Macrogenomic and Metabolomic Analyses Reveal Mechanisms of Gut Microbiota and Microbial Metabolites in Diarrhea of Weaned Piglets

Abstract

Recent studies have shown a correlation between piglet diarrhea and the gut microbiota. However, the precise mechanism by which intestinal microorganisms and their metabolites influence diarrhea in weaned piglets remains unclear. This study explored differences in the gut microbiota and associated metabolites between healthy and diarrheic-weaned piglets using macrogenomic and metabolomic analyses. The histomorphological results showed that diarrheic piglets had shorter jejunal and ileal villi, some of which were shed, compared to healthy piglets. Substantial differences in gut microbial diversity and metabolites were also observed, with Bacteroidaceae bacterium and Caudoviricetes being the main differential organisms that were strongly correlated with host status. Microbial functions, mainly the metabolism of carbohydrates, glycans, lipids, and amino acids, as well as related enzyme activities, were substantially different. The major differential metabolites were carnosine, pantothenic acid (vitamin B₅), pyridoxal, methylimidazoleacetic acid, indole-3-acetaldehyde, and 5-hydroxyindoleacetic acid. These metabolites were enriched in beta-alanine, histidine, tryptophan, and vitamin B₆ metabolism, and in the pantothenate and CoA biosynthesis pathways. Combined macrogenomic and metabolomic analyses revealed that carnosine, vitamin B₅, and pyridoxal were negatively correlated with Caudoviricetes; methylimidazoleacetic acid, indole-3-acetaldehyde, and 5-hydroxyindoleacetic acid were positively correlated with Caudoviricetes. Whereas carnosine and vitamin B5 were positively correlated with Bacteroidaceae bacterium, 5-hydroxyindoleacetic acid was negatively correlated. The decreased abundance of Bacteroidaceae bacterium and the increased abundance of Caudoviricetes and related metabolites likely contribute to post-weaning diarrhea in piglets. Therefore, the abundance of Bacteroidaceae bacterium and Caudoviricetes can likely serve as potential markers for identifying and preventing diarrhea in post-weaning piglets.

Keywords: diarrhea; gut microbiota; macrogenomic; metabolomics; weaned piglets.

Figure 1

Morphological structure of piglet intestine (100 µm): (A) jejunum of weaned healthy piglets; (B) jejunum of weaned diarrhoeic piglets; (C) ileum of weaned healthy piglets; (D) ileum of weaned diarrhoeic piglets.

Figure 2

Diversity of gut microbiota between weaned healthy piglets and weaned diarrhoeic piglets. Alpha diversity including Chao1 (A), Shannon (B) and Simpsons (C) was displayed between weaned healthy piglets and weaned diarrhoeic piglets. Beta diversity including the PCoA (D) and Wilcoxon rank sum test (E) was analyzed between weaned healthy piglets and weaned diarrhoeic piglets.

Figure 3

Composition of gut microbiota between weaned healthy piglets and weaned diarrhoeic piglets. Histogram showing top 10 relative abundance of phylum (A) and top 25 relative abundance of genus (B) and top 35 relative abundance of species (C) among samples or groups.

Figure 4

Differential microbiota between weaned healthy piglets and weaned diarrhoeic piglets at the phylum (A), class (B) and species (C) levels. * p < 0.05 means significant difference.

Figure 5

Differences in gut microbiota between weaned healthy piglets and weaned diarrhoeic piglets: (A) LefSe bar of the weaned healthy piglets and weaned diarrhoeic piglets from phylum to genus level with cladogram showing; (B) LefSe bar of the healthy and diarrhea piglets at species level; (C) LefSe bar of the weaned healthy piglets and weaned diarrhoeic piglets from phylum to family level with cladogram showing; (D) LefSe bar of the healthy and diarrhea piglets at class and phylum level.

Figure 6

Heatmap of correlation between microbial groups and diarrhea status. * means p < 0.05, |r| > 0.6.

Figure 7

Microbial function characteristics and differences between weaned healthy piglets and weaned diarrhoeic piglets: (A) the microbial function characteristics at different levels; the microbial function differences were shown by (B) principal component analysis (PCA), principal coordinate analysis (PCoA) and a one-way analysis of similarity (ANOSIM) test.

Figure 8

Differences in microbial functions between weaned healthy piglets and weaned diarrhoeic piglets revealed by metagenomic analysis: (A) amino acid metabolism; (B) carbohydrate metabolism; (C) glycan biosynthesis and metabolism; (D) lipid metabolism. * p < 0.05 means significant difference, ** p < 0.01 means extremely significant difference.

Figure 9

Diarrhea significantly changed the metabolic profiles of weaned healthy and weaned diarrhoeic piglets: (A) PLS-DA under positive ion modes; (B) PLS-DA under negative ion modes; (C) OPLS-DA under positive ion modes; (D) OPLS-DA under negative ion modes; (E) volcano plot shows the changed metabolites under positive ion modes, weaned diarrhea piglets increased 48 and decreased 43 metabolites when compared with weaned healthy piglets; (F) volcano plot shows the changed metabolites under negative ion modes, weaned diarrhea piglets increased 53 and decreased 40 metabolite when compared with weaned healthy piglets.

Figure 10

Permutation test of OPLS-DA and PLS-DA model between weaned healthy and weaned diarrhoeic piglets. Red and blue represent weaned healthy and weaned diarrhoeic piglets, respectively. PLS-DA permutation test derived from the blood metabolite profiles of weaned healthy and weaned diarrhoeic piglets at positive (A) and negative (B) ions mode. OPLS-DA permutation test derived from the fecal metabolite profiles of weaned healthy and weaned diarrhoeic piglets at positive (C) and negative (D) ions mode.

Figure 11

Pathway impact resulting from the differential metabolites using MetaboAnalyst 3.0. Small p-value and big pathway impact factor indicate that the pathway is greatly influenced.

Figure 12

Spearman correlation coefficients between gut metabolites and microbiota. The x-axis shows top 10 bacteria, while the y-axis shows the 35 significantly changed metabolites. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.01.