Fresh Versus Frozen Stool for Fecal Microbiota Transplantation—Assessment by Multimethod Approach Combining Culturing, Flow Cytometry, and Next-Generation Sequencing

Abstract

The objective of this work was to compare the quality of FMT preparations made from fresh feces with those made from feces frozen at –30°C without any pre-processing or cryopreservation additives. The research hypothesis was that such preservation protocol (frozen whole stool, then thawed and processed) is equipotent to classical fresh FMT preparation. For that, three complementary methods were applied, including: (i) culturing in aerobic and anaerobic conditions, (ii) measuring viability by flow cytometry, and (iii) next-generation sequencing. Flow cytometry with cell staining showed that the applied freezing protocol causes significant changes in all of the observed bacterial fractions. Alive cell counts dropped four times, from around 70% to 15%, while the other two fractions, dead and unknown cell counts quadrupled and doubled, with the unknown fraction becoming the dominant one, with an average contribution of 57.47% per sample. It will be very interesting to uncover what this unknown fraction is (e.g., bacterial spores), as this may change our conclusions (if these are spores, the viability could be even higher after freezing). Freezing had a huge impact on the structure of cultivable bacterial communities. The biggest drop after freezing in the number of cultivable species was observed for Actinobacteria and Bacilli. In most cases, selected biodiversity indices were slightly lower for frozen samples. PCoA visualization built using weighted UniFrac index showed no donor-wise clusters, but a clear split between fresh and frozen samples. This split can be in part attributed to the changes in the relative abundance of Bacteroidales and Clostridiales orders. Our results clearly show that whole stool freezing without any cryoprotectants has a great impact on the cultivability and biodiversity of the bacterial community, and possibly also on the viability of bacterial cells.

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of In Vitro Cultivation and Membrane Integrity Methods

“…We used stool from germ-free (GF) mice, devoid of bacteria, to establish a noise threshold and compared this with stool from conventionally bred (CONV) mice, containing both bacterial and non-bacterial elements (Figure 4). This approach allowed us to define four viability quadrants based on SYTO9 and PI fluorescence, categorizing cells as dead, dying or damaged, viable, or of unknown status (20,21). We observed significantly fewer fluorescent events in GF mouse stool, attributing this to non-bacterial autofluorescence or staining artifacts.…”

“…Fluorescent staining for membrane integrity, typically performed with flow cytometry, is a commonly adopted culture-independent method (35)(36)(37). Nevertheless, its effectiveness can vary across different bacterial taxa, and its application to the heterogeneous matrix of stool presents additional challenges (20,21,29,32) Given these complexities, we compared the two prevalent methods-cultivation and membrane integrity assessment-for their accuracy and clinical applicability. Our adaptation of the LIVE/DEAD™ BacLight™ Bacterial Viability Kit for stool analysis demonstrated the potential to discriminate between microbial DNA signals and background noise effectively.…”

“…Fluorescent staining for membrane integrity, typically performed with flow cytometry, is a commonly adopted culture-independent method (35–37). Nevertheless, its effectiveness can vary across different bacterial taxa, and its application to the heterogeneous matrix of stool presents additional challenges (20,21,29,32)…”

The impact of cefuroxime prophylaxis on human intestinal microbiota in surgical oncological patients

“…Several issues of the current study could be better addressed in future investigations. A recent report highlights alterations in stool microbiome upon sample freezing without the addition of cryoprotectants (Bilinski et al, 2022). In our analysis, we only report data from both fresh and frozen stool samples, and do not account for potential changes caused by cryopreservation.…”

The practice of fecal microbiota transplantation in inflammatory bowel disease

“…On the other hand, freezing of feces is known to impact microbiome composition. It leads to a 4-fold reduction in living bacterial cells as detected by flow cytometry (70% to 15%), similar alpha diversity, and definite differences in beta diversity on principal coordinates analysis from fresh fecal samples [ 53 ]. Prolonged frozen storage leads to an increase in Firmicutes to Bacteroidetes ratio, as some Gram-negative species are depleted during storage and some hypothesize that trials in which frozen FMT was found to be effective also depend on this phenomena of selective depletion of administered Bacteroidetes .…”