Interpersonal Variations in Gut Microbiota Profiles Supersedes the Effects of Differing Fecal Storage Conditions

Abstract

Due to ease of acquisition, fecal samples are often used in studies investigating gut microbiota. Improper handling of these samples can lead to bacterial growth and alter bacterial composition. While freezing samples at −80 °C is considered gold standard, this is not suitable for studies utilizing self-sampling by lay participants or field studies. Thus to effectively prevent bacterial growth, techniques that allow efficient fecal storage outside laboratory facilities are needed. Fecal samples were collected from three donors. From each donor feces, 45 samples were collected and stored either freshly frozen at −80 or −20 °C, or in three separate storage buffers at room temperature or 4 °C for 24 or 72 hours. Bacterial composition was analyzed using Illumina amplicon sequencing of the V4 region of the 16 S rRNA gene. While storage conditions did affect bacterial composition and diversity compared to storage at −80 °C, the variation between donors superseded the variations introduced by storage. Samples stored at −20 °C most closely resembled those stored at −80 °C. When investigating variations in bacterial composition between separate study populations, fecal samples can efficiently be stored in −20 °C freezers or in one of the presented storage buffers, without severe alterations in bacterial composition.

Gut microbiota profiles of autism spectrum disorder and attention deficit/hyperactivity disorder: A systematic literature review.

“…While gut microbiota is robust, differences in storage can lead to growth or disruption of susceptible bacteria, and thus result in differences in bacterial composition between studies. 92,94,95 Extraction of DNA from gram-positive bacteria is problematic, due to the presence of a thick cell wall, that can prevent effective bacterial lysis during DNA extraction. 93,96 This can lead to underrepresentation of gram-positive bacteria in studies investigating gut microbiota.…”

Variations of gut microbiome profile under different storage conditions and preservation periods: A multi-dimensional evaluation

“…Although the efficacy of some commercial stabilizers on maintaining the original status of gut microbiome has been compared, the uncertainty still exists because of the inconsistent observations among some reports (24, 26). Although previous studies suggested that inter-individual variation from donors superseded that introduced by storage conditions (25, 40, 41), the impacts of different storage conditions on microbiome profile need systemic evaluation. Therefore, in our current study, a multi-dimensional comparison among different storage conditions was performed by using a mixture of homogenized feces from rats to minimize the probable artifacts during sampling process.…”

“…Previous studies usually evaluated the impacts of short-term or long-term storage on gut microbial profile using samples stored ranging from 24h to 8 weeks (18, 40, 45). In our current study, the impacts of storage periods under different conditions were further evaluated in a multi-dimensional way after 1 or 2 weeks, and 6 months respectively.…”

The urinary microbiota composition remains stable over time and under various storage conditions

“…Forward reads were quality trimmed using Trimmomatic v 0.32[40] to produce reads with a Phred score of at least 20 and a length of 250 bp. Subsequent bioinformatics followed the UPARSE workflow[41] to remove chimeras, cluster OTUs based on 97 % identity and assign taxonomy using the RDP classifier as previously described[26].…”

“…Despite the growing interest for urinary microbiota research, it appears that the methodologies and study designs, used in different studies, are highly heterogeneous, which makes it difficult to interpret and compare observed findings. Several protocol optimization studies have been conducted on fecal samples, providing valuable guidelines on how to store and process samples for gut microbiota studies[26–31]. Importantly, the chemical content and structure of urine is very different from feces, leaving urinary microbiota research as a bare and unexplored field regarding protocol recommendations.…”