Rapid regrowth and detection of microbial contaminants in equine fecal microbiome samples

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.

尽管当前已在推进基于16S rRNA基因扩增子的研究标准化以实现跨研究比较，但仍存在诸多系统性误差风险，可能导致此类比较失当且具有误导性。此前虽已有针对马粪便微生物组的相关研究，但尚未建立通用的马粪便样本采集方法与样本处理流程。本研究的主要初衷，是确保来自不同地理区域、由不同人员采集（即众包采集）的马粪便样本，不会因采样或保存条件不佳而受到污染。本研究借助v4区16S rRNA扩增子文库，对比了粪便颗粒表面采样与均质化粪便颗粒采样的效果，并探究了排便后不同采样时间对‘增殖类群（bloom taxa）’的影响——增殖类群指可在排便后的马粪便中快速繁殖的微生物类群。本研究从65份马粪便样本中共回收得到1,440,171条序列，在97%相似性阈值下共得到3,422个操作分类单元（Operational Taxonomic Unit, OTU）。无论是采集粪便颗粒表面还是均质化粪便样本，对微生物群落多样性均无显著影响，对微生物组成的影响也极小。在不同时间点（0、2、4、6、12小时）采样，对粪便样本的群落多样性与组成均有显著影响。α多样性（Shannon指数）最初随时间推移而升高，这是因为扩增子文库中检测到了再增殖类群；但到12小时时，群落组成被少数增殖类群科所主导，包括芽孢杆菌科（Bacillaceae）、动球菌科（Planococcaceae）与肠球菌科（Enterococcaceae）等，其他类群科占比相对较低，多样性随之急剧下降。研究结果表明，为确保马粪便样本的微生物群落表征准确，必须在排便后立即进行采样。此外，本研究中发现的部分增殖类群，此前已知可在人类与牛的排便后粪便中存在。此类类群在排便后短时间内的粪便中占据主导地位，这表明粪便正是这些微生物的重要生存环境；而通过检测增殖类群的存在，即可识别出保存不当的马粪便样本。