State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Department of Gastroenterology, Shanghai Key Laboratory of Pancreatic Disease, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 201620, China.
Arch Microbiol. 2022 Aug 16;204(9):557. doi: 10.1007/s00203-022-03169-1.
Stool is the most commonly used sample for gut microbiota analysis in humans and animals. Cryopreservation of stool at - 80 °C is a feasible and simple method in clinics and researches, especially in large-scale cohort studies. However, the viability of bacteria in stool after freezing has yet well-demonstrated quantitatively and compositionally. This study determined the viable microbiota of samples under cryopreservation at - 80 °C, relative to fresh samples and that stored at ambient. Stool samples were collected from three healthy adults. Propidium monoazide treatment combined with quantitative PCR and 16S rRNA gene sequencing was performed to target viable microbiota. After freezing, the number of viable bacteria decreased, though inter-individual difference existed. Notably, the alpha diversity of viable microbiota after freezing did not change significantly, while its composition changed. Freezing significantly reduced the viable bacteria in Gram-negative genera of Bacteroidetes and Firmicutes, and proportionally increased Gram-positive bacteria in genera of Actinobacteria and Firmicutes, including Bifidobacterium, Collinsella and Blautia, implying that the cell envelope structure associated with the bacterial sensitivity to freezing. On the contrary, the room temperature storage not only decreased the number of viable bacteria, but also decreased the microbial alpha diversity, and remarkably enriched facultative anaerobes of Escherichia-Shigella, Enterococcus and Lactococcus, some of which are opportunistic pathogens. Our findings suggested that changes in viable microbiota in stool samples caused by cryopreservation should be paid enough attention for downstream utilization.
粪便是人类和动物肠道微生物群分析中最常用的样本。将粪便在-80°C 下冷冻保存是临床和研究中一种可行且简单的方法，尤其是在大规模队列研究中。然而，冷冻后粪便中细菌的存活率在数量和组成上尚未得到很好的证明。本研究定量和定性地确定了冷冻保存下（-80°C）粪便样本中存活微生物群的情况，与新鲜样本和常温保存的样本进行了比较。从三名健康成年人中收集粪便样本。使用吖啶橙单染色结合定量 PCR 和 16S rRNA 基因测序来靶向存活微生物群。冷冻后，尽管存在个体间差异，但存活细菌的数量减少。值得注意的是，冷冻后存活微生物群的 alpha 多样性没有显著变化，但其组成发生了变化。冷冻显著降低了厚壁菌门和拟杆菌门革兰氏阴性属中的存活细菌数量，并且比例增加了放线菌门和厚壁菌门中革兰氏阳性细菌的数量，包括双歧杆菌、柯林斯菌和布劳特氏菌，这表明与细菌对冷冻敏感相关的细胞包膜结构。相反，室温储存不仅减少了存活细菌的数量，而且降低了微生物的 alpha 多样性，并显著富集了兼性厌氧菌大肠杆菌-志贺氏菌、肠球菌和乳球菌，其中一些是机会致病菌。我们的研究结果表明，冷冻保存引起的粪便样本中存活微生物群的变化应引起足够的重视，以便下游利用。
