Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development.
Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development.
复制标题
DOI：
10.3390/microorganisms10020460
复制
发表时间：
2022-02-17
期刊：
影响因子：
4.5
通讯作者：
McHugh TD
中科院分区：
文献类型：
--
作者：
Evangelopoulos D;Shoen CM;Honeyborne I;Clark S;Williams A;Mukamolova GV;Cynamon MH;McHugh TD
背景：在临床和临床前研究中，用于监测结核分枝杆菌细菌载量的表型检测的周转时间因该微生物在培养基中生长缓慢而延迟。结核分枝杆菌不同可培养群体的存在可能导致对真实数量的低估。此外，培养方法容易受到污染，从而导致关键数据点的丢失。
目的：我们报告了我们针对痰液开发的利用16S核糖体RNA的可靠的无培养检测方法的改进，用于计数动物组织中存在的细菌数量，以此作为一种工具来改进临床前药物疗效研究中的检测结果。
方法：使用掺入已知数量结核分枝杆菌和内部RNA对照的未感染小鼠肺进行初步检测方法的改进。将组织匀浆，提取总RNA，并使用逆转录定量聚合酶链反应（RT - qPCR）进行计数。然后，我们将该检测方法与使用固体和液体培养基上的生长检测估计的细菌计数进行比较，评估其在一组药物组合治疗前后准确反映结核分枝杆菌感染小鼠组织中细菌载量的效用。
结果：在对感染小鼠的肺组织进行检测时，MBL检测产生的结果与固体培养中的细菌计数（菌落形成单位：CFU）相当。值得注意的是，在特定药物治疗下，与CFU相比，MBL检测能够检测到数量显著更多的结核分枝杆菌，这可能表明存在无法在固体培养基上形成菌落的细菌。此外，使用最大可能数（MPN）检测法在液体培养基中的生长恢复能够解释MBL检测和CFU数量之间的差异，这表明MBL检测可检测到结核分枝杆菌不同可培养的亚群。
结论：MBL检测能够实时计数动物组织中的细菌载量，而无需长时间等待培养物生长。检测结果与CFU密切相关。重要的是，我们已经表明MBL能够测量在固体琼脂上未培养出的特定细菌群体。将这种检测方法应用于临床前研究有可能减少从动物实验中获取数据的检测时间，并可能成为结核病药物发现和开发的一种有价值的工具。
Background: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism’s slow growth in culture media. The existence of differentially culturable populations of M. tuberculosis may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. Objectives: We report the adaptation of our robust, culture-free assay utilising 16S ribosomal RNA, developed for sputum, to enumerate the number of bacteria present in animal tissues as a tool to improve the read-outs in preclinical drug efficacy studies. Methods: Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of M. tuberculosis and an internal RNA control. Tissues were homogenised, total RNA extracted, and enumeration performed using RT-qPCR. We then evaluated the utility of the assay, in comparison to bacterial counts estimated using growth assays on solid and liquid media, to accurately inform bacterial load in tissues from M. tuberculosis-infected mice before and during treatment with a panel of drug combinations. Results: When tested on lung tissues derived from infected mice, the MBL assay produced comparable results to the bacterial counts in solid culture (colony forming units: CFU). Notably, under specific drug treatments, the MBL assay was able to detect a significantly higher number of M. tuberculosis compared to CFU, likely indicating the presence of bacteria that were unable to produce colonies in solid-based culture. Additionally, growth recovery in liquid media using the most probable number (MPN) assay was able to account for the discrepancy between the MBL assay and CFU number, suggesting that the MBL assay detects differentially culturable sub-populations of M. tuberculosis. Conclusions: The MBL assay can enumerate the bacterial load in animal tissues in real time without the need to wait for extended periods for cultures to grow. The readout correlates well with CFUs. Importantly, we have shown that the MBL is able to measure specific populations of bacteria not cultured on solid agar. The adaptation of this assay for preclinical studies has the potential to decrease the readout time of data acquisition from animal experiments and could represent a valuable tool for tuberculosis drug discovery and development.
登录
查看更多内容
DOI：
10.4103/ijmr.ijmr_554_18
发表时间：
2018-05
期刊：
The Indian journal of medical research
影响因子：
--
作者：
Singh AK;Gupta UD
通讯作者：
Gupta UD
影响因子：
4.9
作者：
Loraine J;Pu F;Turapov O;Mukamolova GV
通讯作者：
Mukamolova GV
影响因子：
5.2
作者：
Hu Y;Liu A;Ortega-Muro F;Alameda-Martin L;Mitchison D;Coates A
通讯作者：
Coates A
影响因子：
5.2
作者：
Bowness, Ruth;Boeree, Martin J.;Gillespie, Stephen H.
通讯作者：
Gillespie, Stephen H.
DOI：
10.1164/rccm.201604-0769oc
发表时间：
2016-12-15
影响因子：
24.7
作者：
Chengalroyen, Melissa D.;Beukes, Germar M.;Kana, Bavesh D.
通讯作者：
Kana, Bavesh D.