背景:用于监测结核分枝杆菌细菌负荷的表型测试的周转时间,在临床和临床前研究中,被培养基中生物体的缓慢生长所延迟。结核分枝杆菌可差异培养群体的存在可能导致真实数量的低估。此外,培养方法容易受到污染,导致关键数据点的丢失。
目标:我们报告了我们的健壮,利用16S核糖体RNA的无培养测定,开发用于痰,列举动物组织中存在的细菌数量,作为改善临床前药物疗效研究中读数的工具。
方法:使用掺入已知量的结核分枝杆菌和内部RNA对照的初始小鼠肺进行初始测定适应。组织被均匀化,提取的总RNA,并使用RT-qPCR进行计数。然后我们评估了该试验的实用性,与使用固体和液体培养基上的生长测定估计的细菌计数相比,在用一组药物组合治疗之前和期间,准确地告知结核分枝杆菌感染的小鼠组织中的细菌负荷。
结果:在感染小鼠的肺组织上进行测试时,MBL测定产生与固体培养中的细菌计数相当的结果(菌落形成单位:CFU)。值得注意的是,在特定的药物治疗下,与CFU相比,MBL检测能够检测到更高数量的结核分枝杆菌,可能表明存在无法在固体培养物中产生菌落的细菌。此外,使用最可能数量(MPN)测定的液体培养基中的生长恢复能够解释MBL测定和CFU数量之间的差异,这表明MBL检测检测结核分枝杆菌的差异可培养亚群。
结论:MBL测定法可以实时计算动物组织中的细菌负荷,而无需等待长时间培养物生长。读数与CFU良好相关。重要的是,我们已经证明MBL能够测量未在固体琼脂上培养的细菌的特定种群。将该测定法用于临床前研究具有减少从动物实验获取数据的读出时间的潜力,并且可以代表结核病药物发现和开发的有价值的工具。
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.
OBJECTIVE: 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.