Beckers Kalie F, Schulz Christopher J, Childers Gary W
Department of Biological Sciences, Southeastern Louisiana University, Hammond, LA, United States of America.
PLoS One. 2017 Nov 1;12(11):e0187044. doi: 10.1371/journal.pone.0187044. eCollection 2017.
Advances have been made to standardize 16S rRNA gene amplicon based studies for inter-study comparisons, yet there are many opportunities for systematic error that may render these comparisons improper and misleading. The fecal microbiome of horses has been examined previously, however, no universal horse fecal collection method and sample processing procedure has been established. This study was initialized in large part to ensure that samples collected by different individuals from different geographical areas (i.e., crowdsourced) were not contaminated due to less than optimal sampling or holding conditions. In this study, we examined the effect of sampling the surface of fecal pellets compared to homogenized fecal pellets, and also the effect of time of sampling after defecation on 'bloom' taxa (bloom taxa refers to microbial taxa that can grow rapidly in horse feces post-defecation) using v4 16S rRNA amplicon libraries. A total of 1,440,171 sequences were recovered from 65 horse fecal samples yielding a total of 3,422 OTUs at 97% similarity. Sampling from either surface or homogenized feces had no effect on diversity and little effect on microbial composition. Sampling at various time points (0, 2, 4, 6, 12 h) had a significant effect on both diversity and community composition of fecal samples. Alpha diversity (Shannon index) initially increased with time as regrowth taxa were detected in the amplicon libraries, but by 12 h the diversity sharply decreased as the community composition became dominated by a few bloom families, including Bacillaceae, Planococcaeae, and Enterococcaceae, and other families to a lesser extent. The results show that immediate sampling of horse feces must be done in order to ensure accurate representation of horse fecal samples. Also, several of the bloom taxa found in this study are known to occur in human and cattle feces post defecation. The dominance of these taxa in feces shortly after defecation suggests that the feces is an important habitat for these organisms, and horse fecal samples that were improperly stored can be identified by presence of bloom taxa.
在基于16S rRNA基因扩增子的研究标准化方面已取得进展，以便进行研究间的比较，但仍存在许多导致系统误差的机会，这可能会使这些比较不恰当且具有误导性。此前已对马的粪便微生物群进行过研究，然而，尚未建立通用的马粪便采集方法和样本处理程序。本研究在很大程度上是为了确保不同个体从不同地理区域收集的样本（即众包样本）不会因采样或保存条件不理想而受到污染。在本研究中，我们使用v4 16S rRNA扩增子文库，研究了粪便颗粒表面采样与匀浆粪便颗粒采样的效果，以及排便后采样时间对“爆发”类群（爆发类群是指排便后能在马粪便中快速生长的微生物类群）的影响。从65份马粪便样本中总共获得了1,440,171条序列，在97%的相似度下共产生3,422个操作分类单元。从粪便表面或匀浆粪便中采样对多样性没有影响，对微生物组成的影响也很小。在不同时间点（0、2、4、6、12小时）采样对粪便样本的多样性和群落组成都有显著影响。α多样性（香农指数）最初随着时间增加，因为在扩增子文库中检测到了再生类群，但到12小时时，随着群落组成由少数爆发科主导，包括芽孢杆菌科、扁平球菌科和肠球菌科，以及其他程度较轻的科，多样性急剧下降。结果表明，必须对马粪便进行即时采样，以确保准确呈现马粪便样本。此外，本研究中发现的几个爆发类群在人类和牛排便后的粪便中也有出现。这些类群在排便后不久在粪便中的优势表明，粪便对这些生物体来说是一个重要的栖息地，并且可以通过爆发类群的存在来识别储存不当的马粪便样本。
