International Reference Laboratory of Mycobacteriology, Statens Serum Institutgrid.6203.7, Copenhagen, Denmark.
Global Health Section, Department of Public Health, University of Copenhagen, Denmark.
Microbiol Spectr. 2022 Jun 29;10(3):e0031022. doi: 10.1128/spectrum.00310-22. Epub 2022 May 19.
In this study, 28 "historical" clinical freeze-dried nontuberculous mycobacterial isolates collected from 1948 to 1957, were analyzed by investigating their viability and performing whole genome sequencing (WGS) on DNA extracted (i) directly from freeze-dried cells versus (ii) after culturing, to determine cell properties and DNA quality after centuries of freeze-dried storage. The isolated DNA was sequenced on the Illumina MiSeq platform and data quality evaluated analyzing the per-base quality scores of paired-end sequencing reads as well as the overall contiguity of resulting assemblies. After 72 years in storage, all freeze-dried isolates were viable, and showed no signs of cell damage and limited signs of contamination when reculturing. They were recultured without problems and identified through WGS with only four of 13 parameters showing statistical significance based on sequence data obtained directly from the freeze-dried cells versus after reculturing, indicating no DNA degradation. Thus, mycobacteria can be whole genome sequenced successfully directly from freeze-dried material without prior recultivation, saving laboratory time and resources, and emphasizing the value of freeze-drying for long-term storage. Our study lays the groundwork for further genomic investigations of freeze-dried bacterial isolates, and the approximately 4,000 historical isolates in our collection will provide a unique opportunity to investigate mycobacterial DNA from a variety of NTM species unexposed to antimicrobials, some maybe still undescribed species. The genus Mycobacterium was described more than a century ago and new species are continuously identified and described. There is an ongoing discussion about an increase in the incidence of disease caused by nontuberculous mycobacteria (NTM). How the different bacteria looked before exposure to antibiotics can only be investigated by looking at strains from before the antibiotic era. Strains from that era will be stored in different ways, for example by freeze-drying. The question is how to investigate these strains, and if they are still viable, whether they need to be cultured, and if that changes the DNA. Here, we test all these parameters on freeze-dried strains and show that NGS can be applied directly without culturing.
在这项研究中，分析了从 1948 年到 1957 年收集的 28 株“历史”临床冻干非结核分枝杆菌分离株，方法是通过检测其生存能力和对从冻干细胞中提取的 DNA 进行全基因组测序（WGS）（i）直接进行，以及（ii）在培养后进行，以确定经过数百年的冻干储存后细胞特性和 DNA 质量。从冻干细胞中提取的 DNA 在 Illumina MiSeq 平台上进行测序，并通过分析配对末端测序reads 的每个碱基的质量得分以及生成的组装体的整体连续性来评估数据质量。在储存 72 年后，所有冻干分离株均具有生存能力，在重新培养时没有细胞损伤的迹象，仅有有限的污染迹象。它们在没有问题的情况下被重新培养，并通过 WGS 进行鉴定，只有 13 个参数中的 4 个基于直接从冻干细胞中获得的序列数据与培养后相比具有统计学意义，表明没有 DNA 降解。因此，无需事先培养即可直接从冻干材料中成功对分枝杆菌进行全基因组测序，节省了实验室时间和资源，并强调了冻干在长期储存中的价值。我们的研究为进一步研究冻干细菌分离株的基因组奠定了基础，并且我们收集的大约 4000 株历史分离株将提供一个独特的机会来研究从未暴露于抗生素的各种非结核分枝杆菌（NTM）的分枝杆菌 DNA，其中一些可能仍然是未描述的物种。分枝杆菌属在一个多世纪前被描述，新物种不断被发现和描述。关于由非结核分枝杆菌（NTM）引起的疾病发病率增加的讨论一直在进行。只有通过研究抗生素时代之前的菌株，才能了解暴露于抗生素之前细菌的外观。来自那个时代的菌株将以不同的方式储存，例如通过冻干。问题是如何研究这些菌株，以及它们是否仍然具有活力，是否需要培养，以及这是否会改变 DNA。在这里，我们对冻干菌株进行了所有这些参数的测试，并表明无需培养即可直接应用 NGS。
