菌中的变化。S:庆大霉素敏感的大肠杆菌;R:庆大霉素耐药的大肠杆菌。**:p<0.01,two-tailed Mann-Whitney U test。每组6个生物学重复。
图5:火山图显示差异代谢物。图上显示的数据点均为有显著变化的代谢物(p<0.05,two-tailed Mann-Whitney U test)。
图6:大肠杆菌庆大霉素耐药效应评价。分别以甘油、甘油-3-磷酸、棕榈油酸和油酸为变量,基于二元逻辑回归模型,预测大肠杆菌庆大霉素耐药效应。S:庆大霉素敏感的大肠杆菌;R:庆大霉素耐药的大肠杆菌。每组6个生物学重复。
图7:大肠杆菌庆大霉素耐药效应评价的诊断性能。受试者工作特征曲线分析甘油、甘油-3-磷酸、棕榈油酸和油酸分别用于评价大肠杆菌庆大霉素耐药效应的诊断性能。AUC:受试者工作特征曲线下面积。受试者工作特征曲线上的圆圈显示该数据点具有最优的诊断性能(诊断灵敏度和特异性均达到100.0%).
具体实施方式
实施例
下面结合实例进一步描述本发明,但所述实施例仅用于说明本发明,而不是限制本发明。熟悉本领域者在不违背本发明方法及思路的前提下,还可做出各种类似的变型或替换,此类似的变型或替换均包含在本申请权利要求所限定的范围内。
1.大肠杆菌品种和培养
①大肠杆菌K12 BW25113购于广东省微生物菌种保藏中心。庆大霉素耐药大肠杆菌K12 BW25113则经由硫酸庆大霉素连续处理并传代(具体过程见下面庆大霉素耐药大肠杆菌筛选),最后形成耐药菌。作为对照,庆大霉素敏感大肠杆菌K12 BW25113来自于与庆大霉素耐药大肠杆菌同代株,但其未经硫酸庆大霉素处理。大肠杆菌在37℃下培养于50mLLuria-Bertani培养基。
2.生长曲线测定
大肠杆菌单克隆于Luria-Bertani培养基中培养12h后,以1:100(v/v)接种至50mLLuria-Bertani培养基,并在37℃下,以200rpm的速率振荡培养。在14h的培养周期中,每隔2h取样,测定OD600值(样本在可见光600nm处的吸光值),并绘制生长曲线(横坐标是时间,纵坐标是OD600值。如图1B所示。)。实验重复3次。
3.MIC(最小抑制浓度)测定
庆大霉素由Luria-Bertani培养基以2倍的倍数逐级稀释于96孔板中,浓度从0.625到160μg/mL。吸取10μL稀释的菌液至96孔板,菌液数量浓度为5×104CFU/孔。大肠杆菌在37℃下培养12h后,测定庆大霉素能完全抑制大肠杆菌生长的最小浓度,此即为MIC。实验重复3次。
4.庆大霉素耐药大肠杆菌筛选
大肠杆菌K12 BW25113单克隆接种于5mL Luria-Bertani培养基,并在37℃下,以200rpm的速率振荡培养12h。细菌液以1:100(v/v)分别转移至1mL含有和不含有硫酸庆大霉素的Luria-Bertani培养基后,在37℃下培养12h。每12h后,细菌液以1:100(v/v)培养,耐药组用抗生素处理,对照组无抗生素处理。硫酸庆大霉素处理的浓度从0.5倍MIC开始,并以2倍的倍数增加,直至耐药组大肠杆菌产生64倍MIC的耐药效应。每3代测定一次MIC。随后,将庆大霉素敏感和耐药的大肠杆菌保存于-80℃冰箱中备用。
5.代谢组学样本收集
将庆大霉素敏感和耐药的大肠杆菌,以1×108CFU的菌数,分别接种于5mL Luria-Bertani培养基,培养12h后,以1:100(v/v)转移至50mL Luria-Bertani培养基,并分别培养至OD600值为1.0。分别吸取10mL菌液,在8000rpm条件下离心5min后,弃去培养基,保存于-80℃冰箱中备用,得庆大霉素敏感和耐药的大肠杆菌两组,每组各有6个生物学重复样本。
6.代谢组学样本前处理
往大肠杆菌样本中加入1mL80%(v/v)甲醇溶液,涡旋2min后,在13000rpm,4℃条件下离心15min,取出700μL上清,于真空浓缩干燥仪(Thermo Scientific,USA)中浓缩干燥。
往干燥样本中加入50μL甲氧胺吡啶溶液(20mg/mL),涡旋30s后,于37℃水浴中进行1.5h的肟化反应。随后,往样本中加入40μL N-甲基-N-(三甲基硅基)三氟乙酰胺溶液,于37℃水浴中进行1.0h的硅烷化反应。衍生样本于13000rpm,4℃条件下离心15min后,取出上清液用于后续仪器分析。
从所有大肠杆菌样本提取液离心后的剩余上清液中各吸取200μL,并混合成一个QC样本,涡旋5min后,分成每份700μL的QC样本,获得3个QC样本,在后续的浓缩干燥、衍生化和气相色谱-质谱检测过程中,与其它分析样本同样处理。
7.气相色谱-质谱分析方法
气相色谱分析方法:进样量1μL,进样口温度300℃。载气为高纯氦气,采用恒流模式,线速度40cm/s,分流比10:1。色谱柱为DB-5MS毛细管柱,柱长30m,内径250μm,膜厚0.25μm。色谱柱升温程序:初始柱温为70℃,保持3min后,以5℃/min的速率升至300℃,再保持10min。
质谱分析方法:传输线和离子源温度分别为280和230℃,采用电子轰击的电离方式,电离电压70eV。溶剂切割时间为5.3min。质谱扫描频率为5张谱图/s,质荷比扫描范围为33-600。检测电压与调谐电压相同。
8.质谱数据处理
原始质谱数据以NetCDF格式导入R2.3.11软件后,由XCMS程序进行峰匹配和积分,获得样本所有离子的峰面积和保留时间。由ChromaTOF软件对质谱数据进行离子峰识别、重叠峰去卷积、谱库(NIST11,Fiehn和Wiley)检索和匹配后,获得每个代谢物的特征离子,并对代谢物进行鉴定。随后,根据标样的保留指数、保留时间、质谱碎片特征信息,进一步确认定性结果。甘油、甘油-3-磷酸、棕榈油酸和油酸的特征离子(m/z)分别为218,357,117和117,对应的保留时间(min)分别为14.10,26.07,31.40和35.03。甘油、甘油-3-磷酸、棕榈油酸和油酸的含量以其特征离子的原始峰面积除以总峰面积后,乘以1×108的数值表示,并以此数值进行后续的统计分析。
9.统计分析
应用在线软件MetaboAnalyst 5.0进行主成分分析。由two-tailed independentsamples t-test检测庆大霉素敏感与耐药大肠杆菌的生长差异水平。用two-tailed Mann-Whitney Utest测定甘油、甘油-3-磷酸、棕榈油酸和油酸含量在庆大霉素敏感与耐药大肠杆菌间的差异水平。Two-tailed independent samples t-test、二元逻辑回归和受试者工作特征曲线分析均由SPSS软件完成。Two-tailed Mann-Whitney U test分析由MultiExperiment Viewer4.9.0软件执行。差异显著性水平设为0.05。
10.庆大霉素耐药大肠杆菌相关表型
由图1A可知,筛选出的庆大霉素耐药大肠杆菌MIC达80μg/mL,为对照的64倍。生长曲线分析表明,庆大霉素耐药大肠杆菌在2,4,6,8,10和12h的生长显著慢于对照(p<0.05,two-tailed independent samples t-test),而在14h时,两种大肠杆菌生长无显著差异(图1B)。
11.检测方法稳定性和重复性评价
从样本主成分分析的得分图可知,3个QC样本聚集于一起(图2)。再者,由QC样本中离子峰相对标准偏差分布可知,在6580个离子中,4925个离子相对标准偏差小于15%,占总离子数的74.85%;5345和5793个离子相对标准偏差分别小于20和30%,各占总离子数的81.23和88.04%(图3)。综合主成分得分图中QC样本分布及QC样本中离子峰相对标准偏差分布,可知,本发明所步及的大肠杆菌代谢组浓缩干燥、衍生化、气相色谱-质谱分析等步骤具有高度的稳定性、可靠性和重复性,适合分析大肠杆菌样本。
12.庆大霉素耐药大肠杆菌代谢变化
将含有所有代谢物相对含量信息的峰表导入在线软件MetaboAnalyst 5.0,经单位方差归一化后,进行主成分分析,由主成分得分图可知,庆大霉素耐药大肠杆菌的代谢轮廓与对照显著不同,表明庆大霉素耐药大肠杆菌的代谢发生了显著变化(图2)。其中,甘油、甘油-3-磷酸、棕榈油酸和油酸在庆大霉素耐药大肠杆菌中的含量均显著高于对照(p<0.05,two-tailed Mann-Whitney U test),并且,变化倍数均大于8(图4)。此外,由火山图可知,庆大霉素耐药大肠杆菌中甘油、甘油-3-磷酸、棕榈油酸和油酸增大的倍数在所有差异代谢物中均位列前5,并且p值均是最小的(图5)。因而,将甘油、甘油-3-磷酸、棕榈油酸和油酸分别作为潜在标志物用于评价大肠杆菌庆大霉素耐药效应。
13.大肠杆菌庆大霉素耐药效应评价及其诊断性能分析
分别以甘油、甘油-3-磷酸、棕榈油酸和油酸作为标志物,将上述代谢物的相对含量分别导入SPSS软件,基于二元逻辑回归分析,获得回归方程相关参数,计算样本的分类预测概率,并以此评价大肠杆菌庆大霉素耐药效应,结果表明,基于本发明试剂盒检测的甘油、甘油-3-磷酸、棕榈油酸和油酸含量,可正确地识别庆大霉素敏感与耐药大肠杆菌样本,正确率为100.0%,表明本发明提供的方法可正确评价大肠杆菌是否产生了庆大霉素耐药(图6)。此外,采用受试者工作特征曲线分析本发明检测方法的诊断性能,结果表明,本发明对大肠杆菌庆大霉素耐药效应的诊断性能优越,AUC达1.0,最优灵敏度和特异性均可达100.%(图7)。
14.结论:利用本发明提供的试剂盒及其检测方法可高特异性、高灵敏地检测大肠杆菌中甘油、甘油-3-磷酸、棕榈油酸和油酸,并可由甘油、甘油-3-磷酸、棕榈油酸和油酸含量信息,高特异性、高灵敏地识别大肠杆菌庆大霉素耐药效应,可提示大肠杆菌是否发生了庆大霉素耐药。本发明具有操作简单,反应条件温和,高稳定性和重复性的特点,可为抗生素耐药机理研究、抗生素耐药诊断和评估、抗生素生态效应评估、抗生素高效使用、阻断耐药菌传播、预防并减少抗生素及其耐药带来的人及生态系统健康危害等提供有效的技术支持。
Claims (10)
1.代谢物在制备用于评价细菌氨基糖苷类抗生素耐药效应的试剂或试剂盒中的应用或者代谢物在评价细菌氨基糖苷类抗生素耐药效应中的应用,其中所述代谢物指甘油、甘油-3-磷酸、棕榈油酸和油酸中的任一种或二种以上代谢物或其组合。
2.根据权利要求1所述的应用,其特征在于,所述的代谢物用于检测细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸的含量。
3.根据权利要求1所述的应用,其特征在于,所述的试剂或试剂盒为采用气相色谱-质谱方法检测细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸含量的试剂的组合;
包括:
①标准品:甘油、甘油-3-磷酸、棕榈油酸和油酸中的一种或二种以上代谢物或其组合,所述标准品分别用于细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸中的任一种或二种以上的定性;
②提取液:甲醇/水溶液(80-100%,v/v),所述提取液用于提取细菌代谢物;
③衍生化试剂:甲氧胺吡啶溶液(10-20mg/mL)、N-甲基-N-(三甲基硅基)三氟乙酰胺。
4.根据权利要求3所述的应用,其特征在于,细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸含量的检测过程,包括以下步骤:①分别培养并收集经和未经氨基糖苷类抗生素处理的两组细菌样本后,分别用提取液提取代谢物;②细菌代谢物提取物经冷冻浓缩干燥后,分别进行衍生化反应;③采用气相色谱-质谱方法分别检测细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸中的一种或二种以上的含量;④基于甘油、甘油-3-磷酸、棕榈油酸和油酸中的任一种或二种以上代谢物含量,建立判别模型,评价细菌氨基糖苷类抗生素耐药效应,若处理后的细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸中的一种或二种以上的含量大于未经处理的细菌中相对应的甘油、甘油-3-磷酸、棕榈油酸和油酸中的一种或二种以上的含量,则可认定处理后的细菌为耐药菌,否则为非耐药菌。
5.根据权利要求4所述的应用,其特征在于,所述的气相色谱条件:进样量1μL,进样口温度260-300℃;色谱柱为DB-5MS或HP-5MS毛细管色谱柱;载气为高纯氦气,采用恒流控制模式;
所述的质谱条件:传输线温度为260-280℃;离子源温度220-260℃;电离方式为电子轰击,电离电压为70eV;质谱扫描模式为全扫描或选择离子扫描;质量扫描速率为2-5张谱图/s;切割溶剂出峰时间;检测器电压与调谐电压一致。
6.根据权利要求4所述的应用,其特征在于,
若处理后的细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸中的一种或二种以上的含量是未经处理的细菌中相对应的甘油、甘油-3-磷酸、棕榈油酸和油酸中的一种或二种以上的含量2倍以上,则可认定处理后的细菌为耐药菌,否则为非耐药菌。
7.根据权利要求1-4任一所述的应用,其特征在于,所述经和未经氨基糖苷类抗生素处理的细菌,包括但不限于:细菌和/或细菌寄生的宿主(如:人、动物、植物等中的一种或二种以上)或环境(如:土壤、水体等中的一种或二种以上)经氨基糖苷类抗生素处理前、后分别获得的细菌;处理是指细菌与所述氨基糖苷类抗生素接触过或细菌在含有所述氨基糖苷类抗生素的环境中存活或生长过。
8.根据权利要求1-4任一所述的应用,其特征在于,所述细菌指革兰氏阴性菌。
9.根据权利要求1-4任一所述的应用,其特征在于,所述的氨基糖苷类抗生素指由二个或三个氨基糖分子和一个非糖部分称苷元的氨基环醇通过醚键连接而成的化合物,包括:链霉素、庆大霉素、妥布霉素、奈替米星、阿米卡星中的一种或二种以上。
10.用于评价细菌氨基糖苷类抗生素耐药效应的试剂或试剂盒,包括:
①标准品:甘油、甘油-3-磷酸、棕榈油酸和油酸中的任一种或二种以上代谢物或其组合,所述标准品分别用于细菌中甘油、甘油-3-磷酸、棕榈油酸和油酸中的任一种或二种以上的定性;
②提取液:甲醇/水溶液(80-100%,v/v),所述提取液用于提取细菌代谢物;
③衍生化试剂:甲氧胺吡啶溶液(10-20mg/mL)、N-甲基-N-(三甲基硅基)三氟乙酰胺。
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