Analysis of the fecal microbiome is now commonly complemented by an additional analysis of microbial metabolites such as SCFA. To ensure these data can be represented together without the impact of spatial and temporal variability of the fecal material, collection and storage methods for stool samples must be considered. Our results found sporadic detection of low abundance bacterial and fungal species in unhomogenized stool. Further, SCFA concentrations were also shown to vary considerably across a single stool.
The level of variability (CV) in microbial diversity and SCFA concentration across a single stool, was compared per participant to the variability across three separate bowel movements. It was expected that temporal shifts in community structure over three timepoints would be larger than replicate sampling from a single stool. While Shannon Diversity was more variable for 5 of 6 individuals among whole stool samples, Richness based on Chao1 was more variable along a single stool for 4 of 6 individuals. As well, SCFA concentrations were more variable within a single stool than across three separate bowel movements for two individuals. When the bacterial and SCFA data was integrated using rCCA, the intraindividual variability between the aliquots was also evident. Clustering of subsamples from individuals 2, 4, and 5 were very tight, indicating little difference in community structure due to sampling method. Although, samples from subjects 1, 3, and 6 were less tightly clustered, implying community structure changes along the surface of the stool in these individuals that are sensitive to sampling method. This demonstrates that for some individuals, heterogeneity of microorganisms and microbial metabolites in stool may be as great as that observed over the course of two days, which will become apparent if samples are collected by sub-sampling a small volume of stool. This is consistent with reports of heterogeneity in mucosal bacteriome 18, fecal microbiome 19,20, and metabolite concentrations 8. As fecal material moves through the colon, the exterior surface is exposed to the mucus layer secreted by epithelial cells. This mucus (which is a niche for commensal microbes) accumulates in fecal material, and has been proposed as a mechanism for the patchy recovery of microbial species along the surface of stool 13.
While the surface of the stool may have more variable richness and diversity, the beta diversity of bacterial communities between individuals was still significantly different, indicating that differences due to subsampling are less pronounced than differences between individuals. This is consistent with similar work, where beta diversity (weighted UniFrac) was compared across 9 stool subsampling locations with no significant differences observed 21. Fungal communities however did not seem to be structured according to the individual to the same extent as bacterial communities, but was structured in one of two ways: Dominated by Saccharomyces cerevisiae (≥99%), or by hosting a more even abundance of genera including S. cerevisiae, and either Kazachstania servazzii and Cyberlindnera jadinii, or Hanseniapora uvarum and Torulaspora delbrueckii. In another study targeting fungi in the gut using the ITS1 region, three main mycobiome types were found: either dominated by Candida albicans, or Saccharomyces cerevisiae, or multi species type 22. In the present study, Candida spp were not found at greater than 1% of the total community in any individual. However, Candida apicola was also not identified in the fungal mock community which could indicate an unknown technical bias against this group, although presumably not due to primer bias as low abundance was detected and this primer set has been used successfully for other Candida species 23.
The microbiome is often scrutinized for small community changes in association with host-related biological factors such as diet or disease. These changes in microbial signatures are often detected in less abundant taxa, or only within particular groups of bacteria and can vary among individuals. Most bacterial ASVs with large differential abundance were found to be enriched in whole stool compared to surface aliquots, and all but one Alphaproteobacteria were classified as Clostridia. The Internal regions of stool have previously been shown to harbor significantly higher abundances of Firmicutes and Bifiobacteria spp compared to the external surface 19. In this study, the external surface of stool was likely targeted by surface aliquot collections, rather than the internal regions of stool, and if the internal regions of stool harbor larger abundances of Firmicutes, this might explain some of the differences seen between the surface aliquots and the homogenized whole stool. On the other hand, half of all fungi with large differential abundance were found to be reduced in whole stool compared to the surface aliquots; and of these all but one Dothideomycetes were classified as Saccharomycetes, indicating Saccharomycetes may be a mucosal associated fungus in the gut.
The long-term view of the healthy human gut microbiome seems to show a dynamic community which retains prolonged stability, but is punctuated by periods of disturbance 24,25 On a shorter timescale, diet has been shown to cause fluctuations in microbial species 26, as well as SCFA concentrations 27, but what those shifts may look like across consecutive stools has not been previously explored. While only a small proportion of women defecate more than once a day, defecation frequency is known to be higher in men 28, and positively associated with vigorous physical activity, as well as plant based or high fiber diets 29. Therefore, the time of day that samples are collected may need to be indicated in sample collection protocols provided to participants. In this study, all women claimed to regularly defecate more than once a day, and the second stool of the day (collected on average 8 hours after) had significantly higher total SCFA concentrations. The second stool also tended to have lower bacterial richness and Shannon diversity index compared to the first stool, although these differences were not significant. Similarly, a recent study assessing the microbiome and SCFA concentrations at a single timepoint in 441 adults found that lower bacterial diversity was associated with higher SCFA concentrations 30. The association between bacteria and SCFA concentration seen in this study supports the idea that bacterial metabolites are linked to the circadian clock 31, and demonstrates why time of stool sample collection may be particularly important in individuals who defecate more than once per day.
Decreasing bacterial richness has also been found to correlate with decreasing stool firmness, or a higher Bristol Stool form value, based on fecal samples from 53 women 32. As well, the BSFS has also been shown to be a good predictor of whole-gut transit time, with high stool form values correlating to longer transit times 28,33,34. A more recent study also found when stool form had a Bristol Stool value of less than three it was correlated with greater transit times, indicating that stool form can help predict whole-gut or colonic transit times 35. While this study had a small sample size, it was interesting to note that both SCFA and bacterial phylogenetic diversity grouped according to stool form, and when these data were integrated through rCCA this trend was also observed. If microbial diversity and SCFA concentration are also linked to stool form and potentially transit time, assessing stool form at the point of sample processing could be a simple way to add valuable information to downstream multivariate analysis, and help explain sample clustering. Further, to reduce within-day variability that could potentially distort a long-term study, participants could be instructed to collect at a similar time, such as the first bowel movement of the day.
Directly freezing stool samples with no additional solution is considered the gold standard method for storing stool, while Norgen and in OMNIgeneGUT tubes offer a convenient method of collecting fecal material from remote participants. Regardless of collection method, all whole stool samples were dominated by Bacteroidaceae, but the second most abundant family Ruminococcaceae were significantly expanded in samples collected with both the O and N methods compared the F method, indicating that the two preservation methods may impact fecal microbiomes in a similar way. Each of the three collection methods was significantly different, although the differences between the directly frozen samples compared to either of the other two other room temperature preservation methods was most obvious.
Two previous studies have also compared fecal bacterial communities collected using OMNIgeneGUT kits which were frozen prior to processing with samples which were immediately frozen. One study found storage methods, contributed to the significant differences between samples based on Bray-Curtis dissimilarity measure, and that those collected in OMNIgeneGUT kits had a significant increase in Proteobacteria 36; while another study found that samples stored in OMNIgeneGUT tubes resulted in microbiome profiles with decreased Actinobacteria and increased Lenthisphaerae compared to those that were frozen without stabilization 15. Within our study, the preservation tubes were kept at room temperature – in accordance with manufacturer’s instructions – and at the phyla level Actinobacteria were also reduced in fecal samples collected with both the O and N methods. It is more likely then, that the reduction in Actinobacteria is a result of storing in a preservation liquid, rather than the storage temperature.
Stool sample collection methods must not sacrifice sample “viability” for convenience, therefore, where possible we recommend collecting stool in bulk and freezing immediately. As well, during sample processing technicians can record the stool form according to BSFS, and homogenize the entire sample prior to subsampling for analysis. This method eliminates any subsampling bias due to heterogenous distribution of microbes in stool, and provides enough material for multiple assays. Additionally, because this method is less hands-on for participants, it may increase compliance if multiple collections are required. For studies where it is not possible to store a large quantity of bulk stool or where frozen transportation of stool is not viable, commercial preservation tubes may be an attractive alternative. In this circumstance it is recommended to only use a single tube type and insure a standard protocol. Furthermore, if OMNIgeneGUT or Norgen collection kits are used, researchers should be cautious in interpreting the reduced abundance of Firmicutes and Actinobacteria. Lastly, collection protocols should consider that some individuals can regularly have more than one bowel movement per day, and those participants should be instructed, where practical, to collect stool at a similar time.