Evaluation of fecal matter preservation methods and their effect on composition of microbiota assessed through DNA sequencing and analysis

Abstract

Fecal matter transplants (FMT) are an effective, yet underutilized, treatment for potentially life-threatening Clostridium difficile infections. Following antibiotic treatment, an imbalance between the types of colonic microbiota naturally present in a person's gut may occur, allowing the opportunistic pathogenic bacterium C. difficile to proliferate and reach virulent levels. Despite a 90% success rate, and patient reports of immediate improvement, FMTs are approved only as a last resort due to strict US Food and Drug Administration (FDA) restrictions. This study aimed to compare microbial preservation methods to determine the method with the least detrimental effects on the composition of stool microbes. Fecal matter samples, from dogs, were homogenized with either sterile deionized water or 0.85% NaCl. The homogenized mixtures were then partitioned for immediate DNA extraction or for preservation with or without 25% glycerol prior to -80°C storage. After 3 weeks, and again after 10 months, DNA extraction was performed on stored samples. All extracted DNA was subjected to PCR amplification and sequenced. After pairing and filtering, 88.3% of data were retained. Changes in taxa richness over time for each treatment were not significant; changes in taxonomic composition over time were detected in water only and saline only treatments (p = 0.016, p = 0.049 respectively). The water-glycerol treatment resulted in the least amount of change in taxonomic composition and proportions when compared to the sample prior to preservation. Information gained from this study could be used to further improve FMTs and help fuel FMT related research in hopes of attenuating FDA restrictions.