Other Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses (Jul 2024, n=3) "changes induced in fecal microbiota with different storage conditions are marginal compared to the variations between horses"

[Michael Harrop]

https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-024-04166-w

Abstract​

Background​

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.

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The second most notable finding of this study was the difference in community composition observed when using the two separate extraction methods (DNA and cDNA).

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Standard metagenomic analysis of DNA, although providing a comprehensive view of the bacteria present within a sample, does not directly assess activity or potential for metabolic activity of the members of the microbiome, because it cannot differentiate genes from live cells, live but inactive cells, or dead cells [33, 43, 44]. It is for these reasons that cDNA-based sequencing was chosen to assess the potential for metabolic activity in equine fecal transplant preparations, since activity of transplanted bacteria is important for clinical effect in the patient.

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In our study, we investigated the effects of storage conditions including temperature at which samples are stored (-20 °C and − 80 °C) to represent shallow or deep-freezing techniques, buffers such as saline or saline plus glycerol, and length of frozen storage.

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Clearly, saline was not an ideal buffer to preserve metabolically active populations, as changes were noted by buffer in saline preserved samples at both storage temperatures, However, saline plus glycerol stored samples were relatively stable across both temperatures

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FYI, adding saline makes freezing damage worse. It would be interesting to see these studies test pure stool without anything added. Or maybe they did?

Interestingly, no obvious differences were noted from fresh samples at either storage temperature (-20 °C–− 80 °C).

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I'm not sure what they mean by that. I think they did though:

For fresh (Day 0) sample a 400 µl volume of slurry was used for DNA extraction at this time (described below), and the remainder was stored in a -80 °C freezer until DNA extraction on day 90.

While the RNA from fresh samples of each horse was processed immediately to prevent RNA degradation, DNA extraction from fresh samples awaited until all samples were processed and archived at the above-described storage conditions. The stored samples were retrieved from the respective temperatures after the specified stored period had been completed and left to thaw on the benchtop at room temperature. As the volume was small, samples were thawed in approximately 15 min, after which the samples were extracted for DNA and RNA immediately.

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