Bacteria, phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

Abstract

Disturbance of the beneficial gut microbial community is a potential collateral effect of antibiotics, which have many uses in animal agriculture (disease treatment or prevention and feed efficiency improvement). Understanding antibiotic effects on bacterial communities at different intestinal locations is essential to realize the full benefits and consequences of in-feed antibiotics. In this study, we defined the lumenal and mucosal bacterial communities from the small intestine (ileum) and large intestine (cecum and colon) plus feces, and characterized the effects of in-feed antibiotics (chlortetracycline, sulfamethazine and penicillin (ASP250)) on these communities. 16S rRNA gene sequence and metagenomic analyses of bacterial membership and functions revealed dramatic differences between small and large intestinal locations, including enrichment of Firmicutes and phage-encoding genes in the ileum. The large intestinal microbiota encoded numerous genes to degrade plant cell wall components, and these genes were lacking in the ileum. The mucosa-associated ileal microbiota harbored greater bacterial diversity than the lumen but similar membership to the mucosa of the large intestine, suggesting that most gut microbes can associate with the mucosa and might serve as an inoculum for the lumen. The collateral effects on the microbiota of antibiotic-fed animals caused divergence from that of control animals, with notable changes being increases in Escherichia coli populations in the ileum, Lachnobacterium spp. in all gut locations, and resistance genes to antibiotics not administered. Characterizing the differential metabolic capacities and response to perturbation at distinct intestinal locations will inform strategies to improve gut health and food safety.

Figure 1

Swine intestinal bacterial communities differentiate along longitudinal and radial axes. (a) Phylum-level distribution of lumenal bacteria through the intestinal tract. The mean per gut location is plotted. (b) Spatial distribution of bacteria in the swine intestinal tract based on assigned 16S rRNA sequences. The mean percent of assigned reads per treatment group is plotted. A dendrogram shows clustering of OTU data (Bray–Curtis dissimilarity measure), averaged by treatment and location. M, mucosa samples; L, lumen contents; red, medicated; black, non-medicated.

Figure 2

CAZy family abundances per sample. Heatmap depicts the relative abundances of several CAZy families (x-axis) by sample (y-axis). Sample names are coded by treatment status (Med, medicated; Non, non-medicated), pig number and sampling site. Relative abundance was calculated as the fraction of reads with statistically significant sequence similarity to sequences in a particular CAZy gene family, multiplied by a scaling factor (11 000). CAZy families are colored for functions highlighted in the results: starch breakdown (red), and the digestion of PCW components such as pectin (green) and hemicellulose (purple).

Figure 3

Relative abundance of dormancy (a) and phage (b) functional genes in swine gut metagenomes. (a) Biological replicates are plotted showing the standard error (s.e.) around the mean. The results of multiple pairwise comparisons (Mann–Whitney t-test) are shown, with a being significantly different from b, b from c, and so on. at P<0.05.

Figure 4

Nonmetric multidimensional scaling analysis of OTU-based bacterial 16S rRNA gene sequence abundances in individual pig intestinal samples. Environmental variables are plotted as vectors. The length of each vector is arbitrarily scaled, so only their directions and relative lengths should be considered. Lumen samples are solid and mucosal samples are outlined. Diamond, ileum; triangle, cecum; square, colon; and circle, feces.

Figure 5

Bacterial genera that are differentially present due to antibiotic treatment, based on taxonomic inference of bacteria (16S rRNA sequences) from (a) cecal and (b) colonic intestinal samples (P<0.01, q<0.05). Acholeplasma and Holdemania (higher in medicated cecum); Rhodopirellula and Hydrogenoanaerobacterium (higher in non-medicated colon) was not shown because of low abundance.