Effects of Dietary Antimicrobial Growth Promoters on Performance Parameters and Abundance and Diversity of Broiler Chicken Gut Microbiome and Selection of Antibiotic Resistance Genes

Abstract

Antimicrobial growth promoters (AGPs) are commonly used in broiler production. There is a huge societal concern around their use and their contribution to the proliferation of antimicrobial resistance (AMR) in food-producing animals and dissemination to humans or the environment. However, there is a paucity of comprehensive experimental data on their impact on poultry production and the AMR resistome. Here, we investigated the effect of five antimicrobial growth promoters (virginiamycin, chlortetracycline, bacitracin methyl disalicylate, lincomycin, and tylosin) used in the commercial broiler production in the Indian subcontinent and in the different parts of the world for three consecutive production cycles on performance variables and also the impact on gut bacteria, bacteriophage, and resistome profile using culture-independent approaches. There was no significant effect of AGPs on the cumulative growth or feed efficiency parameters at the end of the production cycles and cumulative mortality rates were also similar across groups. Many antibiotic resistance genes (ARGs) were ubiquitous in the chicken gut irrespective of AGP supplementation. In total, 62 ARGs from 15 antimicrobial classes were detected. Supplementation of AGPs influenced the selection of several classes of ARGs; however, this was not correlated necessarily with genes relevant to the AGP drug class; some AGPs favored the selection of ARGs related to antimicrobials not structurally related to the AGP. AGPs did not impact the gut bacterial community structure, including alpha or beta diversity significantly, with only 16-20 operational taxonomic units (OTUs) of bacteria being altered significantly. However, several AGPs significantly reduced the population density of some of the potential pathogenic genera of bacteria, such as Escherichia coli. Chlortetracycline increased the abundance of Escherichia phage, whereas other AGPs did not influence the abundance of bacteriophage significantly. Considering the evidence that AGPs used in poultry production can select for resistance to more than one class of antimicrobial resistance, and the fact that their effect on performance is not significant, their use needs to be reduced and there is a need to monitor the spread of ARGs in broiler chicken farms.

Keywords: amplicon sequencing; antibiotic growth promoter; antimicrobial resistance; broiler–chicken; chickens; gut microbiome; shotgun sequencing.

FIGURE 1

Barplots of the average normalized relative abundance of the 15 most abundant bacterial taxa identified to class level, found in different groups. “Not-assigned” are taxa identified to a lower taxonomic level than class, “Others” taxa not included in the 15 most abundant taxa.

FIGURE 2

UpSet diagram visualizing intersections of sets of OTUs between different groups.

FIGURE 3

Rarefaction curves based on Chao1, ACE, and observed OTUs. Bacterial sequences were rarefied to the minimum library size (at 76,07,352 sequences per sample) without data filtering for rare OTUs.

FIGURE 4

Beta diversity among treatments. Beta diversity plots visualized using Non-metric multidimensional scaling-based ordination at OTU level for different beta diversity metrics (A) Jaccard index, (B) Jensen-Shannon. A stress value of less than 0.1 represents a satisfactory-quality ordination. C, control; V, Virginiamycin; CT, Chlortetracycline; B, Bacitracin Methylene Disalicylate; L, Lincomycin; T, Tylosin.

FIGURE 5

Differential abundance of gut microbiota in different groups at OTU level. OTUs with significant difference in abundance among groups identified with DESeq2 and passing false discovery rate (FDR) filter, were plotted. The size of the bubbles in the bubble plot indicates the log- transformed (LN(2)) normalized (cumulative sum scaling) abundance of each OTU.

FIGURE 6

Differential abundance of gut bacteriophages in different groups. The size of the bubbles in the bubble plot indicates the normalized (normalized to per million reads) abundance.

FIGURE 7

Differential abundance of different categories of gut antimicrobial resistance genes in different experimental groups. The size of the bubbles in the bubble plot indicates the normalized (normalized to per million reads) abundance. MLS, macrolide-lincosamide-streptogramin B.