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On the robustness of inference of association with the gut microbiota in stool, rectal swab and mucosal tissue samples.

ABSTRACT: The gut microbiota plays an important role in human health and disease. Stool, rectal swab and rectal mucosal tissue samples have been used in individual studies to survey the microbial community but the consequences of using these different sample types are not completely understood. In this study, we report differences in stool, rectal swab and rectal mucosal tissue microbial communities with shotgun metagenome sequencing of 1397 stool, swab and mucosal tissue samples from 240 participants. The taxonomic composition of stool and swab samples was distinct, but less different to each other than mucosal tissue samples. Functional profile differences between stool and swab samples are smaller, but mucosal tissue samples remained distinct from the other two types. When the taxonomic and functional profiles were used for inference in association with host phenotypes of age, sex, body mass index (BMI), antibiotics or non-steroidal anti-inflammatory drugs (NSAIDs) use, hypothesis testing using either stool or rectal swab gave broadly significantly correlated results, but inference performed on mucosal tissue samples gave results that were generally less consistent with either stool or swab. Our study represents an important resource for determination of how inference can change for taxa and pathways depending on the choice of where to sample within the human gut.

SUBMITTER: Sun S

PROVIDER: S-EPMC8295290 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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On the robustness of inference of association with the gut microbiota in stool, rectal swab and mucosal tissue samples.

Sun Shan S Zhu Xiangzhu X Huang Xiang X Murff Harvey J HJ Ness Reid M RM Seidner Douglas L DL Sorgen Alicia A AA Blakley Ivory C IC Yu Chang C Dai Qi Q Azcarate-Peril M Andrea MA Shrubsole Martha J MJ Fodor Anthony A AA

Scientific reports 20210721 1

The gut microbiota plays an important role in human health and disease. Stool, rectal swab and rectal mucosal tissue samples have been used in individual studies to survey the microbial community but the consequences of using these different sample types are not completely understood. In this study, we report differences in stool, rectal swab and rectal mucosal tissue microbial communities with shotgun metagenome sequencing of 1397 stool, swab and mucosal tissue samples from 240 participants. Th ...[more]

PMID: 34290321

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Project description:BackgroundSample collection for gut microbiota analysis from in-patients can be challenging. Collection method and storage conditions are potential sources of variability. In this study, we compared the bacterial microbiota from stool stored under different conditions, as well as stool and swab samples, to assess differences due to sample storage conditions and collection method.MethodsUsing bacterial 16S rRNA gene sequence analysis, we compared the microbiota profiles of stool samples stored and collected under various conditions. Stool samples (2 liquid, 1 formed) from three different patients at two hospitals were each evaluated under the following conditions: immediately frozen at -80°C, stored at 4°C for 12-48 hours before freezing at -80°C and stored at -20°C with 1-2 thaw cycles before storage at -80°C. Additionally, 8 stool and 30 rectal swab samples were collected from 8 in-patients at one hospital. Microbiota differences were assessed using the Yue and Clayton dissimilarity index (θYC distance) and analysis of molecular variance (AMOVA).ResultsRegardless of the storage conditions, the bacterial communities of aliquots from the same stool samples were very similar based on θYC distances (median intra-sample θYC distance: 0.035, IQR: 0.015-0.061) compared to aliquots from different stool samples (median inter-sample θYC distance: 0.93, IQR: 0.85-0.97) (Wilcoxon test p-value: <0.0001). For the stool and rectal swab comparison, samples from different patients, regardless of sample collection method, were significantly different (AMOVA p-values: <0.001-0.029) compared to no significant difference between all stool and swab samples (AMOVA p-value: 0.976). The θYC dissimilarity index between swab and stool samples was significantly lower within individuals (median 0.17, IQR: 0.10-0.27) than between individuals (median 0.93, IQR: 0.85-0.97) (Wilcoxon test p-value: <0.0001), indicating minimal differences between stool and swab samples collected from the same individual over the sampling period.ConclusionFor gastrointestinal microbiota studies based on bacterial 16S rRNA gene sequence analysis, interim stool sample storage at 4 °C or -20 °C, rather than immediate storage at -80 °C, does not significantly alter results. Additionally, stool and rectal swab microbiotas from the same subject were highly similar, indicating that these sampling methods could be used interchangeably to assess the community structure of the distal GI tract.

Project description:BackgroundStudies of the gut microbiome are becoming increasingly important. Such studies require stool collections that can be processed or frozen in a timely manner so as not to alter the microbial content. Due to the logistical difficulties of home-based stool collection, there has been a challenge in selecting the appropriate sample collection technique and comparing results from different microbiome studies. Thus, we compared stool collection and two alternative clinic-based fecal microbiome collection techniques, including a newer glove-based collection method.ResultsWe prospectively enrolled 22 adult men from our prostate cancer screening cohort SABOR (San Antonio Biomarkers of Risk for prostate cancer) in San Antonio, TX, from 8/2018 to 4/2019. A rectal swab and glove tip sample were collected from each participant during a one-time visit to our clinics. A single stool sample was collected at the participant's home. DNA was isolated from the fecal material and 16 s rRNA sequencing of the V1-V2 and V3-V4 regions was performed. We found the gut microbiome to be similar in richness and evenness, noting no differences in alpha diversity among the collection methods. The stool collection method, which remains the gold-standard method for the gut microbiome, proved to have different community composition compared to swab and glove tip techniques (p< 0.001) as measured by Bray-Curtis and unifrac distances. There were no significant differences in between the swab and glove tip samples with regard to beta diversity (p> 0.05). Despite differences between home-based stool and office-based fecal collection methods, we noted that the distance metrics for the three methods cluster by participant indicating within-person similarities. Additionally, no taxa differed among the methods in a Linear Discriminant Analysis Effect Size (LEfSe) analysis comparing all-against-all sampling methods.ConclusionThe glove tip method provides similar gut microbiome results as rectal swab and stool microbiome collection techniques. The addition of a new office-based collection technique could help easy and practical implementation of gut microbiome research studies and clinical practice.

Dataset Information

On the robustness of inference of association with the gut microbiota in stool, rectal swab and mucosal tissue samples.

Publications

On the robustness of inference of association with the gut microbiota in stool, rectal swab and mucosal tissue samples.

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