Effect of cryopreservation medium conditions on growth and isolation of gut anaerobes from human faecal samples.

BACKGROUND: 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. RESULTS: 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 10-4) 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 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). CONCLUSION: 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.

Fast quantification of gut bacterial species in cocultures using flow cytometry and supervised classification.

A bottleneck for microbial community experiments with many samples and/or replicates is the fast quantification of individual taxon abundances, which is commonly achieved through sequencing marker genes such as the 16S rRNA gene. Here, we propose a new approach for high-throughput and high-quality enumeration of human gut bacteria in a defined community, combining flow cytometry and supervised classification to identify and quantify species mixed in silico and in defined communities in vitro. We identified species in a 5-species in silico community with an F1 score of 71%. In addition, we demonstrate in vitro that our method performs equally well or better than 16S rRNA gene sequencing in two-species cocultures and agrees with 16S rRNA gene sequencing data on the most abundant species in a four-species community. We found that shape and size differences alone are insufficient to distinguish species, and that it is thus necessary to exploit the multivariate nature of flow cytometry data. Finally, we observed that variability of flow cytometry data across replicates differs between gut bacterial species. In conclusion, the performance of supervised classification of gut species in flow cytometry data is species-dependent, but is for some combinations accurate enough to serve as a faster alternative to 16S rRNA gene sequencing.

Design of synthetic microbial consortia for gut microbiota modulation.

The use of and interest in probiotics to modulate the human intestinal microbiota have strongly increased in recent years. However, most of the current probiotic products have been limited to single-strain formulations of easily culturable food-grade microorganisms and often resulted in mixed results or limited effects on host health. Therefore, a revision of current probiotic strategies by using synthetic human-derived microbial multispecies consortia is necessary. In light of this ongoing evolution of the field, novel approaches are needed to design and assemble bacterial cocktails targeted to restore dysbiotic states in microbiota-associated diseases. This review discusses the steps in the process for identifying effective targets, predicting putative multistrain communities, assembling ecosystems in silico and in vitro and monitoring stability and outputs before in vivo trials.