O'Donnell Michelle M, Rea Mary C, O'Sullivan Órla, Flynn Cal, Jones Beth, McQuaid Albert, Shanahan Fergus, Ross R Paul
Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland; APC Microbiome Institute, University College Cork, Ireland.
Kerry Ingredients, Tralee Road, Listowel, Co. Kerry, Ireland.
J Microbiol Methods. 2016 Oct;129:109-116. doi: 10.1016/j.mimet.2016.08.002. Epub 2016 Aug 4.
In-vitro gut fermentation systems provide suitable models for studying gut microbiota composition and functionality. However, such methods depend on the availability of donors and the assumption of reproducibility between microbial communities before experimental treatments commence. The aim of this study was to develop a frozen standardised inoculum (FSI) which minimizes inter-individual variation and to determine its stability over time using culture-dependent and culture-independent techniques.
A method for the preparation difference of a FSI is described which involves pooling the faecal samples, centrifugation and pelleting of the cell biomass and finally homogenising the cell pellets with phosphate buffer and glycerol. Using this approach, no significant difference in total anaerobe cell viability was observed between the fresh standardised inoculum (before freezing) and the 12days post freezing FSI. Moreover, Quantitative PCR revealed no significant alterations in the estimated bacterial numbers in the FSI preparations for any of the phyla. MiSeq sequencing revealed minute differences in the relative abundance at phylum, family and genus levels between the FSI preparations. Differences in the microbiota denoted as significant were limited between preparations in the majority of cases to changes in percentage relative abundance of ±0.5%. The independently prepared FSIs revealed a high degree of reproducibility in terms of microbial composition between the three preparations.
This study provides a method to produce a standardised human faecal inoculum suitable for freezing. Based on culture-dependent and independent analysis, the method ensures a degree of reproducibility between preparations by lessening the effect of inter-individual variation among the donors, thereby making the system more suitable for the accurate interpretation of the effects of experimental treatments.
体外肠道发酵系统为研究肠道微生物群的组成和功能提供了合适的模型。然而，此类方法依赖于供体的可得性以及在实验处理开始前微生物群落之间可重复性的假设。本研究的目的是开发一种可将个体间差异降至最低的冷冻标准化接种物（FSI），并使用依赖培养和不依赖培养的技术确定其随时间的稳定性。
描述了一种制备FSI的方法，该方法包括汇集粪便样本、对细胞生物质进行离心和沉淀，最后用磷酸盐缓冲液和甘油将细胞沉淀均质化。使用这种方法，新鲜标准化接种物（冷冻前）与冷冻12天后的FSI之间在总厌氧菌细胞活力方面未观察到显著差异。此外，定量PCR显示，FSI制剂中任何门的估计细菌数量均无显著变化。MiSeq测序显示，FSI制剂在门、科和属水平的相对丰度存在微小差异。在大多数情况下，制剂之间被认为显著的微生物群差异仅限于相对丰度百分比变化±0.5%。独立制备的FSI在三种制剂之间的微生物组成方面显示出高度的可重复性。
本研究提供了一种生产适合冷冻的标准化人粪便接种物的方法。基于依赖培养和不依赖培养的分析，该方法通过减少供体之间个体间差异的影响来确保制剂之间的一定程度的可重复性，从而使该系统更适合准确解释实验处理的效果。
