PDF(Pubmed)
Abstract：
BACKGROUND: Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region.
RESULTS: The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities.
CONCLUSIONS: Frozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.
RESULTS: The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities.
CONCLUSIONS: Frozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.
摘要：
背景：目前，马实践中粪便微生物群移植(FMT)缺乏标准化，导致技术高度可变，并且没有关于所施用产品的细菌代谢活性或活力的数据。这项研究的目的是比较马FMT中总的和潜在的代谢活性细菌种群，并评估不同冷冻储存时间的效果，缓冲区,以及马FMT产品的温度。对从三匹健康成年马收集的新鲜粪便进行不同的储存方法。这包括不同的保存溶液（盐水加甘油或仅盐水），温度（-20°C或-80°C），和时间（新鲜，30、60或90天)。样品进行DNA提取以评估总细菌种群（活的和死的组合）和RNA提取，然后逆转录为cDNA作为评估活细菌的替代。然后使用V1-V2区域进行16srRNA基因扩增子测序。
结果：在评估基于DNA（总）和基于cDNA（潜在代谢活性）的提取方法的结果时，可以看到在属水平上的种群指数和分类组成的最大差异。在社区层面,阿尔法多样性(观察到的物种，Shannon多样性）在基于DNA的分析的冷冻样品中显着降低（P<0.05），对于基于cDNA的测序，差异较小。使用基于DNA的分析，储存时间对细菌群落谱有显著影响(P<0.05)。对于潜在代谢活跃的人群，储存总体上对细菌群落组成的影响较小，具有显著的缓冲作用(P<0.05)。个体马在DNA和cDNA细菌群落中的作用最显著。
结论：马FMT材料的冷冻储存可以保留马粪便微生物组的潜在代谢活性细菌，用盐水加甘油保存比单独用盐水保存更有效。需要更大规模的研究来确定这些发现是否适用于其他个体马。冷冻用于马患者的FMT材料的能力可以允许在肠道微生物组紊乱的马中更容易地临床使用粪便移植。
结果：在评估基于DNA（总）和基于cDNA（潜在代谢活性）的提取方法的结果时，可以看到在属水平上的种群指数和分类组成的最大差异。在社区层面,阿尔法多样性(观察到的物种，Shannon多样性）在基于DNA的分析的冷冻样品中显着降低（P<0.05），对于基于cDNA的测序，差异较小。使用基于DNA的分析，储存时间对细菌群落谱有显著影响(P<0.05)。对于潜在代谢活跃的人群，储存总体上对细菌群落组成的影响较小，具有显著的缓冲作用(P<0.05)。个体马在DNA和cDNA细菌群落中的作用最显著。
结论：马FMT材料的冷冻储存可以保留马粪便微生物组的潜在代谢活性细菌，用盐水加甘油保存比单独用盐水保存更有效。需要更大规模的研究来确定这些发现是否适用于其他个体马。冷冻用于马患者的FMT材料的能力可以允许在肠道微生物组紊乱的马中更容易地临床使用粪便移植。
