Rapid regrowth and detection of microbial contaminants in equine fecal microbiome samples

Abstract

Advances have been made to standardize 16S rRNA gene amplicon based studies for inter-study comparisons, yet there are many opportunities for systematic error that may render these comparisons improper and misleading. The fecal microbiome of horses has been examined previously, however, no universal horse fecal collection method and sample processing procedure has been established. This study was initialized in large part to ensure that samples collected by different individuals from different geographical areas (i.e., crowdsourced) were not contaminated due to less than optimal sampling or holding conditions. In this study, we examined the effect of sampling the surface of fecal pellets compared to homogenized fecal pellets, and also the effect of time of sampling after defecation on ‘bloom’ taxa (bloom taxa refers to microbial taxa that can grow rapidly in horse feces post-defecation) using v4 16S rRNA amplicon libraries. A total of 1,440,171 sequences were recovered from 65 horse fecal samples yielding a total of 3,422 OTUs at 97% similarity. Sampling from either surface or homogenized feces had no effect on diversity and little effect on microbial composition. Sampling at various time points (0, 2, 4, 6, 12 h) had a significant effect on both diversity and community composition of fecal samples. Alpha diversity (Shannon index) initially increased with time as regrowth taxa were detected in the amplicon libraries, but by 12 h the diversity sharply decreased as the community composition became dominated by a few bloom families, including Bacillaceae, Planococcaeae, and Enterococcaceae, and other families to a lesser extent. The results show that immediate sampling of horse feces must be done in order to ensure accurate representation of horse fecal samples. Also, several of the bloom taxa found in this study are known to occur in human and cattle feces post defecation. The dominance of these taxa in feces shortly after defecation suggests that the feces is an important habitat for these organisms, and horse fecal samples that were improperly stored can be identified by presence of bloom taxa.

Faecal bacterial composition in horses with and without free faecal liquid: a case control study

“…Due to that participating horses were located all over Sweden and Norway, and sampling periods were the same for all participants, it was not possible to achieve the exact same number of days between sampling and arrival at laboratory. In a study conducted by Beckers et al 37 , the results showed that equine faecal samples changed in microbial composition rapidly post defecation. Changes included changed diversity and community composition, with increase of several bacterial families including Bacillaceae , Planococcaeae and Enterococcaceae 37 .…”

“…In a study conducted by Beckers et al 37 , the results showed that equine faecal samples changed in microbial composition rapidly post defecation. Changes included changed diversity and community composition, with increase of several bacterial families including Bacillaceae , Planococcaeae and Enterococcaceae 37 . Although the time from sampling to arrival at the laboratory varied in the current study, samples from each matched pair were taken on the same day and sent in the same postal package resulting in no difference in number of days between sampling and analysis between case and control horse samples.…”

“…One limitation of the study was that time from sampling to preparation for analysis may have affected the faecal microbiota composition, either by rapid regrowth of microorganisms and/or microbial contaminants 37 . Due to that participating horses were located all over Sweden and Norway, and sampling periods were the same for all participants, it was not possible to achieve the exact same number of days between sampling and arrival at laboratory.…”

Utility of a portable desiccant system for preservation of fecal samples for downstream 16S rRNA amplicon sequencing

“…That said, future studies are needed to determine the maximal moisture capacity of the desiccant cartridges and effects of variable moisture content on efficacy. Notably, one study examining the effects of time post-defecation at ambient temperature on the composition of equine feces also detected significant increases in the relative abundance of microbes in the families Bacillaceae and Planococcaceae (as well as Enterococcaceae ), and a trend ( p = 0.055) toward increased abundance of family Moraxellaceae (Beckers et al, 2017). The close agreement of the current room temperature samples with those results (generated in Southern Louisiana) suggests that the changes observed with sub-optimal sample handling of equine feces are relatively conserved across geographical regions, and that the DriBank instrument was capable of abrogating the majority of those changes.…”

Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome

“…For example, a meta-analysis has shown that human rectal swabs stored at room temperature experience characteristic growths of Gammaproteobacteria (Amir et al, 2017). A similar increase in the abundance of proteobacteria was also seen in the microbiota of tadpoles (Nanorana parker) gut samples left thawing at ambient temperature for 12 h (Anslan et al, 2019), while equine feces collected 12 h post-defecation showed sharp increases of families from the Firmicutes phylum (Beckers, Schulz & Childers, 2017). Though the effect of sample storage at ambient temperature has been well-documented across study systems, it has remained unclear whether freezing at −80 C is overly conservative.…”