Publication
Effect of cryopreservation medium conditions on growth and isolation of gut anaerobes from human faecal samples
Publisher:
Springer Science and Business Media LLC
Date:
30-05-2022
DOI:
10.1186/S40168-022-01267-2
Abstract: Novel strategies for anaerobic bacterial isolations from human faecal s les 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 s le preservation is essential to safeguard the viability of the broadest taxonomic ersity range possible. In contrast to the much more established faecal storage conditions for meta-omics applications, the impact of stool s le preservation conditions on bacterial growth recovery and isolation remains largely unexplored. In this study, aliquoted faecal s les 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 s le dilution and richness of the uncultured source s les 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 ( .01% abundance, dilution 10 −4 ) 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 s le 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 ersity of gut anaerobes recovered from faecal s les. Complementing the current standard practice of cryopreserving stool s les in dry conditions with other preservation conditions, such as Cary-Blair medium with DMSO, could increase the species ersity of gut-associated culture collections.