Objectives
Stool storage is key to researching the association between gastrointestinal dysbiosis and disease states. Fecal microbiota transplantation using frozen filtrate is used for patients with recurrent Clostridioides difficile infection. This study determined the impact of storing stool and frozen filtrate on microbiome composition.
Methods
Fresh stool was obtained from a high-diversity (HD) and low-diversity (LD) donor. Aliquots were stored at room temperature (RT), 5°C, and at −20°C for 24 and 48 hours, or processed immediately. Fresh stool was also homogenized with both 0.9N-sterile-saline and 0.9N-sterile-saline containing 10%-glycerol. Resulting filtrate aliquots were frozen at −20°C and at −80°C. At baseline and after 7, 9, 12, 18, and 24M storage, gDNA was isolated using the MO BIO PowerSoil® DNA Isolation Kit. 16s rRNA gene amplicon sequencing targeting the V4 hypervariable region was performed on the Illumina MiSeq. Low abundant OTUs were excluded.
Results
Differences in microbiota profiles were observed in both HD and LD stool when stored immediately at −20°C, more so with the LD stool. Differences were also noted for LD stool stored at room temperature and less so at 5°C. Storage at RT or 5°C had no impact for the HD stool. Long-term filtrate storage of the HD stool filtrate at −80°C with 10%-glycerol best preserved the bacterial microbiota profile followed by −20°C with 10%-glycerol then −80°C and −20°C without 10%-glycerol. Differences were also observed in the LD stool filtrate when stored in any conditions, with the least impact being −80°C with glycerol.
Conclusions
To preserve microbiota profiles, storing stool at 5°C until received in the laboratory is optimal. For researchers assessing microbiota correlations with disease states, immediate gDNA extraction from stool upon receipt is ideal. For those using frozen filtrate from healthy donors for fecal transplants, long-term storage is best at −80°C with 10%-glycerol.
Figure P01-1:
Impact of storing stool and frozen filtrate on microbiome composition