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BMC veterinary research2024; 20(1); 306; doi: 10.1186/s12917-024-04166-w

Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses.

Abstract: Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region. Results: The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities. Conclusions: Frozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.
© 2024. The Author(s).
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Publication Date: 2024-07-10 PubMed ID: 38987780PubMed Central: PMC11234551DOI: 10.1186/s12917-024-04166-wGoogle Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research investigates the effect of various storage methods and buffers on the bacterial viability and diversity in equine fecal microbiota transplant (FMT) product. It finds that frozen storage can preserve potentially metabolically active bacteria in equine feces, with saline plus glycerol proving more effective than saline alone.

Objective and Methodology

  • The study was undertaken to understand the impact of different storage times, buffers and temperatures on the bacterial metabolic activity and viability in equine FMT product. The need for this research arises from the lack of standardization in equine FMT procedures, currently leading to highly variable results.
  • The study used fresh feces collected from three healthy adult horses and subjected it to different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days).
  • The researchers used two types of extraction methods to assess the bacterial populations: DNA extraction to assess total bacterial populations and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria.
  • Bacterial viability and diversity were determined by 16s rRNA gene amplicon sequencing (a common molecular method for studying microbial communities).

Observations and Findings

  • Significant differences in population indices and taxonomic composition were observed when comparing results from DNA and cDNA extraction methods.
  • DNA-based analysis showed a significant decrease in alpha diversity (observed species, Shannon diversity) in frozen samples, indicating the impact of freezing on total bacterial populations.
  • Conversely, cDNA-based sequencing, representing potentially metabolically active populations, showed less difference in alpha diversity indicating that frozen storage had less of an effect on the viability of the bacterial community.
  • The type of buffer used had a significant effect on potentially metabolically active populations, with saline plus glycerol proving more effective than saline alone.
  • Individual differences between horses had the most significant effect within both DNA and cDNA bacterial communities, indicating the influence of the individual horse’s microbiome on the effectiveness of FMT.

Conclusions and Implications

  • Frozen storage can preserve potentially metabolically active bacteria of the equine fecal microbiome.
  • This has significant implications for the use of FMT in horses with disturbances in their intestinal microbiome, as it could potentially allow for easier clinical usage of fecal transplants.
  • However, the results of the study are preliminary and require further research and validation through larger studies to confirm these findings apply to other individual horses.

Cite This Article

APA
Long AE, Pitta D, Hennessy M, Indugu N, Vecchiarelli B, Luethy D, Aceto H, Hurcombe S. (2024). Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses. BMC Vet Res, 20(1), 306. https://doi.org/10.1186/s12917-024-04166-w

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 20
Issue: 1
Pages: 306

Researcher Affiliations

Long, Alicia E
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA. lalicia@vet.upenn.edu.
Pitta, Dipti
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Hennessy, Meagan
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Indugu, Nagaraju
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Vecchiarelli, Bonnie
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Luethy, Daniela
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Aceto, Helen
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Hurcombe, Samuel
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
  • Veterinary Innovative Partners, New York, NY, USA.

MeSH Terms

  • Animals
  • Horses / microbiology
  • Feces / microbiology
  • Bacteria / classification
  • Bacteria / genetics
  • Bacteria / isolation & purification
  • RNA, Ribosomal, 16S / genetics
  • Fecal Microbiota Transplantation / veterinary
  • Freezing
  • Microbial Viability
  • Cryopreservation / veterinary
  • DNA, Bacterial / genetics

Grant Funding

  • 580-5805-1-461612-xxxx-2000-5872 / Raymond Firestone Trust
  • 580-5805-1-461612-xxxx-2000-5872 / Raymond Firestone Trust

Conflict of Interest Statement

The authors declare no competing interests.

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