Changes in Microbiota Profiles After Prolonged Frozen Storage of Stool Suspensions. Changes in fecal microbiota profiles during frozen storage

Introduction. Faecal microbiota transplantation is recommended as a more efficacious alternative to standard vancomycin treatment for recurrent C. difficile infections. Freezing a donor’s stool samples simplifies the process, allowing a single stool sample to be used across multiple receivers and over an extended period of time. The aim was to assess the effect of long-term frozen storage on bacterial taxonomic profiles of frozen stool sample suspension prepared according to the standardized protocol.Methods. DNA was extracted from a stool sample suspension before freezing and sequentially during storage at –80°C for 1 week, 1 month, and then every three months from month 3 to month 18. Two different protocols were used for DNA extraction. The first relied on a combined mechanical and chemical cell disruption to extract both intra- and extracellular DNA; the second included specific pre-treatments aimed at removing DNA from human and damaged bacterial cells.Results. Taxonomic profiling of bacterial communities was performed by sequencing of V3–V4 16S rRNA gene amplicons. Microbiota similarity between fresh and frozen samples progressively decreased with longer frozen storage times when DNA extraction procedure included pre-treatments; notably, Bacteroidetes and Spirochaetes, tended to decrease in the relative abundance in a storage-duration dependent manner. In contrast, microbiota profiles obtained by whole DNA extraction procedure remained relatively stable for different frozen storage periods.Conclusion. Our data show that metataxonomic analysis of frozen stool suspensions subjected to specific pre-treatments prior to DNA extractions may provide an indirect measure of bacterial viability and/or susceptibility to survive stress conditions.