Identifying Dormant Growth State of Mycobacteria by Orthogonal Analytical Approaches on a Single Cell and Ensemble Basis.
Identifying Dormant Growth State of Mycobacteria by Orthogonal Analytical Approaches on a Single Cell and Ensemble Basis.
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基于单细胞和整体的正交分析方法识别分枝杆菌的休眠生长状态
DOI：
10.1021/acs.analchem.8b03646
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发表时间：
2019
影响因子：
7.4
通讯作者：
Wieser
中科院分区：
文献类型：
--
作者：
Neumann;Hoelscher;Haisch;Wieser
结核病是目前世界上最致命的传染病，也是世界卫生组织确定的公共卫生优先事项。虽然这种疾病一般是可以治愈的，但治疗成功受到长期和副作用倾向治疗的必要性的阻碍。治疗效率低可能部分是由于分枝杆菌进入特殊的生长状态，以避免被抗生素杀死，并在宿主体内持续更长时间。这种生长状态最近被定义为休眠或持续。我们使用酸化模型生产休眠模式生物培养物，并通过使用质谱法（MALDI-TOF）、显微镜（SEM、拉曼）和微生物学技术（CFU、OD 600、ATP水平）的多层方法对其进行表征。通过快速和96良好适应的提取方案，可以灭活分枝杆菌并提取用于MALDI-TOF分析。这是第一次，我们展示了生长状态依赖的文化质量签名的变化，允许一个可靠的区分休眠状态和指数增长。我们还证明复苏从休眠状态回到指数增长。将活分枝杆菌固定化，并通过拉曼显微镜单独分析单个生物体。对于单细胞拉曼显微术，使用新的快速温和的单步固定技术将耻垢分枝杆菌培养物固定在疏水载玻片上。我们能够区分处于休眠状态的单个活细菌与快速生长的遗传相同的对应物，根据单个生物体光谱确定培养物的生长状态。这允许根据它们的生长状态使用无破坏的光学方法的拉曼显微镜的异质培养物的分离。
Tuberculosis is currently the single most deadly infectious disease in the world and a public health priority as defined by WHO. Although the disease is in general curable, treatment success is hampered by the necessity of a long and side effect prone treatment. Low treatment efficiency may be partly due to the special growth states that mycobacteria enter to avoid being killed by antibiotics and to persist longer within the host. Such growth states have been recently defined as dormant or persistent. We produced dormant model-organism cultures using an acidification model and characterized those by a multilayered approach using mass spectrometry (MALDI-TOF), microscopy (SEM, Raman), and microbiological techniques (CFU, OD600, ATP-levels). With a fast and 96-well-adapted extraction protocol, mycobacteria could be inactivated and extracted for MALDI-TOF analysis. For the first time, we demonstrate growth-state-dependent changes in the mass signatures of the culture, allowing for a reliable differentiation of dormant state and exponential growth. We also demonstrate resuscitation from dormant state back to exponential growth. Viable mycobacteria were immobilized, and single organisms were analyzed individually by Raman microscopy. For single-cell Raman microscopy,Mycobacterium smegmatiscultures were fixed using a new fast and gentle single-step immobilization technique on a hydrophobic glass slide. We were able to distinguish single viable bacteria in the dormant state from their rapidly growing, genetically identical counterparts, identifying the growth state of the culture based on single-organism spectra. This allows for the separation of heterogeneous cultures depending on their growth state using the destruction-free optical method of Raman microscopy.
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影响因子：
5.4
作者：
Shiloh, Michael U.;Champion, Patricia A. DiGiuseppe
通讯作者：
Champion, Patricia A. DiGiuseppe
影响因子：
3.7
作者：
El Khéchine A;Couderc C;Flaudrops C;Raoult D;Drancourt M
通讯作者：
Drancourt M
揭示宿主生态位：通过拉曼显微光谱通过巨噬细胞中的类胡萝卜素对戈登分枝杆菌进行无标记原位检测
影响因子：
3.4
作者：
A. Silge;E. Abdou;Kilian R. A. Schneider;S. Meisel;T. Bocklitz;Hui;R. Heintzmann;P. Rösch;J. Popp
通讯作者：
J. Popp
影响因子：
48
作者：
Schindelin, Johannes;Arganda-Carreras, Ignacio;Frise, Erwin;Kaynig, Verena;Longair, Mark;Pietzsch, Tobias;Preibisch, Stephan;Rueden, Curtis;Saalfeld, Stephan;Schmid, Benjamin;Tinevez, Jean-Yves;White, Daniel James;Hartenstein, Volker;Eliceiri, Kevin;Tomancak, Pavel;Cardona, Albert
通讯作者：
Cardona, Albert
影响因子：
2.2
作者：
Rabodoarivelo, Marie Sylvianne;Aerts, Maarten;Martin, Anandi
通讯作者：
Martin, Anandi