Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development: Source Data

The turnaround times for phenotypic tests used to monitor the bacterial load of <i>Mycobacterium tuberculosis</i>, in both clinical and preclinical studies, are delayed by the organism’s slow growth in culture media. The existence of differentially culturable populations of <i>M.</i> <i>tuberculosis</i> may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. 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. Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i>-infected mice before and during treatment with a panel of drug combinations. 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 <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i>. The MBL 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.<br> This dataset contains all the data that were collected as part of this project. These data include the cycle thresholds (Cq) from real time quantitative PCR reactions of the internal control RNA and the <i>M. tuberculosis</i>-specific 16S rRNA derived either from spiked naïve mouse tissues or infected animals with <i>M. tuberculosis</i>. Molecular bacterial load values are compared with Colony Forming Units from solid agar-based plates and Most Probable Number counts using liquid-based media.

用于监测结核分枝杆菌（Mycobacterium tuberculosis）细菌载量的表型检测，在临床与临床前研究中均因该菌体在培养基中生长缓慢而导致周转时间延长。结核分枝杆菌存在差异可培养亚群，可能导致真实细菌载量被低估。此外，培养方法易受污染，造成关键数据点丢失。

本研究报道了我们此前针对痰液开发的、基于16S核糖体RNA（16S ribosomal RNA）的无培养稳健检测方法的适配改造，以实现动物组织中细菌数量的定量，作为改善临床前药物疗效研究检测结果的工具。初步的检测适配实验使用接种了已知数量结核分枝杆菌与内参RNA的未感染小鼠肺组织开展。将组织匀浆后提取总RNA，通过逆转录定量PCR（RT-qPCR）完成细菌定量。随后，我们将该检测方法与基于固体和液体培养基的生长检测估算的细菌计数进行对比，评估其在结核分枝杆菌感染小鼠给药前后，准确反映组织细菌载量的效用。

在感染小鼠肺组织的检测中，MBL（分子细菌载量）检测法所得结果与固体培养的菌落形成单位（CFU）计数具有可比性。值得注意的是，在特定给药方案下，MBL检测法检出的结核分枝杆菌数量显著高于CFU计数，这大概率提示存在无法在固体培养基上形成菌落的菌体。此外，采用最大概率数法（MPN）的液体培养基生长回收实验可解释MBL检测法与CFU计数间的差异，表明MBL检测法可检出结核分枝杆菌的差异可培养亚群。MBL检测法可实时定量动物组织中的细菌载量，无需等待培养基菌体生长的漫长周期，其检测结果与CFU计数相关性良好。重要的是，我们已证实MBL检测法可定量无法在固体琼脂上培养的特定细菌群体。该检测方法适配临床前研究后，有望缩短动物实验的数据获取周期，有望成为结核病药物研发的重要工具。

本数据集包含本项目收集的全部实验数据。这些数据包括内参RNA与结核分枝杆菌特异性16S核糖体RNA的实时定量PCR循环阈值（Cq），样本来源包括接种的未感染小鼠组织以及感染结核分枝杆菌的动物组织。分子细菌载量数值将与固体琼脂平板的菌落形成单位计数、基于液体培养基的最大概率数计数进行对比。