不同保存液和保存期限下肠道微生物组的变化

Variations of gut microbiome composition under different preservation solutions and periods

摘要:

肠道菌群与人体健康的关系密切，通过检测肠道菌群可以获得有关健康的信息。新鲜粪便不易获得，很难做到快速低温冷冻，在进行标准化和大规模人群采样时，可用于常温条件下采集和保存样本的保存液可以弥补采集样本数量多、地域分布广、现场采样条件多样、工作量大、运输条件差等条件不足。本研究招募了5名健康志愿者，采集他们的粪便样本后，通过对比不同市售微生物样本采集常温保存液对新鲜粪便样本的影响，评估了各类保存液的保存效果。在室温下把粪便放置于5种保存液，在第0、1、3、7、15、30天提取元基因组DNA，进行16S rRNA V3–V4区高通量测序，来分析不同保存液，在不同时间段对肠道菌群组成的影响。结果显示，不同保存液对肠道菌群的影响存在差异，与对照组相比，不同保存液对样本中的OUT数量影响不大；保存液A、B和C在菌群构成上更接近对照组，保存液D会明显改变肠道菌群组成，使放线菌门 (Actinobacteria) 和厚壁菌门 (Firmicutes) 增加；随着时间延长，各类保存液都有降低菌群多样性趋势，保存液E降低菌群多样性更明显；第30天时，5种保存液都会改变肠道菌群构成；肠道菌群组成存在个体差异，是影响各样本相似性的主要因素，来自不同志愿者的粪便样本，无论何种保存液和保存时间，彼此之间更接近。不同保存液对革兰氏阳性杆菌、革兰氏阳性球菌和革兰氏阴性菌的含量存在不同影响，保存液C、E会降低双歧杆菌 (Bifidobacterium) 丰度，而保存液D则相对升高；除保存液E相对降低乳酸杆菌 (Lactobacillus) 丰度之外，保存液A、B、C、D与对照都相当。各类保存液对链球菌 (Streptococcus) 的影响，除保存液D差异较大之外，保存液C与对照组最接近；保存液D比对照组降低瘤胃球菌科UCG 003 (Ruminococcaceae UCG 003)，其他各类保存液与对照组差异不大；不同保存液都比对照组增加大肠-志贺氏杆菌 (Escherichia-Shigella) 丰度，保存液A、B会增加克雷伯氏菌 (Klebsiella) 丰度，而保存液C、D和E与对照组相当。整体来看，保存液C在稳定肠道菌群的组成上表现较好。文中通过对比国内外常用保存液在不同时间点对肠道菌群的影响，为进行大规模微生物样本采集、保存提供了参考，也为标准化的微生物组项目提供了可参考的数据。后续的研究可在此基础上选择针对性的保存液和保存时间。

Abstract:

Gut microbiota is closely related to human health, and its composition can give us health information. The large-scale population sampling is required on gut microbiome research; however, fresh feces samples are not easy to obtain, and rapid low-temperature freezing is difficult to achieve. With the development of technology, preservation solutions are widely used for sample collection, storage, and transport under normal temperature conditions. Preservation solutions can be used in large scale sample collection, wide geographical distribution, diverse on-site sampling conditions, heavy workload, and poor transportation conditions. In this study, five healthy volunteers were recruited. After collecting their fresh stool samples, effect of 5 different commercial preservation solutions was evaluated at room temperature. Samples in different preservation solutions after placing fresh stool samples at the 0, 1, 3, 7, 15, and 30 days were collected. All samples were tested by 16S rRNA V3–V4 high-throughput sequencing to analyze the influence of microbiome composition in different preservation solutions. The results show that different preservation solutions had distinct effects on the gut microbiome composition. Compared with the control, different preservation solutions had little effect on the amount of OUTs; preservation solutions A, B and C were closer to the control in the composition of the gut microbiota, but preservation solution D significantly changed the composition by increasing Actinobacteria and Firmicutes abundance. With the time, all solutions tended to reduce the diversity of the microbiota. Preservation solution E significantly reduced the diversity of the flora; on the 30th day, all five solutions changed the composition; the individual differences in the composition of the gut microbiome were the main factors affecting the similarity of each sample, and were derived from different stools donors. The same samples, no matter which storage solution and storage time, were directly closer to each other. Different storage solutions had different effects on the content of Gram-positive bacilli, Gram-positive cocci and Gram-negative bacteria. Storage solutions C and E reduced the abundance of Bifidobacterium, whereas storage solution D increased; except that preservation solution E relatively reduced the abundance of Lactobacillus, but the preservation solution A, B, C, and D were all closer to the control. Except for the greater difference in preservation solution D, preservation solution C was the closest to the control group on Streptococcus; preservation solution D reduced Ruminococcaceae UCG 003 than the control group. However, other preservation solutions were not much different from the control group; different preservation solutions increased the abundance of Escherichia-Shigella than the control group, and preservation solutions A and B increased the abundance of Klebsiella, but preservation solution C, D, and E were closer to the control group. Overall, preservation solution C performed better in stabilizing the composition of the gut microbiota. This study provides reference for standardized microbiome projects. Subsequent research can choose a targeted preservation solution and preservation time based on this study.