Changes in Microbiota Profiles After Prolonged Frozen Storage of Stool Suspensions

Abstract

Introduction: Fecal microbiota transplantation (FMT) is recommended as safe and effective treatment for recurrent Clostridioides difficile infections. Freezing the FMT preparation simplifies the process, allowing a single stool sample to be used for multiple receivers and over an extended period of time. We aimed to assess the effect of long-term frozen storage on bacterial taxonomic profiles of a stool suspension prepared for FMT. Methods: DNA was extracted from a stool suspension before freezing and sequentially during the 18-month storage period at −80 • C. Two different protocols were used for DNA extraction. The first relied on a classical mechanical and chemical cell disruption to extract both intra-and extracellular DNA; the second included specific pre-treatments aimed at removing free DNA and DNA from human and damaged bacterial cells. Taxonomic profiling of bacterial communities was performed by sequencing of V3-V4 16S rRNA gene amplicons. Results: Microbiota profiles obtained by whole DNA extraction procedure remained relatively stable during frozen storage. When DNA extraction procedure included specific pre-treatments, microbiota similarity between fresh and frozen samples progressively decreased with longer frozen storage times; notably, the abundance of Bacteroidetes decreased in a storage duration-dependent manner. The abundance of Firmicutes, the main butyrate producers in the colon, were not much affected by frozen storage for up to 1 year. Conclusion: Our data show that metataxonomic analysis of frozen stool suspensions subjected to specific pre-treatments prior to DNA extractions might provide an interesting indication of bacterial resistance to stress conditions and thus of chances of survival in FMT recipients.

Complementarity of Selective Culture and qPCR for Colistin Resistance Screening in Fresh and Frozen Pig Cecum Samples

“…In this study, we compared both molecular and culture-based methods, focusing on those samples showing growth of ACRB carrying mcr-1 , the main colistin-resistance determinant that has been quantified by SYBR ® Green qPCR. The amount of mcr-1 gene detected was similar between frozen and fresh samples, suggesting that the freeze-thaw process did not significantly modify the availability of functional DNA for the qPCR, in line with other studies that compared PCR results before and during different freezing periods ( Bassis et al, 2017 ; Dorsaz et al, 2020 ). However, PCR may overestimate the content in viable cells showing colistin resistance of cecal samples, since dead cells could also be detected ( Dorsaz et al, 2020 ).…”

“…Therefore, the use of traditional methods, such as solid or liquid culture, could be challenging and may not provide representative data on the amount and diversity of bacteria in these samples prior to being frozen. On the other hand, DNA remains more stable after the freeze-thaw process as shown in different microbiome studies using Next Generation Sequencing (Bassis et al, 2017;Dorsaz et al, 2020). Similarly, the bacterial composition of stools was not dramatically affected after a freezing process using molecular methods, although this has been also associated with freezing conditions (Shao et al, 2012).…”

“…In addition, the freeze-thaw process could be an important issue for bacterial recovery since it could dramatically affect bacterial survival because of the physical changes in the frozen material, assuming that at least a 74% decrease in cell viability may occur (Phalakornkule et al, 2017). This is of paramount importance when the aim is testing bacterial diversity of frozen samples, as previous studies have demonstrated that the freeze-thaw process can decrease bacterial diversity in the samples (Phalakornkule et al, 2017;Dorsaz et al, 2020). Therefore, the use of traditional methods, such as solid or liquid culture, could be challenging and may not provide representative data on the amount and diversity of bacteria in these samples prior to being frozen.…”

Impact of Terrestrial Input on Deep-Sea Benthic Archaeal Community Structure in South China Sea Sediments

“…An estimate of the absolute abundance of 16S rRNA gene copies of SCG, MG-I, and Thermoprofundales per gram of sediment was calculated by multiplying the absolute archaeal 16S rRNA gene abundance (obtained by qPCR) by their respective relative abundance from 16S rRNA gene sequencing ( Lou et al, 2018 ; Elovitz et al, 2019 ; Dorsaz et al, 2020 ).…”

Potential Applications of Human Viral Metagenomics and Reference Materials: Considerations for Current and Future Viruses

“…This may be the case for stool samples, where the majority of the viruses are temperate bacteriophages and have been shown to be resistant to profile changes after long-term preservation at −80°C ( 21 ). This may also be explained by the stability in bacterial community composition after long-term storage at −80°C ( 22 ).…”