Bassis Christine M, Moore Nicholas M, Lolans Karen, Seekatz Anna M, Weinstein Robert A, Young Vincent B, Hayden Mary K
Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Medical Laboratory Science, Rush University Medical Center, Chicago, IL, 60612, USA.
BMC Microbiol. 2017 Mar 31;17(1):78. doi: 10.1186/s12866-017-0983-9.
Sample collection for gut microbiota analysis from in-patients can be challenging. Collection method and storage conditions are potential sources of variability. In this study, we compared the bacterial microbiota from stool stored under different conditions, as well as stool and swab samples, to assess differences due to sample storage conditions and collection method.
Using bacterial 16S rRNA gene sequence analysis, we compared the microbiota profiles of stool samples stored and collected under various conditions. Stool samples (2 liquid, 1 formed) from three different patients at two hospitals were each evaluated under the following conditions: immediately frozen at -80°C, stored at 4°C for 12-48 hours before freezing at -80°C and stored at -20°C with 1-2 thaw cycles before storage at -80°C. Additionally, 8 stool and 30 rectal swab samples were collected from 8 in-patients at one hospital. Microbiota differences were assessed using the Yue and Clayton dissimilarity index (θ distance) and analysis of molecular variance (AMOVA).
Regardless of the storage conditions, the bacterial communities of aliquots from the same stool samples were very similar based on θ distances (median intra-sample θ distance: 0.035, IQR: 0.015-0.061) compared to aliquots from different stool samples (median inter-sample θ distance: 0.93, IQR: 0.85-0.97) (Wilcoxon test p-value: <0.0001). For the stool and rectal swab comparison, samples from different patients, regardless of sample collection method, were significantly different (AMOVA p-values: <0.001-0.029) compared to no significant difference between all stool and swab samples (AMOVA p-value: 0.976). The θ dissimilarity index between swab and stool samples was significantly lower within individuals (median 0.17, IQR: 0.10-0.27) than between individuals (median 0.93, IQR: 0.85-0.97) (Wilcoxon test p-value: <0.0001), indicating minimal differences between stool and swab samples collected from the same individual over the sampling period.
For gastrointestinal microbiota studies based on bacterial 16S rRNA gene sequence analysis, interim stool sample storage at 4 °C or -20 °C, rather than immediate storage at -80 °C, does not significantly alter results. Additionally, stool and rectal swab microbiotas from the same subject were highly similar, indicating that these sampling methods could be used interchangeably to assess the community structure of the distal GI tract.
对住院患者进行肠道微生物群分析的样本采集可能具有挑战性。采集方法和储存条件是潜在的变异性来源。在本研究中，我们比较了在不同条件下储存的粪便中的细菌微生物群，以及粪便和拭子样本，以评估由于样本储存条件和采集方法导致的差异。
使用细菌16S rRNA基因序列分析，我们比较了在各种条件下储存和采集的粪便样本的微生物群谱。来自两家医院的三名不同患者的粪便样本（2份液体样本、1份成形样本）在以下条件下分别进行评估：立即在-80°C冷冻、在4°C储存12 - 48小时后在-80°C冷冻以及在-20°C进行1 - 2次解冻循环后在-80°C储存。此外，从一家医院的8名住院患者中采集了8份粪便样本和30份直肠拭子样本。使用Yue和Clayton差异指数（θ距离）和分子方差分析（AMOVA）评估微生物群差异。
无论储存条件如何，基于θ距离，来自同一粪便样本的等分样本的细菌群落非常相似（样本内θ距离中位数：(0.035)，四分位距：(0.015 - 0.061)），相比之下，来自不同粪便样本的等分样本（样本间θ距离中位数：(0.93)，四分位距：(0.85 - 0.97)）（Wilcoxon检验p值：(<0.0001)）。对于粪便和直肠拭子的比较，来自不同患者的样本，无论样本采集方法如何，均存在显著差异（AMOVA p值：(<0.001 - 0.029)），而所有粪便和拭子样本之间无显著差异（AMOVA p值：(0.976)）。拭子和粪便样本之间的θ差异指数在个体内显著低于个体间（中位数(0.17)，四分位距：(0.10 - 0.27)）（Wilcoxon检验p值：(<0.00
