The effect of storage at ambient temperature on the feline fecal microbiota

BACKGROUND: Feline fecal microbiota analyses can potentially be impacted by a variety of factors such as sample preparation, sequencing method and bioinformatics analyses. Another potential influence is changes in the microbiota from storage of samples prior to processing. This study examined the effect of ambient temperature exposure on the feline fecal microbiota composition. Fecal samples were collected from 12 healthy cats, within 15 min after defecation. Samples were aliquoted and the first aliquot was frozen at −80 °C within 1 hour of defecation. Remaining aliquots were maintained at ambient temperature (20 to 23 °C) and frozen at −80 °C at 6, 12, 24, 36, 48, 72 and 96 h after collection. DNA was extracted from all aliquots, and polymerase chain reaction (PCR). The PCR products were sequenced with next-generation sequencing (Illumina MiSeq). RESULTS: No significant differences were observed in alpha and beta biodiversity indexes, as well as relative abundance of different taxa over time (P > 0.05 for all tests between time points). Principal coordinate analyses demonstrated that samples cluster mainly by cat, with no significant differences between time points (AMOVA, P > 0.05; HOMOVA, P > 0.05). Linear discriminant analysis effect size method was performed and failed to detect any enriched taxa, between time points. Random forest algorithm analysis indicated homogeneity across time points. CONCLUSIONS: Although existing evidence from human fecal storage studies is contradictory, a recent study in companion animals agreed with the current study, demonstrating that maintenance of feline fecal samples at ambient temperature for up to 4 days has no effect on the bacterial membership and structure.