Laboratory of Molecular Bacteriology, Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium.
Center for Microbiology, VIB, Leuven, Belgium.
Microbiome. 2022 May 30;10(1):80. doi: 10.1186/s40168-022-01267-2.
Novel strategies for anaerobic bacterial isolations from human faecal samples and various initiatives to generate culture collections of gut-derived bacteria have instigated considerable interest for the development of novel microbiota-based treatments. Early in the process of building a culture collection, optimal faecal sample preservation is essential to safeguard the viability of the broadest taxonomic diversity range possible. In contrast to the much more established faecal storage conditions for meta-omics applications, the impact of stool sample preservation conditions on bacterial growth recovery and isolation remains largely unexplored. In this study, aliquoted faecal samples from eleven healthy human volunteers selected based on a range of physicochemical and microbiological gradients were cryopreserved at - 80 °C either without the addition of any medium (dry condition) or in different Cary-Blair medium conditions with or without a cryoprotectant, i.e. 20% (v/v) glycerol or 5% (v/v) DMSO. Faecal aliquots were subjected to bulk 16S rRNA gene sequencing as well as dilution plating on modified Gifu Anaerobic Medium after preservation for culturable fraction profiling and generation of bacterial culture collections.
Analyses of compositional variation showed that cryopreservation medium conditions affected quantitative recovery but not the overall community composition of cultured fractions. Post-preservation sample dilution and richness of the uncultured source samples were the major drivers of the cultured fraction richness at genus level. However, preservation conditions differentially affected recovery of specific genera. Presence-absence analysis indicated that twenty-two of the 45 most abundant common genera (>0.01% abundance, dilution 10) were recovered in cultured fractions from all preservation conditions, while nine genera were only detected in fractions from a single preservation condition. Overall, the highest number of common genera (i.e. 35/45) in cultured fractions were recovered from sample aliquots preserved without medium and in the presence of Cary-Blair medium containing 5% (v/v) DMSO. Also, in the culture collection generated from the cultured fractions, these two preservation conditions yielded the highest species richness (72 and 66, respectively).
Our results demonstrate that preservation methods partly determine richness and taxonomic diversity of gut anaerobes recovered from faecal samples. Complementing the current standard practice of cryopreserving stool samples in dry conditions with other preservation conditions, such as Cary-Blair medium with DMSO, could increase the species diversity of gut-associated culture collections. Video abstract.
从人类粪便样本中分离厌氧细菌的新策略以及生成肠道衍生细菌培养物集的各种举措激发了人们对新型基于微生物组治疗方法的极大兴趣。在建立培养物集的早期过程中，优化粪便样本的保存对于保护尽可能广泛的分类多样性的生存能力至关重要。与元组学应用中更为成熟的粪便储存条件相比，粪便样本保存条件对细菌生长恢复和分离的影响在很大程度上仍未得到探索。在这项研究中，根据理化和微生物梯度选择的 11 名健康人类志愿者的粪便样本被分装并在-80°C 下冷冻保存，要么不添加任何培养基（干燥条件），要么在不同的 Cary-Blair 培养基条件下添加或不添加冷冻保护剂，即 20%（v/v）甘油或 5%（v/v）DMSO。在保存后进行可培养分数分析和细菌培养物集的生成之前，对粪便等分试样进行了批量 16S rRNA 基因测序和改良吉富厌氧培养基的稀释平板培养。
组成变化分析表明，冷冻保存培养基条件影响定量回收，但不影响培养分数的总体群落组成。保存后样品的稀释和未培养源样品的丰富度是属水平培养分数丰富度的主要驱动因素。然而，保存条件对特定属的回收有不同的影响。存在-缺失分析表明，在所有保存条件下的培养分数中都可回收 45 个最丰富的常见属中的 22 个（丰度>0.01%，稀释度为 10），而 9 个属仅在单个保存条件下的分数中检测到。总体而言，在培养分数中可回收的最常见属（即 45/45）的数量最多是从无培养基保存的样本等分试样中回收的，并且在含有 5%（v/v）DMSO 的 Cary-Blair 培养基中回收的。此外，从培养分数生成的培养物集中，这两种保存条件分别产生了最高的物种丰富度（分别为 72 和 66）。
我们的结果表明，保存方法部分决定了从粪便样本中回收的肠道厌氧菌的丰富度和分类多样性。除了目前在干燥条件下冷冻保存粪便样本的标准做法外，补充其他保存条件，如含有 DMSO 的 Cary-Blair 培养基，可增加与肠道相关的培养物集的物种多样性。
