Prolonged Storage Reduces Viability of and Core Intestinal Bacteria in Fecal Microbiota Transplantation Preparations for Dogs
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Introduction: Fecal microbiota transplantation (FMT) has been described useful as an adjunct treatment for chronic enteropathy in dogs. Different protocols can be used to prepare and store FMT preparations, however, the effect of these methods on microbial viability is unknown. We aimed (1) to assess the viability of several core intestinal bacterial species by qPCR and (2) to assess () viability through culture to further characterize bacterial viability in different protocols for FMT preparations.
Methods: Bacterial abundances were assessed in feces from six healthy dogs by qPCR after propidium monoazide (PMA-qPCR) treatment for selective quantitation of viable bacteria. Conservation methods tested included lyophilization (stored at 4°C and at -20°C) and freezing with glycerol-saline solution (12.5%) and without any cryoprotectant (stored at -20°C). Additionally, the abundance of was quantified using bacterial culture.
Results: Using PMA-qPCR, the viability of , , , , , and was reduced in lyophilized fecal samples kept at 4°C and -20°C up to 6 months ( < 0.05). In frozen feces without cryoprotectant, only and were not significantly reduced for up to 3 months ( > 0.05). Lastly, no differences were observed in the viability of those species in glycerol-preserved samples up to 6 months (p > 0.05). When using culture to evaluate the viability of , we observed that abundance was lower in lyophilized samples kept at 4°C than -20°C; and abundance was higher in glycerol-preserved samples for up to 6 months than in samples preserved without glycerol for up to 3 months. Moreover, the highest abundance of was observed in glycerol-preserved feces. After 3 months, was undetectable by culture in 83% (5/6) of the frozen samples without glycerol.
Discussion: While the lyophilization procedure initially reduced abundance, viability was stable thereafter for up to 6 months at -20°C. The higher bacterial viability detected in fecal samples preserved with glycerol confirms the use of this cryoprotectant as a reliable method to keep bacteria alive in the presence of fecal matrix for FMT purposes.