Changes in bacterial viability after preparation and storage of fecal microbiota transplantation solution using equine feces

Abstract

Background: Fecal microbiota transplantation (FMT) has been used as a treatment option for horses (Equus caballus) with gastrointestinal diseases. Several preparation and conservation protocols to improve bacterial survival have been studied in other species. Methods: This study aimed to evaluate the impact of oxygen exposure and different protectant solutions on bacterial viability before and after freezing using horse feces. Fecal samples from 10 healthy horses were aliquoted and diluted in cryoprotectant solutions containing antioxidants (n = 40) or 10% glycerol (n = 40). Half of the aliquots from each dilution condition were prepared inside an anaerobic chamber, while the other half were prepared under ambient air conditions. Each sample was also analyzed fresh and after freezing at −20 ̊C for 90 days. Bacterial viability was assessed using flow cytometry. A mixed linear model and the Friedman and Wilcoxon tests were used depending on data distribution. Results: Freeze-thawing decreased bacterial viability by 47% (mean ± SD: 51 ± 27% before, 27 ± 8% after; p < 0.001). Glycerol was superior to the cryoprotectant after freezing (32 ± 8% glycerol, 24 ± 8% cryoprotectant; p < 0.001). Oxygen exposure did not affect viability (p = 0.13). There was no statistical difference between protectant solutions in fresh samples (p = 0.16). Conclusions: Fresh FMT solutions may be better for treating horses with dysbiosis, but if freezing cannot be avoided, glycerol should be used to dilute feces.