On the variability of microbial populations and bacterial metabolites within the canine stool. An in-depth analysis

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. Firmic-utes, 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) concentra-tions. 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 differ-ences 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.