Impact of Bacillus spp. spores and gentamicin on the gastrointestinal microbiota of suckling and newly weaned piglets

Abstract

Administrating antibiotics to young piglets may have short- and long-term consequences on the gut microbiota. We hypothesised that these consequences may be alleviated by concurrent probiotic administration. The study objective was to investigate the effect of administrating gentamicin and a mixture of Bacillus (B.) licheniformis, B. subtilis and B. amyloliquefaeceans spores on the gut microbiota of piglets pre- and post-weaning. Twenty-four sows and their litters were randomly allocated to four treatment groups receiving; a) Bacillus spore mixture (six B. subtilis, two B. amyloliquefaeceans, and one B. licheniformis) fed to sows and piglets (PRO); b) gentamicin (5 mg per day) administered to piglets on day 4, 5, and 6 of age (AB); c) Bacillus spore mixture fed to sows and piglets, and gentamicin to piglets (PRO+AB); or d) no administration of probiotics or antibiotics (CTRL). Faecal and digesta samples were collected repeatedly during the study. Selected samples were subjected to 16S rRNA gene sequencing, culture counts, and organic acid, biogenic amine and tissue gene expression analysis. Treatment had a significant effect on the faecal microbial community composition on day 28 and 42, and colonic community on day 28. Faecal species richness (observed and estimated) and Shannon index, and colonic species richness, were higher in AB compared to PRO piglets on day 28, and were not significantly different from day 42. PRO piglets had the highest faecal concentration of iso-butyric acid on day 7 and a higher butyric acid concentration compared to CTRL piglets. We conclude that gentamicin and Bacillus spores influence the gut microbial diversity of piglets, although administration of gentamicin did not result in dysbiosis as hypothesised.

Fig 1. Microbial culture of Bacillus spores.

Bacillus spores in digesta (log cfu/g wet weight) from stomach, ileum, caecum, and mid colon, sampled on day 3, 28, and 42 of age from piglets administered gentamicin (AB; n = 71), piglets administered Bacillus spores (PRO; n = 68), piglets administered both gentamicin and Bacillus spores (PRO+AB; n = 71), and control piglets receiving neither gentamicin nor Bacillus spores (CON; n = 72). Values are presented as least-square means and the 95% confidence intervals presented as vertical bars. Bars embraced by horisontal brackets market by *(0.01≤p<0.05), **(0.001≤p<0.01) or ***(p<0.001) are significantly different.

Fig 2. Heatmaps showing bacterial phyla and genera; faecal samples.

Heatmaps of faecal samples collected at 7, 28, and 42 days of age from piglets administered gentamicin (AB; n = 53), Bacillus spores (PRO; n = 50), both gentamicin and Bacillus spores (PRO+AB; n = 53), and control piglets not receiving gentamicin or Bacillus spores (CTRL; n = 51). Heatmaps show the relative abundances (%) of (A) the ten most abundant phyla and (B) the 20 most abundant genera in faecal samples. Colours represent the relative abundances on a logarithmic scale.

Fig 3. Species richness and diversity; faecal samples.

Observed and estimated species richness and Shannon diversity index of faecal samples collected at 7, 28, and 42 days of age from piglets administered gentamicin (AB; n = 53), Bacillus spores (PRO; n = 50), both gentamicin and Bacillus spores (PRO+AB; n = 53), and control piglets receiving neither gentamicin nor Bacillus spores (CTRL; n = 51). Boxplots show the (A) observed and (B) estimated species richness and (C) Shannon diversity index.

Fig 4. Heatmaps showing bacterial phyla and genera; digesta samples.

Heatmaps of ileal and colonic digesta samples collected at 3, 28, and 42 days of age from piglets administered gentamicin (AB; n = 33), Bacillus spores (PRO; n = 33), both gentamicin and Bacillus spores (PRO+AB; n = 31), and control piglets receiving neither gentamicin nor Bacillus spores (CTRL; n = 29). Heatmaps show the relative abundances (%) of (A) the eight most abundant phyla in ileal digesta, (B) the 20 most abundant genera in ileal digesta, (C) the eight most abundant phyla in colonic digesta, and (D) the 20 most abundant genera in colonic digesta. Colours represent the relative abundances on a logarithmic scale.

Fig 5. Species richness and diversity; digesta samples.

Observed and estimated species richness and Shannon diversity index of ileal and colonic digesta samples collected at 3, 28, and 42 days of age from piglets administered gentamicin (AB; n = 33), Bacillus spores (PRO; n = 33), both gentamicin and Bacillus spores (PRO+AB; n = 31), and control piglets receiving neither gentamicin nor Bacillus spores (CON; n = 29). Boxplots show the (A) observed and (B) estimated species richness, and (C) Shannon diversity index.