Chemical and Biological Signature Sciences Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, United States of America.
Subsurface Science and Technology Group, Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, United States of America.
PLoS One. 2019 Sep 6;14(9):e0221831. doi: 10.1371/journal.pone.0221831. eCollection 2019.
The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F. novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22-40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S. aureus. This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F. novicida and S. aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures.
在初始采集后，将生物样本保存数天至数周的时间对于细菌病原体的鉴定、筛选和特征分析非常重要。传统上，保存依赖于冷链基础设施；然而，在许多情况下，这是不切实际的或不可能的。因此，我们的目标是开发替代的细菌样本保存和运输培养基，这些培养基在没有冷藏或外部仪器的情况下仍然有效。我们评估了炭疽杆菌 Sterne 34F2、弗朗西斯菌 novicida U112、金黄色葡萄球菌 ATCC 43300 和鼠疫耶尔森菌 KIM D27（pgm-）的存活能力、核酸稳定性和蛋白质稳定性，最长可达 28 天。在 40°C 的高温下，用 L-半胱氨酸制备的 PBS 中的黄原胶（XG）比商业试剂和缓冲液保持了更多的 F. novicida U112 活细胞。在 22-40°C 的温度范围内，XG 中含有 0.9 mM L-半胱氨酸的四种细菌的存活能力都得到了维持。有趣的是，将 L-半胱氨酸的浓度增加到 9 mM 会导致金黄色葡萄球菌的迅速死亡。当在建筑物环境中收集样本时，这可能是有利的，因为在建筑物环境中，金黄色葡萄球菌的收集和稳定的可能性比其他感兴趣的生物体更大。F. novicida 和 S. aureus 的 DNA 在 22°C 或 40°C 下储存长达 45 天是稳定的，并且无需 DNA 提取，即可在 XG 制剂中直接通过实时 qPCR 进行分析。由于 XG 中存在大量的野油菜黄单胞菌残留蛋白，XG 与通过 LC-MS/MS 进行的蛋白质组学分析不兼容。我们的结果表明，多糖基制剂，特别是含有 L-半胱氨酸的 XG，在环境和高温下，可维持多种革兰氏阴性和革兰氏阳性细菌的存活能力和核酸完整性。
