Temporal dynamics of the cecal and litter microbiome of chickens raised in two separate broiler houses

Abstract

In this study, we investigated the dynamics of the ceca and litter microbiome of chickens from post-hatch through pre-harvest. To achieve this, six hundred one-day old Cobb 500 broiler chicks were raised on floor pens for 49 days in two separate houses. We performed short-read and full-length sequencing of the bacterial 16S rRNA gene present in the meconium and in cecal and litter samples collected over the duration of the study. In addition, we determined the antimicrobial resistance (AMR) phenotype of Escherichia coli and Enterococcus spp. isolated from the meconium and the ceca of 49-day old chickens. We monitored the relative humidity, temperature, and ammonia in each house daily and the pH and moisture of litter samples weekly. The overall microbial community structure of the ceca and litter consistently changed throughout the course of the grow-out and correlated with some of the environmental parameters measured (p < 0.05). We found that the ceca and litter microbiome were similar in the two houses at the beginning of the experiment, but over time, the microbial community separated and differed between the houses. When we compared the environmental parameters in the two houses, we found no significant differences in the first half of the growth cycle (day 0-21), but morning temperature, morning humidity, and ammonia significantly differed (p < 0.05) between the two houses from day 22-49. Lastly, the prevalence of AMR in cecal E. coli isolates differed from meconium isolates (p < 0.001), while the AMR phenotype of cecal Enterococcus isolates differed between houses (p < 0.05).

Keywords: antimicrobial resistance (AMR); broiler chickens; environmental condition; microbiome; pre-harvest.

FIGURE 1

Graphical representation of the experimental design.

FIGURE 2

Change of alpha diversity indices from rarefied 16S rRNA gene libraries over time. Boxes indicate the interquartile range (75th to 25th) of the data. The median value is shown as a line within the box. Whiskers extend to the most extreme value within 1.5 * interquartile range and dots represent outliers. Only significant changes are shown with asterisks: *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001. The color of asterisks shows pairwise comparisons between ceca (orange) or litter (green) samples.

FIGURE 3

Shifts of microbial community composition in cecal and litter samples. Beta diversity was calculated using a non-metric multidimensional scaling (NMDS) ordination of 16S rRNA gene libraries based on Bray-Curtis distances. Colors show samples obtained on different days and the shape displays samples from different houses. Only significant (p < 0.05) environmental variables were fitted onto the ordination.

FIGURE 4

Heatmaps illustrating ASVs that were significantly differentially abundant between houses. (A) Ceca samples. (B) Litter samples. The effect size depicts the negative log of the q-value multiplied by the sign of the coefficient. A positive effect size denotes higher abundance in house 2. A negative effect size denotes higher abundance in house 1. Taxonomy of the ASVs is indicated at the genus levels or at the lowest rank that could be assigned confidently (Bootstrap support above 50). Only significant changes are shown.

FIGURE 5

Phylum-level classification of 16S rRNA gene sequence reads. Samples were grouped by days and split by sample type (Meconium, Ceca, Litter). Relative abundance was normalized by total sum scaling and the mean for each sample group is depicted.

FIGURE 6

Heatmap showing the relative abundance of the 50 most abundant ASVs. Samples were grouped by days and split by sample type (Meconium, Ceca, Litter). Relative abundance was normalized by total sum scaling and the mean for each sample group is depicted. Taxonomy of the ASVs is indicated at the genus levels or at the lowest rank that could be assigned confidently (Bootstrap support above 50).

FIGURE 7

Antimicrobial susceptibility testing of (E) coli and Enterococcus isolates. (A) Percentage of E. coli isolates in the meconium (n = 25) and ceca (n = 96) that are resistant to 0–10 antibiotics. (B) Percentage of cecal E. coli isolates from house 1 (n = 48) and house 2 (n = 48) that are resistant to 0–10 antibiotic drugs (C) Percentage of Enterococcus isolates in the meconium (n = 25) and ceca (n = 90) that are resistant to 0–10 antibiotics (D) Percentage of cecal Enterococcus isolates from house 1 (n = 42) and house 2 (n = 48) that are resistant to 0–10 antibiotic drugs.