Metagenomic functional profiling to propose the anti-inflammatory properties of probiotic supplementation

Abstract

The role of the microbiome on equine health and performance is increasingly recognized, with advancements in technologies driving rapid progress. In a recent trial, supplementation of Bacillus coagulans GBI-30, 6086 to horses reduced the inflammatory response to exercise. Based on these findings, the objective of this trial was to investigate mechanisms underlying this effect using metagenomic functional profiling. In a complete crossover trial, horses (n = 18) were stratified by age, BW, BCS, breed, and sex and then randomly assigned to one of 6 starting treatments including a negative and positive control (0.23 mg/kg BW flunixin meglumine at 0 h), or one of 4 probiotics (B. coagulans GBI-30, 6086; Bacillus subtilis-1; B. subtilis-2; or Bacillus amyloliquefaciens) top-dressed to their daily ration at 8 billion cfu/d in dried whey. All horses received a diet of grass hay at 2.0% of BW daily and 4.54 kg of a commercially available feed. Each 3-wk phase consisted of a 2-wk dietary acclimation followed by a 1-wk exercise challenge and sampling. On the day of exercise, horses were offered their AM ration and then subjected to a 2-h exercise test. Rectal swabs were obtained before starting exercise and then again at 0, 2, 4, 6, 8, 24, 48, and 72 h after exercise. Swabs were extracted using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA) and the V3-V4 hypervariable region of the 16S rRNA gene was sequenced (Illumina, San Diego, CA). Data were denoized with DADA2 within the QIIME2 pipeline to identify amplicon sequence variants (ASVs). PICRUSt2 was used to predict microbial functions from ASVs, employing phylogenetic placement via epa-ng and MetaCyc pathway analysis. Pathway abundances were compared using Aldex2 Kruskal–Wallis test followed by Dunn's test for multiple comparisons. Only horses receiving the B. coagulans GBI-30, 6086 displayed a reduced inflammatory pattern therefore, only samples from that treatment and the controls were evaluated. Two pathways were identified as significantly reduced at 4 h after exercise in the B. coagulans GBI-30, 6086 and positive control groups (P < 0.05). Both pathways (PWY-4361 and PWY-7527) are related to methionine biosynthesis, which is associated with regulation of inflammation. It appears that through regulation of these pathways, the probiotic administration is attenuating the inflammation. In addition, this is the first known use of metagenomic pathway analysis to suggest a mechanism behind anti-inflammatory probiotic efficacy in horses.