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Cryopreservation of stool samples altered the microbial viability quantitively and compositionally
Aolei Chen1, Yingxin Hu1, Yajie Zhang1
1State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Abstract:
Stool is the most commonly used sample for gut microbiota analysis in humans and animals. Cryopreservation of stool at - 80 °C is a feasible and simple method in clinics and researches, especially in large-scale cohort studies. However, the viability of bacteria in stool after freezing has yet well-demonstrated quantitatively and compositionally. This study determined the viable microbiota of samples under cryopreservation at - 80 °C, relative to fresh samples and that stored at ambient. Stool samples were collected from three healthy adults. Propidium monoazide treatment combined with quantitative PCR and 16S rRNA gene sequencing was performed to target viable microbiota. After freezing, the number of viable bacteria decreased, though inter-individual difference existed. Notably, the alpha diversity of viable microbiota after freezing did not change significantly, while its composition changed. Freezing significantly reduced the viable bacteria in Gram-negative genera of Bacteroidetes and Firmicutes, and proportionally increased Gram-positive bacteria in genera of Actinobacteria and Firmicutes, including Bifidobacterium, Collinsella and Blautia, implying that the cell envelope structure associated with the bacterial sensitivity to freezing. On the contrary, the room temperature storage not only decreased the number of viable bacteria, but also decreased the microbial alpha diversity, and remarkably enriched facultative anaerobes of Escherichia-Shigella, Enterococcus and Lactococcus, some of which are opportunistic pathogens. Our findings suggested that changes in viable microbiota in stool samples caused by cryopreservation should be paid enough attention for downstream utilization.
Insights
Cryopreservation of stool at -80°C reduces viable bacteria, altering gut microbiota composition. Room temperature storage is worse, decreasing diversity and increasing pathogens.
Area of Science:
- Microbiology
- Gut Microbiome Research
- Molecular Biology
Background:
- Stool analysis is crucial for understanding gut microbiota in humans and animals.
- Cryopreservation at -80°C is a common method for storing stool samples for research.
- Quantitative and compositional effects of cryopreservation on viable gut bacteria remain unclear.
Purpose of the Study:
- To quantitatively and compositionally assess the viable gut microbiota in stool samples after cryopreservation at -80°C.
- To compare the effects of -80°C cryopreservation with ambient temperature storage on viable gut bacteria.
- To identify specific bacterial groups affected by freezing and ambient storage.
Main Methods:
- Stool samples from three healthy adults were analyzed.
- Propidium monoazide (PMA) treatment was used to distinguish viable from non-viable bacteria.
- Quantitative PCR (qPCR) and 16S rRNA gene sequencing were employed to analyze viable microbiota.
Main Results:
- Cryopreservation at -80°C led to a decrease in the overall number of viable bacteria, with inter-individual variations.
- Alpha diversity of viable microbiota remained largely unchanged after freezing, but composition was altered.
- Freezing reduced Gram-negative bacteria (Bacteroidetes, Firmicutes) while proportionally increasing Gram-positive bacteria (Actinobacteria, Firmicutes), including Bifidobacterium.
- Ambient temperature storage significantly decreased viable bacteria, reduced alpha diversity, and enriched opportunistic pathogens like Escherichia-Shigella.
Conclusions:
- Cryopreservation at -80°C alters the composition of viable gut microbiota, with Gram-positive bacteria showing increased relative abundance.
- Bacterial cell envelope structure may influence sensitivity to freezing.
- Ambient temperature storage poses a greater risk to sample integrity, reducing viable bacteria and diversity.
- Researchers must consider the impact of cryopreservation on viable microbiota for accurate downstream analysis.
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