Assessing the impact of dietary interventions on the resistomes of broiler chickens

Antimicrobial resistance (AMR) is a major One Health concern, and while natural feed additives such as probiotics and phytotherapeutics are increasingly used as alternatives to antimicrobial growth promoters (AGPs) in poultry production, their potential effects on the selection of antibiotic resistance genes remain poorly understood. Therefore, our objective was to characterize the effects of a probiotic and an essential oils blend on the broiler resistome. Cobb 500 1-day-old chicks (N=320) were randomly allocated in 32 cages, with eight replicates of ten broilers per cage per treatment and were raised until day 21. Treatments consisted of four diets: a basal diet (negative control), a basal diet with Bacitracin Methylene Disalicylate (BMD) at 50 g/ton, a basal diet with an essential oil blend at 100 g/ton, and a basal diet with a probiotic (Bacillus subtilis) at 226.8 g/ton. Excreta samples were collected at three-time points (1, 10, and 21 days) to characterize broilers' resistome. The DNA extracted from these samples was sequenced using shotgun metagenomics on the NovaSeq platform and statistical analyses were done using Kruskal-Wallis and PERMANOVA to assess gene diversity. Across all samples, 823 unique ARGs were identified. These genes spanned a broad spectrum of classes, including multi-compound, metals, drugs, and biocides resistance. No significant differences in alpha diversity of these genes (P = 0.51) were observed between treatment groups; however, AMR gene diversity varied by age (P < 0.001). A statistically significant difference was observed in beta diversity across ages (P = 0.001), but not between treatments (P = 0.95). While age impacted AMR gene diversity, under our experimental conditions, antibiotics or other in-feed additives did not significantly alter broiler resistomes. This study advances poultry AMR surveillance by demonstrating that resistome diversity and composition in broiler chickens are predominantly shaped by age-dependent microbial succession, while neither in-feed antibiotics nor non-antibiotic feed additives induced persistent or treatment-specific alterations in ARG profiles under the conditions tested.