Abstract
Simple SummaryThe present study investigated for the first time the impact that different sampling points have on the abundance of microbial populations and metabolites within the canine stool. We found that inner stool subsamples resulted in higher concentrations of bacterial metabolites but not of microbial populations. These findings suggest that stool subsampling is unlikely to represent the canine microbiota and metabolome uniformly. We believe that complete homogenisation of the whole stool prior to analysis may improve the final outcome when investigating the canine gut microbiome.Canine faecal microbial populations and metabolome are being increasingly studied to understand the interplay between host and gut microbiome. However, the distribution of bacterial taxa and microbial metabolites throughout the canine stool is understudied and currently no guidelines for the collection, storage and preparation of canine faecal samples have been proposed. Here, we assessed the effects that different sampling points have on the abundance of selected microbial populations and bacterial metabolites within the canine stool. Whole fresh faecal samples were obtained from five healthy adult dogs. Stool subsamples were collected from the surface to the inner part and from three equally sized areas (cranial, central, caudal) along the length axis of the stool log. All samples were finally homogenised and compared before and after homogenisation. Firmicutes, Bacteroidetes, Clostridium cluster I, Lactobacillus spp., Bifidobacterium spp. and Enterococcus spp. populations were analysed, as well as pH, ammonia and short-chain fatty acids (SCFA) concentrations. Compared to the surface of the stool, inner subsamples resulted in greater concentrations of SCFA and ammonia, and lower pH values. qPCR assay of microbial taxa did not show any differences between subsamples. Homogenisation of faeces does not affect the variability of microbial and metabolome data. Although the distribution patterns of bacterial populations and metabolites are still unclear, we found that stool subsampling yielded contradictory result and biases that can affect the final outcome when investigating the canine microbiome. Complete homogenisation of the whole stool is therefore recommended.
Highlights
Faecal Concentration of Bacterial Metabolites and pH Are Affected by Inner and Outer
To assess whether the surface differs from the centre of the stool, specimens were collected from the inside and the outside the stool log
We found large differences in number of metabolites between the inner and outer portion of stools, since inner subsamples resulted in greater concentrations of short-chain fatty acids (SCFA) and ammonia compared to the surface of the stool
Summary
Due to ethical reasons and the feasibility of collection of the samples, characterization of the intestinal microbial community is most often conducted on faecal specimens by molecular tools, which requires the bacterial DNA extraction from the faecal matrix. Several authors reported discrepancies in the abundances of bacterial taxa between studies, attributed mainly to inter-individual differences [8,9], different DNA extraction protocols and sample storage conditions [10,11,12], and the application of different Generation Sequencing protocols [13,14]. Few papers focused the attention on the impact that different sampling methods have on the variability of the human [15,16,17,18,19] and canine [20] stool microbial community, while only Gratton et al
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